「生体分子情報による次世代型がん個別最適化治療法の開発」

平成 25 年度～平成 29 年度
私立大学戦略的研究基盤形成支援事業
研究成果報告書

平成 30 年 3 月
学校法人名 東京医科大学
大学

名 東京医科大学

研 究 組 織 名 医学総合研究所
研究代表者 大屋敷 純子（東京医科大学医学部医学科教授）

目

刊行にあたって次････････････････････････････････････････････････････････ 1

プロジェクトの概要
・ 学校法人名 ･･･････････････････････････････････････････････････････ 3
・ 大学名 ･･･････････････････････････････････････････････････････････ 3･･･････････････････････････････････････････････････････ 3

・ 研究組織名・ プロジェクト所在地･･･････････････････････････････････････････････ 3

・ 研究プロジェクト名･････････････････････････････････････････････････ 3
・ 研究観点 ･････････････････････････････････････････････････････････ 3･･･････････････････････････････････････････････････････ 3

・ 研究代表者

・ プロジェクト参加研究者数 ･････････････････････････････････････････ 3･････････････････････････････････････････････････････ 3

・ 該当審査区分

・ 研究プロジェクトに参加する主な研究者 ･････････････････････････････ 3
・ 研究の概要･･･････････････････････････････････････････････････････ 4

・ キーワード･･･････････････････････････････････････････････････････ 10

・ 研究発表の状況 ･･･････････････････････････････････････････････････ 10・ その他の研究成果等･･･････････････････････････････････････････････ 29

・ 「選定時」及び「中間評価時」に付された留意事項及び対応･････････････ 30
・ 施設・装置・設備・研究費の支出状況 ･･･････････････････････････････ 31
・ 施設・装置・設備の整備状況
・ 研究費の支出状況･･･････････････････････････････････････ 32･････････････････････････････････････････････････ 32

プロジェクトの成果・

主要論文････････････････････････････････････････････････････････ 45

・

その他･････････････････････････････････････････････････････････ 315

私立大学戦略的研究基盤形成支援事業「生体分子情報による次世代型がん個別最適化治療法の開発」研究成果報告書刊行にあたって東京医科大学においては、平成 10 年度に文部科学省・私立大学学術研究高度化推進事業のハイテク・リサーチ・センター整備事業に選定され、学内の研究基盤を強化するために「難病治療研究センター」が設立されました。以後、継続的に文部科学省私学研究高度化事業を軸に着実に研究を推進し、平成 22 年には「医学総合研究所」に発展しました。本事業は平成 20 年

24 年に選定された文部科学省・戦略的研究基盤形成支援事業「分子情報に基づく難病研究拠点
形成」（課題番号 S0801020）を「がん研究」に特化させ飛躍的に発展させた先端研究であると同時に、平成 10 年度から一貫して文部科学省私学研究高度化事業に従事してきた医学総合研究所の２部門（分子腫瘍研究部門、免疫制御研究部門）の研究の集大成であるとも言えます。専門性の極めて高い２部門が協力することによって、テーマが散逸することなく、学内の研究基盤を揺るぎないものとし、出口戦略に繋げることができました。また、新しい研究シーズの発
見や若手研究者の活躍は本学における研究の輝かしい未来を予感させるものと考えます。ご支援ご協力を賜りました文部科学省、学校法人東京医科大学、国内外の共同研究者の皆様に心より厚く御礼申し上げます。
東京医科大学医学総合研究所
教授 大屋敷純子-1-プロジェクトの概要

（様式 2）
法人番号プロジェクト番号131054
S1311016

平成２５年度～平成２９年度「私立大学戦略的研究基盤形成支援事業」
研究成果報告書概要
１ 学校法人名

東京医科大学２ 大学名 東京医科大学

３ 研究組織名 生体分子先端研究センター
４ プロジェクト所在地 東京都新宿区西新宿 6-7-1
５ 研究プロジェクト名 生体分子情報による次世代型がん個別最適化治療法の開発
６ 研究観点研究拠点を形成する研究７ 研究代表者
研究代表者名

所属部局名

職名

大屋敷純子

医学総合研究所教授

８ プロジェクト参加研究者数
９ 該当審査区分

48 名

理工・情報

生物・医歯

人文・社会

１０ 研究プロジェクトに参加する主な研究者
研究者名

大屋敷純子

善本 隆之

大屋敷一馬

梅津 知宏

黒田 雅彦

所属・職名

医学総合
研究所・教
授
医学総合
研究所・教
授
血液内科
学分野・教
授
血液内科
学分野・講
師
分子病理
学分野・教
授

プロジェクトでの研究課題

プロジェクトでの役割

次世代シーケンス技術の応 プロジェクトの立案と総用
括免疫評価法確立とその免疫
免疫制御法の開発
治療への応用個別化医療のための分子マ
がん細胞の変容解明
ーカー探索
エクソソームを標的とした診 がん環境にかかわる生断治療技術開発
体分子の解明人工知能を用いた新規診断 出口戦略と個別化医療技術および DDS の開発の確立＜研究者の変更状況（研究代表者を含む）＞
旧
ﾌﾟﾛｼﾞｪｸﾄでの研究課題（変更の時期：平成

所属・職名

年

月

研究者氏名

日）
-3-

プロジェクトでの役割

（様式 2）
法人番号プロジェクト番号131054
S1311016

新変更前の所属・職名変更（就任）後の所属・職名

研究者氏名

プロジェクトでの役割

１１ 研究の概要（※ 項目全体を１０枚以内で作成）（１）研究プロジェクトの目的・意義及び計画の概要
近年、次世代シーケンス技術の台頭により膨大な DNA 情報の高速処理により個人ゲノムを解読できるようになった。このような生命科学の進歩を医療に役立てるためには医学、薬学、工学、物質科学などの分野横断的アプローチが必須である。一方、近年がん多死化社会の到来が大きな課題となっているが、がん克服のためにはがん細胞の撲滅と同時に副作用を最小限にし、二次発癌を予防することが重要である。このためにはがんの特性や患者の薬剤感受性・免疫学的背景により個別化し、薬剤投与量・投与方法を最適化した医療の確立が望まれている。本研究では新たに次世代シーケエンス技術の導入により、今までの文部科学省
戦略的研究基盤形成支援事業により構築された分野横断的研究体制を飛躍的に発展させ、がん多死化社会に立ち向かう若手人材の育成とともに、がんの個別最適化医療の実現を目的とする。基礎医学と臨床医学の有機的ブリッジングによる新たなドラッグデリバリーシステムの構築や抗がん剤過剰投与回避指針の提案は医療経済学的効果も大きく、社会への還元が期待される。
（２）研究組織本プロジェクトでは、医学総合研究所二部門（分子腫瘍研究部門; 以下「医総研腫瘍」、免疫制御研究部門；以下「医総研免疫）をハブとし、基礎医学（分子病理学分野）、血液内科学分野（以下血内）を中心に臨床医学のブリッジングとともに、産学連携、医薬工連携による人的交流、技術支援などを積極的に行い、下図に示した通り研究基盤構築を行う。実現に際しての最大の問題点と考えられるデータの高速処理については三井情報開発および工学院大学
と共に簡便な高速情報解析システムを共同開発する。先端分子探索寄附講座は平成 28 年
以後、「医総研腫瘍」に統合されたのち、平成 29 年度にロシア発ベンチャー企業（KINTAROCELLS POWER 社）との産学連携講座「細胞治療研究開発講座」に発展した。

（３）研究施設・設備等
基礎―臨床ブリッジングによる情報共有と有効利用のため、東京医科大学病院内の教育研究棟 14 階フロアー（125 ㎡）に生体分子先端研究センターを設置し、副都心を中心とした研究拠点を形成するための研究施設を活用し、平成 25 年に同センターに次世代シーケンス(NGS)
システムを、平成 26 年に同じフロアーにある細胞センターに生細胞動画撮影システム（Incucyte）などの設備を整備し、臨床各科で共同利用している。
-4-

（様式 2）
法人番号プロジェクト番号131054
S1311016（４）研究成果の概要 ※下記、１３及び１４に対応する成果には下線及び＊を付すこと。
＜現在までの進捗状況及び達成度＞
本事業により研究基盤が強化された共同研究の成果を中心にその概要と達成度を列記する。３年目に研究進捗状況、費用対効果などを鑑み、重点領域を絞り込むとともに、病理診断に人工知能を用いた新規アプローチを加えるなど、若干の軌道修正を行なった。それぞれのテーマは縦割りでなく密接に関係しているが、役割分担と共同研究体制を明確化するため、研究組織に示した各分野の役割を付記し、中間報告時の課題であった次世代シーケン
サを用いた共同研究の成果についても報告する。( )内は 13,14 に対応する成果で、最終報告であるので、論文（＊）を中心に、現時点で論文化されてないものなどについては学会発表（＊＊）を引用した（番号は 13 および１4 に対応）。特許、その他社会連携に関する成果についても記載した。
なお、中間報告以後の軌道修正により、テーマが散逸しないように、がん環境、遺伝的背景の解明、免疫制御を重点領域とし、成果が集約することを目指した。ことに中間報告の際の課題であった「次世代シーケンスシステムの臨床応用」に関しては本事業で整備されたター
ゲットシーケンス用の解析装置（MiSeq）をフル稼働すると同時に、全エクソーム解析に関しては京都大学 小川誠司教授に助言をいただき、手探りではあったが当初の目的に近い成果をあげることができた（詳細は後述）。これは、ポスドク、RA をはじめとする若手研究者の精力的な研究活動によるものである。一方、加齢という要素を加味し、人工知能を応用した病理診断など技術的進歩を活用して研究を展開した結果、新しいシーズも見出されたので、以下、主たる成果について記述する。
【がん幹細胞とがん環境の分子基盤】
1. がん幹細胞を標的とした治療戦略：新たな分子標的・バイオマーカーの探索(1) 造血器腫瘍にける新規分子標的に関する研究成果： 血内 医総研腫瘍
慢性骨髄性白血病(CML)根治に関する一連の研究では、MAPK 阻害剤（＊28. Okabe S et al.,J Haematol Oncol. 2015）、polo-like kinase 阻害剤（＊29. Okabe S et al., Oncotarget 2015）,
HDAC 阻害剤（＊42. Okabe S et al., J Haematol Oncol. 2014）、PI3K および mTOR 阻害剤（＊
45. ＊Okabe S et al., Cancer Biol Ther. 2014）について、ぞれぞれの有用性を報告した（達成率 100% ） 。 こ れ ら の 幹 細 胞 研 究 を 一 歩 進 め 、 平 成 27 年 度 よ り iPS 誘 導 ベ ク タ ー(CytoTune-Ips2.0)を用いて、primary 癌細胞を re-programing する癌幹細胞システムを確立した 。 そ し て 骨 髄 異 形 成 症 候 群 に お い て は 患 者 検 体 よ り CD34 陽 性 細 胞 を 分 離 し 、CytoTune-iPS2.0 によるがん幹細胞システム(MDS-iPSC)の作成に成功した。現在、先行する分子標的薬(BBI608)をリード Compound に据えて、新規がん幹細胞分子標的薬開発の基盤形成を目指す新しいアプローチを試みた（＊18. Tauchi T et al., Journal of Cancer Scienceand Therapy. 2017）。幹細胞システムは現時点では実用化まではまだ遠いものの、新しいシーズとして期待される (達成率 80%)。
(2) miRNA によるがん診断：分子病理 医総研腫瘍 血内 婦人科 消化器科
臨床各科でバイオマーカーとしてリキッドバイオプシーを用いた解析が精力的に行われた。
例えば、血清 miR-215 レベル（＊22. Ohyashiki K et al., Int J Mol Sci. 2016）や末梢血リンパ球の miR-148b(＊47. Ohyashiki JH et al., Drug Des Devel Ther. 2014) が CML の投薬中止基準の指標となりうること、胃癌における miR-200c (＊20. Kurata A et al., Oncol Rep. 2018)、大腸癌におけるワクチン療法の有用性の予測マーカーとしての miR-125b-1 and miR-378a (＊Tanaka H et al., Cancer Sci. 2017)、子宮頸癌の重症度と関連する miRNA 発現様式（＊27.
-5-（様式 2）
法人番号プロジェクト番号131054
S1311016

Nagamitsu Y et al., Mol Med Rep. 2016））などを報告した。これらの成果は血清を試料として用いたアッセイにより得られたものであるが、リキッドバイオプシーの特性を活かし、循環がん細胞や残存する微小病変の変化を早期に正確に判定する目的で、エクソソーム内の miRNA 測
定へとアッセイ方法の軌道修正を試みた。血液内科学分野の骨髄移植チームは、造血幹細胞移植を受けた患者の血清よりエクソソームを抽出し、Graft vs host disease 発症時に免疫関連の数種類の miRNA が変動することを見いだし、現在投稿中である（＊＊51. Yoshizawa S
et al., 第 77 回日本血液学会学術集会、2015）。
(3) エピジェネティック修飾薬の分子機構解明と耐性克服: 医総研腫瘍 血内
がん克服の上で、避けて通れない問題として、薬剤耐性の獲得がある。アザシチジンは DNAの脱メチル化をもたらすエピジェネティック修飾薬で、造血器腫瘍に有用とされているが、早期に薬剤耐性を獲得するためその分子機構の解明が急務とされている。アザシチジン耐性
白血病細胞株を用いた解析の結果、DNA 損傷/修復機構の異常が耐性獲得の本体であることを報告した（＊48. Imanishi S et al., Biochem Pharmacol. 2014）。また、市販リウマチ治療薬であるレフルノミフドの活性代謝物テリフルノミドがアザシチジン耐性細胞にも有用であることを見いだし(＊17. Imanishi S et al., Oncotarget, 2017)、ドラッグリポジショニングの一例として特許を出願した（発明者: 今西 哲ほか
出願人: 東京医科大学、出願番号： 特願2015-183778）＊。ヘテロクロマチン１（HP１）やブロモドメイン蛋白の阻害剤に着目しアザシチジン耐性克服に対して多角的アプローチを試みている(＊＊34. Imaniashi S, et al., The 78th
Annual Meeting of Japanese Society of Hematology, 2016)。これらの知見のうち市販薬のテリ
フルノミドの併用薬としての有用性は実用化が期待される。
(4) がん環境を標的とした治療戦略・エクソソームの役割：医総研腫瘍 血内
近年、「がん微小環境」内におけ る細胞間相互作用機序に「エクソソーム」と呼ばれる
30-120nm の細胞外小胞が関与していることが注目されている。たとえば、低酸素環境におけるがん細胞由来エクソソームががん環境を構築する周辺細胞、なかでも、血管内皮細胞に及ぼす影響について検討し、短期間の低酸素環境でも、腫瘍由来エクソソームの血管新生誘導作用を認めることを見いだした(＊59. Tadokoro H et al., J Biol Chem 2013)。さらに、生体内のように長期に持続する低酸素環境では特異的に miR-135b が発現しており、miR-135bがエクソソームを介して血管内皮細胞に取り込まれて、その血管新生を亢進することを明らかにした（＊49. Umezu T et al., Blood 2014）（達成率 95%）。この事実は、「情報の運び屋」としてのエクソソームが治療の標的として注目を集めるきっかけとなり、日本経済新聞にも「がんの新しい治療戦略」として大きく取り上げられた。このがん環境における一連のエクソソーム研究については英国王立協会（Royal Society）の科学会議に招聘され、その概要を紀要に報告した（＊4. Ohyashiki JH et al., Philosophical Transactions of the Royal Society B: Biological
Sciences. 2018）*。平成 28 年度からは骨髄微小環境の主たる構成要素である骨髄間質細胞に着目し、がん細胞と間質細胞のクロストークにおけるエクソソームの役割について解析を進めた。多発性骨髄腫において、患者由来骨髄間質細胞から放出されるエクソソームが腫瘍 血 管 新 生 を 促 進 さ せ る こ と を 見 出 し た （ ＊ ＊ 2. Umezu T et al., Annual Meeting ofInternational Society for Extracellular Vesicles 2018）（論文投稿中）*。このことより、多発性骨髄腫においても固形腫瘍における Cancer associated fibroblast(CAF)同様にがん環境が腫瘍の発育を促進し、その鍵を握っているのがエクソソームであると言える。現在、多発性骨髄腫患者由来の骨髄間質細胞から分泌されるエクソソーム以外の液性因子についてもセクレトーム解析を行い、「がん環境を標的とした治療戦略」へ向けて標的分子を探索中である。
(5) 分子基盤から出口戦略へ-6-

（様式 2）
法人番号プロジェクト番号131054
S1311016

本事業では miRNA 補充療法について研究を進めると同時に、miRNA を包括し、その情報伝搬を担っているエクソソームの二方向から研究を展開した。そして最終的に骨髄間質細胞の有用性について研究を展開することになった。結果的には細胞治療として健常人由来骨髄間質細胞を扱う KINTARO CELLS POWER 社との産学連携研究に発展し、新しいシーズの実用化の第一歩を進むことになった。
(1) miRNA 補充療法の新展開：分子病理 眼科
miRNA 補充療法はがん化の過程で発現が低下したがん抑制性の miRNA を細胞外から補充することで、その miRNA が標的とする複数のがん遺伝子の発現を抑制する革新的な治療法であるが、実用化には核酸に対する免疫応答を抑制する技術の開発が必須である。そこで、
生体分子情報に基づき miRNA の中で唯一 DICER に依存しないで発現する Pre-miR-451 の構造に着目し、研究を進めた。他の miRNA より若干短い一本鎖ヘアピン構造を有する
Pre-miR-451 の構造を基盤として熱力学的に安定なヘアピン構造を保つように短縮化した 30
塩基の「短縮型 mimic miRNA」作製に成功した。さらに「短縮型 mimic miRNA」は RNAi 干渉を誘導する無修飾核酸として最短であり、in vivo および in vitro の実験から免疫応答を誘導しにくい核酸であることが明らかとなった(＊23. Taketani Y et al. Mol Ther Nucleic Acids 2016)。
この短縮化技術は他の miRNA や siRNA にも広く応用できたことから、副作用のない RNA 医
薬品のプラットフォームとして期待されるため、ボナック社と連携して臨床応用の実現をめざしている（発明者:黒田雅彦ほか 出願人: 東京医科大学出願番号： 特願 2016-168499）。
(2) 健康人由来骨髄間質細胞によるエクソソーム治療：医総研腫瘍 血内 産学連携講座
骨髄間質細胞は、免疫原性が低い事より細胞治療のソースとして注目されている。本事業では健常者由来骨髄間質細胞が放出するエクソソームに着眼して検討を進め、健常者由来骨髄間質細胞、特に若年者由来骨髄間質細胞が放出するエクソソームに抗腫瘍血管新生能を有する miRNA が内包されていることを見出した。これらの治療効果の高い miRNA とエクソソーム改変技術を応用してがん環境の改善を目指した治療戦略を構築した(＊16. Umezu T etal., 2017)。 若年者と高齢者ではエクソソーム内容物の変化が治療効果と大きく関係すること
より、加齢という要素に着目し、KINTARO CELLS POWER 社と連携し、新たな研究シーズとして「加齢による骨髄間質細胞機能の分子生物学的解析」に積極的に取り組んでいる。このことは骨髄間質細胞を用いた細胞治療の品質評価のみならず、骨髄間質細胞由来のエクソソームなどの分泌物を用いた次世代型細胞治療につながると考えられる。実際に骨髄間質細胞由来のエクソソーム投与が造血幹細胞移植後の Graft versus host disease に有効であったという報告がある。したがって、より安全な、より効率の良い、エクソソーム治療の実現のためにはエクソソーム改変技術の応用のみならず、エクソソームのバイオロジーに関する基礎的検討が急務と考えられる。
【免疫制御】 医総研免疫 血内 医総研腫瘍
免疫学的側面からのアプローチとしては、従来の基盤研究に加えて、免疫パラメーターの適正評価法を確立し、抗がん剤減量や中止のパラメーターとなりうるエビデンスを蓄積することを目指して、研究を進めた。基盤研究の成果としては IL-7 含有卵白アルブミン（OVA）-ポリエ
チレングリコール（PEG）-ポリアミノ酸ブロック共重合体ミセルによる抗腫瘍効果(35. Toyota H
et al., Oncol Rep. 2015)、IL-27 の抗腫瘍効果(＊33. Yoshimoto T et al., Cancer Sci 2015, 33.
＊Mizoguchi I et al., Oncoimmunology 2015, ＊62. Chiba Y et al., PLoS ONE 2013)について次々と報告した。また、IL-27 の骨髄造血幹細胞に対する作用については、骨髄系前駆細胞へ誘導することにより感染やがんを抑制することを見出した（＊24. Furusawa J et al., PLoS

-7-

（様式 2）
法人番号プロジェクト番号131054
S1311016

Pathog 2016, 10. ＊Chiba Y et al., Cancer Res. 2017, ＊3. Orii N et al., Oncoimmunology2018）＊。一方、眼科領域では眼内リンパ腫とウイルス感染症との関係を解析し、慢性炎症反応に伴う免疫制御機構の異常がリンパ発症に深く係ることを見いだした（2. Usui Y et al.,Scientific Reports 2018）。
一方、本事業における最大の成果は CML 患者における個別最適化判断基準の提唱である。すなわち、 健常人、イマチニブ投与中で寛解を維持できている患者、イマチニブ中止にもかかわらず後寛解を維持できている患者群の末梢血単核球の FACS 解析を用いた比較検討
より、NK 細胞や CD8+T 細胞の活性化がイマチニブ中止の判断基準になり得る可能性を世界に先駆けて明らかにした (＊63. Mizoguchi I et al., Cancer Sci 2013, ＊52. Yoshimoto T et al.,Oncoimmunology 2014)（達成率 100%）。CML 寛解維持のためには終生イマチニブを飲み続けていなければならないとされていた時代から、「ストップイマチニブ」に大きく時代が変わろうとしている今日、イマチニブ中止の免疫学的判断基準の提唱は国内外で大きな反響を呼んだ。チロシンキナーゼ阻害剤と免疫学的背景については国内の多施設共同研究の結果、ダサチニブによる制御性 T 細胞の抑制が有効性と関係していることが報告され（＊1. Najima Y
et al., Leukemia Research, 2018）、免疫制御という側面からのアプローチは今後ますます重要
性を増すと考えられた。
【遺伝的背景】(1) BIM 遺伝子多型を指標とした個別最適化医療の確立：医総研腫瘍 血内 小児科
がん診療における遺伝的素因という大きなテーマのうち、本事業では薬剤感受性を予知する
コンパニオン診断の実用化という視点から、まず BIM 遺伝子多型に的を絞り、研究を進めた。すなわち、シンガポールのグループから BIM 遺伝子多型とチロシンキナーゼ阻害剤感受性との関係が報告された事に注目し、「ストップイマチニブ」患者で BIM 遺伝子多型を解析し再発例には BIM 遺伝子多型が存在することを報告した（＊61. Katagiri S et al., 2013）。また
BIM 遺伝子欠失と一塩基多型を同時に検出できる簡便なアッセイ法を株式会社 BML と連携して開発し（＊50. Ohyashiki K et al., J Hematol Transfus. 2014）*、多発性骨髄腫、悪性リンパ腫、アルツハイマー病などで解析を行った。その結果、悪性リンパ腫のうち、ろ胞性リンパ腫
では BIM 遺伝子多型を有する症例では化学療法抵抗性で再発しやすいことを見いだした（＊＊3. 片桐誠一朗ほか、第 115 回日本内科学会講演会、2018）（達成率 95%）。
(2) がんクリニカルシーケンスの確立 医総研腫瘍 眼科 血内本学におけるがんクリニカルシーケンスの導入を目的として、平成 25 年度に次世代シーケン
サ（MiSeq）を整備し、臨床各科で共同研究を展開した。平成 26 年度には稼働時間が飛躍的に増加し、平成 30 年 3 月の平均稼働時間は 480 時間/月で、若手研究者中心に情報を共有しながら研究を展開した。データ解析のパイプラインが構築された後は、エクソーム解析とターゲットシーケンス解析をそれぞれの利点を生かして使い分けて研究を進め、前者では白血病晩期再発例における微小クローンの推移による再発予知（＊12. Katagiri S et al., Leuk Lym
2018）、後者では骨髄系腫瘍の骨髄間質細胞の遺伝的多様性の解明(＊19. Azuma K et al.,
Leukemia Res., 2017)、眼科領域の腫瘍のエクソーム解析など、臨床のニーズに応じて整備された次世代シーケンスシステムにより学内の研究基盤がより強固なものとなった（達成度80%）。加齢に伴って発生する遺伝子変異が二次発癌の発症にどのようにかかわってくるかについて、悪性リンパ腫を中心に研究をさらに進めている。ことに造血器腫瘍においては健康人においてしばしば認められる clonal hematopoiesis with indeterminate potential (CHIP)が加齢に伴い骨髄系腫瘍に発展することが注目されており、ますます加速される高齢化社会において CHIP をパラメーターとしたがんの危険因子の評価法を確立することが必要と考えられる。
-8-

（様式 2）
法人番号プロジェクト番号131054
S1311016＜特に優れた研究成果＞
本事業の研究成果で特筆すべき点は①世界をリードするエクソソーム研究、②CML における個別最適化医療の分子・免疫マーカーの提唱、③骨髄間質細胞の臨床応用に関する産学連
携講座への発展、の３点である。エクソソームは情報伝達物質である miRNA を内包しており、核酸医薬という側面から、ボナックとの産学連携により基盤研究から出口戦略に直結している。②は治癒のためにはイマチニブという高価な薬剤を終生内服する必要があった CML という疾患を、「ストップイマチニブ」の判定をする分子マーカー（miRNA、BIM）、免疫マーカー（NK 細胞等）によって選別し、真の個別最適化医療に直結させている点で社会的貢献度が大きい。また適正な薬剤中止基準の提唱は医療経済学的側面からも意義が大きい。③は本事業を展開する上で新たに見つかったシーズを実現化するため平成 29 年度に産学連携講座の開設し、もっとも期待される成果である。腫瘍由来骨髄間質細胞はがん環境を構築しがんの発育を助けるため、「腫瘍由来骨髄間質細胞」と標的とした治療戦略の構築につながる。一方、「健康人由来骨髄間質細胞」はがん抑制効果があり、細胞治療としてあるいはエク
ソソームなどの細胞分泌物を用いた次世代型細胞治療として、今後のがん治療の一翼を担うものと考えている。「腫瘍由来骨髄間質細胞」と「健康人由来由来骨髄間質細胞」の中間に位置するのが「高齢者健康人由来骨髄間質細胞」で、加齢という要素を加味した独自のアプ
ローチ及び学内外の共同研究および企業との連携が成功したと考えている。
＜課題となった点＞
期間内に出口戦略へと連結させるため、研究テーマを絞り込んだ結果、一部の各個研究の研究支援が十分ではなかった。なかでも臨床各科のクリニカルシーケンスデータに関しては解析が期間内に解析が完了しなかったものもあった。当初、データ解析のパイプライン構築
に際しては三井情報が 2015 年から開始した癌遺伝子解析と薬剤情報の提供をコアとした診
療支援サービス事業「OncoPrime」を活用する予定であったが、工学院大学との連携がより実用的で、最終的には OncoPrime は用いなかった。このようにパイプラインの構築に手間取った背景には本事業では臨床レベルでの次世代シーケンスへの取り組みが早すぎたことは否めない。臨床各科のデータには隠されたシーズが含まれていることが想定され、事業終了
後も解析を続ける予定である。
＜自己評価の実施結果と対応状況＞
毎月１回の運営会議で進捗状況を報告し、達成率、費用対効果について審議し、必要に応じて研究計画の見直しを行ってきた。平成 28 年度に重点領域を設定し、研究を集約していく方
針で軌道修正を行った。
＜外部（第三者）評価の実施結果と対応状況＞
平成 28 年 1 月 26 日に外部評価委員２名を招いて研究成果の中間報告会（公開）を開催し、外部評価委員からは A 評価を得たが、学内での共同研究における役割分担が不明確であることを指摘されたため、研究計画を見直し、軌道修正を行った。平成 30 年１月 26 日には外部
評価員 3 名を招いて最終成果報告会を開催した。限られた予算のなかで重点領域を集約したことが成果に繋がったこと、次世代シーケンスシステムを早期より導入し臨床各科の若手研究者を活用したこと、研究テーマに加齢などの新しい切り口でアプローチしシーズ発見に繋がったこと、国際シンポジウムの主催(Role of Aging and Cancer, 2017, Tokyo:
http://team.tokyo-med.ac.jp/ims_onc/symposium/index.html)などが評価された。

-9-（様式 2）
法人番号プロジェクト番号131054
S1311016＜研究期間終了後の展望＞
miRNA およびエクソソームに関する基盤研究を発展させた核酸医薬およびエクソソーム療法の実現、ドラッグリポジショニングや分子・免疫マーカーによる個別最適化療法のもたらす社
会貢献はいうまでもないが、最も期待されるのは「骨髄間質細胞の役割解明と臨床応用」である。実現に向けて産学連携講座で研究を展開する予定である。なお、本事業により次世代
シーケンスシステムを中心に若手研究者の育成が格段に進んだので、引き続き学内の研究基盤形成を推進する方針である。
＜研究成果の副次的効果＞
1.*特許出願：「細胞増殖抑制剤、抗がん剤、およびメチル基転移酵素阻害剤に対する感受性増強剤」 発明者: 今西 哲ほか
出願人: 東京医科大学
出願番号： 特願 2015-183778
2. *特許出願：「遺伝子発現制御のための発現制御核酸分子およびその用途」 発明者:黒田雅彦ほか
出願人: 東京医科大学
出願番号： 特願 2016-168499

１２ キーワード（当該研究内容をよく表していると思われるものを８項目以内で記載してください。）（１） miRNA（２） エクソソーム（３） がん環境（４） 核酸医薬（５） 免疫制御（６） 遺伝的素因（７） 次世代シーケンサ（８） 骨髄間質細胞
１３ 研究発表の状況（研究論文等公表状況。印刷中も含む。）
上記、１１(４)に記載した研究成果に対応するものには＊を付すこと。
＜雑誌論文＞ 全て査読有
1. *Najima Y, Yoshida C, Iriyama N, Fujisawa S, Wakita H, Chiba S, , Okamoto S, Kawakami K,
Takezoe N, Kumagai T, Ohyashiki K, Taniguchi J, Yano S, Igarashi T, Kouzai Y, Morita S,
Sakamoto J, Sakamaki H, Inokuchi K: Regulatory T cells inhibition by dasatinib isassociated with natural killer cell differentiation and a favorable molecular response The final results of the D-first study. 2018, Leukemia Research, 2018, 66:66-72 [IF=2.501].
2. ＊Usui Y, , Narsing A. Rao, Takase H, Tsubota K, Umazume K, Aguilar D, Kezuka K,
Mochizuki M, Goto H, Sugita S. Comprehensive polymerase chain reaction assay for
detection of pathogenic DNA in lymphoproliferative disorders of the ocular adnexa.
Scientific Reports. 6:36621 | DOI: 10.1038/srep36621 [IF=4.259]
3. ＊Orii N, Mizoguchi I, Chiba Y, Hasegawa H, Ohashi M, Xu M, Nagai T, Ochiai M, Mochizuki
Y, Owaki T, Yoshimoto T. Protective effects against tumors and infection by IL-27
through promotion of expansion and differentiation of hematopoietic stem cells into
myeloid progenitors. Oncoimmunology. 2018; doi.org/10.1080/2162402X.2017.1421892.
[IF=7.719]
4. ＊Ohyashiki JH, Umezu T, Ohyashiki K: Extracellular vesicle (EV)-mediated cell-cellcommunication in hematologic neoplasms. Philosophical Transactions of the Royal-10-（様式 2）
法人番号プロジェクト番号

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

131054
S1311016

Society B: Biological Sciences. 2018, 373:1737 [IF 5.50]＊Inokuchi K, Nakayama K, Tauchi T, Takaku T, Yamaguchi H, Kumagaya T, Komatsu N,
Ohyashiki K: Molecular biological effects of tyrosine kinase inhibitors and subtype of
BCR-ABL1 transcripts of Japanese chronic myeloid leukemia patients with three-way
chromosomal translocation. Leukemia Research, 2018, 65:74-79. [IF 2.501].
* Kurata A, Yamada M, Ohno SI, Inoue S, Hashimoto H, Fujita K, Takanashi M, Kuroda M
Expression level of microRNA-200c is associated with cell morphology in vitro andhistological differentiation through regulation of ZEB1/2 and E-cadherin in gastriccarcinoma. Oncol Rep. 2018;39(1):91-100. doi: 10.3892/or.2017.6093. [IF 2.662]
Mizoguchi I, Ohashi M, Chiba Y, Hasegawa H, Xu M, Owaki T, Yoshimoto T. Prediction ofchemical respiratory and contact sensitizers by OX40L expression in dendritic cells using
a novel 3D co-culture system. Front Immunol. 2017; 8: 929. [IF=6.429]
Umemoto T, Matsuzaki Y, Shiratsuchi Y, Yoshimoto T, Nakamura-Ishizu A, Petrich B,
Yamato Y, Suda T. Integrin αvβ3 enhances the suppressive effect of interferon-γ on
the maintenance of hematopoietic stem cells. EMBO J. 2017; 36(16): 2390-403.
[IF=9.792]
Shimoura N, Nagai H, Fujiwara S, Jimbo H, Yoshimoto T, Nishigori C. Interleukin (IL)-18,
cooperatively with IL-23, induces prominent inflammation and enhances psoriasis-like
epidermal hyperplasia. Arch Dermatol Res. 2017; 309(4): 315-21. [IF=2.327]＊Chiba Y, Mizoguchi I, Furusawa J, Hasegawa H, Ohashi M, Xu M, Owaki T, Yoshimoto T.
Interleukin-27 exerts its antitumor effects by promoting differentiation of hematopoietic
stem cells to M1 macrophages. Cancer Res. 2017; pii: canres.0960.2017. [IF=9.122]
Chiba Y, Mizoguchi I, Hasegawa H, Ohashi M, Orii N, Nagai T, Sugahara M, Miyamoto Y, Xu
M, Owaki T, Yoshimoto T. Regulation of myelopoiesis by proinflammatory cytokines in
infectious diseases. Cell Mol Life Sci. 2017; doi: 10.1007/s00018-017-2724-5. [IF=5.788]＊Katagiri S, Umezu T, Asano M, Akahane D, Azuma K, Makishima H*, Yoshida K*,
Watatani Y*, Chiba K*, Miyano *S, Ogawa S*, Ohyashiki JH, Ohyashiki K: Selective clonal
sweeping of FLT3-D835Y in very late relapse after allogeneic hematopoietic stem celltransplantation. Leukemia and Lymphoma, 2017, Oct 3:1-4. [IF 2.755].
Asano M, Umezu T, Katagiri S, Kobayashi C, Tauchi T, Gotoh M, Ando K, Okabe S,
Ohyashiki JH, Ohyashiki K: Up-regulated exosomal miRNA-140-3p in CML patients withmusculoskeletal pain associated with discontinuation of tyrosine kinase inhibitors.
International Journal of Hematology, 2017, 105(4):419-422 [IF 1.610].
Katagiri S, Tauchi T, Ando K, Okabe S, Gotoh M, Ohyashiki K: Low body weight and bodymass index may be associated with musculoskeletal pain following imatinib
discontinuation in chronic myeloid leukemia. Leukemia Research Reports, 2017, 7:33-35[IF 0.98].
Okabe S, Tauchi T, Tanaka Y, Ohyashiki K: Targeting peroxisome proliferator-activatedreceptors A novel strategy for Philadelphia- positive leukemia cells. Leukemia andLymphoma, 2017, 58(11): 2762-2764 [IF 2.755].
＊Umezu T, Imanishi S, Azuma K, Kobayashi C, Yoshizawa S, Ohyashiki K, Ohyashiki JH:
Replenishing exosomes from older bone marrow stromal cells with miR-340 inhibits
multiple myeloma-related angiogenesis. Blood Advances, 2017, 1(13):812-823.[2017 年に-11-

（様式 2）
法人番号プロジェクト番号

17.

18.

19.

20.

21.
22.

23.

24.

25.

26.

27.

28.

131054
S1311016

発刊された Blood(IF=10.452)のオンラインジャーナルで現時点では IF がない]
＊Imanishi S, Takahashi R, Katagiri S, Kobayashi C, Umezu T, Ohyashiki K, Ohyashiki JH:
Teriflunomide restores 5-azacytidine sensitivity via activation of pyrimidine salvage in
5-azacytidine-resistant leukemia cells. Oncotarget, 2017, (41):69906-69915. [IF 5.168]＊Tauchi T, Okabe S, Katagiri S, Tanaka Y, Tohyama K, Ohyashiki K: Targeting theHedgehog signaling pathway by glasdegib limits the self-renewal of MDS-derived induced
potent stem cells (iPSC). Journal of Cancer Science and Therapy. 2017, 9(5): 479-484 [IF
3.09]＊Azuma K, Umezu T, Imanishi S, Asano M, Yoshizawa S, Katagiri S, Ohyashiki K,
Ohyashiki JH: Genetic alterations are uncommon in bone marrow mesenchymal stromalcells derived from myeloid malignancies. Leukemia Research, 2017, 62:23-28 [IF 2.501].
*Tanaka H, Hazama S, Iida M, Tsunedomi R, Takenouchi H, Nakajima M, Tokumitsu Y,
Kanekiyo S, Shindo Y, Tomochika S, Tokuhisa Y, Sakamoto K, Suzuki N, Takeda S,
Yamamoto S, Yoshino S, Ueno T, Hamamoto Y, Fujita Y, Tanaka H, Tahara K, Shimizu R,
Okuno K, Fujita K, Kuroda M, Nakamura Y, Nagano H miR-125b-1 and miR-378a are
predictive biomarkers for the efficacy of vaccine treatment against colorectal cancer.
Cancer Sci. 2017;108(11):2229-2238. doi: 10.1111/cas.13390. [IF 3.974]
Ohyashiki JH, Umezu T, Ohyashiki K. Exosome promote bone marrow angiogenesis inhematologic neoplasia：Role of hypoxia. Curr Opin Hematol. 2016; 23(3): 268-73. [IF 3.97]*Ohyashiki1 K, Umezu T, Katagiri S, Kobayashi C, Azuma K, Tauchi T, Okabe S, Fukuoka
Y, Ohyashiki JH. Down-regulation of plasma miR-215 in chronic myeloid leukemia
patients with successful imatinib discontinuation. Int J Mol Sci. 2016; 17(4). pii: E570. doi:
10.3390/ijms17040570 [IF 2.862]*Taketani Y, Usui T, Toyono T, Shima N, Yokoo S, Kimakura M, Satoru Yamagami S,
Ohno S, Onodera R, Tahara K, Takeuchi H, Kuroda M Topical use of angiopoietin-likeprotein 2 RNAi-loaded lipid nanoparticles suppresses corneal neovascularization. Mol
Ther Nucleic Acids. 2016; 5:e292. doi: 10.1038/mtna.2016.1. [IF 4.512]＊Furusawa J, Mizoguchi I, Chiba Y, Hisada M, Kobayashi F, Yoshida H, Nakae S, TsuchidaA, Matsumoto T, Ema H, Mizuguchi J, Yoshimoto T. Promotion of expansion anddifferentiation of hematopoietic stem cells by interleukin-27 into myeloid progenitors tocontrol infection in emergency myelopoiesis. PLoS Pathog 2016; 12(3): e1005507.
[IF=6.608]
Kimura Y, Nagai N, Tsunekawa N, Sato-Matsushita M, Yoshimoto T, Cua D, Iwakura Y,
Yagita H, Okada F, Tahara H, Saiki I, Irimura T, Hayakawa Y. IL-17A-producing CD30+
V 1 T cells drive inflammation-induced cancer progression. Cancer Sci 2016;
107(9):1206-14. [IF=3.974]
Hasegawa H, Mizoguchi I, Chiba Y. Ohashi M, Xu M, Yoshimoto T: Expanding diversity inmolecular structures and functions of the IL-6/IL-12 heterodimeric cytokine family.
Front Immunol 2016 Nov 4;7:479 (IF=6.429)*Nagamitsu Y, Nishi H, Sasaki T, Takaesu Y, Terauchi F, Isaka K. Profiling analysis of
circulating microRNA expression in cervical cancer. Mol Med Rep. 2016; 35(2): 992-8 [IF1.554]*Okabe S, Tauchi T, Tanaka Y, Sakuta J, Ohyashiki K. Anti-leukemic activity of axitinib
against cells harboring the BCR-ABL T315I point mutation. J Hematol Oncol. 2015; 8:97.
doi: 10.1186/s13045-015-0190-9. [IF 4.81]-12-

（様式 2）
法人番号プロジェクト番号131054
S1311016

29. *Okabe S, Tauchi T, Tanaka Y, Sakuta J, Ohyashiki K. Efficacy of the polo-like kinase
inhibitor rigosertib, alone or in combination with Abelson tyrosine kinase inhibitors,
against break point cluster region-c-Abelson-positive leukemia cells. Oncotarget. 2015;
6(24): 20231-40. [IF 6.359]
30. Iriyama N, Fujisawa S, Yoshida C, Wakita H, Chiba S, Okamoto S, Kawakami K, Takezako
N, Kumagai T, Inokuchi K, Ohyashiki K, Taguchi J, Yano S, Igarashi T, Kouzai Y, Morita S,
Sakamoto J, Sakamaki H. Early cytotoxic lymphocyte expansion contributes to a deep
molecular response to dasatinib in patients with newly diagnosed chronic myeloid
leukemia in the chronic phase: results of the D-first study. Am J Hematol. 2015; 90(9):819-24. [IF 3.798]
31. Iriyama N, Fujisawa S, Yoshida C, Wakita H, Chiba S, Okamoto S, Kawakami K, Takezako
N, Kumagai T, Inokuchi K, Ohyashiki K, Taguchi J, Yano S, Igarashi T, Kouzai Y, Morita S,
Sakamoto J, Sakamaki H. Shorter halving time of BCR-ABL1 transcripts is a novel
predictor for achievement of molecular responses in newly diagnosed chronic-phasechronic myeloid leukemia treated with dasatinib: Results of the D-first study of Kanto
CML study group. Am J Hematol. 2015; 90(4): 282-7. [IF 3.798]
32. Imanishi S, Takahashi R, Ohsuga M, Ohyashiki K, Ohyashiki JH. Effect of combined
deferasirox and 5-azacytidine treatment on human leukemia cells in vitro. Ann Hematol.
2015; 94(9): 1601-2. [IF 2.634]
33. *Yoshimoto T, Chiba Y, Furusawa J, Xu M, Tsunoda R, Higuchi K, and Mizoguchi I.
Potential clinical application of interleukin-27 as an antitumor agent. Cancer Sci. 2015;
106 (9), 1103-1110. doi: 10.1111/cas.12731. [IF 3.534]
34. *Mizoguchi I, Chiba Y, Furusawa J, Xu M, Tsunoda R, Higuchi K, and Yoshimoto T.
Therapeutic potential of interleukin-27 against cancers in preclinical mouse models.
Oncoimmunology, 2015; 27; 4(10): e1042200. DOI:10.1080/2162402X..1042200 [IF 6.266]
35. *Toyota, H., Yanase, N., Yoshimoto, T., Harada, M., Kato, Y., and Mizuguchi, J. Vaccination
with OVA-bound nanoparticles encapsulating IL-7 inhibits the growth of OVA-expressing
E.G7 tumor cells in vivo. Oncol. Rep. 2015; 33(1), 292-296. [IF 2.301]
36. Toyono T, Usui T, Yokoo S, Taketani Y, Nakagawa S, Kuroda M, Yamagami S, Amano S.
Angiopoietin-like 7 is an anti-angiogenic protein required to prevent vascularization of
the cornea. PLoS One. 2015; 10(1):e0116838. [IF 3.234]
37. Takanashi M, Sudo K, Ueda S, Ohno S, Yamada Y, Osakabe Y, Goto H, Matsunaga Y,
Ishikawa A, Usui Y, Kuroda M. Novel Types of Small RNA Exhibit Sequence- andTarget-dependent Angiogenesis Suppression Without Activation of Toll-like Receptor 3
in an Age-related Macular Degeneration (AMD) Mouse Model. Mol Ther Nucleic Acids.
2015 4:e258. doi: 10.1038/mtna.2015; 34. [IF 4.512]
38. Yoon JH, Sudo K, Kuroda M, Taniguchi T, Kato M, Lee I, Han JS, Nakae S, Imamura T, Kim
J, Ju JH, Kim DK, Matsuzaki K, Weinstein M, Nakao A, Matsumoto I, Sumida T, Mamura
M.. Phosphorylation status determines the opposing functions of Smad2 and Smad3 as
STAT3 cofactors in TH17 differentiation. Nature commun. 2015; 21:6:7600. [IF 11.470]
39. Nakama T, Yoshida S, Ishikawa K, Kobayashi Y, Zhou Y, Nakao S, Sassa Y, Oshima Y,
Takao K, Shimahara A, Yoshikawa K, Hamasaki T, Ohgi T, Hayashi H, Matsuda A, Kudo A,
Nozaki M, Ogura Y, Kuroda M, Ishibashi T.
Inhibition of choroidal fibrovascularmembrane formation by new class of RNA interference therapeutic agent targeting
periostin. Gene Ther. 2015; 22(2): 127-37. [IF 3.104]
40. Tajima K, Miyake T, Koike N, Hattori T, Takahashi H, Matsumoto T, Fujita K, Kuroda M, Ito-13-

（様式 2）
法人番号プロジェクト番号

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

131054
S1311016

N, Goto H. Two Different Concentrations of Topical Levofloxacin for the Treatment ofMultidrug-Resistant Pseudomonas aeruginosa Keratitis. J Ocul Pharmacol Ther. 2015;31(10): 636-41. [IF 1.47]
Nakajima J Okamono N, Tohyama J, Kato M, Arai H, Funahashi O, Tsurusaki Y, Nakashima
M, Kawashima H, Saitsu H, Matsumoto N, Miyake N. De novo EEF1A2 mutations inpatients with characteristic facial features, intellectual disability, autistic behavior andepilepsy. Clinical Genetics 2014; 87(4): 356-61. [IF 3.390]*Okabe S, Tauchi T, Katagiri S, Tanaka Y, Ohyashiki K. Combination of the ABL kinase
inhibitor imatinib with the Janus kinase 2 inhibitor TG101348 for targeting residual
BCR-ABL-positive cells. J Hematol Oncol. 2014; 7:37. doi: 10.1186/1756-8722-7-37. [IF4.81]
Okabe S, Tauchi T, Kimura S, Maekawa T, Kitahara T, Tanaka Y, Ohyashiki K. Combining
the ABL1 kinase inhibitor ponatinib and the histone deacetylase inhibitor vorinostat: apotential treatment for BCR-ABL-positive leukemia. PLoS One. 2014; 9(2):e89080. doi:
10.1371/journal.pone.0089080. [IF 3.234]
Nishimura J, Yamamoto M, Hayashi S, Ohyashiki K, Ando K, Brodsky AL, Noji H, Kitamura
K, Eto T, Takahashi T, Masuko M, Matsumoto T, Wano Y, Shichishima T, Shibayama H,
Hase M, Li L, Johnson K, Lazarowski A, Tamburini P, Inazawa J, Kinoshita T, Kanakura Y.
Genetic variants in C5 and poor response to eculizumab. N Engl J Med. 2014; 370(7):632-9. [IF 55.873]*Okabe S, Tauchi T, Tanaka Y, Kitahara T, Kimura S, Maekawa T, Ohyashiki K. Efficacy ofthe dual PI3K and mTOR inhibitor NVP-BEZ235 in combination with nilotinib against
BCR-ABL-positive leukemia cells involves the ABL kinase domain mutation. Cancer Biol
Ther. 2014; 15(2): 207-15. [IF 3.072]
Yoshizawa K, Mukai HY, Miyazawa M, Miyao M, Ogawa Y, Ohyashiki K, Katoh T, Kusumoto
M, Gemma A, Sakai F, Sugiyama Y, Hatake K, Fukuda Y, Kudoh S. Bortezomib
therapy-related lung disease in Japanese patients with multiple myeloma: incidence,mortality and clinical characterization. Cancer Sci. 2014; 105(2): 195-201. [IF 3.534]*Ohyashiki JH, Ohtsuki K, Mizoguchi I, Yoshimoto T, Katagiri S, Umezu T, Ohyashiki K.
Downregulated microRNA-148b in circulating PBMCs in chronic myeloid leukemia
patients with undetectable minimal residual disease: a possible biomarker to discontinue
imatinib safely. Drug Des Devel Ther. 2014; 8:1151-9. [IF 3.028]*Imanishi S, Umezu T, Ohtsuki K, Kobayashi C, Ohyashiki K, Ohyashiki JH. Constitutive
activation of the ATM/BRCA1 pathway prevents DNA damage-induced apoptosis in
5-azacytidine-resistant cell lines. Biochem Pharmacol. 2014; 89(3): 361-9. [IF 5.009]*Umezu T, Tadokoro H, Azuma K, Yoshizawa S, Ohyashiki K, Ohyashiki JH. Exosomal
miR-135b shedded from hypoxic multiple myeloma cells enhances angiogenesis bytargeting factor inhibiting HIF-1 (FIH-1). Blood 2014; 124(25), 3748-57. [IF 10.452]*Ohyashiki K, Tadokoro K, Yamaguchi R, Katagiri S, Umezu T, Ohtsuki K, Tauchi T,
Yamamoto T, Ohyashiki JH. Detection of BIM (BCL2L11) polymorphic variants in chronic
myeloid leukemia by Q-Invader assay and their clinical significance. J Hematol Transfus.
2014; e2(3): 1032
Mizoguchi, I., Yoshimoto, T., Katagiri, S., Furusawa, J., Chiba, Y., Mizuguchi, J., Tauchi, T.,
Ohyashiki, J. H., and Ohyashiki, K. Immunological control of chronic myeloid leukemia
leading to treatment-free remission. J. Hematol. Transfus. 2014; 2(3):1024..
*Yoshimoto, T., Mizoguchi, I., Katagiri, S., Tauchi, T., Furusawa, J., Chiba, Y., Mizuguchi, J.,-14-

（様式 2）
法人番号プロジェクト番号

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

131054
S1311016

Ohyashiki, J. H., and Ohyashiki, K. Immunosurveillance markers may predict patients who
can discontinue imatinib therapy without relapse. Oncoimmunology, 2014; 14;3: e28861.
[IF 6.266]
Nakaya T, Ogawa S, Manabe I, Tanaka M, Sanada M, Sato T, Taketo MM, Nakao K,
Clevers H, Fukayama M, Kuroda M, Nagai R. KLF5 regulates the integrity andoncogenicity of intestinal stem cells. Cancer Res. 2014; 74(10): 2882-91. [IF 9.329]
Kato Y, Yoshino I, Egusa C, Maeda T, Pili R, Tsuboi R: Combination of HDAC inhibitor
MS-275 and IL-2 increased anti-tumor effect in a melanoma model via activatedcytotoxic T cells. J Dermatol Sci. 2014; 75:140-7. [IF 3.419]
Okabe S, Tauchi T, Tanaka Y, Ohyashiki K. Efficacy of ponatinib against ABL tyrosine
kinase inhibitor-resistant leukemia cells. Biochem Biophys Res Commun. 2013; 435(3):506-11. [IF 2.297]
Okabe S, Tauchi T, Tanaka Y, Katagiri S, Kitahara T, Ohyashiki K. Activity of omacetaxine
mepesuccinate against ponatinib-resistant BCR-ABL-positive cells. Blood. 2013;
122(17): 3086-8. [IF 10.452]
Okabe S, Tauchi T, Tanaka Y, Kimura S, Maekawa T, Ohyashiki K. Activity of histone
deacetylase inhibitors and an Aurora kinase inhibitor in BCR-ABL-expressing leukemia
cells: Combination of HDAC and Aurora inhibitors in BCR-ABL-expressing cells. Cancer
Cell Int. 2013; 13(1): 32 [IF 2.77]
Katagiri S, Tauchi T, Okabe S, Minami Y, Kimura S, Maekawa T, Naoe T, Ohyashiki K.
Combination of ponatinib with Hedgehog antagonist vismodegib for therapy-resistant
BCR-ABL1-positive leukemia. Clin Cancer Res. 2013; 19(6):1422-32. [IF 8.722]*Tadokoro H, Umezu T, Ohyashiki K, Hirano T, Ohyashiki JH. Exosomes derived fromhypoxic leukemia cells enhance tube formation in endothelial cells. J Biol Chem. 2013;
288(48), 34343-51. [IF 4.573]
Soeda S, Ohyashiki JH, Ohtsuki K, Umezu T, Setoguchi Y, Ohyashiki K. Clinical relevanceof plasma miR-106b levels in patients with chronic obstructive pulmonary disease. Int J
Mol Med. 2013; 31(3):533-9. [IF 2.088]*Katagiri S, Umezu T, Ohyashiki JH, Ohyashiki K. The BCL2L11 (BIM) deletion
polymorphism is a possible criterion for discontinuation of imatinib in chronic myeloid
leukaemia patients. Br J Haematol. 2013; 160(2):269-71. [IF 4.971]*Chiba, Y., Mizoguchi, K. Mitobe, I., Higuchi, K., Nagai, H., Nishigori, C., Mizuguchi, J. andYoshimoto, T. IL-27 enhances the expression of TRAIL and TLR3 in human melanomas
and inhibits their tumor growth in cooperating with a TLR3 agonist poly(I:C) partly in
TRAIL
dependent
manner.
PLoS
ONE.
2013;
8(10):
e76159.
doi:
10.1371/journal.pone.0076159. [IF 3.234]*Mizoguchi I, Yoshimoto T, Katagiri S, Mizuguchi J, Tauchi T, Kimura Y, Inokuchi K,
Ohyashiki J H, and Ohyashiki K. Sustained up-regulation of effector natural killer cells inchronic myeloid leukemia after discontinuation of imatinib. Cancer Sci. 2013;
104:1146-1153. [IF 3.534]
Mizoguchi, I., Higuchi, K., Mitobe, K., Tsunoda, R., Mizuguchi, J., and Yoshimoto, T.
Interleukin-27: Regulation of immune responses and disease development by apleiotropic cytokine with pro- and anti-inflammatory properties. In Cytokine Frontiers:
Regulation of Immune Responses in Health and Disease. Editors: Yoshimoto, T. andYoshimoto, T. Publisher: Springer, 2013; pp. 353-375.
Shimizu M, Ogura K, Mizoguchi I, Chiba Y, Higuchi K, Ohtsuka H, Mizuguchi J, and-15-

（様式 2）
法人番号プロジェクト番号131054
S1311016

Yoshimoto T. IL-27 promotes nitric oxide production induced by LPS through STAT1,
NF-κB and MAPKs. Immunobiology 2013; 218, 628-634. [IF 3.044]
66. Murakami Y, Tamori T, Itami S, Tanahashi T, Toyoda H, Tanaka M, Wu W, Brojigin N,
Kaneoka Y, Maeda A, Kumada T, Kawada N, Kubo S, Kuroda M. The expression level ofmiR-18b in hepatocellular carcinoma is associated with the grade of malignancy andprognosis. BMC Cancer (Section: Genetics, genomics and epigenetics) 2013; 4;13:99.
doi: 10.1186/1471-2407-13-99. [IF 3.362]
67. Ohno S, Takanashi, Sudo K, Ueda S, Ishikawa A, Matsuyama M, Fujita K, Mizutani T, Ohgi
T, Ochiya T, Gotoh, N, Kuroda K. Systemically Injected Exosomes Targeted to EGFR
Deliver Antitumor MicroRNA to Breast Cancer Cells. Mol Ther. 2013; 21(1): 185-191. [IF
6.227]
68. Hashimoto T, Ohno Y, Nakashima J, Gondo T, Ohori M, Tachibana M. Clinicalsignificanceof preoperative peripheral blood neutrophil count in patients withnon-metastatic upper
urinary tract carcinoma. World J Urol. 2013; 31(4): 953-8. [IF 2.666]
69. Wu W, Takanashi M, Borjigin N, Ohno SI, Fujita K, Hoshino S, Osaka Y, Tsuchida A, Kuroda
M. MicroRNA-18a modulates STAT3 activity through negative regulation of PIAS3 during
gastric adenocarcinogenesis. Br J Cancer. 2013; 108(3): 653-61. [IF 4.336]＜図書＞
1. Ohyashiki K, Kuroda M, Ohyashiki JH: Chromosomes and chromosomal instability in humancancer. The Molecular Basis of Human Cancer (2nd Edition). (Coleman WB, Tsongalis GJ,
eds). Splinger 2017, 241-262.
2. Ohno S, Kuroda M. Exosome-Mediated Targeted Delivery of miRNAs. Methods Mol Biol.
2016;1448:261-70. doi: 10.1007/978-1-4939-3753-0_19.
3. 大屋敷一馬：レナリドミドによる 5ｑ-症候群の治療。造血器腫瘍アトラス 改訂第 5 版、2016 年（谷脇雅史,横田昇平,黒田純也 編）日本医事新報社 2016,704 707.
4. 大屋敷一馬、大屋敷純子：細胞死：テロメア・テロメラーゼ。入門腫瘍内科学（2016 年 第2 版）。（監修：日本臨床腫瘍学会、編集：「入門腫瘍内科学改訂第 2 版」編集委員会）、篠原出版 2015 年、39-41.
5. 大屋敷一馬：骨髄異形成症候群のリスク別治療方針と新薬の動向。Annual Review 血液。中外医学社（編集：高久史麿、小澤敬也、坂田洋一。金倉譲、小島勢二）2014 年、
130-135.

＜学会発表＞
1. Umezu T, Imanishi S, Gladkova N, Ohyashiki K, Ohyashiki K: Bone marrow mesenchymal
stromal cells from healthy donors secrete anti-angiogenic EVs as well as soluble factors
having growth inhibitory effect in MM. European Hematology Association (Stockholm,
Sweden, June 14-17, 2018)
2. *Umezu T, Imanishi S, Ohyashiki K, Ohyashiki K:The role of extracellular
vesicle-mediated miR-10a transfer in bone marrow microenvironment of patients withmultiple myeloma. International Society for Extracellular Vesicles 2018, (Barcelona,
Spain, May 2-6, 2018)
3. ＊＊片桐誠一朗、伊藤良和、梅津知宏、田所健一、吉澤成一朗、赤羽大悟、藤本博昭、
大屋敷一馬：濾胞性リンパ腫において BCL2L11(BIM)欠失多型のみられない女性は生存期間の予後良好因子である。第 115 回日本内科学会講演会（2018 年 4 月 13 日～15 日、-16-（様式 2）
法人番号プロジェクト番号131054
S1311016

京都）
4. Suguru Saito 、 Shinichiro Ohno 、 Yuichiro Harada 、 Keiki Oikawa, Yoshihiko Kanno,
Masahiko Kuroda: AAV-mediated miRNA-29b delivary suppuress renal fibrosis 第 9 回日
本 RNAi 研究会(広島) 2017 年 9 月 1 日
5. Yoshihiro Sanbu, Shinichiro Ohno 、 Masahiko Kuroda: Up-regulation of BLU tumor
suppuressor by miR-34a 第 9 回日本 RNAi 研究会(広島) 2017 年 9 月 1 日
6. 黒田雅彦: エクソソームの臨床応用、第 33 回日本 DDS 学会学術集会 招待講演 、
2017 年 7 月 7 日
7. 黒田雅彦: 核酸医薬の臨床応用、第 384 回 CBI 学会講演会－核酸医薬－、東京工業大学キャンパスイノベーションセンター（東京）(2017 年 5 月 25 日)
8. 池畑直樹、高梨正勝、上田しのぶ、里見貴史、渡辺正人、長谷川温、河野通秀、榎本愛、黒田雅彦、近津大地: Activation of TLR2 regulates CARD10 gene expression by
miR-146a-5p in oral squamous cell carcinoma. 第 106 回日本病理学会総会、東京、201
年 4 月 27 日
9. 大野慎一郎、原田裕一郎、村上善基、黒田雅彦: RNA 干渉核酸に働く短鎖ヘアピン RNAの探索と機能解析.(Search and characterization of novel hairpin RNAs acting on RNA
interference. 第 106 回日本病理学会総会、東京、2017 年 4 月 28 日
10. 老川桂生、淺田浩太朗、大野慎一郎、黒田雅彦: miRNA 生成経路を欠損する肺がん細胞株の解析. (Analysis of microRNA-defict lung cancer cell line.) 第 106 回日本病理学会総会、東京、2017 年 4 月 28 日
11. 三部芳裕、大野慎一郎、黒田雅彦: Mir-34a によるがん抑制遺伝子 BLU の発現制御機
構.(Up-regulation of expression of BLU tumor suppressor by miR-32a.) 第 106 回日本病理学会総会、東京、2017 年 4 月 29 日
12. 鈴木未来、梅津知宏、小林千晶、吉澤成一郎、大屋敷一馬、東剣紅、今西哲、大屋敷純子：多発性骨髄腫細胞の増殖に影響を与える骨髄間質細胞由来因子の同定。第 180 回
東京医科大学医学会総会（2017 年 11 月 4 日、東京）。
13. 小澤ひとみ、梅津知宏、川名千晶、東剣紅、今西哲、大屋敷純子：多発性骨髄腫細胞の浸潤様式の解明を目的とした in vivo および in vitro モデル系の構築。第 180 回東京医科大学医学会総会（2017 年 11 月 4 日、東京）。
14. 内山のぞみ、梅津知宏、川名千晶、東剣紅、今西哲、大屋敷純子：多発性骨髄腫細胞の低酸素応答性に関与する lncRNA の解析。第 180 回東京医科大学医学会総会（2017年 11 月 4 日、東京）。
15. Kubota S, Tokunaga K, Oshima M, Umezu T, Kanai A, Ton Tan K, Yang H, Iwanaga E, Asou
N, Maeda T, Iwama A, Ohyashiki K, Osato M, Sashida G. RUNX2 super enhancer promotes
the development of blastic plasmacytoid dendritic cell neoplasia. 79th Annual Meeting of
the Japanese Society of Hematology (Tokyo, Oct 22-24, 2017).
16. Katagiri S, Tauchi T, Ohnishi A, Saito Y, Suguro T, Asano M, Yoshizawa S, Sakuta J,
Akahane D, Tanaka Y, Furuya N, Ando K, Fujimoto H, Okabe S, Gotoh M, Ito Y, Ohyashiki
K: Retrospective analysis of CML patients receiving second TKIs as initial therapy. 79th
Annual Meeting of the Japanese Society of Hematology (Tokyo, Oct 22-24, 2017).
17. Okabe S, Tauchi T, Tanaka Y, Ohyashiki K: Targeting peroxisome proliferator-activatedreceptors: a novel strategy for BCR-ABL positive cells. 79th Annual Meeting of theJapanese Society of Hematology (Tokyo, Oct 22-24, 2017).
18. Saito Y, Azuma K, Imanishi S, Katagiri S, Suguro T, Yoshizawa S, Akahane D, Fujimoto H,
Gotoh M, Itoh Y, Ohyashiki JH, Ohyashiki K: Significance of T cell receptor
rearrangement of bone marrow in extranodal NK/T cell lymphoma, nasal type. 79th Annual-17-（様式 2）
法人番号プロジェクト番号131054
S1311016

Meeting of the Japanese Society of Hematology (Tokyo, Oct 22-24, 2017).
19. Nannya Y*, Takeda J*, Sato S*, Shinozawa Y*, Shiraishi Y*, Kataoka K*, Chiba K*,
Tanaka H*, Sanada M*, Chiba S*, Asou N*, Kiyoi H*, Nakao S*, Ohyashiki K, Miyazaki Y*,
Naoe T*, Makishima H*, Yoshida K*, Miyano S*, Ogawa S*: Efficacy of azacitidine for
TP53-mutated myelodysplastic syndromes (MDS). 79th Annual Meeting of the JapaneseSociety of Hematology (Tokyo, Oct 22-24, 2017).
20. Tanaka Y, Okabe S, Tauchi T, Ohyashiki K: A novel dual inhibitor (PI3K/HDAC),CUDC-907 and combination with carfilzomib inhibits myeloma cells. 79th Annual Meetingof the Japanese Society of Hematology (Tokyo, Oct 22-24, 2017).
21. Nannya Y, Takeda J* Sato S, Shinozawa Y, Shiraishi Y, Kataoka K*, Chiba K, Tanaka H,
Sanada M, Chiba S, Asou N, Kiyoi H, Nakao S, Ohyashiki K, Miyazaki Y, Naoe T Makishima
H, Yoshida K, Miyano S, Ogawa S: Efficacy of azacitidine for TP53-mutatedmyelodysplastic syndromes (MDS). 第 76 回日本癌学会学術総会（2017 年 9 月 28 日～
30 日、横浜）。
22. Umezu T, Imanishi S, Yoshizawa Sm Ohyashiki K, Ohyashiki JH: Inhibition of extracellular
vesicle secretion induces apoptosis of bone marrow stroma cells. 第 76 回日本癌学会学術総会（2017 年 9 月 28 日～30 日、横浜）。
23. 今西 哲、大屋敷 純子、梅津知宏、小林千晶、大屋敷 一馬：クロマチン制御因子 HP1のアザシチジン耐性白血病細胞における役割の解明。第 179 回東京医科大学医学会総会（2017 年 6 月 3 日、東京）
24. 片桐誠一朗、田内哲三、安藤恵子、岡部聖一、後藤守孝、大屋敷一馬：慢性骨髄性白血病において body mass index 低値は imatinib 中止後筋骨格痛と関連する。第 114 回日本内科学会総会・講演会（2017 年 4 月 14～16 日、東京）
25. Katagiri S, Tauchi T, Tanaka Y, Ando K, Okabe S, Gotoh M, Ito Y, Ohyashiki K: Long term
follow-up after tyrosine kinase inhibitors cessation in chronic myeloid leukemia with deep
molecular response. 日本臨床腫瘍学会（2017 年 7 月 27 日～２９日、神戸）
26. 齋藤 優、片桐 誠一朗、勝呂 多光子、吉澤 成一郎、赤羽 大悟、藤本 博昭、後藤 守
孝、伊藤 良和、大屋敷 一馬、東 剣虹、今西 哲、大屋敷 純子：節外性 NK/T 細胞リンパ腫・鼻型における骨髄 T 細胞受容体再構成の意義。第 179 回東京医科大学医学会総会（2017 年 6 月 3 日、東京）
27. Ohno S, Itano K, Harada Y, Asada K, Oikawa K, Kashiwazako M, Okuyama H, Kumagai K,
Takanashi M, Sudo K, Ikeda N, Kuroda M. Development of novel Dicer- andAgo2-independent small hairpin RNAs. American Society of Gene & Cell
Therapy(ASGCT) 19th Annual Meeting. (Washington,DC, USA. May 4-9 2016).
28. 黒田雅彦: RNA ワールドと肺がん発がん機構. (教育講演Ⅲ) 第 108 回 ACCP 日本部会
定期教育講演会. 東京医科大学病院 6 階臨床講堂. 東京.2016 年 10 月 29 日
29. 山田侑子、高梨正勝、上田しのぶ、黒田雅彦、須藤カツ子: 肺線維症に対する miR-29b
補充療法. (医学会奨励賞受賞講演）第 177 回東京医科大学医学会総会、2016 年 6 月 4
日.
30. 黒田雅彦: miRNA 補充療法の開発、第 12 回日本臨床プロテオーム研究会、2016 年 5月 21 日31. 善本隆之：骨髄造血と生体防御 サイトカインによる新たな造血制御 ．第 5 回医総研
シンポジウム（2016 年 1 月 19 日、東京）
32. 善本隆之：免疫学的側面から：サイトカインによる骨髄造血の制御と抗腫瘍効果．平成25 年度選定戦略的研究基盤形成支援事業「生体分子情報による次世代型がん個別最適化治療法の開発」中間報告会（2016 年 1 月 26 日、東京）-18-

（様式 2）
法人番号プロジェクト番号131054
S1311016

33. Umezu T, Imanishi S, Azuma K, Yoshizawa S, Ohyashiki K, Ohyashiki JH: Anti-angiogenic
effect of normal bone marrow stromal cell-derived exosomes rejuvenated by miR-340.
The 78th Annual Meeting of Japanese Society of Hematology (JSH2016) (2016.10.13-15)
Yokohama
34. ＊＊Imanishi S, Takahashi R, Umezu T, Kobayashi C, Asano M, Ohyashiki K, Ohyashiki
JH: Distinct histone modification patterns in 5-azacitidine sensitive human leukemia
cells. The 78th Annual Meeting of Japanese Society of Hematology (2016.10.13-15)
Yokohama
35. Azuma K, Umezu T, Imanishi S, Asano M, Yoshizawa S, Katagiri S, Ohyashiki K, Ohyashiki
JH: Mutation analysis of bone marrow stromal cells derived from AML and MDS. The 78th
Annual Meeting of Japanese Society of Hematology (2016.10.13-15) Yokohama
36. Umezu T, Imanishi S, Azuma K, Kobayashi C, Ohyashiki K, Ohyashiki JH: Bone marrowstromal cells from young healthy donors secrete exosomes that inhibit in vivo
angiogenesis in multiple myeloma. 10th AACR-JCA Joint Conference (Maui, Hawaii,
February 16-20, 2016)37. Ohyashiki K, Umezu T, Katagiri S, Kobayashi C, Azuma K, Tauchi T, Okabe S, Fukuoka Y,
Ohyashiki JH: Down-regulation of plasma miR-215 in chronic myeloid leukemia patientswith successful imatinib discontinuation. 10th AACR-JCA Joint Conference (Maui,
Hawaii, February 16-20, 2016)38. 善本隆之，千葉佑規乃，角田廉，徐明利，溝口出：化学物質の呼吸器感作性 in vitro 評価法の開発．LRI シンポジウム、第 28 回日本動物実験代替法学会（2015 年 12 月 10-12日、横浜）
39. Tauchi T, Okabe S, Katagiri S, Tanaka Y, Tohyama K*, Ohyashiki K: Targeting thehedgehog signaling pathway by PF-04449913 limits the self-renewal of MDS-derived
induced potent stem cells (iPSC). 57th Annual Meeting of American Society of
Hematology (Florida, USA, Dec 5-8, 2015).
40. Okabe S, Tauchi T, Tanaka Y, Sakuta J, Ohyashiki K: Anti-leukemic activity of axitinib
against cells harboring the BCR-ABL point mutation. 57th Annual Meeting of American
Society of Hematology (Florida, USA, Dec 5-8, 2015).
41. Schanz J*, Bennett JM*, Slovak ML*, Fenaux P*, Malcovati L*, Cazzola M*, Valent P*,
Ohyashiki K, Levis A*, Sekeres MA*, Tauro A*, Magalhaes S*, an de Loosdrecht
AA*. Prognostic impact of rare single abnormalities in myelodysplastic syndromes. 57th
Annual Meeting of American Society of Hematology (Florida, USA, Dec 5-8, 2015).
42. Mizoguchi I, Chiba Y, Xu M, Yoshimoto T: Binding of Epstein-Barr virus-induced gene 3 tocalnexin enhances its chaperone activity and augments interleukin-23 receptor
expression, leading to development of colitis. 第 44 回日本免疫学会総会・学術集会（2015 年 11 月 18 日 20 日、札幌）
43. 武内 健, 梅津知宏, 東 剣虹, 小林千晶, 大屋敷一馬, 大屋敷純子：低酸素耐性骨髄腫細胞株 IM-9-HR の樹立と性状解析 第 176 回医学会総会（2015 年 11 月 7 日, 東
京）
44. 大須賀美穂, 高橋諒子, 今西 哲, 小林千晶, 梅津知宏, 片桐誠一朗, 大屋敷一馬,
大屋敷純子：アザシチジン耐性白血病細胞における BET ファミリー阻害剤の分子機構の解明 第 176 回医学会総会（2015 年 11 月 7 日, 東京）
45. 関口昌孝，内田萌々，千葉祐規乃，溝口出，角田廉，徐明利，善本隆之：IL-27 は骨髄由来抑制細胞への分化誘導を阻害する．第 176 回東京医科大学医学会総会（2015 年
11 月 7 日、東京）-19-

（様式 2）
法人番号プロジェクト番号131054
S1311016

46. 加藤利奈、西 洋孝、永光雄造、佐々木 徹、井坂惠一：血清 miR-100 は子宮頸癌の早期発見のマーカーとなりうる． 第 53 回癌治療学会学術集会（2015 年 10 月 29 日～31
日、京都）
47. 上田俊一郎, 山川直之, 坪田欣也, 後藤 浩：次世代シーケンサーを用いた脈絡膜メラノーマの腫瘍特異的遺伝子変異の解析. 第 69 回日本臨床眼科学会（2015 年 10 月 22 日
25 日、名古屋）
48. Okabe S, Tauchi T, Tanaka Y, Ohyashiki K: Phosphoinositide 3-kinase inhibitor, copanlisib
for the treatment of Ph positive leukemia cells. 第 77 回日本血液学会学術集会（2015年 10 月 16 日～18 日、金沢）
49. Tauchi T, Okabe S, Katagiri S, Tanaka Y, Ohyashiki K: Activity of stemness inhibitor,
BBI608, on the self-renewal of BCR-ABL1 positive leukemia cells. 第 77 回日本血液学会学術集会（2015 年 10 月 16 日～18 日、金沢）
50. Azuma K, Takeuchi K, Umezu T, Ohyashiki K, Ohyashiki JH: Establishment andcharacterization of a novel chronic hypoxia-resistant multiple myeloma cell line, IM-9R.
第 77 回日本血液学会学術集会学術集会（2015 年 10 月 16 日～18 日, 金沢）
51. *＊Yoshizawa S, Umezu T, Saitoh Y, Gotoh M, Akahane D, Kobayashi C, Ohyashiki JH,
Ohyashiki K: Exosomal miRNA signature of late onset acute graft-versus-host disease inallogenic hematopoietic stem cell transplantation. 第 77 回日本血液学会学術集会（2015年 10 月 16 日～18 日, 金沢）
52. Katagiri S, Tauchi T, Umezu T, Tadokoro K, Mizoguchi I, Yoshimoto T, Ohyashiki JH,
Ohyashiki K: Predictive factors for successful treatment discontinuation of CML
patients. 第 77 回日本血液学会学術集会 (2015 年 10 月 16 日～18 日, 金沢）
53. Saitoh Y, Imanishi S, Umezu T, Yoshizawa S, Asano M, Fujimoto H, Akahane D, Yamamoto
Y, Kobayashi C, Ohyashiki JH, Ohyashiki K: Exosomal miRNA signature of bone marrowmesenchymal stromal cells derived from MDS patients. 第 77 回日本血液学会学術集会（2015 年 10 月 16 日～18 日, 金沢）
54. Imanishi S, Takahashi R, Ohsuga M, Umezu T, Kobayashi C, Katagiri S, Asano M, Saitoh Y,
Ohyashiki K, Ohyashiki JH: Targeting bromodomain protein in azacytidine resistantleukemia cells. 第 77 回日本血液学会学術集会（2015 年 10 月 16 日～18 日, 金沢）
55. Ito Y, Umezu T, Tadokoro K, Saitoh Y, Katagiri S, Suguro T, Asano M, Yoshizawa S,
Akahane D, Fujimoto H, Okabe S, Gotoh M, Tauchi T, Ohyashiki JH, Ohyashiki K: BIM
deletion polymorphism may be associated with resistance to chemotherapy for follicularlymphoma. 第 77 回日本血液学会学術集会（2015 年 10 月 16 日～18 日, 金沢）
56. Umezu T, Imanishi S, Azuma K, Yoshizawa S, Ohyashiki K, Ohyashiki JH: Exosome-based
therapeutic approach using exosomal miRNAs derived from bone marrow mesenchymal
stromal cells. 第 74 回日本癌学会学術総会（2015 年 10 月 8 日 10 日, 名古屋）
57. Umezu T, Imanishi S, Azuma K, Yoshizawa S, Ohyashiki K, Ohyashiki JH: Exosome-based
therapeutic approach using exosomal miRNAs derived from bone marrow mesenchymal
stromal cells. 第 74 回日本癌学会学術総会（2015 年 10 月 8 日 10 日, 名古屋）
58. 黒田雅彦: エクソソームが開く新しい医療．特別講演．第 19 回日本統合医療学会、山口、2015 年 12 月 13 日
59. 及川恒輔, 高梨正勝, 佐藤冬樹, 黒田雅彦, 村垣泰光：TLS-CHOP を発現する粘液型脂肪肉腫細胞の増殖に必須な新規分子経路の探索 第 74 回日本癌学会学術総会（2015 年 10 月 8 日 10 日, 名古屋）
60. 黒田雅彦：体液中マイクロ RNA 測定とがん検査 第 74 回日本癌学会学術総会（2015年 10 月 8 日 10 日, 名古屋）
-20-

（様式 2）
法人番号プロジェクト番号131054
S1311016

61. 上田俊一郎, 山川直之, 坪田欣也, 後藤 浩：次世代シーケンサーを用いた脈絡膜悪性黒色腫の遺伝子解析. 第 34 回眼腫瘍学会（2015 年 10 月 3 日 4 日、島根）
62. 大屋敷一馬, 溝口 出, 善本隆之, 田内哲三, 岡部聖一, 片桐誠一朗, 梅津知宏,
大屋敷純子：Stop-imatinib CML 患者末梢血単核球における免疫関連 miRNA の意義
第 7 回血液疾患免疫療法研究会学術総会（2015 年 9 月 26 日, 東京）
63. Ohyashiki K, Umezu T, Imanishi S, Ohyashiki JH: Leukemia cells under persistent hypoxia
secrete exosomes enhancing angiogenesis in mice. ISEH 44th Annual Scientific Meeting(Kyoto, Japan, September 17, 2015)
64. Ohki T, Umezu T, Ohyashiki JH, Fukuoka Y: Integrative analysis of expression levels of
miRNA and mRNA: Investigation of hypoxic tolerance in leukemia. 37th International
Annual Conference of IEEE Engineering in Medicine and Biology Society (Milan, Italy, Aug
25-29, 2015)
65. 溝口出，千葉佑規乃，角田廉，徐明利，善本隆之：化学物質の呼吸器感作性 in vitro 評価法の開発．第 3 期 LRI 研究報告会（2015 年 8 月 28 日、東京）
66. 関野知聖, 梅津知宏, 今西 哲, 東 剣虹, 武内 健, 福岡 豊, 大屋敷一馬, 大屋敷純子：骨髄間質細胞由来の老化関連エクソソーム miRNA の解析 第４回医薬工 3 大学
包括連携推進シンポジウム（2015 年 6 月 20 日, 東京）
67. 大木拓也，梅津知宏，大屋敷純子，福岡 豊：異種データの統合による多発性骨髄腫の
低酸素耐性機序の解析．第 4 回医薬工 3 大学包括連携推進シンポジウム （2015 年 6月 20 日、東京）
68. 太田祥貴，松井一馬，梅津知宏，大屋敷純子，福岡 豊：次世代シーケンサーデータの高速処理法の開発．第 4 回医薬工 3 大学包括連携推進シンポジウム （2015 年 6 月 20
日、東京）
69. Tauchi T, Okabe S, Ohyashiki K: The stemness inhibitor, BB1608, reduces theself-renewal of BCR-ABL positive leukemia cells. 20th European Hematology
Association Congress (Wien, Austria, June 11~14, 2015).
70. Umezu T, Imanishi S, Azuma K, Yoshizawa S, Ohyashiki K, Ohyashiki JH: Bone marrowmesenchymal stromal cells from healthy donors secrete exosomes that inhibit in vivo
tumor growth and angiogenesis in multiple myeloma. 20th Congress of European
Hematology Association (EHA) (Vienna, Austria, June 11-14, 2015)
71. Yoshizawa S, Umezu T, Saitoh Y, Gotoh M, Akahane D, Kobayashi C, Ohyashiki JH,
Ohyashiki K: Distinct exosomal miRNA expression of late onset graft-versus-hostdisease in allogeneic hematopoietic stem cell transplantation. 20th Congress of European
Hematology Association (EHA) (Vienna, Austria, June 11-14, 2015)72. 梅津知宏, 吉澤成一郎, 東 剣虹, 大屋敷一馬, 大屋敷純子：がん微小環境を制御す
る新たな機構の解明 細胞外小胞エクソソームを介した骨髄腫細胞 骨髄内間質細胞
間相互作用 第 175 回医学会総会（2015 年 6 月 6 日, 東京）
73. 大屋敷純子, 梅津知宏：エピジェネティックな変化およびテロメア動態を指標とした体細胞初期化品質の評価系の確立 第 175 回医学会総会 （2015 年 6 月 6 日, 東京）
74. 東 剣虹, 今西 哲, 梅津知宏, 大屋敷一馬, 大屋敷純子：次世代シーケンサーによる
クリニカルシーケンスの実際：骨髄系腫瘍の体細胞変異解析 第 175 回医学会総会（2015 年 6 月 6 日, 東京）
75. 千葉佑規乃，溝口 出，角田廉，徐明利，樋口要，善本隆之：IL-27 による M1 マクロファージへの分化増殖と動員を介した抗腫瘍効果の増強．第 175 回東京医科大学医学会総会（2015 年 6 月 6 日、東京）
76. 藤本博昭，片桐誠一朗，大屋敷一馬，溝口出，善本隆之，梅津知宏，大屋敷純子：本邦-21-

（様式 2）
法人番号プロジェクト番号131054
S1311016

健常者における BCL2L11(BIM)遺伝的多型の頻度と免疫細胞との関係．第 175 回東京
医科大学医学会総会（2015 年 6 月 6 日、東京）
77. 溝口 出，金子幸太郎，角田廉，水戸部佳奈，古澤純一，千葉佑規乃，徐明利，樋口要，水口純一郎，善本隆之：IL-27/IL-35 非依存的な EBI3 による IL-23 レセプター発現の安定化による炎症性腸炎発症促進．第 91 回東京医科大学・東京薬科大学・免疫アレ
ルギー研究会（2014 年 6 月 3 日、東京）
78. Umezu T, Imanishi S, Yoshizawa S, Saitoh Y, Yamamoto Y, Ohyashiki K, Ohyashiki JH:
Age-related alterations in exosomal miRNAs derived from bone marrow mesenchymal
stromal cells. 6th JSH International Symposium 2015 (Karuizawa, Japan, May 22-23,
2015)
79. Imanishi S, Ohsuga M, Takahashi R, Umezu T, Kobayshi C, Katagiri S, Asano M, Saitoh Y,
Ohyashiki K, Ohyashiki JH. BET protein inhibition interferes DNA integrity in azacytidine
resistant cells through ATM signaling. 6th JSH International Symposium 2015 (Karuizawa,
Japan, May 22, 2015)
80. Asano M, Kobayashi C, Umezu T, Imanishi S, Azuma K, Akahane D, Fujimoto H, Ohyashiki
JH, Ohyashiki K: The genome-wide DNA methylation status as a predictor of
5-azacytidine in clinical responses. 6th JSH International Symposium 2015 (Karuizawa,
Japan, May 22-23, 2015)
81. Azuma K, Umezu T, Imanishi S, Kobayashi C, Asano M, Katagiri S, Akahane D, Ohyashiki
K, Ohyashiki JH: Telomere repeat-containing RNA (TERRA), a possible epigenetic
biomarker of DNA damage phenotype in 5-azacytidine-resistant leukemia cells. 6th JSH
International Symposium 2015 (Karuizawa, Japan, May 22-23, 2015)
82. Ohno Y, Nakashima J, Ohori M, Tachibana M, et al Clinical significance of nutritional
prognostic index in patients with clear cell renal cell carcinoma., 2015.May 15-19, 110th
Annual meeting of American Urological Association (moderated poster session)
83. 大木拓也，梅津知宏，大屋敷純子，福岡 豊：miRNA と mRNA の統合解析によるがん細胞の低酸素適応のメカニズム解析．第 54 回日本生体医工学会大会 （2015 年 5 月 7 日～9 日、名古屋）
84. 老川 桂生, 浅田 浩太朗, 大野 慎一郎, 黒田 雅彦：microRNA 生成経路を欠損する
肺がん細胞株の解析 第 104 回日本病理学会総会（2015 年 5 月 2 日, 名古屋）
85. 藤田 浩司, 山田 正俊, 高梨 正勝, 黒田 雅彦：浸潤性乳癌に出現する let-7d 発現細胞の検討 第 104 回日本病理学会総会（2015 年 5 月 2 日, 名古屋）
86. 大野 慎一郎, 板野 華蓮, 原田 裕一郎, 浅田 浩太朗, 老川 桂生, 柏迫 幹絵,高梨正勝, 須藤 カツ子, 池田 徳彦, 黒田 雅彦：肺がん治療を想定した副作用の少ない短鎖型新規核酸医薬の開発 第 104 回日本病理学会学会（2015 年 5 月 2 日, 名古屋）
87. 浅田 浩太朗, 老川 桂生, 大野 慎一郎, 黒田 雅彦：肺がんにおける Argonaute ファミリーの発現・機能解析 第 104 回日本病理学会総会（2015 年 5 月 2 日, 名古屋）
88. 山田 侑子, 高梨 正勝, 須藤 カツ子, 上田 しのぶ, 黒田 雅彦：肺線維症に対する
miR-29b 補充療法 第 104 回日本病理学会総会（2015 年 5 月 1 日, 名古屋）
89. 倉田 厚, 山田 正俊, 大野 慎一郎, 橋本 浩次, 藤田 浩司, 高梨 正勝, 黒田雅彦：
胃癌細胞株の形態変化における、上皮間葉移行に関わるマイクロ RNA200c とその標的の検出 第 104 回日本病理学会総会（2015 年 4 月 30 日, 名古屋）
90. 山田 正俊齋藤 彰山本 陽一郎立石 文子黒田 雅彦：核形態測定に基づく乳管内増殖病変診断補助の試み 第 104 回日本病理学会総会（2015 年 4 月 30 日, 名古屋）
91. Miki K, Nagaoka K, Bohnenkamp H, Yoshimoto T, Kamigaki T, Maekawa R: Induction of
antigen specific T cell responses using PepTivator OVA-loaded DCs in mouse model.
-22-

（様式 2）
法人番号プロジェクト番号131054
S1311016

第 12 回日本免疫治療学研究会（2015 年 2 月 28 日、東京）
92. Furusawa J, Chiba Y, Mizoguchi I, Xu M, Mizuguchi J, Yoshimoto T: IL-27 promotes thedifferentiation of hematopoietic stem cells into multipotent myeloid progenitor cells. 第
43 回日本免疫学会総会・学術集会（2014 年 12 月 10-12 日、京都）
93. Chiba Y, Furusawa J, Mizoguchi I, Xu M, Mizuguchi J, Yoshimoto T: IL-27 exerts potent
antitumor activity by promoting the differentiation of hematopoietic stem cells to M1-like
antitumor macrophages and their mobilization. 第 43 回日本免疫学会総会・学術集会（2014 年 12 月 10 日 12 日、京都）
94. Katagiri S, Tauchi T, Saito Y, Suguro T. Asano M, Yoshizawa S, Sakuta J, Akahane D,
Tanaka Y, Furuya N, Ando K, Fujimoto H, Okabe S, Gotoh M, Ito Y, Umezu T, Ohyashiki
JH, Ohyashiki K: BCL2L11 (BIM deletion polymorphism is associated with molecularrelapse after ABL tyrosine kinase inhibitors discontinuation in patients with chronic
myeloid leukemia with complete molecular response. 56th American Society of
Hematology (San Francisco, CA, Dec 6-Dec 9, 2014).
95. Okabe S, Tauchi T, Tanaka Y, Katagiri S, Ohyashiki K: Anti-leukemic activity ofphosphoinositide 3-kinase inhibitor, copanlisib, in ABL tyrosine kinase resistant cells.
56th American Society of Hematology (San Francisco, CA, Dec 6-9, 2014).
96. Tauchi T, Katagiri S, Okabe S, Tanaka Y, Ohyashiki K: Activity of the stemness inhibitor,
BBI608, on the self-renewal of BCR-ABL1 positive leukemia cells: Molecular mechanism.
56th American Society of Hematology (San Francisco, CA, Dec 6-9, 2014).
97. 寺山隼人，内藤宗和，善本隆之，平井宗一，曲寧，畑山直之，隅山香織，坂部貢，伊藤正裕：精巣内インターロイキン-35 の役割．第 28 回日本生殖免疫学会総会・学術集会（2013 年 11 月 30-12 月 1 日、兵庫）
98. 大屋敷純子、梅津知宏、今西 哲、東 剣虹、小林千晶、大屋敷一馬：テロメア RNA(TelRNA)を指標とした５―アザシチジン有効性の予測。第 59 回日本人類遺伝学会・第21 回日本遺伝子診療学会合同大会（2914 年 11 月 20 日～22 日、船堀、東京）.
99. 大屋敷一馬、福岡 豊*、片桐誠一朗、田内哲三、東 剣高、武内 健、梅津知宏、大屋敷純子：イマチニブ中止可能な慢性骨髄性白血病患者におけるマイクロ RNA プロファイリング。第 59 回日本人類遺伝学会・第 21 回日本遺伝子診療学会合同大会（2914 年 11
月 20 日～22 日、船堀、東京）.
100. 梅津知宏、大屋敷一馬、大屋敷純子：エピジェネティク変化およびテロメア動態を指標とした体細胞初期化品質の評価。第 59 回日本人類遺伝学会・第 21 回日本遺伝子診療学会合同大会（2914 年 11 月 20 日～22 日、船堀、東京）.
101. 許 文聰、黒田雅彦、上田しのぶ、田子友哉、西村絵美、中島哲史、瀧下智恵、佐原八束、細川勇一、永川裕一、粕谷和彦、土田明彦：分泌型 microRNA を用いた膵癌診断の開発. 第 76 回日本臨床外科学会総会（2014 年 11 月 21 日、郡山）
102. Kuroda M. The development of short form of mimic microRNA for lung cancer therapy.
26th EORTC-NCI-ACR Symposium. (Barcelona, SPAIN, Nov 18-21、 2014)
103. 寺山隼人，善本隆之，内藤宗和，平井宗一，曲寧，畑山直之，林省吾，倉升三幸，水戸部佳奈，古澤純一，溝口 出，毛塚剛司，後藤浩，金沢輝久，坂部貢，伊藤正裕：インターロイキン-35 による精巣免疫特権の維持．第 92 回東京医科大学・東京薬科大学・免疫アレルギー研究会（2014 年 11 月 18 日、東京）
104. 金子幸太郎，溝口 出，角田廉，千葉佑規乃，樋口要，須藤カツ子，藤田浩司，黒田雅彦，水口純一郎，善本隆之：炎症性腸炎発症における IL-27/IL-35 非依存的な EBI3 による IL-23 レセプター発現の安定化．第 174 回東京医科大学医学会総会（2014 年 11 月
1 日、東京）-23-

（様式 2）
法人番号プロジェクト番号131054
S1311016

105. 川口謙太郎，堀内由莉乃，溝口出，金子幸太郎，角田廉，千葉佑規乃，樋口要，前田龍郎，川口敦子，原田和俊，大久保ゆかり，水上潤哉，坪井良治，水口純一郎，善本隆
之：イミキモド誘導性乾癬モデルにおけるストレス応答性 MAP キナーゼ ASK1 の役割．
第 174 回東京医科大学医学会総会（2014 年 11 月 1 日、東京）
106. 大須賀美穂、今西 哲、大屋敷純子、高橋諒子、梅津知宏、小林千晶、片桐誠一郎、大屋敷一馬：クロマチン制御標的剤によるアザシチジン耐性克服法の開発。第 174 回東京
医科大学医学会総会。（2014 年 11 月 1 日、東京）
107. 高橋諒子、大須賀美穂、今西 哲、大屋敷純子、梅津知宏、小林千晶、片桐誠一郎、大屋敷一馬：アザシチジン耐性白血病細胞におけるヒストン化学修飾の役割の解明。第
174 回東京医科大学医学会総会。（2014 年 11 月 1 日、東京）
108. 関野 知聖、梅津 知宏、小林 千晶、大屋敷一馬、東 剣虹、大屋敷純子：加齢に伴う骨髄間質細胞由来エクソソームの量的・質的変化の解析。第 174 回東京医科大学医学会総会。（2014 年 11 月 1 日、東京）
109. 武内 健、梅津 知宏、小林 千晶、大屋敷一馬、大屋敷純子、東 剣虹：低酸素耐性骨髄腫細胞株の樹立と解析。第 174 回東京医科大学医学会総会。（2014 年 11 月 1 日、東京）110. 吉澤成一郎、齋藤 優、後藤守孝、赤羽大悟、大屋敷一馬、梅津知宏、小林千晶、大屋敷純子：同種造血幹細胞移植の GVHD に特異的な exosomal miRNA の解析。第 174 回
東京医科大学医学会総会。（2014 年 11 月 1 日、東京）
111. Imanishi S, Umezu T, Kobayashi C, Osuga M, Asano M, Ohyashiki K, Ohyashiki JH: A
possible association between global DNA hypomethylation and azacitidine resistance inleukemia cells. 第 76 回 日本血液学会学術集会学術集会（2014 年 10 月 31 日～11 月
2 日、大阪）112. Okabe S, Tauchi T, Katagiri S, Tanaka Y, Ohyashiki K: The PLK inhibitor rigosertib in
combination with ABL kinase inhibitor against BCR-ABL-positive cells。第 76 回 日本血液学会学術集会学術集会（2014 年 10 月 31 日～11 月 2 日、大阪）
113. Tauchi T, Katagiri S, Okabe S, Minami Y*, Naoe T*, Ohyashiki K: Targeting the Dopamine
receptor signal limits the self-renewal of BCR-ABL1 positive leukemia cells. 第 76 回 日本血液学会学術集会学術集会（2014 年 10 月 31 日～11 月 2 日、大阪）
114. Kastagiri S, Tauchi T, Saito Y, Suguro T, Asano M, Yoshizawa S, Kitahara T, Akahane D,
Tanaka Y, Fujimoto H, Okabe S, Gotoh M, Ito Y, Ohyashiki K: Long term follow-up after
Imatinib cessation for patients in deep molecular response。第 76 回 日本血液学会学術集会学術集会（2014 年 10 月 31 日～11 月 2 日、大阪）
115. Asano M, Ohyashiki JH, Imanishi S, Kobayashi C, Yamamoto Y, Osuga M, Takahashi R,
Umezu T, Ohyashiki K: The Global DNA methylation as a predictor of 5-azacytidine inclinical responses. 第 73 回日本癌学会学術総会（2014 年 9 月 25-27 日、横浜）
116. Umezu T, Azuma K, Takeuchi K, Sekino C, Ohyashiki K, Ohyashiki JH: がん微小環境を制御するエクソソーム含有 miRNA：長期低酸素曝露した骨髄腫細胞由来 exosomal
miR-135b の機能解析. 第 73 回日本癌学会学術総会（2014 年 9 月 25-27 日、横浜）
117. Chiba Y, Mizoguchi I, Hisada M, Tsuchida A, Mizuguchi J, Yoshimoto T: Regulation of
antitumor immune responses through differentiation and mobilization of hematopoietic
stem cells by IL-27. 第 73 回日本癌学会学術総会（2014 年 9 月 25-27 日、横浜）
118. Miki K, Nagaoka K, Yoshimoto T, Kamigaki T, Maekawa R: PepTivator OVA-pulsed DCs
can induce both OVA specific CD8+ and CD4+ T cells in mouse model. 第 73 回日本癌学会学術総会（2014 年 9 月 25 日 27 日、横浜）
119. 大野慎一郎、板野華蓮、原田裕一郎、高梨正勝、須藤カツ子、池田徳彦、黒田雅彦. 肺-24-

（様式 2）
法人番号プロジェクト番号131054
S1311016

がんを効果的に抑制する MimicmicroRNA の新規形状の探索. 第 73 回日本癌学会学術総会. （2014 年 9 月 25-27 日、横浜）
120. Furusawa J, Chiba Y, Mitobe K, Mizoguchi I, Mizuguchi J, Yoshimoto T: IL-27 promotes
the differentiation of hematopoietic stem cells into DC progenitor cells. 13th International
Symposium on Dendritic Cells, DC2014 (Tours, France, Sep 14-18, 2014)121. 黒田雅彦、高梨正勝、大野慎一郎. エクソソームによるドラッグデリバリーシステムの応用(Application of drug delivery systems by exosomes). 第 6 回日本 RNAi 研究会(JARI)、
第 1 回日本細胞外小胞学会(JSEV). (2014 年 8 月 28-30 日、広島)
122. Okabe S, Tauchi T, Tanaka Y, Kitahara T, Ohyashiki K: Antileukemic effect of PLK
inhibitor and in combination with ABL tyrosine kinase inhibitors against
BCR-ABL-positive cells. 19th Congress of European Hematology Association, (Milan,
Italy、2014, June, 12-15).
123. Terayama H, Naito M, Yoshimoto T, Hirai S, Qu N, Kuramasu M, Hatayama N, Kanazawa T,
Suyama K, Sakabe K, Itoh M: Contribution of IL-35 to maintaining the testicular immune
privilege. 第 33 回日本アンドロロジー学会学術大会（2014 年 6 月 12 日 13 日、軽井沢）
124. 片桐誠一朗、田内哲三、勝呂多光子、浅野倫代、吉澤成一郎、北原俊彦、赤羽大悟、田中裕子、岡部聖一、後藤守孝、伊藤良和、大屋敷一馬、藤本博昭、梅津 知宏、大屋敷純子、田所健一＊：慢性骨髄性白血病患者における BCL2L11 (BIM)遺伝子多型はイマ
チニブ不耐応と関係する。第 173 回東京医科大学医学会総会（2014 年 6 月 7 日、東京）
125. 梅津知宏、今西 哲、大屋敷純子、大屋敷一馬：本学における次世代シーケンサー
MiSeq システムの使用経験。第 173 回東京医科大学医学会総会（2014 年 6 月 7 日、東京）126. 角田廉，溝口 出，金子孝太郎，樋口要，水口純一郎，善本隆之：IL-27/IL-35 の共通サ
ブユニット EBI3 によるオートファジーを介した腫瘍増殖の増強．第 173 回東京医科大学
医学会総会（2014 年 6 月 7 日、東京）
127. 溝口 出，善本隆之，片桐誠一朗，田内哲三，木村之彦，水口純一郎，大屋敷純子，大屋敷一馬：イマチニブ治療を安全に中止できる慢性骨髄性白血病患者の新しい免疫学的抽出法の開発．第 173 回東京医科大学医学会総会（2014 年 6 月 7 日、東京）
128. 角田廉，溝口 出，金子孝太郎，樋口要，水口純一郎，善本隆之：IL-27/IL-35 の共通サ
ブユニット EBI3 によるオートファジーを介した腫瘍増殖の増強．第 173 回東京医科大学
医学会総会（2014 年 6 月 7 日、東京）
129. 溝口出，善本隆之，片桐誠一朗，田内哲三，木村之彦，水口純一郎，大屋敷純子，大屋敷一馬：イマチニブ治療を安全に中止できる慢性骨髄性白血病患者の新しい免疫学的抽出法の開発．第 173 回東京医科大学医学会総会（2014 年 6 月 7 日、東京）
130. 溝口 出、善本隆之、片桐誠一朗、田内哲三、木村之彦、大屋敷一馬、水口純一郎、大屋敷純子：イマチニブ治療を安全に中止できる慢性骨髄性白血病の新しい免疫学的抽出法の開発。第 173 回東京医科大学医学会総会。（2014 年 6 月 7 日、東京）
131. Aoyama K, Yoshimoto T: In vivo and in vitro models for prediction of respiratory
sensitizers. ILSI-HESI Workshop on the Assessment of Respiratory Sensitization(Alexandria, VA, USA, May 28-29, 2014)132. 梅津知宏、大屋敷一馬、大屋敷純子：がん微小環境ネットワークにおけるエクソソーム含
有 miRNA の機能解析。第 8 回エピジェネティクス研究会。（2014 年 5 月 25 日～27 日、
東京）
133. Okabe S, Tauchi T, Tanaka Y, Katagiri S, Kitahara T, Ohyashiki K: Activity of omacetaxine
mepesuccinate against Ponatinib-resistant BCR-ABL-positive leukemia cells. 5th JSH
International Symposium, (Hamamatsu , May/25, 2014).
-25-

（様式 2）
法人番号プロジェクト番号131054
S1311016

134. Imanishi S, Umezu T, Ohtsuki K, Kobayshi C, Ohyashiki K, Ohyashiki JH. Towards
comprehensive understanding of the mechanism underlying resistance to 5-azacytidine.
5th JSH International Symposium, (Hamamatsu , May/25, 2014).
135. Umezu T, Ohyashiki K, Ohyashiki JH: Hypoxia enhances angiogenesis via exosomal
miR-135b derived from multiple myeloma cells. International Society for Extracellular
vesicles. (Rotterdam, April 30th-May 3rd, 2014).
136. Takahara M, Goto S, Miki K, Saiwaki S, Nagaoka K, Matsushita H, Kondo T, Bohnenkamp
H, Yoshimoto T, Maekawa R, Kamigaki T: Induction of antigen specific T cells using
PepTivator -pulsed DCs. International Society For Cellular Therapy 20th Annual
Meeting 2014 (Paris, France, Apr 23-26, 2014)137. Kato R, Nish H, Nagamitsu Y, Sasaki T, Isaka K: miR-100 mediates resistance topaclitaxel in cervical cancer cells. AACR Annual Meeting 2014 (San Diego, USA, May 5-9,
2014)
138. 高梨 正勝、上田 しのぶ、須藤 カツ子、石川 章夫、黒田 雅彦. 自己細胞由来のエクソソームによるドラッグデリバリーシステムへの応用. 第 103 回日本病理学会総会 (2014
年 4 月 24 日～26 日、広島)
139. 古澤純一，善本隆之：IL-27 による造血幹細胞からのミエロイド系前駆細胞の分化・増殖
誘導．新学術領域細胞運命若手の会（2014 年 4 月 18 日 19 日、浜名湖）
140. 片桐誠一朗, 梅津 知宏, 大槻和重, 田所健一*, 山口敏和*, 山本善規*, 田内哲三,
大屋敷 純子,大屋敷 一馬：BCL2L11 (BIM)遺伝子多型は慢性骨髄性白血病患者のイ
マチニブ不耐応と関係する。内科学会学術集会。（2014 年 4 月 11 日～13 日、東京）
141. Miki K, Nagaoka K, Bohnenkamp H, Yoshimoto T, Kamigaki T, Maekawa R: Induction ofboth OVA specific CD4+ and CD8+ T cells by using PepTivator OVA-pulsed DCs inmouse model. American Association for Cancer Research 105th Annual Meeting 2014
(San Diego, California, USA, Apr 5-9, 2014)142. Murakami Y, Ohno S, Kuroda M. Novel RNAi agent can control HCV replication. AACR
Annual Meeting 2014(San Diego, CA, April 5-9, 2014)
143. 赤羽大悟、後藤守孝、片桐誠一朗、勝呂多光子、浅野倫代、吉澤成一郎、伊藤良和、大屋敷一馬: ハイリスク骨髄系腫瘍に対する同種造血幹細胞移植後の 5-azacytidine を用いた後療法に関する検討。第 36 回造血細胞移植学会（2014 年 3 月 12 日、沖縄）
144. 古澤純一，溝口 出，水戸部佳奈，樋口要，金子幸太郎，角田廉，水口純一郎，久田将之，粕谷和彦，土田明彦，善本隆之：癌 DC ワクチン療法への応用を目指した IL-27 を用いた DC 前駆細胞の分化・増殖誘導法の開発．医薬工３大学包括的連携推進シンポジ
ウム（2013 年 12 月 14 日、東京）
145. Tauchi T, Katagiri S, Okabe S, Minami Y, Naoe T, Ohyashiki K: Targeting the dopamine
receptor signaling limits the self-renewal of BCR-ABL1 positive leukemia cells. Molecularmechanisms. 55th Annual Meeting of ASH (New Orleans, USA, Dec 7-10, 2013).
146. Yoshizawa S, Umezu T, Ohyashiki JH, Iida S, Ohyashiki K: Lower plasma miR-92a levels
predict shorter progression-free survival in newly diagnosed symptomatic multiple
myeloma patients. 55th Annual Meeting of ASH (New Orleans, USA, Dec 7-10, 2013).
147. *田所 弘子、梅津 知宏、大屋敷純子：低酸素暴露した K562 細胞由来エクソソームの血管新生に対する影響 第 3 回医薬工 3 大学包括連携シンポジウム (2013 年 12 月 14 日、
東京)
148. 永井宏，藤原進，下浦典子，善本隆之，錦織千佳子：メラノーマ組織内 CD4+T 細胞除去による抗腫瘍免疫反応機序の解析. 第 25 会日本色素細胞学会学術大会（2013 年 11
月 16 日 17 日、大阪）-26-

（様式 2）
法人番号プロジェクト番号131054
S1311016

149. Furusawa J, Mizoguchi I, Mizuguchi J, Yoshimoto T: IL-27 promotes the differentiation ofbone marrow cells into DC progenitor cells. 第 42 回日本免疫学会総会（2013 年 12 月
11 日 13 日、幕張）
150. Mizoguchi I, Mizuguchi J, Yoshimoto T: EBI3 promotes IL-23-mediated signaling resulting
in the development of colitis by enhancing IFN- production. 第 42 回日本免疫学会総会（2013 年 12 月 11 日 13 日、幕張）
151. Kimura M, Sato-Matsushita M, Yoshimoto T, Tahara H, Saiki I, Irimura T, Hayakawa Y:
Cancer immune escalation process triggered by activation of γδT17 cells through
MyD88/IL-23 cascade. 第 42 回日本免疫学会総会（2013 年 12 月 11-13 日、幕張）
152. 古澤純一，溝口出，水戸部佳奈，樋口要，金子幸太郎，角田廉，水口純一郎，久田将之，粕谷和彦，土田明彦，善本隆之：IL-27 による DC 前駆細胞の分化・増殖誘導．第 90
回東京医科大学・東京薬科大学・免疫アレルギー研究会（2013 年 11 月 5 日、東京）
153. 梅 津 知 宏 、 大 槻 和 重 、 大 屋 敷 純 子 、 大 屋 敷 一 馬 ： IPA デ ー タ ベ ー ス を 用 い た
microRNA-mRNA 統合的な分子間ネットワーク解析 第 172 回東京医科大学医学会総会（2013 年 11 月 2 日、東京）
154. 樋口要，森健太郎，溝口出，水口純一郎，善本隆之：接触性皮膚炎誘導における IL-22の役割．第 172 回東京医科大学医学会総会（2013 年 11 月 2 日、東京）
155. Tauchi T, Katagiri S, Okabe S, Minami Y, Naoe T, Ohyashiki K: Targeting the hedgehog
signaling pathway limits the self-renewal of BCR-ABL1 positive leukemia cells. 第 76 回
日本血液学会学術集会 (2013 年 10 月 11 日~13 日、札幌)
156. Okabe S, Tauchi T, Katagiri S, Tanaka Y, Ohyashiki K: Activity of imatinib and Jak kinase
inhibitor: A potential treatment for Ph-positive leukemia cells. 第 76 回日本血液学会学術集会 (2013 年 10 月 11 日~13 日、札幌)
157. Yoshizawa S, Umezu T, Gotoh M, Fujimoto H, Gotoh A, Tauchi T, Ito Y, Ohyashiki JH, Iida
S, Ohyashiki K: Feasibility of plasma MiR-92a as a prognostic marker in newly diagnosed
multiple myeloma patients. 第 75 回日本血液学会学術総会 (2013 年 10 月 11 13 日、札幌)
158. Katagiri S, Mizoguchi I, Yoshimoto T, Ohyashiki JH, Tauchi T, Inokuchi K, Ohyashiki K:
Long-term up-regulation of effector NK cells in CML after stopping imatinib 第 75 回日本血液学会学術総会 (2013 年 10 月 11 13 日、札幌)
159. Ohtsuki K, Katagiri S, Mizoguchi I, Yoshimoto T, Ohyashiki JH, Tauchi T, Inokuchi K,
Ohyashiki K: Clinical implication of down-regulated miR-148b in CML patients stopping
imatinib. 第 75 回日本血液学会学術総会 (2013 年 10 月 11 13 日、札幌)
160. Asano M, Imanishi S, Umezu T, Ohyashiki JH, Ohyashiki K: Clinical and experimental
assessment of 5-azacytidine resistance in patients with MDS. 第 75 回日本血液学会学術総会 (2013 年 10 月 11 日 13 日、札幌)
161. Tadokoro H, Umezu T, Ohyashiki K, Hirano T, Ohyashiki JH: Exosomes derived fromhypoxic K562 cells enhances tube formation in endothelial cells. 第 75 回日本血液学会
学術総会 (2013 年 10 月 11 日 13 日、札幌)
162. Tauchi T, Tojo A, Nakamae H, Kobayashi Y, Miyamura K, Kyo T, Iwasaki H, Hatake K, Usui
N, Narasimhan N, Zhang J, Naoe T, Ohyashiki K: Preliminary report of a phase 1/2 studyof ponatinib in Japanese patients with Ph+ leukemias. 第 76 回日本血液学会学術集会(2013 年 10 月 11 日~13 日、札幌)
163. 黒田 雅彦. シンポジウム 6.マイクロ RNA のがん治療および癌診断への応用 新規のlipid conjugate RNA を用いたがん治療法の開発． 第 72 回日本癌学会学術総会 （2013年 10 月５日、横浜）-27-

（様式 2）
法人番号プロジェクト番号131054
S1311016

164. Okabe S, Tauchi T, Katagiri S, Tanaka Y, Ohyashiki K: Activity of the Aurora kinase
inhibitor, MLN8237 (alisertib) and ponatinib against BCR-ABL-positive cells. 第 72 回日本癌学会学術総会（2013 年 10 月 3 日～5 日、横浜）
165. Imanishi S, Umezu T, Ohtsuki K, Kobayashi C, Ohyashiki K, Ohyashiki JH: A possible roleof DNA damage/repair in 5-azacytidine resistant leukemia cells. 第 72 回日本癌学会学術総会（2013 年 10 月 3～5 日、横浜）
166. Umezu T, Tadokoro H, Hirano T, Ohyashiki K, Ohyashiki JH: Hypoxia myeloma cells
modulates its microenvironment via exosome. 第 72 回日本癌学会学術総会（2013 年 10
月 3～15 日、横浜）
167. Yoshimoto T, Mizoguchi I, Hisada M, Mizuguchi J: New approach for DC-mediated cancer
vaccine using IL-27-expanded myeloid progenitor cells. 第 72 回日本癌学会学術総会（2013 年 10 月 3 日 5 日、横浜）
168. Kimura Y, Sato-Matsushita M, Yoshimoto T, Tahara H, Saiki I, Irimura T, Hayakawa Y:
Cross-talk between host and cancer cells amplify inflammation triggered by γδT cells
to promote malignant progression. 第 72 回日本癌学会学術総会（2013 年 10 月 3 日 5日、横浜）
169. Mizoguchi I, Mitobe K, Tsunoda R, Higuchi K, Mizuguchi J, Yoshimoto T: EBI3 functions as
an intracellular molecule to regulate the development of colitis in naive CD4+CD25- T
cells. 15th International Congress of Immunology (Milan, Italy, Aug 22-27, 2013)170. 黒田 雅彦、大野慎一郎、高梨 正勝. Exosome によるがん特異的 miRNA デリバリー法の開発. 第 29 回日本ＤＤＳ学会学術集会、シンポジウム（招待講演）（2013 年７月５日、京都）
171. Katagiri S, Mizoguchi I, Yoshimoto T, Mizuguchi J, Ohyashiki JH, Tauchi T, Inokuchi K,
Ohyashiki K: The percentage of effector populations of NK cells correlated highly withsustained CMR with or without imatinib in chronic myeloid leukemia patients. 18th
Congress of the European Hematology Association (Stockholm, Sweden, Jun 13-16,
2013)
172. 高梨 正勝、須藤カツ子、松永 芳径、石川 章夫、大木 忠明、濱崎 智洋、後藤 浩、
黒田 雅彦．新規核酸を用いた血管新生網膜症に対する分子標的治療法の開発．第102 回日本病理学会総会（2013 年 6 月 7 日、札幌）
173. 山田 正俊、大野慎一郎、藤田 浩司、倉田 厚、黒田 雅彦．前立腺癌におけるmicroRNA-200c の発現解析．第 102 回日本病理学会総会 (2013 年 6 月 7 日、札幌)
174. 藤田 浩司、仲矢 丈雄、池田 徳彦、黒田 雅彦．細気管支肺胞上皮癌における RASと let-7 の発現の特徴．第 102 回日本病理学会総会 (2013 年 6 月 7 日、札幌)
175. 角田廉，溝口 出，樋口要，久田将之，粕谷和彦，土田明彦，水口純一郎，善本隆之：
IL-27 together with stem cell factor induces differentiation of bone marrow cells into
myeloid progenitor cells and expands them. 第 171 回東京医科大学医学会総会（2013年 6 月 1 日、東京）
176. 溝 口 出 ， 片 桐 誠 一 朗 ， 水 口 純 一 郎 ， 大 屋 敷 純 子 ， 大 屋 敷 一 馬 ， 善 本 隆 之 ：Up-regulation of effector natural killer cells and effector memory CD8+ T cells inmaintaining complete molecular response after imatinib discontinuation in chronic
myeloid leukemia. 第 171 回東京医科大学医学会総会（2013 年 6 月 1 日、東京）＜研究成果の公開状況＞（上記以外）-28-

（様式 2）
法人番号プロジェクト番号131054
S1311016

シンポジウム・学会等の実施状況、インターネットでの公開状況等＜既に実施しているもの＞
1. 国際シンポジウム（主催）Tokyo Medical University, Institute of Medical Science
1st International Symposium "Role of Aging and Cancer"
Date: August 28, 2017 14:00~19:00
Venue: Tokyo Medical University Hospital,
Education and Research Building Meeting Room (3rd floor)
Organized by Masahiko Kuroda, Takayuki Yoshimoto and Junko H. Ohyashiki
http://team.tokyo-med.ac.jp/ims_onc/symposium/index.html
2. 研究成果のインターネットによる公開
http://team.tokyo-med.ac.jp/ims_onc/research/project_nano.html＜これから実施する予定のもの＞
なし

１４ その他の研究成果等
プレスリリース(1) エクソソームを「若返えらせる」ことによる多発性骨髄腫の血管新生抑制効
果を証明～細胞分泌物を利用した「がんの次世代細胞治療」へ新たな道筋～
2017 年 6 月 16 日
*科学新聞：多発性骨髄腫 癌血管新生効果証明～エクソソームが若返り～ 2017 年 7月 14 日(2) 抗がん剤に対する薬剤耐性の解除と予防方法を見出す～既存の関節リウマチ等治療薬で血液がんの薬剤耐性を世界で初めて克服へ～2017 年 7 月 25 日
*科学新聞：抗ガン剤「薬剤耐性」解除と予防法発見 2017 年 9 月 1 日-29-

（様式 2）
法人番号プロジェクト番号
１５ 「選定時」及び「中間評価時」に付された留意事項及び対応＜「選定時」に付された留意事項＞
なし＜「選定時」に付された留意事項への対応＞なし＜「中間評価時」に付された留意事項＞
なし＜「中間評価時」に付された留意事項への対応＞
なし

-30-

131054
S1311016

（様式2）
法人番号プロジェクト番号131054
S1311016

１６

（千円）
内
年度・区分 支出額 法 人
平 施 設
成
2 装 置
5 設 備
年
度 研究費
平 施 設
成
2 装 置
6 設 備
年
度 研究費
平 施 設
成
2 装 置
7 設 備
年
度 研究費
平 施 設
成
2 装 置
8 設 備
年
度 研究費

負 担

私 学
助 成

21,666

7,222

14,444

8,331

4,661

3,670

11,880

3,960

7,920

14,029

8,201

5,828

13,716

11,785

14,844

10,657

訳

共同研
受託
究機関
寄付金 その他（
研究等
負担

）

0
0

0
0

0
0
0
25,50100025,501

平 施 設
成
2 装 置
9 設 備
年
度 研究費

25,33014,411

10,919

施 設

0

0

0

0

0

0

0

総

装 置

0

0

0

0

0

0

0

額

設 備

33,546

11,182

22,364

0

0

0

0

研究費

98,692

55,83342,8590000132,238

67,01565,223

0

0

0

0

総

計

0
0
0

-31-

備 考

（様式2）
法人番号131054

１７《施設》 （私学助成を受けていないものも含め、使用している施設をすべて記載してください。）
研究施設面積整備年度

施 設 の 名 称

研 究 室 等 数 使用者数

事業経費

補助金額

0
99.29㎡
1
15名
1575
125㎡
1
10名
血液内科学分野
055.31㎡
1
7名
臨床共同研究センター 培養室
016.32㎡
1
22名
医学総合研究所免疫制御
0
135㎡
4
15名
分子病理学分野
2
15名
107.78㎡
0※ 私学助成による補助事業として行った新増築により、整備前と比較して増加した面積0
医学総合研究所分子腫瘍

生体分子先端研究センター

H25

0
0
0
0
0
0
㎡

《装置・設備》 （私学助成を受けていないものは、主なもののみを記載してください。）
整備年度

装置・設備の名称

型

番

台

数

稼働時間数

事業経費

（千円）
補助主体

（千円）
補助金額

補助主体

（研究装置）
IncuCyte ZOOM 生細胞イメージングシステム

次世代シークエンサーシステム一式
HSオールインワン蛍光顕微鏡
VENTANA DISCOVERY ULTRA特別仕様
Applied Biosystems 7900HT Fast リアルタイムPCRシステム

Applied Biosystems 3130X1ｼﾞｪﾈﾃｨｯｸｱﾅﾗｲｻﾞ

自動ｱﾚｲﾊｲﾌﾞﾘﾀﾞｲｾﾞｰｼｮﾝｼｽﾃﾑ

H26
H25
H24
H23
H22
H20
H15

１式
１式
１式
１式
１式
１式
１式
１式
１式
１式

MS-J-001
BZ-9000
免疫染色&in Situ hybridization 全自動システム

7900HT-41F
3130X1
VersaDoc1000ｼｽﾃﾑ

GenePix 4000B
GeneTAC Hybridization Station

遺伝子解析ｼｽﾃﾑ

H14

ABI PRISM 3100特型 他

120/週
96/週
72/週
60/週
72/週
72/週
48/週
48/週
48/週
6/日

7,920 私学助成
14,444 私学助成
6,400 私学助成
0013,671 私学助成
15,808 私学助成
私学助成
私学助成
25,000 私学助成

11,88021,666
9,600
19,95017,76020,506
24,960

ｈ
ｈ
ｈ
ｈ
ｈ
ｈ
ｈ
ｈ
ｈ
ｈ

37,500（情報処理関係設備）
ｈｈｈｈ

１８ 研究費の支出状況
年
度
平成
小 科 目
消 耗 品 費光 熱 水 費
通信運搬費
印刷製本費
旅費交通費
報酬・委託料（ 会議会合費 ）（ 謝金 ）
計
人件費支出（兼務職員）
教育研究経費支出
計
教育研究用機器備品

図

（千円）
25

年度

支 出 額

主 な 使 途教
育研
究
6,354 実験材料

37 会議費100 謝金
6,491
ア
ル

バ

イ

金

積 算 内 訳
額
主 な
経
費
支
出
6,354 試薬、実験器具

37 会議飲食代
100 講演者謝金
6,491
関
係
支

ト

出

0
設 備 関 係 支 出（１個又は１組の価格が５００万円未満のもの）
1,840 機器備品
1,840 超低温槽、実験台

書
計

1,840
研
究

ス

タ

ッ

リサーチ・アシスタント
ポスト・ドクター
研究支援推進経費

計

0

-32-

フ

1,840
関

係

支

出

内

容

（様式2）
法人番号
年

度

小 科 目

年度

積 算 内 訳主 な 使 途
金 額
主 な
教
育
研
究
経
費
支
出
8,280 実験材料
8,280 試薬、実験器具
0
0
0
0
0
0
0
0
376 保守料
376 機器保守料
120 謝金120 講演者謝金
1,996 雑費
1,996 ソフトウェア
10,772
10,772
ア
ル
バ
イ
ト
関
係
支
出

支 出 額

消 耗 品 費光 熱 水 費
通信運搬費
印刷製本費
旅費交通費
報酬・委託料（ 謝金 ）（ 雑費 ）
計
人件費支出（兼務職員）
教育研究経費支出
計内容0
設 備 関 係 支 出（１個又は１組の価格が５００万円未満のもの）
3,257 機器備品
3,257 培養器

教育研究用機器備品

図

26

平成

131054

書
3,257
研
究

計
リサーチ・アシスタント
ポスト・ドクター
研究支援推進経費

計
年

度

小 科 目
消 耗 品 費光 熱 水 費
通信運搬費
印刷製本費
旅費交通費
報酬・委託料（ 雑費 ）（ 修理費 ）
計
人件費支出
（兼務職員）
教育研究経費支出
計
教育研究用機器備品

図

ス

タ

ッ

3,257
フ
関

係

4,112 研究補助

4,112 学内1人

4,112

4,112 学内1人

27

平成

支

出

年度

積 算 内 訳主 な 使 途金 額
主 な 内 容
教
育
研
究
経
費
支
出
16,328 実験材料
16,328 試薬、実験器具
000020 ポスター印刷
20 印刷費1,608 学会費
1,608 学会旅費、参加費
4,609 委託料
4,609 機器保守料、補助業務費
351 雑費351 マウス飼育管理費、英文校閲料
1,225 修理費
1,225 機器備品修理費
24,141
24,141
ア
ル
バ
イ
ト
関
係
支
出
511 時給1,400円，年間時間数 180時間
511 研究補助支 出 額

実人数 2人511
設 備 関 係 支 出（１個又は１組の価格が５００万円未満のもの）
849 機器備品
849 多本架冷却遠心機

書計

リサーチ・アシスタント
ポスト・ドクター
研究支援推進経費

計

849
研
究

ス

タ

ッ

4,211 研究補助

フ

関

係

4,211 学内1人

4,211

学内1人

-33-

支

出

（様式2）
法人番号
年

度

小 科 目

年度

支 出 額

主 な 使 途教
育研
究
19,033 実験材料

消 耗 品 費光 熱 水 費
通信運搬費
印刷製本費
旅費交通費
報酬・委託料（ 雑費 ）（ 修理費 ）
（ 雑務費 ）
計金

積 算 内 訳
額
主 な
経
費
支
出
19,033 試薬、実験器具

内

容

論文投稿料
学会旅費、参加費
機器保守料
ソフトウェア
機器備品修理費
解析、英文校正料

63 印刷製本費
218 学会費
3,138 委託料
201 雑費828 修理費1,420 雑務費
24,901
アル
バ

人件費支出（兼務職員）
教育研究経費支出
計

イ

ト

関

係

支

出

0
設 備 関 係 支 出（１個又は１組の価格が５００万円未満のもの）
600 機器備品
600 マルチガスインキュベーター

教育研究用機器備品

図

28

平成

131054

書
600
研
究

計
リサーチ・アシスタント
ポスト・ドクター
研究支援推進経費

ッ

フ

小 科 目
消 耗 品 費光 熱 水 費
通信運搬費
印刷製本費
旅費交通費
報酬・委託料（ 雑費 ）（ 修理費 ）
（ 雑務費 ）
計
人件費支出
（兼務職員）
教育研究経費支出
計
教育研究用機器備品

関

29

平成

係

支

出

学内1人

3,890

度

図

タ

3,890 研究補助

計

年

ス

3,890

年度

支 出 額

主 な 使 途教
育研
究
17,305 実験材料

128 印刷製本費
410 学会費
3,967 委託料
980 雑費
67 修理費982 雑務費
23,839
ア
ル
バ

イ

金

ト

積 算 内 訳
額
主 な
経
費
支
出
17,305 試薬、実験器具

リサーチ・アシスタント
ポスト・ドクター
研究支援推進経費

計容128 抄録印刷、ポスター印刷
410 学会旅費、参加費
3,967 機器保守料、補助業務費980 論文掲載料、ソフトウェア
67 機器備品修理費
982 解析、英文校正料
23,839関係
支出

0
設 備 関 係 支 出（１個又は１組の価格が５００万円未満のもの）
1,491 機器備品
1,491 微量分光光度計、微量高速遠心機

書
計

内

1,491
研
究

ス

タ

ッ

フ

1,491
関

係

4,734 研究補助

4,734 学内1人

4,734

4,734

-34-

支

出

プレスリリース
報道関係各位
平成 29 年 6 月 16 日
東京医科大学
エクソソームを「若返えらせる」ことによる多発性骨髄腫の血管新生抑制効果を証明～細胞分泌物を利用した「がんの次世代細胞治療」へ新たな道筋～
東京医科大学の大屋敷純子教授、梅津知宏講師を中心とする研究チームは若年者骨髄由来細胞に特徴的なマイクロ RNA を同定しました。改変エクソソーム技術を使って、必要なマイクロ RNA をエクソソームに直接導入し、「エクソソームそのものを若返らせる」ことによって、血液がんの一種である多発性骨髄腫のがん血管新生が抑制されることを見いだしました。正常の骨髄間質細胞は細胞治療のソースとして長らく注目されていましたが、細胞から分泌されるエクソソームの加齢による機能低下については、今まで不明でした。高齢者に多い多発性骨髄腫では骨髄間質細胞の老化と共に分泌するエクソソームも機能低下し、がんの進展に係っていると考えられ、改変エクソソーム技術による「エクソソームの若返り」は、細胞自体ではなく細胞分泌物を利用する「がんの次世代細胞治療」の新しいアプローチとして期待できると考えています。この成果は 2017 年 5月 23 日（米国東部標準時間）に米国科学誌 Blood のオンライン姉妹誌である Blood
Advances に掲載されました。
【研究の背景】
がんの発症が年齢とともに増加することは広く知られており、その要因としては免疫能の低下をはじめとして、加齢による生体防御機構の機能低下が想定されています。多発性骨髄腫は高齢者に多い血液のがんで、感染症、骨折等さまざまな症状で、高齢化社
会に伴い近年増加しています。一方、広い意味での骨髄間質細胞は骨髄間葉系幹細胞を含んだ細胞集団で、免疫原性が低いことより、細胞治療のソースとして有用であることが知られています。また、健康人の骨髄間質細胞にはがん細胞の抑制効果があり、その効果については加齢による影響があるのではないかと漠然と考えられていました。エク
ソソームとは細胞が分泌する細胞外小胞の一種で、その中にタンパク質、DNA、RNA、マ
イクロ RNA などの遺伝情報が内包されており、エクソソームが細胞から細胞へ移動する
ことよって、細胞同士のコミュニケーションが行われていることが最近明らかになりました。そこで、健康人の骨髄間質細胞が分泌するエクソソームの多発性骨髄腫に対する効果を調べるとともに、骨髄を提供する健康人の年齢によるエクソソームの差を明らかにし、がん抑制機能が低下したエクソソームを改変することによって、治療に応用できないかと考え、研究を行いました。
【本研究の概要と意義】
本研究ではまず、若年者骨髄間質細胞と高齢者骨髄間質細胞の双方で、その分泌物であるエクソソームのがん抑制効果がどう違うかを検討しました（図１）。正常骨髄間質細胞としてはロンザ社から購入した様々な年齢層の細胞を用いました。そして、若年者骨-35-

髄間質細胞由来エクソソーム（若年者エクソソーム）と高齢者骨髄間質細胞由来エクソ
ソーム（高齢者エクソソーム）の機能を比較しました。腫瘍細胞は低酸素耐性多発性骨髄腫細胞株（名称：RPMI8226-HR）を用い、マトリゲルという大豆大の丸い透明のゲルに腫瘍細胞とエクソソームを同時に加え、マウスの皮下に接種し、３週間後のゲルの変化を観察しました。図１上段は生理食塩水を接種したコントロールを、中段は細胞と一緒に若年者エクソソームを下段は高齢者エクソソームを加えています。そして、そのマト
リゲルをマウスの背中の皮下に接種し、３週間後に取り出すと、生理的食塩水を加えた
場合はマトリゲル内にがん血管と腫瘍細胞が充満してどす黒い赤色になっているのに対して、エクソソームを加えた場合はマトリゲルには何も起こらず、白いままです。このことよりエクソソームががん血管新生と腫瘍細胞の発育を阻止していることを意味しています。さらに、よく見ると高齢者エクソソームではうっすらと茶色い部分、すなわち
がん血管新生と腫瘍細胞のわずかな発育が見られ、そのがん抑制効果は若年者エクソソームに劣ることがわかります。右端の２つの図はマトリゲルの組織を染めたものです。
多発性骨髄腫特異的マーカーである CD138 と血管特異的マーカーである CD31 の二重染色で、若年者エクソソームと比べて高齢者エクソソームでは抑制効果が弱く、部分的に腫瘍細胞やがん血管が認められます。右端はがん血管新生の度合いを蛍光染色したもので、
DAPI という色素で青く染まっているのが細胞の核、赤く染まっているのが CD31 陽性のがん血管です。同様に若年者エクソソームの方がすぐれたがん血管抑制効果を示していることがわかります。

図１：若年者エクソソームの血管新生抑制効果
生理的食塩水（コントロール）

CD138 / CD31 DAPI / CD31
マトリゲルプラグ

腫瘍細胞
若年者エクソソーム

腫瘍細胞

高齢者エクソソーム

腫瘍細胞

マトリゲル
次に、この若年者エクソソームの優れたがん抑制効果はエクソソームの中味の何によるものかを明らかにするため、385 個のマイクロ RNA について検討しました。その結果、特定のマイクロ RNA（miR-411, miR-374a, miR-340, miR-365 など）が若年者エクソソー
ムに多く含まれていることがわかりました。
そこで、高齢者エクソソームにこれらのマイクロ RNA 類似体を単独、または混合して直接導入し、「エクソソームの若返り」を試みたところ、導入するマイクロ RNA によっては高齢者エクソソームの若返りにより若年者エクソソームと同程度のがん抑制効果が得られることがわかりました。

-36-

これらの事実から得られる結論を図２に示しました。

図２： 仮説、改変エクソソームによるがん血管新生抑制とがん抑制効果（多発性骨髄腫モデル）
血
がん血管
管
内
若年者エクソソーム
皮細そのまま投与胞
エクソソーム
増回収
殖
抑制
マイクロRNA補充による
骨髄間質細胞を培養 「若返りエクソソーム」投与

腫瘍塊

図２の左下に示したのは健康人の骨髄間質細胞をシャーレの中で培養している様子です。
培養した骨髄間質細胞は大量のエクソソームを分泌しますので、それを回収します。培養した骨髄間質細胞が若年者由来であれば、若年者エクソソームとしてそのまま投与することで（図２ 中央上）、血管内皮細胞の増殖を抑えます。もし、培養した骨髄間質細胞が高齢者由来であれば、改変エクソソーム作製手法を用いてマイクロ RNA を補充し「若返りエクソソーム」にしてから、投与すると若年者エクソソームと同様の血管内皮細胞抑制効果がみられます。図２右は多発性骨髄腫細胞が増殖した腫瘍塊とそれに栄養を供給するがん血管（腫瘍塊の左上）を示します。さらに、これらのエクソソームを投与することで、栄養が断たれることを黄色のマークで表しています。すなわち腫瘍細胞を直接攻撃するのではなく、腫瘍が増えるための栄養血管を遮断するといういわばがんの兵
糧攻めのような構図が浮かび上がります。本研究はマウスを用いたモデルですが、エク
ソソームのデリバリーの問題を含めて、「がんの次世代細胞治療」へと発展させたいと思います。
【今後の展望】
エクソソームの臨床応用については診断、治療とも日々飛躍的に研究が進んでいます。
本研究ではエクソソーム治療のソースである骨髄間質細胞の加齢という点に着目し、新規技術を用いてエクソソームの若返りによる機能強化を実現し、その作用機序を明らかにしました。この新規技術ならびにエクソソームによる「がんの次世代細胞治療」を実現するためにはデリバリーの問題はあります。近年、エクソソームが目的の臓器に到達するための郵便番号のような目安として、インテグリンが鍵をにぎっていることが明らかになってきました。したがって、インテグリンをめやすとしたデリバリーシステムが将来確立されれば、多発性骨髄腫のみならず、多種類の臓器の固形癌においても「がんの次世代細胞治療」として期待されます。
【用語の解説】
注１） 骨髄間質細胞
骨髄の支持組織を構成する線維芽細胞、骨芽細胞、脂肪細胞、間葉系幹細胞など雑多な細胞の総称。
注２） エクソソーム細胞が分泌する細胞外小胞（エクストラセリュラーベシクルス：EV）のうち、30 から 100
ナノメーターのもの。なお、現在一般的に用いられている回収法ではエクソソーム分画-37-

のみを分離することは難しく、厳密に言えば、細胞外小胞という呼称が適切で、エクソソームは便宜上の標記
注３） がん血管がん組織、腫瘍組織に栄養を供給する血管。
注４） マイクロ RNA
遺伝子の転写制御にかかわる 20-25 塩基の一本差 RNA。
注５） マトリゲルプラグ
マウスの皮下に注入して血管新生を判定するための増殖因子を含んだゲル状の構造物。
【発表雑誌】
雑誌名：Blood Advances 2017 1(13): 812-823
論文名：Replenishing exosomes from older bone marrow stromal cells with miR-340
inhibits myeloma-related angiogenesis
掲載日：米国東部標準時間５月 23 日、オンラインで掲載。
【参照 URL】
Blood Advances のホームページ：
http://www.bloodadvances.org/content/1/13?current-issue=y
DOI: http://doi.org/10.1182/bloodadvances.2016003251【研究支援】
本研究は「文部科学省私立大学戦略的研究基盤形成支援事業：「生体分子情報による次世代型がん個別最適化治療法の開発：事業番号 S1311016」および日本学術振興会科研費の支援を受けています。

【お問い合わせ】
東京医科大学研究に関するお問い合わせ
責任著者 大屋敷 純子（おおやしき じゅんこ）
東京医科大学医学総合研究所 分子腫瘍研究部門 教授
電話：03-3342-6111 (内線 5171)
FAX : 03-3345-0185
E-mail: junko@tokyo-med.ac.jp〒160-0023 東京都新宿区西新宿 6-7-1 東京医科大学病院
梅津 知宏（うめず ともひろ）
東京医科大学医学総合研究所 分子腫瘍研究部門 講師
電話：03-3342-6111 (内線 5171)
FAX : 03-3345-0185
E-mail: t_umezu@tokyo-med.ac.jp〒160-0023 東京都新宿区西新宿 6-7-1 東京医科大学病院
研究以外のお問い合わせ
総務部 広報･社会連携推進課
電話：03-3351-6141（代表）-38-プレスリリース
報道関係各位2017 年 7 月 25 日
東京医科大学

抗がん剤に対する薬剤耐性の解除と予防方法を見出す～既存の関節リウマチ等治療薬で血液がんの薬剤耐性を世界で初めて克服へ～
東京医科大学 医学総合研究所（東京都新宿区）の大屋敷純子教授、今西哲助教を中心とする研究チームは、関節リウマチや多発性硬化症の治療に用いられる薬が、血液がんの一種である骨随異形成症候群の薬剤耐性を解除、克服する可能性があることを見出しました。抗がん剤への耐性について、ある疾患の治療に使用されている既存の薬を、他の疾患の治療に応用する“ドラッグリポジショニング”手法を用いることで解除と予防方法を見出したのは世界で初めてのことになります。
この成果は 2017 年 7 月 22 日（米国東部標準時間）米国科学誌「Oncotarget」電子版に掲載されました。
【研究の背景】
骨髄異形成症候群は高齢者に多い血液のがんで、貧血、出血傾向や感染しやすい等の症状を示し、高齢化に伴い近年増加している疾患の一つです。また、およそ 30%の患者が急性骨髄性白血病へと進展する疾患です。現在、これらの症状や予後を改善するためにアザシチジンという抗がん剤が使用されています。しかし、骨髄異形成症候群の患者のおよそ半数はアザシチジンが無効で、またアザシチジンが有効でも長い間には効果が期待できなくなってくる患者がみられることが臨床上の問題となっています。
【研究の成果と意義】
今回、研究チームでは、関節リウマチや多発性硬化症に用いられる既存薬のテリフル
ノミドを併用することで、耐性を示していたアザシチジンの代謝を促し、耐性を解除することを見出しました。
がんの化学療法において耐性化は依然大きな問題です。耐性化を克服するため新規の抗がん剤の開発研究が産学を問わず精力的に展開されていますが、新薬の開発には膨大な時間と費用がかかってしまいます。本研究では耐性の機序に着目し、ドラッグリポジ
ショニングの手法を採用することで、薬剤の耐性を解除・予防できる薬剤を見出す事に成功しました。
既知のメカニズムに基づいて、ドラッグリポジショニングを試みるアプローチは、時間と費用の大幅な削減につながります。特に、がんの薬剤耐性のような喫緊の課題の解

1

-39-

決には極めて有効であることが期待できます。加えて耐性の予防や解除は、がん化学療法における新しい治療戦略となることが期待されます。
【本研究に至る経緯】
研究チームは、以前の研究でアザシチジンへの耐性を獲得した白血病細胞では、DNAや RNA の原料であるピリミジンの代謝が変化していることを見出していました。通常、白血病細胞は「サルベージ経路」と呼ばれる方法でピリミジンを利用可能な状態に代謝します。アザシチジンもこのサルベージ経路を通して代謝されることで効果を発揮します。ところが、アザシチジン耐性白血病細胞ではサルベージ経路で重要な役割を果たす
タンパク質が減っていました。この結果を受けて、研究チームはもう一つのピリミジン
代謝経路である「de novo 合成経路」に着目しました。De novo 合成経路は関節リウマチや多発性硬化症に用いられるテリフルノミドで阻害できることが知られています。すな
わちテリフルノミドを併用することで、その存在下では de novo 合成経路が阻害される
ためピリミジンが不足し、これを補うために再度サルベージ経路が活性化されます。そこにアザシチジンが存在すれば、これが再び代謝されるようになり、薬剤としての効果をもたらすのではないか、ということです。
そして下記の詳細のような検討を行った結果、テリフルノミドが de novo 合成経路を阻害することで、サルベージ経路が活性化され、アザシチジンへの耐性を解除することが分かりました。さらにはテリフルノミドは既に獲得されたアザシチジンへの耐性を解除するだけでなく、耐性の獲得を予防できる可能性があることも示唆されました。
【本研究の経緯詳細】
本研究ではまずアザシチジン感受性白血病細胞とアザシチジン耐性白血病細胞のテリ
フルノミドへの感受性を比較しました。アザシチジン耐性細胞はアザシチジン感受性細胞よりもテリフルノミドに高い感受性を示し、アザシチジン耐性細胞では de novo 合成経路が重要な役割を担っていることが示唆されました。サルベージ経路の活性が低下しているアザシチジン耐性細胞は 5-エチニルウリジンという物質の取り込みが低下していますが、細胞増殖に影響しない濃度（1M）のテリフルノミドの存在下では 5-エチニル
ウリジンを取り込んでいる細胞が増加しました。この結果はテリフルノミドによる denovo 合成経路の阻害が、サルベージ経路を活性化出来る事を意味しています（図１）。

2

-40-

そこで 1M のテリフルノミドと臨床的濃度のアザシチジンを一緒にアザシチジン耐性細胞の培養液に加え、72 時間後の生存細胞数を計測したところ、アザシチジンの濃度に依存して生存細胞数が減少しました（図２左）。同様の結果はアザシチジンへの耐性を獲得した患者由来の白血病細胞でも観察されました（図２右）。以上のことからテリフルノミ
ドはアザシチジンへの耐性を解除し、感受性を回復させると考えられます。

さらに研究チームは、アザシチジン耐性の獲得をテリフルノミドとの併用で予防できるのではないかと考えました。この可能性を検証するため、5M のアザシチジンのみを含む培養液（AZA 群）、1M のテリフルノミドのみを含む培養液（TFN 群）、1M のテリフ
ルノミドと 5M アザシチジンを含む培養液（AZA+TFN 群）のそれぞれで、アザシチジン
感受性細胞を長期間培養する実験を行いました。実験開始から 42 日後には AZA 群で、アザシチジン存在下でも増殖する細胞が出現していることが確認できました。しかし

3

-41-

AZA+TFN 群の細胞では増殖が抑えられたままでした。実験開始から 49 日目に全て群の細胞を回収し、アザシチジンへの感受性を評価したところ、AZA+TFN 群の細胞では、予想通りアザシチジン感受性が保たれていることが明らかになりました（図３）。このことから、テリフルノミドは既に獲得されたアザシチジンへの耐性を解除するだけでなく、耐性の獲得を予防できると考えられます。

今回の実験で使用したテリフルノミドの濃度は、関節リウマチや多発性硬化症の患者さんの血中濃度のおよそ 1/10 という低濃度で、臨床でも安全に投与できると思われます。
テリフルノミドとアザシチジンの併用は、骨髓異形成症候群の新たな治療戦略になると期待されます。

【用語の解説】
注１） 骨髓異形成症候群
全ての血球のもとになる造血幹細胞のがん化が原因で発症すると考えられ、赤血球、白血球、巨核球（血小板を作る細胞）の形態や機能の異常を生じる疾患。正常な血球を作れないため、貧血、易感染性、出血傾向といった症状を示す。高齢者に多く約 30%が最終的に急性骨髄性白血病に進展する。
注２） アザシチジン
国内では 2011 年に骨髄異形成症候群への適応が承認された。DNA や RNA の材料であるシチジンと類似した構造をしているため、シチジンの代わりに DNA や RNA に取り込まれる。
タンパク質合成の阻害、DNA メチル化の阻害による遺伝子発現の調節など、広範な活性
を示す。

4

-42-

注３） ピリミジン
化学的には化合物の名称だが、ここではピリミジン塩基であるシトシン、チミン、ウラシルの総称として使用している。各ピリミジンがリボースと結合したものを、それぞれシチジン、チミジン、ウリジンとよぶ。これらの物質は通常、細胞内に備蓄されており、サルベージ経路を通してリン酸化されて DNA、RNA の材料となる。また、備蓄以外に一部のアミノ酸から合成される経路が存在し、de novo 合成経路と呼ばれている。
注４） テリフルノミド
ピリミジン de novo 合成経路の阻害剤で免疫を調整する活性をもつ。欧米では多発性硬化症への適応がみとめられている。国内ではテリフルノミド自体は未承認だが、関節リウマチに使用されるレフルノミドは体内でテリフルノミドに変換されて効果を発揮することが知られている。
【発表雑誌】
雑誌名：Oncotarget
論文タイトル：Teriflunomide Restores 5-Azacytidine Sensitivity via Activation of
Pyrimidine Salvage in 5-Azacytidine-Resistant Leukemia Cells.
掲載日：米国東部標準時間 7 月 22 日、オンラインで掲載。
【参照 URL】
Oncotarget のホームページ：
http://www.impactjournals.com/oncotarget/index.php?journal=oncotarget&page=article&op=view&path[]=19436&author-preview=ezw【研究支援】
本研究は文部科学省私立大学戦略的研究基盤形成支援事業：「生体分子情報による次世代型がん個別最適化治療法の開発：事業番号 S1311016」、日本学術振興会科研費の支援を受けています。

【本件に関するお問い合わせ】
責任著者 大屋敷 純子（おおやしき じゅんこ）
東京医科大学医学総合研究所 分子腫瘍研究部門 教授
電話：03-3342-6111 (内線 5171) FAX : 03-3345-0185
E-mail: junko@tokyo-med.ac.jp
今西 哲（いまにし さとし）
東京医科大学医学総合研究所 分子腫瘍研究部門 助教
電話：03-3342-6111 (内線 5171) FAX : 03-3345-0185
E-mail: s-ima@tokyo-med.ac.jp
研究内容以外のお問い合わせ
総務部 広報･社会連携推進課
電話：03-3351-6141（代表） FAX : 03-6302-0289

5

-43-

プロジェクトの成果

Leukemia Research 66 (2018) 66–72

Contents lists available at ScienceDirect

Leukemia Research
journal homepage: www.elsevier.com/locate/leukres

Research paper

Regulatory T cell inhibition by dasatinib is associated with natural killer cell
diﬀerentiation and a favorable molecular response—The ﬁnal results of the
D-ﬁrst study

T⁎

Yuho Najimaa, Chikashi Yoshidab, Noriyoshi Iriyamac, , Shin Fujisawad, Hisashi Wakitae,
Shigeru Chibaf, Shinichiro Okamotog, Kimihiro Kawakamih, Naoki Takezakoi, Takashi Kumagaij,
Kazuma Ohyashikik, Jun Taguchil, Shingo Yanom, Tadahiko Igarashin, Yasuji Kouzaio,
Satoshi Moritap, Junichi Sakamotoq, Hisashi Sakamakia, Koiti InokuchiraHematology Division, Tokyo Metropolitan Cancer and Infectious Diseases Center, Komagome Hospital, Tokyo, Japan
Department of Hematology, National Hospital Organization, Mito Medical Center, Higashiibarakigun, Japanc
Division of Hematology and Rheumatology, Department of Medicine, Nihon University School of Medicine, 30-1 Oyaguchi Kami-cho, Itabashi-ku, Tokyo 173-8610,
Japand
Department of Hematology, Yokohama City University Medical Center, Yokohama, Japane
Departmet of Hematology and Oncology, Japanese Red Cross Society, Narita Red Cross Hospital, Narita, Japanf
Department of Hematology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japang
Division of Hematology, Department of Medicine, Keio University School of Medicine, Tokyo, Japanh
Department of Hematology and Clinical Oncology, Kagawa Prefectural Central Hospital, Kagawa, Japani
Department of Hematology, National Hospital Organization Disaster Medical Center, Tachikawa, Japanj
Department of Hematology, Ohme Municipal General Hospital, Tokyo, Japank
Department of Hematology, Tokyo Medical University, Tokyo, Japanl
Department of Hematology, Japanese Red Cross Shizuoka Hospital, Shizuoka, Japanm
Division of Clinical Oncology and Hematology, Department of Internal Medicine, Jikei University School of Medicine, Tokyo, JapannDivision of Hematology and Oncology, Gunma Prefectural Cancer Center, Ota, Japano
Department of Hematology, Tokyo Metropolitan Tama Synthesis Medical Center, Tokyo, Japanp
Department of Biomedical Statistics and Bioinformatics, Kyoto University Graduate School of Medicine, Kyoto, Japanq
Tokai Central Hospital, Kakamigahara, Japanr
Department of Hematology, Nippon Medical School, Tokyo, Japanb

A R T I C L E I N F O

A B S T R A C T

Keywords:
Chronic myeloid leukemia
Regulatory T cell
Natural killer cell
Dasatinib

We evaluated the eﬀects of regulatory T cell (Treg) inhibition during dasatinib treatment on the anticancer
immune response, particularly on natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Fifty-two newly
diagnosed Japanese patients with chronic myeloid leukemia (CML) in the chronic phase were enrolled in the Dﬁrst study; all received 100 mg of dasatinib once daily and were followed for at least 36 months. The cumulative
deep molecular response (DMR, MR4) rate was 65% by 36 months; the 3-year overall survival was 96%. CD4+ T
cell counts were stable, whereas the proportion of CD4+CD25+CD127low (Treg) cells decreased in a time-dependent manner. The DMR rate by18 months was signiﬁcantly better in low Treg patients (< 5.7% at 12
months) compared to the remaining patients (odds ratio 4.07). NK cell and CTL counts at several time points
were inversely correlated with Treg counts. Furthermore, the degree of NK cell diﬀerentiation (CD3−CD57+/
CD3−CD56+) was closely and inversely correlated with the proportion of Treg cells throughout the observation
period, and showed a gradually increasing trend. In conclusion, our results demonstrate that Treg inhibition by
dasatinib contributes to better treatment response through enhancement of the immune system, particularly via
NK cell diﬀerentiation.

⁎

Corresponding author.
E-mail address: iriyama.noriyoshi@nihon-u.ac.jp (N. Iriyama).

https://doi.org/10.1016/j.leukres.2018.01.010
Received 11 November 2017; Received in revised form 17 January 2018; Accepted 19 January 2018
Available online 03 February 20180145-2126/ © 2018 Elsevier Ltd. All rights reserved.

-45-

Leukemia Research 66 (2018) 66–72

Y. Najima et al.

1. Introduction

transcript per microgram of RNA, normalized to GAPDH, which ensured
a BCR-ABL1 IS of < 0.01% (MR4). Immunophenotyping of lymphocyte
fractions in the peripheral blood samples was performed by ﬂow cytometry before and 1, 2, 3, 6, 9, 12, 15, 18, 24, and 36 months after
commencing dasatinib treatment at a centralized laboratory (BioMedical Laboratories (BML), Tokyo, Japan).

Dysregulation of the immune system plays a critical role in malignant disease progression [1]. Regulatory T cells (Tregs) have a pivotal
role in cancer immunology [2]; several studies revealed that Treg inﬁltration into the tumor microenvironment is strongly associated withpoor clinical outcomes [3–9]. Therefore, several cancer immunotherapeutic approaches based on the inhibition of Tregs are being
clinically tested. Successful clinical trials that evaluated the beneﬁt of
immune-checkpoint inhibitors, including an anti-CTLA-4 monoclonal
antibody, support a new strategy of targeting regulatory immune cells,
which can achieve drastic anti-tumor results in selected patients withadvanced tumors [10,11]. However, the impact of peripheral blood
Tregs on long-term outcomes of patients with leukemic diseases, including chronic myeloid leukemia (CML), has not been fully evaluated.
The development of tyrosine kinase inhibitors (TKIs) has dramatically improved the outcomes of patients with CML in the chronic phase(CP-CML), which is caused by a BCR-ABL1 chromosomal translocation(the Philadelphia chromosome) [12–14]. Dasatinib, a second generation BCR-ABL1 kinase inhibitor, is more eﬀective against CP-CML thanthe ﬁrst generation inhibitor imatinib [15]. In addition to direct antitumor eﬀects by blocking tyrosine kinase, dasatinib also possesses animmunomodulating eﬀect that may enhance its anti-tumor activity[16,17]. Importantly, it has been shown that the proportion of Tregs isreduced during dasatinib therapy, particularly in patients with lymphocytosis; this is evidence of an immune response against Philadelphia
chromosome-positive leukemias [16].
Achievement of a deep molecular response (DMR) is an important
CP-CML treatment goal, as it enables the achievement of long-term
treatment-free remission in some patients who are receiving TKIs suchas imatinib [18–21], dasatinib [22], and/or nilotinib [23]. However,the prognostic markers for predicting DMR have not been established.
We previously reported that a shorter halving time of BCR-ABL1 transcripts and early cytotoxic lymphocyte expansion were associated withthe achievement of DMR in newly diagnosed CP-CML patients treatedwith dasatinib [24,25]. Herein, the long-term results of the D-ﬁrst study
were analyzed after a minimum follow-up period of 36 months. We
prospectively investigated the impact of the proportions of lymphocyte
subsets on the response to dasatinib treatment, as well as the association between Tregs, cytotoxic lymphocyte proliferation, and naturalkiller (NK) cell diﬀerentiation.

2.3. Flow cytometric analysis of lymphocytes
Isotype controls were used to set gating boundaries. The CD4+lymphocyte count was calculated by multiplying the total lymphocyte
count by the percentage of the CD4+ lymphocyte subset.
Immunophenotyping of lymphocyte fractions in the fresh peripheral
blood samples was performed before and 1, 2, 3, 6, 9, 12, 15, 18, 24, 36
months after commencing dasatinib treatment at BML. Lymphocytes
were classiﬁed according to their immunophenotypes as determined by
ﬂow cytometry (FACSCalibur, Becton–Dickinson, NJ), as described
previously [26]. Cytotoxic T lymphocyte (CTL) lineages were deﬁned
according to the expression of CD3+CD8+; NK cells were identiﬁed as
CD3−CD56+, and diﬀerentiated NK cells were CD3−CD57+. Tregs
were deﬁned as lymphocytes with CD4+CD25+CD127low expression.
2.4. Statistical analysis
The results are shown as mean values ± standard errors of themean. The correlation between 2 factors was evaluated with thePearson product-moment correlation coeﬃcient. The 2-tailed Fisher’s
exact test was used to determine statistical signiﬁcance; a p-value lessthan 0.05 was considered a statistically signiﬁcant result. Receiver
operating characteristic (ROC) curves were generated, and area under
the curve (AUC) values were used to evaluate the correlations between
the expression of Tregs and prognosis. The optimal thresholds along the
ROC curves were determined using the Youden index. The statisticalanalyses were performed using EZR (Saitama Medical Center, Jichi
Medical University), which is a graphical user interface for R (The R
Foundation for Statistical Computing) [27].
3. Results
3.1. Eﬃcacy and safety proﬁle of ﬁrst-line dasatinib treatment in Japanese
CP-CML patients

2. Patients and methods

The ﬁndings following the ﬁrst 18 months of dasatinib treatment inour patients have been reported previously [24]. In this study, we
analyzed the ﬁnal outcomes after at least 36 months of follow-up, at
which time 40 of the 52 patients (77%) were still undergoing dasatinib
treatment. The reasons for drug discontinuation in the remaining patients are listed in Table 1; 9 patients (18%) had discontinued dasatinibby 36 months due to drug-related non-hematologic adverse events,
while another 3 (6%) requested switching from dasatinib to another

2.1. Study design
This open-label, multicenter, prospective phase II clinical trial, the‘D-ﬁrst study’ (ClinicalTrials.gov identiﬁer: NCT01464411), was conducted by the Kanto CML study group. Detailed information regarding
the study design and patient characteristics were described previously[24]. Brieﬂy, 52 patients with newly diagnosed CP-CML were enrolled
between June 2011 and June 2012, and were treated with dasatinib
100 mg once daily. All patients were followed for a minimum of 36
months. This study was approved by the research ethics boards of allparticipating institutions, and was conducted in accordance with the
Declaration of Helsinki. Written informed consent was obtained fromall patients prior to enrollment.

Table 1Treatment status and reasons for dasatinib discontinuation at 36 months.

2.2. Evaluation of treatment response and molecular analysis
Patients were assessed for molecular response before and 1, 3, 6, 9,
12, 15, 18, 24, and 36 months after commencing dasatinib treatment by
real-time quantitative polymerase chain reaction (PCR) analysis of BCRABL1 transcripts standardized on an International Scale (BCR-ABL1 IS).
The primary endpoint of the study was the rate of DMR achievement by
18 months; DMR was deﬁned as less than 50 copies of BCR-ABL1
67

-46-

Status at 36 months

Patients, n (%)

Received treatment
Still on treatment.
Discontinued treatment
Drug-related adverse event
Pleural eﬀusion
Pericardial eﬀusion
Proteinuria & systemic edema
Pulmonary hypertension
MalaiseElevation of intraocular pressure
Interstitial pneumonia
Requested to change medication

52 (100)
40 (77)12 (23)
3 (6)
1 (2)
1 (2)
1 (2)
1 (2)
1 (2)
1 (2)
3 (6)

Leukemia Research 66 (2018) 66–72

Y. Najima et al.

Fig. 1. Dynamics of CD4+ T lymphocytes and the proportion of Tregs in the peripheral blood. (A) The cumulative rates of major and deep molecular responses at each time point. (B) The
mean CD4+ T lymphocyte counts were plotted (n = 52). The total numbers of CD4+ lymphocytes remained stable except for the ﬁrst month after dasatinib treatment. (C) The proportionof CD4+CD25+CD127low cells (Tregs) among all CD4+ T cells in the peripheral blood decreased gradually during the ﬁrst 12 months. Bars indicate the mean ± SEM.

TKI. The median interval between the day of dasatinib initiation anddiscontinuation was 23 months (range, 1–33 months). The cumulative
DMR rates were 49% by 12 months, 59% by 18 months, 59% by 24months, and 65% by 36 months (Fig. 1a). No patient experienced disease progression to the accelerated or blastic phase. Two patients diedof ovarian cancer and a transplant-related complication, respectively.
There were no patients who developed grade ≥3 infections during theobservation period in this study. The 3-year overall survival was 96%.
Of note, no patients opted to discontinue dasatinib during the observation period. These data support the use of dasatinib (100 mg QD)
as a frontline treatment for Japanese patients with newly diagnosed CPCML.

3.3. Determination of the treg frequency threshold for predicting DMR
We analyzed the association between the proportion of Tregs in
CD4+ T cells at each time point and the rate of DMR achievement by 18
months (the primary endpoint). The ROC curve indicated mild correlations between the Treg frequency in CD4+ T cells at 12 months andthe DMR rate by 18 months, with an AUC of 0.64 and the highest
Youden index at 5.7%. Using this cutoﬀ value, patients were divided
into low and high Treg groups. The DMR rate by 18 months was signiﬁcantly better in the low Treg group than in the high Treg group, withan odds ratio of 4.07 (p < 0.05; Fig. 2). The DMR rates by 12, 24, and36 months also tended to be favorable in the low Treg group, althoughthese diﬀerences were statistically insigniﬁcant. These results suggestthat Treg inhibition contributes to the achievement of DMR.

3.2. The proportion of tregs in CD4+ T cells predicts treatment response
3.4. The proportion of Tregs is inversely associated with the presence of
diﬀerentiated NK cells

Subsets of lymphocytes in peripheral blood were analyzed in 47 of
52 patients to assess the association between Treg proportion and otherlymphocyte fractions such as NK cells and CTLs. Flow cytometric analysis of CD4+ T cell counts showed them to be stable throughout the
study
period(Fig.
1B),
whereastheproportionof
CD4+CD25+CD127low Tregs decreased in a time-dependent manner
during the follow-up period (Fig. 1C).

Our previous study revealed that higher NK cell and CD8+ T
lymphocyte counts at 1-month post-dasatinib treatment predict favorable odds of achieving DMR [26]. We analyzed the associations between the proportion of Tregs and cytotoxic eﬀector cells counts, including NK cells and CTLs, in the peripheral blood. Furthermore, the
68

-47-

Leukemia Research 66 (2018) 66–72

Y. Najima et al.

proﬁles of Japanese patients to those of a worldwide cohort [29,30].
Our results suggest that dasatinib is a suitable therapeutic agent for
newly diagnosed CP-CML in Japan.
In 2009, Fei et al. [29] demonstrated that dasatinib inhibits Treg
proliferation and function in a dose-dependent manner via cell cycle
arrest and downregulation of the master transcription regulator Forkhead box P3 in vitro. Furthermore, Mustjoki et al. [16] reported thatsome CML patients who received dasatinib and developed large granular lymphocytic (LGL) lymphocytosis had lower Treg numbers compared to patients without LGL lymphocytosis. These data led us to
conduct the prospective study described herein. Consistent with previous studies, our analysis of Treg and eﬀector cell subsets revealed that
the Treg proportion decreased during dasatinib therapy. Furthermore,
the proportion of Tregs at 12 months of dasatinib treatment was associated with achievement of DMR; this association was signiﬁcant at 18
months. Notably, Treg inhibition by dasatinib was time-dependent andcorrelated with NK cell diﬀerentiation. However, it should be notedthat the gap in the DMR rates between the low and high Treg groups
was shortened by 36 months. Due to the relatively low sensitivity of
PCR for major BCR-ABL1 transcripts, DMR in this study was deﬁned asless than MR4.0, but not MR4.5 or deeper. Slow responders with gradually decreasing minimal residual disease markers could eventuallyachieve MR4.0. This might be a reason that the impact of Treg number
on treatment response was reduced after long-term follow-up.
While a lower number of Tregs is linked to the incidence of LGL
lymphocytosis, the mechanism of Treg inhibition by dasatinib has remained unclear. In this study, we found that Tregs likely impede theimmune response against CML cells by inhibiting NK cell diﬀerentiation
based on the following observations: First, the Treg proportion anddegree of NK cell diﬀerentiation were strongly and inversely correlated
throughout the study period, while the associations between the Treg
proportion and NK cell count were signiﬁcant only at 9 and 15 months
after commencing dasatinib. Second, a previous study also found that
the Treg proportion decreased in a time-dependent manner after the
initiation of dasatinib, while an increase in lymphocyte counts wasobserved within 3 months, thus validating our ﬁndings [30]. Third, theincrease of LGL cell counts following dasatinib treatment was closely
associated with plasma drug concentrations, but Treg counts were notaﬀected by the oral intake of dasatinib [17]. These ﬁndings suggest that
Treg inhibition by dasatinib is time-dependent and contributes to NK
cell diﬀerentiation, leading to the improved cytotoxic activity of NK
cells. The observation that lower numbers of Treg cells are associatedwith improved treatment outcomes further validates the notion that
Tregs inhibit the immune system response.
The functional signiﬁcance of CD57 expression in NK cells and T
cells was established recently [28]. On T cells, CD57 expression indicates terminal diﬀerentiation towards anergy and senescence. In
contrast, CD57+ NK cells are highly cytotoxic and play an important
active role in tumor immunity. High frequencies of peripheral or tumorassociated CD57+ NK cells predict less severe disease and better outcomes in various cancers, including acute lymphoblastic leukemia andHodgkin’s lymphoma [28,31,32]. Consistent with these previous observations, our results indicate that the accumulation of CD57+ NK
cells is associated with a favorable outcome, which in turn is strongly
associated with a decrease in Treg frequency upon dasatinib treatment.
Several studies investigated the detailed mechanism of NK cell activation by dasatinib treatment. Kreutzman et al. [33] revealed thatdasatinib promotes T-helper (Th)-1 activity. Furthermore, NK cells in
the peripheral blood of CML patients undergoing dasatinib treatment
have high levels of phosphorylated AKT, STAT1, and STAT3 comparedto those receiving other TKIs [25]. Based on these results, we positedthat dasatinib potentiates NK cell reactivity by stimulating cell signaling pathways via increasing the secretion of Th1 cytokines. In aseries of in vitro and in vivo experiments, Pedroza-Pacheco et al. [34]demonstrated that Tregs inhibit NK cell diﬀerentiation from CD34+
hematopoietic stem cells through contact-dependent signaling.

Fig. 2. Cumulative deep molecular response (DMR) rates according to the percentage ofTregs among all CD4+ lymphocytes 12 months after dasatinib initiation. The cumulative
DMR rate by 18 months was signiﬁcantly better in the low Treg group (< 5.7%) compared to the high Treg group (≥5.7%). The error bars indicate the 95% conﬁdence intervals.

degree of NK cell diﬀerentiation was evaluated using CD57 as a marker
for mature NK cells at stage 5 [28].
To evaluate the eﬀect of Treg inhibition on the normal immune
system, the correlations between the proportion of Tregs and the cytotoxic eﬀector cell count or the degree of NK cell diﬀerentiation wereanalyzed throughout the observation period. NK cell and CTL counts atseveral time points showed weak but signiﬁcant correlations with Treg
proportions, as indicated by the relative number of CD4+25+CD127low
cells among all CD4+ lymphocytes as well as the total number of NK
cells and CTLs (Table 2 and Fig. 3). Moreover, the degree of NK cell
diﬀerentiation, as revealed by the ratio of CD3−CD57+ to CD3−CD56+
cells, was distinctly correlated with the proportion of Tregs. The ratio ofdiﬀerentiated NK cells tended to gradually increase during the observation period (Fig. 4A). Moreover, the Treg proportion was strongly
correlated with the degree of NK cell diﬀerentiation throughout the
study period except for the pretreatment time point (Table 2), as shownin the representative scatter-plots at 6, 9, and 18 months (Fig. 4B).
These prospective observations indicate that NK cell diﬀerentiation canbe induced through Treg inhibition by dasatinib.
4. Discussion
In this ﬁnal study of our prospective D-ﬁrst multicenter clinical trial
for ﬁrst-line dasatinib treatment, we reconﬁrmed the drug’s high eﬃcacy in Japanese patients, who achieved a DMR rate of greater than60%; this is consistent with previous studies comparing the clinical
Table 2
Associations between the probability of Treg in CD4+ lymphocyte and counts of naturalkiller (NK) cells, cytotoxic T lymphocytes (CTLs), or NK cell diﬀerentiation indicated by
CD3−CD57+ to CD3−CD56+ ratio in the peripheral blood. The calculated Pearson’s
product-moment correlation coeﬃcients (r) and p-values are shown.
Time point(months)

Pretreatment
1
2
3
6
9
12
15
18
21
24
36

NK cells

CTLs

CD3-CD57+ to CD3CD56+ ratior

p valuer

p valuer

p value−0.0517−0.0327−0.2180.0304−0.0979−0.319−0.232−0.461−0.253−0.164−0.22−0.439

0.724
0.824
0.166
0.834
0.499
0.03270.12
0.0277
0.102
0.324
0.219
0.036

0.03470.166
0.149−0.118−0.139−0.368−0.239−0.0603−0.0606
0.023−0.0479
0.0045

0.813
0.256
0.346
0.413
0.334
0.0128
0.109
0.712
0.699
0.891
0.791
0.984

−0.179−0.482−0.465−0.463−0.543−0.474−0.344−0.488−0.499−0.625−0.454−0.6980.218< 0.001
0.0019< 0.001< 0.001
0.001
0.01780.0014< 0.001< 0.001
0.008< 0.001

69-48-

Leukemia Research 66 (2018) 66–72

Y. Najima et al.

Fig. 3. Scatter plots for the frequency of Tregs and counts of natural killer (NK) cells (A) or cytotoxic T lymphocytes (CTLs) (B) in the peripheral blood. Plots of measurements 9 months
after dasatinib treatment (right panels) revealed signiﬁcant inverse correlations; these were not signiﬁcant at the pretreatment time point. The calculated Pearson’s product-moment
correlation coeﬃcients (r) and p-values are shown.

Furthermore, results of stopping TKI trials indicated that NK cell immunity might be a more valuable indicator of the achievement oftreatment-free remission of CML than CTL counts, as higher NK cellcounts are predictive of successful treatment-free remission [22,35].
Taken together, these data show that Treg inhibition might be associated with NK cell diﬀerentiation rather than proliferation. We lackdirect evidence of these postulated mechanisms, which is one of the
major limitations of our study; therefore, further investigations are required to clarify the immunological foundation of dasatinib treatment.
It has been shown that infectious complication is a critical concern
during dasatinib therapy, as, in the DASISION study, infection-related
mortality was more frequent in the dasatinib arm than in the imatinibarm [36]. However, no cases with severe infection were reported in ourstudy. Further, we reinvestigated the incidence of cytomegalovirus reactivation, which is reported to be associated with dasatinib therapy(particularly with lymphocytosis) [37]. Among 48 evaluable patients,
there were no cases of clinical cytomegalovirus reactivation during theobservation period. We conclude that patients with CP-CML treatedwith ﬁrst-line dasatinib are less predisposed to cytomegalovirus infection complications, probably due to immunocompetency.
In conclusion, 36 months of data collection from the D-ﬁrst study
validates the use of 100 mg QD of dasatinib as a ﬁrst-line treatment for
newly diagnosed Japanese CML-CP patients. Inhibition of Treg in the
peripheral blood due to dasatinib treatment is associated with theachievement of DMR, which is predictive of favorable outcomes. Our
prospective analyses of patient samples suggested that Treg inhibition
by dasatinib exerted an immunotherapeutic eﬀect through the induction of NK cell diﬀerentiation, providing a possible explanation of theresults of previous in vivo and in vitro studies.

Conﬂicts of interest
Y.N. received speaker fees from Novartis Pharma K.K. C.Y. received
honoraria and speaker fees from Bristol-Myers Squibb and Pﬁzer, as
well as honoraria, speaker fees and grants from Otsuka Pharmaceutical.
N.I. received honoraria and speaker fees from Bristol-Myers Squibb andNovartis Pharma K.K. S.F. received honoraria from Chugai
Pharmaceutical and Pﬁzer, as well as speaker fees from Pﬁzer. S.C.
received grants, speaker fees, and research grants from Bristol-Myers
Squibb and Novartis Pharma K.K. S.O. received lecture fees and a grantfrom Bristol-Myers Squibb. T.K. received a lecture fee from BristolMyers Squibb, Novartis Pharma, and Pﬁzer. K.O. is a paid consultant for
Ariad. S.M. received a speaker fee from Bristol-Myers Squibb. K.I. received honoraria and grants from Bristol-Myers Squibb, and also received honoraria from Novartis Pharma K.K. and Pﬁzer.

70

-49-

Leukemia Research 66 (2018) 66–72

Y. Najima et al.

Fig. 4. Dynamics of natural killer (NK) cell diﬀerentiation in the peripheral blood following dasatinib treatment. (A) The ratios of CD3−CD57+ to CD3−CD56+ NK cells were plotted at
the indicated time points. Bars indicate the mean ± SEM. (B) Scatter plots for the frequency of Tregs and CD3−CD57+ to CD3−CD56+ cell ratios at pretreatment (upper left), 6 months(upper right), 9 months (lower left), and 18 months (lower right) are shown. Pearson’s product-moment correlation coeﬃcients (r) and p-values are also shown.

71

-50-

Leukemia Research 66 (2018) 66–72

Y. Najima et al.

Author contributions

maintained complete molecular remission for at least 2 years: the prospective,
multicentre Stop Imatinib (STIM) trial, Lancet Oncol. 11 (2010) 1029–1035.
[19] S. Mori, E. Vagge, P. le Coutre, E. Abruzzese, B. Martino, E. Pungolino, et al., Ageand dPCR can predict relapse in CML patients who discontinued imatinib: the ISAV
study, Am. J. Hematol. 90 (2015) 910–914.
[20] D.M. Ross, S. Branford, J.F. Seymour, A.P. Schwarer, C. Arthur, D.T. Yeung, et al.,Safety and eﬃcacy of imatinib cessation for CML patients with stable undetectable
minimal residual disease: results from the TWISTER study, Blood 122 (2013)
515–522.
[21] P. Rousselot, A. Charbonnier, P. Cony-Makhoul, P. Agape, F.E. Nicolini, B. Varet,
et al., Loss of major molecular response as a trigger for restarting tyrosine kinase
inhibitor therapy in patients with chronic-phase chronic myelogenous leukemia
who have stopped imatinib after durable undetectable disease, J. Clin. Oncol. 32(2014) 424–430.
[22] J. Imagawa, H. Tanaka, M. Okada, H. Nakamae, M. Hino, K. Murai, et al.,Discontinuation of dasatinib in patients with chronic myeloid leukaemia who havemaintained deep molecular response for longer than 1 year (DADI trial): a multicentre phase trial, Lancet Haematol. 2 (2015) e528–e535.
[23] D. Rea, F.E. Nicolini, M. Tulliez, F. Guilhot, J. Guilhot, A. Guerci-Bresler, et al.,Discontinuation of dasatinib or nilotinib in chronic myeloid leukemia: interim
analysis of the STOP 2G-TKI study, Blood 129 (2017) 846–854.
[24] N. Iriyama, S. Fujisawa, C. Yoshida, H. Wakita, S. Chiba, S. Okamoto, et al., Shorter
halving time of BCR-ABL1 transcripts is a novel predictor for achievement of molecular responses in newly diagnosed chronic-phase chronic myeloid leukemia
treated with dasatinib: results of the D-ﬁrst study of Kanto CML study group, Am. J.
Hematol. 90 (2015) 282–287.
[25] N. Iriyama, Y. Hatta, M. Takei, Direct eﬀect of dasatinib on signal transduction
pathways associated with a rapid mobilization of cytotoxic lymphocytes, Cancer
Med. 5 (2016) 3223–3234.
[26] N. Iriyama, S. Fujisawa, C. Yoshida, H. Wakita, S. Chiba, S. Okamoto, et al., Early
cytotoxic lymphocyte expansion contributes to a deep molecular response to dasatinib in patients with newly diagnosed chronic myeloid leukemia in the chronic
phase: results of the D-ﬁrst study, Am. J. Hematol. 90 (2015) 819–824.
[27] Y. Kanda, Investigation of the freely available easy-to-use software ‘EZR' for medical
statistics, Bone Marrow Transplant. 48 (2013) 452–458.
[28] C.M. Nielsen, M.J. White, M.R. Goodier, E.M. Riley, Functional signiﬁcance of CD57
expression on human NK cells and relevance to disease, Front. Immunol. 4 (2013)422.
[29] F. Fei, Y. Yu, A. Schmitt, M.T. Rojewski, B. Chen, M. Gotz, et al., Dasatinib inhibits
the proliferation and function of CD4 + CD25+ regulatory T cells, Br. J. Haematol.
144 (2009) 195–205.
[30] T. Kumagai, E. Matsuki, K. Inokuchi, K. Ohashi, A. Shinagawa, J. Takeuchi, et al.,Relative increase in lymphocytes from as early as 1 month predicts improved response to dasatinib in chronic-phase chronic myelogenous leukemia, Int. J.
Hematol. 99 (2014) 41–52.
[31] R. Ortac, S. Aktas, G. Diniz, A. Erbay, C. Vergin, Prognostic role of natural killer
cells in pediatric mixed cellularity and nodular sclerosing Hodgkin's disease, Anal.
Quant. Cytol. Histol. 24 (2002) 249–253.
[32] D. Sorskaar, O. Forre, S.O. Lie, Increased natural killer cell activity and numbers of
Leu-7 and Leu-11b (CD 16)-positive cells in bone marrow of children in remission
from acute lymphoblastic leukaemia, Scand. J. Immunol. 29 (1989) 65–72.
[33] A. Kreutzman, M. Ilander, K. Porkka, J. Vakkila, S. Mustjoki, Dasatinib promotes
Th1-type responses in granzyme B expressing T-cells, Oncoimmunology 3 (2014)
e28925.
[34] I. Pedroza-Pacheco, D. Shah, A. Domogala, M. Luevano, M. Blundell, N. Jackson,
et al., Regulatory T cells inhibit CD34+ cell diﬀerentiation into NK cells byblocking their proliferation, Sci. Rep. 6 (2016) 22097.
[35] I. Mizoguchi, T. Yoshimoto, S. Katagiri, J. Mizuguchi, T. Tauchi, Y. Kimura, et al.,Sustained upregulation of eﬀector natural killer cells in chronic myeloid leukemia
after discontinuation of imatinib, Cancer Sci. 104 (2013) 1146–1153.
[36] J.E. Cortes, G. Saglio, H.M. Kantarjian, M. Baccarani, J. Mayer, C. Boqué, et al.,Final 5-year study results of DASISION: the dasatinib versus imatinib study intreatment-naïve chronic myeloid leukemia patients trial, J. Clin. Oncol. 34 (2016)
2333–2340.
[37] A. Kreutzman, K. Ladell, C. Koechel, E. Gostick, M. Ekblom, L. Stenke, et al.,Expansion of highly diﬀerentiated CD8+ T-cells or NK-cells in patients treated withdasatinib is associated with cytomegalovirus reactivation, Leukemia 25 (2011)
1587–1597.

Study conception and design: TK, JS, and HS. Acquisition of data:
All authors. Analysis and interpretation of data: YN, NI, SF, CY, TK, SM,
JS, and HS. Drafting of manuscript: YN, NI, SF, TK, JS, and HS. Finalapproval of manuscript: All authors.
Acknowledgments
This study was supported by the non-proﬁt organization
Epidemiological and Clinical Research Information Network (ECRIN).
The authors thank Yumi Miyashita at ECRIN for collecting the data andYoshinori Yamamoto at BML for analyzing the data.
References[1] O.J. Finn, Cancer immunology, N. Engl. J. Med. 358 (2008) 2704–2715.
[2] H. Nishikawa, S. Sakaguchi, Regulatory T cells in cancer immunotherapy, Curr.
Opin. Immunol. 27 (2014) 1–7.
[3] T.J. Curiel, G. Coukos, L. Zou, X. Alvarez, P. Cheng, P. Mottram, et al., Speciﬁc
recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege andpredicts reduced survival, Nat. Med. 10 (2004) 942–949.
[4] N. Hiraoka, K. Onozato, T. Kosuge, S. Hirohashi, Prevalence of FOXP3+ regulatory
T cells increases during the progression of pancreatic ductal adenocarcinoma and its
premalignant lesions, Clin. Cancer Res. 12 (2006) 5423–5434.
[5] N. Kobayashi, N. Hiraoka, W. Yamagami, H. Ojima, Y. Kanai, T. Kosuge, et al.,FOXP3+ regulatory T cells aﬀect the development and progression of hepatocarcinogenesis, Clin. Cancer Res. 13 (2007) 902–911.
[6] F. Pages, A. Berger, M. Camus, F. Sanchez-Cabo, A. Costes, R. Molidor, et al.,Eﬀector memory T cells early metastasis, and survival in colorectal cancer, N. Engl.
J. Med. 353 (2005) 2654–2666.
[7] N. Redjimi, C. Raﬃn, I. Raimbaud, P. Pignon, J. Matsuzaki, K. Odunsi, et al.,CXCR3+ T regulatory cells selectively accumulate in human ovarian carcinomas tolimit type I immunity, Cancer Res. 72 (2012) 4351–4360.
[8] T. Saito, H. Nishikawa, H. Wada, Y. Nagano, D. Sugiyama, K. Atarashi, et al., Two
FOXP3(+)CD4(+) T cell subpopulations distinctly control the prognosis of colorectal cancers, Nat. Med. 22 (2016) 679–684.
[9] E. Sato, S.H. Olson, J. Ahn, B. Bundy, H. Nishikawa, F. Qian, et al., Intraepithelial
CD8+ tumor-inﬁltrating lymphocytes and a high CD8+/regulatory T cell ratio areassociated with favorable prognosis in ovarian cancer, Proc. Natl. Acad. Sci. U. S. A.
102 (2005) 18538–18543.
[10] F.S. Hodi, S.J. O'Day, D.F. McDermott, R.W. Weber, J.A. Sosman, J.B. Haanen,
et al., Improved survival with ipilimumab in patients with metastatic melanoma, N.
Engl. J. Med. 363 (2010) 711–723.
[11] S.L. Topalian, F.S. Hodi, J.R. Brahmer, S.N. Gettinger, D.C. Smith, D.F. McDermott,et al., Safety activity, and immune correlates of anti-PD-1 antibody in cancer, N.
Engl. J. Med. 366 (2012) 2443–2454.
[12] G.Q. Daley, R.A. Van Etten, D. Baltimore, Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome, Science
247 (1990) 824–830.
[13] T.G. Lugo, A.M. Pendergast, A.J. Muller, O.N. Witte, Tyrosine kinase activity andtransformation potency of bcr-abl oncogene products, Science 247 (1990)
1079–1082.
[14] C.L. Sawyers, Chronic myeloid leukemia, N. Engl. J. Med. 340 (1999) 1330–1340.
[15] H. Kantarjian, N.P. Shah, A. Hochhaus, J. Cortes, S. Shah, M. Ayala, et al., Dasatinib
versus imatinib in newly diagnosed chronic-phase chronic myeloid leukemia, N.
Engl. J. Med. 362 (2010) 2260–2270.
[16] S. Mustjoki, M. Ekblom, T.P. Arstila, I. Dybedal, P.K. Epling-Burnette, F. Guilhot,
et al., Clonal expansion of T/NK-cells during tyrosine kinase inhibitor dasatinib
therapy, Leukemia 23 (2009) 1398–1405.
[17] S. Mustjoki, K. Auvinen, A. Kreutzman, P. Rousselot, S. Hernesniemi, T. Melo, et al.,Rapid mobilization of cytotoxic lymphocytes induced by dasatinib therapy,
Leukemia 27 (2013) 914–924.
[18] F.X. Mahon, D. Rea, J. Guilhot, F. Guilhot, F. Huguet, F. Nicolini, et al.,Discontinuation of imatinib in patients with chronic myeloid leukaemia who have

72

-51-

www.nature.com/scientificreports

OPEN

received: 27 July 2016
accepted: 17 October 2016
Published: 10 November 2016

Comprehensive polymerase
chain reaction assay for
detection of pathogenic DNA inlymphoproliferative disorders of
the ocular adnexa
Yoshihiko Usui1,*, Narsing A. Rao2, Hiroshi Takase3, Kinya Tsubota1, Kazuhiko Umazume1,
Daniel Diaz-Aguilar4, Takeshi Kezuka1, Manabu Mochizuki3,5, Hiroshi Goto1 & Sunao Sugita6,7,*
Infectious agents have been identified as a major cause of specific types of human cancers worldwide.
Several microorganisms have been identified as potential aggravators of ocular adnexal neoplasms;
however, given the rarity of these neoplasms, large epidemiological studies are difficult to coordinate.
This study aimed to conduct an exhaustive search for pathogenic DNA in lymphoproliferative disorders(LPD) of the ocular adnexa in a total of 70 patients who were diagnosed with LPD of the ocular adnexa
between 2008 and 2013. Specimens were screened for bacterial, viral, fungal, and parasitic DNA bymultiplex polymerase chain reaction (PCR) and quantitative real-time PCR. Among cases of conjunctival
mucosa-associated lymphoid tissue lymphoma, human herpes virus (HHV)-6, HHV-7, chlamydia,
Epstein-Barr virus (EBV) and bacterial 16S ribosomal DNA were detected. In cases of IgG4-related oculardisease, similar pathogens were detected but in a larger number of patients. Our PCR assays detected
DNAs of various infectious agents in tumor specimens, especially HHV6, HHV7, and EBV, with different
positive rates in various types of LPD. Chronic inflammatory stimulation or activation of oncogenes
from these infectious agents might be involved in the pathogenesis of LPD of the ocular adnexa.
The link between chronic viral, bacterial, or parasitic stimulation and the development of specific malignancies
in humans are well described1. Long-standing infection with specific pathogens is implicated in the origin anddevelopment of several benign and malignant lymphoproliferative disorders (LPD). Examples of well-studied
malignant associations include Helicobacter pylori infection in mucosa-associated lymphoid tissue (MALT)
lymphoma; hepatitis B virus (HBV) and hepatitis C virus (HCV) in hepatic carcinoma; and Epstein-Barr virus(EBV) in Hodgkin’s and Burkitt’s lymphoma2. Malignant transformation of lymphocytes in the context of viralor bacterial infection is a multi-dimensional process. Certain viruses such as EBV and HTLV1 directly infectlymphocytes, inducing lymphoid hyperplasia and malignant transformation over time. However, direct antigen
stimulation is not always necessary for the development of lymphoma. For instance, H. pylori-induced chronic
gastritis progresses to MALT lymphoma via chronic inflammation and indirect stimulation of lymphocytes.
These direct and indirect stimulations of lymphocytes through chronic infectious disease may also play a role inthe development of less well-studied benign LPDs, such as IgG4-related disease3.
LPDs of the ocular adnexa comprise a wide spectrum of disorders from benign lymphoid hyperplasia
to monoclonal malignant lymphoma, with a range of incidence and potential origins. In Japan, MALT lymphoma is the most frequent primary malignancy of the ocular adnexa region, whereas IgG4-related ophthalmic
1

Department of Ophthalmology, Tokyo Medical University, Tokyo, Japan. 2Department of Ophthalmology, Universityof Southern California, USA. 3Department of Ophthalmology & Visual Science, Tokyo Medical and Dental University,
Tokyo, Japan. 4David Geffen School of Medicine, University of California, 10833 Le Conte Ave, Los Angeles, CA 90095.
5
Miyata Eye Hospital, Miyakonojo, Japan. 6Laboratory for Retinal Regeneration, RIKEN Center for Developmental
Biology, Kobe, Japan. 7Department of Ophthalmology, Kobe City Medical Center General Hospital, Kobe, Japan.
*
These authors contributed equally to this work. Correspondence and requests for materials should be addressed toY.U. (email: usuyoshi@gmail.com)

1

Scientific Reports | 6:36621 | DOI: 10.1038/srep36621-52-

www.nature.com/scientificreports/

Figure 1. Representative real-time PCR results of a tumor sample obtained from a case of IgG4-related
ophthalmic disease. The sample is positive for HHV7-DNA, with 1.24 ×​  106 copies/μ​g DNA. Control DNA:
standard HHV7-DNA (106, 104, 102 copies).

disease (IgG4-ROD) and reactive lymphoid hyperplasia (RLH) are the most common benign LPDs of the ocularadnexa4,5. The etiologies of most LPDs of the ocular adnexa have been widely investigated, but several otherdisorders including IgG4-ROD require further study. IgG4-ROD is a recently recognized syndrome with clinicalfeatures that mimic malignant lymphoma, including local and systemic lymphadenopathy and vascular proliferation, which persist and relapse over time3,5. Although long-term outcomes of patients with IgG4-ROD are
generally favorable, there are reports of increased mortality due to systemic effects including multiple organ
involvement and damage6.
The natural history and pathogenesis of several LPDs are not completely understood. A potentially promisingline of study, however, implicates chronic antigen stimulation as a causative agent in the development of benign
or malignant lymphoproliferative processes7–9.
Several studies have investigated the possible association between LPDs of the ocular adnexa and infectious
agents such as herpes simplex virus (HSV)-1, HSV-2, adenovirus 8, HCV, Helicobacter pylori, Chlamydia psittaci,
Chlamydia trachomatis, Chlamydophila abortus, and Chlamydophila pneumoniae10,11. The findings suggestthat infectious agents are associated with the pathogenesis of LPDs of the ocular adnexa. This hypothesis led
us to examine the possible association of infectious agents and LPDs of the ocular adnexa. The multiplex andbroad-range polymerase chain reaction (PCR) system has been used clinically for simultaneous detection ofmany pathogens in a very small sample volume of ocular fluids and the diagnosis of infectious uveitis12. Using thismethod, we investigated the presence of 23 pathogenic DNA in tissue specimens of ocular adnexa with various
LPDs.
The aim of the present study was to comprehensively examine various infectious agents in ocular adnexa with
LPDs using the multiplex and broad-range PCR systems, which might be useful for detecting unexpected infectious associations and elucidating the origins of LPDs of the ocular adnexa.

Results

Demographic characteristics and identification of infectious agents in lymphoproliferative
disorders of the ocular adnexa. The patients studied showed no significant difference in age or gender

distribution, while patients with conjunctival MALT lymphoma, orbital MALT lymphoma or orbital DLBCL
demonstrated IgH gene rearrangement. PCR results of a sample obtained from a representative case are shownin Fig. 1. The results of biopsied specimens in each LPD of the ocular adnexa, in which our comprehensive PCRsystem showed positivity, are presented in Table 1. Orbital MALT lymphoma tissues revealed a very rate of positivity for infectious agents, with only 1/15 patients demonstrating an HHV-6 infection from the collected biopsy.
However, conjunctival MALT lymphoma demonstrated a much higher, but non-specific association with invasive
pathogens including EBV, HHV-6, HHV-7 chlamydia and non-specific bacteria. Orbital DLBCL demonstrated
a moderate association with EBV presence in 28.6% of all cases. However, IgG4-ROD showed a much stronger
association with EBV (31.8%), HHV-6 (22.7%) and HHV-7 (36.4%), and similar values were seen in orbital RLH.

2

Scientific Reports | 6:36621 | DOI: 10.1038/srep36621-53-

www.nature.com/scientificreports/
Multiplex PCR and Real-time PCR
Infectious agent

Conjunctival MALT

Orbital MALT

Orbital DLBCL

IgG4-ROD

EBV

3/19 (15.8%)

0/15 (0%)

2/7 (28.6%)

7/22 (31.8%)

Orbital RLH
2/7 (28.6%)

HHV-6

1/19 (5.2%)

1/15(6.7%)

0/7 (0%)5/22 (22.7%)

2/7 (28.6%)
3/7 (42.9%)

HHV-7

1/19 (5.2%)

0/15 (0%)

0/7 (0%)8/22 (36.4%)

Chlamydia

1/19 (5.2%)

0/15 (0%)

0/7 (0%)0/22 (0%)

0/7 (0%)Bacteria

2/19 (10.5%)

0/15 (0%)

1/7 (14.3%)

1/22 (4.5%)

1/7 (14.3%)

Table 1. Positive rates for various microorganisms detected by comprehensive polymerase chain reaction
assays in tumor specimens of 70 patients with lymphoproliferative disorders of the ocular adnexa. PCR,
polymerase chain reaction; MALT, mucosa-associated lymphoid tissue lymphoma; DLBCL, diffuse large B-cell
lymphoma; IgG4-ROD, IgG4-related ophthalmic disease; RLH, reactive lymphoid hyperplasia; EBV, EpsteinBarr virus; HHV, human herpes virus.

Number of patients withdouble positive (%)

Number of patients withtriple positive (%)

Number of patients withquadruple positive (%)

2/19 (10.5)

0/19 (0)0/19 (0)Conjunctival MALT

HHV-6 +​ EBV: 1 case
HHV-7 +​ EBV: 1 case
Orbital MALT

0/15 (0)0/15 (0)0/15 (0)Orbital DLBCL

1/7 (14.3)

0/7 (0)0/7 (0)7/22 (31.8)

1/22 (4.5)

0/22 (0)HHV-6 +​ HHV-7: 2 cases

HHV-6 +​  HHV-7 +​  EBV:
1 caseEBV +​ Bacteria: 1 case
IgG4-ROD

HHV-6 +​ EBV: 2 cases
HHV-7 +​ EBV: 2 cases
EBV +​ Bacteria: 1 case
RLH

0/7 (0)0/7 (0)1/7 (14.3)
HHV-6 +​  HHV7 +​  EBV +​ Bacteria: 1 case

Table 2. Multiple positive rates detected by comprehensive polymerase chain reaction assays in biopsy
specimens of 70 patients with lymphoproliferative disorders of the ocular adnexa. MALT, mucosaassociated lymphoid tissue lymphoma; DLBCL, diffuse large B-cell lymphoma; IgG4-ROD, IgG4-related
ophthalmic disease; RLH, reactive lymphoid hyperplasia; HHV, human herpes virus; EBV, Epstein-Barr virus.
Only one sample of conjunctival MALT lymphoma tested positive for chlamydia 23S (1/19; 6.39 ×​  101 copies/μ​g DNA). Our multiplex real-time PCR analyses identified 29 PCR-positive patients. HSV-1, HSV-2, VZV,
CMV, HHV-8, BK virus, JC virus, HTLV-1, Coxsackie virus, enterovirus, influenza, Bartonella, chlamydia,
Acanthamoeba, toxocara, toxoplasma, and fungal 18S/28S were not detected in any of the samples. No positivity
was found for these microorganisms in peripheral blood mononuclear cells obtained from these patients or in
conjunctiva obtained from 23 controls.

Prevalence of infectious pathogen and presence of multiple co-infections in biopsied specimensof LPD. Members of the herpes family were most commonly associated with the collected samples of LPD.

EBV was the most commonly associated agent with 14 samples tested positive for EBV: conjunctival MALT
lymphoma (3/19; mean 3.87 ×​  102 copies/μ​g DNA), orbital DLBCL (2/7; mean 2.96 ×​  103 copies/μ​g DNA),
IgG4-ROD (7/22; mean 3.88 ×​  103 copies/μ​g DNA), and orbital RLH (2/7; mean 1.22 ×​  102 copies/μ​g DNA).
Twelve samples tested positive for HHV-7: conjunctival MALT lymphoma (1/19; 1.36 ×​  102 copies/μ​g DNA),
IgG4-ROD (8/22; mean 5.03 ×​  103 copies/μ​g DNA), and orbital RLH (3/7; mean 1.20 ×​  104 copies/μ​g DNA).
Nine samples tested positive for HHV-6: conjunctival MALT lymphoma (1/19; 1.61 ×​  102 copies/μ​g DNA), orbital
MALT lymphoma (1/15; 6.10 ×​  101 copies/μ​g DNA), IgG4-ROD (5/22; mean 1.71 ×​  103 copies/μ​g DNA), andorbital RLH (2/7; mean 1.46 ×​  103 copies/μ​g DNA). Five samples tested positive for bacterial 16S: conjunctival MALT lymphoma (2/19; mean 3.60 ×​  103 copies/μ​g DNA), orbital DLBCL (1/7; 4.30 ×​  103 copies/μ​g DNA),
IgG4-ROD (1/22; 2.54 ×​  102 copies/μ​g DNA), and orbital RLH (1/7; 1.02 ×​  103 copies/μ​g DNA).
As shown in Table 2, double infections (HHV-6 and EBV: 1 case, HHV-7 and EBV: 1 case) were detected intumor specimens from 2 of 19 patients with conjunctival MALT lymphoma. Furthermore, double infections(HHV-6 and HHV-7: 2 cases, HHV-6 and EBV: 2 cases, HHV-7 and EBV: 2 cases, EBV and bacterial 16S: 1 case),and even triple infection (HHV-6, HHV-7, EBV: 1 case) were detected in the tumor specimens from 8 of 22
patients in IgG4-ROD. In addition, quadruple infection (HHV-6, HHV-7, EBV, and bacterial 16S) was detected
in only one patient with orbital RLH among all patients studied.

3

Scientific Reports | 6:36621 | DOI: 10.1038/srep36621-54-www.nature.com/scientificreports/

Discussion

Demonstrating the presence of pathogens in patients with lymphoma tissues is challenging. To our knowledge,
this is the first multiple virus detection in LPD samples performed using multiplex and quantitative real-time
PCR assays that allow simultaneous determination of various genomic DNA pathogens. In addition, our study isthe first to report the presence of HHV-6, HHV-7, and EBV in orbital inflammatory tumors such as IgG4-ROD
and orbital RLH, demonstrating an association between viral antigen expression and LPDs. This result supports
the hypothesis that certain infections, particularly those caused by HHV-6, HHV-7, and EBV, are related to specific cases of malignant and benign LPDs of the ocular adnexa.
Malignant tumors typically linked to EBV include Burkitt’s lymphoma nasopharyngeal carcinoma, T-cell
lymphoma, and Hodgkin’s lymphoma. Evidence for the oncogenic potential of EBV derives from its ability toinfect and transform normal human B cells in vitro, manipulating differentiation signaling pathways and leading
to continuously growing lymphoblast cell lines and potentially immortal cell lines13. This germ cell expansion
can lead to oncogenic rearrangements such as a t(8:14) c-myc translocation and result in the development ofmalignant lymphoma14–16. Additionally, the presence of EBV has a significant effect on the clinical outcome oflymphomas, including diffuse large B-cell lymphoma. These observations raise a question of whether the presenceof EBV in ocular LPD can be predictive of clinical outcome13. Our study demonstrated an even stronger association between EBV and IgG4-ROD or RLH compared to conjunctival MALT lymphoma (31.8% or 28.6% versus
15.8%), suggesting that the malignant potential of EBV occurs in conjunction with a primarily increased risk ofthe development of benign LPDs of the ocular adnexa. One investigation of the association of EBV and benign
LPDs identified a 58% rate of EBV reactivation in patients with IgG4-ROD and an 18% rate of reactivation inpatients with RLH3. Our study also demonstrated a similar association between HHV-6 and HHV-7 viruses of
the herpes family and LPDs.
HHV-6 and HHV-7 are established oncogenic viruses that can induce tumors in an animal model throughthe inactivation of p53 and deregulation of nuclear factor kappa-B17,18. Known malignant tumors typically linked
with HHV-6 include T-cell lymphoma and Hodgkin’s lymphoma. Although HHV-6 displays tropism for T cells,
the prevalence of HHV-6 DNA in biopsied specimens with B-cell lymphoma or Hodgkin’s disease is 22.2% or
35.1%, respectively19. Reactivation of HHV-6 is also observed in gastrointestinal polyps, with one group reporting HHV-6 antigen expression in biopsy specimens from 62.5% of patients with gastric adenomas and 87.5% of
patients with tubulovillous adenoma, with no detection in mucosal samples from healthy controls20. Although theincidence of HHV-6 detection in conjunctival and orbital MALT lymphoma was lower (5.2% and 6.7%, respectively) in the present study, its association with IgG4-ROD was more robust. In cases of IgG4-ROD, 5 of 22
cases (23%) were positive for HHV-6 DNA, ranging from 8.1 ×​  102 to 6.7 ×​  104 copies (mean, 1.7 ×​  103 copies/μ​g DNA). The number of RLH patients was relatively low with 7 samples studied; however, 3 of these samples(43.9%) contained detectable HHV-6, establishing a correlation that warrants further evaluation. HHV-7 DNA
is closely related to HHV-6 and establishes a life-long infection following exposure, primarily within the salivary glands and lymphoreticular system, similar to HHV-6. Our study revealed the HHV-7 genome in 8 of 22
IgG4-ROD patients studied (36.4%), 3 of 7 RLH samples, and 1 of 19 MALT lymphoma samples. These results
taken together indicate that HHV-6 and HHV-7 have a stronger relationship with benign orbital LPDs than
MALT lymphomas. Further studies are necessary to examine whether HHV-6 and HHV-7 DNA are also related
to other malignant LPDs such as Mantle and T-cell lymphoma, as well as the pathophysiology of their association
with LPDs.
If an association between infectious agents and LPDs exists, it is unclear how the infectious agents induce
these LPDs of the ocular adnexa. One possibility is that infectious antigens expedite or are required for the expansion of the monoclonal/oligoclonal B cells that are found in either benign or malignant LPDs during the germinal
center reaction. As HHV-6, HHV-7, and EBV continuously infect the salivary glands21, it is conceivable that they
may also continuously infect lacrimal glands and conjunctiva. Through local and chronic stimulation, these continuous infections as exogenous or endogenous antigens may trigger infectious oncogenes. Further studies areneeded to examine whether these infectious antigens consequently induce the proliferation of lymphocytes andwhether they play a primary or secondary pathogenic role.
Moreover, although continuous infections of lymphocytes by HHV-6, HHV-7, and EBV were reported in thispresent case, pathogenic DNA was not detected in peripheral blood (data not shown), suggesting local infectionof the tumor. Taking into account the prevalence rate for these viruses in the salivary glands of healthy individuals, we can postulate that the interaction between these viral infections and other environmental risk factors
increases the risk for developing LPDs in conjunction with genetic risk factors (related to immune/infectious
responses) through undetermined underlying mechanisms.
Mechanisms consistent with the pathogenesis of MALT lymphoma, such as the expression of viral oncogenes
leading to the stimulation of a Th2 immune response, may play a role in the polyclonal or monoclonal proliferation of LPDs22. The predominantly Th2 immune response and increased production of Th2-type cytokines, suchas interleukin (IL)-4, IL-5, IL-10, and IL-13, have also been reported in patients with IgG4-ROD23,24; but, thesource of the putative antigen-reacting Th2 cells (CD4+ lymphocytes) involved in this process remains largely
unknown. Virus-induced IL-10 plays an important role in Th2 and regulatory T cells, acts as a potent factor of B
cell survival, and is produced after viral stimulation25. Although many investigators reported that IgG4 disease isTh2 dominant, the identity of the antigen that these Th2 cells are responding to is unclear. Thus, it is possible thatthese detected viruses may activate the Th2 cells that cause the pathology of IgG4, RLH, and MALT lymphoma.
In fact, HHV-6 and HHV-7 can infect latently CD4+ T cells, and they proliferate in the cells.
Infectious agents were more common in patients with conjunctival MALT lymphoma than in those withorbital MALT lymphoma, indicating that there may be different pathways leading to lymphomagenesis. Thisobservation is similar to what we reported after using high-resolution genomic copy number profiling by single
nucleotide polymorphism microarrays26. These infectious agent-driven polyclonal B-cell proliferations could be

4

Scientific Reports | 6:36621 | DOI: 10.1038/srep36621-55-

www.nature.com/scientificreports/
an initial molecular event or process that could lead to or increase the probability of a real clonal expansion overtime. If these infections play an etiologic role in benign and malignant LPDs, the elimination of these infectious
agents might facilitate control of these diseases. Double and triple infections were detected in some of the patientswith LPDs of the ocular adnexa. However, the clinical relevance of multiple infections is currently unclear, andwarrants further studies, which may lead to a novel method to reduce the probability of benign and malignant
lymphadenopathy. For example, considering that HHV-6 is the cause of exanthema subitum, treatment for HHV6, such as ganciclovir, may also be useful for treating IgG4-related diseases by suppressing viral RNA expression.
Similarly, other antiviral therapies might be used as prophylaxis or treatment for patients at higher risk for developing LPDs from these ubiquitous pathogens.
For a large part of the population, these infections are acquired at a young age and remain latent in the bodyfor life27,28. It is speculated that most individuals are latently infected by these viruses at levels that are insufficient
to determine viral load in peripheral blood mononuclear cells until they become reactivated, such as in case of
immunosuppression. This concept is consistent with previous reports indicating that molecular detection of theseviruses by PCR is not always associated with the presence of lymphocytic infiltrates in tissue29. Because theseviruses are latent in lymphocytes, false-positive PCR detection in the biopsy specimens could have occurred.
Furthermore, symptomatic viral reactivations are associated with high levels of viral loads (>​500 copies/μ​g ofDNA), while values of <​100 copies/μ​g of DNA are probably indicative of latent virus infections30. Our analysis,
however, could not directly distinguish whether these viruses persist in a latent or reactivated state. Because of thenature of biopsy sampling, there is a high level of difficulty in obtaining lacrimal tissue from healthy individuals.
This lack of control tissue prevented us from directly comparing the incidence of virus detection between patientswith LPD and healthy controls and from establishing a baseline of latent viral expression. Therefore, it is possiblethat the detection of viral antigens may be related to a latent or non-contributory infection, potentially of the infiltrating inflammatory cells rather than the adnexal tumor itself, whose presence may be expected in a common
infection acquired early in life. Our PCR system is less likely to detect positive latent samples, however, than aqualitative PCR assay, which picks up more latent viruses. By using the multiplex PCR system as an initial screening followed by an additional quantitative PCR analysis, we reduced the probability of latent or non-contributory
infections being detected and reported31. Finally, although our protocol allowed us to evaluate the presence of the
majority of known oncogenic pathogens, our PCR system does not include every potentially oncogenic pathogen.
Some notable examples worth evaluating in the future for completeness includes the human papilloma virus family, parvovirus, hepatitis B and C viruses, Merkel cell polyomavirus, and simian virus 40.
In summary, HHV-6, HHV-7, and EBV may be associated with the pathogenesis of benign and malignant
LPDs of the ocular adnexa. The exact mechanism of the role of these viruses, and thus the clinical significance, is
not yet clear. Presence of DNA of the infectious agents could also be co-incidental/epithenomenon. This possibility is unlikely, as normal conjunctival and orbital tissues did not reveal the presence of DNA of agents. Thesefindings, however, open a new perspective for further research of LPDs of the ocular adnexa. Further efforts areneeded to elucidate the relevance of the molecular detection of these viruses in the context of LPDs of the ocularadnexa. Additionally, further studies of the mechanisms underlying malignant and benign LPDs are needed toclarify the potentially inductive roles of environmental and pathogenic stimuli.

Methods

Patients, Sample Collection, and Diagnosis. We studied 70 patients (32 men and 38 women) diagnosed
with LPDs of the ocular adnexa, including 19 conjunctival MALT lymphomas (7 men and 12 women, mean age
50.8 ±​ 18.0 years), 15 orbital MALT lymphomas (7 men and 8 women, mean age 70.8 ±​ 13.9 years), 7 orbital
difuse large B-cell lymphomas (DLBCL; 4 men and 3 women, mean age 64.4 ±​ 14.2 years), 22 IgG4-ROD (12 men
and 10 women, mean age 60.5 ±​ 15.5 years), and 7 orbital RLH (2 men and 5 women, mean age 55.7 ±​  13.4 years)
from 2008 to 2013 at the Department of Ophthalmology, Tokyo Medical University. This study was approved by
Tokyo Medical University institutional review board. All experimental methods were conducted in accordancewith the Declaration of Helsinki. All patients were Asian and immunocompetent adults. Surgically resected or
biopsied specimens of LPDs of the ocular adnexa were delivered immediately to the Pathology and Molecular
Laboratory. All patients gave informed consent to the collection and analysis of samples.
The diagnosis of each LPD of the ocular adnexa was established on the basis of clinical, radiographic, histologic, and molecular genetic analyses such as gene rearrangement. Specifically, the diagnosis of IgG4-ROD
was made in accordance with recently published criteria5,32. Conventional histologic and immunohistochemicalevaluations were performed on resected tissues fixed in 10% formaldehyde to evaluate features of the LPDs. B-cellmonoclonal expansion was confirmed by flow cytometry analysis and immunoglobulin heavy chain (IgH) generearrangements using Southern blot analysis or PCR for conjunctival MALT lymphoma (SRL, Tokyo, Japan).
IgG4 values higher than the normal range of 4.8–105 mg/dl were used as diagnostic criteria to establish the presence of IgG4-ROD. Patient characteristics and presence of monoclonality are summarized in Table 3.
Polymerase Chain Reaction Analyses. Specimens were screened for bacterial, viral, fungal, and parasitic DNA by multiplex and real-time PCR assays, and the quantity of microorganism DNA was measured using
broad-range real-time PCR, as described previously12. Briefly, DNA was extracted from the tumor specimens
using a DNA Mini Kit (Qiagen, Valencia, CA). Genomic DNA of eight human herpes viruses (HHV1–8), toxoplasma, bacteria, and fungi in the tumor specimens was measured by two independent PCR assays: (1) multiplex
and real-time PCR and (2) broad-range real-time PCR.
The multiplex PCR was designed to qualitatively measure genomic DNA of 8 HHVs (HSV-1, HSV-2,
varicella-zoster virus (VZV), EBV, cytomegalovirus (CMV), HHV-6, HHV-7, and HHV-8), BK virus, JC virus,
human T-lymphotrophic virus (HTLV-1), Coxsackie virus, enterovirus, influenza virus, Bartonella, chlamydia
23S, Acanthamoeba, toxocara, and toxoplasma. If the multiplex PCR results were positive, real-time PCR was then5

Scientific Reports | 6:36621 | DOI: 10.1038/srep36621-56-

www.nature.com/scientificreports/
Conjunctival MALT

Orbital MALT

Orbital DLBCL

IgG4-ROD

Number

19

15

7

22

Orbital RLH
7

Age (yrs)

50.8 ±​  18.0

70.8 ±​  13.9

64.4 ±​  14.2

60.5 ±​  15.5

55.7 ±​  13.4

Men/Women

7/12

7/8

4/3

12/10

2/5

IgH gene rearrangementall (+​)all (+​)all (+​)all (−​)all (−​)

Table 3. Patient characteristics and monoclonality status of IgH gene rearrangement. MALT, mucosaassociated lymphoid tissue lymphoma; DLBCL, diffuse large B-cell lymphoma; IgG4-ROD, IgG4-related
ophthalmic disease; RLH, reactive lymphoid hyperplasia; IgH, immunoglobulin heavy chain.

performed. The broad-range real-time PCR was performed to detect the genome for bacterial 16S rDNA or fungal
18S/28S rDNA33. PCR was performed with a LightCycler 480 II instrument (Roche, Basel, Switzerland). Real-time
PCR was performed using the AmpliTaq Gold and the Real-Time PCR 7300 system (Applied Biosystems, Foster
City, CA) or LightCycler 480 II. Primers and probes for each pathogen and the PCR conditions were described
previously33–36. Amplification of the human gamma-globulin gene served as an internal positive extraction andamplification control. Copy number values of more than 50 copies/μ​g DNA (virus and parasite) or 100 copies/μ​g
DNA (bacteria and fungi) in the biopsy sample were considered to be significant34.

References

1. Efird, J. T., Davies, S. W., O’Neal, W. T. & Anderson, E. J. Animal viruses, bacteria, and cancer: a brief commentary. Front Public
Health. 2, 14 (2014).
2. Herrington, C. S., Coates, P. J. & Duprex, W. P. Viruses and disease: emerging concepts for prevention, diagnosis and treatment. J
Pathol. 235,149–152 (2015).
3. Takeuchi, M., Sato, Y., Yasui, H. et al. Epstein-Barr virus-infected cells in IgG4-related lymphadenopathy with comparison withextranodal IgG4-related disease. Am J Surg Pathol. 38, 946–955 (2014).
4. Japanese Study Group for IgG4-Related Ophthalmic Disease. A prevalence study of IgG4-related ophthalmic disease in Japan. Jpn J
Ophthalmol. 57, 573–579. (2013).
5. Goto, H., Takahira, M. & Azumi, A., Japanese Study Group for IgG4-Related Ophthalmic Disease. Diagnostic criteria for IgG4related ophthalmic disease. Jpn J Ophthalmol. 19, 12–1926 (2014).
6. Khosroshahi, A. & Stone, J. H. Treatment approaches to IgG4-related systemic disease. Curr Opin Rheumatol. 23, 67–71 (2011).
7. Chan, C. C., Shen, D., Mochizuki, M., Gonzales, J. A., Yuen, H. K. et al. Detection of Helicobacter pylori and Chlamydia pneumoniae
genes in primary orbital lymphoma. Trans Am Ophthalmol Soc. 104, 62–70 (2006).
8. Collina, F., De Chiara, A., De Renzo, A., De Rosa, G. Botti, G. et al. Chlamydia psittaci in ocular adnexa MALT lymphoma: a possible
role in lymphomagenesis and a different geographical distribution. Infect Agent Cancer. 7, 8 (2012).
9. Oh, D. E. & Kim, Y. D. Lymphoproliferative diseases of the ocular adnexa in Korea. Arch Ophthalmol. 125, 1668–1673 (2007).
10. Ferreri, A. J., Ernberg, I. & Copie-Bergman, C. Infectious agents and lymphoma development: molecular and clinical aspects. J
Intern Med. 265, 421–438 (2009).
11. Chan, C. C., Smith, J. A., Shen, D. F. et al. Helicobacter pylori (H. pylori) molecular signature in conjunctival mucosa-associatedlymphoid tissue (MALT) lymphoma. Histol Histopathol. 19, 1219–1226 (2004).
12. Sugita, S., Ogawa, M., Shimizu, N., Morio, T., Ohguro, N. et al. Use of a comprehensive polymerase chain reaction system fordiagnosis of ocular infectious diseases. Ophthalmology. 120, 1761–1768 (2013).
13. Park, S., Lee, J., Ko, Y. H., Han, A. & Jun, H. J. The impact of Epstein-Barr virus status on clinical outcome in diffuse large B-cell
lymphoma. Blood. 110, 972–978 (2007).
14. Cesarman, E. Epstein-Barr virus (EBV) and lymphomagenesis. Front Biosci. 7, e58–e65 (2002).
15. Shannon-Lowe, C., Adland, E., Bell, A. I., Delecluse, H. J., Rickinson, A. B. et al. Features distinguishing Epstein-Barr virus
infections of epithelial cells and B cells: viral genome expression, genome maintenance, and genome amplification. J Virol. 83,
7749–7760 (2009).
16. Isaacson, P. G. & Du, M. Q. MALT lymphoma: from morphology to molecules. Nat Rev Cancer. 4, 644–653 (2004).
17. Luppi, M., Barozzi, P., Garber, R. et al. Expression of human herpesvirus-6 antigens in benign and malignant lymphoproliferative
diseases. Am J Pathol. 153, 815–823 (1998).
18. Kofman, A., Marcinkiewicz, L., Dupart, E., Lyschev, A., Martynov, B. et al. The roles of viruses in brain tumor initiation andoncomodulation. J Neurooncol. 105, 451–466 (2011).
19. Collot, S., Petit, B., Bordessoule, D., Alain, S. & Touati, M. Real-time PCR for quantification of human herpesvirus 6 DNA fromlymph nodes and saliva. J Clin Microbiol. 40, 2445–2451 (2002).
20. Halme, L., Loginov, R., Arola, J., Turunen, T. & Lautenschlager, I. HHV-6 antigen and HHV-6 DNA expression in sporadic
adenomatous polyps of the colon. Scand J Gastroenterol. 48, 1423–1427 (2013).
21. Klussmann, J. P., Muller, A., Wagner, M. et al. Human herpesvirus type 8 in salivary gland tumors. J Clin Virol. 16, 239–246 (2000).
22. Isaacson, P. G. Mucosa-associated lymphoid tissue lymphoma. Semin Hematol. 36, 139–147 (1999).
23. Kosaka, N., Hayashi, H., Mito, T., Nagasu, A., Sakuta, T. et al. Predominance of Th2 and regulatory cytokines in the serum of apatient with IgG4-related lymphadenopathy. Int J Rheum Dis. 16, 486–488 (2013).
24. Umehara, H., Nakajima, A., Nakamura, T., Kawanami, T., Tanaka, M. et al. IgG4-related disease and its pathogenesis-cross-talkbetween innate and acquired immunity. Int Immunol. 26, 585–595 (2014).
25. Saraiva, M. & O’Garra, A. The regulation of IL-10 production by immune cells. Nat Rev Immunol. 10, 170–181 (2010).
26. Takahashi, H., Usui, Y., Ueda, S., Yamakawa, N., Sato-Otsubo, A. et al. Genome-wide analysis of ocular adnexal lymphoproliferative
disorders using high-resolution single nucleotide polymorphism array. Invest Ophthalmol Vis Sci. 56, 4156–65 (2015).
27. Flano, E., Jia, Q., Moore, J., Woodland, D. L., Sun, R. et al. Early establishment of gamma-herpesvirus latency: implications for
immune control. J Immunol. 174, 4972–4978 (2005).
28. Zerr, D. M. Human herpesvirus 6: a clinical update. Herpes. 13, 20–24 (2006).
29. Lozinski, G. M., Davis, G. G., Krous, H. F., Billman, G. F., Shmizu, H. et al. Adenovirus myocarditis: retrospective diagnosis by gene
amplification from formalin-fixed, paraffin-embedded tissues. Hum Pathol. 25, 831–834 (1994).
30. Kimura, H., Morita, M., Yabuta, Y. et al. Quantitative analysis of Epstein-Barr virus load by using a real-time PCR assay. J Clin
Microbiol. 37, 132–136 (1999).
31. Pradeau, K., Couty, L., Szelag, J. C., Tulure, P., Rolle, F. et al. Multiplex real-time PCR assay for simultaneous quantitation of humancytomegalovirus and herpesvirus-6 in polymorphonuclear and mononuclear cells of transplant recipients. J Virol Methods. 132,
77–84 (2006).

6

Scientific Reports | 6:36621 | DOI: 10.1038/srep36621-57-

www.nature.com/scientificreports/
32. Andrew, N. H., Sladden, N., Kearney, D. J. & Selva, D. An analysis of IgG4-related disease (IgG4-RD) among idiopathic orbital
inflammations and benign lymphoid hyperplasias using two consensus-based diagnostic criteria for IgG4-RD. Br J Ophthalmol. 250,
391–398 (2014).
33. Sugita, S., Kamoi, K., Ogawa, M., Watanabe, K., Shimizu, N. et al. Detection of Candida and Aspergillus species DNA using broadrange real-time PCR for fungal endophthalmitis. Graefes Arch Clin Exp Ophthalmol. 250, 391–398 (2012).
34. Sugita, S., Shimizu, N., Watanabe, K., Mizukami, M., Morio, T. et al. Use of multiplex PCR and real-time PCR to detect humanherpes virus genome in ocular fluids of patients with uveitis. Br J Ophthalmol. 92, 928–932 (2008).
35. Ogawa, M., Sugita, S., Watanabe, K., Shimizu, N. & Mochizuki,, M. Novel diagnosis of fungal endophthalmitis by broad-range realtime PCR detection of fungal 28S ribosomal DNA. Graefes Arch Clin Exp Ophthalmol. 250, 1877–1883 (2012).
36. Sugita, S., Ogawa, M., Inoue, S., Shimizu, N. & Mochizuki, M. Diagnosis of ocular toxoplasmosis by two polymerase chain reaction(PCR) examinations: qualitative multiplex and quantitative real-time. Jpn J Ophthalmol. 55, 495–501 (2011).

Acknowledgements

This study was supported by the Tokyo Medical University Cancer Research Foundation; a Grand-in-Aid for
Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; the‘Strategic Research-Based Support’ Project for private universities; with matching funds from MEXT (Ministry of
Education, Culture, Sports, and Science), Japan; Health and Labour Sciences Research Grants. Research on rare
and intractable diseases; and the Practical Research Project for Rare / Intractable Diseases from Japan Agency for
Medical Research and development, AMED.

Author Contributions

Study design: Y.U. Collection, management, analysis, and interpretation of the data: Y.U., K.T., H.T., K.U., T.K.,
M.M. and H.G. Preparation of manuscript: Y.U., N.A.R., D.D.A. and S.S. Review, and approval of manuscript: all.

Additional Information

Competing financial interests: The author(s) declare that there is no conflict of interest associated with thisstudy and publication.
How to cite this article: Usui, Y. et al. Comprehensive polymerase chain reaction assay for detection ofpathogenic DNA in lymphoproliferative disorders of the ocular adnexa. Sci. Rep. 6, 36621; doi: 10.1038/
srep36621 (2016).
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps andinstitutional affiliations.
This work is licensed under a Creative Commons Attribution 4.0 International License. The images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of thislicense, visit http://creativecommons.org/licenses/by/4.0/© The Author(s) 2016

7

Scientific Reports | 6:36621 | DOI: 10.1038/srep36621-58-

OncoImmunology

ISSN: (Print) 2162-402X (Online) Journal homepage: http://www.tandfonline.com/loi/koni20

Protective effects against tumors and infection byinterleukin-27 through promotion of expansion
and differentiation of hematopoietic stem cells
into myeloid progenitors
Naoko Orii, Izuru Mizoguchi, Yukino Chiba, Hideaki Hasegawa, Mio Ohashi,
Mingli Xu, Taro Nagai, Nakaba Ochiai, Yuki Mochizuki, Toshiyuki Owaki &Takayuki Yoshimoto
To cite this article: Naoko Orii, Izuru Mizoguchi, Yukino Chiba, Hideaki Hasegawa, Mio
Ohashi, Mingli Xu, Taro Nagai, Nakaba Ochiai, Yuki Mochizuki, Toshiyuki Owaki & Takayuki
Yoshimoto (2018) Protective effects against tumors and infection by interleukin-27 throughpromotion of expansion and differentiation of hematopoietic stem cells into myeloid progenitors,
OncoImmunology, 7:5, e1421892, DOI: 10.1080/2162402X.2017.1421892
To link to this article: https://doi.org/10.1080/2162402X.2017.1421892

Accepted author version posted online: 28
Dec 2017.
Published online: 15 Jan 2018.
Submit your article to this journal

Article views: 16

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=koni20-59-

ONCOIMMUNOLOGY
2018, VOL. 7, NO. 5, e1421892 (3 pages)
https://doi.org/10.1080/2162402X.2017.1421892

AUTHOR’S VIEW

Protective effects against tumors and infection by interleukin-27 through promotionof expansion and differentiation of hematopoietic stem cells into myeloid progenitors
Naoko Oriia, Izuru Mizoguchia, Yukino Chibaa, Hideaki Hasegawaa, Mio Ohashia, Mingli Xua, Taro Nagaib, Nakaba Ochiaia,
Yuki Mochizukia, Toshiyuki Owakia, and Takayuki YoshimotoaaDepartment of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo, Japan; bDepartment of
Immunology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo, Japan

ABSTRACTARTICLE HISTORYInterleukin (IL)-27 is a multifunctional cytokine that belongs to the IL-6/IL-12 family and has potent
antitumor activity through various mechanisms. Our novel ﬁndings indicate that IL-27 directly acts onhematopoietic stem cells and promotes their expansion and differentiation into myeloid progenitors tocontrol infection and to eradicate tumors.

Received 19 December 2017
Accepted 20 December 2017

Hematopoiesis is tightly orchestrated by hematopoietic stem
cells (HSCs) in the bone marrow (BM) niche to ultimately produce mature myeloid and lymphoid blood cells. During infection, HSCs, which are predominantly dormant, brieﬂy expand,proliferate, and differentiate in response to inﬂammatory signals such as pro-inﬂammatory cytokines. This process is called
emergency myelopoiesis and plays a critical role in counterbalancing the loss of myeloid cells and replenishing them to control the infection, because myeloid cells generally have very low
proliferative activity.
Macrophages are characterized by high plasticity and diversity, with two major phenotypes. M1 macrophages can augment helper T (Th)1 immune responses, resulting in strong
microbicidal and tumoricidal activity, and M2 macrophages
have an immunoregulatory function that helps to promote tissue remodeling and tumor progression. Under various pathogenic conditions, immature myeloid cells designated as
myeloid-derived suppressor cells emerge and exert strong
immunosuppressive functions. These cells are generated as anormal physiological response and are developed by stimulation with several tumor- or infection-derived cytokines, such as
granulocyte macrophage colony-stimulating factor, granulocytecolony-stimulating factor, and macrophage colony-stimulating
factor, but blocked from differentiating. Generally, tumor-bearing hosts and cancer patients have increased inﬁltration of
immunosuppressive myeloid cells, such as M2 macrophages
and myeloid-derived suppressor cells.
Interleukin (IL)-27, a member of the IL-6/IL-12 family ofcytokines, is a multifunctional cytokine with both pro-inﬂammatory and anti-inﬂammatory properties. It promotes the early
induction of Th1 differentiation and generation of cytotoxic T
lymphocytes, but it inhibits the differentiation of naive CD4C T

CONTACT Takayuki Yoshimoto, Ph.D
yoshimot@tokyo-med.ac.jp
6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160–8402, Japan.

KEYWORDS

IL-27; hematopoietic stem
cells; myeloid progenitors;
emergency myelopoiesis;
antitumor activity

cells into Th2 and Th17 cells and suppresses the production of
pro-inﬂammatory cytokines.1,2 IL-27 is composed of EpsteinBarr virus-induced gene 3 and p28, and its receptor (R) consistsof IL-27Ra and glycoprotein130, a common receptor subunit
for the IL-6 family of cytokines.1,2 We ﬁrst demonstrated theantitumor effects of IL-27 in 2004 using a mouse transplantable
tumor model.3 Accumulating evidence obtained using several
preclinical mouse and human tumor models indicates that IL27 has potent antitumor activity against various types of tumors
without apparent adverse effects; IL-27 acts via multiple mechanisms including cytotoxic T lymphocytes and natural killer
cells, depending on the characteristics of individual tumors.4,5
In addition, we ﬁrst clariﬁed its promoting effects on the expansion and differentiation of HSCs into myeloid progenitors by
establishing IL-27-overexpressing transgenic mice.6 We also
recently elucidated the protective effects of IL-27 on a mousemodel of blood-stage malaria infection by promoting emergency myelopoiesis (Fig. 1A),7 as well as its antitumor effects
through enhanced differentiation into antitumorigenic M1
macrophages (Fig. 1B).8
The IL-27-transgenic mice show enhanced myelopoiesis
with splenomegaly and extramedullary hematopoiesis in the
spleen.6 Consistent with this, IL-27 together with stem cell factor synergistically and vigorously expands mouse BM cells for along period.7 Among the various types of HSCs and progenitors in the BM, IL-27 and stem cell factor predominately act onlong-term repopulating HSCs and promote their differentiation
into myeloid progenitors, which have a unique potential to differentiate into migratory dendritic cells, neutrophils, and mast
cells and less so into macrophages and basophils, but not into
plasmacytoid dendritic cells, conventional dendritic cells, T
cells, or B cells. Among the cytokines, IL-27 in synergy with

Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University,© 2018 Taylor & Francis Group, LLC-60-

e1421892-2

N. ORII ET AL.

to remove infected red blood cells.9 IL-27Ra-deﬁcient miceshow a greater increase in parasitemia during the early infection.7 The infection enhances IL-27 expression through IFN-g
production in the BM and spleen, and IL-27 then promotes the
expansion and differentiation of LSK cells into myeloid progenitors, enhancing the production of neutrophils to control theinfection (Fig. 1A).7 Thus, IL-27 is one of the few cytokinesthat directly acts on HSCs and promotes emergency
myelopoiesis.
In tumor-bearing mice, IL-27 was revealed to augment theinﬁltration of myeloid cells into tumors. Intriguingly, however,deletion experiments of these myeloid cells and admixture
experiments of them with parental tumors show that the IL-27mediated tumor-inﬁltrating myeloid cells have reduced immunosuppressive activity and potent antitumor activity, rather
than protumor activity as usually observed in non-treatedtumor-bearing mice (Fig. 1B).8 The myeloid cells express ahigher level of inducible nitric oxide synthase and directly killtumors, mainly in a nitric oxide–dependent manner. In the BMof tumor-bearing mice, IL-27 increases the LSK cell population,
which has an enhanced ability to differentiate into antitumorigenic M1 macrophages.8 These M1 macrophages then inﬁltrate
into tumors and eradicate them.
In analogy with the effects of IL-27 on HSCs, some researchers
have proposed the possibility of IL-27 inducing the expansion anddifferentiation of leukemic stem cells. Aberrant IL-27Ra signalingor IL-27 stimulation of leukemic stem cells that contain proliferative mutations like BCR/ABL may enhance myeloid cell growth,potentially leading to myeloproliferative neoplasms. However, IL27-overexpressing transgenic mice never show any spontaneousdevelopment of leukemia during the entire life span under normal
conditions.10 Moreover, supporting the effect of IL-27 on hematopoiesis in the BM, the p28 subunit of IL-27 is unique in having apolyglutamic acid domain, which is the acidic domain withhydroxyapatite-binding ability and bone tropism to bone sialoprotein. Therefore, therapeutic applications of IL-27 targeting hematologic tumor and solid tumor metastasis with bone tropism
could prove beneﬁcial.
Figure 1. Protective effects against tumors and infection by IL-27 through promotion of expansion and differentiation of HSCs into myeloid progenitors. (A) The
mouse model of blood-stage malaria infection enhances IL-27 expression throughIFN-g production in the BM and spleen, and IL-27 then promotes the expansion
and differentiation of HSCs into myeloid progenitors, enhancing the production ofneutrophils to control the infection. Thus, IL-27 is a novel cytokine that directly
acts on HSCs and promotes emergency myelopoiesis. (B) Tumor-bearing mice generally have increased inﬁltration of immunosuppressive myeloid cells such as M2
macrophages and myeloid-derived suppressor cells. In mouse transplantable
tumor models, however, IL-27 directly acts on HSCs in the BM and promotes their
expansion and differentiation into myeloid progenitors, which have an enhanced
ability to further differentiate into antitumorigenic M1 macrophages. These M1
macrophages then inﬁltrate into tumors and eradicate them mainly in a nitricoxide–dependent manner. CTL, cytotoxic T lymphocyte; DC, dendritic cell; GM-CSF,
granulocyte macrophage colony-stimulating factor; MF, macrophage; MDSC, myeloid-derived suppressor cell; NK, natural killer.

Abbreviations
BM
HSC
IFN
IL
LSK
R
Th

bone marrow
hematopoietic stem cellinterferoninterleukin
lineage¡Sca-1Cc-KitC
receptorhelper T

Disclosure of potential conﬂicts of interest
No potential conﬂicts of interest were disclosed.

stem cell factor has the strongest ability to augment the expansion of lineage¡Sca-1Cc-KitC (LSK) cells, which is a population
highly enriched in HSCs, and their differentiation into myeloid
progenitors retaining the same LSK phenotype.7
In the mouse model of blood-stage malaria infection, interferon (IFN)-g production induced by IL-12 and phagocytic
cells in the spleen play critical roles in controlling parasitemia

Funding
Grant-in-Aid forScientiﬁc Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan Research Grant from Princess
Takamatsu Cancer Research Fund Private University Strategic Research
Based Project from the Ministry of Education, Culture, Sports, Science,
and Technology, Japan.

-61-

ONCOIMMUNOLOGY

References
1. Mizoguchi I, Higuchi K, Mitobe K, Tsunoda R, Mizuguchi J, Yoshimoto T.
Interleukin-27: regulation of immune responses and disease development
by a pleiotropic cytokine with pro- and anti-inﬂammatory properties. In:
Yoshimoto T, Yoshimoto T, eds. Cytokine frontiers: regulation of immune
responses in health and disease. Tokyo: Springer; 2013. p. 353–375.
2. Yoshida H, Hunter CA. The immunobiology of interleukin-27. Annu
Rev Immunol. 2015;33:417–443. doi:10.1146/annurev-immunol032414-112134.
3. Hisada M, Kamiya S, Fujita K, Belladonna ML, Aoki T, Koyanagi Y,
et al. Potent antitumor activity of interleukin-27. Cancer Res.
2004;64:1152–1156. doi:10.1158/0008-5472.CAN-03-2084.
4. Mizoguchi I, Chiba Y, Furusawa JI, Xu M, Tsunoda R, Higuchi K,
Yoshimoto T. Therapeutic potential of interleukin-27 against cancers
in preclinical mouse models. Oncoimmunology. 2015;4:e1042200.
doi:10.1080/2162402X.2015.1042200.
5. Yoshimoto T, Chiba Y, Furusawa J, Xu M, Tsunoda R, Higuchi K, Mizoguchi I. Potential clinical application of interleukin-27 as an antitumor agent.
Cancer Sci. 2015;106:1103–1110. doi:10.1111/cas.12731.
6. Seita J, Asakawa M, Ooehara J, Takayanagi S, Morita Y, Watanabe N,
Fujita K, Kudo M, Mizuguchi J, Ema H, et al. Interleukin-27 directly-62-

e1421892-3

induces differentiation in hematopoietic stem cells. Blood.
2008;111:1903–1912. doi:10.1182/blood-2007-06-093328.
7. Furusawa J, Mizoguchi I, Chiba Y, Hisada M, Kobayashi F, Yoshida H,
Nakae S, Tsuchida A, Matsumoto T, Ema H, et al. Promotion of expansion
and differentiation of hematopoietic stem cells by interleukin-27 into myeloid progenitors to control infection in emergency myelopoiesis. PLoS
Pathog. 2016;12:e1005507. doi:10.1371/journal.ppat.1005507.
8. Chiba Y, Mizoguchi I, Furusawa J, Hasegawa H, Ohashi M, Xu M,
Owaki T, Yoshimoto T. Interleukin-27 exerts its antitumor effects bypromoting differentiation of hematopoietic stem cells to M1 macrophages. Cancer Res. 2017; Nov 1:pii: canres.0960.2017. doi:10.1158/
0008-5472.CAN-17-0960.
9. Yoneto T, Waki S, Takai T, Tagawa Y, Iwakura Y, Mizuguchi J, Nariuchi
H, Yoshimoto T. A critical role of Fc receptor-mediated antibody-dependent phagocytosis in the host resistance to blood-stage Plasmodium
berghei XAT infection. J Immunol. 2001;166:6236–6241. doi:10.4049/
jimmunol.166.10.6236.
10. Yoshimoto T, Yoshimoto T, Yasuda K, Mizuguchi J, Nakanishi K. IL27 suppresses Th2 cell development and Th2 cytokines production
from polarized Th2 cells: a novel therapeutic way for Th2-mediatedallergic inﬂammation. J Immunol 2007;179:4415–4423. doi:10.4049/
jimmunol.179.7.4415.

Downloaded from http://rstb.royalsocietypublishing.org/ on January 5, 2018

rstb.royalsocietypublishing.org

Extracellular vesicle-mediated cell – cellcommunication in haematologicalneoplasms
Junko H. Ohyashiki1, Tomohiro Umezu1,2 and Kazuma Ohyashiki2

Review
Cite this article: Ohyashiki JH, Umezu T,
Ohyashiki K. 2017 Extracellular vesiclemediated cell– cell communication in
haematological neoplasms. Phil. Trans. R.
Soc. B 373: 20160484.
http://dx.doi.org/10.1098/rstb.2016.0484
Accepted: 15 April 2017
One contribution of 13 to a discussion meeting
issue ‘Extracellular vesicles and the tumour
microenvironment’.
Subject Areas:
molecular biology
Keywords:
extracellular vesicles, leukaemia, multiple
myeloma, hypoxia, bone marrow mesenchymal
stromal cells, miRNA
Author for correspondence:
Junko H. Ohyashiki
e-mail: junko@hh.iij4u.or.jp

1

Department of Molecular Oncology, Institute of Medical Science, and 2Department of Hematology, Tokyo
Medical University, Tokyo, Japan
JHO, 0000-0002-5997-2790
Crosstalk between bone marrow tumour cells and surrounding cells, including bone marrow mesenchymal stromal cells (BM-MSCs), endothelial cells
and immune cells, is important for tumour growth in haematological neoplasms. In addition to conventional signalling pathways, extracellular
vesicles (EVs), which are endosome-derived vesicles containing proteins,
mRNAs, lipids and miRNAs, can facilitate modulation of the bonemarrow microenvironment without directly contacting non-tumourous
cells. In this review, we discuss the current understanding of EV-mediatedcell –cell communication in haematological neoplasms, particularly leukaemia and multiple myeloma. We highlight the actions of tumour and
BM-MSC EVs in multiple myeloma. The origin of EVs, their tropism andmechanism of EV transfer are emerging issues that need to be addressedin EV-mediated cell –cell communication in haematological neoplasms.
This article is part of the discussion meeting issue ‘Extracellular vesicles
and the tumour microenvironment’.

1. Introduction
The tumour microenvironment (TME) plays an essential role in cancer development, metastasis and drug resistance [1]. Cancer-associated fibroblasts (CAFs)
are considered as a major component of the TME [2–5]. However, the origin of
CAFs is still controversial [2,6]. Relative to solid tumours, the bone marrow microenvironment (BME), where haematopoietic stem cells (HSCs) are located, isconsidered much more complicated (figure 1). First, HSCs reside in a specialized
microenvironment called the haematopoietic niche [7]. The endosteal niche, hallmarked by osteoblasts lining the bone marrow cavity, has been proposed as animportant source of HSC quiescence (figure 1, top right), while the vascularniche has been implicated in HSC maintenance and regeneration (figure 1,
bottom right). Second, the definition of CAFs is not generally used to indicate
the tumour-surrounding cell population in haematological neoplasms. Such cells
are referred to as bone marrow mesenchymal stromal cells (BM-MSCs) [8].
While BM-MSCs and CAFs may share biological properties, especially in multiple
myeloma (MM) [9] and chronic lymphocytic leukaemia (CLL) [10], the differences
and similarities between BM-MSCs and CAFs are not fully elucidated in leukaemias [8]. In addition to BM-MSCs, immune cells, such as T cells, B cells andnatural killer (NK) cells, also interact with tumour cells. Myeloid-derived suppressor cells (MDSCs) represent another population of regulatory cells, which impair
anti-tumour innate and adaptive immune responses [11].
It is believed that cell–cell communication between tumour cells and their surrounding cells is modulated by direct cell contact and soluble factors such as
cytokines. Emerging evidence suggests that extracellular vesicles (EVs) shedfrom either tumour cells or their surrounding cells act as a mediator of crosstalkin the TME [12] because EVs contain complex cargo including proteins, mRNAs,
lipids and microRNAs (miRNAs). The current generally recognized categories of
EVs are exosomes (the most well-known category) with a size of 30–100 nm,
microvesicles with a size of 100–1000 nm, and large oncosomes ranging in size

& 2017 The Author(s) Published by the Royal Society. All rights reserved.
-63-

Downloaded from http://rstb.royalsocietypublishing.org/ on January 5, 20182

endosteal niche
bone
MDSC

tumour cell
T cellhaematopoietic
stem cells

NK cellmacrophage

mesenchymal
stem cell

endothelial cell

RBC

RBC

sinusoidbone marrow mesenchymal
stromal cell (BM-MSC)

extracellular vesicle (EV)vascular niche

Figure 1. Bone marrow microenvironment in haematological neoplasms. The bone marrow microenvironment is located in bone. Two types of niches, the endosteal
niche and vascular niche, are located adjacent to the bone cortex. Tumour cells secrete extracellular vesicles (small green circles) towards their surrounding cells such asbone marrow mesenchymal stromal cells (BM-MSCs), endothelial cells and immune cells. Various types of cell–cell communication are assumed, such as tumour cells to
BM-MSCs, tumour cells to endothelial cells, tumour cells to immune cells, and others. MDSC, myeloid-derived suppressor cell; NK, natural killer; RBC, red blood cell.
proteins, mRNAs and miRNAs, have been identified [17,21], it isdifficult to focus on a single pathway for AML. Another important issue is that AML-EVs suppress immune cells such as
NK cells [18]. In serum obtained from AML patients, AMLmicrovesicles mediate suppression of NK cell activity via thetransforming growth factor-b1 signalling pathway, and interleukin (IL)-15 protects NK cells from the adverse effects of
AML-microvesicles [18].
To date, there is no appropriate in vitro model to elucidate
the complex cell–cell interactions in the bone marrow niche
where leukaemic stem cells exist. Crosstalk via EVs between
osteoblasts and HSCs or between BM-MSCs and HSCs also
remains to be resolved. For this reason, much effort has been
made to determine the diagnostic value of circulating EVs
rather than the mechanism of cell–cell communication in
AML [23]. A recent report by Viola et al. demonstrated thatEVs derived from BM-MSCs induce tyrosine kinase inhibitor
resistance in AML [24], suggesting a new therapeutic approachtargeting BM-MSCs in AML. Considering the critical roles of
EVs in AML, understanding the mechanisms regulating signalling pathways in recipient cells may provide additional insights
into the use of EVs as therapeutic agents for treating AML.

from 1 to 10 mm [13,14]. To date, most EV studies in haematologic neoplasms refer to exosomes or microvesicles. However, a
recent study by Johnson et al. focused on the heterogeneity of
EVs and characterized large EVs in acute lymphoblastic leukaemia [15]. In this review, we highlight and discuss recent studies
of EV-mediated cell–cell communication in haematologicalneoplasms, particularly leukaemia and MM.

2. Acute myeloid leukaemia-derived extracellular
vesicles in the bone marrow
microenvironment
Leukaemia is a type of haematological neoplasm which arises
from HSCs. Leukaemia types are subdivided by cell lineage(myeloid or lymphoid) and clinical course (acute or chronic).
High throughput genomic analyses have revealed that genetic
alteration of leukaemic cells is likely a more important determinant of disease severity [16]. However, increasing evidence
suggests that leukaemic cell-derived EVs affect their surrounding
cells in autocrine and/or paracrine manners [17–19].
In acute myeloid leukaemia (AML), Kumar et al. found evidence that AML-EVs alter the BME to facilitate leukaemic cell
growth and suppress normal haematopoiesis in a mousemodel [20]. The unique miRNA profile of AML-EVs, including
miR-155, has the potential to increase leukaemic fitness by dysregulation of other cell types in the BME [21]. Myelodysplastic
syndrome (MDS) is a clonal myeloid neoplasm characterized
by ineffective haematopoiesis, and approximately 30% of
patients develop AML. In addition to AML-EVs, MDS-EVs arelinked to stromal cell dysfunction. Therefore, EV-mediatedcell–cell interaction is also involved in bone marrow failure syndrome [22]. Although various components of AML-EVs, such as

3. Bone marrow angiogenesis and extracellular
vesicles derived from chronic myeloid
leukaemia
Chronic myelogenous leukaemia (CML) characterized by the
BCR-ABL chimeric protein is another type of myeloid leukaemia,
which tends to progress more slowly than AML. Increased
microvessel density and clinicopathological correlations withbone marrow angiogenesis have been reported in CML patients-64-

Phil. Trans. R. Soc. B 373: 20160484

osteoclast

rstb.royalsocietypublishing.org

osteoblastDownloaded from http://rstb.royalsocietypublishing.org/ on January 5, 2018

control
HUVECs24 h

20% O2

24 h

1% O2

normoxia

K562

K562

hypoxia[25]. We and others have shown that EVs secreted by CML cells
can potentially influence in vitro and/or in vivo angiogenesis bydirectly affecting the properties of endothelial cells [26,27].
Taverna and colleagues first provided direct evidence that fluorescent-labelled EVs released by K562 cells are internalized byhuman umbilical vein endothelial cells (HUVECs) during tubular differentiation on Matrigel, thereby enhancing angiogenesis[26,28]. They also demonstrated that functional transfer of
CML-EV-miR126 targets C-X-C motif chemokine ligand 12 andvascular cell adhesion molecule in HUVECs [29]. These findings
indicate that exogenous miRNAs transferred via EVs function
similarly to endogenous miRNAs in HUVECs. CML-EVs
also induce increased secretion of IL-8 in BM-MSCs, thereby
promoting leukaemic cell growth in vitro and in vivo [30].
Because hypoxia is known to be a regulator of angiogenesis,
we investigated how hypoxia triggers EV-mediated angiogenesis using the human leukaemic cell line K562. We found that
K562 cell-EVs under hypoxic conditions (1% O2 for 24 h) significantly enhance tube formation of HUVECs compared withEVs produced under normoxic conditions (20% O2 for 24 h)[31] (figure 2). These experiments employed artificial in vitro conditions for short-term exposure to hypoxia. However, we foundthat miR-210 in EVs (EV-miR-210) downregulates ephrin A3 in
HUVECs and alters EV components under hypoxic conditions,
thereby affecting the behaviour of HUVECs [31]. EVs shedfrom leukaemic cells act in the following manners. First,
leukaemia-EVs educate BM-MSCs for their own cell survival.
As a result, normal haematopoiesis is suppressed. Second, leukaemia-EVs induce immune suppression by targeting T cells, B
cells and NK cells. Third, leukaemia-EVs induce angiogenesis,
especially in CML.

4. Extracellular vesicle-mediated cell –cellcommunication in multiple myeloma
MM is a blood cancer formed by malignant plasma cells thatcreate tumour masses in bone. In contrast to leukaemia, thetumour masses are predominantly located in the bone

-65-

Phil. Trans. R. Soc. B 373: 20160484

Figure 2. Hypoxia and angiogenesis in leukaemia. Human umbilical vein
cells (HUVECs) were cultured with or without extracellular vesicles (EVs)secreted from a human leukaemia cell line (K562). The cells were cultured
for 24 h and then EVs were collected. In normoxia, tube formation of
HUVECs was increased in the presence of K562-derived EVs compared with
control HUVECs (middle). When EVs obtained from K562 cells cultured
under hypoxic conditions (K562-EV-hypoxia) were added to HUVECs, tube formation was much more prominent (bottom). These findings indicate possibleroles of EVs, hypoxia and angiogenesis in leukaemia.

3

rstb.royalsocietypublishing.org

marrow. Therefore, cell –cell communication in the BME has
a key role in the pathogenesis of MM. Because targeting
MM cells alone is insufficient to establish curable treatment
strategies for MM, the interaction between MM cells andtheir surrounding cells has been studied extensively [32]. In
terms of clinical practice, circulating EV-miRNAs fromnewly diagnosed MM patients are a potential biomarker for
prognosis [33]. Because MM appears to be pathophysiologically similar to solid tumours, a cell-free specimen has great
benefit for evaluating minimal residual disease. We discuss
the characteristic BME in MM, especially hypoxia, in thefollowing section. We then summarize the role of MM-EVs,
BM-MSC-EVs and the mechanism of EV transfer in MM.
Bone marrow is hypoxic in nature, and oxygen tension in
MM-infiltrated BM is lower than that in normal BM [34]. The
massive proliferation of MM cells produces hypoxic conditions in the tumour, which may lead to more rapid cell
growth, drug resistance and angiogenesis [35]. Therefore,
we focused on the effect of hypoxia in the BME andestablished three MM cell lines that could be adapted tolong-lasting hypoxia, namely hypoxia-resistant MM cells(HR-MM cells). Mutation analysis of HR-MM cells revealed
no acquired genetic variation compared with their parental
cells, while the gene expression profile of HR-MM cells was
modified during the adaptation process [36]. We consideredthat HR-MM cells may be suitable for analyses of the BME,
which mimics the in vivo state because most in vitro hypoxia
studies use short-term hypoxic exposure (3–24 h) which may
reflect acute hypoxic shock rather than the MM-BME.
Although no significant difference existed in the size of EVs
between HR-MM and parental cells, the amount of EVs
released from HR-MM cells was approximately twofold
greater. We also found that HR-MM cells enhanced in vitro
tube formation and increased angiogenesis in vivo in a Matrigel assay. It was notable that EV-miR-135b from HR-MM
cells enhanced angiogenesis by direct suppression of factorinhibiting hypoxia-inducible factor 1 (FIH-1) in endothelial
cells. It remains uncertain how other components within
HR-MM cell-EVs, such as proteins, mRNAs and miRNAs,
may be involved in EV-mediated angiogenesis.
Regarding the cargo of MM-EVs, three independent
proteomic studies revealed the significance of proteins in
EV-mediated intercellular communication [37–39]. Harshman
et al. identified 583 MM-EV proteins in total by liquid chromatography coupled with tandem mass spectrometry [37]. They
found that MM-EVs contained molecules related to a distinct
pathway, such as antigen-presenting molecules (major histocompatibility complex class I and II) and adhesion molecules(tetraspanins and integrins), in MM cell lines MM.1S andU266 [37]. MM-EVs contain the biologically active form of
CD147, which drives MM cell proliferation [38]. In addition,
MM-EVs harbour CD138, which is known to be an angiogenic
regulator [39], and the angiogenic activity has been confirmed
using mouse MM-EVs in vivo [40].
MM-EVs also interact with BM-MSCs to establish afavourable BME for MM: they promote an increase of
miR-146a in BM-MSCs, which induces more cytokines andchemokines, such as IL-6, and support MM cell growth [41](figure 3). Another example of tumour cell and BM-MSC
cooperation has been reported in another type of lymphoidmalignancy, CLL. CLL-derived EVs are internalized by
BM-MSCs and deliver functionally active components to
BM-MSCs [10]. Taken together, tumour-derived EVs haveDownloaded from http://rstb.royalsocietypublishing.org/ on January 5, 2018

osteolysis

MM cells

immunosuppression

rstb.royalsocietypublishing.org

osteoclast

NK cellmacrophage

T cellsoluble factors

MDSC

IL-6, VEGF, TNF-a,
TGF-b, RANKL,CXCL12, etc.

miR-135b

hypoxia
FIH-1

BM-MSC

endothelial cell
HIF-1

MM cell growth, survival
and drug resistanceangiogenesis

Figure 3. Cell– cell communication by extracellular vesicles in multiple myeloma. Massive proliferation of abnormal plasma cells is shown in the centre. The centreof the mass has pale blue plasma cells, indicating the hypoxic state. In addition to conventional signalling pathways, such as direct cell adhesion and soluble factors,
either extracellular vesicles (EVs) derived from multiple myeloma (MM) cells (MM-EVs; small green circles) or EVs derived from bone marrow mesenchymal stromalcells (BM-MSC-EVs; small orange circles) modify the behaviour of recipient cells as follows. MM-EVs modulate BM-MSCs and induce a favourable microenvironmentfor MM cells. Conversely, BM-MSC-EVs also induce MM cell growth, survival and drug resistance (bottom right). MM-EVs induce angiogenesis. In a long-lasting
hypoxic state, EV-miR-135b acts as a mediator of angiogenesis through the FIH-1/hypoxia-inducible factor-1 signalling pathway (bottom left). BM-MSC-EVs interact
with myeloid-derived suppressor cells (MDSCs), and MM-EVs modulate immune cells, such as natural killer (NK) cells, resulting in immunosuppression (top right).
MM-EVs activate osteoclasts which may be linked to osteolysis in MM (top left).
cells [42]. In solid tumours, tumour-derived EVs use tropism
to transfer into organ-specific recipient cells, and this tropism
depends on distinct integrin expression patterns [43]. The
mechanism by which EVs are captured and why EVs canselect recipient cells remain to be elucidated in MM. Although
the BME in MM is controlled by either a cytokine/chemokine
network or cell adhesion molecules [32], interactions between
conventional and EV-mediated pathways are much more
complex than ever imagined.

the ability to transform BM-MSCs to CAF-like cells. Of note isthat MM-EVs induce prolonged survival of MDSCs whichhave a strong immune-suppressive activity in cancer patients[40]. Raimondi et al. demonstrated the effect of MM-EVs on
both human primary osteoclasts and murine pre-osteoclastic
RAW264.7 cells [42]. These results indicate that MM-EVs are
key regulators that educate BME-targeting endothelial cells,
BM-MSCs, MDSCs and osteoclasts.
In addition to MM-EVs, EVs derived from BM-MSCsfacilitate MM cell growth by maintaining a favourable BME
for MM. Roccaro et al. reported the difference in EV-miRNA
contents between MM-BM-MSCs and normal BM-MSCs [43].
EVs derived from MM-BM-MSCs have less tumour suppressor miR-15a and higher levels of oncogenic proteins,
cytokines and adhesion molecules, whereas normal-BMMSCs have the potential to inhibit MM cell growth. Theirfindings indicate a CAF-like ability of MM-BM-MSCs andthe possible use of normal BM-MSCs for MM cell therapy.
We also found that BM-MSCs from healthy young donors
inhibit MM angiogenesis through EV-miRNA [44]. From
the aspect of CAFs, BM-MSC-derived EVs induce proliferation, survival and drug resistance of MM cells [45], andthey are able to activate MDSCs [46]. As a result, BM-MSCderived EVs contribute to not only the BME supporting
MM cell growth but also immunosuppression related todisease progression.
Recent studies have focused on the mechanisms of EV
release and uptake in MM. EV-fibronectin has recently been
identified as a key binding ligand for heparan sulfate that
can act as a receptor for EV uptake by MM cells [41]. Conversely, Bcl-xL is an exosomal caspase-3 substrate, and its
processing is required for the uptake of exosomes by recipient

5. Perspectives
Despite the fact that most studies have focused on tumourcell- and BM-MSC-derived EVs, how EVs derived fromother types of cells act in the BME is largely unknown.
Because a category of EVs includes components other than
exosomes, including oncosomes, the type of EV and thecells from which EVs originate should be clarified to elucidate the precise mechanism of EV-mediated cell –cellcommunication. We identified EV-miRNA as an angiogenic
factor in our experimental model. However, analysis ofplasma EV-miR-135b did not simply reflect MM disease
severity. It is likely that EV-miRNA in the proximal area (i.e.
bone marrow) is not linked to EV-miRNA in remote areas
such as circulating blood. The manner of cell–cell communication in the proximal area might be different from that in
the distal area. Therefore, careful attention is needed for diagnostic use of EV-miRNA. To better understand EV-mediatedcell–cell communication in haematological neoplasms, theorigin of EVs, their tropism, and mechanism of EV transfer
are emerging issues that need to be addressed.

-66-

Phil. Trans. R. Soc. B 373: 20160484

MM-EV
BM-MSC-EV

direct celladhesion

4

Downloaded from http://rstb.royalsocietypublishing.org/ on January 5, 2018

Data accessibility. This article has no additional data.
Authors’ contributions. T.U. performed part of the study discussed in thisCompeting interests. We have no competing interests.
Funding. This review was supported by the Private University Strategic

5

review. J.H.O. drafted the manuscript. K.O. helped to draft themanuscript. All authors read and approved the final manuscript.

Research-Based Support Project (grant no. S1311016) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

rstb.royalsocietypublishing.org

References
1.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

J. Extracell. Vesicles 3, 26913. (doi:10.3402/jev.v3.
26913)
Johnson SM, Dempsey C, Parker C, Mironov A,
Bradley H, Saha V. 2017 Acute lymphoblastic
leukaemia cells produce large extracellular vesicles
containing organelles and an active cytoskeleton.
J. Extracell. Vesicles 6, 1294339. (doi:10.1080/
20013078.2017.1294339)
White BS, DiPersio JF. 2014 Genomic tools in acute
myeloid leukemia: from the bench to the bedside.
Cancer 120, 1134–1144. (doi:10.1002/cncr.28552)
Zhou J, Wang S, Sun K, Chng WJ. 2016 The
emerging roles of exosomes in leukemogeneis.
Oncotarget 7, 50 698– 50 707. (doi:10.18632/
oncotarget.9333)
Szczepanski MJ, Szajnik M, Welsh A, Whiteside TL,
Boyiadzis M. 2011 Blast-derived microvesicles insera from patients with acute myeloid leukemia
suppress natural killer cell function via membraneassociated transforming growth B1. Haematologica
96, 1302–1309. (doi:10.3324/haematol.2010.
039743)Raimondo S, Saieva L, Corrado C, Fontana S, Flugy
A, Rizzo A, De Leo G, Alessandro R. 2015 Chronic
myeloid leukemia-derived exosomes promote tumor
growth through an autocrine mechanism. Cell
Commun. Signal. 13, 8. (doi:10.1186/s12964-0150086-x)
Kumar B, Lewis X, Murakami J, Hu X, McDonald T,
Kumar A, DiGiusto D, Bhatis R, Chen C-CC. 2013
Leukemia-derived exosomes recognize bone marrow
microenvironment in AML. Blood 122, 2455. (http://
www.bloodjournal.org/content/122/21/2455)
Hornick NI, Doron B, Abdelhamed S, Huan J,
Harrington CA, Shen R, Cambronne XA,
Chakkaramakkil Verghese S, Kurre P. 2016 AML
suppresses hematopoiesis by releasing exosomesthat contain microRNAs targeting c-MYB. Sci. Signal.
9, ra88. (doi:10.1126/scisignal.aaf2797)
Horiguchi H et al. 2014 Exosomes derived from
AML/MDS cells is involved in stromal dysfunction
and bone marrow failure. Blood 124, 4627. (http://
www.bloodjournal.org/content/124/21/4627)
Hornick NI, Huan J, Doron B, Goloviznina NA,
Lapidus J, Chang BH, Kurre P. 2015 Serum exosome
microRNA as a minimally-invasive early biomarker
of AML. Sci. Rep. 5, 11295. (doi:10.1038/srep11295)
Viola S, Traer E, Huan J, Hornick NI, Tyner JW,
Agarwal A, Loriaux M, Johnstone B, Kurre P. 2016
Alterations in acute myeloid leukaemia bonemarrow stromal cell exosome content coincide withgains in tyrosine kinase inhibitor resistance.
Br. J. Haematol. 172, 983–986. (doi:10.1111/bjh.
13551)-67-

25. Korkolopoulou P et al. 2003 Clinicopathologic
correlations of bone marrow angiogenesis in chronic
myeloid leukemia: a morphometric study. Leukemia
17, 89 – 97. (doi:10.1038/sj.leu.2402769)
26. Mineo M, Garfield SH, Taverna S, Flugy A, De Leo G,
Alessandro R, Kohn EC. 2012 Exosomes released by
K562 chronic myeloid leukemia cells promoteangiogenesis in a Src-dependent fashion. Angiogenesis
15, 33–45. (doi:10.1007/s10456-011-9241-1)
27. Umezu T, Ohyashiki K, Kuroda M, Ohyashiki JH.
2013 Leukemia cell to endothelial cellcommunication via exosomal miRNAs. Oncogene 32,
2747– 2755. (doi:10.1038/onc.2012.295)
28. Taverna S, Flugy A, Saieva L, Kohn EC, Santoro A,
Meraviglia S, De Leo G, Alessandro R. 2012 Role of
exosomes released by chronic myelogenous
leukemia cells in angiogenesis. Int. J. Cancer 130,2033– 2043. (doi:10.1002/ijc.26217)
29. Taverna S, Amodeo V, Saieva L, Russo A,
Giallombardo M, De Leo G, Alessandro R. 2014
Exosomal shuttling of miR-126 in endothelial cells
modulates adhesive and migratory abilities ofchronic myelogenous leukemia cells. Mol. Cancer
13, 169. (doi:10.1186/1476-4598-13-169)
30. Corrado C, Raimondo S, Saieva L, Flugy AM, De Leo
G, Alessandro R. 2014 Exosome-mediated crosstalk
between chronic myelogenous leukemia cells andhuman bone marrow stromal cells triggers aninterleukin 8-dependent survival of leukemia cells.
Cancer Lett. 348, 71 – 76. (doi:10.1016/j.canlet.
2014.03.009)
31. Tadokoro H, Umezu T, Ohyashiki K, Hirano T,
Ohyashiki JH. 2013 Exosomes derived from hypoxic
leukemia cells enhance tube formation inendothelial cells. J. Biol. Chem. 288, 34 343–
34 351. (doi:10.1074/jbc.M113.480822)
32. Bianchi G, Munshi NC. 2015 Pathogenesis beyond
the cancer clone(s) in multiple myeloma. Blood 125,
3049–3058. (doi:10.1182/blood-2014-11-568881)
33. Manier S et al. 2017 Prognostic role of circulating
exosomal miRNAs in multiple myeloma. Blood
129, 2429– 2436. (doi:10.1182/blood-2016-09742296)
34. Asosingh K, De Raeve H, de Ridder M, Storme GA,
Willems A, Van Riet I, Van Camp B, Vanderkerken K.
2005 Role of the hypoxic bone marrow
microenvironment in 5T2MM murine myeloma
tumor progression. Haematologica 90, 810– 817.
35. Martin SK, Diamond P, Gronthos S, Peet DJ,
Zannettino AC. 2011 The emerging role of hypoxia,
HIF-1 and HIF-2 in multiple myeloma. Leukemia 25,
1533– 1542. (doi:10.1038/leu.2011.122)
36. Umezu T, Tadokoro H, Azuma K, Yoshizawa S,
Ohyashiki K, Ohyashiki JH. 2014 Exosomal miR-135b

Phil. Trans. R. Soc. B 373: 20160484

2.

Quail DF, Joyce JA. 2013 Microenvironmental
regulation of tumor progression and metastasis.
Nat. Med. 19, 1423–1437. (doi:10.1038/nm.3394)
Augsten M. 2014 Cancer-associated fibroblasts asanother polarized cell type of the tumor
microenvironment. Front. Oncol. 4, 62. (doi:10.3389/
fonc.2014.00062)
Farahani M, Rubbi C, Liu L, Slupsky JR, Kalakonda
N. 2015 CLL exosomes modulate the transcriptome
and behaviour of recipient stromal cells and are
selectively enriched in miR-202-3p. PLoS ONE 10,
e0141429. (doi:10.1371/journal.pone.0141429)
Gascard P, Tlsty TD. 2016 Carcinoma-associatedfibroblasts: orchestrating the composition ofmalignancy. Genes Dev. 30, 1002 –1019. (doi:10.
1101/gad.279737.116)
Kalluri R. 2016 The biology and function offibroblasts in cancer. Nat. Rev. Cancer 16, 582–598.
(doi:10.1038/nrc.2016.73)
Xing F, Saidou J, Watabe K. 2010 Cancer associatedfibroblasts (CAFs) in tumor microenvironment. Front.
Biosci. 15, 166– 179. (doi:10.2741/3613)
Adams GB, Scadden DT. 2006 The hematopoietic
stem cell in its place. Nat. Immun. 7, 333– 337.
(doi:10.1038/ni1331)
Raffaghello L, Vacca A, Pistoia V, Ribatti D. 2015
Cancer associated fibroblasts in hematologicalmalignancies. Oncotarget 6, 2589 –2603. (doi:10.
18632/oncotarget.2661)
Frassanito MA et al. 2016 Halting pro-survival
autophagy by TGFb inhibition in bone marrow
fibroblasts overcomes bortezomib resistance inmultiple myeloma patients. Leukemia 30, 640–
648. (doi:10.1038/leu.2015.289)
Paggetti J et al. 2015 Exosomes released by chronic
lymphocytic leukemia cells induce the transition ofstromal cells into cancer-associated fibroblasts.
Blood 126, 1106 –1117. (doi:10.1182/blood-201412-618025)
Solito S, Pinton L, Mandruzzato S. 2017 In brief:
myeloid-derived suppressor cells in cancer. J. Pathol.
(doi:10.1002/path.4876)
Tkach M, Thery C. 2016 Communication by
extracellular vesicles: where we are and where weneed to go. Cell 164, 1226– 1232. (doi:10.1016/j.
cell.2016.01.043)
Raposo G, Stoorvogel W. 2013 Extracellular
vesicles: exosomes, microvesicles, and friends.
J. Cell Biol. 200, 373– 383. (doi:10.1083/jcb.
201211138)
Lotvall J et al. 2014 Minimal experimental
requirements for definition of extracellular vesicles
and their functions: a position statement from theInternational Society for Extracellular Vesicles.

Downloaded from http://rstb.royalsocietypublishing.org/ on January 5, 2018

-68-

44. Umezu T, Imanishi S, Azuma K, Kobayashi C,
Yoshizawa S, Ohyashiki K, Ohyashiki JH. 2017
Replenishing exosomes from older bone marrowstromal cells with miR-340 inhibits myelomarelated angiogenesis. Blood Adv. 1, 812 –823.
(doi:10.1182/bloodadvances.2016003251)
45. Wang J, Hendrix A, Hernot S, Lemaire M, De Bruyne
E, Van Valckenborgh E, Lahoutte T, De Wever O,
Vanderkerken K, Menu E. 2014 Bone marrow stromalcell-derived exosomes as communicators in drug
resistance in multiple myeloma cells. Blood 124,
555–566. (doi:10.1182/blood-2014-03-562439)
46. Wang J, De Veirman K, De Beule N, Maes K, De
Bruyne E, Van Valckenborgh E, Vanderkerken K,
Menu E. 2015 The bone marrow microenvironment
enhances multiple myeloma progression by
exosome-mediated activation of myeloid-derived
suppressor cells. Oncotarget 6, 43 992–44 004.
(doi:10.18632/oncotarget.6083)

6

Phil. Trans. R. Soc. B 373: 20160484

40. Wang J, De Veirman K, Faict S, Frassanito MA,
Ribatti D, Vacca A, Menu E. 2016 Multiple myeloma
exosomes establish a favourable bone marrow
microenvironment with enhanced angiogenesis andimmunosuppression. J. Pathol. 239, 162 –173.
(doi:10.1002/path.4712)
41. De Veirman K et al. 2016 Induction of miR-146a
by multiple myeloma cells in mesenchymal
stromal cells stimulates their pro-tumoral activity.
Cancer Lett. 377, 17 – 24. (doi:10.1016/j.canlet.
2016.04.024)
42. Raimondi L et al. 2015 Involvement of multiple
myeloma cell-derived exosomes in osteoclastdifferentiation. Oncotarget 6, 13772–13789.
(doi:10.18632/oncotarget.3830)
43. Roccaro AM et al. 2013 BM mesenchymal stromalcell-derived exosomes facilitate multiple myeloma
progression. J. Clin. Invest. 123, 1542–1555.
(doi:10.1172/JCI66517)

rstb.royalsocietypublishing.org

shed from hypoxic multiple myeloma cells enhances
angiogenesis by targeting factor-inhibiting HIF-1.
Blood 124, 3748 –3757. (doi:10.1182/blood-201405-576116)
37. Harshman SW et al. 2013 Characterization ofmultiple myeloma vesicles by label-free relative
quantitation. Proteomics 13, 3013 –3029. (doi:10.
1002/pmic.201300142)
38. Arendt BK, Walters DK, Wu X, Tschumper RC,
Jelinek DF. 2014 Multiple myeloma cell-derived
microvesicles are enriched in CD147 expression
and enhance tumor cell proliferation.
Oncotarget 5, 5686 – 5699. (doi:10.18632/
oncotarget.2159)
39. Liu Y, Zhu XJ, Zeng C, Wu PH, Wang HX, Chen ZC, Li
QB. 2014 Microvesicles secreted from humanmultiple myeloma cells promote angiogenesis. Acta
Pharmacol. Sin. 35, 230–238. (doi:10.1038/aps.
2013.141)

ONCOLOGY REPORTS 39: 91-100, 2018

Expression level of microRNA-200c is associated with cellmorphology in vitro and histological differentiation throughregulation of ZEB1/2 and E-cadherin in gastric carcinoma
Atsushi Kurata1, Masatoshi Yamada1, Shin-ichiro Ohno1, Shigeru Inoue2,
Hirotsugu Hashimoto3, Koji Fujita1, Masakatsu Takanashi1 and Masahiko Kuroda1
Departments of 1Molecular Pathology and 2Preventive Medicine and Public Health, Tokyo Medical University,
Tokyo 160-8402; 3Department of Diagnostic Pathology, NTT Medical Center Tokyo, Tokyo 141-8625, Japan
Received June 27, 2017; Accepted October 30, 2017
DOI: 10.3892/or.2017.6093
Abstract. Scirrhous type gastric cancer is characterized bydiffuse infiltration of poorly differentiated adenocarcinoma
cells and poor prognosis. Although association of poorly
differentiated histology with reduction in E-cadherin expression, as well as association of microRNA (miR)-200c with
E-cadherin through regulation of ZEB1/2, has been reported,
participation of miR-200c in gastric carcinogenesis is not
fully understood. We used 6 cell lines originating from gastriccancers, and investigated levels of miR-200c along with its
target mRNAs ZEB1/2 and E-cadherin by qRT-PCR. ZEB1 andE-cadherin protein expression was also assessed via western
blotting. Furthermore, we investigated the expression levels ofmiR‑200c by in situ hybridization, along with the expressionof ZEB1 and E-cadherin by immunohistochemistry, in 97
gastric adenocarcinoma tissues. Inverse correlation between
miR‑200c and ZEB1 levels were obtained by qRT-PCR in celllines (P<0.05). Cell lines with low miR-200c and high ZEB1
exhibited low E-cadherin expression in both qRT-PCR andwestern blotting, and exhibited spindle-shaped morphology,
in contrast to round cell morphology in those cell lines withhigh miR-200c levels. Inverse correlations were also obtained
between miR-200c and ZEB1 as well as between ZEB1 andE-cadherin levels in tissue samples (P<0.001). Cancer tissues
with low miR-200c, high ZEB1, and low E-cadherin expression were associated with poorly differentiated histology,
in contrast to tubular form in cancers with high miR-200c
expression levels (P<0.001). Our data revealed that downregulation of miR-200c primarily regulated cell morphology by

Correspondence to: Dr Atsushi Kurata, Department of MolecularPathology, Tokyo Medical University, 6-1-1, Shinjuku, Shinjuku-ku,
Tokyo 160-8402, Japan
E-mail: akurata@tokyo-med.ac.jp

Key words: gastric adenocarcinoma, microRNA-200c, E-cadherin,
epithelial-mesenchymal transition, cell morphology

downregulation of E-cadherin through upregulation of ZEB1,
leading to poorly differentiated histology in gastric cancer.
Introduction
Gastric cancer is the fourth most common cancer and thesecond leading cause of cancer-related deaths in the world (1).
More than 980,000 new gastric cancer cases are diagnosed
annually, and the disease causes approximately 730,000
deaths per year, with the highest incidence rates in Eastern
Asia (1). Scirrhous cancer, which accounts for approximately 10% of all gastric cancers, is characterized by diffuse
infiltration of poorly differentiated adenocarcinoma cellswith extensive stromal fibrosis, leading to frequent peritoneal
dissemination and extremely poor prognosis (2,3). Molecularanalyses have revealed that reduction in the expression of
the cell-cell adhesion molecule, E-cadherin, was involvedin the development and infiltrative growth of scirrhous typegastric carcinomas (4,5). Reduction in E-cadherin expression
observed in poorly differentiated carcinomas of the lungs andendometrium (6,7), promotes detachment of cells from eachother and increases cell motility; thus, leading to epithelialmesenchymal transition (EMT) (8).
On the other hand, epigenetic regulation including therole of microRNAs (miRNAs, miRs) in carcinogenesis has
recently been intensively studied. miRNAs are endogenous
18-24-nucleotide (nt) single-stranded RNA molecules that act
as post-transcriptional regulators of gene expression (9). They
stabilize target mRNA transcripts through post-transcriptional
gene silencing via either inhibition of the translation process orcleavage of target mRNAs (10,11). miRNAs sharing the sameseed sequence are grouped into families and are theorized totarget overlapping sets of genes (12). miRNAs play diverseroles in numerous cellular processes; in particular, theirexpression is altered during tumorigenesis, and they can act as
oncogenes or tumor suppressors (13).
Recent studies have shown the importance of certain
miRNAs in modulating EMT, and the miR-200 family has
been described to be a key regulator of this process (7,8,14).
The miR-200 family consists of 5 members: miR-200a,
miR-200b and miR-429 which are located on chromosome

-69-

92

Kurata et al: miR-200c in gastric cancer

1p36 while miR-200c and miR-429 are located on chromosome 12p13 (14). The miR-200 family suppresses zinc-finger
E-box binding homeobox (ZEB) 1 and ZEB2, which in turninhibit the expression of E-cadherin; thus, the miR-200
family maintains the epithelial phenotype through upregulation of E-cadherin (7,8,14). Among the miR-200 family, lossof miR‑200c has been reported to be pivotal to inducing
aggressive, invasive and chemoresistant phenotypes in several
cancers (15-17). Although downregulation of miR-200a andmiR-200b have been reported to be associated with poor
prognosis or EBV-association in gastric cancers (18,19), little
is known about miR-200c in this context. Recent reports
demonstrated that miR-200c may affect E-cadherin expression
through regulation of ZEB1/2 expression (20,21); however, theinfluences of miR-200c on cell morphology and its association
with histological type have so far not been reported.
In the present study, we investigated the expressionof miR‑200c, as well as corresponding targets at both themRNA and protein levels, in several gastric carcinoma
cell lines. In addition, we examined the expression level ofmiR‑200c and respective target proteins in gastric carcinoma
tissues and adjacent non-tumor tissues. Our results revealed
that the expression level of miR-200c was associated withthe morphology of gastric carcinoma cell lines, as well as
histological differentiation in cancer tissues, along with theupregulation or downregulation of ZEB 1/2 and E-cadherin.
Materials and methods
Cell culture and RNA preparation. Six human gastriccarcinoma cell lines, H-111-TC, HGC-27, Kato-III, MKN-1,
MKN-45 and NU-GC-4 were purchased from Riken Cell Bank
(Tsukuba, Japan) and used in the present study. According to
the description by the Riken Cell Bank and literature (22-27),
these cell lines originated from 1 unknown origin (H-111-TC)
and 5 metastatic carcinoma, as well as 2 tubular (well differentiated or adenosquamous) carcinoma (H-111-TC and MKN-1)
and 4 poorly differentiated carcinoma including signet ringcell type. HGC-27 and MKN-1 cell lines were maintained in
Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL,
Rockville, MD, USA), whereas NU-GC-4, Kato-III, MKN-45
and H-111-TC cell lines were maintained in RPMI-1640(Gibco-BRL), supplemented with 10% fetal calf serum (FCS),
penicillin, and streptomycin, at 37˚C in a humidified atmosphere of 5% CO2. When they reached 80-90% confluence,the cells were washed with phosphate-buffered saline (PBS)
and homogenized immediately in Isogen reagent (Nippon
Gene, Osaka, Japan). Total RNA was extracted according to
the manufacturer's instructions.
Quantitative real-time reverse transcription polymerase
chain reaction (qRT-PCR) analysis. The RNA samples weresuspended in 20 µl of nuclease-free water, and miRNA-200c
levels were quantified using TaqMan MicroRNA Assay technology (Applied Biosystems; Life Technologies, Carlsbad, CA,USA), as previously described (28). miRNA-200c levels werenormalized against levels of RNU6B. The hsa-miR-200c
oligonucleotide was a synthetic double-stranded 23-nt RNA,
5'-UAAUACUGCCGGGUAAUGAUGGA-3' and 5'-CGUCUUACCCAGCAGUGUUUGG-3' purchased from BONAC

Corp. (Fukuoka, Japan). To assess the mRNA levels of ZEB1,
ZEB2 and E-cadherin, reverse-transcription reactions were
performed using M-MLV reverse transcriptase (Invitrogen,
Carlsbad, CA, USA). Quantitative PCR (FastStart Universal
SYBR-Green Master; Roche, Basel, Switzerland) reactions
were run on an Mx3005P thermocycler (Stratagene, La Jolla,
CA, USA) and analyzed using MxPro QPCR software, version
4.01 (Stratagene, Agilent Technologies, Santa Clara, CA,
USA). The level of gene expression relative to glyceraldehyde
3-phosphate dehydrogenase (GAPDH) was determined. The
primer sequences were as follows: ZEB1 forward, 5'-TGTCACCATGAAACCATTGC-3' and reverse, 5'-AGGTAAAGTG
CGCTTCCTCA-3'; ZEB2 forward, 5'-GGGCATTCAGTGA
CCTGACA-3' and reverse, 5'-GCATTGTTCCCATAGAGT
TC-3'; E-cadherin forward, 5'-TCCTCGGATTCTCTGCT
CTC-3' and reverse, 5'-CTCTGACCTTTTGCCAGGAG-3';
GAPDH forward, 5'-ATGGGGAAGGTGAAGGTCG-3' andreverse, 5'-GGGTCATTGATGGCAACAATATC-3'. The
experiments were performed 3 times, with the average levels
and standard deviations from the mean being obtained.
Correlations between average levels of miR-200c, ZEB1,
ZEB2 and E-cadherin in all cell lines were estimated by
Spearman's rank correlation using the SPSS software package(ver17.0; SPSS Japan Inc., Tokyo, Japan). The results wereconsidered significant if the P<0.05.
Western blot analysis. Cell lines were lysed in RIPA buffer.
After boiling with sodium dodecyl sulfate loading buffer, equalamounts (10 µg) of the proteins were electrophoresed on 10%
sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) andtransferred to Immobilon membranes (Millipore Inc., Bedford,
MA, USA) by semidry blotting. Five percent milk powder
dissolved in PBS buffer containing 0.1% Tween-20 was used toblock non-specific binding. Then, using standard techniques,
the membranes were probed with the following antibodies:anti-E-cadherin antibody (mouse monoclonal antibody tohuman E-cadherin; clone: HECD-1; dilution: 1:200; Takara,
Tokyo, Japan), anti-ZEB1 antibody (rabbit monoclonal antibody to ZEB1; clone: D80D3; dilution: 1:100; Cell Signaling
Technology, Danvers, MA, USA), and anti-β-actin antibody(mouse monoclonal antibody to β -actin; clone: C4; dilution: 1:1,000; Santa Cruz Biotechnology, Dallas, TX, USA).
Antibody-antigen complexes were detected using Immobilon
Western chemiluminescent horseradish peroxidase substrate(Millipore), and signals were recorded on a LAS-3000 mini
system (Fujifilm, Tokyo, Japan).
Human tissue specimens and tissue microarrays. Human tissue
specimens were obtained from 97 patients who underwent
gastric cancer surgery at Tokyo Medical University Hospital
between 2003 and 2010. The Tokyo Medical University
institutional review board approved the present study, andall patients provided written informed consent. The patient
cohort included 74 males and 23 females, ranging in age from40 to 92 years (average 66.3 years). Other clinicopathologicalcharacteristics are listed in Table I. The tumors were cut into
~5-mm sections after fixation in 10% formalin and embedded
in paraffin. From these paraffin blocks, tissue microarrays(TMAs) were constructed, each consisting of 12 specimensof 6-mm diameter on one slide. As previously described (29),-70-

ONCOLOGY REPORTS 39: 91-100, 2018

Table I. Clinical and histopathological features of 97 patients.
Characteristics	Data
Mean age (years)
Sex (male/female)
Lymph nodes metastasis (+/-)
Tumor diameter (mm)Depth (M/SM/MP/SS/SE/SI)
Vessel infiltration (+/-)
Histology of tubular formation (+/-)

66.3±11.9
74/23
33/64
47.8±28.2
25/26/8/25/9/4
50/47
67/30

M, mucosa; SM, submucosa; MP, muscularis propria; SS, subserosa;
SE, serosa exposed; SI, serosa infiltrating (neighboring organ ororgans involved).

TMAs allow the relocation of multiple tissue samples fromconventional histologic paraffin blocks so that tissues frommultiple patients can be analyzed on the same slide.
In situ hybridization. In situ hybridization (ISH) was
performed on TMAs containing 3-µm consecutive sectionsof formalin-fixed, paraffin-embedded (FFPE) samples.
Probes utilized were based on 5' digoxigenin (DIG)-labeled
ISH locked nucleic acid (LNA) technology (miRCURY-LNA
detection probe; Exiqon A/S, Vedbaek, Denmark). Slides
were prepared using the Ventana HX System BenchMark(Ventana Medical Systems, Inc., Tucson, AZ, USA). We
used the following 5' DIG-labeled LNA probes for ISH:
hsa-miR-200c, 5'-TCCATCATTACCCGGCAGTATTA-3';
and non-target negative control sequence, 5'-GTGTAACACGTCTATACGCCCA-3'.
Immunohistochemistry. Immunohistochemistry (IHC) was
performed on TMAs containing 4-µm consecutive sectionsof FFPE gastric tissue samples. After deparaffinization andrehydration, endogenous peroxidases were blocked by incubation in 0.3% hydrogen peroxide solution for 20 min. To
expose antigens, the sections were autoclaved in EDTA buffer(pH  9.0) for 10 min and cooled for 30 min. After rinsing
in 0.05 M Tris-buffered saline containing 0.1% Tween-20(pH 7.6), the sections were incubated with an affinity-purified
anti-ZEB1 antibody (clone: D80D3; dilution: 1:200; Cell
Signaling Technology) and anti-E-cadherin antibody (clone:
HECD-1; dilution: 1:1,000; Takara) for 3 h at RT. Samples
were then washed three times in PBS and incubated with
Dako secondary antibody for 15 min at RT. Horseradish
peroxidase-labeled polymer conjugated to a mixture of goatanti-mouse and anti-rabbit immunoglobulin antibodies (Code:K5007; prediluted; Dako, Glostrup, Denmark) was used as
the secondary antibody. 3,3'-Diaminobenzidine tetrachloride(DAB) was used for color development and sections werecounterstained with hematoxylin.
Evaluation of tissue images. ISH- or IHC-stained slides
were examined by light microscopy using an Olympus BX50
microscope. Three different fields (x200) in each tissue
were randomly selected. Digital images of ISH and IHC

93

were captured by the NY-D5000 super system (Microscope
Network; Nikon, Tokyo, Japan) and were printed by a truecolor printer (IPSiO SP C420; Ricoh, Tokyo, Japan). Positive
ISH results caused the cytoplasm to appear blue, whereas positive IHC results appeared brown. Nuclear immunostaining wasassessed for ZEB1, while cytomembranous immunostaining
was assessed for E-cadherin. Images were interpreted semiquantitatively by assessing the extent and intensity of staining
on the entirety of each tissue section present on the slides,
as previously described (30). Briefly, the total percentage of
positively-stained tumor cells was first determined. Then, thepercentage of weakly-, moderately- and strongly-stained cells
was determined, so that the sum of these categories equatedwith the overall percentage of positivity. A final staining score
was then calculated as the sum of 1 x percentage of weak, 2 x
percentage of moderate, and 3 x percentage of strong staining(maximum score=300).
Statistical analyses of data from human tissue studies.
Prior to statistical analyses, clinicopathlogical data as wellas tissue image results were dichotomized into two groupsof ‘high’ and ‘low’ levels, according to the median values.
Thus, the final score for staining of miR-200c was used todefine high-level expression (final score ≥201) and low-level
expression (final score <201). Likewise, the final score forstaining of ZEB1 was used to define high-level expression(final score ≥1) and low-level expression (final score <1), as
well as that of E-cadherin used to define high-level expression (final score ≥111) and low-level expression (final score<111). Tumor diameter was dichotomized as either highlevel (diameter ≥40 mm) or low-level (diameter <40 mm).
High-age (≥65 years old) and low-age (<65 years old)groups were established according to convention, as wereadvanced (depth ≥mp) and early (depth <mp) cancer groups.
Histologically, both a well/moderately differentiated tubular
adenocarcinoma group and a poorly differentiated adenocarcinoma group were established. Then, statistical analyses
including Chi-square test and multivariate analysis using
logistic regression models were performed using the SPSS
software package (ver17.0; SPSS). Results were consideredsignificant if the P<0.05.
Results
Quantitative analysis of miR-200c and target mRNA
expression in gastric carcinoma cell lines. We investigatedmiR-200c expression in 6 gastric carcinoma cell lines by
qRT-PCR analysis. As shown in Fig. 1A, the expression levelof miR-200c descended in the following order (from high tolow): Kato-III > MKN-45 > NU-GC-4 > H-111-TC > HGC-27> MKN-1. Of note, the latter 2 cell lines HGC-27 and MKN-1
expressed very low levels of miR-200c. We also investigatedthe expression of particular target mRNAs, namely ZEB1and ZEB2, along with E-cadherin, in the same cell lines. The
expression level of ZEB1 descended in the following order(from high to low): HGC-27 > MKN-1 > NU-GC-4 > H-111-TC> MKN-45 > Kato-III (Fig. 1B). Of note, the 2 cell lines inwhich miR-200c expression level was very low exhibited thegreatest expression level of ZEB1. Similarly, the expression
level of ZEB2 descended in the following order (from high to

-71-

94

Kurata et al: miR-200c in gastric cancer

Figure 1. The level of miR-200c and respective target mRNAs in gastric carcinoma cell lines. (A) Gastric carcinoma cell lines were arranged according to theexpression level of miR-200c. The expression levels of (B) ZEB1, (C) ZEB2 and (D) E-cadherin expression in gastric carcinoma cell lines arranged according
to the level of miR-200c expression.

low): HGC-27 > H-111-TC > NU-GC-4 > MKN-45 > MKN-1> Kato-III (Fig. 1C). Here, one of the 2 cell lines in which
miR-200c expression level was very low exhibited the greatest
expression level of ZEB2. On the other hand, the expression
level of E-cadherin descended in the following order (fromhigh to low): H-111-TC > MKN-45 > NU-GC-4 > Kato-III >MKN-1 > HGC-27 (Fig. 1D). Of note, the 2 cell lines in which
the expression level of miR-200c was very low exhibited thelowest expression level of E-cadherin. As shown in Table II,
an inverse correlation was observed between miR-200c and
ZEB1 (P<0.05; by Spearman's rank correlation).
ZEB1 and E-cadherin protein expression in gastric carcinoma cell lines. The expression of ZEB1 and E-cadherin
proteins was examined in the 6 human gastric carcinoma celllines mentioned thus far by western blot analysis. As shown in
Fig. 2, expression of ZEB1 was only detected in HGC-27 andMKN-1 cells, which exhibited the lowest expression level ofmiR-200c determined by qRT-PCR analysis. Conversely, theexpression of E-cadherin was not detected in these 2 cell lines;

Figure 2. Expression of ZEB1, E-cadherin and β -actin proteins in gastriccarcinoma cell lines, presented by western blot analysis performed on 10 µgof the cell extract.

-72-

ONCOLOGY REPORTS 39: 91-100, 201895

Table II. Correlations between the average expression levels of miR-200c, ZEB1, ZEB2 and E-cadherin in all cell lines estimatedby Spearman's rank correlation.
ZEB1-----------------------------------------------------------------Correlation coefficient
P-value

ZEB2--------------------------------------------------------------Correlation coefficient
P-valuemiR-200c-0.8860.019-0.4290.397
ZEB1			0.6000.208ZEB2					E-cadherin-----------------------------------------------------------------Correlation coefficient
P-value
0.429-0.600
0.029

0.397
0.208
0.957

Bold indicates P<0.05.

Figure 3. Six gastric carcinoma cell lines arranged according to expression the level of miR-200c. This order is compared with the origin (primary cancer ormetastatic cancer), differentiation state (tubular or poorly differentiated), and shape (round/intermediate/spindle) of the cell lines.

instead, E-cadherin expression was detected in NU-GC-4 andH-111-TC cell lines, although very weak expression was also
detected in Kato-III cells.
Interpretation of divergent expression levels of miR-200c,
ZEB1/2, and E-cadherin in different gastric carcinoma celllines. The results of qRT-PCR analyses and western blot
analysis were in agreement. Cell lines with a high expression
level of miR-200c exhibited low ZEB1/2 and high E-cadherin
expression, whereas those with a low expression level ofmiR‑200c exhibited high ZEB1/2 and low E-cadherin expression. Therefore, we investigated characteristics that illustrated
differences between the group of cell lines with high miR-200c
expression and those with low miR-200c expression. As shownin Fig. 3, we first compared the tumor origin (primary or metastatic tumor) of the cell lines. However, the tumor origin did
not clearly correspond to the levels of miR-200c expression, as
cell lines derived from metastatic tumors exhibited both low
and high miR-200c expression levels. Secondly, we compared

the histological differentiation state of the tumors from which
the cell lines were derived was compared. Although cell lines
from poorly differentiated tumors exhibited high miR-200c
expression, the results were inconclusive as one of the celllines with low miR-200c expression (HGC-27) was originallyfrom a poorly differentiated tumor. Thirdly, we revealed thatthe morphology of the cell lines was well correlated with theexpression level of miR-200c. Cell lines with high miR-200c
expression (Kato-III, MKN-45 and NU-GC-4) were roundshaped, those with low miR-200c expression (HGC-27 andMKN-1) were spindle-shaped, and those with intermediate
miR-200c expression (H-111-TC) exhibited a phenotype which
was a mid-way between round- and spindle-shaped (polygonalor short spindle-shaped).
miR-200c expression patterns in gastric adenocarcinoma andadjacent non-tumor tissues. In human tissue samples, positivemiR-200c expression, as determined by ISH, was denoted bycytoplasmic granular blue staining. Most of the normal tubules

-73-

96

Kurata et al: miR-200c in gastric cancer

Figure 5. Upregulation/downregulation of miR-200c affects cell morphology
and histology.

protein expression was denoted by nuclear staining. Overall
positivity of ZEB1 was relatively low in normal tubules andgastric carcinomas. ZEB1 was almost negative in tubular
adenocarcinomas; however, positive expression of ZEB1,
although generally weak, was sporadically observed in poorly
differentiated adenocarcinomas. The average staining score
for ZEB1 was 0.84±2.93 in tubular adenocarcinomas, while itwas 5.8±8.6 in poorly differentiated adenocarcinomas. Positive
E-cadherin protein expression was indicated by cytomembranous staining. Normal tubules and tubular adenocarcinomas
were strongly stained, while poorly differentiated adenocarcinomas were weakly stained (Fig. 4). The average staining score
for E-cadherin was 146±52 in tubular adenocarcinomas, while
it was 39±38.6 in poorly differentiated adenocarcinomas.
Figure 4. Representative photomicrographs including in situ hybridizationand immunohistochemistry. (A and B) Staining with H&E and staining for (Cand D) miR-200c, (E and F) ZEB1 and (G and H) E-cadherin in (A, C, E andG) tubular adenocarcinoma and (B, D, F and H) poorly differentiated adenocarcinoma. Original magnification, x200. (A) Left side are normal glands,
right side is carcinoma. (B) Left side is carcinoma, right side is normal
foveolar epithelium. (C) Cytoplasmic granular staining in tubular adenocarcinoma was stronger than that found in normal glands. (D) Cytoplasmic
granular staining in poorly differentiated adenocarcinoma was weaker thanthat observed in normal epithelium. (E) Nuclear immunostaining was negative in tubular adenocarcinoma. (F) Nuclear immunostaining was weak but
positive in poorly differentiated adenocarcinoma. (G) Cytomembranous
immunostaining in tubular adenocarcinoma was as strong as that found innormal glands. (H) Cytomembranous immunostaining in poorly differentiated adenocarcinoma was weaker than that observed in normal epithelium.

stained positive for miR-200c, whereas stromal tissue was notstained. As shown in Fig. 4, miR-200c expression patterns ingastric adenocarcinoma differed substantially between tubular
adenocarcinoma and poorly differentiated adenocarcinoma. In
tubular adenocarcinoma, miR-200c expression was observedwith an intensity level either stronger than or the same asnormal tubules. Conversely, in poorly differentiated adenocarcinoma, miR-200c expression was either not identified orobserved with weaker staining patterns than normal tubules.
The average staining score for miR-200c was 265±81.6 intubular adenocarcinomas, while it was 104±75.2 in poorly
differentiated adenocarcinomas.
ZEB1 and E-cadherin expression patterns in gastric adenocarcinoma and adjacent non-tumor tissues. Positive ZEB1Interpretation of statistical analyses of human tissue studies.
Use of the Chi-square test revealed a correlation between
miR-200c and E-cadherin expression levels (P<0.001) and an
inverse correlation between miR-200c and ZEB1 expression
levels (P<0.001) as well as between ZEB1 and E-cadherin
expression levels (P<0.001). We further compared theexpression levels of miR-200c, ZEB1 and E-cadherin withclinicopathological factors (Table III). High expression levelsof miR-200c were correlated with older age (≥65 years old)
(P= 0.002) and tubular histology (P<0.001). High expression
levels of ZEB1 were correlated with poorly differentiatedhistology (P<0.001) and advanced stage (P= 0.040). Highexpression levels of E-cadherin were correlated with older age(≥65 years old) (P=0.039) and tubular histology (P<0.001). In
the multivariate analysis (Table IV), among the clinicopathological factors significantly correlated with the expression
levels of miR-200c, ZEB1 and E-cadherin, there was a significant association between the expression level of ZEB1 andadvanced stage (P= 0.031), as well as a significant correlation
between the expression level of ZEB1 and histology (P<0.001).
Discussion
In the present study, we revealed inverse correlations between
miR-200c and ZEB1 in both cell lines and tissues, as well as
inverse correlations between ZEB1 and E-cadherin in tissues,in gastric carcinoma. These well known correlations which
are associated with EMT (8) have been demonstrated bothin vitro and in vivo in several cancers including non-small celllung cancer and pancreatic cancer (15,16) and recently also in-74-

ONCOLOGY REPORTS 39: 91-100, 201897

Table III. Correlations of the expression levels of miR-200c, ZEB1 and E-cadherin with the clinicopathological factors as determined by Chi-squared test.
miR-200c
ZEB1
E-cadherin--------------------------------------------------------------------------------------------------------------------------------------------------------------------	HighLow
P-value	HighLow
P-value	HighLow
P-value
Sex			
0.474			0.419			
Male
37
37		
18
56		
35
39
Female
9
14		
8
15		
12
11

Age (years old)			
0.002			0.063			
≥65
35
22		
11
46		
33
24
<65
11
29		
15
25		
14
26

0.812
0.039

LN metastasis			0.289			1.000			Positive
13
20		
9
24		
15
18
Negative
33
31		
17
47		
32
32

0.830

0.224			0.360			Tumor size (mm)			
≥40
19
28		
15
32		
20
27
<40
27
23		
11
39		
27
23

0.312

Vessel invasion			
0.157			0.259			Positive
20
30		
16
34		
25
25
Negative
26
21		
10
37		
22
25

0.840

Histology			<0.001			
<0.001			
<0.001Tubular
45
22		
9
58		
45
22
Poor differentiation
1
29		
17
13		
2
28

Stage			0.310			0.040			0.840Advanced
19
27		
17
29		
23
23
Early
27
24		
9
42		
24
2

LN, lymph node. Bold indicates P<0.05.

Table IV. Multivariate analyses of correlations between the clinicopathological factors and positive expression of miR-200c,
ZEB1 and E-cadherin.

Clinicopathological factors

miR-200c-----------------------------------------------OR (95% CI)
P-value

ZEB1-----------------------------------------------------------OR (95% CI)
P-valueE-cadherin-----------------------------------------------OR (95% CI)
P-valueSexmale vs. female

1.822
0.359
(0.505-6.568)		

0.294
0.082
(0.074-1.170)		

0.438(0.124-1.553)

0.201

LN metastasis
positive vs. negative

1.038
0.959
(0.249-4.320)		

0.347
0.125
(0.090-1.342)		

0.458(0.111-1.888)

0.280

Age (years old)
≥65 vs. <65

Histology
tubular vs. poor differentiation
Tumor size (mm)≥40 vs. <40Stage
advanced vs. early

Vessel invasion
positive vs. negative

2.742
0.071
(0.916-8.202)		

0.505
0.261
(0.153-1.662)		

1.085(0.370-3.184)

0.882

0.023
<0.001(0.003-0.182)		

7.556<0.001(2.471-23.109)		

0.025(0.005-0.137)<0.0011.073
0.923(0.258-4.473)		

5.522
0.031
(1.166-26.148)		

2.197(0.558-8.653)

0.260

1.087
0.889
(0.337-3.509)		

0.484
0.354
(0.105-2.240)		

0.809
0.730
(0.242-2.703)		

0.998
0.998
(0.227-4.378)		

LN, lymph node; OR, odds ratio; CI, confidence interval. Bold indicates P<0.05.

-75-

0.533
(0.172-1.652)

0.276

2.182
(0.489-9.732)

0.306

98

Kurata et al: miR-200c in gastric cancer

gastric cancer (21,31). However, this study is the first to havedemonstrated that this phenomenon was associated with themorphology of cell lines and histological differentiation, thelatter as visualized by ISH, in gastric carcinoma.
qRT-PCR analysis of 6 cell lines in the present study
revealed that the expression level of miR-200c descended
in the following order: Kato-III > MKN-45 > NU-GC-4> H-111-TC > HGC-27 > MKN-1. The expression level of
ZEB1 or ZEB2 corresponded to the reverse order, and that ofE-cadherin corresponded to this order. Indeed, significantly
inverse correlations between miR-200c and ZEB1 wereobtained by Spearman's rank correlation. Our results are inline with a previous study in which E-cadherin gene expression was present in MKN-45 cells but absent in HGC-27
cells (32), along with a recent study which revealed that
Kato-III, MKN-45 and NU-GC-4 cells had high expressionof E-cadherin and low expression of ZEB1, while MKN-1
cells had low expression of E-cadherin and high expression of
ZEB1 (33). However, these authors did not mention any differences in cell morphology in their studies. In the present study,
western blot analysis coincided with qRT-PCR data, albeit
E-cadherin expression in Kato-III and MKN-45 cells wasalmost absent contrary to positive expression as determined
by qRT-PCR analysis. This result was compatible with anotherstudy that revealed that E-cadherin protein expression wasundetectable by western blot analysis, while it was detected
at the DNA and mRNA level by southern and northern blot
analysis, respectively, in Kato-III and MKN-45 cells (4). They
mentioned that the possibility of post-translational modification or minor abnormalities in antibody-recognition sites may
be involved in this discrepancy.
We searched for key differences in the characteristics
between cell lines displaying divergent expression levels of
miR-200c which also corresponded to differences in theexpression levels of ZEB1/2 and E-cadherin. Although histological differences were conspicuous in the tissue studies
mentioned below, cell line groupings based on the expression of miR-200c and associated targets did not correspondto histological differentiation of the derived tumors. Other
authors have also demonstrated that E-cadherin was ‘unexpectedly’ detected in cell lines that originated from poorly
differentiated gastric carcinomas (26). This may be due tothe fact that cell lines have generally been established fromtumors exhibiting heterogenous histology. Indeed, it has beenreported that histological difference in original tumors did not
influence the growth rate of various cell lines in gastric carcinomas (25). Instead, we established that cell lines with highmiR-200c expression (Kato-III, MKN-45 and NU-GC-4),
low miR-200c expression (HGC-27 and MKN-1), and intermediate miR-200c expression (H-111-TC) exhibited round,spindle, and intermediate, respectively, cell morphologies.
Although studies concerning cell morphology in association
with expression of miR-200 family members are scant, one
research group demonstrated that expression of miR-200c
was associated with round cell morphology whereas that of
miR-200a was associated with spindle cell morphology inmelanoma cell lines (34). The phenomenon where E-cadherin(+) cells are round and E-cadherin (-) cells are spindle in shapehas been reported in esophageal cancer cells and, recently,
in colon cancer cells (35,36). Furthermore, it was formerly

reported that cell morphology of E-cadherin-deficient celllines was generally altered from the fibroblastic cell type tothe epithelial cell type by transfection with complementary
DNA that codes for E-cadherin (37). Therefore, the influenceof miR-200c and E-cadherin on cell morphology may be auniversal phenomenon.
In the tissue samples, miR-200c expression, as visualized
by ISH, was strongly expressed in tubular adenocarcinomas but
weakly expressed in poorly differentiated adenocarcinomas.
This result was in agreement with a recent study in which low
expression levels of each member of the miR-200 family wereassociated with histological grade in gastric carcinoma tissues,
as assessed by qRT-PCR analysis (31). Analysis of non-small
cell lung cancer tissue also revealed that low miR-200c
expression levels were associated with poorly differentiatedhistology (15). Assessment of endometrial carcinosarcoma
tissues revealed that members of the miR-200 family were
downregulated in the mesenchymal part of the tumor (38).
Therefore, it is likely that the expression of miR-200c is associated with epithelial histology of tumors in general.
IHC analysis revealed that tubular adenocarcinomas were
negative for ZEB1 and positive for E-cadherin, while poorly
differentiated adenocarcinomas were sporadically positive for
ZEB1 and negative for E-cadherin. Notably, it has long been
pointed out that the level of E-cadherin is significantly lower
in poorly differentiated adenocarcinomas compared to differentiated adnocarcinomas and non-neoplastic mucosa of thestomach (4,32). The reason for this phenomenon was proposed
to be inactivation of the E-cadherin gene due to either a classic
two-hit mechanism or hypermethylation (32). In the present
study, we add EMT mediated by miR-200c as a possible causal
mechanism, since miR-200c and ZEB1 expression levels, aswell as ZEB1 and E-cadherin expression levels, were inversely
correlated (P<0.001). Other authors have also demonstrated
an inverse correlation between ZEB1 and E-cadherin mRNA
expression in gastric cancer tissue (33). Association of poorly
differentiated histology with high expression of ZEB1 and low
expression of E-cadherin, as determined by IHC, has also been
demonstrated in endometrioid carcinoma and non-small celllung cancers (6,7).
In addition to histology, older age was also correlated withhigh expression levels of miR-200c and E-cadherin as assessedby Chi-squared test. However, in terms of multivariate analyses, age was not correlated with miR-200c and E-cadherin
expression, although histology remained correlated. These
results coincide with a well-known fact that poorly differentiated scirrhous gastric carcinoma is more frequent in younger
patients (2,3). Conversely, a high expression level of ZEB1 wascorrelated with advanced stage by multivariate analyses. As
other authors have also reported, gastric cancer patients withhigh ZEB1 mRNA expression displayed significantly poorer
survival than those with low expression (33), ZEB1 may be
independently associated with advanced stage.
Concerning miR-200c expression in tumors in general,a meta-analysis revealed that low expression of miR-200c inprimary cancer tissue from early-stage patients was significantly associated with poor survival (39). Association of low
miR-200c expression with high propensity to metastasis, alongwith highly invasive/aggressive ability, has been demonstrated
in several cancers including non-small cell lung cancer and-76-

ONCOLOGY REPORTS 39: 91-100, 2018

pancreatic cancer (15,16). Recent studies have also demonstrated that migration/invasion abilities of gastric cancer cells
were promoted by decreased expression of miR-200c, while
they were inhibited by ZEB1 siRNA (21,33). In addition, thepresent study demonstrated an association of miR-200c withcell morphology and histological differentiation of the tumor.
Since it has been proposed that the ability of miR-200c toregulate morphological plasticity may be important in relation to dissemination capacity in melanoma (34), we postulatethat downregulation of miR-200c primarily regulates cellmorphology by downregulation of E-cadherin through upregulation of ZEB1/2, with this morphological change causing
poorly differentiated histology (Fig. 5), producing enhanced
migration/invasion abilities, and finally leading to scirrhous
stromal reaction and poor prognosis (2,3).
In summary, using cell culture models and histologicalassessment of gastric carcinoma, we demonstrated an inverse
correlation in the expression level between miR-200c and
ZEB1 in vitro and in vivo, as well as between ZEB1 andE-cadherin in vivo. Low miR-200c expression was associatedwith spindle cell morphology in vitro and poorly differentiated histology in vivo; thus, this factor may be involved in the
carcinogenesis of scirrhous gastric carcinoma.
Acknowledgements
The present study was supported by Grants-in-Aid for scientific research (C) (grant no. 24590431).
References
1.	Jemal A, Bray F, Center MM, Ferlay J, Ward E and Forman D:
Global cancer statistics. CA Cancer J Clin 61: 69-90, 2011.
2.	Duarte I and Llanos O: Patterns of metastases in intestinal anddiffuse types of carcinoma of the stomach. Hum Pathol 12:237-242, 1981.
3. Yokota T, Teshima S, Saito T, Kikuchi S, Kunii Y and Yamauchi H:
Borrmann's type IV gastric cancer: Clinicopathologic analysis.
Can J Surg 42: 371-376, 1999.
4. Yasui W, Kuniyasu H, Akama Y, Kitahara K, Nagafuchi A,
Ishihara S, Tsukita S and Tahara E: Expression of e-cadherin,alpha-catenins and Beta-catenins in human gastric carcinomas- correlation with histology and tumor progression. Oncol Rep 2:
111-117, 1995.
5.	Hippo Y, Yashiro M, Ishii M, Taniguchi H, Tsutsumi S,
Hirakawa K, Kodama T and Aburatani H: Differential gene
expression profiles of scirrhous gastric cancer cells with highmetastatic potential to peritoneum or lymph nodes. Cancer Res
61: 889-895, 2001.
6. Matsubara D, Kishaba Y, Yoshimoto T, Sakuma Y, Sakatani T,
Tamura T, Endo S, Sugiyama Y, Murakami Y and Niki T:
Immunohistochemical analysis of the expression of E-cadherin
and ZEB1 in non-small cell lung cancer. Pathol Int 64: 560-568,
2014.
7. Romero-Pérez L, López-García MÁ, Díaz-Martín J, Biscuola M,
Castilla MÁ, Tafe LJ, Garg K, Oliva E, Matias-Guiu X,
Soslow RA, et al: ZEB1 overexpression associated with
E-cadherin and microRNA-200 downregulation is characteristicof undifferentiated endometrial carcinoma. Mod Pathol 26:1514‑1524, 2013.
8. Burk U, Schubert J, Wellner U, Schmalhofer O, Vincan E,
Spaderna S and Brabletz T: A reciprocal repression between
ZEB1 and members of the miR-200 family promotes EMT andinvasion in cancer cells. EMBO Rep 9: 582-589, 2008.
9. Poliseno L, Salmena L, Riccardi L, Fornari A, Song MS,
Hobbs RM, Sportoletti P, Varmeh S, Egia A, Fedele G, et al:
Identification of the miR-106b~25 microRNA cluster as a protooncogenic PTEN-targeting intron that cooperates with its hostgene MCM7 in transformation. Sci Signal 3: ra29, 2010.

99

10. Sethi S, Kong D, Land S, Dyson G, Sakr WA and Sarkar FH:
Comprehensive molecular oncogenomic profiling and miRNA
analysis of prostate cancer. Am J Transl Res 5: 200-211, 2013.
11.	Hassan O, Ahmad A, Sethi S and Sarkar FH: Recent updates on
the role of microRNAs in prostate cancer. J Hematol Oncol 5: 9,
2012.
12. Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, Aravin A,
Pfeffer S, Rice A, Kamphorst AO, Landthaler M, et al: Amammalian microRNA expression atlas based on small RNA
library sequencing. Cell 129: 1401-1414, 2007.
13. Ventura A and Jacks T: MicroRNAs and cancer: Short RNAs goa long way. Cell 136: 586-591, 2009.
14. Bojmar L, Karlsson E, Ellegård S, Olsson H, Björnsson B,
Hallböök O, Larsson M, Stål O and Sandström P: The role ofmicroRNA-200 in progression of human colorectal and breast
cancer. PLoS One 8: e84815, 2013.
15. Ceppi P, Mudduluru G, Kumarswamy R, Rapa I, Scagliotti GV,
Papotti M and Allgayer H: Loss of miR-200c expression
induces an aggressive, invasive, and chemoresistant phenotype
in non-small cell lung cancer. Mol Cancer Res 8: 1207-1216,
2010.
16. Yu J, Ohuchida K, Mizumoto K, Sato N, Kayashima T, Fujita H,
Nakata K and Tanaka M: MicroRNA, hsa-miR-200c, is anindependent prognostic factor in pancreatic cancer and its
upregulation inhibits pancreatic cancer invasion but increases
cell proliferation. Mol Cancer 9: 169, 2010.
17. Marchini S, Cavalieri D, Fruscio R, Calura E, Garavaglia D,
Fuso Nerini I, Mangioni C, Cattoretti G, Clivio L, Beltrame L,
et al: Association between miR-200c and the survival of patientswith stage I epithelial ovarian cancer: A retrospective study of
two independent tumour tissue collections. Lancet Oncol 12:273-285, 2011.
18. Kurashige J, Mima K, Sawada G, Takahashi Y, Eguchi H,
Sugimachi K, Mori M, Yanagihara K, Yashiro M, Hirakawa K,
et al: Epigenetic modulation and repression of miR-200b by
cancer-associated fibroblasts contribute to cancer invasion andperitoneal dissemination in gastric cancer. Carcinogenesis 36:
133-141, 2015.
19. Shinozaki A, Sakatani T, Ushiku T, Hino R, Isogai M, Ishikawa S,
Uozaki H, Takada K and Fukayama M: Downregulation of
microRNA-200 in EBV-associated gastric carcinoma. Cancer
Res 70: 4719-4727, 2010.
20. Song F, Yang D, Liu B, Guo Y, Zheng H, Li L, Wang T, Yu J,
Zhao Y, Niu R, et al: Integrated microRNA network analyses
identify a poor-prognosis subtype of gastric cancer characterized
by the miR-200 family. Clin Cancer Res 20: 878-889, 2014.
21. Zhou X, Wang Y, Shan B, Han J, Zhu H, Lv Y, Fan X, Sang M,
Liu XD and Liu W: The downregulation of miR-200c/141
promotes ZEB1/2 expression and gastric cancer progression.
Med Oncol 32: 428, 2015.
22. Akagi T and Kimoto T: Human cell line (HGC-27) derived fromthe metastatic lymph node of gastric cancer. Acta Med Okayama
30: 215-219, 1976.
23. Akiyama S, Amo H, Watanabe T, Matsuyama M, Sakamoto J,
Imaizumi M, Ichihashi H, Kondo T and Takagi H: Characteristicsof three human gastric cancer cell lines, NU-GC-2, NU-GC-3
and NU-GC-4. Jpn J Surg 18: 438-446, 1988.
24. Kakinuma N, Sato M, Yamada T, Kohu K, Nakajima M,
Akiyama T, Ohwada S and Shibanaka Y: Cloning of novel
LERGU mRNAs in GPR30 3' untranslated region and detectionof 2 bp-deletion polymorphism in gastric cancer. Cancer Sci 96:
191-196, 2005.
25. Motoyama T, Hojo H and Watanabe H: Comparison of seven celllines derived from human gastric carcinomas. Acta Pathol Jpn
36: 65-83, 1986.
26. Konno-Shimizu M, Yamamichi N, Inada K, Kageyama-Yahara N,
Shiogama K, Takahashi Y, Asada-Hirayama I, YamamichiNishina M, Nakayama C, Ono S, et al: Cathepsin E is a marker ofgastric differentiation and signet-ring cell carcinoma of stomach:
A novel suggestion on gastric tumorigenesis. PLoS One 8:
e56766, 2013.
27. Yamada Y, Yoshida T, Hayashi K, Sekiya T, Yokota J,
Hirohashi S, Nakatani K, Nakano H, Sugimura T and Terada M:
p53 gene mutations in gastric cancer metastases and in gastriccancer cell lines derived from metastases. Cancer Res 51: 58005805, 1991.
28. Tanaka M, Oikawa K, Takanashi M, Kudo M, Ohyashiki J,
Ohyashiki K and Kuroda M: Down-regulation of miR-92 in
human plasma is a novel marker for acute leukemia patients.
PLoS One 4: e5532, 2009.

-77-

100

Kurata et al: miR-200c in gastric cancer

29. Wu W, Takanashi M, Borjigin N, Ohno SI, Fujita K, Hoshino S,
Osaka Y, Tsuchida A and Kuroda M: MicroRNA-18a modulates
STAT3 activity through negative regulation of PIAS3 during
gastric adenocarcinogenesis. Br J Cancer 108: 653-661, 2013.
30. Acs G, Zhang PJ, McGrath CM, Acs P, McBroom J, Mohyeldin A,
Liu S, Lu H and Verma A: Hypoxia-inducible erythropoietin
signaling in squamous dysplasia and squamous cell carcinoma of
the uterine cervix and its potential role in cervical carcinogenesis
and tumor progression. Am J Pathol 162: 1789-1806, 2003.
31. Chang L, Guo F, Huo B, Lv Y, Wang Y and Liu W: Expression
and clinical significance of the microRNA-200 family in gastriccancer. Oncol Lett 9: 2317-2324, 2015.
32. Tamura G, Sakata K, Nishizuka S, Maesawa C, Suzuki Y,
Iwaya T, Terashima M, Saito K and Satodate R: Inactivation of
the E-cadherin gene in primary gastric carcinomas and gastriccarcinoma cell lines. Jpn J Cancer Res 87: 1153-1159, 1996.
33. Murai T, Yamada S, Fuchs BC, Fujii T, Nakayama G, Sugimoto H,
Koike M, Fujiwara M, Tanabe KK and Kodera Y: Epithelial-tomesenchymal transition predicts prognosis in clinical gastriccancer. J Surg Oncol 109: 684-689, 2014.
34. Elson-Schwab I, Lorentzen A and Marshall CJ: MicroRNA-200
family members differentially regulate morphological plasticity
and mode of melanoma cell invasion. PLoS One 5: e13176, 2010.

35.	Doki Y, Shiozaki H, Tahara H, Inoue M, Oka H, Iihara K,
Kadowaki T, Takeichi M and Mori T: Correlation between
E-cadherin expression and invasiveness in vitro in a humanesophageal cancer cell line. Cancer Res 53: 3421-3426, 1993.
36.	Ji M, Lee EJ, Kim KB, Kim Y, Sung R, Lee SJ, Kim DS andPark SM: HDAC inhibitors induce epithelial-mesenchymal transition in colon carcinoma cells. Oncol Rep 33: 2299-2308, 2015.
37. Takeichi M: The cadherins: Cell-cell adhesion molecules
controlling animal morphogenesis. Development 102: 639-655,
1988.
38. Castilla MÁ, Moreno-Bueno G, Romero-Pérez L, Van De
Vijver K, Biscuola M, López-García MÁ, Prat J, Matías-Guiu X,
Cano A, Oliva E, et al: Micro-RNA signature of the epithelialmesenchymal transition in endometrial carcinosarcoma. J Pathol
223: 72-80, 2011.
39. Shao Y, Geng Y, Gu W, Huang J, Pei H and Jiang J: Prognostic
role of tissue and circulating microRNA-200c in malignant
tumors: A systematic review and meta-analysis. Cell Physiol
Biochem 35: 1188-1200, 2015.

-78-

This work is licensed under a Creative Commons
Attribution-NonCommercial-NoDerivatives 4.0
International (CC BY-NC-ND 4.0) License.

Published OnlineFirst November 1, 2017; DOI: 10.1158/0008-5472.CAN-17-0960

CancerResearch

Tumor Biology and Immunology

Interleukin-27 Exerts Its Antitumor Effects by
Promoting Differentiation of Hematopoietic
Stem Cells to M1 Macrophages
Yukino Chiba, Izuru Mizoguchi, Junichi Furusawa, Hideaki Hasegawa,
Mio Ohashi, Mingli Xu, Toshiyuki Owaki, and Takayuki Yoshimoto

Abstract
The interleukin IL27 promotes expansion and differentiation ofhematopoietic stem cells into myeloid progenitor cells. Manytumor-inﬁltrating myeloid cells exert immunosuppressive effects,
but we hypothesized that the myeloid cells induced by IL27 would
have antitumor activity. In this study, we corroborated this hypothesis as investigated in two distinct mouse transplantable tumor
models. Malignant mouse cells engineered to express IL27 exhibited reduced tumor growth in vivo. Correlated with this effect was asigniﬁcant increase in the number of tumor-inﬁltrating CD11bþ
myeloid cells exhibiting a reduced immunosuppressive activity.
Notably, these CD11bþ cells were characterized by an activated M1
macrophage phenotype, on the basis of increased expression of
inducible nitric oxide synthase and other M1 biomarkers. In vivo
depletion of these cells by administering anti–Gr-1 eradicated theantitumor effects of IL27. When admixed with parental tumors,
CD11bþ cells inhibited tumor growth and directly killed the tumor
in a nitric oxide-dependent manner. Mechanistically, IL27 expanded LineageSca-1þc-Kitþ cells in bone marrow. Transplant experiments in Ly5.1/5.2 congenic mice revealed that IL27 directly acted
on these cells and promoted their differentiation into M1 macrophages, which mobilized into tumors. Overall, our results illustrated how IL27 exerts antitumor activity by enhancing the generation of myeloid progenitor cells that can differentiate into antitumorigenic M1 macrophages.
Signiﬁcance: These ﬁndings show how the interleukin IL27
exerts potent antitumor activity by enhancing the generation of
myeloid progenitor cells that can differentiate into antitumorigenic M1 macrophages. Cancer Res; 78(1); 182–94. 2017 AACR.

Introduction

M2 macrophage phenotype is characterized by immunoregulatory functions leading to the promotion of tissue remodelingand tumor progression.
IL27 is an IL12 family cytokine that consists of two distinct
subunits, Epstein-Barr virus-induced gene 3 and p28, and its
receptor (R) is composed of IL27Ra (WSX-1/TCCR) and glycoprotein 130 (4, 5). It is one of the pleiotropic cytokines withboth pro- and anti-inﬂammatory properties acting on various
cell types. IL27 promotes the early induction of Th1 differentiation and generation of cytotoxic lymphocytes, but it inhibits
the differentiation to Th2 and Th17 cells and suppresses the
production of pro-inﬂammatory cytokines (4, 5). Because weﬁrst demonstrated the antitumor efﬁcacy of IL27 in 2004 using
a transplanted mouse tumor genetically engineered to secrete
IL27 as a preclinical tumor model (6), accumulating evidence
suggests that IL27 has potent antitumor activity through multiple mechanisms, depending on the characteristics of individual tumors (7, 8). Moreover, we recently demonstrated that
IL27 can promote the expansion and differentiation of hematopoietic stem cells (HSC) into myeloid progenitor cells and thatit plays a critical role in the control of parasite infection during
emergency myelopoiesis (9, 10).
IL27 was also demonstrated to augment the productionof immunosuppressive cytokine IL10 and enhance expressionof the immune checkpoint molecules such as programmed
death-1 ligand 1 (PD-L1) and T-cell immunoglobulin andmucin domain-3 (TIM-3) in lymphocytes and macrophages(11–15). In addition, IL27 was recently shown to upregulate
another immunosuppressive molecule, CD39, together with
PD-L1 and IL10 in macrophages, resulting in acquisition of

Although immunotherapeutic approaches against cancer are
promising new treatments, their effectiveness can be further
improved by overcoming the immunosuppression induced bytumors. These immunosuppressive mechanisms include
immune checkpoint molecules, regulatory T cells, and immunosuppressive myeloid cells. Generally, tumor-bearing hosts
and cancer patients have increased inﬁltration of immunosuppressive myeloid cells, including tumor-associated macrophages, M2 macrophages, and myeloid-derived suppressor
cells (MDSC; refs. 1, 2). Macrophages are characterized byhigh plasticity and diversity with two major phenotypes,
namely M1 (classically activated) and M2 (alternatively activated) macrophages (3). The M1 macrophage phenotype ischaracterized by the production of pro-inﬂammatory cytokines, reactive oxygen species, and nitric oxide (NO) and thepromotion of helper T (Th) 1 immune responses, resulting instrong microbicidal and tumoricidal activity. In contrast, theDepartment of Immunoregulation, Institute of Medical Science, Tokyo Medical
University, Shinjuku-ku, Tokyo, Japan.
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Author: Takayuki Yoshimoto, Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku,
Shinjuku-ku, Tokyo 160-8402, Japan. Phone: 81-3-3351-6141; Fax: 81-3-33516250; E-mail: yoshimot@tokyo-med.ac.jp
doi: 10.1158/0008-5472.CAN-17-0960
2017 American Association for Cancer Research.

182

Cancer Res; 78(1) January 1, 2018

Downloaded from cancerres.aacrjournals.org on April 28, 2018. © 2018 American Association for Cancer Research.

-79-

Published OnlineFirst November 1, 2017; DOI: 10.1158/0008-5472.CAN-17-0960

Differentiation to Antitumorigenic M1 Macrophages by IL27

immunoregulatory properties by tumor-associated macrophages in various cancer patients (16, 17). These results indicate that macrophages may play an inhibitory role in theexertion of antitumor activity by IL27. However, myeloablation by treosulfan, a chemotherapy drug used to induce a severeor complete depletion of bone marrow (BM) cells including
cells other than macrophages, was recently shown to mostly
abolish the antitumor effects of IL27 in mice bearing nonsmall cell lung cancer (18). Moreover, IL27 inhibited thedevelopment of M2 macrophages from the human monocytic
cell line U937 differentiated by phorbol 12–myristate 13–
acetate and subsequently polarized by IL4 (19). Therefore, ourunderstanding of the role of macrophages in the exertion of
antitumor activity by IL27 remains incomplete.
Although the mouse transplantable tumor model geneticallyengineered to secrete IL27 signiﬁcantly decreased tumor growth(20, 21), we initially noticed that the number of tumor-inﬁltrating cells, including CD11bþ myeloid cells greatly increased.
Therefore, we wondered whether these myeloid cells would be
protumorigenic or antitumorigenic. In the present study, toclarify this, we used two distinct mouse transplantable tumor
models, B16F10 melanoma and MC38 colon adenocarcinoma.
Our results clearly revealed a novel mechanism whereby IL27
exerts antitumor activity by promoting expansion and differentiation of HSCs into antitumorigenic M1 macrophages intumor-bearing mice.

Materials and Methods
Cell lines and mice
Mouse melanoma cell line B16F10 and colon carcinoma cellline MC38 were kindly provided by Dr. Nariuchi (University ofTokyo, Tokyo, Japan) in 2007 and by Dr. Kawakami (Keio
University, Tokyo, Japan) in 2014, respectively. These cell lines
were expanded in our laboratory, frozen immediately, andstored in liquid nitrogen to ensure that cells used for experiments were passaged for fewer than 6 months. Early passage
cells were used for all experiments. Cell line authentication was
not routinely performed. But cell lines were tested routinely for
identity by appearance, staining with Hoechst 33258 (Dojindo,
Kumamoto, Japan), or growth curve analysis in 3H-thymidine
incorporation assay, and validated to be mycoplasma free.
These cell lines were cultured at 37 C under 5% CO2/95% air
in RPMI-1640 (Sigma) containing 10% FCS and 100 mg/mL
kanamycin (Meiji Seika, Tokyo, Japan). BM cells and LineageSca-1þc-Kitþ (LSK) cells were cultured in the same RPMI1640 medium additionally supplemented with 50 mmol/L
2 mercaptoethanol (Gibco). OT-I mice, C57BL/6 (CD45.2) mice,and C57BL/6 (CD45.1) mice were maintained in speciﬁc pathogen-free conditions. All experiments were approved by the Animal
Care and Use Committee of Tokyo Medical University and were
performed in accordance with our institutional guidelines.
Antibodies and reagents
Monoclonal antibodies (mAb) for mouse c-Kit (2B8), Sca-1(D7), CD3e (145-2C11), CD8a (53-6.7), CD19 (6D5), CD49b(DX5), Gr-1 (RB6-8C5), TER119/erythroid cell (TER-119),CD11c (N418), CD11b (M1/70), F4/80 (BM8), NK1.1(PK136), B220 (RA3-6B2), FceRIa (MAR-1), macrophage colony-stimulating factor receptor (M-CSFR; AFS98), Flt3
(A2F10), CD16/32 (2.4G2), CD150 (TC15-12F12.2), MHC

class II I-A/I-E (M5/114.15.2), Ly6G (1A8), Ly6C (HK1.4),
CD45.1 (A20), and CD45.2 (104) were purchased from BioLegend. mAbs against mouse CD4 (GK1.5), CD34 (RAM34),
and CD86 (GL1) were purchased from eBioscience. APC-Cy7–
conjugated streptavidin, PerCP/Cy5.5-conjugated streptavidin,
and Brilliant Violet 510-conjugated streptavidin were purchased from BioLegend and used to reveal staining with biotinylated Abs. Mouse recombinant IL27 was prepared as atagged single-chain fusion protein by ﬂexibly linking EBI3 to
p28 using HEK293-F cells (Life Technologies), as described
previously (22). Mouse recombinant stem cell factor (SCF)
and IL3 were purchased from PeproTech. Depletion mAb to
Gr-1 (RB6-8C5) was obtained from BioXCell. NG-monomethyl
L-arginine (L-NMMA) was purchased from Sigma-Aldrich.
Preparation of transfectants
B16F10 and MC38 tumor cells were transfected with theexpression vector of a FLAG-tagged single-chain fusion proteinof IL27–expression vector in p3xFLAG-CMV-14 vector (SigmaAldrich; ref. 6) or empty vector as control using Fugene 6 (Roche)and selected with Geneticin (G418). Resultant respective transfectants were designated as B16F10-IL27 or MC38-IL27 andB16F10-Vector or MC38-Vector, respectively.
Transplantable mouse tumor models
Tumor cells (1  106 cells/mouse) of B16F10 or MC38 were
injected subcutaneously into the right ﬂank of mice (n ¼ 3–5).
Tumor growth was monitored twice a week with an electronic
caliper and expressed as volume (mm3) by calculating using thefollowing volume equation: 0.5(ab2), where a is the long diameterand b is the short diameter.
Flow cytometry
On days 13 to 22 after tumor implantation, tumor was harvested from mice (n ¼ 3–5), minced into small pieces, and treatedwith collagenase. Subsequently, tumor-inﬁltrating mononuclear
cells were puriﬁed from it by Percoll (GE Healthcare) density
gradient centrifugation. Then, ﬂow cytometry was performed on a
FACS Canto II (BD Biosciences), and data were analyzed using
FlowJo Software (Tree Star). Cell number was counted by using
ﬂow cytometry. The following cell surface markers were used to
identify M1 and M2 macrophages (Supplementary Fig. S1): M1
macrophages, Class IIhighCD86þCD45.2þCD11bþF4/80þ; M2
macrophages, Class IIlowCD86þCD45.2þCD11bþF4/80þ (1, 23).
Quantitative real-time RT-PCR
Total RNA was prepared from the tumor-inﬁltrating mononuclear cells of mice (n ¼ 4) using an RNeasy Mini Kit (Qiagen), andcDNA was prepared using oligo(dT) primer and SuperScript III RT(Thermo Fischer Scientiﬁc). Real-time quantitative PCR was performed using SYBR Premix Ex Taq II and a Thermal Cycler Dicereal-time system according to the manufacturer's instructions(Takara). Glyceraldehyde-3-phosphate (GAPDH) was used as ahousekeeping gene to normalize mRNA. Relative expression ofreal-time PCR products was determined by using the DDCt method to compare target gene and GAPDH mRNA expression. Primers
used in this study are listed in Supplemental Table S1.
Measurement of immunosuppressive activity
On days 13 to 20 after tumor implantation, tumor was harvested from mice (n ¼ 3 or 4), minced into small pieces, andwww.aacrjournals.org

Cancer Res; 78(1) January 1, 2018

Downloaded from cancerres.aacrjournals.org on April 28, 2018. © 2018 American Association for Cancer Research.

-80-

183

Published OnlineFirst November 1, 2017; DOI: 10.1158/0008-5472.CAN-17-0960

Chiba et al.

treated with collagenase; tumor-inﬁltrating mononuclear cells
were puriﬁed from it by using Percoll density gradient centrifugation. Tumor-inﬁltrating myeloid cells were further isolated by
AutoMACS Pro after staining with anti-CD11b magnetic beads(Miltenyi Biotec). Immunosuppressive activity was then assessedby the ability to inhibit ovalbumin (OVA)-speciﬁc CD8þ T-cellproliferation. Spleen cells (5  105 cells/200 mL) obtained from
OT-I T-cell receptor transgenic mice were stimulated with OVA(0.3 mg/mL) in the presence of increasing cell numbers (1, 3, and10  104 cells) of CD11bþ myeloid cells for 48 h and pulsed with3
H-thymidine for last 8 hours, and the incorporation of 3Hthymidine was measured in triplicate.
Depletion experiment
After tumor (2  105 cells/mouse) implantation, 0.3 mg ofanti–Gr-1 mAb (RB6-8C5) or control rat IgG was intraperitoneally administered to mice (n ¼ 5) three times a week to deplete
myeloid cells. Tumor growth was monitored twice a week. The
depletion level was conﬁrmed by analyzing blood samples
obtained from the tail vein.
Admixture experimentCD11bþ myeloid cells (1  106 cells) were puriﬁed from tumorof mice (n ¼ 4–6) on days 13 to 22 after tumor implantation. The
cells were admixed with parental tumor (2  105 cells) in 200 mL
PBS and then immediately injected into the right ﬂank of mice.
Tumor growth was monitored twice a week.
Killing assay
CD11bþ myeloid cells were puriﬁed from tumor of mice (n ¼4 or 5) on days 13 to 22 after tumor implantation. Parental
tumor cells (1  106 cells/50 mL of 50% fetal calf serum/RPMI1640) were labeled by incubating in 100 mCi 51Cr for 45 min
at 37 C and then washed three times. Labeled target cells(1  104 cells/200 mL) were incubated with puriﬁed CD11bþ
cells (1  105 cells) in a 96-well U-bottomed plate at 37 C for
4 hours. Supernatants then were analyzed in a scintillation
counter. The percentage of lysis was determined in triplicate
as (experimental count  spontaneous count)  100/(maximal
count  spontaneous count). Results were expressed as thepercentage of speciﬁc lysis. The effect of inducible nitric oxide
synthase (iNOS) inhibitor was analyzed in the presence orabsence of L-NMMA (100 mmol/L).
Measurement of NO production
NO production by CD11bþ myeloid cells (1  105 cells/200mL) puriﬁed from tumor of mice (n ¼ 4 or 5) on days 17 to 21 after
tumor implantation was determined by measuring nitrite(NO2), a stable and non-volatile by-product of NO, producedin culture supernatants for 48 hours using a Griess reagent kit(Thermo Fischer Scientiﬁc). The Griess reagent and nitrite standards or supernatants were mixed, incubated, and then read at
520 and 590 nm as a reference to measure nitrite levels. The effectof iNOS inhibitor was analyzed in the presence or absence of
L-NMMA (100 mmol/L).
Preparation of LSK cells
Spleen and BM Lineage cells were enriched by negative selection using an AutoMACS Pro with a combination of magnetic
beads conjugated with mAbs against CD3e, CD4, CD8a, Gr-1,
TER119, CD11b, CD11c, NK1.1, B220, and FceRIa. Cells werethen stained with mAbs against c-Kit, and Sca-1 was used for LSK.
Sorting was performed on a MoFlo XDP (Beckman Coulter).
In vitro differentiation assay
For analysis of multipotent myeloid potential, LSK cells were
puriﬁed from BM cells in tumor-bearing mice (n ¼ 3) on days
17 to 20 after tumor implantation or untreated mice. The sorted
LSK cells (2.5  102/200 mL) were cultured with 10 ng/mL
SCF and 10 ng/mL IL3 in 96-well plates for 7 days. Half ofthe medium was replaced every 3 days, with cytokines added.
The following cell surface markers were used to identify respective cells (Supplementary Fig. S1): neutrophil, Ly6GþCD11bþ
or Gr-1þCD11bþ; macrophage, F4/80þCD11bþ; mast cell,
c-KitþFceR1aþCD11b; basophil, CD49bþFceR1aþCD11b.
Adoptive cell transfer
BM LSK cells (5  103) puriﬁed from C57BL/6 (CD45.1)
congenic mice were intravenously transferred into sublethally
irradiated (4 Gy) C57BL/6 (CD45.2) recipient mice (n ¼ 4). On
the next day, tumor cells (1  106 cells) of B16F10-IL27 or
B16F10-Vector were injected subcutaneously into the right ﬂankof resulting mice. Tumor growth was monitored twice a week.
ELISA
Serum protein level of TGFb1 was determined by ELISA according to the manufacturers' instructions (R&D Systems).
Statistical analysis
Data are presented as mean  SEM. Statistical analyses were
performed by using the two-tailed Student t test for comparisonsof two groups and one-way ANOVA and Tukey–Kramer multiple
comparison test for comparing more than three groups using
GraphPad Prism 7 (GraphPad Software Inc.). P < 0.05 wasconsidered to indicate a statistically signiﬁcant difference.

Results
IL27 augments the inﬁltration of CD11bþ myeloid cells intotumor
As reported previously (20), melanoma B16F10 tumor cells
transfected with IL27–expression vector (B16F10-IL27) showed
great inhibition of tumor growth compared with those transfected with control empty vector (B16F10-Vector; Fig. 1A and
B). During analysis of antitumor activity of IL27, we initially
noticed that the percentage and total number of mononuclear
cells inﬁltrating into tumors were signiﬁcantly increased by
IL27 (Fig. 1C and D). Implantation of tumors also increased thespleen weight regardless of B16F10-IL27 or B16F10-Vector (Fig.
1B), implying augmented myelopoiesis by tumor growth.
Among the various types of cells, FACS analyses revealed that
IL27 greatly increased the percentage and number of not only
CD8þ T cells but also CD11bþ myeloid cells in tumor pertumor volume, as well as per tumor weight (Fig. 1E and F). A
similar increase of tumor-inﬁltrating CD11bþ myeloid cells
was observed with another tumor model, MC38 colon carcinoma (Supplementary Fig. S2).
Given that tumor-inﬁltrating myeloid cells are generally immunosuppressive cells, such as M2 macrophages and MDSCs, it isimportant to investigate the properties of IL27–medaited inﬁltrating myeloid cells, their role in induction of antitumor effects,

184 Cancer Res; 78(1) January 1, 2018

Cancer Research

Downloaded from cancerres.aacrjournals.org on April 28, 2018. © 2018 American Association for Cancer Research.

-81-

Published OnlineFirst November 1, 2017; DOI: 10.1158/0008-5472.CAN-17-0960

Differentiation to Antitumorigenic M1 Macrophages by IL27

Figure 1.
IL27 augments the inﬁltration of CD11bþ myeloid cells into tumor. A, B16F10-IL27 or B16F10-Vector tumor was implanted and tumor growth was monitored.
B–D, On day 13, tumor and spleen were harvested, and their weights were measured (B), single-cell suspension was then prepared from the tumor
and subjected to ﬂow cytometer analysis (C), and total number of tumor-inﬁltrating mononuclear cells was counted (D). E, Representative dot plotsof individual cell populations are shown. F, Their cell numbers were counted, and calculated per tumor volume and also per tumor weight. Data are shownas mean  SEM (n ¼ 3 or 4) and are representative of six independent experiments.  , P < 0.05;    , P < 0.001.

www.aacrjournals.org

Cancer Res; 78(1) January 1, 2018

Downloaded from cancerres.aacrjournals.org on April 28, 2018. © 2018 American Association for Cancer Research.

-82-

185

Published OnlineFirst November 1, 2017; DOI: 10.1158/0008-5472.CAN-17-0960

Chiba et al.

and the molecular mechanism whereby IL27 induces such aphenomenon.
IL27 promotes the differentiation into M1 macrophages intumor-bearing miceM1 and M2 macrophages have antitumorigenic activity andprotumorigenic activity, respectively (3). Therefore, we next
examined the phenotype of these IL27–mediated tumor-inﬁltrating myeloid cells. On day 13 after tumor implantation,mononuclear cells were puriﬁed from tumor and subjected tocell surface analysis by using FACS. IL27 greatly increased thepercentage and number of monocytic (Mo)MDSCs (1, 2) andmacrophages, but decreased those of granulocytic (Gr)MDSCs(1, 2), in tumor per tumor volume, as well as per tumor
weight (Fig. 2A and B). Among these tumor-inﬁltrating macrophages, IL27 greatly increased the percentage and number of M1
macrophages (Class IIhighCD86þCD45.2þCD11bþF4/80þ) as
compared with those of M2 macrophages (Class
IIlowCD86þCD45.2þCD11bþF4/80þ; Fig. 2C–E). The ratio of
M1/M2 macrophages was signiﬁcantly increased, indicating
the enhanced bias toward M1 macrophages by IL27 (Fig. 2D
and E). Consistent with this, mRNA expression analyses
revealed that CD11bþ myeloid cells puriﬁed from tumor of
B16F10-IL27 tumor-bearing mice express more M1 macrophage markers, such as iNOS, interferon regulatory factor 8(IRF8), and IL12p40, but less M2 macrophage markers, such asariginase-1 (Arg-1), chitinase 3-like-3 (Ym1), and found ininﬂammatory zone 1 (Fizz1), compared with those of B16F10Vecter tumor-bearing mice (Fig. 2F). Similar results wereobtained with the MC38 tumor model (Supplementary Fig.
S3). These results suggest that IL27 promotes differentiation
into M1 macrophages in tumor-bearing mice.
Because IL27–transduced tumor grows at a much slower rate
than control tumor, not only the tumor size but also the tumor
microenvironment might be different between them and therefore affect the number and phenotype of tumor-inﬁltrating
cells. To examine this possibility, we tried to adjust the tumor
size to reach the same size by changing the conditions such as
the cell number of tumors to inject, and then analyzed thetumor-inﬁltrating cells by ﬂow cytometry. Under these conditions, we could still observe that IL27 much less but signiﬁcantly enhances the percentage and number of tumor-inﬁltrating cells including macrophages, especially with M1 phenotype(Supplementary Fig. S4).
IL27 inhibits differentiation into immunosuppressive
macrophages in tumor-bearing mice
Generally, tumor-inﬁltrating myeloid cells are immunosuppressive (1). Therefore, we next examined immunosuppressive
activity of tumor-inﬁltrating myeloid cells. On day 16 after
tumor implantation, mononuclear cells were puriﬁed fromtumor, and CD11bþ myeloid cells were further puriﬁed fromthem and subjected to immunosuppressive activity, which wasassessed by the ability to inhibit OVA-speciﬁc CD8þ T-cellproliferation. Myeloid cells puriﬁed from tumor of B16F10Vector tumor-bearing mice showed marked inhibitory activity
against the antigen-speciﬁc proliferation (Fig. 3). In contrast,
myeloid cells puriﬁed from tumor of B16F10-IL27 tumorbearing mice showed signiﬁcantly reduced inhibitory activity
compared with those of control tumor-bearing mice (Fig. 3).
Similar results were obtained with the MC38 tumor model(Supplementary Fig. S5). Thus, IL27 inhibits the generationof immunosuppressive myeloid cells such as M2 macrophages
in tumor-bearing mice, but it promotes the generation of M1
macrophages.
IL27-induced tumor-inﬁltrating myeloid cells have potent
antitumor effects
To further explore the role of myeloid cells in B16F10-IL27
tumor-bearing mice in the IL27-mediated antitumor activity,
depletion experiments of myeloid cells by treatment with antiGr-1 mAb were performed. Treatment of mice with anti–Gr-1
mAb almost completely depleted Gr-1þCD11bþ myeloid
cells in blood (Fig. 4A). Under these conditions, depletion of
myeloid cells in B16F10-Vector tumor-bearing mice greatly
inhibited the tumor growth, as generally expected (Fig. 4A).
In contrast, depletion of myeloid cells in B16F10-IL27 tumorbearing mice signiﬁcantly promoted the tumor growth(Fig. 4B). These results suggest that IL27 exerts antitumor
activity via myeloid cells.
IL27-induced tumor-inﬁltrating myeloid cells exert direct
antitumor effects by killing tumor through NO
To further examine the effect of IL27 on myeloid cells in tumorbearing mice, admixture experiments of myeloid cells with parental B16F10 tumor were performed. Consistent with the results ofdepletion experiments, admixture of CD11bþ myeloid cells puriﬁed from tumor of B16F10-Vector tumor-bearing mice withparental B16F10 tumor greatly promoted the tumor growth (Fig.
5A). In contrast, admixture of CD11bþ myeloid cells puriﬁed
from tumor of B16F10-IL27 tumor-bearing mice with parental
B16F10 tumor markedly inhibited the tumor growth (Fig. 5A).
Again, tumor-inﬁltrating myeloid cells are quite different between
B16F10-IL27 and B16F10-Vector tumor-bearing mice, being antitumorigenic versus protumorigenic cells.
To further evaluate the therapeutic potential of the CD11bþ
myeloid cells puriﬁed from tumors of B16F10-IL27 tumorbearing mice, the antitumor efﬁcacy of these myeloid cells on
pre-existing tumor model was examined. When mice were
implanted with parental B16F10 tumor and their tumor size
reached 5 mm in diameter on day 5, these CD11bþ myeloid
cells were both intravenously and intratumorally injected into
these mice with the pre-existing tumor. By using these injections, we could observe a tendency to inhibit the tumor growth(Supplementary Fig. S6).
Next, to investigate the molecular mechanism whereby IL27
augments antitumor activity via myeloid cells, the possibilityof direct killing of tumors by macrophages was examined byusing a 51Cr-releasing assay. CD11bþ myeloid cells puriﬁed
from tumor of B16F10-IL27 tumor-bearing mice showed greatly augmented killing activity compared with those of B16F10Vector tumor-bearing mice (Fig. 5B). Correlating with theaugmented killing activity, myeloid cells puriﬁed from tumorof B16F10-IL27 tumor-bearing mice produced more NO thanthose of B16F10-Vector tumor-bearing mice (Fig. 5C). Treatment with NOS inhibitor, L-NMMA, almost completely
blocked the killing activity of myeloid cells puriﬁed fromtumor of B16F10-IL27 tumor-bearing mice (Fig. 5D). Similar
results were obtained with the MC38 tumor model (Supplementary Fig. S7).
Thus, IL27 promotes the generation of antitumorigenic myeloid cells rather than protumorigenic myeloid cells in tumor-

186 Cancer Res; 78(1) January 1, 2018

Cancer Research

Downloaded from cancerres.aacrjournals.org on April 28, 2018. © 2018 American Association for Cancer Research.

-83-

Published OnlineFirst November 1, 2017; DOI: 10.1158/0008-5472.CAN-17-0960

Differentiation to Antitumorigenic M1 Macrophages by IL27

Figure 2.
IL27 promotes the differentiation into M1 macrophages in tumor-bearing mice. A and B, Single-cell suspensions prepared from tumor on day 13 after
tumor implantation were analyzed by ﬂow cytometry. Representative dot plots of Gr-MDSCs (CD45.2þCD11bþLy6GþLy6Cint), Mo-MDSCs(CD45.2þCD11bþLy6GLy6Chi), and macrophages (CD45.2þCD11bþF4/80þ) are shown, and their cell numbers were counted and calculated per tumor volume
and also per tumor weight. C, Representative dot plots of M1 macrophages (Class IIhighCD86þCD45.2þCD11bþF4/80þ) and M2 macrophages (Class
IIlowCD86þCD45.2þCD11bþF4/80þ) are shown. D and E, Their cell numbers were counted, and calculated per tumor volume (D) and also per tumor
weight (E), together with respective ratio of M1/M2 macrophages. Data are shown as mean  SEM (n ¼ 3 or 4) and are representative of more than six
independent experiments. F, mRNA expression of markers for M1 and M2 macrophages in puriﬁed CD11bþ myeloid cells from tumor-bearing mice on day 17
was analyzed by real-time RT-PCR. Data are shown as mean  SEM (n ¼ 4) and are representative of more than six independent experiments.  , P < 0.05;
, P < 0.01;    , P < 0.001.

www.aacrjournals.org

Cancer Res; 78(1) January 1, 2018

Downloaded from cancerres.aacrjournals.org on April 28, 2018. © 2018 American Association for Cancer Research.

-84-

187

Published OnlineFirst November 1, 2017; DOI: 10.1158/0008-5472.CAN-17-0960

Chiba et al.

bearing mice, and the antitumorigenic myeloid cells exert direct
killing activity against tumor through NO.

the promotion of myelopoiesis in the BM and spleen (1). Consistent with this phenomenon, tumor-bearing mice have splenomegaly (Fig. 1B). Therefore, we investigated the effects of IL27 on
the myelopoiesis in tumor-bearing mice. Similar to the results
obtained by using the malaria infection model (10), IL27
increased the percentage and number of LSK cells in both BM
and spleen (Fig. 6A). Moreover, IL27 also increased the percentage
and number of the most primitive HSC population, long-term
HSC (LT-HSC; ref. 24), but not multipotent progenitor (MPP)in both BM and spleen (Fig. 6B). Thus, IL27 directly affects
LSK cells and LT-HSCs in the BM to promote the myelopoiesis
in transplantable tumor models.
Next, the multipotency of LSK cells in the BM of tumorbearing mice was examined under in vitro myeloid celldifferentiation conditions. LSK cells puriﬁed from the BM of
B16F10-Vector tumor-bearing mice had less ability to differentiate into myeloid cells such as mast cells, basophils, neutrophils, and macrophages compared with those puriﬁed from
untreated mice (Fig. 6C and D). However, LSK cells puriﬁed
from the BM of B16F10-IL27 tumor-bearing mice had agreater ability to differentiate into these myeloid cells compared with those of B16F10-Vector tumor-bearing mice (Fig. 6C
and D). Interestingly, BM LSK cells from B16F10-IL27 tumorbearing mice had an enhanced ability to differentiate into
M1 macrophages compared with those from B16F10-Vector
tumor-bearing mice (Fig. 6E). These results suggest that IL27
promotes the expansion and generation of LSK cells, whichhave an enhanced ability to differentiate into myeloid cells,
especially with the M1 macrophage phenotype.

IL27 induces the expansion of LSK cells with enhanced ability todifferentiate into M1 macrophages
Generally, tumor-bearing mice have increased production oftumor-derived cytokines such as G-CSF and GM-CSF, resulting in

IL27 directly acts on BM LSK cells and promotes the expansion
and differentiation into M1 macrophages in vivo
To further examine the direct effect of IL27 on BM HSCs
to differentiate into M1 macrophages in vivo, adoptive cell

Figure 3.
IL27 inhibits the differentiation into immunosuppressive macrophages in tumorbearing mice. On day 16 after tumor implantation, mononuclear cells
were puriﬁed from tumor, and CD11bþ myeloid cells were further puriﬁed
and subjected to immunosuppressive activity, which was assessed by theability to inhibit OVA-speciﬁc CD8þ T-cell proliferation. Data are shown asmean  SEM (n ¼ 3 or 4) and are representative of more than six
independent experiments.  , P < 0.05;   , P < 0.01;    , P < 0.001.

Figure 4.
IL27–induced tumor-inﬁltrating myeloid cells havepotent antitumor effects. A, Mice were treated with anti–
Gr-1 mAb or control IgG, and deletion level of neutrophils
was analyzed in blood by ﬂow cytometry on day 7 after
tumor implantation. B, Tumor growth was monitoredwith time course. Data are shown as mean  SEM (n ¼ 5)
and are representative of three independent
experiments.  , P < 0.05.

188 Cancer Res; 78(1) January 1, 2018

Cancer Research

Downloaded from cancerres.aacrjournals.org on April 28, 2018. © 2018 American Association for Cancer Research.

-85-

Published OnlineFirst November 1, 2017; DOI: 10.1158/0008-5472.CAN-17-0960

Differentiation to Antitumorigenic M1 Macrophages by IL27

Figure 5.
IL27-induced tumor-inﬁltrating myeloid cells exert
direct antitumor effects by killing tumor throughNO. A, CD11bþ myeloid cells puriﬁed from tumor of
B16F10-IL27 or B16F10-Vector tumor-bearing mice
were admixed with parental B16F10 tumor, and tumor
growth was monitored with time course. Data areshown as mean  SEM (n ¼ 6) and are representativeof more than three independent experiments. B and C,
On day 18 after tumor implantation, killing activity of
CD11bþ myeloid cells against parental B16F10 tumor
was measured by 51Cr release assay (B), and NO
production in the culture supernatant was measured
in the presence or absence of NOS inhibitor, L-NMMA(C). D, The effect of inhibition of NOS activity by
L-NMMA on the killing activity was examined. Data
are shown as mean  SEM (n ¼ 4 or 5) and are
representative of more than ﬁve independent
experiments.  , P < 0.05;    , P < 0.001.

transfer of BM LSK cells puriﬁed from CD45.1 congenic mice
into sublethally irradiated CD45.2 recipient mice was performed. Tumor cells of B16F10-IL27 or B16F10-Vector werethen implanted, and tumor growth was monitored. As shown
before (Fig. 1A), IL27 signiﬁcantly inhibited tumor growth (Fig.
7A). On day 20 after tumor implantation, tumor-inﬁltrating
cells were analyzed. IL27 greatly increased the percentage andnumber of CD45.1þ cells and macrophages inﬁltrating into
tumors (Fig. 7B and C). Consistent with the in vivo results (Fig.
2) and in vitro differentiation results (Fig. 6C–E), IL27 markedly
increased the percentage and number of M1 macrophages into
tumor (Fig. 7D). Thus, IL27 directly acts on the BM LSK cells
and promotes the expansion and differentiation into antitumorigenic M1 macrophages in tumor-bearing mice.

Discussion
Accumulating evidence suggests that IL27 possesses potent
antitumor activity through multiple mechanisms depending on
the characteristics of individual tumors (7, 8). The present results
indicate that IL27 has another novel mechanism to exert potent
antitumor activity. IL27 promotes the expansion and differentiation of HSCs into myeloid progenitor cells, which have enhanced
potential to differentiate into antitumorigenic M1 macrophages
with increased expression of iNOS, IRF8, and IL12p40 but
reduced expression of Arg-1, Ym1, and Fizz1 in tumor-bearing
mice. Blocking experiments using the iNOS inhibitor, L-NMMA,
further revealed that the CD11bþ myeloid cells puriﬁed from
IL27-tumor-bearing mice directly kill tumors in a highly NOdependent manner, whereas whether other potential mechanisms
may also exist remains to be elucidated. This is consistent with ourprevious report showing that IL27 augments the production of
NO in thioglycollate-elicited peritoneal macrophages in synergy
with lipopolysaccharide through STAT1, NF-kB, and MAP kinases(25). We also reported that IL27 greatly induced IRF1 and IRF8
expression in mouse melanoma B16F10-expressing WSX-1, leading to inhibition of its tumor growth (21). Importantly, IRF8 is a
critical transcription factor for the development of monocytes,
dendritic cells, eosinophils, and basophils in synergy with PU.1
(26), whereas IRF8 inhibits CCAAT-enhancer-binding protein aactivity to suppress the generation of neutrophils (26). IRF8 is alsoa critical transcription factor for induction of iNOS expression incollaborating with IRF1 (27) or NF-kB (28), resulting in enhanced
NO production. In addition, IRF8 is an integral negative regulator(29) against the development of MDSCs by promoting spontaneous apoptosis and turnover in myeloid cells (30) and byrepressing GM-CSF expression in T cells (31). Thus, IL27-mediated promotion of HSCs to expand and differentiate into antitumorigenic M1 macrophages with enhanced iNOS expression
presumably is highly dependent on IRF8, but this possibility
remains to be further clariﬁed.
Of note, the present results indicate that properties of tumorinﬁltrating myeloid cells in the presence or absence of IL27 arequite different. Myeloid cells in IL27 tumor-bearing mice are
antitumorigenic with the M1 macrophage phenotype, whereas
those in control tumor-bearing mice are protumorigenic with theM2 macrophage phenotype (Figs. 2–5A). Myeloid cells in IL27
tumor-bearing mice were demonstrated to kill parental tumors
directly in a NO-dependent manner (Fig. 5B–D). Moreover, IL27
increased the percentage and number of LSK cells in both BM andspleen of tumor-bearing mice (Fig. 6A and B). We previously
demonstrated that IL27 directly acts on LSK cells, especially
LT-HSCs, and promotes their expansion and differentiation into
phenotypically similar LSK cells but distinct myeloid progenitors(10). LSK cells in the BM of control tumor-bearing mice have a
reduced ability to ex vivo differentiate into myeloid cells comparedwith LSK cells in the BM of untreated mice, possibly due to theimmunosuppressive effects of myeloid cells on LSK cells in the

www.aacrjournals.org

Cancer Res; 78(1) January 1, 2018

Downloaded from cancerres.aacrjournals.org on April 28, 2018. © 2018 American Association for Cancer Research.

-86-

189

Published OnlineFirst November 1, 2017; DOI: 10.1158/0008-5472.CAN-17-0960

Chiba et al.

Figure 6.
IL27 induces the expansion of LSK cellswith enhanced ability to differentiate into
M1 macrophages. A and B, On day 13 after
tumor implantation, cell populations of LSK(A), LT-HSC, and MPP (B) were analyzed
by ﬂow cytometry. Data are shown asmean  SEM (n ¼ 3 or 4) and are
representative of more than six
independent experiments. C and D, The
multipotency of LSK cells in the BM of
B16F10-IL27 or B16F10-Vector tumorbearing mice on day 19 after tumor
implantation together with untreated micewas examined under in vitro myeloid celldifferentiation conditions into mast cells,
basophils, neutrophils, and macrophages.
E, Cell number of M1 and M2 macrophages
was counted and the ratio of M1/M2
macrophages was calculated. Data areshown as mean  SEM (n ¼ 3) and are
representative of more than three
independent experiments.  , P < 0.05;
, P < 0.01;    , P < 0.001.

tumor-bearing mice (Fig. 6C and D). In contrast, LSK cells in the
BM of IL27-tumor-bearing mice have a greatly increased ability to
ex vivo differentiate into myeloid cells including M1 macrophages
compared with those of control tumor-bearing mice (Fig. 6C–E).
Finally, adaptive cell transfer experiments revealed that IL27 acts
directly on the LSK cells in the BM in vivo and promotes the
expansion and differentiation into M1 macrophages, mobilizingthem in tumor (Fig. 7).

Although the inﬁltration of CD8þ T cells into IL27–treatedtumors is dominant in B16F10 melanoma model (Fig. 1F),
the inﬁltration of CD4þ T cells is dominant in MC38 colon
carcinoma model (Supplementary Fig. S2C). Although themolecular mechanism whereby IL27 induces such distinct
dominancy remains elusive, IL27 also greatly induces theinﬁltration of CD11bþ myeloid cells into tumor in both models irrespectively of CD8þ or CD4þ T cell inﬁltration. One of the

190 Cancer Res; 78(1) January 1, 2018

Cancer Research

Downloaded from cancerres.aacrjournals.org on April 28, 2018. © 2018 American Association for Cancer Research.

-87-

Published OnlineFirst November 1, 2017; DOI: 10.1158/0008-5472.CAN-17-0960

Differentiation to Antitumorigenic M1 Macrophages by IL27

possible reasons for the different effects by IL27 on the tumorinﬁltrating cells may be ascribed to the different microenvironment situations such as distinct places and stages where
IL27 acts on these cells. In the case of myeloid cells, IL27
directly acts on the BM cells and promotes the differentiation
into M1 macrophages following the inﬁltration into tumor(Fig. 6 and 7). On the other hand, IL27 likely acts on naive
T cells in the spleen or lymph node to promote the differen-

tiation and proliferation of them (32). Moreover, IL27 seemingly induced high M1/M2 ratio and NO production of thetumor-inﬁltrating CD11bþ myeloid cells in the MC38 modelcompared with those in the B16F10 model (Fig. 1C and 5C;
Supplementary Figs. S3C and S7C). Correlating to these results,
the killing activity of the CD11bþ myeloid cells augmented
by IL27 in relation to that by control vector in the MC38
model was higher than that in the B16F10 model (41.8 

Figure 7.
IL27 directly acts on BM LSK cells and promotes
the expansion and differentiation into M1
macrophages in vivo. A, BM LSK cells puriﬁed
from CD45.1 congenic untreated mice wereadoptively transferred into sublethally irradiatedCD45.2 recipient mice, and then tumor cells of
B16F10-IL27 or B16F10-Vector were implanted,
and tumor growth was monitored with timecourse. B and C, On day 20 after tumor
implantation, percentage and number ofCD45.1þ or CD45.2þ cell population (B) andmacrophages (C) were measured. D,
Representative dot plots of M1 and M2
macrophages are shown, their cell numbers
were counted, and ratio of M1/M2 macrophages
was calculated. Data are shown as mean  SEM(n ¼ 4) and are representative of more than
three independent experiments.  , P < 0.05.

www.aacrjournals.org

Cancer Res; 78(1) January 1, 2018

Downloaded from cancerres.aacrjournals.org on April 28, 2018. © 2018 American Association for Cancer Research.

-88-

191

Published OnlineFirst November 1, 2017; DOI: 10.1158/0008-5472.CAN-17-0960

Chiba et al.

18.9-folds vs. 7.1  4.2-folds; Fig. 5B; Supplementary Fig. S7B).
Further studies are necessary to clarify the different effects by
IL27 between these two tumor models.
Because IL27 induces upregulation of immunosuppressive
molecules such as PD-L1, CD39, TIM-3, and IL10 (11–17), thepossibility that IL27 may induce protumorigenic effects has
been discussed (8, 33). However, by using histopathologicalanalyses, Airoldi and colleagues (18) observed that IL27 treatment increases the inﬁltration of granulocytes and macrophages into tumor using xenograft models of human lungadenocarcinoma and squamous cell carcinoma cell lines
Calu-6 and SK-MES in immunodeﬁcient mice. To assess whether these granulocytes and macrophages may account for the
antitumor effects of IL27, mice were pretreated with myeloablative doses of treosulfan. The myeloablation signiﬁcantly
abolished the antitumor effect of IL27, although this treatment
induced a severe or complete depletion of BM cells, which
affects the cellularity of peripheral cells other than macrophages (18). Yao and colleagues (19) also recently reported
that IL27 inhibits the development of M2 macrophage fromthe human monocytic cell line U937 differentiated by phorbol
12–myristate 13–acetate and subsequently polarized by IL4
in vitro. Consistent with the present results, these two reports
further support the possibility that macrophages play a criticalrole in exerting antitumorigenic effects rather than protumorigenic effects by IL27 in tumor-bearing mice.
Given that IL27 is a multifunctional cytokine with a doubleedged sword, both antitumor and protumor effects of IL27 areconceivably expected. Indeed, we observed enhanced mRNA
expression of immune checkpoint molecules such as PD-L1 andTIM-3 by in tumor-inﬁltrating CD11bþ myeloid cells (Supplementary Fig. S8A and S8B) as reported (14, 15, 17). Similartendency of the up-regulation of PD-1 mRNA expression by IL27
was also observed, although its expression level even in steadystate of myeloid cells seemed to be low (Supplementary Fig. S8A
and S8B; ref. 34). Similar upregulation of TGFb1 mRNA expression by IL27 was observed in tumor-inﬁltrating CD11bþ myeloid
cells, but its serum level was rather decreased by IL27 (Supplementary Fig. S8C). Because IL27 was recently reported to inhibit
the functions of TGFb1, including induction of ﬁbrosis andepithelial–mesenchymal transition (35, 36), which contributeto antitumor effects, further study is necessary to clarify the roleof TGFb1 in the exertion of antitumor effect by IL27. Because
B16F10 tumor does not express endogenous one of IL27R subunits, WSX-1, IL27 does not directly act on B16F10 tumor cells to
inhibit the immunosuppressive functions of the tumors (21).
Rather, IL27 induces antitumor activity via acting on other cells
including T cells, NK cells, macrophages, and endothelial cells in
the tumor microenvironment (7, 8). Thus, the overall antitumor
potency of IL27 is highly likely to result from the sum of its
positive and negative effects on tumors within the tumor microenvironment. Therefore, if the IL27 therapy could be combinedwith the immune checkpoint blockade by using antibody (37), wewould expect a synergistic antitumor effect between them. Thispossibility is currently under investigation.
IL27 belongs to the IL12 cytokine family including IL12,
IL23, and so on. IL12 plays a critical role in the induction of
Th1-type immune responses through STAT4 activation, and it
was demonstrated to induce strong antitumor effects via
mainly IFN-g production in various preclinical models (38).
However, the clinical trials of IL12 have been limited bysystemic toxicities such as hepatomegaly, leukopenia, andgastrointestinal toxicity (39–42). IL23 plays a key role inautoimmune and inﬂammatory diseases through STAT3/
STAT4 activation via IL23/IL17 immune axis (43). IL23 wasdemonstrated to promote tumorigenesis by inducing inﬂammation, which inhibits adaptive immune surveillance (44).
Nevertheless, multiple studies have also shown the antitumor
effects by IL23 in various mouse tumor models (45–47).
Thus, the role of IL23 in tumor procession is still controversial.
On the other hand, IL27 activate STAT1 and STAT3 throughits respective receptor subunits, WSX-1 and gp130, respectively(48, 49). IL27 was previously reported to induce direct antiproliferative effects on melanoma cells via STAT1 signaling as
does IFN-g (21). IL27 has very low toxicity, probably becauseof low induction of IFN-g (6, 47). IL27 treatment does not
induce severe liver injury with increased levels of aspartate andalanine aminotransferases (6, 47), and transgenic mice constitutively overexpressing IL27 can survive for almost thewhole life span (50). Such a low toxic property of IL27 is of
beneﬁt to its clinical application.
Taken together, our present ﬁndings clearly indicate thattumor-inﬁltrating myeloid cells play a critical role in exertionof antitumor effects by IL27, although these myeloid cells showhigh expression of immune checkpoint molecules. To ensure
patient safety, therefore, combination therapy of IL27 withmonoclonal antibodies against these immune checkpoint
molecules could be a more promising and effective therapeuticstrategy against cancer.

Disclosure of Potential Conﬂicts of Interest
No potential conﬂicts of interest were disclosed.

Authors' Contributions
Conception and design: Y. Chiba, T. Yoshimoto
Development of methodology: Y. Chiba, I. Mizoguchi, T. Yoshimoto
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): Y. Chiba, I. Mizoguchi, H. Hasegawa, M. Ohashi
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): Y. Chiba, I. Mizoguchi, H. Hasegawa, M. Ohashi
Writing, review, and/or revision of the manuscript: Y. Chiba, M. Xu, T. Owaki,
T. Yoshimoto
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): J. Furusawa, T. Owaki
Study supervision: T. Yoshimoto

Acknowledgments
The authors thank Dr. H. Nariuchi (University of Tokyo) and
Dr. Y. Kawakami (Keio University) for the B16F10 mouse melanoma
cell line and MC38 colon carcinoma cell line, respectively, and also thank
R. Tsunoda (Tokyo Medical University) for his encouragement. This study
was supported in part by a grant-in-aid (to T. Yoshimoto) and the Private
University Strategic Research Based Support Project (to T. Yoshimoto)from the Ministry of Education, Culture, Sports, Science, and Technology,
Japan, by Research Grant from Princess Takamatsu Cancer Research Fund
(to T. Yoshimoto), and by Research Fellowship for Young Scientists (DC2)from Japan Society for the Promotion of Science (to Y. Chiba).
The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received April 4, 2017; revised August 25, 2017; accepted October 27, 2017;
published OnlineFirst November 1, 2017.

192 Cancer Res; 78(1) January 1, 2018

Cancer Research

Downloaded from cancerres.aacrjournals.org on April 28, 2018. © 2018 American Association for Cancer Research.

-89-

Published OnlineFirst November 1, 2017; DOI: 10.1158/0008-5472.CAN-17-0960

Differentiation to Antitumorigenic M1 Macrophages by IL27

References
1. Gabrilovich DI, Ostrand-Rosenberg S, Bronte V. Coordinated regulation of
myeloid cells by tumours. Nat Rev Immunol 2012;12:253–68.
2. Trikha P, Carson WE III. Signaling pathways involved in MDSC regulation.
Biochim Biophys Acta 2014;1846:55–65.
3. Murray PJ, Allen JE, Biswas SK, Fisher EA, Gilroy DW, Goerdt S, et al.
Macrophage activation and polarization: nomenclature and experimental
guidelines. Immunity 2014;41:14–20.
4. Yoshida H, Hunter CA. The immunobiology of interleukin-27. Annu Rev
Immunol 2015;33:417–43.
5. Mizoguchi I, Higuchi K, Mitobe K, Tsunoda R, Mizuguchi J, Yoshimoto
T. Interleukin-27: Regulation of immune responses and disease development by a pleiotropic cytokine with pro- and anti-inﬂammatory
properties. In:Yoshimoto T, Yoshimoto T, editors. Cytokine frontiers:
regulation of immune responses in health and disease Tokyo: Springer;
2013. p 353–75.
6. Hisada M, Kamiya S, Fujita K, Belladonna ML, Aoki T, Koyanagi Y,
et al. Potent antitumor activity of interleukin-27. Cancer Res 2004;
64:1152–6.
7. Mizoguchi I, Chiba Y, Furusawa JI, Xu M, Tsunoda R, Higuchi K, et al.
Therapeutic potential of interleukin-27 against cancers in preclinicalmouse models. Oncoimmunology 2015;4:e1042200.
8. Yoshimoto T, Chiba Y, Furusawa J, Xu M, Tsunoda R, Higuchi K, et al.
Potential clinical application of interleukin-27 as an antitumor agent.
Cancer Sci 2015;106:1103–10.
9. Seita J, Asakawa M, Ooehara J, Takayanagi S, Morita Y, Watanabe N, et al.
Interleukin-27 directly induces differentiation in hematopoietic stem cells.
Blood 2008;111:1903–12.
10. Furusawa J, Mizoguchi I, Chiba Y, Hisada M, Kobayashi F, Yoshida H, et al.
Promotion of expansion and differentiation of hematopoietic stem cells byinterleukin-27 into myeloid progenitors to control infection in emergency
myelopoiesis. PLoS Pathog 2016;12:e1005507.
11. Stumhofer JS, Silver JS, Laurence A, Porrett PM, Harris TH, Turka LA, et al.
Interleukins 27 and 6 induce STAT3-mediated T cell production of interleukin 10. Nat Immunol 2007;8:1363–71.
12. Awasthi A, Carrier Y, Peron JP, Bettelli E, Kamanaka M, Flavell RA,
et al. A dominant function for interleukin 27 in generating interleukin 10-producing anti-inﬂammatory T cells. Nat Immunol 2007;
8:1380–9.
13. Iyer SS, Ghaffari AA, Cheng G. Lipopolysaccharide-mediated IL-10 transcriptional regulation requires sequential induction of type I IFNs and IL27 in macrophages. J Immunol 2010;185:6599–607.
14. Hirahara K, Ghoreschi K, Yang XP, Takahashi H, Laurence A, Vahedi G, et al.
Interleukin-27 priming of T cells controls IL-17 production in trans via
induction of the ligand PD-L1. Immunity 2012;36:1017–30.
15. Zhu C, Sakuishi K, Xiao S, Sun Z, Zaghouani S, Gu G, et al. An IL-27/NFIL3
signalling axis drives Tim-3 and IL-10 expression and T-cell dysfunction.
Nat Commun 2015;6:6072.
16. d'Almeida SM, Kauffenstein G, Roy C, Basset L, Papargyris L, Henrion D,
et al. The ecto-ATPDase CD39 is involved in the acquisition of theimmunoregulatory phenotype by M-CSF-macrophages and ovarian cancer
tumor-associated macrophages: Regulatory role of IL-27. Oncoimmunology 2016;5:e1178025.
17. Horlad H, Ma C, Yano H, Pan C, Ohnishi K, Fujiwara Y, et al. An IL-27/
Stat3 axis induces expression of programmed cell death 1 ligands (PDL1/2) on inﬁltrating macrophages in lymphoma. Cancer Sci 2016;107:
1696–704.
18. Airoldi I, Tupone MG, Esposito S, Russo MV, Barbarito G, Cipollone G,
et al. Interleukin-27 re-educates intratumoral myeloid cells and downregulates stemness genes in non-small cell lung cancer. Oncotarget
2015;6:3694–708.
19. Yao L, Wang M, Niu Z, Liu Q, Gao X, Zhou L, et al. Interleukin-27
inhibits malignant behaviors of pancreatic cancer cells by targeting
M2 polarized tumor associated macrophages. Cytokine 2017;89:
194–200.
20. Shimizu M, Shimamura M, Owaki T, Asakawa M, Fujita K, Kudo M, et al.
Antiangiogenic and antitumor activities of IL-27. J Immunol 2006;176:7317–24.
21. Yoshimoto T, Morishima N, Mizoguchi I, Shimizu M, Nagai H, Oniki S,
et al. Antiproliferative activity of IL-27 on melanoma. J Immunol
2008;180:6527–35.

22. Yoshimoto T, Okada K, Morishima N, Kamiya S, Owaki T, Asakawa M, et al.
Induction of IgG2a class switching in B cells by IL-27. J Immunol
2004;173:2479–85.
23. Calderon B, Carrero JA, Ferris ST, Sojka DK, Moore L, Epelman S, et al.
The pancreas anatomy conditions the origin and properties of resident
macrophages. J Exp Med 2015;212:1497–512.
24. Cai X, Gaudet JJ, Mangan JK, Chen MJ, De Obaldia ME, Oo Z, et al. Runx1
loss minimally impacts long-term hematopoietic stem cells. PLoS ONE
2011;6:e28430.
25. Shimizu M, Ogura K, Mizoguchi I, Chiba Y, Higuchi K, Ohtsuka H,
et al. IL-27 promotes nitric oxide production induced by LPS
through STAT1, NF-kB and MAPKs. Immunobiology 2013;218:
628–34.
26. Tamura T, Kurotaki D, Koizumi S. Regulation of myelopoiesis by thetranscription factor IRF8. Int J Hematol 2015;101:342–51.
27. Xiong H, Zhu C, Li H, Chen F, Mayer L, Ozato K, et al. Complex formationof the interferon (IFN) consensus sequence-binding protein with IRF-1 isessential for murine macrophage IFN-g-induced iNOS gene expression. J
Biol Chem 2003;278:2271–7.
28. Simon PS, Sharman SK, Lu C, Yang D, Paschall AV, Tulachan SS, et al.
The NF-kB p65 and p50 homodimer cooperate with IRF8 to activate
iNOS transcription. BMC Cancer 2015;15:770.
29. Netherby CS, Abrams SI. Mechanisms overseeing myeloid-derived suppressor cell production in neoplastic disease. Cancer Immunol Immunother 2017;66:989–96.
30. Waight JD, Netherby C, Hensen ML, Miller A, Hu Q, Liu S, et al. Myeloidderived suppressor cell development is regulated by a STAT/IRF-8 axis.
J Clin Invest 2013;123:4464–78.
31. Paschall AV, Zhang R, Qi CF, Bardhan K, Peng L, Lu G, et al. IFN regulatory
factor 8 represses GM-CSF expression in T cells to affect myeloid cell lineage
differentiation. J Immunol 2015;194:2369–79.
32. Pﬂanz S, Timans JC, Cheung J, Rosales R, Kanzler H, Gilbert J, et al.
IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein,induces proliferation of naive CD4þ T cells. Immunity 2002;16:
779–90.
33. Li MS, Liu Z, Liu JQ, Zhu X, Liu Z, Bai XF. The Yin and Yang aspects of IL-27
in induction of cancer-speciﬁc T-cell responses and immunotherapy.
Immunotherapy 2015;7:191–200.
34. Cho HY, Lee SW, Seo SK, Choi IW, Choi I, Lee SW. Interferon-sensitive
response element (ISRE) is mainly responsible for IFN-a-induced upregulation of programmed death-1 (PD-1) in macrophages. Biochim Biophys Acta 2008;1779:811–9.
35. Dong Z, Tai W, Lei W, Wang Y, Li Z, Zhang T. IL-27 inhibits the TGF-b1induced epithelial-mesenchymal transition in alveolar epithelial cells.
BMC Cell Biol 2016;17:7.
36. Dong Z, Zhao X, Tai W, Lei W, Wang Y, Li Z, et al. IL-27 attenuates the TGFb1-induced proliferation, differentiation and collagen synthesis in lungﬁbroblasts. Life Sci 2016;146:24–33.
37. Sharma P, Allison JP. Immune checkpoint targeting in cancer therapy:
toward combination strategies with curative potential. Cell 2015;161:
205–14.
38. Colombo MP, Trinchieri G. Interleukin-12 in anti-tumor immunity andimmunotherapy. Cytokine Growth Factor Rev 2002;13:155–68.
39. Marshall E.Cancer trial of interleukin-12 halted. Science 1995;268:1555.
40. Car BD, Eng VM, Schnyder B, LeHir M, Shakhov AN, Woerly G, et al. Role ofinterferon-g in interleukin 12-induced pathology in mice. Am J Pathol
1995;147:1693–707.
41. Ryffel B.Interleukin-12: role of interferon-gamma in IL-12 adverse effects.
Clin Immunol Immunopathol 1997;83:18–20.
42. Car BD, Eng VM, Lipman JM, Anderson TD. The toxicology of interleukin12: a review. Toxicol Pathol 1999;27:58–63.
43. Cua DJ, Sherlock J, Chen Y, Murphy CA, Joyce B, Seymour B,
et al. Interleukin-23 rather than interleukin-12 is the criticalcytokine for autoimmune inﬂammation of the brain. Nature 2003;
421:744–8.
44. Langowski JL, Zhang X, Wu L, Mattson JD, Chen T, Smith K, et al. IL-23
promotes tumour incidence and growth. Nature 2006;442:461–5.
45. Shan BE, Hao JS, Li QX, Tagawa M. Antitumor activity and immune
enhancement of murine interleukin-23 expressed in murine colon carcinoma cells. Cell Mol Immunol 2006;3:47–52.

www.aacrjournals.org

Cancer Res; 78(1) January 1, 2018

Downloaded from cancerres.aacrjournals.org on April 28, 2018. © 2018 American Association for Cancer Research.

-90-

193

Published OnlineFirst November 1, 2017; DOI: 10.1158/0008-5472.CAN-17-0960

Chiba et al.

46. Hu P, Hu HD, Chen M, Peng ML, Tang L, Tang KF, et al. Expression ofinterleukins-23 and 27 leads to successful gene therapy of hepatocellular
carcinoma. Mol Immunol 2009;46:1654–62.
47. Oniki S, Nagai H, Horikawa T, Furukawa J, Belladonna ML, Yoshimoto T,
et al. Interleukin-23 and interleukin-27 exert quite different antitumor andvaccine effects on poorly immunogenic melanoma. Cancer Res 2006;66:
6395–404.
48. Kamiya S, Owaki T, Morishima N, Fukai F, Mizuguchi J, Yoshimoto T. An
indispensable role for STAT1 in IL-27-induced T-bet expression but notproliferation of naive CD4þ T cells. J Immunol 2004;173:3871–7.

49. Owaki T, Asakawa M, Morishima N, Mizoguchi I, Fukai F, Takeda
K, et al. STAT3 is indispensable to IL-27-mediated cell proliferation but not to IL-27-induced Th1 differentiation and suppressionof proinﬂammatory cytokine production. J Immunol 2008;180:2903–11.
50. Yoshimoto T, Yoshimoto T, Yasuda K, Mizuguchi J, Nakanishi K.
IL-27 suppresses Th2 cell development and Th2 cytokines production from polarized Th2 cells: A novel therapeutic way
for Th2-mediated allergic inﬂammation. J Immunol 2007;179:
4415–23.

194 Cancer Res; 78(1) January 1, 2018

Cancer Research

Downloaded from cancerres.aacrjournals.org on April 28, 2018. © 2018 American Association for Cancer Research.

-91-

Published OnlineFirst November 1, 2017; DOI: 10.1158/0008-5472.CAN-17-0960

Interleukin-27 Exerts Its Antitumor Effects by Promoting
Differentiation of Hematopoietic Stem Cells to M1 Macrophages
Yukino Chiba, Izuru Mizoguchi, Junichi Furusawa, et al.
Cancer Res 2018;78:182-194. Published OnlineFirst November 1, 2017.

Updated version

Access the most recent version of this article at:doi:10.1158/0008-5472.CAN-17-0960

Supplementary
Material

Access the most recent supplemental material at:
http://cancerres.aacrjournals.org/content/suppl/2017/11/01/0008-5472.CAN-17-0960.DC1

Cited articles

This article cites 49 articles, 14 of which you can access for free at:
http://cancerres.aacrjournals.org/content/78/1/182.full#ref-list-1

E-mail alerts

Sign up to receive free email-alerts related to this article or journal.

Reprints andSubscriptions
Permissions

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at
pubs@aacr.org.
To request permission to re-use all or part of this article, use this link
http://cancerres.aacrjournals.org/content/78/1/182.
Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
Rightslink site.

Downloaded from cancerres.aacrjournals.org on April 28, 2018. © 2018 American Association for Cancer Research.

-92-

Leukemia & Lymphoma

ISSN: 1042-8194 (Print) 1029-2403 (Online) Journal homepage: http://www.tandfonline.com/loi/ilal20

Hidden FLT3-D835Y clone in FLT3-ITD-positive
acute myeloid leukemia that evolved into very laterelapse with T-lymphoblastic leukemia
Seiichiro Katagiri, Tomohiro Umezu, Kenko Azuma, Michiyo Asano, Daigo
Akahane, Hideki Makishima, Kenichi Yoshida, Yosaku Watatani, Kenichi
Chiba, Satoru Miyano, Seishi Ogawa, Junko H. Ohyashiki & Kazuma
Ohyashiki
To cite this article: Seiichiro Katagiri, Tomohiro Umezu, Kenko Azuma, Michiyo Asano, Daigo
Akahane, Hideki Makishima, Kenichi Yoshida, Yosaku Watatani, Kenichi Chiba, Satoru Miyano,
Seishi Ogawa, Junko H. Ohyashiki & Kazuma Ohyashiki (2017): Hidden FLT3-D835Y clone in
FLT3-ITD-positive acute myeloid leukemia that evolved into very late relapse with T-lymphoblastic
leukemia, Leukemia & Lymphoma, DOI: 10.1080/10428194.2017.1382696
To link to this article: https://doi.org/10.1080/10428194.2017.1382696

View supplementary material

Published online: 03 Oct 2017.

Submit your article to this journal

Article views: 62

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ilal20-93-

LEUKEMIA & LYMPHOMA, 2017https://doi.org/10.1080/10428194.2017.1382696

LETTER TO THE EDITOR

Hidden FLT3-D835Y clone in FLT3-ITD-positive acute myeloid leukemia thatevolved into very late relapse with T-lymphoblastic leukemia
Seiichiro Katagiria, Tomohiro Umezua,b, Kenko Azumab, Michiyo Asanoa, Daigo Akahanea,
Hideki Makishimac, Kenichi Yoshidac, Yosaku Watatanic, Kenichi Chibad, Satoru Miyanod, Seishi Ogawac,
Junko H. Ohyashikib and Kazuma OhyashikiaaDepartment of Hematology, Tokyo Medical University, Tokyo, Japan; bDepartment of Molecular Oncology, Institute of Medical
Science, Tokyo Medical University, Tokyo, Japan; cDepartment of Pathology and Tumor Biology, Kyoto University, Kyoto, Japan;dLaboratory of DNA Information Analysis, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan

ARTICLE HISTORY Received 17 June 2017; revised 19 August 2017; accepted 16 September 2017

Late relapse of acute myeloid leukemia (AML), occurring
over five years after successful treatment, is rare [1,2].
Recently, a large retrospective multicenter analysis fromthe European Society for Blood and Marrow
Transplantation indicated the possibility of very laterelapse (>10 years) of AML after autologous hematopoietic stem cell transplantation [3]. However, there havebeen few reports of very late relapse after allogeneic
hematopoietic stem cell transplantation (allo-HSCT) [4].
To our knowledge, the largest case series study was by
Watts et al., who reported three patients with very laterelapse of AML after allo-HSCT, all of whom showed
extramedullary involvement [4]. Herein, we report apatient with acute myelomonocytic leukemia (AMMoL)
with internal tandem duplication mutation of FMS-liketyrosine kinase 3 (FLT3-ITD), who maintained complete
remission (CR) for 15 years after bone marrow transplantation (BMT) but subsequently relapsed with T-lymphoblastic leukemia (T-ALL). Given the long delay between
BMT and relapse, and the different phenotype of therelapsed disease compared with AMMoL, we analyzed theorigin of the relapsed leukemia by whole-exome sequencing (WES) and droplet digital polymerase chain reaction(ddPCR) analysis. Our results showed that a very small
clone with the FLT3-D835Y mutation from the AMMoL
phase expanded in the T-ALL phase, despite the disappearance of FLT3-ITD.
A 46-year-old man with shortness of breath wasreferred to our hospital and diagnosed with AMMoL.
Blood tests revealed white blood cell (WBC) count of
3.8  109/L (with 16% blasts), hemoglobin (Hb) level of
6.2 g/dL and platelet (PLT) count of 156  109/L. Bonemarrow (BM) examination showed 85.2% myeloblasts andmonoblasts with positivity for myeloperoxidase, andmonoblasts were positive for nonspecific esterase. The

leukemic cells were also positive for CD2, CD7, CD13,
CD15, CD117 and human leukocyte antigen (HLA)-D
related (DR) by flow cytometric analysis. Cytogenetic analysis of 10 metaphases revealed one normal metaphase,
two 46,XY,der(14)t(14;?)(q32;?), three 46,XY,i(8)(q10), andfour 46,XY,i(8)(q10),der(14)t(14;?)(q32;?). Chromosome
8p11.2 translocations, including t(8;13)(p11;q12), were notdetected. Mutational analysis revealed an FLT3-ITD. The
patient failed to achieve hematological remission despite
two courses of induction by idarubicin and cytarabine. He
then received two courses with cytarabine, aclarubicinand granulocyte-colony-stimulating factor (CAG) andachieved hematological and cytogenetic CR. After two
consolidation therapies (CAG and mitoxantrone plus
high-dose cytarabine), the patient underwent a BMT froman HLA-matched sibling donor after conditioning withhigh-dose cyclophosphamide and total body irradiation.
The patient received cyclosporine and short-term methotrexate for graft-versus-host disease (GVHD) prophylaxis.
The patient maintained CR without acute (grade II–IV) or
chronic GVHD after BMT and was followed up.
After 15 years, the patient (aged 61 years) was found tohave bicytopenia (WBC count of 2.9  109/L without peripheral blood blasts, Hb level of 8.8 g/dL and PLT count of
234  109/L) with 60% BM blasts. These cells were negative
for myeloperoxidase or esterase staining; positive for CD2,
cytoplasmic CD3, CD7, CD11b, CD13, CD71, CD117 andHLA-DR by flow cytometric analysis; and positive for terminal deoxynucleotidyl transferase and CD99 by immunostaining. The patient was diagnosed with T-ALL according
to the criteria of the WHO. Cytogenetic analysis of 21 metaphases revealed normal results for two, and 46,XY,4,der(5)t(5;?)(q31;?),-13,der(14)t(14;?)(q32;?),þ2mar for 19.
Whole BM chimerism analysis showed mixed chimerism(donor chimerism: 50%). Molecular analysis revealed

CONTACT Seiichiro Katagiri
patchsei@yahoo.co.jp
Department of Hematology, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo
160-0023, Japan
Supplemental data for this article can be accessed here.
ß 2017 Informa UK Limited, trading as Taylor & Francis Group

-94-

2

S. KATAGIRI ET AL.

Figure 1. Analysis between acute myelomonocytic leukemia at onset and relapsed leukemia by whole-exome sequencing anddroplet digital polymerase chain reaction. (A) Diagram of variant allele frequency distribution of somatic mutations between
AMMoL and the T-ALL phase. Brown dots represent somatic mutations detected in both the AMMoL and T-ALL phases. Thirty-four
mutations detected only in the AMMoL phase, including TET2-T4997G, became undetectable in the T-ALL phase. The FLT3-D835Y
mutation was detected in the T-ALL phase with high frequency, while the mutation was detected with very low frequency in the
AMMoL phase. RPTN-M538I was consistently detected with VAF 0.029–0.098, demonstrating the common genetic change with low
frequency in the affected patient and his sibling. (B) Droplet digital polymerase chain reaction of the FLT3-D835Y mutation. The
FLT3-D835Y mutation was detected using a QuantStudio 3D AnalysisSuite system (Thermo Fisher Scientific, Waltham, MA) and a
FLT3-D835Y probe set derived from the Bio-Rad FLT3 WT and D835Y Assay Kit (Bio-Rad Laboratories, Hercules, CA). Red dots, VICFLT3 WT; blue dots, FAM-FLT3-D835Y; green dots, FAM-FLT3-D835Y þ VIC-FLT3 WT; yellow dots, no amplification. FLT3-D835Y-positive clones were detected at low levels during the AMMoL phase but at higher levels in the T-ALL phase (detection limit: 0.1%).
AMMoL: acute myelomonocytic leukemia; BMMCs: bone marrow mononuclear cells; BMSCs: bone marrow stroma cells; T-ALL:
T-lymphoblastic leukemia; VAF: variant allele frequency.

-95-

CLONAL SWEEPING IN RELAPSE AML AFTER BMT

reconstitution of T-cell receptor c without the presence of
the FLT3-ITD mutation. The patient was treated with CAG
but failed to achieve hematological remission. He underwent a second BMT from the same sibling donor after conditioning with high-dose cytarabine, fludarabine andmelphalan and achieved hematological remission 36 days
post-transplantation. Cytogenetic analysis of BM showed anormal karyotype with complete donor chimerism. The
patient received cyclosporine for GVHD prophylaxis andremained in CR for two years without acute or chronic
GVHD after his second BMT.
To determine the origin of T-ALL, we performed WESof BM mononuclear cells (BMMCs) collected at four time
points (onset [AMMoL], CR at six months, CR at 2.5 years
after the first BMT, and at the T-ALL phase) and of BM
stromal cells (BMSCs) collected at the BAL phase. DNA
from the buccal mucosa was used as a germline control.
Written informed consent was obtained from the patient
for sequencing of normal and leukemic tissues, and thestudy was approved by the institutional review board ofTokyo Medical University (no. 2151). We detected eight
nonsynonymous mutations (EP300, CUL3, KCNMA1, TONSL,SERPINB4, SMC5, CCDC50, C3orf80) and four synonymous
mutations (Supplementary Table 1), shared by BMMCs at
the AMMoL and T-ALL phases (Figure 1(A)), which were
not detected in the other samples tested. The FLT3D835Y mutation was detected only in the T-ALL phase(variant allele frequency (VAF), 0.469), while the mutation
was detectable with VAF of 0.026 at the time of the first
AMMoL phase (Figure 1(A)). Because the VAF of less than
0.05 is usually considered to be negative, we performed
ddPCR on the BMMCs at AMMoL onset and the T-ALL
phase, as well as on BMSCs at relapse. FLT3-D835Y wasdetected in 0.43% and 40.89% of BMMCs at AMMoL
onset and at the T-ALL phase, respectively, but wasabsent from BMSCs (0.024%) (Figure 1(B)).
The pathogenesis of AML involves an initial mutationin hematopoietic stem cells, and subsequent acquisitionof driver and passenger mutations in the preleukemic
clones. Thus, a main clone and minor subclones are present at disease onset [5]. When a subclone evolves tobecome the dominant clone at relapse, the relapsed
clone also retains the mutations in the founding clone[6]. In this study, we detected eight nonsynonymous
mutations that were present at both disease onset andrelapse, indicating that the primary and relapsed leukemic clones were derived from the same founding
clone. A common FLT3-D835Y mutation was detected atboth onset and the T-ALL phase, demonstrating that the
T-ALL clone was present as an extremely low-frequency
subclone at disease onset. Kindler et al. demonstrated
that the FLT3-D835Y mutation causes constitutive activation of the FLT3 receptor, promoting tumor growth in
AML [7]. In earlier reports of FLT3-ITD-positive AML, FLT3ITD was not always responsible for relapse, and othersubclones may have evolved at relapse [8–10]. Baker
et al. identified new FLT3-tyrosine kinase domain

3mutations at relapse after FLT3 tyrosine kinase inhibitor
treatment for FLT3-ITD-positive AML patients [11].
In the present case, ddPCR revealed the presence of
the FLT3-D835Y mutation in the AMMoL phase, suggesting that a small clone with the FLT3-D835Y mutation
remained present for 15 years after BMT and evolved as
the dominant clone at the T-ALL phase. Clonal sweeping
is a phenomenon whereby a minor clone emerges as the
predominant clone by replacing other clones present at
the time of diagnosis. Clonal sweeping has been demonstrated in myelodysplastic syndrome that evolved into
AML [12]. In this study, we detected eight nonsynonymous mutations (EP300, CUL3, KCNMA1, TONSL, SERPINB4,
SMC5, CCDC50, C3orf80) that were identified in both the
AMMoL and T-ALL phases. Among them, EP300, encoding
a histone acetyltransferase, is closely related to CREBbinding protein (CREBBP) and acts as a transcriptional
coactivator. EP300/CREBBP mutations were recently
reported to be related to the pathogenesis of B-cell nonHodgkin lymphoma [13] and the progression from severecongenital neutropenia to AML [14]. We therefore speculate that the EP300 mutation might have been a driver
mutation of the founding clone in our patient. However,during the long-lasting CR state, clonal hematopoiesis ofindeterminate potential-related mutations [15], such as
TET2 and DNMT3A, were not detectable in the leukemic
cells or BMSCs at the T-ALL phase.
In a previous report, patients with very late relapses of
AML after allo-HSCT showed extramedullary involvement,suggesting that the graft-versus-leukemia effect may be
important in controlling dormant leukemia and impaired
immune surveillance may be a risk factor for late relapse[4]. The present report indicates that clonal sweeping of
subclones may be an important factor for late relapse insome AML patients after allo-HSCT. Further knowledge
may clarify the molecular mechanisms underlying verylate relapse of AML.

Acknowledgements
This study was supported by the Platform Project for
Practical Research for Innovative Cancer Control from theJapan Agency for Medical Research and Development(AMED) (#15Ack0106073h0002) (KO: Tokyo, SO: Kyoto) and
the Private University Strategic Research-Based Support
Project (S1311016) from the Ministry of Education, Culture,
Sports, Science, and Technology (Tokyo, Japan).

Potential conflict of interest: Disclosure forms provided
by the authors are available with the full text of this article
online at https://doi.org/10.1080/10428194.2017.1382696.

References

-96-

[1]

€hner K, Krauter J, et al. Mutations andSchlenk RF, Do
treatment outcome in cytogenetically normal acute myeloid
leukemia. N Engl J Med. 2008;358:1909–1918.

4

S. KATAGIRI ET AL.

[2]

[3]

[4]

[5]
[6]

[7]

[8]

Medeiros BC, Minden MD, Schuh AC, et al. Characteristics
and outcomes of acute myelogenous leukemia patients withvery late relapse (>5 years). Leuk Lymphoma. 2007;48:65–71.
Czerw T, Labopin M, Gorin NC, et al. Long-term follow-up ofpatients with acute myeloid leukemia surviving and free of
disease recurrence for at least 2 years after autologous stem
cell transplantation: a report from the Acute Leukemia
Working Party of the European Society for Blood and Marrow
Transplantation. Cancer. 2016;122:1880–1887.
Watts JM, Wang XV, Swords RT, et al. Very late relapse of AML
after allogeneic hematopoietic cell transplantation is often
extramedullary. Bone Marrow Transplant. 2016;51:1013–1015.
Welch JS, Ley TJ, Link DC, et al. The origin and evolution of
mutations in acute myeloid leukemia. Cell. 2012;150:264–278.
Ding L, Ley TJ, Larson DE, et al. Clonal evolution in relapsed
acute myeloid leukaemia revealed by whole-genome
sequencing. Nature. 2012;481:506–510.
Kindler T, Lipka DB, Fischer T. FLT3 as a therapeutic target in
AML: still challenging after all these years. Blood. 2010;116:
5089–5102.
Tiesmeier J, M€
uller-Tidow C, Westermann A, et al. Evolutionof FLT3-ITD and D835 activating point mutations in relapsing
acute myeloid leukemia and response to salvage therapy.
Leuk Res. 2004;28:1069–1074.

[9]

[10]

[11]

[12]

[13]

[14]

[15]

-97-

Warren M, Luthra R, Yin CC, et al. Clinical impact of changeof FLT3 mutation status in acute myeloid leukemia patients.
Mod Pathol. 2012;25:1405–1412.
Meloni G, Mancini M, Gianfelici V, et al. Late relapse of acute
myeloid leukemia with mutated NPM1 after eight years: evidence of NPM1 mutation stability. Haematologica. 2009;94:
298–300.
Baker SD, Zimmerman EI, Wang YD, et al. Emergence ofpolyclonal FLT3 tyrosine kinase domain mutations during
sequential therapy with sorafenib and sunitinib in FLT3-ITDpositive acute myeloid leukemia. Clin Cancer Res. 2013;
19:5758–5768.
Makishima H, Yoshizato T, Yoshida K, et al. Dynamics of
clonal evolution in myelodysplastic syndromes. Nat Genet.
2017;49:204–212.
Pasqualucci L, Dominguez-Sola D, Chiarenza A, et al.
Inactivating mutations of acetyltransferase genes in B-cell
lymphoma. Nature. 2011;471:189–195.
Beekman R, Valkhof MG, Sanders MA, et al. Sequential gainof mutations in severe congenital neutropenia progressing toacute myeloid leukemia. Blood. 2012;119:5071–5077.
Steensma DP, Bejar R, Jaiswal S, et al. Clonal hematopoiesis
of indeterminate potential and its distinction from myelodysplastic syndromes. Blood. 2015;126:9–16.

REGULAR ARTICLE

Replenishing exosomes from older bone marrow stromal cells with
miR-340 inhibits myeloma-related angiogenesis
Tomohiro Umezu,1,2 Satoshi Imanishi,2 Kenko Azuma,2 Chiaki Kobayashi,1,2 Seiichiro Yoshizawa,1 Kazuma Ohyashiki,1 andJunko H. Ohyashiki2
1

Department of Hematology and 2Department of Molecular Oncology, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan

Key Points• Exosomal miR-340 derived from young
BMSCs inhibited tumor
angiogenesis via the
HGF/c-MET signaling
pathway.
• The anti-angiogenic effect of exosomes fromolder BMSCs was restored by direct transfection of young
BMSC-derived exosomal miRNAs.

The study of bone marrow stromal cells (BMSCs) and the exosomes they secrete is considered
promising for cancer therapy. However, little is known about the effect of donor age on
BMSCs. In the present study, we investigated the therapeutic potential of BMSC exosomes
derived from donors of different ages using an in vivo model of hypoxic bone marrow in
multiple myeloma (MM). We found that donor age was strongly related to senescent changes
in BMSCs. Exosomes derived from young BMSCs signiﬁcantly inhibited MM-induced
angiogenesis in Matrigel plugs. The exosomal microRNA (miRNA) expression proﬁle wasdifferent between young and older BMSCs, despite similarities in the size and quantity of
exosomes. Of note was the observation that the antiangiogenic effect of older BMSCs wasenhanced by direct transfection of miR-340 that was preferentially expressed in exosomes
derived from young BMSCs. We found that miR-340 inhibited angiogenesis via the hepatocyte
growth factor/c-MET (HGF/c-MET) signaling pathway in endothelial cells. Our data provide
new insights into exosome-based cancer therapy by modiﬁcation of BMSC-derived exosomes.

Introduction
The tumor microenvironment has gradually been recognized as a key contributor to cancer progression,angiogenesis, and metastasis1 and to the development of drug resistance.2 Therefore, future
development of anticancer therapies3 should consider the tumor microenvironment. Bone marrowstromal cells (BMSCs) interact with multiple myeloma (MM) cells in the bone marrow (BM). BMSCs also
create a permissive microenvironment for MM cell growth and survival.4 An increase of angiogenesis is
involved in the progression of MM and the cell-to-cell contact between MM cells, whereas BMSCs
regulate the secretion of key mediators of angiogenesis, such as vascular endothelial growth factor andbasic fibroblast growth factor.5
It is well established that MM is an incurable disease. It commonly occurs in older people with a medianage at diagnosis of approximately 70 years.6,7 A previous study by Andre et al demonstrated that
BMSCs in MM patients have an early senescent profile.8 Furthermore, BMSC aging results in adecrease of their ability to maintain the BM microenvironment, leading to increases in the incidenceof hematological malignancies, anemia, and immune dysfunction.9,10 Recent evidence indicates that
exosome-mediated cell-to-cell communication plays an important role in maintaining the BM
microenvironment.11
Exosomes are 30- to120-nm membrane-bound vesicles secreted naturally by almost all cells and exist in
all body fluids.12 Accumulating evidence has shown that exosomes contain proteins, lipids, DNA,
messenger RNA (mRNA), microRNA (miRNA), and long noncoding RNA that can be transferred fromproducer cells to recipient cells, thereby facilitating cell-to-cell communication.13-16 More recent studies
in animal models have indicated that healthy BMSC-derived exosomes are effective for treatment ofSubmitted 28 November 2016; accepted 13 April 2017. DOI 10.1182/
bloodadvances.2016003251.

© 2017 by The American Society of Hematology

The full-text version of this article contains a data supplement.

812

23 MAY 2017 x VOLUME 1, NUMBER 13-98-

cardiovascular disease,17 lung injury,18,19 kidney injury,20,21 andliver injury.22 These findings suggest that the contents transferredby healthy BMSC-derived exosomes have a therapeutic potential.
Kordelas et al demonstrated that the clinical symptoms of graftversus-host disease are significantly improved after treatment withhealthy BMSC-derived exosomes.23 In a manner similar to thatof BMSCs, BMSC-derived exosomes have lower antigenicity
than do various cell types used for cell therapy, such as activated T cells.24,25 Although aging of BMSCs has been studied
extensively, the effect of exosomes derived from older BMSCs on
the BM microenvironment remains unclear. Therefore, we hypothesized that the therapeutic potential of healthy BMSC-derived
exosomes depends largely on the donor age, possibly because ofage-specific differences in their contents (eg, mRNA, miRNA, andprotein).
To test this hypothesis, we investigated the donor age–relateddifferences and therapeutic effects of healthy BMSC-derived
exosomes by using a heterotransplant mouse model of chronic
hypoxia-resistant (HR) MM cells.26 HR-MM cells showed continuous growth under persistent hypoxic conditions, and exosomes
derived from HR-MM cells affected angiogenic functions in bothin vitro and in vivo models.26 In the present study, we modified
BMSC-derived exosomes from older healthy donors by directlytransfecting them with miRNA expressed specifically in young
BMSC exosomes. This approach resulted in exosomes derived
from older BMSCs acquiring antiangiogenic properties, suggesting that exosomal miRNA replacement has a therapeuticpotential.

Methods
Cell lines and culture conditions
Human MM cell lines (RPMI8226, KMS-11, and IM-9) werepurchased from the Health Science Research Resource Bank
(Osaka, Japan). Sublines that survived well under long-term hypoxia(HR-MM cell lines; RPMI8226-HR, KMS-11-HR, and IM-9-HR)26
were maintained under hypoxic conditions (1% O2, 5% CO2, and
94% N2) in a humidified, gas-sorted hypoxic incubator (MCO-5M;
Panasonic, Osaka, Japan). Pooled human umbilical vein endothelial
cells (HUVECs) and human BMSCs from young (donor ages: 19
and 20 years old; young BMSCs) and older (donor ages: 68 and 72
years old; older BMSCs) healthy donors were purchased from
Lonza Inc. (Allendale, NJ). BMSCs were harvested by trypsinizationand either passaged for expansion or subjected to analyses(passages 1-5). During days 1 to 5, the number of viable cells wasdetermined using a commercial kit (Cell Counting Kit-8; Dojindo,
Kumamoto, Japan). See the supplemental experimental proceduresfor details.

Measurement of terminal restriction
fragments (TRFs)
Genomic DNA were extracted from each group of BMSCs by using
the Gentra Puregene Cell kit (Qiagen, Hiden, Germany) anddigested by restriction enzymes (Hinf I or Rsa I). Digested DNA
were fractionated by electrophoresis on 0.7% agarose gel and thentransferred to nylon membranes. Hybridization was performed with
the Telo TTAGGG Telomere Length Assay kit (Roche, Mannheim,
Germany) according to the manufacturer’s instructions. Smears ofthe developed films were captured on a VersaDoc 1000 imaging
system (Bio-Rad, Hercules, CA).

Senescence-associated (SA) b-galactosidase(b-gal) staining
BMSCs were fixed in 0.2% glutaraldehyde, washed with phosphatebuffered saline (PBS), and incubated overnight at 37°C (without
CO2) in staining solution (1 mg/mL of X-gal in 40 mM of citric acid/sodium phosphate, pH 6.0, 5 mM of potassium ferrocyanide, 5 mMof potassium ferricyanide, 150 mM of NaCl, and 2 mM of MgCl2).
After incubation, the cells were washed with PBS and imaged for thepresence of blue staining.

Isolation and characterization of exosomes
BMSCs (4 3 104 cells/cm 2 ) were cultured in T-25 flasks (BDBiosciences, San Jose, CA) containing 5 mL of serum-free AIM-V
medium (Invitrogen, Carlsbad, CA). The culture supernatants were
harvested after 48 h of incubation, and the exosome fraction was
purified using Exoquick-TC reagent (System Biosciences, Mountain
View, CA) according to the manufacturer’s instructions. Exosomes
were quantified by nanoparticle tracking analysis (NanoSight LM10;
Malvern, Herrenberg, Germany) and observed under a transmission
electron microscope (JEM-1200EX; JEOL, Tokyo, Japan), as described previously.27 See the supplemental experimental proceduresfor details.

Direct transfection of miRNA mimics into exosomes
miRNA mimics (Ambion; Invitrogen) were labeled with the Label
IT small interfering RNA Tracker Cy3 kit (Mirus, Madison, WI)
according to the manufacturer’s instructions. mirVana miRNA mimics(see supplemental Table 1 for mimic ID) (Applied Biosystems) weretransfected directly into exosomes using Exo-fect (System Biosciences). See the supplemental experimental procedures for details.

miRNA expression profiles
Cellular and exosomal miRNAs were isolated using the miRNeasykit (Qiagen), and miRNA profiling of both cells and exosomes was
performed using the TaqMan low-density miRNA array (Thermo
Fisher Scientific, Carlsbad, CA) according to the manufacturer’s
recommendations. The relative expression of each gene was calculated using the comparative threshold cycle method, as described
previously.28 See the supplemental experimental procedures fordetails.

Tube formation assay
The formation of capillary-like structures was assessed, as described previously.27 HUVECs (2 3 104 cells/well) were seeded on
Matrigel (growth factor reduced, 280 mL/well; BD) in 24-well plates
and treated with exosomes (1.2 3 107 particles) in endothelial basal
medium-2 (480 mL/well; Lonza Inc.), supplemented with or without
endothelial growth medium-2 SingleQuots and 5% fetal bovine
serum for 24 h. BMSC-derived exosomes were titrated to determine
the optimal concentration for tube formation assay (supplemental
Figure 1). The total area of tube formation was quantified as themean pixel density obtained from image analysis of 5 random
microscopic fields using ImageJ software (http://rsb.info.nih.gov/
nih-image/).

In vivo Matrigel plug assay
A mixture of growth factor–reduced Matrigel (200 mL) and 4 3 106
HR-MM cells was admixed with exosomes derived from BMSCs(4 3 107 particles). The Matrigel mixtures were injected subcutaneously

23 MAY 2017 x VOLUME 1, NUMBER 13

EXOSOMAL miR-340 INHIBITS TUMOR ANGIOGENESIS

-99-

813

older BMSCs(donor age: 19 years)(donor age: 68 years)

young BMSCs

older BMSCs(donor age: 20 years)(donor age: 72 years)

B

E
10

M
ar
k
yo er
un
g
B
M
ol
SC
de
s
rB
M
SC
s

young BMSCs

Cell number (x105/ml, log)A

young BMSCs
older BMSCs

(kB)
20
1
8
5
0

24

48

72

96 120hrs
2D
older BMSCs(donor age: 19 years)(donor age: 68 years)

β-gal

β-gal

F
60

20

*
Telomere length (mean TRF, kbp)

young BMSCs

SA-β-gal positive cells (%)

C

40

20

s
SC

SC

M

(donor age: 72 years)

M(donor age: 20 years)

15

10

5

0

yo

G

rB
de

β-gal

β-gal

ol

un

g

B

M

M

SC

SC

s

s

yo

ol

un

de

g

rB

B

older BMSCss

0

young BMSCs*

older BMSCs

H

Nanoparticle concentration (x 108 particle/ml)Iyoung BMSCs

3

2

young BMSCs
older BMSCs

1

0
CD63

0

100

200

300

Particle size (nm)CD81

young BMSCs

older BMSCs
Figure 1.

814

UMEZU et al

23 MAY 2017 x VOLUME 1, NUMBER 13

-100-

400

into 8-week-old male nude mice (BALB/c-nu/nu; CLEA Japan, Tokyo,
Japan). All experimental groups included 3 mice. Three weeks after
implantation, the Matrigel plugs were harvested and processed foranalysis. The Matrigel plugs were fixed with 4% paraformaldehyde
and embedded in paraffin. Then, 8-mm-thick paraffin sections
were prepared using a microtome (RM2245; Leica Biosystems,
Heidelberg, Germany). Neovascularization was evaluated byimmunohistochemistry. The total CD31-positive area was quantified as the mean pixel density obtained from image analysis of 6
random microscopic fields by using ImageJ software. See thesupplemental experimental procedures for details on analysis ofcapillary density.

Western blotting
Western blots were probed with antibodies against cMET (rabbit
polyclonal, 1:500; C-28; Santa Cruz Biotechnology, Santa Cruz,
CA), phosphorylated cMET (p-cMET) (rabbit polyclonal, 1:100;
ab5662; Abcam, Cambridge, MA), hepatocyte growth factor (HGF)(rabbit polyclonal, 1:100; ab83760; Abcam), and b-actin (mousemonoclonal, 1:2500; Chemicon, Temecula, CA). CD63 (mousemonoclonal anti-CD63 antibody, 1:100, sc-5275; Santa Cruz
Biotechnology) and CD81 (mouse monoclonal anti-CD81 antibody; 1:100; sc-23962; Santa Cruz Biotechnology) were usedas markers of exosomes. Horseradish peroxidase–conjugatedsecondary antibodies were purchased from GE Healthcare Life
Science (Uppsala, Sweden). See the supplemental experimental
procedures for details.

EthicsAll animal experiments were conducted in compliance with theinstitutional guidelines of the Animal Experimental Center of Tokyo
Medical University/Animal Biosafety Level II Laboratory for Useof Animals. The experimental protocols were approved by theInstitutional Animal Care and Use Committee of Tokyo Medical
University. The use of patient samples was approved by theInstitutional Review Board of Tokyo Medical University (no. 2648,
approved 22 April 2014). Written informed consent was obtained
from all participants before specimen collection, according to the
Declaration of Helsinki.

Results
Donor age–dependent senescent changes in BMSCs
There were no morphological differences between BMSCs derived
from young or older healthy donors (Figure 1A). The cell proliferation rate was not significantly different between young andolder BMSCs for at least 72 h in culture, although older BMSCs
had a gradual reduction in their proliferative capability (Figure 1B).
The number of SA-b gal-positive cells among older BMSCs was
significantly higher than that among young BMSCs at the same
passage (Figure 1C-D; P , .01). Similarly, TRFs in older BMSCs(11 6 0.99 kbp) were shorter than were those in young BMSCs(14.8 6 1.41 kbp) (Figure 1E-F), suggesting that senescenceassociated changes in BMSCs are dependent on the donor age.

Characterization of exosomes derived from young
and older BMSCs
The size and structure of exosomes appeared to be similar between
young and older BMSCs (Figure 1G). Additionally, the expression ofcommon exosomal markers (CD63 and CD81) was similar between
young and older BMSCs (Figure 1H). The particle size of BMSCderived exosomes was approximately 50 nm. On average, after 48 hof culture, 1.9 3 108/mL exosomes and 2.3 3 108/mL exosomes
were found in the culture supernatants of young BMSCs and older
BMSCs, respectively (Figure 1I).

In vivo model of the MM hypoxic niche
We previously established HR-MM cells derived from MM cell lines(RPMI8226-HR, KMS-11-HR, and IM-9-HR) to evaluate thepotential role of MM cells in the hypoxic BM environment. We foundthat HR-MM cells induce tumor angiogenesis under chronic hypoxic
conditions.26 In this study, we used an in vivo Matrigel plug angiogenesis assay to analyze the angiogenic responses to HR-MM
cells in vivo (supplemental Figure 2). HR-MM cells formed tumors
with increased amounts of CD31-positive endothelial cells in the
Matrigel plug (supplemental Figure 2A,C), whereas parental cells did
not grow (supplemental Figure 2B-C). Among them, RPMI8226-HR
cells induced higher levels of angiogenesis in comparison with other
HR-MM cell lines (supplemental Figure 2A). Therefore, we primarily
used RPMI8226-HR cells in this study.

Statistical analyses
Data are presented as the mean 6 standard deviation (SD).
Comparisons between 2 groups were made with the Student t test.
Multiple group comparisons were made by analysis of variance.
GraphPad Prism (version 5c) for Macintosh (GraphPad Inc., La
Jolla, CA) was used for statistical analyses. Results were considered
statistically significant at P , .05.

Antiangiogenic effects of BMSC-derived exosomesfrom young donors
BALB/c nude mice were injected subcutaneously with HR-MM cells(RPMI8226-HR) seeded in Matrigel plugs containing exosomes(4 3 10 7 particles per 200 mL Matrigel) derived from young
BMSCs (young BMSC exosomes) or older BMSCs (older BMSC

Figure 1. Characterization of BMSCs. (A) Morphology of BMSCs derived from 2 young healthy donors (young BMSCs; passage 2; donor ages: 19 and 20 years old) and 2
older healthy donors (older BMSCs; passage 2; donor age: 68 and 72 years) by phase-contrast inverted microscopy. Scale bar, 50 mm. (B) BMSC growth measured after 24, 48,
72, 96, and 120 h in culture. Cell proliferation of young BMSCs (pink line) and older BMSCs (blue) was evaluated. Each value represents the mean 6 SD (n 5 6). (C-D)
Representative images of SA-b-gal staining (arrows indicate positive cells) and quantification of cell senescence. Data represent the mean 6 SD. *P , .01 in comparison with young
BMSCs using a Student t test. Scale bar, 50 mm. (E-F) Telomere lengths in young and older BMSCs determined by Southern blotting of TRFs. Data represent the mean 6 SD.
*P , .05 in comparison with young BMSCs using a Student t test. (G) Transmission electron micrographs of exosomes derived from young and older BMSCs. Scale bar, 50 nm.
(H) Western blots of exosomal lysates (CD63 and CD81 are common exosomal markers) derived from young and older BMSCs. Exosomes were isolated from 5 mL of culture
medium collected from BMSCs (4 3 104 cells/cm2) grown for 48 h. Equal volumes of exosomal lysate (30 mL) were loaded in the lanes of gels. (I) Nanoparticle concentration andsize distribution of exosomes derived from young BMSCs (pink line) and older BMSCs (blue line).

23 MAY 2017 x VOLUME 1, NUMBER 13

EXOSOMAL miR-340 INHIBITS TUMOR ANGIOGENESIS

-101-

815

A

B
RPMI8226-HR+
PBS (control)

RPMI8226-HR
Matrigel plug

RPMI8226-HR+
older BMSC
exosomes

RPMI8226-HR+
young BMSC
exosomes

PBS (control)oryoung BMSC exosomesorolder BMSC exosomes

BALB/c-nu/nuC
Vessel density(relative CD31+ area)

1.5

1.0

*
0.5

0.0
RPMI8226-HR
young BMSC
exosomes
older BMSC
exosomes

CD138 / CD31

CD138 / CD31

DAPI / CD31

DAPI / CD31

DAPI / CD31

**
+

+

+

–

+

–

–

–

+

E

D
1.2

Cell viability (fold of control)

CD138 / CD31

Control

young BMSC
exosomes

older BMSC
exosomes

1.0
0.8
0.6
0.4

F
P = 0.038

0.0
young BMSC
exosomes
older BMSC
exosomes

–

+

–

–

–

+

Relative tube formation(fold of control)

0.2

1.5

P < 0.001

P = 0.007

1.0
0.5

0.0
young BMSC exosomes
older BMSC exosomes

–
–

+
–

–
+

Figure 2. Healthy BMSC exosomes inhibit HR-MM cell-induced angiogenesis in Matrigel plugs. (A) Schematic of the Matrigel plug assay. A mixture of Matrigel and
4 3 106 RPMI8226-HR cells was admixed with exosomes derived from young BMSCs (young BMSC exosomes; 4 3 107 particles per 200 mL Matrigel) or older BMSCs(older BMSC exosomes; 4 3 107 particles per 200 mL Matrigel). (B) After 3 weeks, the Matrigel plugs were harvested and photographed. Paraffin-embedded sections of Matrigel
plugs were stained with hematoxylin and eosin and then subjected to immunostaining for CD138 (brown) and CD31 (red). Cell nuclei were stained with 49,6-diamidino-2phenylindole (DAPI) (blue). Scale bar, 500 mm. (C) Quantification of vessel density in Matrigel plugs by pixel density. Young and older BMSC exosomes inhibited tumor angiogenesis
in Matrigel plugs compared with the control (RPMI8226-HR only) (*P , .01; **P , .001 vs control, Student t test, n 5 6). Values represent the mean 6 SD. (D) Effect of
BMSC exosomes on the viability of HR-MM cells. RPMI8226-HR cells were cultured with young or older BMSC exosomes (2.5 3 107 particles/mL). Cell viability was assayed after

816

UMEZU et al

23 MAY 2017 x VOLUME 1, NUMBER 13

-102-

B
g

B
ol
d

yo
un

(B) Schematic of the modification of older BMSC exosomes.

• Tube formation
Assay (in vitro)• Matrigel plug
assay (in vivo)

older BMSC exosomesSC

exosomes was determined by TaqMan miRNA array analysis.

M

M

SC

Differential miRNA expression of young and older BMSC

s

s

A
B

exosomes derived from young and older BMSCs. (A)erFigure 3. Differential miRNA expression profiles ofYoung BMSC exosome-specific miRNA mimics weretransfected directly into older BMSC exosomes by Exo-fect.

older BMSCs

antiangiogenic effect of modified exosomes. Older BMSC
exosomes transfected with miR-340 mimics (miR-340
exosomes) and older BMSC exosomes transfected with
miR-365 mimics (miR-365 exosomes) significantly inhibited
HUVEC tube formation in comparison with the control (older

24 upregulated
exosomal miRNAs(C) Quantification of tube-like structures to determine thedirecttransfection

young BMSC exosomes-specific miRNA mimics

C
1.5
ΔCt

miR) (control vs miR-340 exosomes; *P , .01, Student t test;
the mean 6 SD. Ct, comparative threshold cycle.

8

12 downregulated
exosomal miRNAs**P , .001, control vs miR-365 exosomes). Values represent-8Relative tube formation(fold of control)

BMSC exosomes transfected with negative control miR; control

1.0

*
0.5

**

co

nt

ro
l
m miR
iR
m 10
iR a
m -41
iR 1
-4
m 87
iR a
m -1
iR 4
-2 5
m 8-5
iR p
-3
7
m 4a
iR
m -24
iR
m -34
iR 0
-1
m 33
iR a
m 365
iR
m 328
iR
-1
85

0.0

exosomes) (Figure 2A). We observed hyperproliferative CD138positive RPMI8226-HR cells and CD31-positive endothelial cells
attracted into the Matrigel plug (Figure 2B, left). However, older
BMSC exosomes inhibited the proliferation of RPMI8226-HR
cells and angiogenesis in the Matrigel plug (Figure 2B, right) in comparison with the control (RPMI8226-HR 1 PBS) (Figure 2B-C; P , .05).
In addition, the antiangiogenic effect was more evident with young
BMSC exosomes than with older BMSC exosomes (Figure 2B, middle,
and Figure 2C; P , .001).
To test whether BMSC exosomes directly affect the cell viabilityof HR-MM cells, we measured the cell proliferation of RPMI8226HR cells treated with young or older BMSC exosomes (2.5 3 107
particles/mL). Neither older BMSC exosomes nor young BMSC
exosomes affect the survival of RPMI8226-HR cells (Figure2D). We assessed effector caspase activity in RPMI8226-HR
cells treated with BMSC exosomes (2.5 3 10 7 particles/mL).
There was no significant difference in caspase 3/7 activity in
RPMI8226-HR cells treated with and without BMSC exosomes(supplemental Figure 3B). Furthermore, we performed experiments to assess nonapoptotic cell death mechanisms in HR-MM
cells, including autophagic cell death. The expression of p62
protein showed no significant difference between RPMI8226-HR
cells treated with BMSC exosomes and without (supplemental
Figure 3B).

We next performed an endothelial tube formation assay with
HUVECs treated with young or older BMSC exosomes (2.5 3 107
particles/mL). Consistent with the results obtained from the in vivo
Matrigel plug assay, HUVEC tube formation was inhibited to ahigher degree by young BMSC exosomes than they were by older
BMSC exosomes (young BMSC exosomes vs older BMSC exosomes;
P 5 .007) (Figure 2E-F).

Identification of young BMSC exosome-specificmiRNAs
To identify the exosomal miRNAs associated with the antiangiogenic effect of young BMSC exosomes, we first comparedexosomal miRNA expression profiles between young and older
BMSCs (National Center for Biotechnology Information [NCBI],
gene expression omnibus; GSE78865). A subset of 12 miRNAs
was significantly downregulated in older BMSC exosomes. ThesemiRNAs were specifically expressed in young BMSC exosomes(Figure 3A). Subsequently, we replenished the 12 miRNAs in older
BMSC exosomes by direct transfection with miRNA mimics (Figure3B) and investigated whether young BMSC exosome-specific miRNAs
affect HUVEC tube formation. Exosomes transfected with miR-340 or
miR-365 mimics significantly inhibited angiogenesis in comparison
with scrambled control miRNA-containing exosomes (control miR
exosomes) (Figure 3C; P , .01, P , .001).

Figure 2. (continued) 48 h using Cell Counting Kit-8. Values represent the mean 6 SD. (E) The formation of tube-like structures was observed using the cell-permeable
dye Calcein AM (green). Endothelial tube formation of HUVECs treated with young BMSC exosomes, older BMSC exosomes, or the control (without exosomes). Scale bar,
500 mm. (F) Quantification of tube-like structures by pixel density. Young and older BMSC exosomes significantly inhibited HUVEC tube formation in comparison withthe control (control vs young BMSC exosomes; P , .001, control vs older BMSC exosomes; P 5 .038, Student t test). Values represent the mean 6 SD.

23 MAY 2017 x VOLUME 1, NUMBER 13

EXOSOMAL miR-340 INHIBITS TUMOR ANGIOGENESIS

-103-

817

A

quantified by reverse transcription polymerase chain reaction(young BMSC exosomes vs older BMSC exosomes; *P , .01,

4

young BMSC exosomes vs miR-340 exosomes; **P , .001,*

Student t test). Values represent the mean 6 SD. (B-C) Recipient2

cells were treated with exosomes directly transfected with Cy3miR-340 mimics. Cy3-miR-340 signals were detected in theexSCCy3-miR-340 / DAPI

Cy3-miR-340 / DAPI
Calcein AM

cytoplasm of RPMI8226-HR cells (B) and HUVECs (C). Nuclear
and cytoplasmic staining were performed with DAPI (blue) andCalcein AM (green), respectively. Scale bar, 25 mm. (D) A mixture

m

SC

M
B
er

ol
d

B

M

ex

iR
o.
co -340nt
exrol m o.
iR
ex
o.

0

E

of Matrigel and 4 3 106 RPMI8226-HR cells was admixed with

1.5

older BMSC exosomes directly transfected with miR-340 mimics(miR-340 exosomes; 4 3 107 particles per 200 mL Matrigel), miR-

RPMI8226-HR RPMI8226-HR RPMI8226-HR+++
control miRmiR-340miR-365exosomes
exosomes
exosomess

Vessel density(relative CD31+ area)D

365 mimics (miR-365 exosomes; 4 3 107 particles per 200 mL
Matrigel), or negative control miR (control miR exosomes). The

1.0

Matrigel mixture was injected subcutaneously into nude mice.
Three weeks after implantation, the Matrigel plugs were harvested.
Paraffin-embedded sections of Matrigel plugs were stained with0.5

hematoxylin and eosin and then subjected to immunostaining for*
0.0
RPMI8226-HR
control miR-exo.
miR-340-exo.
miR-365-exo.
DAPI / CD31

DAPI / CD31

+
+
–
–

+
–
+
–

CD31 (red). Scale bar, 500 mm. (E) Quantification of vessel density
in Matrigel plugs by pixel density. Both mir-340 and miR-365*

exosomes inhibited tumor angiogenesis in Matrigel plugs in

+
–
–
+

comparison with the control (*P , .001 vs control, Student t test,n 5 6). Values represent the mean 6 SD. Exo, exosomes.

DAPI / CD31

Enrichment of older BMSC exosomes with young
BMSC exosome-specific miRNAs inhibits
angiogenesis in vivo
Older BMSC exosomes transfected with miR-340 mimics(miR-340 exosomes) exhibited a 6-fold increase in the expressionof miR-340 in relation to young BMSC exosomes (Figure 4A). We
also visualized with an in vitro model the uptake of exosomal Cy3labeled miR-340 into HR-MM cells (RPMI8226-HR; Figure 4B) and
HUVECs (Figure 4C) via exosomes. Matrigel plug assays were performed
using Cy3-labeled miR-340. We checked the colocalization of Cy3labeled miR-340 and HR cells in the Matrigel plug. Cy3-miR-340
signals were detected in not only the cytoplasm of HR cells(supplemental Figure 4A-B) but also the cytoplasm of endothelial
cells (supplemental Figure 4C-F).
Matrigel plug assays confirmed that older BMSC exosomestransfected with miR-340 mimics (miR-340 exosomes) or miR365 mimics (miR-365 exosomes) significantly inhibited angiogenesis in the Matrigel plug in comparison with scrambled control
miRNA-containing exosomes (control miR exosomes) (Figure 4D-E;
P , .001). We also observed inhibition of the hyperproliferationof HR-MM cells in conjunction with the antiangiogenic effectof older BMSC exosomes transfected with miR-340 andmiR-365 mimics (Figure 4D). These findings suggested that theantiangiogenic effect of older BMSC exosomes was restored
by transfection with young BMSC exosome-specific miRNAs (ie,miR-340 and miR-365). We performed Matrigel plug assays with
KMS-11-HR and IM-9-HR cells and primary myeloma cells of MM
818

exosomes, older BMSC exosomes, and miR-340 exosomes were

HUVECs6gyounvivo. (A) Exosomal miR-340 expression levels in young BMSC

RPMI8226-HRo.

Relative expression level(mIR-340/ath-miR-159)**
8

Figure 4. Antiangiogenic effects of modified exosomes inCBpatients. As in the experiment using RPMI8226-HR cells, miR-340
exosomes significantly inhibited angiogenesis induced by HR-MM
cells (supplemental Figure 5A) or CD1381 cells from a MM
patient with a high tumor burden in the Matrigel plug (supplemental Figure 5B).

Phosphorylated cMET and HGF protein levels vary in
HUVECs cocultured with HR-MM cells
We selected various confirmed targets of miR-340 and miR-365
according to reporter assays or Western blotting using miRTarBase.29 Within this list, we searched for a candidate target of miR340 with a high predicted efficacy as calculated by the context
scores. We focused on the cMET tyrosine kinase receptor thatexerts context-dependent effects on endothelial cells. cMET expression was significantly higher in HUVECs than in MM cells (RPMI8226
and RPMI8226-HR) (Figure 5A). There was no difference in cMET
expression between parental and HR-MM cells (RPMI8226-HR)(Figure 5A-B; P , .01). HGF protein expression was higher in HRMM cells than in parental and endothelial cells (Figure 5A-B;
P , .01). We next investigated changes in the expression of cMET,
phosphorylated cMET (p-cMET), and HGF in HUVECs cultured
with or without MM cells using a noncontact coculture system(Figure 5C). Although there were no significant differences in thelevels of cMET, p-cMET was significantly upregulated in HUVECs
cocultured with HR-MM cells (Figure 5D-E; P , .01, P , .001).
Additionally, HGF expression in HUVECs was induced by coculture
with HR-MM cells (Figure 5D-E; P , .001).

UMEZU et al

23 MAY 2017 x VOLUME 1, NUMBER 13

-104-

C

A

RPMI8226
or
RPMI8226-HR

cMET

D
cMET
p-cMET

HGF
HGF
sβ-actin

EC

−

+

−

RPMI8226-HR

−

−

+

UV

RPMI8226

H

RP
M

I8

RP
M

22

I8

6H

22

R

6

β-actin

HUVECs (1% O2)

HUVECs

4
2
s
UV
H

4

1.0

0.5

5

0
+ c
+R RP ontr
PM MI ol
8
I8 22
22 6
6H
R

s

0

H

UV

H
622

2

RP

M

I8

4

EC

R

6

0
I8

6

+ co
+R RP ntr
PM MI8 ol
I8 22
22 6
6H
R

1

+ c
+R RP ontr
PM MI ol
8
I8 22
22 6
6H
R

2

M

**

**

*
0.0

RP

8

10

*

3

22

Relative expression level(HGF/β-actin)

RP

M

I8

RP

M

22

I8

6-

H

EC

R

6

0

15

1.5

Relative expression level(HGF/β-actin)

6

Relative expression level(cMET/β-actin)*

Relative expression level(p-cMET/β-actin)

E

8

22

Relative expression level(cMET/β-actin)

B

Figure 5. p-cMET and HGF protein levels vary in HUVECs cocultured with HR-MM cells. (A) Western blots showing the expression of cMET and HGF in MM cells(RPMI8226 and RPMI8226-HR) and HUVECs. (B) The intensities of cMET and HGF bands in panel A were quantified and normalized to the levels of b-actin (*P , .01 vs
RPMI8226, Student t test, n 5 3). (C) Schematic of the nonadherent coculture system of MM cells (blue) and HUVECs (green). MM cells and HUVECs were cocultured separately
using a Transwell filter (polycarbonate membrane insert, 0.45-mm pores). (D) cMET, p-cMET, and HGF levels in HUVECs were measured by Western blotting after coculture
with RPMI8226 or RPMI8226-HR cells under hypoxic conditions (1% O2). (E) The intensities of cMET, p-cMET, and HGF bands in panel D were quantified and normalized to thelevels of b-actin (*P , .01; **P , .001 vs control (HUVECs only), Student t test, n 5 3).

Exosomal miR-340 suppresses tube formation via
inhibition of the cMET signaling pathway

directly transfected with miR-340 mimics; 45% reduction) incomparison with the control (HUVECs only) (Figure 6C).

To determine whether cMET expression was suppressed by theaddition of older BMSC exosomes transfected with miR-340
mimics (miR-340 exosomes), we examined cMET protein levels
in HUVECs treated with miR-340 exosomes (Figure 6A). cMET
protein levels were reduced by approximately 60% in HUVECs
treated with miR-340 exosomes in comparison with control miR
exosomes (Figure 6A). These results indicated that exogenous miR340 interacted with cMET for translational suppression of cMET in
HUVECs via exosomes, whereas inhibition of cMET expression by
miR-340 exosomes did not affect the expression of HGF (Figure 6A).
To confirm direct binding between miR-340 and the cMET 39untranslated region (UTR), we performed a luciferase reporter
assay using a reporter plasmid containing the cMET 39-UTR withthe miR-340-binding site (Figure 6B). A reduction of the luciferase
activity from the pLuc-cMET-39-UTR plasmid was observed in
HUVECs treated with miR-340 exosomes (older BMSC exosomes

In the in vitro assay (Figure 6D), endothelial cell tube formation wasenhanced by treatment with conditioned medium derived from HRMM cells or HGF. Such enhanced tube formation was inhibitedby the addition of miR-340 exosomes (Figure 6E-F; P , .01).
Furthermore, we assessed endothelial cMET activation in vivo inMatrigel plugs by immunohistochemistry. In Matrigel plugs, CD31positive endothelial cells attracted by HR-MM cells expressed
p-cMET (Figure 6G).

Discussion
The advantages of exosome therapy are its stability, biocompatibility, biological barrier permeability, low toxicity, and native
mechanism of cell-to-cell transfer. Exosomes derived from BMSCs
have low inherent immunogenicity because of composition similar
to that of the body’s own cells.30 The use of BMSC exosomes istherefore especially promising for exosome-based cancer therapy.

23 MAY 2017 x VOLUME 1, NUMBER 13

EXOSOMAL miR-340 INHIBITS TUMOR ANGIOGENESIS

-105-

819

B

A

hsa-miR-340

3’ UUAGUCAGAGU -AACG-AAAUAUU 5’

Human cMET 3’UTR(1487-1508)

5’ UA - AAGUGUUAUAUUU-UUUAUAAA 3’

cMET
HGF
β-actin

H
+c UV
on EC
tro s
lm
iR

ex
o.
H
+m UV
iR EC
-3 s
40
ex
o.

C

Mutated sensor vector

Sensor vector#

1.2

Relative luciferase activity

Relative luciferase activity

1.2
1.0

*

0.8
0.6
0.4
0.2

1.0
0.8
0.6
0.4
0.2
0.0

0.0
young BMSC exosomes
Control miR exosomes
miR-340 exosomes

–
–
–

+
–
–

–
+
–

–
–
–

–
–
+

HUVECs/Luc/β-gal

D

E

–
+
–

–
–
+

HUVECs/Luc/β-gal

HUVECsHUVECs+control miR exo. +miR-340 exo.

miR-340 exo.

+
–
–

G

Basal medium
Basal medium+
Conditioned med.
or
HGF

Basal medium+
conditioned med.
from HR cells

p-cMET

Basal medium+
HGF

HUVECson Matrigel

Relative tube formation(Fold of control)

F

*

10

CD31

*

8
6
4
2

0
miR-340 exo.
Conditioned med. from HR cells
HGF

–
–
–

+
–
–

–
+
–

+
+
–

–
–
+

+
–
+

DAPI / p-cMET / CD31

Figure 6. Regulation of HGF/cMET signaling in endothelial cells by exosomal miR-340 derived from young BMSCs. (A) Representative Western blots of the expressionof cMET and HGF in HUVECs after treatment with older BMSC exosomes transfected with miR-340 mimics (miR-340 exo.) or negative control miR (control miR exo.). (B) The
hsa-miR-340-binding site spanning nucleotides 1487–1508 (accession no. NM_000245) of the human cMET 39-UTR was predicted to be evolutionarily conserved. The seedsequence of hsa-miR-340 (AAAUAU) is shown in red. (C) Luciferase reporter assay using a reporter plasmid containing the cMET 39-UTR with the miR-340-binding site. The
luciferase reporter vector to assess miR-340-specific activity contained complementary miR-340 sequences in its 39-UTR. Normalized firefly luciferase activity was obtained

820

UMEZU et al

23 MAY 2017 x VOLUME 1, NUMBER 13-106-

There are 2 purposes in our study. One is to clarify the difference in
BMSC and BMSC exosome changes by aging. The other is to findexosomes with therapeutic effects and develop exosome-based
therapy.
Target miRNA-overexpressing cells have generally been used as
exosome-producing cells to obtain target miRNA-enriched exosomes. However, the expression of exosomal factors other than thetarget miRNA also changes using this method, because the biology
of exosome-producing cells is modified by either transfected miRNAor electroporation.31 To overcome this problem, we adopted the“Exo-fect” system, which enables direct transfection of miRNAs into
isolated exosomes. The transfection efficiencies achieved by Exofect were approximately 75% (supplemental Figure 6). As a result,
we found that only 1 exosomal miRNA of young healthy donors
restored the antiangiogenic function of exosomes derived from older
healthy donors.
We initially hypothesized that the function of exosomes is highly
dependent on the BMSC donor age. We found that young BMSC
exosomes and miRNA had significant antiangiogenic effects. In thisstudy, we first investigated the differential gene expression profilesof young BMSCs and older BMSCs (NCBI, gene expression
omnibus; GSE78235) and found that 128 genes were upregulated
and 117 genes were downregulated in older BMSCs in comparison
with young BMSCs (supplemental Figure 7A). By performing gene
ontology (GO) term analysis using the Functional Annotation Tool of
the Database for Annotation, Visualization, and Integrated Discovery (http://david.ncifcrf.gov), we identified major changes between
the functions of young and older BMSCs, such as developmental
processes and cell proliferation and adhesion (supplemental Figure 7B).
Although we did not identify GO terms related to cellular senescence, we found that older BMSCs displayed downregulationof aldehyde dehydrogenase 1, which is involved in self-renewal
and differentiation, indicating that the donor age might affect thedifferentiation potential of BMSCs. Previous studies on age-relatedchanges in BMSCs clearly show that the age of the BMSC donor
directly influences BMSC differentiation, proliferation, and metabolic profiles.32,33 Furthermore, the immunoregulatory potential ofyoung BMSCs is higher than that of older BMSCs.34 Age-related
increases in the expression of apoptotic and senescent genes witha concomitant decrease in Sirt1 gene expression also inhibits stem
cell function to some degree.35 These findings suggest that the ageof BMSC donors should be considered in regard to their therapeutic
efficacy. This interference further suggests that the loss of young
BMSC exosome-specific miRNAs is related to cancer, because thedevelopment of the majority of cancers is considered to be related
to age.

In the present study, we extracted several exosomal miRNAs specificto young BMSCs and identified 2 miRNAs (miR-340 and miR-365)
that exerted an antiangiogenic effect (Figures 3C and 4D). Our insilico analysis showed that miR-365 targeted interleukin-6 (IL-6), andwe conducted a luciferase reporter assay with a reporter vector
containing the 39-UTR of IL-6 with the predicted miR-365 bindingsite (supplemental Figure 8A). Unexpectedly, overexpression of
miR-365 did not decrease the activity of the luciferase reporter(supplemental Figure 8B).
We therefore focused on miR-340, which restored and evenincreased the antiangiogenic effect of older BMSC exosomes. The
first identified target of miR-340 was microphthalmia-associatedtranscription factor, a master regulator of melanocyte development
and melanogenesis in melanoma cells.36 Although several miR-340
target genes have been identified to date, the HGF receptor, cMET,
has been reported as a target of miRNA several times.37 It is known
that the HGF/cMET interaction plays a key role in the regulationof angiogenesis, and high levels of HGF expression and cMET
activation (p-cMET) have been found in BM endothelial cells of MM
patients in comparison with normal endothelial cells of healthy
donors.38 Consequently, miR-340 might act as a negative regulatorof angiogenesis by inhibiting cMET expression. On the basis of aprevious report,37 we assessed direct binding between the cMET
39-UTR and miR-340 by a luciferase reporter assay (Figure 6C).
Furthermore, Moschetta et al demonstrated that resistant R5 cells
and plasma cells from MM patients at relapse with drug resistancehave a high HGF-independent p-cMET content with constitutive
activation of the cMET receptor.39 The expression of cMET andp-cMET did not show a difference between parental and HR-MM cells.
We also did not observe constitutive activation of cMET in CD1381
cells obtained from MM patients in this study. Unfortunately, using
our system, we could not analyze the effect of miR-340 on
overcoming drug resistance by inhibition of cMET expression.
One of the reasons why we replenished miR-340 in older BMSC
exosomes was to use older BMSC exosomes as an experimental
control (miR-340-insufficient exosomes) to demonstrate miR-340
functions. However, the principal reason for modifying exosomes bydirect transfection of miRNA was establishment of novel strategies
for exosome-based therapy. An important issue that should be
addressed is whether miR-340-enriched exosomes have a direct
inhibitory effect on tumor growth. Although we cannot completely
rule out the possibility of either direct growth inhibition of tumor cells
or remaining natural killer cell activity in the nude mouse, miR-340enriched exosomes did not induce HR cell proliferation in vitro(Figure 2D). We believe the inhibition of tumor angiogenesis by
miR-340-enriched exosomes was due to tumor growth suppression

Figure 6. (continued) according to firefly luciferase activity/b-gal activity. Sensor vector: the luciferase activity of HUVECs/Luc/b-gal (HUVECs co-transfected with luciferase
reporter and b-gal control vectors) treated with young BMSC exosomes was significantly reduced in comparison with control cells (HUVECs only, *P , .01, n 5 3). miR-340
exosomes (older BMSC exosomes transfected with miR-340 mimics) also reduced the luciferase activity in HUVECs in comparison with control miR exosomes (older BMSC
exosomes transfected with negative control miR) (#P , .01, Student t test, n 5 3). Using the mutated sensor vector, there was no difference in luciferase activity with or without exosomes.
(D) Schematic of the endothelial cell tube formation assay to confirm the antiangiogenic effect of miR-340 exosomes. HUVECs were cultured on Matrigel in 3 types of medium (basal
medium, basal medium supplemented with conditioned medium from RPMI8226-HR cells, or basal medium supplemented with HGF). (E) Endothelial tube formation in response to older
BMSC exosomes transfected with miR-340 mimics (miR-340 exo.) observed under a bright field by phase-contrast microscopy. Scale bar, 500 mm. (F) Quantification of tube formation asshown in panel E. The conditioned medium from HR-MM (RPMI8226-HR) cells and HGF induced endothelial tube formation in vitro. Induction of tube formation by conditioned medium of
HR-MM cells and HGF was inhibited by older BMSC exosomes transfected with miR-340 mimics (miR-340 exosomes) in comparison with the control (older BMSC exosomes transfectedwith negative control miR; control miR exosomes) (*P , .01, Student t test). Values represent the mean 6 SD. (G) Immunohistochemical staining of CD31 (red) and p-cMET (green) in
Matrigel plugs seeded with RPMI8226-HR cells at 3 weeks after transplantation into nude mice. Scale bar, 200 mm. med., medium.

23 MAY 2017 x VOLUME 1, NUMBER 13

EXOSOMAL miR-340 INHIBITS TUMOR ANGIOGENESIS

-107-

821

in the Matrigel plug, although further studies are required to test thishypothesis.
In conclusion, we provide new information on the age-relatedchanges of BMSC-derived exosomes and the inhibitive effectof exosomal miRNA on tumor angiogenesis. The present study
provides evidence of the therapeutic potential of exosomal miRNAs
derived from young BMSCs and offers a foundation for the development of novel strategies for exosome-based therapy via replenishment of exosomal miRNAs.

of Japan (15H04303, 15K06841, and 16K07183); and the Tokyo
Biochemical Research Foundation.

Authorship
Contribution: T.U., K.O., and J.H.O. designed the study and wrote themanuscript; T.U., S.I., K.A., and C.K. performed experiments andanalyzed data; and S.Y. provided patient samples.

Acknowledgments

Conflict of interest disclosure: K.O. received research support
from Celgene KK, Chugai Pharmaceutical KK, and Janssen Pharma
KK. The remaining authors declare no competing financial interests.

This study was supported in part by the Private University Strategic
Research-Based Support Project (S1311016); grants-in-aid from
the Ministry of Education, Culture, Sports, Science, and Technology

Correspondence: Tomohiro Umezu, Tokyo Medical University,
6-7-1 Nishi-shinjuku, Shinjuku, Tokyo 160-0023, Japan; e-mail:t_umezu@tokyo-med.ac.jp.

References
1.

Quail DF, Joyce JA. Microenvironmental regulation of tumor progression and metastasis. Nat Med. 2013;19(11):1423-1437.

2.

Tsai MJ, Chang WA, Huang MS, Kuo PL. Tumor microenvironment: a new treatment target for cancer. ISRN Biochem. 2014;2014:351959.

3.

Chen F, Zhuang X, Lin L, et al. New horizons in tumor microenvironment biology: challenges and opportunities. BMC Med. 2015;13:45.

4.

Manier S, Sacco A, Leleu X, Ghobrial IM, Roccaro AM. Bone marrow microenvironment in multiple myeloma progression. J Biomed Biotechnol.
2012;2012:157496.

5.

Gupta D, Treon SP, Shima Y, et al. Adherence of multiple myeloma cells to bone marrow stromal cells upregulates vascular endothelial growth factor
secretion: therapeutic applications. Leukemia. 2001;15(12):1950-1961.

6.

Palumbo A, Anderson K. Multiple myeloma. N Engl J Med. 2011;364(11):1046-1060.

7.

Kyle RA, Rajkumar SV. Multiple myeloma. Blood. 2008;111(6):2962-2972.

8.

André T, Meuleman N, Stamatopoulos B, et al. Evidences of early senescence in multiple myeloma bone marrow mesenchymal stromal cells. PLoS One.
2013;8(3):e59756.

9.

Rossi DJ, Jamieson CH, Weissman IL. Stems cells and the pathways to aging and cancer. Cell. 2008;132(4):681-696.

10. Dorshkind K, Montecino-Rodriguez E, Signer RA. The ageing immune system: is it ever too old to become young again? Nat Rev Immunol. 2009;9(1):57-62.
11. Roccaro AM, Sacco A, Maiso P, et al. BM mesenchymal stromal cell-derived exosomes facilitate multiple myeloma progression. J Clin Invest. 2013;
123(4):1542-1555.
12. Vlassov AV, Magdaleno S, Setterquist R, Conrad R. Exosomes: current knowledge of their composition, biological functions, and diagnostic andtherapeutic potentials. Biochim Biophys Acta. 2012;1820(7):940-948.
13. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic
exchange between cells. Nat Cell Biol. 2007;9(6):654-659.
14. Kosaka N, Iguchi H, Yoshioka Y, Takeshita F, Matsuki Y, Ochiya T. Secretory mechanisms and intercellular transfer of microRNAs in living cells. J Biol
Chem. 2010;285(23):17442-17452.
15. Camussi G, Deregibus MC, Bruno S, Grange C, Fonsato V, Tetta C. Exosome/microvesicle-mediated epigenetic reprogramming of cells. Am J Cancer
Res. 2011;1(1):98-110.
16. Kosaka N, Iguchi H, Yoshioka Y, Hagiwara K, Takeshita F, Ochiya T. Competitive interactions of cancer cells and normal cells via secretory microRNAs.
J Biol Chem. 2012;287(2):1397-1405.
17. Lai RC, Chen TS, Lim SK. Mesenchymal stem cell exosome: a novel stem cell-based therapy for cardiovascular disease. Regen Med. 2011;6(4):481-492.
18. Sdrimas K, Kourembanas S. MSC microvesicles for the treatment of lung disease: a new paradigm for cell-free therapy. Antioxid Redox Signal. 2014;21(13):1905-1915.
19. Zhu YG, Feng XM, Abbott J, et al. Human mesenchymal stem cell microvesicles for treatment of Escherichia coli endotoxin-induced acute lung injury
in mice. Stem Cells. 2014;32(1):116-125.
20. Reis LA, Borges FT, Simões MJ, Borges AA, Sinigaglia-Coimbra R, Schor N. Bone marrow-derived mesenchymal stem cells repaired but did not prevent
gentamicin-induced acute kidney injury through paracrine effects in rats. PLoS One. 2012;7(9):e44092.
21. Tomasoni S, Longaretti L, Rota C, et al. Transfer of growth factor receptor mRNA via exosomes unravels the regenerative effect of mesenchymal stem
cells. Stem Cells Dev. 2013;22(5):772-780.

822

UMEZU et al

23 MAY 2017 x VOLUME 1, NUMBER 13-108-

22. Tan CY, Lai RC, Wong W, Dan YY, Lim SK, Ho HK. Mesenchymal stem cell-derived exosomes promote hepatic regeneration in drug-induced liver injury
models. Stem Cell Res Ther. 2014;5(3):76.
23. Kordelas L, Rebmann V, Ludwig AK, et al. MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus-host disease. Leukemia. 2014;
28(4):970-973.
24. Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat
Biotechnol. 2011;29(4):341-345.
25. Ha D, Yang N, Nadithe V. Exosomes as therapeutic drug carriers and delivery vehicles across biological membranes: current perspectives and futurechallenges. Acta Pharm Sin B. 2016;6(4):287-296.
26. Umezu T, Tadokoro H, Azuma K, Yoshizawa S, Ohyashiki K, Ohyashiki JH. Exosomal miR-135b shed from hypoxic multiple myeloma cells enhances
angiogenesis by targeting factor-inhibiting HIF-1. Blood. 2014;124(25):3748-3757.
27. Tadokoro H, Umezu T, Ohyashiki K, Hirano T, Ohyashiki JH. Exosomes derived from hypoxic leukemia cells enhance tube formation in endothelial cells.
J Biol Chem. 2013;288(48):34343-34351.
28. Umezu T, Ohyashiki K, Kuroda M, Ohyashiki JH. Leukemia cell to endothelial cell communication via exosomal miRNAs. Oncogene. 2013;32(22):2747-2755.
29. Hsu SD, Lin FM, Wu WY, et al. miRTarBase: a database curates experimentally validated microRNA-target interactions. Nucleic Acids Res. 2011;
39(Database issue):D163-D169.
30. Battiwalla M, Hematti P. Mesenchymal stem cells in hematopoietic stem cell transplantation. Cytotherapy. 2009;11(5):503-515.
31. Marcus ME, Leonard JN. FedExosomes: engineering therapeutic biological nanoparticles that truly deliver. Pharmaceuticals (Basel). 2013;6(5):659-680.
32. Fafián-Labora J, Fernández-Pernas P, Fuentes I, et al. Influence of age on rat bone-marrow mesenchymal stem cells potential. Sci Rep. 2015;5:16765.
33. Siegel G, Kluba T, Hermanutz-Klein U, Bieback K, Northoff H, Schäfer R. Phenotype, donor age and gender affect function of human bone marrowderived mesenchymal stromal cells. BMC Med. 2013;11:146.
34. Wu LW, Wang YL, Christensen JM, et al. Donor age negatively affects the immunoregulatory properties of both adipose and bone marrow derived
mesenchymal stem cells. Transpl Immunol. 2014;30(4):122-127.
35. Choudhery MS, Khan M, Mahmood R, Mehmood A, Khan SN, Riazuddin S. Bone marrow derived mesenchymal stem cells from aged mice have reduced
wound healing, angiogenesis, proliferation and anti-apoptosis capabilities. Cell Biol Int. 2012;36(8):747-753.
36. Goswami S, Tarapore RS, Teslaa JJ, Grinblat Y, Setaluri V, Spiegelman VS. MicroRNA-340-mediated degradation of microphthalmia-associatedtranscription factor mRNA is inhibited by the coding region determinant-binding protein. J Biol Chem. 2010;285(27):20532-20540.
37. Wu ZS, Wu Q, Wang CQ, et al. miR-340 inhibition of breast cancer cell migration and invasion through targeting of oncoprotein c-Met. Cancer.
2011;117(13):2842-2852.
38. Ferrucci A, Moschetta M, Frassanito MA, et al. A HGF/cMET autocrine loop is operative in multiple myeloma bone marrow endothelial cells and may
represent a novel therapeutic target. Clin Cancer Res. 2014;20(22):5796-5807.
39. Moschetta M, Basile A, Ferrucci A, et al. Novel targeting of phospho-cMET overcomes drug resistance and induces antitumor activity in multiple myeloma.
Clin Cancer Res. 2013;19(16):4371-4382.

23 MAY 2017 x VOLUME 1, NUMBER 13

EXOSOMAL miR-340 INHIBITS TUMOR ANGIOGENESIS

-109-

823

www.impactjournals.com/oncotarget/

Oncotarget, 2017, Vol. 8, (No. 41), pp: 69906-69915
Research Paper

Teriflunomide restores 5-azacytidine sensitivity via activationof pyrimidine salvage in 5-azacytidine-resistant leukemia cells
Satoshi Imanishi1, Ryoko Takahashi1, Seiichiro Katagiri2, Chiaki Kobayashi1,2,
Tomohiro Umezu1,2, Kazuma Ohyashiki2 and Junko H. Ohyashiki1
1

Department of Molecular Oncology, Institute for Medical Science, Tokyo Medical University, Nishi-Shinjuku, Shinjuku, Tokyo,
Japan

2

Department of Hematology, Tokyo Medical University, Nishi-Shinjuku, Shinjuku, Tokyo, Japan

Correspondence to: Satoshi Imanishi, email: s-ima@tokyo-med.ac.jp
Keywords: azacytidine, teriflunomide, pyrimidine metabolism, sensitization
Abbreviations: AZA, azacytidine; UCK, uridine cytidine kinase; DNMT, DNA methyltransferase; MDS, myelodysplastic syndromes;
AML, acute myeloid leukemia
Received: November 09, 2016     Accepted: June 19, 2017     Published: July 22, 2017
Copyright: Imanishi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License
3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author andsource are credited.

ABSTRACTPrevious studies showed that downregulation of pyrimidine salvage underlies
resistance against 5-azacytidine (AZA), indicating an important role for de novo
pyrimidine synthesis in AZA resistance. Because de novo pyrimidine synthesis isinhibited by the immunomodulator teriflunomide and its pro-drug leflunomide,
we examined the effect of combined treatment with AZA and teriflunomide on
AZA resistance to develop a novel strategy to cancel and prevent AZA resistance.
Teriflunomide markedly inhibited the growth of AZA-resistant human leukemia celllines (R-U937 and R-HL-60) in comparison with their AZA-sensitive counterparts(U937 and HL-60). In the presence of a non-toxic concentration of teriflunomide(1 μM), AZA induced apoptosis in AZA-resistant cells and leukemia cells from AZAresistant patients. AZA acted as a DNA methyltransferase 3A inhibitor in AZA-resistantcells in the presence of 1 μM teriflunomide. Although AZA-sensitive cells acquired
AZA resistance after continuous treatment with AZA for 42 days, the growth of AZAsensitive cells continuously treated with the combination of AZA and teriflunomide
was significantly inhibited in the presence of AZA, demonstrating that the combined
treatment prevented AZA resistance. These results suggest that combined treatment
with AZA and teriflunomide can be a novel strategy to overcome AZA resistance.

phosphorylated by other enzymes to produce 5-AZAdiphosphate (azaCDP) and 5-AZA-triphosphate, andincorporated into RNA. Part of azaCDP is converted
to 2ʹ-deoxy-5-AZA-triphosphate (d-azaCTP) byribonucleotide reductase [5]. d-azaCTP is incorporated
into DNA and forms irreversible DNA methyltransferase(DNMT)/DNA complexes. Removal of DNMT/DNA
complexes and repair of DNA result in degradation of
DNMT proteins accompanying demethylation of DNA [6].
Previously, we reported that AZA resistance in
R-U937 cells and R-HL-60 cells (AZA-resistant cells),
which we originally created from U937 cells and HL60 cells, respectively [7], involves downregulationof UCK2 expression and conversion of UTP to CTP.

INTRODUCTION
5-azacytidine (AZA) is approved for the treatment ofpatients with myelodysplastic syndrome (MDS) in Europe,
the United States, Japan, and other countries. AZA yields
a 40–60% response rate in these patients [1]. However,some patients treated with AZA develop resistance to thedrug after various treatment durations [2, 3]. Although theprognosis of MDS patients after AZA treatment failure ispoor, with a median overall survival time of 5.6 months[4], a strategy to cancel resistance against AZA has notbeen developed.
AZA taken up by cells is mono-phosphorylated byuridine-cytidine kinase (UCK) 1 or UCK2, subsequently
www.impactjournals.com/oncotarget

69906

-110-

Oncotarget

Valencia et al. reported that lower expression of UCK1mRNA correlates to primary resistance against AZA inpatients with MDS [8]. Because of the roles of UCK1
and UCK2 in pyrimidine salvage, these findings indicate
the important role of the alternative pyrimidine-supplying
mechanism—de novo pyrimidine synthesis—in AZA
resistance in cell lines and patients. It is well establishedthat de novo pyrimidine synthesis is inhibited byteriflunomide and its pro-drug leflunomide, which are
the immunomodulators approved for the treatment ofmultiple sclerosis and rheumatoid arthritis, respectively[9, 10]. Therefore, we examined the effect of combined
treatment of AZA and teriflunomide on AZA sensitivity
and resistance.

was significantly decreased in comparison with their AZAsensitive counterparts (U937 and HL-60 cells; Figure 1A,
1B). Treatment with 1 μM teriflunomide notably increased
the fraction with a higher signal for 5-ethynyluridine(EU), which is phosphorylated via pyrimidine salvage
and is incorporated with RNA [11] in both types of AZAresistant cells (Figure 1C), whereas the mRNA expressionof UCK1 and UCK2 was not affected by the presence ofteriflunomide (Figure 1D).

AZA-induced apoptosis in AZA-resistant cells in
the presence of teriflunomide
Next, we examined the effect of teriflunomide on
the growth inhibitory activity of 1, 5, and 10 μM AZA,
which are the relevant doses to the serum concentrationof AZA in MDS patients [12, 13]. Although treatment
with teriflunomide up to 1.25 μM for 72 h did not show
significant growth-inhibitory effects in the cells usedin this study (Figure 1), 1 μM teriflunomide enhanced
the cytotoxic effects of 1 μM AZA in U937 and HL-60
cells (Figure 2A, 2B). In R-U937 and R-HL-60 cells,
we detected a significant decrease in the viability in an
AZA dose-dependent manner in the presence of 1 μM

RESULTS
Teriflunomide-activated pyrimidine salvage in
AZA-resistant cells
After treatment with teriflunomide at a dose
equivalent to the serum concentration of teriflunomide(1.25–10 μM) in patients with rheumatoid arthritis treatedwith leflunomide, viability of R-U937 and R-HL-60 cells

Figure 1: Viability of U937 cells and R-U937 cells (A) and of HL-60 cells and R-HL-60 cells (B) after treatment with teriflunomide(TFN) for 72 h. The p-values are indicated. N.S. indicates nonsignificant results of the t-test following two-way ANOVA. The results ofthree independent experiments are shown. (C) Analysis of EU availability in R-U937 cells (left panel) and R-HL-cells (right panel) without
and with 1 μM TFN treatment for 72 h. Green lines: without treatment; red lines: with treatment. (D) Relative mRNA expression levelsof UCK1 and UCK2 normalized with the mRNA expression level of ACTB in R-U937 and R-HL-60 cells without and with 1 μM TFN
treatment for 72 h. The results of three independent experiments are shown.
www.impactjournals.com/oncotarget

69907

-111-

Oncotargetteriflunomide (Figure 2C, 2D), demonstrating that thepresence of teriflunomide restored AZA sensitivity in
AZA-resistant cells.
To examine whether the effects of the combined
treatment with AZA and teriflunomide was the true
synergic effect, we calculated combined index of 25%
inhibition of cell growth (CI 25) using the Chou-Talalay
method [14]. CI 25 of the combined treatment with AZA
and teriflunomide was 0.72 for U937 cells, 0.48 for HL-60
cells, 0.54 for R-U937 cells, and 0.47 for R-HL-60 cells,suggesting that the effect of the combined treatment with
AZA and teriflunomide was the true synergic effect.
In the analyses of annexin V-propidium iodide
double staining, we detected induction of apoptosis in
AZA-resistant cells after combined treatment for 96 h in
R-U937 cells (Figure 3, upper panels) and R-HL-60 cells(Figure 3, lower panels), but not in those treated with AZAor teriflunomide alone, implying that the AZA-dependent
decrease of viability in AZA-resistant cells after combined
treatment was accompanied by apoptosis.

Because R-U937 and R-HL-60 cells showed lower
sensitivity to decitabine (5-aza-2'-deoxycytidine: DAC)
in comparison with their AZA-sensitive counterparts [7],we examined the effects of treatment with teriflunomide
on DAC sensitivity. The presence of 1 μM teriflunomide
did not affect the viability of U937 and HL-60 cells
cultured with 0.2 μM DAC for 72 h, whereas greater
growth repression by 0.2 μM DAC in the presence of 1
μM teriflunomide was observed in R-U937 and R-HL-60
cells (Figure 4A, 4B).

AZA acted as a DNMT inhibitor in AZAresistant cells in the presence of teriflunomide
To clarify whether AZA acted as a DNMT inhibitorin AZA-resistant cells in the presence of teriflunomide,
we examined DNMT3A and DNMT1 protein amounts.
(DNMT3B was not examined because its expression
was not detected in R-U937 and R-HL-60 cells in ourprevious study [7].) In AZA-sensitive cells, treatment with

Figure 2: Viability of U937 cells (A), HL-60 cells (B), R-U937 cells (C), and R-HL-60 cells (D) after treatment with AZA (0, 1, 5, 10 μM)
for 72 h in the absence or presence of 1 μM teriflunomide (TFN). The p-values of significance are indicated. N.S. indicates nonsignificant
results of the t-test and two-way ANOVA. The results of three independent experiments are shown.
www.impactjournals.com/oncotarget

69908

-112-

Oncotarget

1 μM AZA for 48 h decreased DNMT1 and DNMT3A
proteins and the presence of 1 μM teriflunomide enhanced
degradation of DNMT3A, but not DNMT1, by AZA
treatment (Figure 5A). The effects of treatment with
1 μM teriflunomide alone on these proteins were notsignificant. Combined treatment with 5 μM AZA and 1
μM teriflunomide for 48 h significantly decreased theamount of DNMT3A protein in AZA-resistant cells,
whereas the amount of DNMT3A was not affected

when these cells were treated with 5 μM AZA or 1 μM
teriflunomide alone (Figure 5B). The amount of DNMT1
protein did not change after treatment with each agent or
their combination.
To examine why the amount of DNMT1 did notchange after the combined treatment, we compared DNA
amount in between AZA-resistant and AZA-sensitive cells
by using a chromatin immunoprecipitation (ChIP) assay
with anti-DNMT1 antibody or anti-DNMT3A antibody

Figure 3: Analysis of apoptosis in R-U937 cells. (upper panels) and R-HL-60 cells (lower panels) of mock treatment, treatment with 10
μM AZA alone, treatment with 1 μM teriflunomide (TFN) alone, and combination treatment with AZA and TFN for 96 h.

Figure 4: Viability of U937 and R-U937 cells (A) and HL-60 and R-HL-60 cells (B) after treatment with 0.2 μM DAC for 72 h in theabsence or presence of 1 μM teriflunomide (TFN). The results of three independent experiments are shown.
www.impactjournals.com/oncotarget

69909

-113-

Oncotarget

(Supplementary Figure 1). With anti-DNMT1 antibody,the amount of precipitated DNA from AZA-resistantcells was significantly less than that from AZA-sensitive
cells, indicating that recruitment of DNMT1 on DNA isless frequent in AZA-resistant cells. No such significant
difference was detected in the experiments using antiDNMT3A antibody.

teriflunomide. In leukemia cells with AZA resistancefrom four patients (nos. 1, 3, 4, 5), decreased viability was
detected when they were treated with 1 μM AZA combinedwith teriflunomide at 1 μM; however, 1 μM AZA and 1
μM teriflunomide individually were not cytotoxic (Figure
6A). Leukemia cells from patient 2 showed relatively
higher sensitivity to teriflunomide, resulting in decreased
viability after treatment with teriflunomide alone;
however, combined treatment reduced their viability much
more so (Figure 6A). The viability of CD34-posisitve cells
from a healthy donor was not affected by treatment with
1 μM AZA or 1 μM teriflunomide or by the combined
treatment, indicating that treatment with AZA and/or
teriflunomide did not influence the proliferation of healthy
hematopoietic stem/progenitor cells (Figure 6B).

Teriflunomide canceled AZA resistance inleukemia cells obtained from patients
The experiments using cell lines demonstrated
that teriflunomide could cancel AZA resistance, so we
examined whether AZA resistance in leukemia cells frompatients is canceled by combined treatment of AZA with

Figure 5: (A) Amounts of DNMT3A and DNMT1 in U937 cells and HL-60 cells after treatment with the indicated agent for 48 h. Values

indicate the relative amounts of DNMT3A and DNMT1 normalized with β-ACTIN. (B) Amounts of DNMT3A and DNMT1 in R-U937 and
R-HL-60 cells after treatment with the indicated agent for 48 h. Values indicate the relative amounts of DNMT3A and DNMT1 normalizedwith β-ACTIN.

www.impactjournals.com/oncotarget

69910

-114-

Oncotarget

Teriflunomide prevented AZA-resistance in vitro

treatment of multiple sclerosis, to be an agent that canovercome and prevent AZA resistance in leukemia byusing an approach based on the molecular mechanism
underlying AZA resistance. Recent attention to drug
repositioning has mainly focused on computational
approaches using virtual screening of the comprehensive
libraries of approved compounds based on threedimensional structures of target proteins [16–18].
However, mechanism-based drug repositioning may be
another approach to finding drugs that can overcome
drug resistance in cancer. To date, several moleculartargeted drugs, including HDAC inhibitors, have beenused to overcome AZA resistance, but none of them
restores the action of AZA in leukemia cells that creates
AZA resistance. We demonstrated for the first time thatmechanism-based drug repositioning could resensitize
AZA-resistant human leukemia cells to AZA.
Despite the fact that downregulated UCK1
or UCK2 expression in AZA resistance has been
extensively studied [7, 8], the role of de novo pyrimidine
synthesis in AZA resistance is not fully understood.
In this study, we demonstrated that sensitivity toteriflunomide in AZA-resistant cells was higher thanthat in their AZA-sensitive counterparts, suggesting that
AZA-resistant cells more strongly depend on de novo
pyrimidine synthesis. Treatment with teriflunomide at a
non-cytotoxic dose increased the availability of EU in
AZA-resistant cells, but it did not induce re-expressionof UCK1 or UCK2. Therefore, inhibition of de novo
pyrimidine synthesis might activate pyrimidine salvage
without induction of UCK1 or UCK2. Teriflunomide
might reduce the pool size of CTP and dCTP and thus
decrease competition for the incorporation of azaCTP

Finally, we examined whether teriflunomide canprevent the acquisition of AZA resistance in AZAsensitive cells. After continuous treatment with 5 μM
AZA for 42 days, the growth of U937 and HL-60 cells was
detected even in the presence of 5 μM AZA, indicatingthat the cells had acquired AZA resistance. Continuous
treatment with 1 μM teriflunomide for 42 days did notaffected in the growth of U937 and HL-60 cells, and their
growth when treated with a combination of 5 μM AZA and1 μM teriflunomide for 42 days was significantly slower
than that of AZA- or teriflunomide-treated cells (Figure
7A, 7B).
To assess the sensitivity to AZA in U937 and HL60 cells after continuous treatment, we cultured them inthe presence of 5 and 10 μM AZA for 72 h. As expected,
U937 and HL-60 cells after continuous treatment with
AZA showed lower sensitivity to AZA, whereas thesecells after continuous combined treatment showeddecreased viability, as did control cells without treatment
and cells continuously treated with teriflunomide for 42days (Figure 7C, 7D). These findings demonstrate that the
combined treatment prevented the acquisition of resistanceagainst AZA in U937 and HL-60 cells.

DISCUSSION
Drug repositioning, which is a pharmaceutical
approach to finding novel activity of existing drugs,
has become popular because it reduces development
costs and accelerates regulatory approval [15]. We
found teriflunomide, an immunomodulator approved for

Figure 6: (A) Viability of leukemia cells from patients with AZA resistance after treatment with 1 μM AZA and/or 1 μM teriflunomide(TFN) for 24 h. *p < 0.01. (B) Viability of healthy CD34+ cells after treatment with 1 μM AZA and/or 1 μM TFN for 48 h.

www.impactjournals.com/oncotarget

69911

-115-

Oncotargetinto RNA and d-azaCTP into DNA. It is noteworthy
that AZA also inhibited cell growth in the presence ofteriflunomide in freshly obtained leukemia cells frompatients with AZA resistance. Therefore, inhibition of
de novo pyrimidine synthesis by teriflunomide may
play a key role in the restoration of AZA sensitivity infreshly obtained leukemia cells from patients as well ascell lines.
Another important finding is the notable decreaseof DNMT3A in AZA-resistant cells after combined
treatment with AZA and teriflunomide. As the ChIP assay
demonstrated, the recruitment of DNMT1 on DNA wasless frequent in AZA-resistant cells than AZA-sensitive
counterparts. Given the hypomethylation of DNA in AZAresistant cells [7], DNMT3A might be recruited on DNA
more frequently than DNMT1 in AZA-resistant cells. The
decrease of DNMT3A might indicate that AZA acted as a
DNMT inhibitor in AZA-resistant cells in the presence ofteriflunomide. Therefore, combined treatment with AZA
and teriflunomide or leflunomide could be a promisingstrategy to sensitize patients with MDS or acute myeloid
leukemia (AML) who developed AZA resistance. In
addition to restoration of AZA sensitivity in AZA-resistantcells, enhancement of growth inhibitory activity of AZA
at 1 μM accompanied with increase of degradation of
DNMT3A but not DNMT1 was found in AZA-sensitive
cells. Although the reason why teriflunomide did notincrease the degradation of DNMT1 remains unclear, one
possibility is that DNMT1 recruited on DNA in AZAsensitive cells might be nearly exhausted in the presenceof 1 μM AZA.
The cross-resistance against DAC could be
weakened in the presence of teriflunomide in R-U937 and
R-HL-60 cells, suggesting that the combined treatment
with DAC and teriflunomide might be another treatment
choice for myeloid neoplasm with AZA resistance. On
the other hand, inhibition of de novo pyrimidine synthesis
by teriflunomide failed to enhance the growth inhibitory
activity of DAC in AZA-sensitive cells. Unlike our result,
Raynal et al. demonstrated that inhibition of de novo
pyrimidine synthesis by an inhibitor of CTP synthaseenhances the cytotoxic activity of DAC by enhancing
the incorporation of DAC into DNA [19]. Despite thefact that the sensitivity to DAC might correlate with denovo pyrimidine synthesis as well as AZA sensitivity, thekey enzyme determining the sensitivity to AZA and DAC

Figure 7: (A, B) The growth of U937 cells (A) and HL-60 cells (B) treated with 5 μM AZA and/or 1 μM teriflunomide (TFN) for 42 days.

*p < 0.01 in comparison with mock or TFN-treated cells, #p < 0.01 in comparison with AZA-treated cells. (C, D) The AZA sensitivity of
U-937 cells (C) and HL-60 cells (D) treated with 5 μM AZA and/or 1 μM TFN for 42 days.

www.impactjournals.com/oncotarget

69912

-116-

Oncotarget

might be different. More detailed research on the role ofde novo pyrimidine synthesis in the sensitivity to AZA and
DAC is needed.
Evidence suggests that AZA resistance requires a
shift in the pyrimidine-supplying pathway from salvagedependent to de novo synthesis-dependent [7, 20]. In
the current study, we demonstrated that teriflunomide
prevented the acquisition of AZA resistance. This
prevention might be accompanied with maintenance of
pyrimidine salvage activity through inhibition of de novo
pyrimidine synthesis. The combined treatment with AZA
and teriflunomide could be a novel strategy to prevent thedevelopment of AZA resistance.
Some limitations exist in our approach. First,
our results should be confirmed by in vivo studies
using animal model. Similarly, we could not examine
whether teriflunomide or leflunomide may improve theimmunological environment of patients with MDS in
our in vitro study, but the immunomodulating activity ofteriflunomide and leflunomide is well established [9, 10].
In conclusion, we provide evidence of theimportance of de novo pyrimidine synthesis in AZA
resistance and of its potential as a therapeutic target ofde novo pyrimidine synthesis to overcome and prevent
AZA resistance. Anti-cancer activity of teriflunomide
has been reported for various tumors [21–23], including
hematologic malignancies [24]. Therefore, not only
combined use with AZA but also use of a higher doseof teriflunomide might be a potent strategy for thetreatment of AML with AZA resistance. However,some questions still remain, such as whether de novo
pyrimidine synthesis inhibitors can sensitize primary
resistance against AZA and whether de novo pyrimidine
synthesis inhibitors can prevent the development of DAC
resistance. To answer these questions, further studies arenecessary.

Laboratories, Fujioka, Japan). CD34-posistive cells
were obtained from peripheral blood of a healthy donor
treated with G-CSF. After isolation of mononuclear cells
using Lymphosepar I, CD34-positive cells were purified
using CD34 MicroBead Kit UltraPure (Miltenyi Biotech,
Gladbach, Germany) and autoMACS Pro Separator(Miltenyi Biotech). In flow cytometric analysis, thepurity of CD34-positive cells was >98% and nearly 80%of them were CD38-negative.
AZA and DAC were purchased from Wako Pure
Chemical Industries (Osaka, Japan) and teriflunomide
from Selleck Chemicals (Houston, TX, USA). The antiDNMT1 antibody 4H80, the anti-DNMT3A antibody
H-295, and the anti-β-ACTIN antibody C4 were purchasedfrom Santa Cruz Biotechnology (Dallas, TX, USA). The
secondary antibodies, namely horseradish peroxidase(HRP)-labeled anti-mouse IgG antibody and HRP-labeledanti-rabbit IgG antibody, were purchased from GE
Healthcare (Buckinghamshire, UK).

Cell culture and chemical reagent treatment
U937, HL-60, R-U937, and R-HL-60 cells
and leukemia cells from patients were incubated in
RPMI1640 medium (Life Technologies Inc., Carlsbad,
CA, USA) including 10% inactivated fetal bovine serumand 1% penicillin/streptomycin (Life Technologies).
CD34-positive cells from a healthy donor was cultured
in Hematopoietic Progenitor Expansion Medium DXF(PromoCell, Heidelberg, Germany) supplemented
with Cytokine Mix E (PromoCell). For treatment withreagents, cells were collected by centrifugation andsuspended at 1 × 105 cells/ml in fresh medium with theagents or 0.01% DMSO as the vehicle. Cell viability
was measured using the Cell Counting Kit-8 (Dojindo,
Kumamoto, Japan) as previously described [25]. CI
25 was calculated using Chou-Talalay method [14].
For continuous treatment, 5 μM AZA and/or 1 μM
teriflunomide was added to the U937 cells or HL-60 cells
suspended in the fresh medium at 1 × 105 cells/ml at day
0. Then at days 3, 7, 10, 14, 17, 21, 24, 28, 31, 35, 38, 42,
and 49, culture medium was changed to fresh medium
with the added agents and cell viability was measured
using the Cell Counting Kit-8.

MATERIALS AND METHODS
Cells and reagents
U937 and HL-60 cells were purchased from ATCC
(Manassas, VA, USA). AZA-resistant cell lines (R-U937
and R-HL-60) were originally created in our laboratoryfrom U937 and HL-60 cells, respectively [7]. Prior to
specimen collection from patients, the study was approvedby the institutional review board of Tokyo Medical
University (IRB no. 1974), and written informed consent
was obtained from the patients in accordance with the
Declaration of Helsinki.
The peripheral blood samples with >80% blastsof leukemia cells were collected from five patientswith AML that developed from MDS-acquired AZA
resistance. The clinical characters are described in the
Supplementary Table 1. Leukemia cells from patientswere isolated using Lymphosepar I (Immuno-Biologicalwww.impactjournals.com/oncotarget

Measurement of pyrimidine salvage activity
Pyrimidine salvage activity was estimated based on
the availability of EU. Cells were incubated in the medium
with EU for 3 h after treatment with teriflunomide for 72
h. EU incorporated in RNA was labeled with Alexa Fluor
488 using the Click-It RNA Alexa Fluor 488 Imaging
Kit (Thermo Fisher Scientific, Waltham, MA, USA).
The signal for EU was measured using flow cytometricmeasurements performed on a BD Accuri C6 Flow
Cytometer (BD Bioscience, Franklin Lakes, NJ, USA).
69913

-117-

OncotargetA 488-nm blue laser was used for excitation, and thesignal was detected by the FL1 channel (533 nm). The
signals of 30,000 events were obtained. Analyses of theobtained data were performed using C6 software version
1.0 (BD Biosciences).

p < 0.05 was considered significant. Data are shown asmean ± SD in the figures, and they represent the results
obtained from three independent experiments.

Detection of apoptosis

S.I. designed this study and prepared the manuscript.
S.I., R.T., T.U., and C.K. performed experiments. S.K.
collected specimens from patients. K.O. and J.H.O
supervised this study.

Author contributions

The FITC Annexin V Apoptosis Detection Kit I (BD
Bioscience) was used. Cell lines treated with the reagentsfor 4 days were suspended in binding buffer and incubatedwith FITC-labeled annexin V and propidium iodide in thedark.
Flow cytometric measurements were performed on a
BD Accuri C6 Flow Cytometer (BD Biosciences). A 488nm blue laser was used for excitation, and signals weredetected using the FL1 channel (533 nm) for FITC and theFL2 channel (585 nm) for propidium iodide. The signalsof 30,000 events were obtained. Analyses of the obtained
data were performed using C6 software version 1.0 (BDBiosciences).

CONFLICTS OF INTEREST
K.O. received research support from Celegene KK,
served as consultant and advisor of Celegene KK, andreceived honoraria for lecture fees from Nippon Shinyaku.
The other authors have no conflicts to disclose.

FUNDING
This work was supported by the Private University
Strategic Research-Based Support Project (grant no.
S1311016 to J.H.O.) from the Ministry of Education,
Culture, Sports, Science and Technology, Japan, andby a Grant-in-Aid for Young Scientists (B) (grantno. 16K19186 to S.I.) from the Japan Society for the
Promotion of Science.

Western blotting
Western blotting was performed as previously
described [25]. Briefly, the membranes were probed with
antibodies directed against DNMT1 (1:200), DNMT3A(1:200), or β-ACTIN (1:500) and then treated with theappropriate secondary antibodies.

REFERENCES

Chromatin immunoprecipitation (ChIP) assay

1. Tefferi A, Vardiman JW. Myelodysplastic syndromes. N
Engl J Med. 2009; 361: 1872-1885. https://doi.org/10.1056/
NEJMra0902908.

ChIP assay was performed using ChIP-IT Express
kit (Active Motif, Carlsbad, CA, USA) in accordance withthe manufacturer’s protocols. Briefly, the chromatin was
extracted from 1 × 107 formalin-fixed cells and chromatin
including 15 μg DNA was immunoprecipitated using 2
μg anti-DNMT1 antibody or anti-DNMT3A antibody.
For negative control, mouse non-specific IgG was used.
The amount of precipitated DNA was measured using
NanoDrop 1000 (Thermo Fisher Scientific).

2. Fenaux P, Mufti GJ, Hellstrom-Lindberg E, Santini V,
Finelli C, Giagounidis A, Schoch R, Gattermann N, Sanz G,
List A, Gore SD, Seymour JF, Bennett JM, et al. Efficacyof azacitidine compared with that of conventional careregimens in the treatment of higher-risk myelodysplastic
syndromes: a randomised, open-label, phase III study.
Lancet Oncol. 2009; 10: 223-232. https://doi.org/10.1016/
S1470-2045(09)70003-8.
3. Fenaux P, Mufti GJ, Hellstrom-Lindberg E, Santini V,
Gattermann N, Germing U, Sanz G, List AF, Gore S,
Seymour JF, Dombret H, Backstrom J, Zimmerman L,
et al. Azacitidine prolongs overall survival comparedwith conventional care regimens in elderly patients withlow bone marrow blast count acute myeloid leukemia. J
Clin Oncol. 2010; 28: 562-569. https://doi.org/10.1200/
JCO.2009.23.8329.

Quantitative RT-PCR
Quantitative RT-PCR was performed as previously
described [26]. TaqMan gene expression assays (LifeTechnologies) were used for UCK1 (Hs01075618_m1) and
UCK2 (Hs00367072_m1). The TaqMan Pre-Developed
Assay Reagent (Life Technologies) was used for ACTB.
The relative expression level of each gene to the ACTB
expression level was determined by the ΔCT method.

4. Prebet T, Gore SD, Esterni B, Gardin C, Itzykson R,
Thepot S, Dreyfus F, Rauzy OB, Recher C, Ades L,
Quesnel B, Beach CL, Fenaux P, et al. Outcome of highrisk myelodysplastic syndrome after azacitidine treatment
failure. J Clin Oncol. 2011; 29: 3322-3327. https://doi.
org/10.1200/JCO.2011.35.8135.

Statistical analysis
For statistical analyses, two-way ANOVA followedby the t-test were performed using GraphPad PRISM 6
software (GraphPad Software Inc., La Jolla, CA, USA);
www.impactjournals.com/oncotarget

69914

-118-

Oncotarget

5. Stresemann C, Lyko F. Modes of action of the DNA
methyltransferase inhibitors azacytidine and decitabine.
Int J Cancer. 2008; 123: 8-13. https://doi.org/10.1002/
ijc.23607.

15. Harrison C. Genetic signatures uncover new uses. Nat Rev
Drug Discov. 2011; 10: 732-733. https://doi.org/10.1038/
nrd3565.
16. Jenwitheesuk E, Samudrala R. Identification of potential
multitarget antimalarial drugs. JAMA. 2005; 294: 14901491. https://doi.org/10.1001/jama.294.12.1490.

6. Nakano T, Katafuchi A, Matsubara M, Terato H, Tsuboi
T, Masuda T, Tatsumoto T, Pack SP, Makino K, Croteau
DL, Van Houten B, Iijima K, Tauchi H, et al. Homologous
recombination but not nucleotide excision repair playsa pivotal role in tolerance of DNA-protein cross-links inmammalian cells. J Biol Chem. 2009; 284: 27065-27076.
https://doi.org/10.1074/jbc.M109.019174.

17. Jenwitheesuk E, Horst JA, Rivas KL, Van Voorhis WC,
Samudrala R. Novel paradigms for drug discovery:
computational multitarget screening. Trends Pharmacol Sci.
2008; 29: 62-71. https://doi.org/10.1016/j.tips.2007.11.007.
18. Minie M, Chopra G, Sethi G, Horst J, White G, Roy A, Hatti
K, Samudrala R. CANDO and the infinite drug discovery
frontier. Drug Discov Today. 2014; 19: 1353-1363. https://doi.org/10.1016/j.drudis.2014.06.018.

7. Imanishi S, Umezu T, Ohtsuki K, Kobayashi C, Ohyashiki
K, Ohyashiki JH. Constitutive activation of the ATM/
BRCA1 pathway prevents DNA damage-induced
apoptosis in 5-azacytidine-resistant cell lines. Biochem
Pharmacol. 2014; 89: 361-369. https://doi.org/10.1016/j.
bcp.2014.03.008.

19. Raynal NJ, Momparler LF, Rivard GE, Momparler RL.
3-Deazauridine enhances the antileukemic action of
5-aza-2'-deoxycytidine and targets drug-resistance due to
deficiency in deoxycytidine kinase. Leuk Res. 2011; 35:
110-118. https://doi.org/10.1016/j.leukres.2010.04.014.

8. Valencia A, Masala E, Rossi A, Martino A, Sanna A,
Buchi F, Canzian F, Cilloni D, Gaidano V, Voso MT,
Kosmider O, Fontenay M, Gozzini A, et al. Expressionof nucleoside-metabolizing enzymes in myelodysplastic
syndromes and modulation of response to azacitidine.
Leukemia. 2014; 28: 621-628. https://doi.org/10.1038/
leu.2013.330.

20. Sripayap P, Nagai T, Uesawa M, Kobayashi H, Tsukahara
T, Ohmine K, Muroi K, Ozawa K. Mechanisms ofresistance to azacitidine in human leukemia cell lines. Exp
Hematol. 2014; 42: 294-306.e2. https://doi.org/10.1016/j.
exphem.2013.12.004.

9. Bar-Or A, Pachner A, Menguy-Vacheron F, Kaplan J,
Wiendl H. Teriflunomide and its mechanism of action in
multiple sclerosis. Drugs. 2014; 74: 659-674. https://doi.
org/10.1007/s40265-014-0212-x.

21. Huang O, Zhang W, Zhi Q, Xue X, Liu H, Shen D, Geng
M, Xie Z, Jiang M. Teriflunomide, an immunomodulatory
drug, exerts anticancer activity in triple negative breast
cancer cells. Exp Biol Med (Maywood). 2015; 240: 426437. https://doi.org/10.1177/1535370214554881.

10. Cherwinski HM, Cohn RG, Cheung P, Webster DJ, Xu
YZ, Caulfield JP, Young JM, Nakano G, Ransom JT. The
immunosuppressant leflunomide inhibits lymphocyte
proliferation by inhibiting pyrimidine biosynthesis. J
Pharmacol Exp Ther. 1995; 275: 1043-1049.

22. Alhefdhi A, Burke JF, Redlich A, Kunnimalaiyaan M, Chen
H. Leflunomide suppresses growth in human medullary
thyroid cancer cells. J Surg Res. 2013; 185: 212-216.
https://doi.org/10.1016/j.jss.2013.05.089.

11. Jao CY, Salic A. Exploring RNA transcription and turnoverin vivo by using click chemistry. Proc Natl Acad Sci
USA. 2008; 105: 15779-15784. https://doi.org/10.1073/
pnas.0808480105.

23. Hail N Jr, Chen P, Bushman LR. Teriflunomide(leflunomide) promotes cytostatic, antioxidant, andapoptotic effects in transformed prostate epithelial cells:
evidence supporting a role for teriflunomide in prostatecancer chemoprevention. Neoplasia. 2010; 12: 464-475.

12. Marcucci G, Silverman L, Eller M, Lintz L, Beach
CL. Bioavailability of azacitidine subcutaneous versus
intravenous in patients with the myelodysplastic
syndromes. J Clin Pharmacol. 2005; 45: 597-602. https://doi.org/10.1177/0091270004271947.

24. Ringshausen I, Oelsner M, Bogner C, Peschel C, Decker T.
The immunomodulatory drug leflunomide inhibits cell cycle
progression of B-CLL cells. Leukemia. 2008; 22: 635-638.
https://doi.org/10.1038/sj.leu.2404922.

13. Uchida T, Ogawa Y, Kobayashi Y, Ishikawa T, Ohashi H,
Hata T, Usui N, Taniwaki M, Ohnishi K, Akiyama H, Ozawa
K, Ohyashiki K, Okamoto S, et al. Phase I and II studyof azacitidine in Japanese patients with myelodysplastic
syndromes. Cancer Sci. 2011; 102: 1680-1686. https://doi.
org/10.1111/j.1349-7006.2011.01993.x.

25. Umezu T, Ohyashiki K, Kuroda M, Ohyashiki JH.
Leukemia cell to endothelial cell communication via
exosomal miRNAs. Oncogene. 2013; 32: 2747-2755.
https://doi.org/10.1038/onc.2012.295.
26. Ohyashiki JH, Hisatomi H, Nagao K, Honda S, Takaku
T, Zhang Y, Sashida G, Ohyashiki K. Quantitative
relationship between functionally active telomerase andmajor telomerase components (hTERT and hTR) in acuteleukaemia cells. Br J Cancer. 2005; 92: 1942-1947. https://doi.org/10.1038/sj.bjc.6602546.

14. Chou TC. Drug combination studies and their synergy
quantification using the Chou-Talalay method. Cancer Res.
2010; 70: 440-446. https://doi.org/10.1158/0008-5472.
CAN-09-1947.

www.impactjournals.com/oncotarget

69915

-119-

Oncotarget

T herapy

Jour nal of&r Sciennce
ce
Ca

ISSN: 1948-5956

Journal ofCancer Science & Therapy

Tauchi et al., J Cancer Sci Ther 2017, 9:6
DOI: 10.4172/1948-5956.1000462

Research Article

Open AccessTargeting the Hedgehog Signaling Pathway by Glasdegib Limits the SelfRenewal of MDS-Derived Induced Potent Stem Cells (iPSC)
Tetsuzo Tauchi1*, Seiichi Okabe1, Seiichiro Katagiri1, Yuko Tanaka1, Kaoru Tohyama2 and Kazuma Ohyashiki1
Department of Hematology, Tokyo Medical University, Tokyo, Japan

1

Kawasaki Med School, Kurashiki, Japan

2

Abstract
Objective: Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by noefficient hematopoiesis and frequent progression to acute myeloid leukemia (AML). Even in low risk MDS, clonal
hematopoiesis already dominates at diagnosis, and clones found in secondary AML originate from the MDS stageof disease, highlighting the need to specifically target the MDS-initiating clone. Glasdegib is a potent and selective
hedgehog pathway inhibitor that acts by binding Smoothened (SMO) and blocking signal transduction. Glasdegib
demonstrated preliminary antitumor activity in a phase I trial, when given as monotherapy in patients with several
hematopoietic malignancies. In the present study, we investigated the molecular mechanisms by which glasdegib
regulate the self-renewal of MDS-derived iPS cells (iPSCs) in vivo.
Methods: We generated iPSCs from bone marrow mononuclear cells of an MDS patient with chromosome 5deletion and complex karyotypic abnormalities. We examined the activity of glasdegib against MDS-derived iPSCs
transferred NOD/SCID mice in vivo and in vitro. We performed the serial in vivo transplantation to assess the effectsof hedgehog inhibition on long term self-renewal. NOD/SCID mice were injected with MDS-iPSCs then treated withglasdegib.
Results: We observed that glasdegib significantly reduced the self-renewal of NOD/SCID re-populating
cells from MDS-derived iPSC during serial transplantation in vivo. Further, NOD/SCID mice were injected with
MDS-iPSCs, and then treated with glasdegib on day 21 for 7 days. These treatments reduced the population of
CD34+CD38-cells. To investigate the entire the apoptosis-induction pathways by hedgehog inhibition, MSD-L cells
were incubated with 5-azacytidine and glasdegib for 72 hrs. Glasdegib enhanced the expression of cleaved PARP,
cleaved caspase-3, p21 and reduced expression of c-Myc.
Conclusion: Our pre-clinical results indicate that glasdegib have potential as an important option for controllingthe drug-resistant MDS-initiating cells. It is expected that the glasdegib may become extremely useful therapeuticinterventions in a number of hematological neoplasms, including MDS, where the persistence of cancer stem cells.

Keywords: Targeted therapy; Hedgehog; MDS; Leukemia Stem Cell;
iPSC; NOD/SCID; Phase I

Introduction
The majority of hematological disorders involving the myeloid
lineage are thought to be of stem cell origin, including myeloproliferative
disease, myelodysplastic syndrome (MDS), acute myeloid leukemia(AML) and acquired or heritable bone marrow failure syndromes[1,2]. In each instance, dysregulation of normal stem cell function isthought to contribute to the disease phenotype [2]. Moreover, stem cellcharacteristics are modulated by a variety of developmental pathways
and regulators. MDS are clonal hematopoietic disorders characterized
by in inefficient hematopoiesis and frequent progression to AML [3].
Even in low-risk MDS, clonal hematopoiesis already dominates atdiagnosis, and clonal hematopoiesis and clones found in secondary
AML originate from the MDS stage of disease [3], highlighting theneed to specifically targeted the MDS-initiating clone. Althoughmassive parallel sequencing studies have revealed a number of genomic
alterations associated with MDS, functional consequence of thesealterations remain poorly understood, mainly due to a difficulty in the exvivo culture of primary MDS cells and lack of appropriate animal model[4]. The discovery of key transcription factors enabling reprograming
a somatic cell into a pluripotent stem cell, called induced pluripotent
stem cell (iPSC) open new avenues in medicine [5-7]. Since iPSC canbe maintained indefinitely in vitro, they represent an unlimited sourceof cells, which could overcome the difficulty of obtaining sufficientamounts of MDS cells [8]. Thus, iPSCs become an attractive model
J Cancer Sci Ther, an open access journal
ISSN: 1948-5956

for cancer stem cell studying, especially the LSC behavior and thescreening of new therapeutic targets reducing LSC survive. Glasdegib isa potent and selective hedgehog pathway inhibitor that acts by binding
Smoothened (SMO) and blocking signal transduction (Figureure 1A)[9]. In xenograft models of human colorectal and pancreatic cancer,
treatment with glasdegib in combination with other anticancer agents
reduced the tumor growth [10]. Furthermore, glasdegib demonstrated
preliminary antitumor activity in a phase I trial, when given as
monotherapy in patients with several hematopoietic malignancies[11,12]. In the present study, we investigated the molecular mechanisms
by which glasdegib regulate the self-renewal of MDS-derived iPS cells(iPSCs) in vivo. We observed that glasdegib significantly reduced theself-renewal of NOD/SCID re-populating cells from MDS-derived
iPSC during serial transplantation in vivo. In vitro clonogenic growth*Corresponding author: Tetsuzo Tauchi, Department of Hematology, Tokyo
Medical University, 6-7-1 Nishishinjuku, Shinjukuku, Tokyo, 160-0023, Japan, Tel:
81333426111; Fax: 81353816651; E-mail: tauchi@tokyo-med.ac.jp
Received May 16, 2017; Accepted June 13, 2017; Published June 15, 2017
Citation: Tauchi T, Okabe S, Katagiri S, Tanaka Y, Tohyama K, et al. (2017)
Targeting the Hedgehog Signaling Pathway by Glasdegib Limits the Self-Renewalof MDS-Derived Induced Potent Stem Cells (iPSC). J Cancer Sci Ther 9: 479-484.
doi: 10.4172/1948-5956.1000462
Copyright: © 2017 Tauchi T, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author andsource are credited.

Volume 9(6 ) 479-484 (2017) - 479-120-

Citation: Tauchi T, Okabe S, Katagiri S, Tanaka Y, Tohyama K, et al. (2017) Targeting the Hedgehog Signaling Pathway by Glasdegib Limits the SelfRenewal of MDS-Derived Induced Potent Stem Cells (iPSC). J Cancer Sci Ther 9: 479-484. doi: 10.4172/1948-5956.1000462

upon serial re-plating, secondary colony formations were significantly
inhibited by hedgehog pathway inhibitors. These results suggested thatthe hedgehog inhibitors may become extremely useful therapeuticinterventions in a number of hematological neoplasms, including
MDS, where the persistence of cancer stem cells [13,14].

Material and Methods
Cell line and antibodies and reagents
MDS-L cell line was established from MDS patient with del (5q)and complex karyotypic abnormalities [15]. Anti-PARP Ab, anticleaved Caspase-3 Ab, anti-p21 Ab, and ant-c-Myc Abs were purchasedfrom Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Glasdegib was
obtained from Pfizer. 5-axacytidine was obtained from Wako (Tokyo).

Generation and culture of iPSCs from a patient with MDSThis research was approved by the ethics committees at Tokyo
Medical University. Primary samples of MDS bone marrow cells wereobtained after informed consent. Briefly, mononuclear cells wereisolated by Ficoll gradient. CD34+ cells were purified according to the
manufacturer’s instructions (Milteny Biotech). iPSCs generation wereobtained by transduction of CD34+ cells from MDS patients with
CytoTune iPS2.0 Sendai virus vector (DINAVEV, Tsukuba, Japan) ata multiplicity of infection (MOI) of 100. After an additional 2-days
culture, cells were transferred onto mitomycined mouse embryonic
fibroblasts (MEFs) and cultured in ES medium as described below.
Starting from day 14 to 21, the individual iPSC colonies were pickedup for expansion.

Human iPSCs culture
Human iPSC clones were maintained as undifferentiated cells in
cultures with mitomycined MEFs. The ES medium used was from
ReproStem (ReproCELL Inc., Tokyo) containing 20% knockout serum
replacement (KSR; ReproCELL Inc.), and 5 ng/ml of recombinanthuman basic fibroblast growth factor (FGF; ReproCELL Inc.). The ES
medium was changed every day.

iPSCs characterization
For teratoma induction, iPSCs were plated in a 10 cm MEFs
feeder dish. At day 6, approximately 2 × 106 cells were harvested,resuspended in 100 µL of ES medium and injected into 6-week-old
female NOD/SCID mice subcutaneously. This study was carried out instrict accordance with the recommendation of the Institutional Animal
Care and Use Committee in Tokyo Medical University and approved(agreement number: H-27020). Teratomas were harvested 6 to 8 weeks
after injection. Paraffin-embedded tissue was sliced and stained with H.E.

Karyotyping
G-banding analysis was performed following the international
system for human cytogenetic nomenclature (ISCN) 2013. Spectral
Karyotyping (SKY) was performed with Spectral Karyotyping reagents(SKYPaint™, Applied Spectral Imaging Ltd., Israel).

RT-PCR analysis
After extraction of total RNA with RNAeasy reagents (QIAGEN,
Germantown, MD), reverse transcription was performed with
SuperScript III (Invitrogen, Waltham, MA). Primer sequences used forthe detection of stem cell genes were previously describe [16,17].

J Cancer Sci Ther, an open access journal
ISSN: 1948-5956

2nd transplantation
NOD/SCID mice were injected with 5 colonis of MDS-iPSCs
subcutaneously, then treated with glasdegib (100 mg/kg; p.o.; q.d.)(n=6) on day 7 for 21 days. After 56 days, we observed that all mice werealive without body weigh loss. We isolated human CD45+ cells from thespleen of mice and injected equivalent numbers of leukemia cells into
secondary recipients. On 28 days, all mice were sacrificed for evaluated.

Results
Glasdegib enhanced the induction of apoptosis with
5-azacytidine in MDS-L cells
We used the MDS-L, a myelodysplastic cell line established from an
MDS patient with del (5q) and complex karyotypic abnormalities for invitro studies. MDS-L cells are cultured with indicated concentrations ofglasdegib for 72 hrs, extracted RNA were analyzed by Real time PCR.
Treatment with glasdegib reduced the expression of Gli1 and Nanog
at more than 5 nM (Figure 1B). Next, MDS-L cells were cultured
with 5-azacytidine and glasdegib for 72 hrs, then, the induction of
apoptosis was analyzed by Annexin V (Figure 1C). The treatment with
5-azacytidine and glasdegib enhanced the induction of apoptosis in
MDS-L cells. To investigate the entire the apoptosis-induction pathways
by hedgehog inhibition, MSD-L cells were incubated with 5-azacytidine
and glasdegib for 72 hrs, the cell lysates were immunoblotted with antiPARP, anti-cleaved Caspase-3, p21, and c-Myc. Glasdegib enhanced
the expression of cleaved PARP, cleaved caspase-3, p21 and reduced
expression of c-Myc (Figure 1D).

Establishment of in vivo differentiation system of MDS-iPSCs
After obtaining informed consent, CD34+ cells were purified
from bone marrow mononuclear cells of the MDS (RAEB2) patient,harboring 45,XY,-5,add(7)(q22),+8,-12,add(18)(q22),-12,-20. MDSCD34+ cells were cultured with α-minimum essential medium (MEM)
containing 20% fetal calf serum (FCS) supplemented with 100 ng/
ml stem cell factor, 10 ng/ml thrombopoietin, 100 ng/ml FL3L, 10
ng/ml IL-3, and 100 mg/ml IL-6 for 2 days, then, CytoTune iPS2.0
Sendai virus vector transduction with the transcription factors OCT3/4,
SOX2, KLF4, and MYC was performed. Two days after transduction,
we reseeded cells onto mitomycin treated mouse embrionic fibroblast(MEF) cells and cultured them for another 2 days. Then, we replaced
the medium with human ES medium supplemented with 5 ng/ml ofrecombinant human bFGF. As a result, MDS-derived iPSCs (MDSiPSCs) were generated, which were derived from the patient (Figure
2A ). MDS-iPSCs showed the typical morphology as iPSCs (Figure2A) and expresses the embryonic stem cell characteristic transcripts(OCT3/4, NANOG, KLF4, SOX) confirmed by RT-PCR (Figure2B). SKY fluorescence in situ hybridization (FISH) confirmed thesame karyotic anomaly as the patient sample (Figure 2C). Teratoma
formation capacity of iPSCs, which were injected into immunodeficient
NOD/SCID mice, were also confirmed (Figure 2D). iPSCs were plated
in a 10 cm MEFs feeder dish. At day 6, approximately 2 × 106 cells
were harvested, resuspended in 100 µL of ES medium and injected
into 6-week-old female NOD/SCID mice subcutaneously. All micedemonstrated engraftment of CD34+CD38-cells, CD45+CD19+ cells,and CD45+CD3+cells from the teratoma at mesoderm portion by flow
cytometry (Figure 3A and 3B). NOD/SCID mice were injected with
MDS-iPSCs. After 56 days, we isolated human CD45+ cells from theteratoma of mice and injected equivalent numbers of CD45+ cells into
secondary recipients. Following 56 days, all mice received MDS-iPSCs
derived CD45+ cells engrafted with engraftment of CD34+CD38-

Volume 9(6 ) 479-484 (2017) - 480-121-

Citation: Tauchi T, Okabe S, Katagiri S, Tanaka Y, Tohyama K, et al. (2017) Targeting the Hedgehog Signaling Pathway by Glasdegib Limits the SelfRenewal of MDS-Derived Induced Potent Stem Cells (iPSC). J Cancer Sci Ther 9: 479-484. doi: 10.4172/1948-5956.1000462

A

B

Gli1 mRNA Expression
Normalized Ratio

1

PF-04449913(Glasdegib)

0.5

PF

0

1

5

10 100 nM

Nanog mRNA Expression
Normalized Ratio

1

0.5

Glasdegib
PF

0

1

5

10 100 nM

OCT3/4 mRNA Expression
Normalized Ratio

1

0.5
Gli1 promoter
PF

C

0

1

5

10 100 nM

D
Control
11.5%

5-AzaC 10 M
63.9%

Glasdegib 1 M

Glasdegib 5 M

11.0%

10.9%

Glasdegib 1 M +5-AzaC 10 M

Glasdegib 5 M +5-AzaC 10 M

86.3%

85.1%

Figure 1: Hedgehog pathway inhibition enhances the induction of apoptosis in MDS-derived cell line. (A) Chemical structure of glasdegib, a selective smoothened
antagonist. (B) MDS-L cells are cultured with indicated concentrations of glasdegib for 72 hrs, extracted RNA were analyzed by Real time PCR. (C) MDS-L cells were
cultured with 5-azacytidine and glasdegib for 72 hrs, then, the induction of apoptosis was analyzed by Annexin V. (D) MSD-L cells were incubated with 5-azacytidine
and glasdegib for 72 hrs, the cell lysates were immunoblotted with anti-PARP, anti-cleaved Caspase-3, p21, and c-Myc.

cells, CD45+CD19+ cells, and CD45+CD3+cells by flow cytometry(Figure 3A and 3B). These results demonstrated that MDS-iPSCs
derived CD34+CD38-cells have the self-renewal and mutilineage
differentiation capabilities.

Hedgehog inhibition with glasgegib limits self-renewal of
MDS-derived iPSC in vivo and in vitro
We examined the effects of hedgehog pathway modulation on invitro clonogenic growth. MDS-L cells and the CD34+cells derived
from the MDS-iPSCs cells were treated with 1 µM of glasdegib
for 72 hrs, washed free of drugs, and plated in quadruplicate inmethylcellulose. At 14 days, colonies were counted as initial plating(Figure 4A ). The representative plate was then washed and cells
were resuspended and replated. After an additional 14 days, colonies
were counted as secondary replanting (Figure 4A). Upon serial replating, secondary colony formations were significantly inhibited by
glasdegib (Figure 4A). We also examined the secondary replating
assays by using CD34+cells separated from MDS-RAEB2 primary
samples. Clonogenic recovery of MDS-L cells, CD34+ cells derived
from MDS-iPSCs, CD34+cells from primary MDS cells were reduced
by glasdegib-treatments (Figure 4A). Further, we performed the serial
in vivo transplantation to assess the effects of hedgehog inhibition onlong term self-renewal (Figure 4B). NOD/SCID mice were injectedwith MDS-iPSCs then treated with glasdegib on day 7 for 21 days.
All mice demonstrated engraftment of CD45+ cells by flow cytometry.

J Cancer Sci Ther, an open access journal
ISSN: 1948-5956

We isolated human CD45+ cells from the spleen of mice from eachtreatment group and injected equivalent numbers of leukemia cells
into secondary recipients. Following 28 days, all mice received MDSiPSCs from vehicle treated mice engrafted with MDS cells (Figure
4B). In contrast, MDS-engraftment was not detected in recipient
mice receiving MDS-iPSCs from initial glasdegib-treated donors(Figure 4B). We have also used smoothened inhibotor, vismodegib
for the experiments to rule out any off-target effects of glasdegib. We
performed the serial in vivo transplantation to assess the effects ofanother smoothened inhibotor, vismodegib on long term self-renewal(Suplymentary Figure). NOD/SCID mice were injected with MDSiPSCs then treated with vismodegib on day 7 for 21 days. However,
3 mice have been died for vismodegib toxicity (mainly body weigt
loss). Remaining mice demonstrated engraftment of CD45+ cellsby flow cytometry. We isolated human CD45+ cells from the spleenof mice from each treatment group and injected equivalent numbersof leukemia cells into secondary recipients. Following 28 days, allmice received MDS-iPSCs from vehicle treated mice engrafted with
MDS cells (Suplymentary Figure). In contrast, MDS-engraftment
was not detected in recipient mice receiving MDS-iPSCs from initialvismogegib-treated donors (Suplymentary Figure). These results
demonstrate the persistent effects of hedgehog inhibition on long term
self-renewing MDS-iPSCs. Further, NOD/SCID mice were injectedwith MDS-iPSCs, then treated with glasdegib on day 21 for 7 days.
These treatments reduced the population of CD34+CD38-cells (Figure

Volume 9(6 ) 479-484 (2017) - 481-122-Citation: Tauchi T, Okabe S, Katagiri S, Tanaka Y, Tohyama K, et al. (2017) Targeting the Hedgehog Signaling Pathway by Glasdegib Limits the SelfRenewal of MDS-Derived Induced Potent Stem Cells (iPSC). J Cancer Sci Ther 9: 479-484. doi: 10.4172/1948-5956.1000462

A

B

MEF

MEF

MDS-iPSC

MDS-iPSC-actin GAPDH

MEF
MDS-iPSC

MDS-iPSCMEF
MDS-iPSCMEF

MDS-iPSC

MEF

Oct3/4 Sox2 Klf4 Nanog

D

C

Reverse DAPI banding

Ectoderm

Endoderm

Mesoderm

Endoderm

Special Image

Comprehensive Karyotyping

Figure 2: Establishment of iPSCs from MDS patient. (A) iPSCs derived from MDS patients shows the typical morphology as iPSCs. Scale bar=100 µm (B) RT-PCR
shows the embryonic stem cell characteristic transcripts (OCT3/4, NANOG, KLF4, SOX) in MDS-iPSCs. (C) SKY fluorescence in situ hybridization (FISH) from iPSCs
from MDS patient. (D) Histological analyses of teratoma from MDS-iPSCs. Hematoxylin and eosin staining. Original rate of magnification: x100. Scale bar=100 µm.

4C). These results demonstrated that the loss of MDS-engraftment in
NOD/SCID mice was caused by glasdegib-reduced the population of
CD34+CD38-cells.

Discussion and Conclusion
Efforts to analyze distinct human CSCs have become a major forcus
in translational and clinical cancer research. The identification of
distinct human CSCs exerting their potential in patients, including in
MDS, has remain elusive [2,3]. In vitro modeling of human disease has
been greatly facilitated by iPSCs technologies [8,13]. Characterized bytheir ability to self-renew indefinitely and differentiate into various celllineages, iPSCs provide a powerful system for human disease modeling[8,13]. In the present study, we established iPSCs from an MDS patient.
MDS-derived-iPSCs had pluripotent markers, teratoma capability,
particular morphology with sharp-edged like ESCs, characteristics of
iPSC (Figure 2A-2D). In vivo assay using NOD/SCID mice, MDS-derived
iPSCs have differentiated into CD34+CD38-cells, CD45+CD19+ cells,and CD45+CD3+ cells from spleen by flow cytometry (Figure 4A
and 4B). Further, MDS-iPSCs derived CD34+CD38-cells have theself-renewal and mutilineage differentiation capabilities (Figure 3B).
However, the reprogramming efficacy of bone marrow MDS CD34+
cells were especially lower than that of normal CD34+ control cells, anddelayed reprograming period (28 days vs 14 days) (data not shown).
This result might be accounted for the fact that cancer-specific genetic
lesions may be a hindrance for reprogramming cancer cells illustrated
by the rare cases of successful cancer cells reprogramming report. It

J Cancer Sci Ther, an open access journal
ISSN: 1948-5956

will be important to overcome the difficulty of reprogram cancer cells.
Application of other factors in addition to the Yamanaka factors may
be effective, such as exogenous expression of miRNA-302, chemicalcompound such as histone deacetylate inhibitor, and knock down of
p53, p21, and Ink4/Arf [14]. We used Sendai virus system, which isefficient induction of transgene free iPSCs. In retrovirus system, theintegration site of retrovirus in iPSCs may affect the gene expression
and change the disease phenotype after differentiating them into theoriginal lineage. Much more effort will be required to overcome thedifficulty of reprogram cancer cells.
Hedgehog signaling is selectively activated in CSCs compared withbulk tumor cells from several tumor types [10]. Also hedgehog is ahighly conserved developmental pathway involved on organogenesis,
stem cell maintenance, and tissue regeneration [10]. Glasdegib is apotent and selective hedgehog pathway inhibitor that acts by binding
SMO and blocking signal transduction [9]. The phase I study showsthat glasdegib is safe and tolerable, with on-target adverse events, doseproportional pharmacokinetics, and early suggestions of clinical activity
in patients with hematological malignancies [11,12]. 5-azacytidine
is a hypomethylating agent that is used as first-line therapy in MDS.
However, only 30%-50% of MDS patients show some response. In
the present study, we observed that co-treatment with glasdegib and5-azacitidine enhanced the induction of apoptosis in MDS-L cells.
Serial transplantation is widely accepted as an assay measuring longterm self-renewal in normal hematopoietic stem cells and neoplasms.
We used a complementary in vitro assay with serial replating and colony

Volume 9(6 ) 479-484 (2017) - 482-123-

Citation: Tauchi T, Okabe S, Katagiri S, Tanaka Y, Tohyama K, et al. (2017) Targeting the Hedgehog Signaling Pathway by Glasdegib Limits the SelfRenewal of MDS-Derived Induced Potent Stem Cells (iPSC). J Cancer Sci Ther 9: 479-484. doi: 10.4172/1948-5956.1000462

A

B

Figure 3: In vivo differentiation system of MDS-iPSCs. (A) Morphology of differentiated MDS-iPSCs. Wright-Giemsa staining. (B) Surface markers of MDS-iPECsderived progenitor cells from MOD/SCID mice. MDS-derived iPSCs have differentiated into CD34+CD38- cells, CD45+CD19+ cells, and CD45+CD3+ cells.

B

A

C

Figure 4: Hedgehog pathway inhibition with glasdegib limits self-renewal in vitro and in vivo (A) MDS-L cells and the CD34+cells derived from the MDS-iPSCs cells
were treated with 1 µM of glasdegib for 72 hrs, washed free of drugs, and plated in quadruplicate in methylcellulose. After an additional 14 days, colonies were counted
as secondary replating. Clonogenic recovery of untreated cells was normalized to 100% and plating results from all treatment groups were expressed as % control.
*p<0.001 compared with control. Similar results were obtained in three independent experiments. (B) An initial cohort of mice was injected with MDS-iPSCs cells andtreated with either glasdegib or vehicle. All mice were engrafted with MDS. Following transplantation of harvested spleen cells, leukemia engraftment was not detected
in recipient mice receiving MDS-iPSCs cells from initial glasgedib-treated donors. Similar results were obtained in two independent experiments. (C) NOD/SCID mice
were injected with MDS-iPSCs cells, then treated with glasdegib on day 21 for 7 days. The percentage of CD34+CD38- cells was analyzed by flow cytometry.

J Cancer Sci Ther, an open access journal
ISSN: 1948-5956

Volume 9(6 ) 479-484 (2017) - 483-124-

Citation: Tauchi T, Okabe S, Katagiri S, Tanaka Y, Tohyama K, et al. (2017) Targeting the Hedgehog Signaling Pathway by Glasdegib Limits the SelfRenewal of MDS-Derived Induced Potent Stem Cells (iPSC). J Cancer Sci Ther 9: 479-484. doi: 10.4172/1948-5956.1000462

formation, as well as serial in vivo transplantation by using MDS-iPSCs
to assess the effects of hedgehog inhibition on long term self-renewal.
We found that glasdegib significantly reduced MDS-iPSCs derived cells
and MDS-L cell line as well as primary MDS cells (Figure 3A and 3B).
The loss of serial transplantation ability is most consistent with selfrenewal, as similarly seen in serial transplantation experiments withnormal hematopoietic cells. It is notable that such long-lasting effectsof hedgehog inhibition were seen following only short exposure to
glasdegib either 72 hours in colony formation assays or after 28 daysof treatment in primary recipient mice. On the basis of our in vitro
clonogenic data, we believe that this loss of serial colony formation andtransplantation ability is due to the effects of hedgehog inhibition on the
self-renewal properties of MDS-initiating cells. However, it is possiblethat the inhibition of engraftment during secondary transplantation ismediated by the effects of hedgehog inhibition on quiescence of MDSinitiating cells their ability to interact with potential stem cell niches,and proper homing during transplantation. The combined results ofcell-based and in vivo studies suggest that glasdegib exhibits sufficient
activity against MDS-derived cells to warrant consideration for
combination with standard care in MDS. Moreover, clinical importanceof glasdegib mediated sensitization of dormant MDS-initiating cells totherapeutic modalities that target dividing cells will need to assess inclinical trials. Although several hedgehog inhibitors have now enteredclinical evaluation, it is expected that hedgehog inhibitors may become
extremely useful therapeutic innervations in a number of hematopoietic
neoplasms where the persistence of cancer stem cells.
References

Establishment of human Ipsc-based models for the study and targeting ofglioma initiating cells. Nat comm 7: 1-14.
5. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse
embrionic and adult fibroblast cultyres by defined factors. Cell 126: 663-676.
6. Takahashi K, Tanaka K, Ohnuki M, Narita M, Ichisaka T, et al. (2007) Inductionof pluripotent stem cells from adult human fibroblasts by defined factors. Cell
131: 861-872.
7. Lee DF, Su J, Kim HS, Chang B, Papatsenko D, et al. (2015) Modeling familial
cancer with induced pluripotent stem cells. Cell 161: 240-254.
8. Kotini AG, Chang CJ, Boussaad I, Delrow JJ, Dolezal EK, et al. (2015)
Functional analysis of a chromosomal deletion associated with myelodysplastic
syndromes using isogenic human induced pluripotent stem cells. Nat biotech
33: 646-655.
9. Munchhof MJ, Li Q, Shavnya A, Borzillo GV, Boyden TL, et al. (2016) Discovery
of PF-04449913, a potent and orally bioavailable inhibitor of smoothend. Med
Chem lett 3: 106-111.
10. Justilien V, Fields AP (2015) Molecular pathways: Novel approaches for
improved therapeutic targeting of hedgehog signaling in cancer stem cells. Clin
Cancer Res 21: 505-513.
11. Wagner AJ, Messersmith WA, Shaik MN, Li S, Zheng X, et al. (2015) A phaseI study of PF-04449913, an oral hedgehog inhibitor, in patients with advanced
solid tumors. Clin Cancer Res 21: 1044-1051.
12. Martinelli G, Oehler VG, Papayannidis C, CourtneyR, Shaik MN, et al. (2015)
Treatment with PF-04449913, an oral smoothened antagonist, in patients withmyeloid malignancies: A phase 1 safety and pharmacokinetics study. Lancet
Oncol 8: e339-346.
13. Papapetrou EP (2016) Patient-derived induced pluripotent stem cells in cancer
research and precision oncology. Nat Med 22: 1392-1400.

1. Jaiswal S, Ebert BL (2014) MDS is a stem cell disorder after all. Cancer Cell
25: 713-714.

14. Lee MR, Prasain N, Chae HD, Kim YJ, Mantel C, et al. (2013) Epigenetic
regulation of nanog by miR-302 cluster-MBD2 completes induced pluripotent
stem cell reprogramming. Stem Cells 31: 666-681

2. Woll PS, Kjallquist U, Chowdhury O, Doolittle H, Wedge DC, et al. (2014)
Myelodysplastic syndromes are propagated by rare and distinct human cancer
stem cells in vivo. Cancer Cell 25: 794-808.

15. Matsuoka A, Tochiga A, Kishimoto M, Nakahara T, Kondo T, et al. (2010)
Lenalidomide induces cell death in an MDS-derived cell line with deletion ofchromosome 5q by inhibition of cytokinesis. Leukemia 24: 748-755.

3. Pang WW, Pluvinage JV, Price EA, Sridhar K, Arber DA, et al. (2013)
Hematopoietic stem cell and progenitor cell mechanisms in myelodysplastic
syndromes. Proc Natl Acad Sci USA 110: 3011-3016.

16. Sadarangani A, Pineda G, Lennon KM, Chun HJ, Shih A, et al. (2015) Gli2
inhibition abrogates human leukemia stem cell domancy. J Trans Med 13: 98-112.

4. Sancho-Martinez I, Nivet E, Xia Y, Hishida T, Agurrie A, et al. (2016)

J Cancer Sci Ther, an open access journal
ISSN: 1948-5956

17. Liu Z, Zhang J, Kang H, Sun G, Wang B, et al. (2016) Significance of stem cellmarker Nanog gene in the diagnosis and prognosis of lung cancer. Oncol Lett
12: 2507-2510.

Volume 9(6 ) 479-484 (2017) - 484-125-

Leukemia Research 62 (2017) 23–28

Contents lists available at ScienceDirect

Leukemia Research
journal homepage: www.elsevier.com/locate/leukres

Research paper

Genetic variations of bone marrow mesenchymal stromal cells derived fromacute leukemia and myelodysplastic syndrome by targeted deep sequencing

MARK

Kenko Azumaa, Tomohiro Umezub, Satoshi Imanishia, Michiyo Asanob,1, Seiichiro Yoshizawab,⁎
Seiichiro Katagirib, Kazuma Ohyashikib, Junko H. Ohyashikia,ab

Department of Molecular Oncology, Institute of Medical Science, Tokyo Medical University, 160-0023, Japan
Department of Haematology, Tokyo Medical University, 160-0023, JapanA R T I C L E I N F O

A B S T R A C T

Keywords:
Bone marrow mesenchymal stromal cells
Targeted deep sequencing
Genetic variants
Myeloid malignancies

Bone marrow mesenchymal stromal cells (MSCs), which support proliferation and diﬀerentiation of hematopoietic stem cells, may play a crucial role in the pathogenesis of myeloid neoplasms. To determine whether MSCs
in myeloid neoplasms harbor distinct somatic mutations that may aﬀect their function, we used a targeted genesequencing panel containing 50 myeloid neoplasm-associated genes with coverage of ≥500. We compared thegenetic alterations between MSCs and bone marrow hematopoietic (BM) cells from patients with acute leukemia(n = 5) or myelodysplastic syndrome (MDS, n = 5). Non-synonymous somatic mutations, such as DNMT3AR882H and FLT3-D835Y, were only detected in BM cells with high allelic frequency. We found several nonsynonymous genetic variants overlapping BM cells and MSCs, including TP53 and ASXL1, partially owing to theheterogenous cell fraction of MSC samples and lineage ﬁdelity. We also found MSC-speciﬁc genetic variants withvery low allelic frequency (7% to 8%), such as NF1-G2114D and NF1-G140. Further studies in large cohorts areneeded to clarify the molecular properties of MSCs including age-related genetic alterations by targeted deep
sequencing.

1. Introduction
The bone marrow mesenchymal stromal cell (MSC) population, themajor component of hematopoietic microenvironment [1,2], is comprised of a mixture of several adherent cell types including ﬁbroblasts,
endothelial cells, macrophages, osteoclasts [3,4], and mesenchymal
stem cells that can diﬀerentiate into adipocytes, astrocytes, cardiomyocytes, chondrocytes, hepatocytes, muscles, neurons, and osteoblasts [5]. The deﬁnition of MSC is therefore somewhat obscure owing
to the complexity of cells in the stromal cell fraction. The hematopoietic
microenvironment is involved in the pathogenesis of acute myeloid
leukemia (AML) as well as myelodysplastic syndrome (MDS) [6,7].
Accordingly, dysfunction of MSCs leading to insuﬃcient stromal support, impaired osteogenic diﬀerentiation activity, and increased IL-6
secretion has been reported in AML and MDS [8–10]. In addition to thefunctional abnormalities of MSCs, a distinct gene expression proﬁle in
MSCs from AML and MDS has also been reported using either microarray or RNA sequencing [11–13].
Several reports demonstrated cytogenetic abnormalities in stromal
cell fractions derived from AML and MDS patients [14–19]. More recent⁎

1report using whole exome sequencing demonstrated that genetic alterations in the stromal compartment in 16 AML patients were nonspeciﬁc [13], while other studies using mouse models suggested nicheinduced oncogenesis [20,21]. Therefore, whether MSCs derived fromhematologic disorders harbor speciﬁc somatic mutations that may playa key role in the pathogenesis of myeloid neoplasms remains unclear. In
practical terms, genetic analysis of MSCs is challenging because it isdiﬃcult to exclude genetic polymorphisms by simultaneous study ofgerminal cells, in addition to lineage ﬁdelity of MSCs. To address thesequestions and to gain more insight into genetic properties of MSCs
derived from patients with acute leukemia and MDS, we analyzed genetic variants in 50 major genes associated with myeloid malignancies
by a targeted deep sequencing.
2. Materials and methods
2.1. PatientsTen consecutive patients with myeloid malignancies (ﬁve withacute leukemia and ﬁve with MDS) were included in this study

Corresponding author.
E-mail address: junko@hh.iij4u.or.jp (J.H. Ohyashiki).
Current address: Department of Hematology, Erasmus Medical Center, Wytemaweg 80 3015CN, Rotterdam, the Netherland.

http://dx.doi.org/10.1016/j.leukres.2017.09.008
Received 30 March 2017; Received in revised form 15 September 2017; Accepted 17 September 2017
Available online 21 September 20170145-2126/ © 2017 Elsevier Ltd. All rights reserved.

-126-

Leukemia Research 62 (2017) 23–28

K. Azuma et al.

Table 1
Patient characteristics and cytogenetic results.
UPN

Age (years)

GenderDiagnosis

Karyotypes of BM cells

Karyotypes of BMSCs

Acute leukemia
36
74

Male

AML

45,XY,-18[6]/46,XY[27]3755

FemaleAML

Multiple missing chromosomesa [11]/46,XX[7]

38
39

77
61

Male
Male

AML
BAL

46,XY,inv(9)(p12q13)[15]/46,XY[6]46,XY,-4,add(5)(q31),-13, add(14)(q32),+2 ∼ 4mar[19]/46,XY,-4,add(5)(q31),add(14)(q32),-20,+2mar[1]/46,XY,-8,8,+2mar[1]46,XX,inv(16)(p13q22)[4]/47,XX,idem,+22[18]47,XY,+7[2]/46,XY,-15,der(1)t(1;15)(q24;q13)[1]/46,XY[22]46,XX,t(3;12)(q24;q13),t(1;8)(p24;q22)[2]/47,XX,+8[1]/46,XX,-7,+der(7)t(1;7)(q11;p21)[1]/45,XX,-5,6,-12,+2mar[1]/46,XX[21]46,XY[6]46,XY[1]4042FemaleMyelodysplastic syndrome
10
60
Male
18
75
Male

25
13

72
70

Male
Male

35

67

Male

t-MDS/AML
RA (intermediate)
RCMD (very high)

RCMD (intermediate)
RAEB-2(intermediate)
RAEB-2 (high)46, XX[5]46,XY,del(20)(q11)[20]/46,XY[1]41-44,X,-Y,del(5)(q13), del(17)(p12)[16]/-18[14]/t(1;3)(p22;q22),del(7)(p15)[11]/add(2)(q37),-13[10]/-22,+mar[5]/-20[4]/del(11)(q23)[3]46,XY,-7,+mar[21]45,X,-Y[6]/46,XY[16]46,XY[2]46,XY[4]46,XY[10]46,XY,del(3)(p21),add(4)(p15)[8]/multiple deletion

45,XY,del(1)(p12),-15[2]/46,XY[13]No metaphase

UPN, unique patient number; BM, bone marrow; BMSC, bone marrow stromal cell; RA, refractory anemia; RCMD, refractory cytopenias with multilineage dysplasia; RAEB-2, refractory
anemia with excess blasts-2; AML, acute myeloid leukemia; BAL, biphenotypic acute leukemia; MDS, myelodysplastic syndrome; (x), IPSS-R; [n], number of metaphases.
amultiple missing chromosomes: representative karyotype = 42,XX,-5,-7,-8,-10, 43,XX,-6,-15,-17; missing 17 in 6 cells, missing 5 in 4 cells, missing 11 in 3 cells, missing 11b in 3 cells,
missing 19 in 3 cells, missing 6 in 23 cells, and missing 7 in 3 cells.

(Table 1). All patients were treated at Tokyo Medical University, Tokyo,
Japan from April 2011 to September 2015. The patients’ mean age was65.3 years (range 42–77 years). The diagnoses were established according to the 2008 World Health Organization criteria: three patientswere diagnosed with de novo AML, one patients with biphenotypic
acute leukemia (BAL), one patient with therapy-related myelodysplastic
syndrome/acute myeloid leukemia (t-MDS/AML), one patient with refractory anemia (RA), two patients with refractory anemia with excess
blasts (RAEB), and two patients with refractory cytopenia with multilineage dysplasia (RCMD). Cytogenetic analyses of both bone marrow
and MSCs were performed as reported previously [22]. Bone marrow
and MSCs obtained from three patients with non-Hodgkin’s lymphoma(NHL) without bone marrow invasion were also used as non-myeloid
malignancy controls for deep sequencing. Written informed consent
was obtained from all patients. The study was validated by the internal
review boards of Tokyo Medical University and followed both the Declaration of Helsinki and “Guidelines for Genetic Tests and Diagnoses in
Medical Practice” by The Japanese Association of Medical Sciences.

monoclonal antibodies from BD Pharmingen (San Jose, CA, USA). MSCs
diﬀerentiated to adipocytes and osteoblasts after exposure to MSC differentiation media (PromoCell, Heidelberg, Germany) for at least one
week. Adipocyte and osteoblast diﬀerentiation was determined bystaining with Oil Red (Sigma, St. Louis, MO, USA) and Alizarin Red(Wako, Osaka, Japan), respectively (Supplementary Fig. 1).

2.2. Primary MSC culture

2.4. GeneRead DNAseq library preparation

MSCs were obtained by the classical adhesion method with a minor
modiﬁcation [23,24]. Brieﬂy, 0.5–1 mL of freshly obtained bonemarrow aspirates were cultured in an equivalent volume of RPMI1640
medium (Thermo Fisher Scientiﬁc, Waltham, MA, USA) containing 10%of fetal bovine serum (FBS; GE Healthcare UK, Buckinghamshire, England), 1% of penicillin streptomycin (P/S; Thermo Fisher Scientiﬁc),and Dulbecco's Modiﬁed Eagle Medium (DMEM) (Thermo Fisher Scientiﬁc) containing 10% of FBS (GE Healthcare, UK), 1% of P/S, and 1%
of non-essential amino acids (NEAA; Thermo Fisher Scientiﬁc). Primarycultured cells were cultured for 3–5 days, and medium was exchanged
to DMEM (with 10% of FBS, 1% of P/S, 1% of NEAA) for non-hematopoietic expansion, after removing non-adherent cells. Over a period of
1–2 weeks, the adherent cells cultured with DMEM were split bytrypsinization and harvested for cryopreservation with Cell Banker 1(ZENOAQ, Fukushima, Japan) until use.
The cultured MSC population was identiﬁed as CD73+, CD90+,
CD105+, CD34-, CD45- and HLA-DR- by ﬂow cytometry with < 5%
CD34+ and CD45+. Data for all cell surface markers were obtainedusing a BD Accuri C6 (Becton Dickinson, Franklin Lakes, NJ, USA), with

The targeted gene fragments were ampliﬁed from 40 ng of genomic
DNA from each patient using the GeneRead DNAseq Panel PCR Kit V2(QIAGEN) and GeneRead DNAseq Targeted Panel V2 (QIAGEN), resulting in an average amplicon size of 150 bp. Amplicons were puriﬁedusing Agecourt AMP XP Beads (BECKMAN COULTER, Brea, CA, USA).
Following library construction, puriﬁed amplicons were end-repaired(GeneRead DNA Library I Core Kit; QIAGEN) and patient-speciﬁc barcode adapters were ligated (GeneRead Adapter I Set 12-plex; 12 different ones in total; QIAGEN). Constructed libraries were puriﬁed using
the GeneRead Size Selection Kit (QIAGEN) and Agecourt AMP XP Beads(BECKMAN COULTER). Puriﬁed libraries were then ampliﬁed using the
GeneRead DNA I Amp Kit (QIAGEN), and puriﬁed using the QIAquick
PCR Puriﬁcation Kit (QIAGEN), resulting in an average amplicon size of
280 bp. Each library was eventually pooled equimolarly in one tube for
DNA sequencing. The quality of libraries was conﬁrmed by an Agilent
2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) andquantiﬁed using the Qubit 2.0 ﬂuorometer (Thermo Fisher Scientiﬁc)with the Molecular Probes Qubit dsDNA HS Assay Kit (Thermo Fisher
Scientiﬁc).

2.3. Genomic DNA extraction
MSCs were collected for DNA extraction after one passage of culture. Bone marrow mononuclear cells containing hematopoietic cells(hereafter BM cells) were separated using Ficoll-Hypaque gradients asreported previously [25]. Buccal mucosa cells were also collected fromthree patients (from whom informed consent was obtained) by gently
stroking the intraoral cavity three times using sterile foam tipped applicators (GE Healthcare, UK). Genomic DNA was extracted from
BMSCs, BM cells and buccal mucosa cells using a Genta PureGene Cell
Kit (QIAGEN, Hilden, Germany), according to the suppliers’ instruction.

24

-127-

Leukemia Research 62 (2017) 23–28

K. Azuma et al.

2.5. Targeted sequencing and data analysis

variants, tumor protein p53 (TP53-R202H) and additional sex combs
like 1 (ASXL1-L1444R), were found in MSCs of UPN37 in addition to
BM cells. Notably, neuroﬁbromin 1 (NF1-G2114D) in UPN36 was the
only MSC-speciﬁc genetic variant, while the VAF was low compared tothose in leukemia cell-speciﬁc genetic variants (Table 2). We also noteda genetic variant of tet methylcytosine dioxygenase 2 (TET2-S1107P)
with low VAF in UPN39 and UPN40; the genetic variant was detected in
MSC and BM cells and not detected in germ line controls.

The Myeloid Neoplasms Panel (QIAGEN), with 50 known or putative mutation gene targets of hematopoietic diseases, was used for DNA
sequencing with MiSeq (Illumina, San Diego, CA, USA). All sequencing
data were mapped and annotated by QIAGEN cloud-based mapping andannotating tool GeneRead Panel Variant Calling. High-probability oncogenic genetic variants were extracted from annotated data based on
the available database ClinVar (https://www.ncbi.nlm.nih.gov/
clinvar/) and COSMIC (http://cancer.sanger.ac.uk/cosmic).
All other non-polymorphous non-synonymous/synonymous andintronic genetic variants were extracted individually by ﬁltering population frequencies (< 1%) with a coverage of ≥500 using the sequencing data analysis tool VariantStudio (Illumina).

3.3. Non-synonymous mutations detected in patients with MDS
Unlike acute leukemia patients, the VAFs in specimens from MDS
were variable, and we did not detect MSC-speciﬁc genetic variants(Table 3). BM cell-speciﬁc genetic variants DNMT3A-G543C and lysine
methyltransferase 2A (KMT2A-R2890P), with VAFs of approximately
50%, were detected in UPN18. In UPN25, genetic variants of SET
binding protein 1 (SETBP1-G870S) and DNMT3A-F354S were detected,
and the VAFs in BM cells (40% and 38%, respectively) were higher inthose in BMSCs (12% and 13%, respectively), indicating heterogeneityof MSCs obtained by the classical adhesion method. We also noted agenetic variant of KRAS proto-oncogene, GTPase (KRAS-T58I), in
UPN25 with low VAF (10%). Mutation of neuroﬁbromin 1 (NF1E1720D) in BM cells, MSCs, and buccal mucosa cells (germ line controls) was found in UPN35.

3. Results
3.1. Limitation of cytogenetic analysis to identify clonal nature of MSCs
We tried to compare the cytogenetic results between BM cells andMSCs, however we could only obtain suﬃcient metaphases in a limited
number of samples (Table 1). Unfortunately, we could not obtain metaphases in one MSC sample from UPN 13, and ﬁve patients showednormal karyotypes in less than 10 metaphases (UPN 38, 39, 40, 10 and18). Although the majority of metaphases were normal karyotype, threepatients (UPN 36, 37, and 25) harbored a clonal cytogenetic change
distinct from those of BM cells. The remaining one patient (UPN 35)
had multiple chromosome deletions in addition to structural changes in
MSCs; however, BM cells showed normal male karyotype. In brief, we
could not conclude the clonal nature of MSCs by conventional cytogenetic analysis, possibly due to the low mitotic activity of MSCs.
Therefore, we next performed mutation analysis.

3.4. Synonymous and intronic mutations in patients with acute leukemia
and MDSWe next assessed the synonymous genetic variants that do not alter
the amino acid sequence of a protein (Table 4). We found genetic
variants with VAF of approximately 50% in structural maintenance ofchromosomes 1A (SMC1A-Q391) in UPN40 and KMT2A-E3639 in
UPN18. In UPN40, SMCA1Z-Q391 was also detected in germ-line controls. We also found genetic variants of JAK1-E186 in three patients(UPN13 and 18) and JAK2-I214 in four patients (UPN10, 13, 18, and39); however, the VAFs were less than 20%. We also noted MSC-speciﬁc
genetic variants of NF1-G140 with low VAF (8%) in UPN40 (Table 4).
To further elucidate MSC-speciﬁc genetic alterations, we summarized the intronic genetic variants in Table 4, although biological signiﬁcance is poorly understood. Similar to the results in synonymous
genetic variants of NF1-E1720D, shared genetic variants were found in
3 genes: SMC3, RAD21 cohesin complex component (RAD21), and NF1
were detected in three patients (UPN35, 39, and 40), with VAF of approximately 0.5. The polycomb protein SUZ12 (SUZ12-T > T/G) was
BMSC-speciﬁc genetic variants with low VAF (Supplementary ﬁle 1).

3.2. Non-synonymous mutations detected in acute leukemia patients
We were able to obtain buccal mucosa cells as a source in 3 of 10
patients, however germ line controls were not available in the remaining patients. We therefore carefully extracted genetic variants to
exclude polymorphisms using two available databases: ClinVar and
COSMIC. We then focused on non-synonymous genetic variants thatalter the amino acid sequence of a protein in acute leukemia patients(Table 2). Genetic variants were mainly compared between MSC andBM cells, since we could not obtain germ-line controls, except for threepatients (UPN35, 39 and 40). In BM cells with acute leukemia, BM cellspeciﬁc genetic variants with a variant allele frequency (VAF) of approximately 50% corresponding heterogeneous mutations were detected in three of the ﬁve patients: DNA methyltrasnferase 3A(DNMT3A-R882H) and MPL proto-oncogene, thrombopoietin receptor(MPL-Ter636H) in UPN36; fms related tyrosine kinase 3 (FLT3-D836H)
in UPN37; and FLT3-D836Y in UPN39. In contrast, two genetic

3.5. Non-synonymous mutations in non-myeloid malignancies
To clarify whether genetic variants are found in non-myeloid malignancies, we used stage I NHL specimens. We performed targeted

Table 2
Non-synonymous genetic variants detected in patients with acute leukemia.
Gene

UPN36
BM

DNMT3A
TP53
ASXL1
FLT3
MPL
NF1
TET2

UPN37
MSC

BM

MSC

R202H (0.50)
L1444R (0.55)
D835H (0.46)

R202H (0.46)
L1444R (0.50)

UPN38

UPN39

BM

BM

MSC

UPN40
MSC

Bu

BM

MSC

S1107P (0.06)

S1107P (0.07)

Bu

R882H (0.42)

D835Y (0.43)

Ter636H (0.45)
G2114D (0.07)
S1107P (0.06)

S1107P (0.07)

UPN, unique patient number; BM, bone marrow mononuclear cells; MSC, bone marrow stromal cells; Bu, buccal mucosa cells; mutated amino acid position is described in “Nonsynonymous”; Variant allele frequency (VAF) in this table is shown as raw data in the parenthesis. The VAF is the proportion of reads at a site which contain the variant allele. If you have
500 reads total, and 100 of them have the variant, then its frequency is 100/500 = 0.2.

25

-128-

Leukemia Research 62 (2017) 23–28

K. Azuma et al.

Table 3
Non-synonymous genetic variants detected in patients with myelodysplastic syndrome.
Gene

UPN10
BM

UPN13
MSC

BM

UPN18
MSC

SETBP1
DNMT3A
KMT2A
FLT3
NF1
KRAS
TET2

UPN25

BM

MSC

G543C (0.38)
R2890P (0.39)
P336L (0.50)

UPN35

BM

MSC

G870S (0.40)
F354S (0.38)

G870S (0.12)
F354S (0.13)

BM

MSC

Bu

E1720D (0.52)

E1720D (0.50)

E1720D (0.49)

P336L (0.54)
T58I (0.10)
S1107P (0.06)

UPN, unique patient number; BM, bone marrow mononuclear cells; MSC, bone marrow stromal cells, Bu, buccal mucosa cells; mutated amino acid position was described in “Nonsynonymous”; Variant allele frequency (VAF) in this table is shown as raw data in the parenthesis. The VAF is the proportion of reads at a site which contain the variant allele. If you have
500 reads total, and 100 of them have the variant, then its frequency is 100/500 = 0.2.

deep sequencing using the same panel of 50 genes related to myeloid
malignancy. We did not identify any non-synonymous mutations, either
leukemia cell-speciﬁc or overlapping in BM cells and MSCs with highallelic frequency. Further, we did not detect any genetic variants at low
allele frequency, except for TET2. We found that both BM cells andMSCs harbored TET2-S1107P in all three lymphoma patients (aged
65–71 years old).

MSC-speciﬁc mutations, and overlapping genetic variants, possibly due
to the high coverage of reads. We considered that several overlapping
mutations found in BM cells, MSC and buccal mucosa cells may be
germ-line genetic variants. Besides, shared genetic variants in BM cells
and MSC might be due to the involvement of hematopoietic cells during
the separation procedure or lineage ﬁdelity. While the VAF of BMspeciﬁc genetic variants was variable in MDS patients, possibly due toheterogeneity of specimens, it was approximately 50% in acute leukemia patients. In contrast, the MSC-speciﬁc VAF was less than 10% in
both MDS and acute leukemia, indicating that these are possible nonrandom genetic variations but not likely to be clonal. A recent reportdemonstrated non-speciﬁc genetic variation in MSCs using wholeexome analysis with coverage of ≥20 reads [13]. In contrast, we performed deep sequencing with coverage of ≥500, and therefore we
could not ignore the MSC-speciﬁc mutations with low VAF.
Several reports have demonstrated genetic abnormalities of MSCs
[11,14–17]. Flores-Figueroa et al., reported that MSCs derived from 5 of
9 MDS patients harbored abnormal karyotypes [14]. Molecular cytogenetic analysis by Blau et al., revealed that BMSCs from MDS and AML
patients exhibit chromosomal abnormalities, with the majority of cytogenetic aberrations being non-clonal and distinct from chromosomal
markers in hemopoietic cells from the same individual [15]. They also
reported MSC-speciﬁc chromosome abnormalities in 15 of 94 patientswith myeloid neoplasia, including four structural abnormalities andeleven numerical changes (e.g., −Y), although they did not detect MSCspeciﬁc mutations by conventional DNA sequencing [17]. Using ﬂuorescent in situ hybridization (FISH) and identifying genetic changes byarray-based comparative genomic hybridization (array-CGH), LopezVillar et al., demonstrated that MSCs obtained from MDS patients havegenomic abnormalities, with some linked to MDS with isolated del(5q)[16]. More recently, Huang et al. reported that MSCs derived from AML
had cytogenetic abnormalities both distinct and overlapping with theircorresponding blasts. In the current study, we did not ﬁnd clonal

4. Discussion
The interaction between cancer cells and their surrounding tissue
that constructs the tumor microenvironment has been extensively studied in solid tumors as well as hematological malignancies [26–28].
One of the major components of the tumor microenvironment is ﬁbroblasts, namely cancer-associated ﬁbroblasts (CAFs), and somatic
mutations in CAFs from whole tumor biopsies were reported in breast
cancer cells [29,30]. However, Campbell and his colleagues demonstrated that strictly isolated CAFs did not show any evidence of copy
number gain or loss, or loss of heterozygosity of any chromosomes byimprovement of technical limitation [31]. Thus, two important issues
should be addressed when dealing with genetic abnormalities of CAFs:
one is the heterogeneity of CAFs, and the other is establishing a method
to detect genetic variants using expanded CAFs in vitro.
Similar to the obstacles in solid tumors, the heterogeneity of MSCs
and methodological diﬀerences, such as depth of sequencing andlineage ﬁdelity, complicate the interpretation of genetic analyses inhematologic malignancies. For example, early passage MSCs cannot be
separated (as with microdissection), while later passage cells might be
modiﬁed by in vitro culture. Consequently, discussing genetic variationof MSCs in hematologic malignancies is challenging. Lack of germ line
controls also complicates interpretation in some cases. Considering
those complexities, we categorized several types of genetic alternations,such as BM cell-speciﬁc genetic variants reﬂecting neoplastic nature,

Table 4
Synonymous genetic variants detected in patients with acute leukemia and myelodysplastic syndrome.
Acute leukemia
Gene

UPN36
BM

SMC1A
Myelodysplastic syndrome
Gene

UPN10
BM

JAK1
JAK2
KMT2A
NF1

UPN37
MSC

I214 (0.05)

MSC

BM

MSC

UPN13
BM
MSC
E186 (0.05)
I214 (0.16)

BM

UPN38
MSC

UPN18
BM
MSC
E186 (0.05)
I214 (0.09)
E3639 (0.42) E3639 (0.48)

BM

UPN39
MSC

Bu

BM
Q391 (0.52)

BM

MSC

UPN25
BM

UPN40
BM
Q391 (0.50)

Bu
Q391 (0.49)

UPN35
MSC

Bu

I214 (0.06)
G140 (0.08)

UPN, unique patient number; BM, bone marrow mononuclear cells; MSC, bone marrow stromal cells; Bu, buccal mucosa cells; variant allele frequency is indicated in parentheses. Variant
allele frequency (VAF) in this table is shown as raw data in the parenthesis. The VAF is the proportion of reads at a site which contain the variant allele. If you have 500 reads total, and100 of them have the variant, then its frequency is 100/500 = 0.2.

26

-129-

Leukemia Research 62 (2017) 23–28

K. Azuma et al.

cytogenetic abnormalities in MSC corresponding malignant cells;
however, we found both distinct and overlapping mutations by deep
sequencing. Cai et al. reported that the non-synonymous changes inhuman MSCs detected by whole-genome sequencing increased after latepassages of culture; the genetic abnormalities did exist in uncultured
cells with a low allelic frequency but reached up to 36% in passage 13[32]. Similarly, Kouvidi et al. reported that chromosomal abnormalities
in MDS-derived MSCs are more frequently detected in passage 6–8
compared to passage 2, suggesting that MDS-derived MSCs are genetically unstable [19]. Kim et al. also reported that global DNA hypomethylation, which is closely linked to genomic instability, was prominent in AML-derived BM-MSCs compared with MDS-derived BMMSCs [18]. While the earlier report by Lopez-Villar et al., demonstrated
chromosomal abnormalities in uncultured MSCs [16], genetic analysis
results (either cytogenetic or mutation analyses) may be partially
modiﬁed by in vitro culture periods.
The question still remains as to whether clonal sweeping of rare
mutations with low allelic frequency plays some roles in the leukemia
cell-stroma cell interaction. In human disease, there are conceptual
diﬃculties with the genetic coevolution of tumor cells and stroma cells,
since it means a kind of double cancer. However, a murine model by
Raaijmakers et al. demonstrated that deletion of Dicer1 speciﬁcally inmouse osteoprogenitors disrupted the integrity of hematopoiesis [20].
Since osteoprogenitors are a mesenchymal subset of stromal cells, primary stromal dysfunction due to Dicer 1 deletion leads to leukemia as
secondary malignancy. Although the biological relevance of the BMSCspeciﬁc genetic variants detected in the current study is still uncertain,
we completely rule out the possibility that clonal selection of genetically altered BMSCs, even with low allelic frequency, may modify theleukemia cell-BMSC interaction.
This study has several limitations. First, we could not obtain MSCs
from normal individuals due to ethical issues. As an alternative, weused NHL specimens with no bone marrow invasion, and except for
TET2-S1107P, did not identify any genetic variants in AL and MDS
patients. Since all lymphoma patients were aged over 60 years, TET2S1107P at low allelic frequency may reﬂect age-related genetic
changes. However, it is still uncertain whether MSCs derived fromnormal individuals exhibit rare genetic variants, and analysis using alarge number of non-myeloid malignancy controls, as well as normal
controls, must be performed. Second, we used MSCs obtained by theclassical adhesion method instead of by sorting MSCs, and therefore thecell components are heterogeneous. Hence, interpretation of shared
mutations should be performed carefully, especially when germ-line
controls are absent. Third, the number of patients is too small, andtherefore we could not discuss acute leukemia derived-MSCs and MDSderived MSCs separately. Further study should clarify these points.
In summary, our study provides ﬁndings by targeted deep sequencing that could not be identiﬁed by whole exome analysis with limited
read depth. First, we identiﬁed neoplastic cell-speciﬁc mutations, aswell as shared genetic variants, in both BM cells and MSCs. In comparison with germ-line controls, some shared mutations reﬂect pathogenic ones, while others reﬂect germ-line genetic changes. Second, we
found MSC-speciﬁc genetic variants at a very low allele frequency,
which may possibly reﬂect clonal sweeping. Since allelic frequency isvery low, these genetic variants might be undetectable by conventional
DNA sequencing. Finally, we found the TET2-S1107P mutation in BMs
and MSCs, but not germ line control cells. Non-myeloid controls also
harbor this genetic change, indicating the possibility of age-related
clonal hematopoiesis [33]. Theoretically, it would be ideal to perform
deep sequencing using singe cells without any in vitro expansion, but
this strategy is diﬃcult in practical terms. While analysis of genetic
changes of MSCs is still challenging, collecting germ line cells simpliﬁes
interpretation of the results. Another important point is the depth ofsequencing in order to evaluate genetic variants with low allelic frequency. Further studies in large cohorts are needed to clarify the molecular properties of MSCs, especially age-related genetic alterations, bydeep sequencing in addition to transcriptional and epigenetic analyses.
Conﬂicts of interest
All authors have no conﬂicts of interest to disclose.
Acknowledgments
This work was supported by the Private University Strategic
Research Based Support Project from MEXT (Ministry of Education,
Culture, Sports, Science and Technology), Tokyo, Japan [grant no.
S1311016].
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.leukres.2017.09.008.
References[1] P.H. Krebsbach, S.A. Kuznetsov, P. Bianco, P.G. Robey, Bone marrow stromal cells:
characterization and clinical application, Crit. Rev. Oral Biol. Med. 10 (2) (1999)
165–181.
[2] P.E. Boulais, P.S. Frenette, Making sense of hematopoietic stem cell niches, Blood
125 (17) (2015) 2621–2629.
[3] S.J. Morrison, D.T. Scadden, The bone marrow niche for haematopoietic stem cells,
Nature 505 (7483) (2014) 327–334.
[4] B.A. Anthony, D.C. Link, Regulation of hematopoietic stem cells by bone marrowstromal cells, Trends Immunol. 35 (1) (2014) 32–37.
[5] P. Bianco, X. Cao, P.S. Frenette, J.J. Mao, P.G. Robey, P.J. Simmons, C.Y. Wang, The
meaning, the sense and the signiﬁcance: translating the science of mesenchymal
stem cells into medicine, Nat. Med. 19 (1) (2013) 35–42.
[6] C.R. Cogle, N. Saki, E. Khodadi, J. Li, M. Shahjahani, S. Azizidoost, Bone marrowniche in the myelodysplastic syndromes, Leuk. Res. 39 (10) (2015) 1020–1027.
[7] M.Y. Konopleva, C.T. Jordan, Leukemia stem cells and microenvironment: biology
and therapeutic targeting, J. Clin. Oncol. 29 (5) (2011) 591–599.
[8] S. Geyh, S. Oz, R.P. Cadeddu, J. Frobel, B. Bruckner, A. Kundgen, R. Fenk, I. Bruns,
C. Zilkens, D. Hermsen, N. Gattermann, G. Kobbe, U. Germing, F. Lyko, R. Haas,
T. Schroeder, Insuﬃcient stromal support in MDS results from molecular andfunctional deﬁcits of mesenchymal stromal cells, Leukemia 27 (9) (2013)
1841–1851.
[9] S. Geyh, M. Rodriguez-Paredes, P. Jager, C. Khandanpour, R.P. Cadeddu,
J. Gutekunst, C.M. Wilk, R. Fenk, C. Zilkens, D. Hermsen, U. Germing, G. Kobbe,
F. Lyko, R. Haas, T. Schroeder, Functional inhibition of mesenchymal stromal cells
in acute myeloid leukemia, Leukemia 30 (3) (2016) 683–691.
[10] K. O'Hagan-Wong, S. Nadeau, A. Carrier-Leclerc, F. Apablaza, R. Hamdy, D. ShumTim, F. Rodier, I. Colmegna, Increased IL-6 secretion by aged human mesenchymal
stromal cells disrupts hematopoietic stem and progenitor cells' homeostasis,
Oncotarget 7 (12) (2016) 13285–13296.
[11] J.C. Huang, S.K. Basu, X. Zhao, S. Chien, M. Fang, V.G. Oehler, F.R. Appelbaum,
P.S. Becker, Mesenchymal stromal cells derived from acute myeloid leukemia bonemarrow exhibit aberrant cytogenetics and cytokine elaboration, Blood Cancer J. 5
(2015) e302.
[12] S. Chen, N.A. Zambetti, E.M. Bindels, K. Kenswill, A.M. Mylona, N.M. Adisty,
R.M. Hoogenboezem, M.A. Sanders, E.M. Cremers, T.M. Westers, J.H. Jansen,
A.A. van de Loosdrecht, M.H. Raaijmakers, Massive parallel RNA sequencing ofhighly puriﬁed mesenchymal elements in low-risk MDS reveals tissue-context-dependent activation of inﬂammatory programs, Leukemia 30 (9) (2016) 1938–1942.
[13] E.K. von der Heide, M. Neumann, S. Vosberg, A.R. James, M.P. Schroeder, J. OrtizTanchez, K. Isaakidis, C. Schlee, M. Luther, K. Johrens, I. Anagnostopoulos,
L.H. Mochmann, D. Nowak, W.K. Hofmann, P.A. Greif, C.D. Baldus, Molecular alterations in bone marrow mesenchymal stromal cells derived from acute myeloid
leukemia patients, Leukemia (2016).
[14] E. Flores-Figueroa, R.M. Arana-Trejo, G. Gutierrez-Espindola, A. Perez-Cabrera,
H. Mayani, Mesenchymal stem cells in myelodysplastic syndromes: phenotypic andcytogenetic characterization, Leuk. Res. 29 (2) (2005) 215–224.
[15] O. Blau, W.K. Hofmann, C.D. Baldus, G. Thiel, V. Serbent, E. Schumann, E. Thiel,
I.W. Blau, Chromosomal aberrations in bone marrow mesenchymal stroma cells
from patients with myelodysplastic syndrome and acute myeloblastic leukemia,
Exp. Hematol. 35 (2) (2007) 221–229.
[16] O. Lopez-Villar, J.L. Garcia, F.M. Sanchez-Guijo, C. Robledo, E.M. Villaron,
P. Hernandez-Campo, N. Lopez-Holgado, M. Diez-Campelo, M.V. Barbado,
J.A. Perez-Simon, J.M. Hernandez-Rivas, J.F. San-Miguel, M.C. del Canizo, Bothexpanded and uncultured mesenchymal stem cells from MDS patients are genomically abnormal, showing a speciﬁc genetic proﬁle for the 5q- syndrome,
Leukemia 23 (4) (2009) 664–672.
[17] O. Blau, C.D. Baldus, W.K. Hofmann, G. Thiel, F. Nolte, T. Burmeister, S. Turkmen,
O. Benlasfer, E. Schumann, A. Sindram, M. Molkentin, S. Mundlos, U. Keilholz,
E. Thiel, I.W. Blau, Mesenchymal stromal cells of myelodysplastic syndrome andacute myeloid leukemia patients have distinct genetic abnormalities compared with

27

-130-

Leukemia Research 62 (2017) 23–28

K. Azuma et al.
leukemic blasts, Blood 118 (20) (2011) 5583–5592.
[18] Y. Kim, D.W. Jekarl, J. Kim, A. Kwon, H. Choi, S. Lee, Y.J. Kim, H.J. Kim, I.H. Oh,
M. Kim, Genetic and epigenetic alterations of bone marrow stromal cells in myelodysplastic syndrome and acute myeloid leukemia patients, Stem Cell Res. 14 (2)(2015) 177–184.
[19] E. Kouvidi, A. Stratigi, A. Batsali, I. Mavroudi, S. Mastrodemou, M. Ximeri,
H.A. Papadaki, C.G. Pontikoglou, Cytogenetic evaluation of mesenchymal stem/
stromal cells from patients with myelodysplastic syndromes at diﬀerent time-points
during ex vivo expansion, Leuk. Res. 43 (2016) 24–32.
[20] M.H. Raaijmakers, S. Mukherjee, S. Guo, S. Zhang, T. Kobayashi, J.A. Schoonmaker,
B.L. Ebert, F. Al-Shahrour, R.P. Hasserjian, E.O. Scadden, Z. Aung, M. Matza,
M. Merkenschlager, C. Lin, J.M. Rommens, D.T. Scadden, Bone progenitor dysfunction induces myelodysplasia and secondary leukaemia, Nature 464 (7290)(2010) 852–857.
[21] A. Kode, J.S. Manavalan, I. Mosialou, G. Bhagat, C.V. Rathinam, N. Luo,
H. Khiabanian, A. Lee, V.V. Murty, R. Friedman, A. Brum, D. Park, N. Galili,
S. Mukherjee, J. Teruya-Feldstein, A. Raza, R. Rabadan, E. Berman, S. Kousteni,
Leukaemogenesis induced by an activating beta-catenin mutation in osteoblasts,
Nature 506 (7487) (2014) 240–244.
[22] F. Braulke, C. Muller-Thomas, K. Gotze, U. Platzbecker, U. Germing, W.K. Hofmann,
A.A. Giagounidis, M. Lubbert, P.L. Greenberg, J.M. Bennett, F. Sole, M.L. Slovak,
K. Ohyashiki, M.M. Le Beau, H. Tuchler, M. Pfeilstocker, B. Hildebrandt, C. Aul,
R. Stauder, P. Valent, C. Fonatsch, U. Bacher, L. Trumper, D. Haase, J. Schanz,Frequency of del(12p) is commonly underestimated in myelodysplastic syndromes:
results from a German diagnostic study in comparison with an international control
group, Genes. Chromosomes Cancer 54 (12) (2015) 809–817.
[23] S. Aizawa, M. Nakano, O. Iwase, M. Yaguchi, M. Hiramoto, H. Hoshi, R. Nabeshima,
D. Shima, H. Handa, K. Toyama, Bone marrow stroma from refractory anemia of
myelodysplastic syndrome is defective in its ability to support normal CD34-positive cell proliferation and diﬀerentiation in vitro, Leuk. Res. 23 (3) (1999) 239–246.

[24] L. da Silva Meirelles, P.C. Chagastelles, N.B. Nardi, Mesenchymal stem cells residein virtually all post-natal organs and tissues, J. Cell Sci. 119 (Pt 11) (2006)
2204–2213.
[25] J.H. Ohyashiki, H. Hisatomi, S. Shimizu, M. Sugaya, K. Ohyashiki, Detection of low
allele burden of JAK2 exon 12 mutations using TA-cloning in patients with erythrocytosis, Jpn. J. Clin. Oncol. 39 (8) (2009) 509–513.
[26] D.F. Quail, J.A. Joyce, Microenvironmental regulation of tumor progression andmetastasis, Nat. Med. 19 (11) (2013) 1423–1437.
[27] C. Korn, S. Mendez-Ferrer, Myeloid malignancies and the microenvironment, Blood
129 (7) (2017) 811–822.
[28] L. Raﬀaghello, A. Vacca, V. Pistoia, D. Ribatti, Cancer associated ﬁbroblasts inhematological malignancies, Oncotarget 6 (5) (2015) 2589–2603.
[29] K. Kurose, K. Gilley, S. Matsumoto, P.H. Watson, X.P. Zhou, C. Eng, Frequent somatic mutations in PTEN and TP53 are mutually exclusive in the stroma of breast
carcinomas, Nat. Genet. 32 (3) (2002) 355–357.
[30] A. Patocs, L. Zhang, Y. Xu, F. Weber, T. Caldes, G.L. Mutter, P. Platzer, C. Eng,Breast-cancer stromal cells with TP53 mutations and nodal metastases, N. Engl. J.
Med. 357 (25) (2007) 2543–2551.
[31] W. Qiu, M. Hu, A. Sridhar, K. Opeskin, S. Fox, M. Shipitsin, M. Trivett,
E.R. Thompson, M. Ramakrishna, K.L. Gorringe, K. Polyak, I. Haviv, I.G. Campbell,
No evidence of clonal somatic genetic alterations in cancer-associated ﬁbroblasts
from human breast and ovarian carcinomas, Nat. Genet. 40 (5) (2008) 650–655.
[32] J. Cai, X. Miao, Y. Li, C. Smith, K. Tsang, L. Cheng, Q.F. Wang, Whole-genome
sequencing identiﬁes genetic variances in culture-expanded human mesenchymal
stem cells, Stem Cell Rep. 3 (2) (2014) 227–233.
[33] M. Xie, C. Lu, J. Wang, M.D. McLellan, K.J. Johnson, M.C. Wendl, J.F. McMichael,
H.K. Schmidt, V. Yellapantula, C.A. Miller, B.A. Ozenberger, J.S. Welch, D.C. Link,
M.J. Walter, E.R. Mardis, J.F. Dipersio, F. Chen, R.K. Wilson, T.J. Ley, L. Ding, Agerelated mutations associated with clonal hematopoietic expansion and malignancies, Nat. Med. 20 (12) (2014) 1472–1478.

28

-131-

miR-125b-1 and miR-378a are predictive biomarkers
for the efficacy of vaccine treatment against
colorectal cancer
Hironori Tanaka,1 Shoichi Hazama,1,2
Michihisa Iida,1 Ryouichi Tsunedomi,1Hiroko Takenouchi,11111Masao Nakajima, Yukio Tokumitsu, Shinsuke Kanekiyo, Yoshitaro Shindo, Shinobu Tomochika,1
Yoshihiro Tokuhisa,1 Kazuhiko Sakamoto,1 Nobuaki Suzuki,1 Shigeru Takeda,1 Shigeru Yamamoto,1Shigefumi Yoshino,3 Tomio Ueno,1 Yoshihiko Hamamoto,4 Yusuke Fujita,4 Hiroaki Tanaka,5 Ko Tahara,6
Ryoichi Shimizu,7 Kiyotaka Okuno,8 Koji Fujita,9 Masahiko Kuroda,9 Yusuke Nakamura10 and Hiroaki Nagano1
1
Department of Gastroenterological, Breast and Endocrine Surgery, Yamaguchi University Graduate School of Medicine; 2Department of Translational
Research and Developmental Therapeutics against Cancer, Yamaguchi University School of Medicine; 3Oncology Center, Yamaguchi University Hospital;
4
Division of Electrical, Electronic and Information Engineering, Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Ube;
5
Department of Surgical Oncology, Osaka City University Graduate School of Medicine, Osaka; 6Department of Surgery, Kure-Kyosai Hospital, Kure;
7
Department of Surgery, Ogori Daiichi General Hospital, Yamaguchi; 8Department of Surgery, Kinki University Faculty of Medicine, Osaka-Sayama;
9
Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan; 10Department of Medicine and Surgery, The University of Chicago, Chicago,USA

Key words
Biomarker, colorectal cancer, miR-125b-1, miR-378a, peptide vaccine
Correspondence
Hiroaki Nagano, Department of Gastroenterological,
Breast and Endocrine Surgery, Yamaguchi University
Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ubecity, Yamaguchi 755-8505, Japan.
Tel: +81-836-22-2264; Fax: +81-836-22-2263;
E-mail: hnagano@yamaguchi-u.ac.jp
Funding Information
Research program of the Project for Development of
Innovative Research on Cancer Therapeutics (P-DIRECT),(Grant/Award Number: ‘11039020’) The Japan Agency for
Medical Research and Development (AMED), (Grant/Award Number: ‘15cm0106085h0005’) Leading Advanced
Projects for Medical Innovation (LEAP), (Grant/Award
Number: ‘16am0001006h0003’).
Received May 10, 2017; Revised August 24, 2017;
Accepted August 27, 2017
Cancer Sci 108 (2017) 2229–2238

Many clinical trials of peptide vaccines have been conducted. However, these vaccines have provided clinical benefits in only a small fraction of patients. The purpose of the present study was to explore microRNAs (miRNAs) as novel
predictive biomarkers for the efficacy of vaccine treatment against colorectal cancer. First, we carried out microarray analysis of pretreatment cancer tissues in aphase I study, in which peptide vaccines alone were given. Candidate miRNAs
were selected by comparison of the better prognosis group with the poorer
prognosis group. Next, we conducted microarray analysis of cancer tissues in aphase II study, in which peptide vaccines combined with chemotherapy were
given. Candidate miRNAs were further selected by a similar comparison of prognosis. Subsequently, we carried out reverse-transcription PCR analysis of phase II
cases, separating cancer tissues into cancer cells and stromal tissue using laser
capture microdissection. Treatment effect in relation to overall survival (OS) andmiRNA expression was analyzed. Three miRNA predictors were negatively associated with OS: miR-125b-1 in cancer cells (P = 0.040), and miR-378a in both cancer
cells (P = 0.009) and stromal cells (P < 0.001). Multivariate analysis showed that
expression of miR-378a in stromal cells was the best among the three predictors(HR, 2.730; 95% CI, 1.027–7.585; P = 0.044). In conclusion, miR-125b-1 and miR378a expression might be considered as novel biomarkers to predict the efficacy
of vaccine treatment against colorectal cancer.

doi: 10.1111/cas.13390

olorectal cancer is the third most commonly diagnosed
cancer in males and the second most commonly diagnosed
cancer in females, with an estimated 1.4 million cases and693 900 deaths occurring in 2012.(1) In the past decade,chemotherapy and monoclonal antibody treatment have
improved the prognosis of patients with mCRC. However,most of these patients die because the cancer develops tolerance to these agents. There is, therefore, a great need fordevelopment of additional treatment.
Immunotherapy has been used to treat mCRC and has thepotential to eradicate the disease by activating immune
responses.(2) Peptide vaccines are one form of cancer vaccinethat induces tumor-specific T-cell responses using tumor-associated antigen-derived peptides. Many phase I and II clinical

trials using peptide vaccines have been conducted and thesevaccines have provided clinical benefits in a small fraction ofpatients.(3,4) Currently, immune checkpoint blockade therapies
are a very promising cancer immunotherapy. For example,
antibodies blocking PD-1 and PD-L1 have shown durable
responses in a number of different advanced malignancies.(5,6)
Additionally, several recent studies suggest alternative targeting strategies for indoleamine 2,3-dioxygenase blockade at thelevel of gene expression.(7,8) Furthermore, combination
immunotherapy with peptide vaccines and immune-checkpoint
blockade therapies that are designed to concurrently activate
tumor-specific immune responses and inactivate immunosuppression in the tumor microenvironment may lead to the induction of stronger antitumor responses.(9,10)© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.
This is an open access article under the terms of the Creative Commons Attrib
ution-NonCommercial License, which permits use, distribution and reproduction
in any medium, provided the original work is properly cited and is not used forcommercial purposes.

Cancer Sci | November 2017 | vol. 108 | no. 11 | 2229–2238

C

-132-

Original Article
miRNA biomarkers for CRC vaccine

www.wileyonlinelibrary.com/journal/cas

We previously reported a phase Ι and II study in which fiveepitope peptides were applied to advanced-stage colon cancer
patients.(11,12) However, active immunotherapy using epitope
peptides requires preservation of the host immune system.
Hence, it is desirable to identify predictive biomarkers for theselection of patients who are likely to respond well, and toeffectively induce specific CTL to epitope peptides.
miRNAs are endogenously expressed non-coding RNAs, 18–
25 nucleotides in length, which regulate gene expression at the
post-transcriptional level.(13) miRNAs play roles as crucial
modulators of various biological processes such as proliferation, tumor initiation and development in CRC.(14) However,miRNAs are also critical regulators of immune responses,(15)and their aberrant expression or dysfunction in the immune
system has been associated with cancer. For example, p53
modulates the tumor immune response by regulating PD-L1
through miR-34 in non-small-cell lung cancer.(16) In anotherexample, miR-20b, -21 and -130b are upregulated in advanced
CRC and inhibit PTEN expression, resulting in PD-L1 overexpression.(17) In addition, some miRNAs have been mentioned
as possible biomarkers for immunotherapy.(18–20)
Cancer tissues consist of cancer cells and surrounding stromal cells, including inflammatory cells, immunocompetentcells, endothelial cells, and fibroblasts. Stromal cells in thetumor microenvironment play an important role in cancer
metastasis.(21) Cancer stroma interacts with cancer tissues
through miRNAs, creating a niche for the cancer cells.(22) A
study showed that high levels of miR-21 in the stroma predict
short disease-free survival in stage II CRC.(23) In a separate
study, miR-21 suppression with antisense inhibitors was shown
to prevent fibroblast-to-myofibroblast transdifferentiation in
response to TGF-b.(24)The purpose of the present study was to explore miRNAs
as novel predictive biomarkers for the efficacy of
immunotherapies before their treatment. We previously
reported that three miRNAs might be useful biomarkers.(25)As the cellular origin is crucial for determination of miRNAs,in the present study, we used LCM to separate cancer cells
and stromal cells for miRNA analysis. Here we demonstrate
the results of miRNA microarray analysis and qRT-PCR in
CRC cancer and stromal cells. We suggest that miR-125b-1
and miR-378a may serve as novel predictive biomarkers for
active immunotherapies.
Materials and Methods
Summary of the phase I study. Detailed protocol of the phaseI study was described previously.(11) Briefly, patients were eligible for enrolment when: (i) they had histologically confirmed
CRC without indication for surgical resection; (ii) they had
failed to respond to prior standard chemotherapy or could not
tolerate the standard therapy; and (iii) when they had thehuman leukocyte antigen (HLA)-A*2402-allele by DNA typing.
Good Manufacturing Practice grade peptides of RNF43721,(26) TOMM34-299,(27) KOC1 (IMP-3)-508,(28) VEGFR11084,(29) and VEGFR2-169,(30) which are peptides that arerecognized by HLA-A*2402-restricted CTL, were used. Doseescalation was carried out in three patient cohorts using dosesof 0.5 mg, 1 mg, and 3 mg for each peptide. Each peptide wasgiven to patients s.c. into the thigh or axilla regions on days 1,
8, 15, and 22 in a 28-day treatment course. The determinedrecommended dose was 3.0 mg, which was followed by carrying out a single injection of a cocktail of the five peptides.
The cocktail of the five peptides at a dose of 3 mg was given

to six patients. Among 18 HLA-A*2402-matched CRC patients
who were enrolled in this study, eight primary CRC tissues
were available for miRNA microarray analysis.
Summary of the phase II study. To evaluate clinical benefitsof cancer vaccination treatment, we conducted a phase II trial,
non-randomized, HLA-A status double-blind study. Detailed
protocol of this phase II study was described previously.(12)Briefly, the therapy consisted of a cocktail of five peptides in
addition to oxaliplatin-containing chemotherapy. The peptides
used in this study were recognized by HLA-A*2402-restricted
CTL; however, the HLA-A status of all enrolled patients was
masked. The cocktail of 3 mg each of five peptides was mixed
with 1.5 mL incomplete Freund’s adjuvant and given s.c. into
the thigh or axilla regions weekly for 13 weeks, then biweekly
after 14 weeks. Patients were eligible for enrolment when they
were ≥20 years old with a histologically confirmed advanced
CRC, were na€ıve for chemotherapy, had adequate functions ofcritical organs, and had a life expectancy of ≥3 months.
Between February 2009 and November 2012, 96 CRC patientswere enrolled under the masking of their HLA-A status.
Among these 96 patients, 26 cases were available for miRNA
microarray analysis, and 68 primary CRC tissues were available for qRT-PCR analysis.
Sample collection. In the phase I trial, eight primary CRC tissues from 18 patients were obtained by surgery before treatment. Tissues were snap-frozen in liquid nitrogen and stored at
80°C. In the phase II trial, 26 primary CRC tissues wereobtained from 96 patients and were stored as frozen tissues; 16
samples were from the HLA-A*2402-matched group and 10
samples were from the HLA-A*2402-unmatched group. Sixtyeight primary CRC tissues were obtained and stored as FFPE
tissues; 34 samples from each of the HLA-A*2402-matchedand HLA-A*2402-unmatched groups. Primary CRC lesions
were resected in advance of the vaccine treatment studies at
Yamaguchi University Hospital and affiliated hospitals. Written informed consent was obtained from all patients. This
study was approved by the Institutional Ethics Review Boardsof Yamaguchi University and was conducted in accordancewith the Declaration of Helsinki (H20-102, Clinical Trial
Registration: UMIN000001791).
Microarray analysis. Total RNA was extracted from frozen
tissue using a mirVana™ miRNA Isolation Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. Concentrations and purities of total RNAs were assessedusing a spectrophotometer and RNA integrity was verified
using an Agilent RNA 6000 Nano Kit (Agilent Technologies,
Santa Clara, CA, USA). OD260/OD280 ratios of each sample
were between 2.00 and 2.10, which were accepted as adequate
for microarray analysis. The miRNA array analysis was carriedout with 1-lg samples of total RNA using the miRCURY
LNA™ microRNA Array 6th generation (Exiqon, Vedbaek,
Denmark) and the GenePixâ4000B (Molecular Devices, Sunnyvale, CA, USA). Relative intensity of each hybridization signal was evaluated using the Microarray Data Analysis Tool
Ver3.2 (Filgen, Nagoya, Japan).
Laser capture microdissection. FFPE tumor specimens weresectioned into 15-lm-thick slices, and stained with H&E toenable visualization of histology.(31) LCM was carried out using
the LMD system LMD6000 (Leica Microsystems, Tokyo, Japan)
to microdissect cancer cells and stromal cells from CRC tissues.
At least 30 mm2 of cancer cells and 45 mm2 of stromal cells
were collected from each FFPE specimen. Depending on thepercentage of cancer cells and stromal cells in the specimen, thisgenerally required from two to four sections.

© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

-133-

Cancer Sci | November 2017 | vol. 108 | no. 11 | 2230

Original Article
Tanaka et al.

www.wileyonlinelibrary.com/journal/cas
qRT-PCR. Total RNA was extracted from LCM-captured
material using a QIAGEN miRNeasy FFPE Kit (Qiagen,Tokyo, Japan) according to the manufacturer’s protocol. Concentrations and purities of the total RNAs were assessed as
described for microarray analysis using a spectrophotometer.
Expression of miRNAs in both cancer cells and stromal cells
was verified by qRT-PCR analysis using TaqManâ MicroRNA
Assays (Life Technologies). cDNA was synthesized in a 7.5lL reaction volume from total RNA (10 ng) using the
TaqManâ MicroRNA Reverse Transcription kit (Applied Biosystems, Waltham, MA, USA). Reactions were incubated at
16°C for 30 min and then at 42°C for 30 min, and were
inactivated at 85°C for 5 min. Each cDNA was analyzed in
duplicate by qRT-PCR using sequence-specific primers
for hsa-miR-125b-1 (ID002378), hsa-miR-224 (ID002099),
hsa-miR-486 (ID001278), hsa-miR-147b (ID002262), hsa-miR196b (ID002215), rno-miR-422b (ID001314), or RNU6B(ID001093) in an Applied Biosystems 7300 real-time PCR
instrument (Life Technologies). miR-378a was previously
known as miR-422b, and rno-miR-422b has the same sequence
as miR-378a (ACUGGACUUGGAGUCAGAAGGC). Eachindividual assay was carried out in a 20-lL reaction volume
with 1.33 lL cDNA, 1.0 lL TaqMan MicroRNA Assay,
10 lL TaqMan™ Universal PCR Master Mix II (Applied Biosystems) with No AmpErase UNG and 7.67 lL nuclease-freewater (Applied Biosystems). Reactions were incubated for
10 min at 95°C, and then for 55 cycles with denaturation for
15 s at 95°C and annealing/extension for 60 s at 60°C. Data
were analyzed using SDS software v1.3.1 (Life Technologies).
Threshold and baseline settings were set according to protocol
recommendations.
In situ hybridization. ISH was carried out on 5-lm-thick sections of FFPE samples using the RiboMap Kit (Ventana Medical Systems, Tucson, AZ, USA) on the Discovery ULTRA
automated ISH instrument (Ventana Medical Systems). Probes
were 50 DIG-labelled ISH LNAs (miRCURY-LNA detection
probes; Exiqon). Sequences of probe for miR-125b-1 and miR378a were/5DigN/AGCTCCCAAGAGCCTAACCCGT and/
5DigN/CCTTCTGACTCCAAGTCCAGT, respectively.
Sequence of the scramble probe for a negative control was/
5DigN/GTGTAACACGTCTATACGCCCA. The ISH steps
after the deparaffinization step were carried out based on thestandard protocol provided in the manufacturer’s RiboMap
application note. Briefly, after treatment with proteinase K, theslides were hybridized with the detection probe for 8 h at
52°C. The digoxigenins were detected with a polyclonal antiDIG antibody and an alkaline phosphatase-conjugated second
antibody (Ventana Medical Systems). Signal was detectedusing the BlueMap NBT/BCIP substrate kit (Ventana Medical
Systems). The sections were counterstained with Kernechtrot
as a marker stain and covered with a glass coverslip.
Statistical analysis. In microarray analysis, expression signals
were calculated by log2 transformation of the normalized data,
and differentially expressed miRNAs were detected by using
the fold-change value and the Fisher index using Microsoft
Excel 2010 according to a previously reported formula.(32) In
qRT-PCR, relative quantities of miRNA levels were determined using the 2-DDCT method after normalization withRNU6B as a standard reference.(33) Differences between
groups were estimated using the Mann–Whitney U-test. Categorical variables were compared by using the chi-squared andFisher’s exact tests. The cut-off value was determined by Youden’s index for each ROC analysis. OS was measured inmonths from the first vaccination to the day of patient death
Cancer Sci | November 2017 | vol. 108 | no. 11 | 2231

from any cause. Survival curves were analyzed by the Kaplan–
Meier method and the log–rank test, and a Cox’s proportional
hazards model was used to estimate the HR and the 95% CI
for the treatment effect in relation to OS and miRNA expression, other biomarkers, and prognostic clinical information.
Statistical analyses were done with JMP V11 (SAS InstituteInc.). P < 0.05 was considered statistically significant.
Results
Selection of candidate miRNAs to predict the efficacy of vaccination. We first carried out microarray analysis of the expres-sion profiles of 1425 miRNAs using eight frozen tissues frompatients enrolled in a phase I study. We classified the patients
into two groups of four patients each, a responder group and a
non-responder group. Responders were defined according to thefollowing criteria: (i) having a PFS period of more than
150 days; or (ii) having an OS period of longer than 300 days
without any additional chemotherapy. Non-responders were
defined according to the following criteria: (i) having a PFS
period shorter than 90 days; and (ii) having an OS period
shorter than 200 days. Subsequently, on the basis of the relative
comparison of hybridization signals of individual miRNAs, weselected the top 30 miRNAs showing significant differences in
expression levels between the two groups (Table S1). Next, 26
frozen tissues from the phase II study were analyzed using amiRNA microarray. In this analysis, we examined expression
levels of the 30 candidate miRNAs and their association withclinical outcome. The expression levels of six miRNAs weresignificantly associated with prognosis in the HLA-A*2402matched groups. There was no difference in prognosis in HLAA*2402-unmatched groups according to the expression levelsof these six miRNAs. Therefore, these miRNAs had the potential to be predictive biomarkers for the efficacy of the vaccine
treatment. Expression levels of these six miRNAs and survival
analysis are shown in Table 1.
Validation of candidate miRNA expression and association of
the miRNA expression levels with clinical outcome. We carriedout qRT-PCR analysis of the expression of these six miRNAs in
68 FFPE tissues from the phase II study. The FFPE specimens
were separated into cancer cells and stromal cells by LCM.
miRNA expression levels were examined in these two types ofcells, and association of miRNA expression levels with clinicaloutcome was evaluated. Characteristics of these 68 patients arelisted in Table 2. No difference in any characteristic was foundbetween HLA-A*2402-matched and HLA-A*2402-unmatchedgroups. Expression levels of the six miRNAs and survival analysis of the patients according to low or high expression of thesemiRNAs are shown in Table 3. Three predictors were negatively
associated with OS in the HLA-A*2402-matched group only:
miR-125b-1 in cancer cells (P = 0.040), and miR-378a in bothcancer cells (P = 0.009) and stromal cells (P < 0.001). In addition, expression of miR-125b-1 (P = 0.089) and miR-486(P = 0.092) in stromal cells tended to be negatively associatedwith OS. Overall survival of the HLA-A*2402-matched and unmatched groups of patients according to high or low expression of miR-125b-1 or miR-378a is shown in the form of Kaplan
Meier curves in Figure 1. These data suggest that miR-125b-1
and miR-378a have the potential to be negative predictive
biomarkers for the efficacy of the vaccine treatment.
Univariate and multivariate analysis of the association of 125b1 and miR-378a expression with OS. Results of univariate andmultivariate analyses are shown in Table 4. Expression of
miR-378a in both cancer cells (HR, 2.853; 95% CI 1.281–© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

-134-

Original Article
miRNA biomarkers for CRC vaccine

www.wileyonlinelibrary.com/journal/cas

Table 1. Microarray analysis of candidate miRNA expression and phase II study patient survival analysis (log–rank test)
Expression in cancer tissue
miR name

Cut-off value
Mean

SD

Median

689.1

666.5

526.5

196b-5p

400

HLA-A*2402

Group

n

P-value

Matched

Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High

7
9
5
5
9
7
5
5
9
7
5
5
11
5
6
4
8
8
5
5
11
5
5
5

0.001

Unmatched
147b

483.6

185.2

460.0

500

Matched
Unmatched

378a6225.62216.96336.76500

Matched
Unmatched486-5p

345.3

71.0

339.0

365

Matched
Unmatched224-5p

343.2

290.8

272.2

270

Matched
Unmatched125b-1

604.0

312.0

518.0

550

Matched
Unmatched

0.366
0.040
0.366
0.066
0.515
0.006
0.998
0.011
0.657
0.021
0.092

HLA, human leukocyte antigen; miRNA and miR, microRNA; SD, standard deviation.

Table 2. Clinical characteristics of patients in the phase II study
whose tissues were analyzed for miRNA using qRT-PCR
Characteristics

HLA-A*2402
matched (n = 34)

Sex
Male
FemaleAge (years)
Mean
SD
Range
Unresectable site
Liver
Lung
Dissemination
Bone
Lymph node
Other
No. involved organs
One
Two
Three
Primary tumor site
Colon
RectumHLA-A*2402
unmatched (n = 34)

P-value

14
20

19
15

0.225

68
8
47–82

64
8
38–77

0.080

19
11
4
0
5
3

26
10
3
2
8
1

0.073
0.793
0.690
0.151
0.355
0.303

27
6
1

22
8
4

0.302

22
12

25
9

0.431

HLA, human leukocyte antigen; SD, standard deviation.

6.788: P = 0.010) and stromal cells (HR, 3.984; 95% CI
1.661–9.243: P = 0.003) was significantly associated with OS.
The expression of miR-378a in stromal cells was confirmed to

be an independent predictor of OS by multivariate regression
analysis (HR, 2.730; 95% CI, 1.027–7.585; P = 0.044).
miR-125b-1 and miR-378a localization in tumor tissue. For
specific identification of miR-125b-1 and miR-378a expression
in tissue sections, we carried out ISH in tissue sections frompatients with a poor prognosis. miR-125b-1 was highly
expressed in both cancer and stromal cells (Fig. 2a). Within
stromal cells, fibroblast-like cells (Fig. 2b) and mononuclear
cells (Fig. 2c) showed strong miR-125b-1 signals. A weakmiR-378a signal was observed in both cancer and stromal cells(Fig. 2f). Within stromal cells, fibroblast-like cells showed
weak miR-378a signals (Fig. 2g). Such weak signals might bea result of the affinity of LNA probes for miR-378a.
Discussion

Findings of the present study showed that miR-125b-1 andmiR-378a are negative predictive biomarkers for the efficacy
of vaccine treatment against colorectal cancer. To our knowledge, this is the first study to systematically investigate
miRNA biomarkers for cancer immunotherapy and to focus on
separate analysis of cancer cells and stromal cells within cancer tissue. Previous miRNA microarray analysis by our group
showed that miR-196b, miR-378a, and miR-486 are possible
predictive biomarkers. However, it should be noted that the
sample size was small and that the precise origins of thesemRNAs are unknown. In the present study, we used qRT-PCR
in an increasing number of samples and used LCM to separate
the cancer cells and stromal cells. In addition, we carried out
ISH for specific identification of the miRNAs.
First, our results indicated that high miR-125b-1 expression
in cancer cells was significantly associated with poor prognosis© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

-135-

Cancer Sci | November 2017 | vol. 108 | no. 11 | 2232

Original Article
Tanaka et al.

www.wileyonlinelibrary.com/journal/cas
Table 3. qRT-PCR analysis of candidate miRNA expression and phase II study patient survival analysis (log–rank test)
Expression in cancer cells
miR nameMean
196b-5pSD135.079 9 10

2Median169.617 9 10

263.862 9 10

Cut-off value
2108.588 9 10

2

HLA-A*2402

Group

n

P-value

Matched

Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High

24
10
22
12
28
6
27
7
15
19
13
21
18
16
16
18
20
14
18
16
27
7
23
11

0.652

HLA-A*2402

Group

n

P-value

Matched

Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High

22
12
18
16
17
17
17
17
23
11
23
11
19
15
17
17
16
18
13
21
20
14
18
16

0.311

Unmatched147b

1.237 9 1021.254 9 1020.834 9 102

1.527 9 102

Matched
Unmatched

378a

61.995 9 10265.306 9 10240.041 9 10227.856 9 102

Matched
Unmatched486-5p5.450 9 102

20.571 9 1022.299 9 1022.290 9 102

Matched
Unmatched224-5p101.859 9 102

113.0851 9 102

61.063 9 102

68.359 9 102

Matched
Unmatched125b-10.786 9 1021.690 9 1020.289 9 1020.498 9 102

Matched
Unmatched

0.120
0.210
0.046
0.009
0.777
0.165
0.798
0.172
0.512
0.040
0.429

Expression in stromal cells
miR nameMean
196b-5p33.801 9 10SD
240.004 9 10Median2

Cut-off value

18.673 9 10

222.450 9 10

2Unmatched147b

0.599 9 1020.535 9 1020.424 9 1020.413 9 102

Matched
Unmatched

378a

43.009 9 10244.441 9 10228.802 9 102

43.080 9 102

Matched
Unmatched486-5p7.569 9 1028.741 9 1024.725 9 1024.766 9 102

Matched
Unmatched224-5p19.249 9 102

19.626 9 102

13.315 9 102

10.984 9 102

Matched
Unmatched125b-12.311 9 1022.371 9 102

1.554 9 1021.690 9 102

Matched
Unmatched

0.218
0.290
0.515
0.001
0.215
0.092
0.124
0.525
0.990
0.089
0.136

HLA, human leukocyte antigen; miRNA and miR, microRNA; SD, standard deviation.

in HLA-matched patients. Previous studies have shown thatmiR-125b plays crucial roles in immune system development
and immunological host defense.(34) Upregulated expression of
miR-125b may enhance type I interferon expression in airway

epithelial cells in eosinophilic chronic rhinosinusitis with nasal
polyps,(35) and promote classical activation of macrophages.(36,37) miR-125b-1 was also found to be specificallyupregulated in patients with ulcerative colitis,(38) and may

Cancer Sci | November 2017 | vol. 108 | no. 11 | 2233

© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

-136-

Original Article
miRNA biomarkers for CRC vaccine

www.wileyonlinelibrary.com/journal/cas

Fig. 1. Overall survival (OS) according to miRNA expression. OS of the HLA-A*2402-matched (left side) and -unmatched groups (right side) ofphase II study patients according to high or low expression of miR-125b-1 or miR-378a. (a) According to miR-125b-1 in cancer cells. (b) According
to miR-125b-1 in stromal cells. (c) According to miR-378a in cancer cells. (d) According to miR-378a in stromal cells. Solid line shows high miRNA
expression and dashed line shows low miRNA expression. Statistical analysis was carried out using the log–rank test. HLA, human leukocyte antigen; miRNA and miR, microRNA.

© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

-137-

Cancer Sci | November 2017 | vol. 108 | no. 11 | 2234

Original Article
Tanaka et al.

www.wileyonlinelibrary.com/journal/cas
Table 4. Univariate and multivariate analyses of associations between clinical data and overall survival
Univariate analysis
95% CI

FactorCut-off

CRP
NLR
CEA
CA19-9No. involved organs
Relative expression of miR-378a in cancer cells
Relative expression of miR-378a in stromal cells
Relative expression of 125b-1 in cancer cells
Relative expression of 125b-1 in stromal cells

Multivariate analysis

>1
>3
>16
>21
One or more
>27.856 9 102>43.080 9 102>0.498 9 102
>1.690 9 102

95% CI
P-value

HR

1.537
1.298
1.972
2.010
2.487
2.853
3.984
2.586
1.950

Lower

Upper

0.619
0.542
0.871
0.907
0.960
1.281
1.661
0.925
0.876

3.525
2.939
4.784
4.514
5.759
6.788
9.243
6.286
4.279

0.338
0.544
0.105
0.085
0.060
0.010
0.003
0.068
0.100

P-value

HR

1.888
2.730

Lower

Upper

0.695
1.027

5.031
7.585

0.207
0.044

CA19-9; carbohydrate antigen 19-9; CEA, carcinoembryonic antigen; CI, confidence interval; CRP, C-reactive protein; HR, hazard ratio; NLR, neutrophil:lymphocyte ratio.

increase risk of CRC through nuclear factor kappa B (NF-jB)
activation.(39,40) However, miR-125b negatively regulates
TNF-a expression in neonatal monocytes,(41) and inhibits
proper TNF-a production in response to LPS.(42) Hence, miR125b-1 might play the role of an immune suppressor byincreasing immune exhaustion,(43) and resulting in the inhibition of the antitumor immune system. Expression of miR125b-1 in cancer cells seems to be more important than that instromal cells (Fig. 1a,b), although strong signals were also
observed in stromal cells (Fig. 2b,c). miR-125b-1 might beproduced by cancer cells and/or stromal cells, and cross-talkby miRNAs between cancer cells and stromal cells might ultimately work as an immune suppressor.
Second, our results indicated that high expression levels of
miR-378a in both cancer cells and stromal cells were significantly associated with poor prognosis in HLA-matchedpatients. Previous reports showed that miR-378 was downregulated in CRC(44,45) and that miR-378a expression might play
an important role as a tumor suppressor gene.(46) In contrast,
miR-378a was reported to inhibit cytotoxicity of human NK
cells(47) and enhance tumor growth and angiogenesis throughrepression of the expression of two types of tumor suppressors,
Sufu and Fus-1.(48) Hence, after the establishment of tumor
microenvironments, the function of miR-378a was thought tobe as an immune suppressor. Regarding expression of miR378a, there were some discrepancies in miRNA expression
using ISH, miRNA microarray, and qRT-PCR. Expression
level of miR-378a was higher than that of miR-125b-1 in
miRNA microarray and qRT-PCR analyses. However, theopposite phenomenon was observed in ISH analysis. The miR378a signal was weak in the cancer cells and stromal cells,
and miR-125b-1 was highly expressed in both cell types. This
discrepancy might mean that there were some problems withthe affinity of the ISH probe for miR-378a.(49)
In multivariate analysis, miR-378a in stromal cells showed
the best performance among the three predictors for selection ofpatients who are likely to respond well to peptide vaccines(Table 4). We additionally considered whether the combinationof miR-125b-1 and miR-378a gave a better predictive power.
Because the high expression of miR-125b-1 and miR-378a
might be negative predictive factors, we classified 68 patients
into three groups according to the number of predictive factors(i.e. high expression of miR-125b-1 or miR-378a in cancer or
stromal cells). The OS of each group was shown in the form of

Kaplan–Meier curves in Figure S1. There was a significant difference between the “0 factor” group and the “2 factors” group.
Hence, the combination of miR-125b-1 and miR-378a in cancer
cells and stromal cells might have effective prognostic power.
The link between miR-125b-1 and miR-378a withimmunotherapy is unclear, and it is very important to identify
the target genes of these two miRNAs. Hence, we carried out
RNA sequencing using 22 cancer tissues from a phase II study(data not shown). We classified the 22 samples into threegroups according to the expression of each miRNA by qRTPCR analysis: the high expression group in both cancer andstromal cells, the low expression group in both cell types, andthe group of others. Difference in target gene expression
between the “high expression group” and the “low expression
group” was assessed by log2 ratio and Ingenuity Pathways
Analysis (IPA) (Qiagen).(50) Regarding miR-125b-1, receptor
activator of NF-jB (RANK) was downregulated in the “highexpression group”. RANK, a member of the TNF receptor
family, and its ligand RANKL play important roles in the
immune system. T cells activated as a result of RANKL
expression stimulate dendritic cells, expressing RANK, toenhance their survival and enhance induction of T-cell
response.(51,52) RANK is a target gene of miR-125b-1 by IPA(Qiagen), and miR-125b-1 might work as an immune suppressor regulating RANK. Regarding miR-378a, RNF125 was
downregulated in the “high expression group”. RNF125, E3
ubiquitin ligase, is positive regulator of T-cell activation.(53)miR-378a was considered to regulate RNF125 according to
IPA and could also be an immune suppressor. Thus, miR125b-1 and miR-378a might be therapeutic targets.
It must be mentioned that the mean survival time of the“HLA matched-miRNA high” group appeared to be shorter
than that of the “HLA unmatched-miRNA high” group(Fig. 1). However, there was no significant difference between
their survival curves (Fig. S2). Furthermore, we investigatedside-effects in the “HLA matched-miRNA high” group. The
incidence of severe AE in each group of miR-378a is shownin Table S2. There was no significant difference in the incidence of AE between the “HLA matched-miRNA high” groupand other groups. It is quite unlikely that vaccine treatment led
to deleterious effects in “HLA matched-miRNA low” patients.
It is known that the behavior of cancer cells and the development of tumor tissues are modulated by cross-talk throughmiRNAs between cancer cells and stromal cells such as

Cancer Sci | November 2017 | vol. 108 | no. 11 | 2235

© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

-138-

Original Article
miRNA biomarkers for CRC vaccine

www.wileyonlinelibrary.com/journal/cas

Fig. 2. In situ hybridization detecting miR-125b-1
and miR-378a. (a) miR-125b-1 is highly expressed incancer cells and stromal cells. (b) Arrows indicate
miR-125b-1-positive fibroblast-like cells. (c) Arrows
indicate miR-125b-1-positive mononuclear cells. (d)Arrows indicate miR-125b-1-positive cancer cells. (e)Scramble probe. (f) A miR-378a signal was observedin both cancer cells and stromal cells. (g) Arrows
indicate miR-378a-positive cancer cells. (h) Arrows
indicate miR-378a-positive fibroblast-like cells. (i)Scramble probe. Bar, 200 lm. miR, microRNA.

fibroblasts, endothelial cells, pericytes, immune cells, and local
and bone marrow-derived stromal stem cells.(54) Although several studies have reported the importance of miRNAs such as
miR-21,(23) miR-17-92a and the miR-106b-25 cluster(22) incancerous stromal cells, the present study is the first to regard
the role of miR-125b-1 and miR-378a in colorectal cancer
cells and stromal cells for predictive markers of immunotherapy.
In conclusion, miR-125b-1 and miR-378a expression might
be considered novel biomarkers to predict the efficacy of
immunotherapy in CRC. Although our results are preliminary
and the functions of each of the miRNAs in immune responses
are still unclear, the applicability of these miRNAs should be
confirmed in a future large-scale study as biomarkers to selectpatients who can expect a better outcome with the vaccine
treatment.
Acknowledgments
This study was carried out as a research program of the Project forDevelopment of Innovative Research on Cancer Therapeutics(P-DIRECT; 11039020), The Japan Agency for Medical Research andDevelopment (AMED; 15cm0106085h0005). This study was supported
in part by a grant for Leading Advanced Projects for Medical Innovation (LEAP; 16am0001006 h0003) from the Japan Agency for Medical
Research and Development. The authors would like to thank Dr
Takuya Tsunoda and Dr Koji Yoshida, Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The
University of Tokyo, for their excellent advice and cooperation and for© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

-139-

Cancer Sci | November 2017 | vol. 108 | no. 11 | 2236

Original Article
Tanaka et al.

www.wileyonlinelibrary.com/journal/cas
providing all of the peptides. The authors thank Ms Naoko Okayama
for technical support.

Disclosure Statement
Yusuke Nakamura is a stockholder and a scientific advisor of
OncoTherapy Science, Inc. The other authors declare no potential conflicts of interest for this article.

Abbreviations
AE
CA19-9
CEA
CI
CRC
CRP
DIG
FFPE

adverse event
carbohydrate antigen 19-9
carcinoembryonic antigen
confidence intervalcolorectal cancer
C-reactive protein
digoxigenin
formalin-fixed paraffin-embedded

References
1 Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global
cancer statistics, 2012. CA Cancer J Clin 2015; 65: 87–108.
2 Xiang B, Snook AE, Magee MS, Waldman SA. Colorectal cancer
immunotherapy. Discov Med 2013; 15: 301–8.
3 Rosenberg SA, Yang JC, Restifo NP. Cancer immunotherapy: moving
beyond current vaccines. Nat Med 2004; 10: 909–15.
4 Nagorsen D, Thiel E. Clinical and immunologic responses to active specificcancer vaccines in human colorectal cancer. Clin Cancer Res 2006; 12:3064–9.
5 Topalian SL, Hodi FS, Brahmer JR et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 2012; 366: 2443–54.
6 Le DT, Uram JN, Wang H et al. PD-1 blockade in tumors with mismatchrepair deficiency. N Engl J Med 2015; 372: 2509–20.
7 Muller AJ, DuHadaway JB, Donover PS, Sutanto-Ward E, Prendergast GC.
Inhibition of indoleamine 2,3-dioxygenase, an immunoregulatory target ofthe cancer suppression gene Bin1, potentiates cancer chemotherapy. Nat Med
2005; 11: 312–9.
8 Hou D-Y, Muller AJ, Sharma MD et al. Inhibition of indoleamine 2,3-dioxygenase in dendritic cells by stereoisomers of 1-methyl-tryptophan correlateswith antitumor responses. Cancer Res 2007; 67: 792–801.
9 Ali OA, Lewin SA, Dranoff G, Mooney DJ. Vaccines combined withimmune checkpoint antibodies promote cytotoxic T-cell activity and tumor
eradication. Cancer Immunol Res 2016; 4: 95–100.
10 Lemke C, Salem A, Krieg A, Weiner G. Abstract 1417: combination cancer
immunotherapy using checkpoint blockade and intratumoral virus-like particles containing CpG ODN. Cancer Res 2016; 76: 1417.
11 Hazama S, Nakamura Y, Takenouchi H et al. A phase I study of combination vaccine treatment of five therapeutic epitope-peptides for metastatic colorectal cancer; safety, immunological response, and clinical outcome. J
Transl Med 2014a; 12: 63.
12 Hazama S, Nakamura Y, Tanaka H et al. A phase II study of five peptides
combination with oxaliplatin-based chemotherapy as a first-line therapy foradvanced colorectal cancer (FXV study). J Transl Med 2014b; 12: 108.
13 Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function.
Cell 2004; 116: 281–97.
14 Wang S, Wang L, Bayaxi N et al. A microRNA panel to discriminate carcinomas from high-grade intraepithelial neoplasms in colonoscopy biopsy tissue. Gut 2013; 62: 280–9.
15 Lodish HF, Zhou B, Liu G, Chen C-Z. Micromanagement of the immune
system by microRNAs. Nat Rev Immunol 2008; 8: 120–30.
16 Cortez MA, Ivan C, Valdecanas D et al. PDL1 regulation by p53 via miR34. J Natl Cancer Inst 2016; 108: djv303.
17 Zhu J, Chen L, Zou L et al. MiR-20b, -21, and -130b inhibit PTEN expression resulting in B7-H1 over-expression in advanced colorectal cancer. Hum
Immunol 2014; 75: 348–53.
18 Zhang Q, Di W, Dong Y et al. High serum miR-183 level is associated withpoor responsiveness of renal cancer to natural killer cells. Tumour Biol
2015; 36: 9245–9.
19 Galore-Haskel G, Nemlich Y, Greenberg E et al. A novel immune resistancemechanism of melanoma cells controlled by the ADAR1 enzyme. Oncotarget 2015; 6: 28999–9015.

Cancer Sci | November 2017 | vol. 108 | no. 11 | 2237

HLA
HR
ISH
LCM
LNA
mCRC
miRNA
NLR
OS
PD-1
PD-L1
PFS
PTEN
qRT-PCRRNF125
ROC
TGF-b
TNF

human leukocyte antigen
hazard ratioin situ hybridization
laser capture microdissection
locked nucleic acidmetastatic CRC
microRNA
neutrophil lymphocyte ratio
overall survival
programmed death-1
PD-ligand 1
progression-free survival
phosphatase and tensin homolog
quantitative RT-PCR
ring finger protein 125
receiver operating characteristictransforming growth factor beta
tumor necrosis factor

20 Kijima T, Hazama S, Tsunedomi R et al. MicroRNA-6826 and-6875 inplasma are valuable non-invasive biomarkers that predict the efficacy of vaccine treatment against metastatic colorectal cancer. Oncol Rep 2017; 37: 23–30.
21 Zhang Y, Yang P, Wang X-F. Microenvironmental regulation of cancer
metastasis by miRNAs. Trends Cell Biol 2014; 24: 153–60.
22 Nishida N, Nagahara M, Sato T et al. Microarray analysis of colorectal cancer stromal tissue reveals upregulation of two oncogenic miRNA clusters.
Clin Cancer Res 2012; 18: 3054–70.
23 Nielsen BS, Jorgensen S, Fog JU et al. High levels of microRNA-21 in thestroma of colorectal cancers predict short disease-free survival in stage II
colon cancer patients. Clin Exp Metastasis 2011; 28: 27–38.
24 Yao Q, Cao S, Li C, Mengesha A, Kong B, Wei M. Micro-RNA-21 regulates TGF-beta-induced myofibroblast differentiation by targeting PDCD4 intumor-stroma interaction. Int J Cancer 2011; 128: 1783–92.
25 Shindo Y, Hazama S, Nakamura Y et al. miR-196b, miR-378a and miR-486
are predictive biomarkers for the efficacy of vaccine treatment in colorectal
cancer. Oncol Lett 2017; 14: 1355–62.
26 Uchida N, Tsunoda T, Wada S, Furukawa Y, Nakamura Y, Tahara H. Ringfinger protein 43 as a new target for cancer immunotherapy. Clin Cancer
Res 2004; 10: 8577–86.
27 Shimokawa T, Matsushima S, Tsunoda T, Tahara H, Nakamura Y, Furukawa
Y. Identification of TOMM34, which shows elevated expression in the majorityof human colon cancers, as a novel drug target. Int J Oncol 2006; 29: 381–6.
28 Suda T, Tsunoda T, Daigo Y, Nakamura Y, Tahara H. Identification ofhuman leukocyte antigen-A24-restricted epitope peptides derived from geneproducts upregulated in lung and esophageal cancers as novel targets for
immunotherapy. Cancer Sci 2007; 98: 1803–8.
29 Ishizaki H, Tsunoda T, Wada S, Yamauchi M, Shibuya M, Tahara H. Inhibition of tumor growth with antiangiogenic cancer vaccine using epitope peptides derived from human vascular endothelial growth factor receptor 1. Clin
Cancer Res 2006; 12: 5841–9.
30 Wada S, Tsunoda T, Baba T et al. Rationale for antiangiogenic cancer therapy with vaccination using epitope peptides derived from human vascularendothelial growth factor receptor 2. Cancer Res 2005; 65: 4939–46.
31 Espina V, Wulfkuhle JD, Calvert VS et al. Laser-capture microdissection.
Nat Protoc 2006; 1: 586–603.
32 Iizuka N, Oka M, Yamada-Okabe H et al. Oligonucleotide microarray forprediction of early intrahepatic recurrence of hepatocellular carcinoma after
curative resection. Lancet 2003; 361: 923–9.
33 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using
real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods
2001; 25: 402–8.
34 Sun YM, Lin KY, Chen YQ. Diverse functions of miR-125 family in different cell contexts. J Hematol Oncol 2013; 6: 6.
35 Zhang XH, Zhang YN, Li HB et al. Overexpression of miR-125b, a novel
regulator of innate immunity, in eosinophilic chronic rhinosinusitis withnasal polyps. Am J Respir Crit Care Med 2012; 185: 140–51.
36 Essandoh K, Li Y, Huo J, Fan GC. MiRNA-mediated macrophage polarization and its potential role in the regulation of inflammatory response. Shock2016; 46: 122–31.
37 Chaudhuri AA, So AY, Sinha N et al. MicroRNA-125b potentiates macrophage activation. J Immunol 2011; 187: 5062–8.

© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

-140-

Original Article
miRNA biomarkers for CRC vaccine

www.wileyonlinelibrary.com/journal/cas

38 Coskun M, Bjerrum JT, Seidelin JB, Troelsen JT, Olsen J, Nielsen OH.
miR-20b, miR-98, miR-125b-1*, and let-7e* as new potential diagnostic
biomarkers in ulcerative colitis. World J Gastroenterol 2013; 19: 4289–99.
39 Kim S-W, Ramasamy K, Bouamar H, Lin A-P, Jiang D, Aguiar RCT.
MicroRNAs miR-125a and miR-125b constitutively activate the NF-jB pathway by targeting the tumor necrosis factor alpha-induced protein 3(TNFAIP3, A20). Proc Natl Acad Sci USA 2012; 109: 7865–70.
40 Wang S, Liu Z, Wang L, Zhang X. NF-jB signaling pathway, inflammationand colorectal cancer. Cell Mol Immunol 2009; 6: 327.
41 Huang HC, Yu HR, Huang LT et al. miRNA-125b regulates TNF-alpha production in CD14 + neonatal monocytes via post-transcriptional regulation. J
Leuk Biol 2012; 92: 171–82.
42 Tili E, Michaille JJ, Cimino A et al. Modulation of miR-155 and miR-125b
levels following lipopolysaccharide/TNF-alpha stimulation and their possibleroles in regulating the response to endotoxin shock. J Immunol 2007; 179:
5082–9.
43 Li Q, Johnston N, Zheng X et al. miR-28 modulates exhaustive differentiation of T cells through silencing programmed cell death-1 and regulatingcytokine secretion. Oncotarget 2016; 7: 53735–50.
44 Faltejskova P, Svoboda M, Srutova K et al. Identification and functional
screening of microRNAs highly deregulated in colorectal cancer. J Cell Mol
Med 2012; 16: 2655–66.
45 Mosakhani N, Sarhadi VK, Borze I et al. MicroRNA profiling differentiatescolorectal cancer according to KRAS status. Genes Chromosom Cancer
2012; 51: 1–9.

46 Li H, Dai S, Zhen T et al. Clinical and biological significance of miR-378a3p and miR-378a-5p in colorectal cancer. Eur J Cancer 2014; 50: 1207–21.
47 Wang P, Gu Y, Zhang Q et al. Identification of resting and type I IFN-activated human NK cell miRNomes reveals microRNA-378 and microRNA30e as negative regulators of NK cell cytotoxicity. J Immunol 2012; 189:
211–21.
48 Lee DY, Deng Z, Wang CH, Yang BB. MicroRNA-378 promotes cell survival, tumor growth, and angiogenesis by targeting SuFu and Fus-1 expression. Proc Natl Acad Sci USA 2007; 104: 20350–5.
49 Urbanek MO, Nawrocka AU, Krzyzosiak WJ. Small RNA detection byin situ hybridization methods. Int J Mol Sci 2015; 16: 13259–86.
50 Kramer A, Green J, Pollard J Jr, Tugendreich S. Causal analysis approaches
in ingenuity pathway analysis. Bioinformatics 2014; 30: 523–30.
51 Anderson DM, Maraskovsky E, Billingsley WL et al. A homologue of the
TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 1997; 390: 175–9.
52 Renema N, Navet B, Heymann MF, Lezot F, Heymann D. RANK-RANKL
signalling in cancer. Biosci Rep 2016; 36: e00366.
53 Giannini AL, Gao Y, Bijlmakers MJ. T-cell regulator RNF125/TRAC-1
belongs to a novel family of ubiquitin ligases with zinc fingers and a ubiquitin-binding domain. Biochem J 2008; 410: 101–11.
54 Suzuki HI, Katsura A, Matsuyama H, Miyazono K. MicroRNA regulons intumor microenvironment. Oncogene 2015; 34: 3085–94.

Supporting Information
Additional Supporting Information may be found online in the supporting information tab for this article:
Table S1. Top 30 miRNAs upregulated or downregulated in a responder group compared with a non-responder group, as assessed using microarray analysis.
Table S2. Frequency of severe adverse events (CTCAE version 3.0) in four groups classified according to miR-378a expression and HLA-A status.
Fig. S1. Overall survival (OS) according to number of predictive factors. OS of the HLA-A*2402-matched (left side) and -unmatched groups (rightside) of phase II study patients according to the number of predictive factors (high expression of miR-125b-1 and/or miR-378a). (a) Predictive factors in cancer cells. (b) Predictive factors in stromal cells. Dotted line shows the survival curve of patients with 0 factors. Solid line shows the survival curve of patients with one factor and the dashed line shows the survival curve of patients with two factors. Statistical analysis was doneusing the log–rank test. HLA, human leukocyte antigen.
Fig. S2. Overall survival (OS) according to miRNA expression and HLA-A status. (a) According to miR-125b-1 in cancer cells. (b) According tomiR-125b-1 in stromal cells. (c) According to miR-378a in cancer cells. (d) According to miR-378a in stromal cells. Red line shows HLAA*2402-matched group and black line shows -unmatched group of phase II study patients. Solid line shows high miRNA expression and thedashed line shows low miRNA expression. Statistical analysis was done using the log–rank test. HLA, human leukocyte antigen; miR, microRNA.

© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

-141-

Cancer Sci | November 2017 | vol. 108 | no. 11 | 2238

International Journal of

Molecular Sciences
ArticleDownregulation of Plasma miR-215 in Chronic
Myeloid Leukemia Patients with Successful
Discontinuation of Imatinib
Kazuma Ohyashiki 1,2, *, Tomohiro Umezu 2 , Seiichiro Katagiri 1 , Chiaki Kobayashi 2 ,Kenko Azuma 3 , Tetsuzo Tauchi 1 , Seiichi Okabe 1 , Yutaka Fukuoka 4 and Junko H. Ohyashiki 3
1
2
3
4

*

Department of Hematology, Tokyo Medical University, Tokyo 160-0023, Japan; patchsei@yahoo.co.jp (S.K.);
tauchi@tokyo-med.ac.jp (T.T.); okabe@tokyo-med.ac.jp (S.O.)
Department of Molecular Science, Tokyo Medical University, Tokyo 160-0023, Japan;t_umezu@tokyo-med.ac.jp (T.U.); chiaki-k@tokyo-med.ac.jp (C.K.)
Department of Molecular Oncology, Institute of Medical Science, Tokyo Medical University, Tokyo 160-0023,
Japan; k_azuma@tokyo-med.ac.jp (K.A.); junko@hh.iij4u.or.jp (J.H.O.)
Department of Electrical Engineering, Kogakuin University, Tokyo 163-8677, Japan;
fukuoka@cc.kogakuin.ac.jp
Correspondence: ohyashik@rr.iij4u.or.jp; Tel.: +81-3-3342-1510; Fax: +81-3-5381-6651

Academic Editors: William Chi-shing Cho and Nalini Santanam
Received: 8 January 2016; Accepted: 8 April 2016; Published: 15 April 2016

Abstract: Approximately 40% of chronic myeloid leukemia (CML) patients who discontinue imatinib(IM) therapy maintain undetectable minimal residual disease (UMRD) for more than one year
(stopping IM (STOP-IM)). To determine a possible biomarker for STOP-IM CML, we examined
plasma miRNA expression in CML patients who were able to discontinue IM. We first screened
candidate miRNAs in unselected STOP-IM patients, who had sustained UMRD after discontinuing
IM for more than six months, in comparison with healthy volunteers, by using a TaqMan low-density
array for plasma or exosomes. Exosomal miR-215 and plasma miR-215 were downregulated in
the STOP-IM group compared to the control, indicating that the biological relevance of the plasmamiR-215 level is equivalent to that of the exosomal level. Next, we performed real-time quantitative
RT-PCR in 20 STOP-IM patients, 32 patients with UMRD on continued IM therapy (IM group) and28 healthy volunteers. The plasma miRNA-215 level was significantly downregulated in the STOP-IM
group (p < 0.0001); we determined the cut-off level and divided the IM group patients into two groups
according to whether the plasma miR-215 was downregulated or not. The IM group patients witha low plasma miR-215 level had a significantly higher total IM intake, compared to the patients withelevated miR-215 levels (p = 0.0229). Functional annotation of miR-215 target genes estimated by
the Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatic tools
involved cell cycle, mitosis, DNA repair and cell cycle checkpoint. Our study suggests a possible roleof miR-215 in successful IM discontinuation.
Keywords: plasma miR-215; chronic myeloid leukemia; imatinib; discontinuation

1. Introduction
Tyrosine kinase inhibitors (TKIs), including imatinib (IM), are now a standard frontline therapy
for chronic myeloid leukemia (CML) that yields successful outcomes [1]. CML therapy is widely
accepted, and achieving cytogenetic and molecular responses at certain time points after TKI therapy
and switching among TKIs have been proposed as practical guidelines [2]. In addition, clinical trials
for TKI discontinuation are still on-going, with the most famous studies being the STIM study [3] andthe TWISTER study [4]. In the STIM study, approximately 40% of CML patients with a molecularInt. J. Mol. Sci. 2016, 17, 570; doi:10.3390/ijms17040570

www.mdpi.com/journal/ijms-142-Int. J. Mol. Sci. 2016, 17, 570

2 of 11

remission of at least a two years’ duration safely discontinued IM; they maintained undetermined
minimal residual disease (UMRD) for more than two years, and 60% maintained major molecularresponse (MMR: MR4.0 ) [3]. The STIM study showed that male gender, low Sokal score and IM
duration of >72 months were associated with maintaining treatment-free remission [3]. The TWISTER
study, using a patient-specific primer for BCR-ABL, revealed that some CML patients who maintained
UMRD after stopping IM had BCR-ABL DNA, leading to molecular relapse [4]. Early reduction of
BCR-ABL1 transcription by TKIs induces a deep molecular response [5], and expansion of cytotoxic
natural killer (NK) cells [6] could serve as a predictive marker for future treatment-free remission.
We have reported that extracellular microRNAs (miRNAs), including plasma miRNAs andexosomal miRNAs, are altered in hematopoietic neoplasias [7–9], and some support malignant
progression via the microenvironment [10,11]. miRNAs are noncoding single-stranded RNAs of
21–25 nucleotides that have recently been implicated in the regulation of cellular processes, such as
apoptosis, proliferation, development or differentiation, not only in normal hematopoiesis, but also
in hematological malignancies. The biological and clinical implications of cellular miRNAs are now
being extensively studied. Recently, extracellular miRNAs, also known as secretary miRNAs, havebeen proposed as having multiple functions, including immune-modulation, angiogenesis and cancer
progression [11–13]. Extracellular miRNAs, such as exosomal miRNAs, are extensively studied
in non-hematologic diseases, such as cardiovascular diseases, endocrine disorders or pulmonary
diseases [12]. Unlike solid tumors, the cellular component is easily obtained in the context of leukemia;
however, analysis of the cell-free fraction, including plasma, is worthwhile in cases of complete
remission when neoplastic cells are not present in the peripheral blood. We attempted to identify
circulating miRNAs in CML patients who maintained UMRD after stopping IM (STOP-IM), and we
studied target molecules by using bioinformatics tools.
2. Results
2.1. miRNA Expression Profiling by the TaqMan miRNA Array
To identify candidate plasma miRNAs with altered expression in the STOP-IM group, we screened
miRNA expression using a TaqMan miRNA array on seven unselected CML patients from the STOP-IM
group and seven healthy volunteers. Between these two groups, we observed differential expressionof 69 miRNAs identified by using GeneSpring software (Agilent Technologies, Santa Clara, CA,
USA) (Figure 1; Gene Expression Omnibus (GEO) Accession No. GSE75392). Based on the Wilcoxon
rank sum test of the R statistical software, only two miRNAs, miR-215 (p = 0.006841) and miR-134(p = 0.028805), had greater than a 1.5-fold change in expression.
To determine whether miRNA expression in plasma indeed reflects exosomal miRNA,
we compared the expression profiles by using a TaqMan low-density array. We randomly chose
three patients in the STOP-IM group and three healthy volunteers. Eleven miRNAs in plasma and35 exosomal miRNAs were found to be significantly different between the two groups. Among thesemiRNAs, downregulation of miRNA-215 expression was highly significant in the STOP-IM group(plasma miRNAs, p = 0.00311 (Table S1); exosomal miRNAs, p = 0.00039 (Table S2)); therefore,
we concluded that plasma miR-215 expression mirrors exosomal miRNA expression and focusedon expression of this miRNA for further study.

-143-

Int. J. Mol. Sci. 2016, 17, 570

3 of 10Int. J. Mol. Sci. 2016, 17, 570

3 of 11

Int. J. Mol. Sci. 2016, 17, 570

3 of 10Figure 1. miRNAprofiling theby the TaqMan(Thermo(Thermo Fischer
Science,
Waltham,
MA, USA)
FigureFischer
Science,
Waltham,MA,miRNA
USA)miRNA
miRNA
Figure1.1.miRNA
miRNAprofilingprofilingby
by the TaqMan
TaqMan (Thermo Fischer
Science,
Waltham,MA,
USA)
array. A differential expression pattern was found between stopping imatinib (STOP-IM) patients andarray.
patternwas
foundbetweenstopping
imatinib(STOP-IM)
patients
andarray.AAdifferential
differentialexpression
expression
patternwas
foundbetweenstopping
imatinib(STOP-IM)
patients
control subjects. Using Sequence Detection System (SDS, Version 2.4, Thermo Fisher Science, Waltham,control
subjects.
Using
Sequence
Detection
System(SDS,
Version
2.4,
ThermoFisherScience,
Waltham,and control
subjects.
Using
Sequence
Detection
System(SDS,
Version
2.4,
ThermoFisherScience)and
MA, USA) and DataAssist software (Thermo Fisher Science, Waltham, MA, USA), we calculated the
MA,
USA)
and
DataAssist
software(ThermoFisherScience,
Waltham,wecalculated
the
DataAssist
software(ThermoFisherScience),wecalculatedof USA),
miRNAsbased
normalized
by thetheCexpression
t value ofMA,
ath-miR-159.
Data
wereon theirexpressionof miRNAs
basedon theirCt value
analyzed
withGeneSpring
software(Agilent
Technologies,
Santa
Clara,
CA,
USA).
value normalized
by theanalyzed
Ct valuewithof ath-miR-159.
wereexpressionof miRNAsbybased
theirof
Ctath-miR-159.
Ct value normalized
the Con
Data were
GeneSpringData
softwaret value(Agilent Technologies,
Santa
Clara, CA,
USA).Technologies, Santa Clara, CA, USA).
analyzedwith GeneSpring
software(Agilent

2.2. Quantification of Individual miRNA by Real-Time Quantitative Reverse Transcriptase-Polymerase
Chain Reaction

2.2.
QuantitativeReverse
ReverseTranscriptase-Polymerase
Transcriptase-Polymerase
2.2.Quantification
QuantificationofofIndividual
Individual miRNA
miRNA by Real-Time
Real-Time Quantitative
Since
the
expressionofmiR-215
was
typically
downregulated
in
both
the
plasma and exosomes
Chain
Reaction
Chain Reactionof the STOP-IM group compared to healthy individuals, we analyzed the plasma miR-215 expression

Since
the
expression
ofbymiR-215
miR-215
was
typically
downregulated
in
the
and
level
inexpression
CML
patientsof
individual
real-timequantitative
RT-PCR. Plasma
samples
were obtained
Since
the
was
downregulated
in bothboththeplasma
plasmaandexosomes
exosomesfrom20
patients
inthe
STOP-IM
group
and
32
of
the
IM
group,
as
well
as
from
28
healthy
volunteers.
theSTOP-IM
STOP-IMgroup
groupcompared
compared toto healthy
healthy individuals,
individuals, weofofthewe analyzed
analyzed thethe plasma
plasma miR-215
miR-215expression
expression
ACML
validation
analysisusing a large
number ofsamples revealed
that expressionof miR-215 waslevelininCML
patients
by individual
individual
real-timequantitative
RT-PCR.
level
patients
byreal-timequantitative
RT-PCR. Plasma
Plasma samples
sampleswere
wereobtained
obtainedsignificantly lower in the STOP-IM group than in the healthy volunteers (p < 0.0001) or in the IMfrom20
patients
inthe
STOP-IM
group
and
32
of
the
IM
group,
as
well
as
from
28
healthy
volunteers.
from 20 patients
inthe
STOP-IM
group
and
32
of
the
IM
group,
as
well
as
from
28
healthy
volunteers.
group (p = 0.0439) (Figure 2A).
validation analysis
analysis using
using aa large
large number
number ofof samples
AAvalidation
samples revealed
revealed thatthat expression
expression ofof miR-215
miR-215was
was
significantly
lower
in
the
STOP-IM
group
than
in
the
healthy
volunteers(p<0.0001)
or
in
the
IM
group
significantly lower in the STOP-IM group than in the healthy volunteers (p < 0.0001) or in the IM(p = 0.0439)(Figure2A). 2A).
group(p = 0.0439)(FigureFigure 2. Expression of miR-215 (A). The cutoff level for miR-215 expression between the STOP-IM
and control groups (B). The area under the curve level was 0.9204. The cutoff value was thus
determined to be 0.6627, which was used in the following analyses of plasma miR-215 expression.
Abbreviations: IM, imatinib mesylate; CI, confidence interval; CML, chronic myeloid leukemia;
STOP-IM, patients with sustained undetectable minimal residual disease (UMRD) for more than six
months after discontinuing IM; N, healthy volunteers; IM, CML patients taking IM who have a
Figure2.2.
Expression
ofmiR-215
miR-215 (A).
(A); The
The cutoff level for miR-215
FigureExpressionofmiR-215 expression
expression between
betweenthe
theSTOP-IM
STOP-IM
molecular
response.

andcontrol
control groups
groups (B).
(B). The
The area under theandthe curve
curve level
level was
was 0.9204.
0.9204. The
The cutoff
cutoff value
value was
was thus
thus
determinedtotobe
be0.6627,
0.6627, which
which waswas used
used inin the following analyses of plasma
determined
plasma miR-215
miR-215 expression.
expression.
Abbreviations: IM,
IM, imatinib
imatinib mesylate;mesylate; CI,
CI, confidence interval; CML, chronic
Abbreviations:chronic myeloid
myeloid leukemia;
leukemia;
STOP-IM,patients
withsustained
undetectable
minimalresidualdisease(UMRD)
more
than
STOP-IM, patients with sustained undetectable minimal residual disease (UMRD) forfor
more
than
six
six months
discontinuingN, healthy
volunteers;
CML
patients
takingIMIMwho
whohavehavea
monthsafterafter
discontinuing
IM; IM;N, healthy
volunteers;
IM,IM,
CML
patients
takinga molecularresponse.
molecular
response.

-144-

Int. J. Mol. Sci. 2016, 17, 570

4 of 112.3. Clinical and Biological Relevance of miR-215 Expression
To estimate a cutoff value for miR-215 expression in plasma for distinguishing between STOP-IM
and healthy control groups, we performed receiver operating characteristics (ROC) curve analysis(Figure 2B). The area under the ROC curve was 0.9204% with 85.00% sensitivity (95% confidence
interval (CI), 62.22%–96.79%) and 85.71% specificity (95% CI, 42.13%–85.71%). The cutoff value wasthus determined to be 0.6627, which was used in subsequent analyses of miR-215 expression. We next
analyzed the clinical relevance of the plasma miR-215 expression level in CML patients who sustained
MR4.0 while on IM (IM group). There were no significant differences in age (p = 0.5903), sex (p = 1),
Sokal score (p = 0.2719) or prior interferon therapy history (p = 0.7026). Notably, the patients witha low plasma miR-215 level had a significantly higher total IM intake, compared to the patients witha high miR-215 level (p = 0.0229), while no significant differences existed with regard to the daily IM
dose (p = 0.395) or duration of IM therapy (p = 0.0871) between the two CML groups with or withouta low expression level of plasma miR-215 (Table 1). These two CML groups also did not significantly
differ in the International Scale at the time of plasma miR-215 examination (p = 0.4126). The durationof UMRD from CML diagnosis (p = 0.3827) and the duration of stable molecular response (maintain
MMR) (p = 0.7548) did not differ between these two groups (Table 1).
Table 1. Clinico-hematologic characteristics of chronic myeloid leukemia (CML) patients under imatinib
therapy with or without down-regulation of plasma miR-215 expression.
Plasma miR-215 Levels (Number of CML
Patients Who Are Taking Imatinib)<0.6627>0.6627n = 22n = 10Age (years)
Sex (male/female)
Sokal score (low/intermediate/high)Prior interferon therapy (yes/no)
Total IM dose (g)Daily IM dose (mg)
Duration IM (months)
UMRD from diagnosis (months)
Duration of UMRD (months)
International scale percentage at the time ofplasma miRNA examination

55.91 ˘ 2.436
18/4
19/3/0
7/15
931.0 ˘ 65.32
377.3 ˘ 11.27
84.05 ˘ 5.654
53.27 ˘ 8.855
41.82 ˘ 5.283

58.50 ˘ 4.634
8/2
7/2/1
4/6
670.5 ˘ 71.67
360.0 ˘ 16.33
65.30 ˘ 9.656
70.80 ˘ 22.24
38.60 ˘ 9.782

0.59031*
0.2719 *0.7026 *0.02290.395
0.0871
0.38270.75480.0026 ˘ 0.0003

0.0031 ˘ 0.0006

0.4126

p ValueIM, imatinib mesylate; UMRD, undetectable minimum residual disease; MR, molecular response; MMR, major
molecular response; * determined by the chi-square test.

2.4. Functional Annotation of miRNA-215 Target Genes
The clinical relevance of miR-215b was not evident, so we sought experimentally-validated
target genes of miRNA-215 in miRTarBase or determining the biological significance of miR-215.
More than 100 target genes were extracted by miRTarBase and the target genes for which strong
evidence existed are summarized in Table 2. Six genes, including WNK lysine deficient protein kinase
1 (WNK1), activated leukocyte cell adhesion molecule (ALCAM), retinoblastoma 1 (RB1), protein
tyrosine phosphatase receptor type T (PTPRT), activin A receptor type IIB (ACVR2B) and catenin beta
interacting protein 1 (CTNNBIP1), were experimentally validated as genes with strong evidence forbeing targets (Table 2) [14–23]. To estimate the biological implication of miR-215 target genes, wethen used the DAVID bioinformatic tool for functional annotation of target genes and possible related
pathways (p < 0.0001). Approximately 30% of the target genes were related to cellular metabolic
process, and miR-215 target genes were subsequently determined to be involved in the cell cycle,
cellular stress, mitosis, DNA repair and cell cycle checkpoints (Figure 3 and Table S3).

-145-

Int. J. Mol. Sci. 2016, 17, 570

5 of 10

Table 2. Top 10 of the predicted target genes for miR-215 that identified by MiRTarBase 5 of 11

Int. J. Mol. Sci. 2016, 17, 570

Validation Methods
Strong EvidenceLess Strong Evidence
ID
Target
Sum ReferencesTable
2. Top 10of the predicted
ReporterWesterntarget genes for miR-215 that identified by MiRTarBase.
qPCR Microarray NGS pSILAC Other
Assay
Blot
Validation Methods
MIRT005583
WNK1
*
*
*
*
4
[14]
StrongEvidence
Less Strong Evidence
MIRT024327
ALCAM
*
*
*
*
4
[15,16]
TargetID
Sum References
ReporterWestern
Blot*qPCR MicroarrayNGS
pSILAC Other
MIRT024341
RB1* Assay
*
*
4
[15]
4 3
[14] [17]MIRT005583 ZEB2
WNK1
*
*
*
*
MIRT052961***
MIRT024327 PTPRT
ALCAM
*
*
*
4 3 [15,16] [18]MIRT054778****
MIRT024341
RB1
*
*
*
*
4
[15]
MIRT006340
ACVR2B**MIRT052961
ZEB2
*
*
*
3 2
[17] [19]MIRT007364***
MIRT054778CTNNBIP1
PTPRT
*
*
3 2
[18] [15,20]
MIRT006340 DTL
ACVR2B**2 2[19] [15,21]
MIRT024273**
CTNNBIP1**
2[15,20]
MIRT007364
MIRT024412
GABPB1* **2 [15,21][15,22]
MIRT024273
DTL*
2
MIRT024495* ***MIRT024412 SYNGR1GABPB12 2 [15,22][15,23]
SYNGR1**
2[15,23]
MIRT024495
Asterisks
indicate
data from validation methods; NGS, next-generation
sequencing; pSILAC,
stableAsterisks indicate data from validation methods; NGS, next-generation sequencing; pSILAC, stable isotope
isotopelabelinglabeling
by amino
in cell culture.
by aminoacid inacidcell culture.

Figure3. A3.functionalannotation
Bioinformatic
tools
v6.7
demonstrated
FigureA functionalannotationanalysis
analysisusing
using DAVIDDAVID Bioinformatic
tools
v6.7
[24][24]
demonstrated
that thatthe thetargetgenes
miR-215were
werehighlyrepresented
in the categories
cellularprocess
metabolictargetgenesforfor miR-215
highlyrepresented
in the categories
of cellularof
metabolic(Gene
Ontology(GO):(GO):44237),44237),
cell cycle
07049),
stimulusto(GO:51716),(GO:mitosisprocess(Gene
Ontologycell(GO:
cycle(GO:cellular
07049),response
cellular to
response
stimulus
51716),(GO:
07067),
DNA
repair(GO:
06281)
and
cell
cycle
checkpoint(GO:
00075).
The
percentages
ofgenes
mitosis (GO: 07067), DNA repair (GO: 06281) and cell cycle checkpoint (GO: 00075). The percentages
that contributed
to representative
categories
are depicted.
of genesthat contributed
to representative
categories
are depicted.

3. Discussion
3. Discussion
During the last decade, emerging evidence has shown that miRNAs are a key molecular tool for
During the last decade, emerging evidence has shown that miRNAs are a key molecular tool forcancer diagnosis and prognosis. A section of tumor tissue or a circulating cell-free fraction, such as
cancerdiagnosis and prognosis. A section of tumor tissue or a circulating cell-free fraction, such asplasma, may be used for miRNA analysis for finding novel diagnostic and prognostic markers [12].
plasma,may
be majority
used for ofmiRNA
findingnovel
diagnostic
and prognostic
markers
In thevast
reportsanalysis
dealing for
with
miRNA
diagnosis,
investigators
identified
tumor [12].
In thevast majority
ofWhenreports
dealingwithis miRNA
diagnosis, investigators
tumor
cell-derived
miRNA.
a cell-freefractionused, biologically-relevant
miRNA is identified
considered tocell-derived
miRNA.
Whenacell-free
fractionisused,biologically-relevant
miRNA
isconsidered
be exosomal miRNA derived from circulating cancer cells. For miRNA analysis in leukemia, using to
be exosomal
fromcirculating
cancercells. For
in ofleukemia,
leukemia miRNAcells mayderived
indeed be
morereliable fordetermining
the miRNA
biologicalanalysis
relevancethese cellsusing
than using
However,to findreliablea novel for
prognostic
markerthein patients
withrelevancecomplete remission,
leukemiacells plasma.
may indeed
be moredetermining
biologicalof these cells
suchasSTOP-IMpatients,
we
need
to
use
a
different
type
of
miRNA
analysis.
For
instance,
wethan using plasma. However, to find a novel prognostic marker in patients with complete remission,
recently
showedthat
microRNA-148b,
a
type
of
immunemiRNA
incirculating
peripheral
bloodsuch as STOP-IM patients, we need to use a different type of miRNA analysis. For instance, wemononuclear cells (PBMCs), is downregulated in STOP-IM patients [13], in accordance with increased
recently
showed that microRNA-148b, a type of immune miRNA in circulating peripheral blood
NK cells [6]. This finding indicated the possibility that maintaining UMRD after stopping IM might bemononuclear cells (PBMCs), is downregulated in STOP-IM patients [13], in accordance with
due to immune surveillance. In such cases, the miRNA level may reflect the component of circulating
increased NK cells [6]. This finding indicated the possibility that maintaining UMRD after stopping
immune cells; therefore, miRNA analysis requires careful attention and interpretation in a complete
IM might
be dueto immune surveillance. In such cases, the miRNA level may reflect the component
remissionstate.

of circulating immune cells; therefore, miRNA analysis requires careful attention and interpretation
in a complete remission state.
To further elucidate the possible role of miRNA in STOP-IM patients, we analyzed plasma
miRNA. We and others have reported the clinical relevance of plasma or serum miRNA in hematologic
neoplasia [7–9,25]. Recent technical advances in exosome separation and characterization enabled-146-

Int. J. Mol. Sci. 2016, 17, 570

6 of 11

To further elucidate the possible role of miRNA in STOP-IM patients, we analyzed plasma
miRNA. We and others have reported the clinical relevance of plasma or serum miRNA in hematologic
neoplasia [7–9,25]. Recent technical advances in exosome separation and characterization enabled
analyzing exosomal miRNA rather than plasma or serum miRNA [11]. Theoretically, using exosomal
miRNA for analysis would be ideal, but in practical terms, the procedure remains complicated. For thisreason, we compared the results obtained from plasma and exosome. Regardless of the template source,
plasma miR-215 expression was downregulated in STOP-IM patients (Tables S1 and S2). Our CML
patients in the STOP-IM group did not take IM for more than six months before the plasma miRNA
assay, indicating that the downregulation of plasma miR-215 in STOP-IM patients was not due tocurrent IM intake. It is well known that approximately 60% of CML patients who discontinue IM
relapse within 12 months [3–5] and CML stem cells may persist after stopping IM [4]. Since the total
IM intake dose was significantly higher in CML patients with low miR-215 levels in the IM group,
CML stem cells may be more suppressed, although the prognosis in the CML-IM group did not differ
between patients with and without low plasma miR-215 expression. Long-term follow-up of CML-IM
patients may clarify this matter.
In the current study, plasma miR-215 level was significantly low in the STOP-IM group, whencompared to normal subjects, nevertheless, none of these groups took IM for more than six months.
We speculated that the difference of plasma miR-215 level in the IM group (CML patients with UMRD,
but maintaining IM therapy) could predict a subset of CML patients who could stop IM safely, but thecurrent study failed to predict the selection of the candidate correctly. Therefore, we searched miR-215
target genes by miRTarBase or those that may be linked to immunological surveillance or residual
CML stem cells; miR-215 has previously been demonstrated to be dysregulated in several humanmalignancies and to be correlated with tumor progression [19,21,26,27]. Meta-analyses have revealed
that elevated miR-215 expression is correlated with poor prognosis in renal cell carcinomas [28],glioma [29] and pancreatic cancer [30]. In contrast, decreased expression was associated withadvanced disease stage in colorectal cancer [31], and miR-192-5p/215 is a putative tumor suppressor
in non-small cell lung cancer [32]. Taken together, miR-215 may potentially be both an oncomiRand a tumor-suppressive miR, with its actual role depending on the type of cancer and the pathway
involved. In the current study, it was impossible to conduct a functional analysis of miR-215, such asa reporter assay using specimens from patients in a complete remission state. As an alternative, we
utilized bioinformatics tools, such as miRTarBase and DAVID, to identify the miR-215 target gene.
miRTarBase includes information on miRNA–target interactions (MTIs), with the collected MTIs
having been experimentally validated by reporter assay, Western blot, microarray and next-generation
sequencing [33]. Based on miRTarBase, we particularly focused on RB1 and ACVR2, showing strong
evidence in this study.
Deng et al. [32] reported upregulation of miR-215 in gastric cancer tissues, and functional analysis
revealed that inhibition of miR-215 significantly suppressed gastric cancer cell proliferation, possibly
via G1 arrest. In addition, miR-215 was able to target RB1 through its 31 -UTR in gastric cancer
cells [32]. Conversely, Braun et al. [34] reported that p53 induces upregulation of miR-192, miR-194
and miR-215, and miR-192/miR-215 enhanced CDKN1/p21 levels, colony suppression and cell cycle
arrest; therefore, miR-192/miR-215 may suppress carcinogenesis through p21 accumulation and cell
cycle arrest. Senanayake et al. [19] reported that miR-192, miR-215 and miR-194 had significantly lower
expression in nephroblastomas, and they identified the target as activating receptor type 2B (ACVR2B),
indicating that downregulation of miR-215 may affect an important step in the pathogenesis of renal
childhood neoplasms. ACVR2B is known to be associated with human embryonic stem cells [35].
Human embryonic stem cells express receptors for nodal (ACVR1B and ACVR2B) and a co-receptor
for nodal, and they control NANOG expression via SMAD2/SMAD3; therefore, miR-215 targeting
ACVR2B may affect the self-renewal capability of stem cells [36]. Although all of the evidence isprovided by databases, DAVID bioinformatics tools clearly showed that miR-215 targets genes in thepathway involved in cell cycle regulation.

-147-

Int. J. Mol. Sci. 2016, 17, 570

7 of 11The data presented in the current study suggest that the expression level of plasma miRNAs
in CML patients who maintained a deep molecular response to IM is lower than in normal subjects.
Of the 32 CML patients taking IM at the time of plasma miRNAs examination, 16 patients discontinued
IM after the assay, and five have had a molecular relapse. Unfortunately, we observed no significant
differences in the plasma miR-215 level or in the clinical data between these two IM groups (datanot shown), possibly because of the retrospective study for miRNAs assessment. The plasmamiR-215 level in CML patients who had been maintaining molecular remission without imatinib
was actually downregulated; therefore, further prospective studies may provide new insight into
available biomarkers for therapy-free remission in CML patients.
4. Experimental Section
4.1. Patients and Samples
Fifty-two consecutive patients with CML, who maintained MR4.0 or more by IM therapy,
were enrolled in this study. Samples from unselected 20 patients who had sustained UMRD after
discontinuing IM (STOP-IM group) were collected more than 6 months after discontinuation [13].
The STOP-IM patients maintained the negativity of nested PCR for more than two years before
stopping IM therapy. Thirty-two patients who were receiving IM and maintained less than MR4.0
at the time of plasma collection were categorized as the IM group. Twenty-eight healthy volunteers
served as the control group. CML patients who had not received treatment and those who did
not respond to IM were not included in this study. No significant difference existed in sex orage between the CML groups and healthy volunteers. Molecular response was determined by the
BCR-ABL1 transcript, as reported previously [6,13], and was represented as an International Scale(IS). UMRD was confirmed by the absence of BCR-ABL1 transcript as determined by nested reverse
transcription-polymerase chain reaction (RT-PCR) [13]. This study was approved by the institutional
review board of Tokyo Medical University (No. 930, approved 24 June 2008). Written informed
consent was obtained from all patients prior to specimen collection, in accordance with the Declarationof Helsinki.
4.2. TaqMan Low-Density Array Screening
Total RNA was isolated with the mirVana PARIS kit (Ambion, Austin, TX, USA) fromseven randomly-selected healthy volunteers and seven randomly-selected STOP-IM patients.
Five hundred microliters of plasma were diluted with 500 µL of binding solution. After a 5-min
incubation, 1 µL of 1 nM ath-miR-159 (Hokkaido System Science, Hokkaido, Japan) was added,
followed by vortexing for 30 s and incubation on ice for 10 min. Subsequent phenol extraction andcartridge filtration were performed according to the manufacturer’s instructions. The RT reaction
and pre-amplification step were set up according to the recommendations. miRNAs were reverse
transcribed with Megaplex Prime Pools (Human Pools A v2:1; Thermo Fisher Sciences). The miRNA
expression profiles were determined with a TaqMan miRNA Array Human Card A (Thermo Fisher
Sciences). To compare the results between plasma miRNA expression and exosomal miRNA expression,
we extracted the exosome by Total Exosome Isolation Reagent (Invitrogen, Carlsbad, CA, USA) andthen assessed exosomal miRNA expression in a subset of samples, as described previously [11].
Quantitative RT-PCR was performed on an Applied Biosystem 7900 HT thermocycler according to
the manufacturer’s recommended program [13]. With the use of SDS2.2 software (Invitrogen) and
DataAssist (Thermo Fisher Sciences), the expression of plasma miRNAs was calculated based on cycle
threshold (Ct ) values normalized to those of ath-miR-159, which was spiked in each plasma sample.
Data analysis was done using GeneSpring software (Agilent Technologies). The Benjamin-Hochberg
algorithm was used for estimation of false discovery rates, as we reported previously [13].

-148-Int. J. Mol. Sci. 2016, 17, 570

8 of 114.3. Real-Time Quantitative RT-PCR for miR-215
We determined the amounts of the individual miRNA by real-time quantitative RT-PCR witha TaqMan MicroRNA Assay (Thermo Fisher Sciences) and the following miRNA-specific stem-loop
primer: hsa-miR-215 (hsa-miR-215-4373084). Subsequently, quantitative real-time PCR was performed
with an ABI Prism 7000 sequence detection system (Thermo Fisher Sciences). The reaction was initiatedby incubation at 95 ˝ C for 2 min, followed by 50 cycles of 95 ˝ C for 15 s and then 60 ˝ C for 1 min.
All reactions were run in duplicate. Mean (Ct ) values for all miRNAs were quantified with sequence
detection system software (SDS Version 1.02, Thermo Fisher Scientific) [13]. The relative expressionof miR-215 was calculated by using the ∆Ct method as reported previously [8]. The spike control(ath-miR-159) was used as an invariant control for plasma miRNA.
4.4. Statistical and Bioinformatics Analyses
Data were expressed as means ˘ standard deviation (SD). Mann-Whitney U and χ-square tests
were used to determine statistical significance for comparisons between the control and test groups.
Multiple groups were compared by one-way analysis of variance (ANOVA). Statistical analysis
was done using R software and GraphPad Prism software (Version 5c for Macintosh; GraphPad
Software Inc., La Jolla, CA, USA). miRNAs with a ∆Ct of >1.0 or <´1.0 and p-values of >0.05 weredeemed as being differentially expressed.
Following identification of differentially-expressed miRNAs, the predicted target genes for thesealtered miRNAs were subjected to the experimentally-validated miRNA-target interactions database
miRTarBase [37]. Moreover, functional annotation of target genes was determined by the Database forAnnotation, Visualization and Integrated Discovery (DAVID Bioinformatics tools v6.7) [24].
5. Conclusions
In the current study, we found that plasma miR-215 was downregulated in CML patients who
had stopped IM therapy compared to normal subjects. Further studies will reveal the biological andclinical significance of miR-215, including the plasma miR-215 level, and provide further information
for treatment-free remission in CML patients. In conclusion, plasma miRNA analysis, which may
reflect exosomal miRNA, could be a potent prognostic biomarker in hematologic malignancies thathave a current remission status.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/
17/4/570/s1.
Acknowledgments: This work was supported by the Private University Strategic Research Based Support Project(S1311016) and in part by the Grant-in-Aid (15H04303) from MEXT (Ministry of Education, Culture, Sports,
Science and Technology, Tokyo, Japan).
Author Contributions: Kazuma Ohyashiki designed and performed the results. Kazuma Ohyashiki,
Seiichiro Katagiri, Tetsuzo Tauchi and Seiichi Okabe contributed essential reagents or tools. Tomohiro Umezu,
Chiaki Kobayashi and Kenko Azuma performed PCR analysis and bioinformatic analysis. Yutaka Fukuoka
performed bioinformatic analysis. Kazuma Ohyashiki wrote the paper. Junko H. Ohyashiki performed criticalreading. All authors contributed toward data analysis, drafting and revising of the manuscript.
Conflicts of Interest: Kazuma Ohyashiki received research support from Novartis and Bristrol-Meyer Squibb.
The authors report no other conflicts of interest in this work.

References1.

2.

Hochhaus, A.; O’Brien, S.G.; Guilhot, F.; Druker, B.J.; Branford, S.; Foroni, L.; Goldman, J.M.; Müller, M.C.;
Radich, J.P.; Rudoltz, M.; et al. Six-year follow-up of patients receiving imatinib for the first-line treatment ofchronic myeloid leukemia. Leukemia 2009, 23, 1054–1061. [CrossRef] [PubMed]
Baccarani, M.; Deininger, M.W.; Rosti, G.; Hochhaus, A.; Soverini, S.; Apperley, J.F.; Cervantes, F.; Clark, R.E.;
Cortes, J.E.; Guilhot, F.; et al. European LeukemiaNet recommendations for the management of chronic
myeloid leukemia: 2013. Blood 2013, 122, 872–884. [CrossRef] [PubMed]-149-

Int. J. Mol. Sci. 2016, 17, 570

3.

4.

5.

6.

7.

8.

9.

10.
11.

12.
13.

14.

15.

16.

17.

18.

9 of 11Mahon, F.X.; Réa, D.; Guilhot, J.; Guilhot, F.; Huguet, F.; Nicolini, F.; Legros, L.; Charbonnier, A.; Guerci, A.;
Varet, B.; et al. Intergroupe Français des Leucémies Myéloïdes Chroniques. Discontinuation of imatinibin patients with chronic myeloid leukaemia who have maintained complete molecular remission for atleast 2 years: The prospective, multicentre Stop Imatinib (STIM) trial. Lancet Oncol. 2010, 11, 1029–1035.
[CrossRef]
Ross, D.M.; Branford, S.; Seymour, J.F.; Schwarer, A.P.; Arthur, C.; Yeung, D.T.; Dang, P.; Goyne, J.M.;
Slader, C.; Filshie, R.J.; et al. Safety and efficacy of imatinib cessation for CML patients with stable
undetectable minimal residual disease: Results from the TWISTER study. Blood 2013, 122, 515–522. [CrossRef][PubMed]Branford, S.; Yeung, D.T.; Parker, W.T.; Roberts, N.D.; Purins, L.; Braley, J.A.; Altamura, H.K.; Yeoman, A.L.;
Georgievski, J.; Jamison, B.A.; et al. Prognosis for patients with CML and >10% BCR-ABL1 after 3 months ofimatinib depends on the rate of BCR-ABL1 decline. Blood 2014, 124, 511–518. [CrossRef] [PubMed]
Yoshimoto, T.; Mizoguchi, I.; Katagiri, S.; Tauchi, T.; Furusawa, J.I.; Chiba, Y.; Mizuguchi, J.; Ohyashiki, J.H.;
Ohyashiki, K. Immunosurveillance markers may predict patients who can discontinue imatinib therapy
without relapse. Oncoimmunology 2014, 3, e28861. [CrossRef] [PubMed]
Tanaka, M.; Oikawa, K.; Takanashi, M.; Kudo, M.; Ohyashiki, J.; Ohyashiki, K.; Kuroda, M. Down-regulationof miR-92 in human plasma is a novel marker for acute leukemia patients. PLoS ONE 2009, 4, e5532.
[CrossRef] [PubMed]
Ohyashiki, K.; Umezu, T.; Yoshizawa, S.; Ito, Y.; Ohyashiki, M.; Kawashima, H.; Tanaka, M.; Kuroda, M.;
Ohyashiki, J.H. Clinical impact of down-regulated plasma miR-92a levels in non-Hodgkin’s lymphoma.
PLoS ONE 2011, 6, e16408. [CrossRef] [PubMed]
Yoshizawa, S.; Ohyashiki, J.H.; Ohyashiki, M.; Umezu, T.; Suzuki, K.; Inagaki, A.; Iida, S.; Ohyashiki, K.
Downregulated plasma miR-92a levels have clinical impact on multiple myeloma and related disorders.
Blood Cancer J. 2012, 2, e53. [CrossRef] [PubMed]
Tadokoro, H.; Umezu, T.; Ohyashiki, K.; Hirano, T.; Ohyashiki, J.H. Exosomes derived from hypoxic leukemia
cells enhance tube formation in endothelial cells. J. Biol. Chem. 2013, 288, 34343–34351. [CrossRef] [PubMed]
Umezu, T.; Tadokoro, H.; Azuma, K.; Yoshizawa, S.; Ohyashiki, K.; Ohyashiki, J.H. Exosomal miR-135b shedfrom hypoxic multiple myeloma cells enhances angiogenesis by targeting factor-inhibiting HIF-1. Blood 2014,
124, 3748–3757. [CrossRef] [PubMed]
Kosaka, N.; Iguchi, H.; Ochiya, T. Circulating microRNA in body fluid: A new potential biomarker for cancer
diagnosis and prognosis. Cancer Sci. 2010, 101, 2087–2092. [CrossRef] [PubMed]
Ohyashiki, J.H.; Ohtsuki, K.; Mizoguchi, I.; Yoshimoto, T.; Katagiri, S.; Umezu, T.; Ohyashiki, K.
Downregulated microRNA-148b in circulating PBMCs in chronic myeloid leukemia patients with
undetectable minimal residual disease: A possible biomarker to discontinue imatinib safely. Drug Des.
Dev. Ther. 2014, 8, 1151–1159. [CrossRef] [PubMed]
Elvira-Matelot, E.; Zhou, X.O.; Farman, N.; Beaurain, G.; Henrion-Caude, A.; Hadchouel, J.; Jeunemaitre, X.
Regulation of WNK1 expression by miR-192 and aldosterone. J. Am. Soc. Nephrol. 2010, 21, 1724–1731.
[CrossRef] [PubMed]
Georges, S.A.; Biery, M.C.; Kim, S.Y.; Schelter, J.M.; Guo, J.; Chang, A.N.; Jackson, A.L.; Carleton, M.O.;
Linsley, P.S.; Cleary, M.A.; et al. Coordinated regulation of cell cycle transcripts by p53-Inducible microRNAs,
miR-192 and miR-215. Cancer Res. 2008, 68, 10105–10112. [CrossRef] [PubMed]
Jin, Z.; Selaru, F.M.; Cheng, Y.; Kan, T.; Agarwal, R.; Mori, Y.; Olaru, A.V.; Yang, J.; David, S.; Hamilton, J.P.;
et al. MicroRNA-192 and -215 are upregulated in human gastric cancer in vivo and suppress ALCAM
expression in vitro. Oncogene 2011, 30, 1577–1585. [CrossRef] [PubMed]
White, N.M.; Khella, H.W.; Grigull, J.; Adzovic, S.; Youssef, Y.M.; Honey, R.J.; Stewart, R.; Pace, K.T.;
Bjarnason, G.A.; Jewett, M.A.; et al. miRNA profiling in metastatic renal cell carcinoma reveals
a tumour-suppressor effect for miR-215. Br. J. Cancer 2011, 105, 1741–1749. [CrossRef] [PubMed]
Liu, F.; You, X.; Chi, X.; Wang, T.; Ye, L.; Niu, J.; Zhang, X. Hepatitis B virus X protein mutant HBx∆127
promotes proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT. Biochem. Biophys.
Res. Commun. 2014, 444, 128–134. [CrossRef] [PubMed]-150-

Int. J. Mol. Sci. 2016, 17, 570

19.

20.

21.

22.

23.

24.
25.

26.

27.
28.

29.

30.
31.

32.

33.

34.

35.

10 of 11

Senanayake, U.; Das, S.; Vesely, P.; AlzoughbI, W.F.; Fröhlich, L.; Chowdhury, P.; Leuschner, I.; Hoefler, G.;
Guertl, B. miR-192, miR-194, miR-215, miR-200c and miR-141 are downregulated and their common target
ACVR2B is strongly expressed in renal childhood neoplasms. Carcinogenesis 2012, 33, 1014–1021. [CrossRef][PubMed]Mu, J.; Pang, Q.; Guo, Y.H.; Chen, J.G.; Zeng, W.; Huang, Y.J.; Zhang, J.; Feng, B. Functional implicationsof microRNA-215 in TGF-β1-induced phenotypic transition of mesangial cells by targeting CTNNBIP1.
PLoS ONE 2013, 8, e58622. [CrossRef] [PubMed]
Song, B.; Wang, Y.; Titmus, M.A.; Botchkina, G.; Formentini, A.; Kornmann, M.; Ju, J. Molecular mechanismof chemoresistance by miR-215 in osteosarcoma and colon cancer cells. Mol. Cancer 2010, 30, 96. [CrossRef][PubMed]Lipchina, I.; Elkabetz, Y.; Hafner, M.; Sheridan, R.; Mihailovic, A.; Tuschl, T.; Sander, C.; Studer, L.; Betel, D.
Genome-wide identification of microRNA targets in human ES cells reveals a role for miR-302 in modulating
BMP response. Genes Dev. 2011, 25, 2173–2186. [CrossRef] [PubMed]
Skalsky, R.L.; Corcoran, D.L.; Gottwein, E.; Frank, C.L.; Kang, D.; Hafne, M.; Nusbaum, J.D.; Feederle, R.;
Delecluse, H.J.; Luftig, M.A.; et al. The viral and cellular microRNA targetome in lymphoblastoid cell lines.
PLoS Pathog. 2012, 8, e1002484. [CrossRef] [PubMed]
The Database for Annotation, Visualization and Integrated Discovery (DAVIS Bioinformatics Tools v6.7).
Available online: http://david.abcc.ncifcrf.gov (accessed on 13 April 2016).
Grasedieck, S.; Sorrentino, A.; Langer, C.; Buske, C.; Dohner, H.; Mertens, D.; Kuchenbauer, F. Circulating
microRNAs in hematological diseases: Principles, challenges, and perspectives. Blood 2013, 121, 4977–4984.
[CrossRef] [PubMed]
Pichiorri, F.; Suh, S.S.; Rocci, A.; de Luca, L.; Taccioli, C.; Santhanam, R.; Zhou, W.; Benson, D.M.J.;
Hofmainster, C.; Alder, H.; et al. Downregulation of p53-inducible microRNAs 192, 194, and 215 impairs
the p53/MDM2 autoregulatory loop in multiple myeloma development. Cancer Cell 2010, 18, 367–381.
[CrossRef] [PubMed]
Chiang, Y.; Zhou, X.; Wang, Z.; Song, Y.; Liu, Z.; Zhao, F.; Zhu, J.; Xu, H. Expression levels of microRNA-192
and -215 in gastric carcinoma. Pathol. Oncol. Res. 2012, 18, 585–591. [CrossRef] [PubMed]
Gu, L.; Li, H.; Chen, L.; Ma, X.; Gao, Y.; Li, X.; Zhang, Y.; Fan, Y.; Zhang, X. Micro RNAs as prognostic
molecular signatures in renal cell carcinoma: A systematic review and meta-analysis. Oncotarget 2015, 6,32545–32560. [PubMed]
Tong, Y.Q.; Liu, B.; Zheng, H.Y.; Gu, J.; Liu, F.; Tan, B.H.; Hartman, M.; Song, C.; Li, Y. MIR-215, an activatorof the CTNNBIP1/β-catenin pathway, is a marker of poor prognosis in human glioma. Oncotarget 2015, 6,25024–25033. [CrossRef] [PubMed]
Choi, D.H.; Park, S.J.; Kim, H.K. Mir-215 overexpression distinguishes ampullary carcinomas from pancreatic
carcinomas. Hepatobiliary Pancreat. Dis. Int. 2015, 14, 325–329. [CrossRef]
Slattery, M.L.; Ferrick, J.S.; Mullany, L.E.; Valeri, N.; Stevens, J.; Caan, B.J.; Samowitz, W.; Wolff, R.K.
An evaluation and replication of miRNAs with disease stage and colorectal cancer-specific mortality.
Int. J. Cancer 2015, 137, 428–438. [CrossRef] [PubMed]
Deng, Y.; Huang, Z.; Xu, Y.; Jin, J.; Zhuo, W.; Zhang, C.; Zhang, X.; Shen, M.; Yan, X.; Wang, L.; et al. miR-215
modulates gastric cancer cell proliferation by targeting RB1. Cancer Lett. 2014, 342, 27–35. [CrossRef][PubMed]Hsu, S.D.; Lin, F.M.; Wu, W.Y.; Liang, C.; Huang, W.C.; Chan, W.L.; Tsai, W.T.; Chen, G.Z.; Lee, C.J.;
Chiu, C.M.; et al. miRTarBase: A database curates experimentally validated microRNA-target interactions.
Nucleic Acids Res. 2011, 39, gkq107. [CrossRef] [PubMed]
Braun, C.J.; Zhang, X.; Savelyeva, I.; Wolff, S.; Moll, U.M.; Schepeler, T.; Ørntoft, T.F.; Andersen, C.L.;
Dobbelstein, M. p53-Responsive micrornas 192 and 215 are capable of inducing cellcycle arrest. Cancer Res.
2008, 68, 10094–10104. [CrossRef] [PubMed]
Skottman, H.; Mikkola, M.; Lundin, K.; Olsson, C.; Strömberg, A.M.; Tuuri, T.; Otonkoski, T.; Hovatta, O.;
Lahesmaa, R. Gene expression signatures of seven individual human embryonic stem cell lines. Stem Cells
2005, 23, 1343–1356. [CrossRef] [PubMed]-151-

Int. J. Mol. Sci. 2016, 17, 570

36.
37.

11 of 11Pera, M.F.; Tam, P.P. Extrinsic regulation of pluripotent stem cells. Nature 2010, 465, 713–720. [CrossRef][PubMed]
The experimentally-validated miRNA-target interactions database (miRTarBase). Available online:
http://mirtarbase.mbc.nctu.edu.tw/ (accessed on 13 April 2016).
© 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open accessarticle distributed under the terms and conditions of the Creative Commons Attribution(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).

-152-

Citation: Molecular Therapy—Nucleic Acids (2016) 5, e292; doi:10.1038/mtna.2016.1
Official journal of the American Society of Gene & Cell Therapy All rights reserved 2162-2531/16
www.nature.com/mtna

Topical Use of Angiopoietin-like Protein 2
RNAi-loaded Lipid Nanoparticles Suppresses
Corneal Neovascularization
Yukako Taketani1, Tomohiko Usui1, Tetsuya Toyono1, Nobuyuki Shima1, Seiichi Yokoo1, Mikiko Kimakura1, Satoru Yamagami1,
Shinichiro Ohno2, Risako Onodera3, Kohei Tahara3, Hirofumi Takeuchi3 and Masahiko Kuroda2

Corneal neovascularization (CNV) is a sight-threatening condition that is encountered in various inflammatory settings including
chemical injury. We recently confirmed that angiopoietin-like protein 2 (ANGPTL2) is a potent angiogenic and proinflammatory
factor in the cornea, and we have produced a single-stranded proline-modified short hairpin anti-ANGPTL2 RNA interference
molecule that is carried in a lipid nanoparticle (ANGPTL2 Li-pshRNA) for topical application. In this study, we have further
examined the topical delivery and anti-ANGPTL2 activity of this molecule and have found that fluorescence-labeled ANGPTL2
Li-pshRNA eye drops can penetrate all layers of the cornea and that ANGPTL2 mRNA expression was dramatically inhibitedin both epithelium and stroma at 12 and 24 hours after administration. We also examined the inhibitory effect of ANGPTL2
Li-pshRNA on CNV in a mouse chemical injury model and found that the area of angiogenesis was significantly decreased incorneas treated with ANGPTL2 Li-pshRNA eye drops compared to controls. Together, these findings indicate that this modified
RNA interference agent is clinically viable in a topical formulation for use against CNV.
Molecular Therapy—Nucleic Acids (2016) 5, e292; doi:10.1038/mtna.2016.1; published online 8 March 2016
Subject Category: nanoparticles

Introduction
Corneal neovascularization (CNV) is a sight-threatening
condition that is encountered in various inflammatory settings including chemical injury. CNV can lead to corneal
scarring, edema, and lipid deposition that may compromise
visual function and decrease the success rate of subsequent
penetrating keratoplasty.1 Topical corticosteroids have been
the mainstay for suppressing CNV.2 However, the inhibitory
effects of corticosteroids are limited and may be accompanied by adverse effects including delayed wound healing,
infection, posterior subcapsular cataracts, and elevationof intraocular pressure. As an alternative, antibody-based
anti-vascular endothelial growth factor molecular targetingtherapy has recently become available for the treatment of
CNV.3,4 However, this treatment also has several drawbacks,
including high costs and questionable efficacy in terms ofvessel regression and repair of epithelial defects.5
RNA interference (RNAi) occurs when small interfering
RNA (siRNA) molecules, in association with a nucleolytic
cytoplasmic protein complex, mediate sequence-specificdegeneration of target RNA molecules. Theoretically, thismechanism can silence the expression of virtually any
gene in a sequence-directed manner with high specificity.
The potential clinical applications of this phenomenon are
now being explored.
Angiopoietin-like protein 2 (ANGPTL2) is a member of theangiopoietin-like protein family and is essential for various
physiologic activities, including lipid metabolism, angiogenesis, and inflammation.6,7 Kanda et al. have reported that

ANGPTL2 in the ocular tissue was related to acute inflammation in the retina in an endotoxin-induced uveitis model via
activation of an NF-κB signaling pathway, and they indicatedthat ANGPTL2 could be a therapeutic target in acute inflammatory disease.8 In addition, we have previously found that
ANGPTL2 is a potent proangiogenic factor in mouse corneal
inflammation and that subconjunctival injection of ANGPTL2siRNA in mice dramatically suppressed local expression of
ANGPTL2 mRNA and reduced the extent of CNV.9 This finding represents a new approach to the treatment of angiogenesis-associated corneal diseases. However, it is not ideal toperform repeated subconjunctival injections in the clinicalsetting, and a common problem with RNAi agents has been
the lack of an effective in vivo drug delivery system. Moreover, because siRNAs are double stranded, they might act
as ligands for Toll-like receptor 3,10 which may induce innate
immune reactions.
To address these problems, we generated a singlestranded RNAi that can be delivered in a lipid nanoparticle
for use as a topical anti-ANGPTL2 agent in the treatment ofocular diseases. In this study, we have examined its efficacy
in terms of drug delivery and inhibition of CNV in a mousealkali-burn model.
Results
Safety of proline-modified short hairpin RNA-loaded
lipid nanoparticles in vitro
Cell viability was examined using simian virus 40-immortalized human corneal epithelial cells. There was no significant

1
Department of Ophthalmology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; 2Department of Molecular Pathology, Tokyo Medical University,
Tokyo, Japan; 3Laboratory of Pharmaceutical Engineering, Gifu Pharmaceutical University, Gifu, Japan. Correspondence: Tomohiko Usui, Department of Ophthalmology,
Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: tomohiko-tky@umin.ac.jp
Keywords: angiogenesis; cornea; lipid nanoparticles; topical RNAi eye drops
Received 25 September 2015; accepted 20 December 2015; published online 8 March 2016. doi:10.1038/mtna.2016.1-153-

Topical RNAi Suppresses Corneal Neovascularization
Taketani et al.

2**

Cell viability (saline = 100%)

1,000%

100%

10%

1%
SalineANGPTL2
pshRNAANGPTL2
Li-pshRNA

Negative
pshRNA

Negative
Li-pshRNA

Amphotericin

Figure 1 WST-1 assay of Li-pshRNA. WST-1 assay was performed to confirm cell viability after treatment with Li-pshRNA. There was no
significant difference in cell viability among the treatment groups (n = 4, **P < 0.01). ANGPTL2, angiopoietin-like protein 2; Li-pshRNA, prolinemodified short hairpin RNA wrapped with liposome.

a
Epi
St

Epi

Endo

St
Endo

b

Time posteye drops

Control

Li-pshRNA

1 hour200 µm200 µ m200 µ m200 µm200 µm200 µ m3 hours

6 hours

Figure 2 Delivery of fluorescence-labeled pshRNA eye drops. (a) Left panel: bare siRNA with fluorescent label; right panel: fluorescencelabeled pshRNA enclosed in the lipid nanoparticle. The pshRNA was distributed only in the superficial layer of the corneal epithelium, while theLi-pshRNA was observed in almost all layers. (b) Delivery of fluorescence-labeled Li-pshRNA drops to mouse eyes. Samples were extracted at
1, 3, and 6 hours after instillation of the eye drops. Left panel: saline as control. Right panel: fluorescence-labeled Li-pshRNA. The Li-pshRNA
was distributed to all layers as time advanced. Epi, epithelium; Endo, endothelium of cornea; St, stroma. Li-pshRNA, proline-modified short
hairpin RNA wrapped with liposome.
Molecular Therapy—Nucleic Acids-154-

Topical RNAi Suppresses Corneal Neovascularization
Taketani et al.

3difference in posttreatment viability among five groups (control group, negative proline-modified short hairpin RNA(pshRNA) group, ANGPTL2 pshRNA group, negative
pshRNA wrapped with liposome (Li-pshRNA), and ANGPTL2 Li-pshRNA group), and cell viability in all five groups
was greater than that of cells treated with amphotericin
B (P < 0.01 for comparison, Figure 1).
Drug delivery and efficacy in normal mouse eyes
Fluorescence-labeled siRNA was distributed only in the
superficial layer of the corneal epithelium, while fluorescence-labeled Li-pshRNA showed almost full-thickness distribution (Figure 2a) and was detected in the stroma andthe endothelium at 3 and 6 hours after instillation of the eye
drops (Figure 2b).
ANGPTL2 mRNA expression was strongly inhibited in bothepithelial and stromal cells at 12 and 24 hours (P = 0.049 and<0.001, respectively) after instillation of eye drops in mice,but normal expression of ANGPTL2 mRNA was restored by
72 hours (Figure 3, n = 7 for all experiments). In contrast,
there was no change in ANGPTL2 mRNA expression in thecorneas of control mice (Figure 3).

Relative mRNA expression of ANGPTL2

a
*
1e+01

**

1e−01

1e−03

1e−05
Control

Negative 12 hours
Li-pshRNA

24 hours

48 hours

72 hours

48 hours

72 hours

Relative mRNA expression of ANGPTL2

b
50.00

*

*

5.00

0.50

0.05
0.01
Control

Negative 12 hours
Li-pshRNA

24 hours

Figure 3 Reduction of ANGPTL2 mRNA expression in normal
cornea after topical administration of ANGPTL2 Li-pshRNA.
Reverse transcriptase PCR showed that ANGPTL2 mRNA
expression was significantly reduced in the corneal epithelium (a)at 12 and 24 hours (P = 0.049 and <0.001, respectively; n = 7) andin stromal cells (b) at 12 and 24 hours (P = 0.0030 and 0.0021,
respectively, n = 7). Expression returned to baseline levels by 72
hours. *P < 0.05, **P < 0.01. ANGPTL2, angiopoietin-like protein
2; Li-pshRNA, proline-modified short hairpin RNA wrapped withliposome.

CNV in alkali-burn models
The effect of Angptl2 Li-pshRNA on CNV after alkali injury
was investigated. Drug delivery in the alkali model was also
evaluated by fluorescence assay, which confirmed that ANGPTL2 Li-pshRNA was distributed in all layers of the injured
cornea, while ANGPTL2 pshRNA alone was not (­ Figure 4a).
Expression of ANGPTL2 mRNA was inhibited at 36 hours
after alkali injury by topical treatment with ANGPTL2 LipshRNA, but not by topical administration of negative LipshRNA or by pshRNA alone (P = 0.043, n = 6; Figure 4b).
However, there was no significant difference in the levelsof expression of ANGPTL2 mRNA expression between the
ANGPTL2 Li-pshRNA group and the control group at 48
hours after alkali injury (Figure 4c). After 10 days of oncedaily treatment, the area of CNV was markedly reduced in
medicated eyes compared to the controls (P < 0.01, n = 7;
Figure 5).
Discussion
Several researchers have evaluated the therapeutic potential of gene silencing by RNAi for CNV in animal models.
Kim et al., using a corneal micro-pocket assay and herpetic
stromal keratitis models in mice, reported that the subconjunctival injections and systemic administration of vascularendothelial growth factor pathway-specific siRNAs inhibited
CNV,11 and Qazi et al. reported that intrastromal injection ofpoly nanoparticles loaded with plasmid-expressing anti-vascular endothelial growth factor siRNA also inhibited CNV ina more sustained and robust manner than the naked plasmid alone.12 Thus, the concept of using RNAi agents against
CNV seems promising. However, the invasiveness of subconjunctival or intrastromal injections could pose a hurdle torepeated treatments. A topical formulation would be a moredesirable and convenient approach. In this study, we haveshown that effective topical delivery of a modified RNAi agent(ANGPTL2 Li-pshRNA) can be accomplished using a lipid
nanoparticle, and that this agent reduced the expression of
ANGPTL2 mRNA and inhibited CNV in a mouse alkali-burnmodel.
It is crucial to confirm the safety of any therapy before clinical application. RNAi is generally thought to have minimal
cellular toxicity because of its endogenous nature, and in aprevious study, the intravitreal injection of RNAi did not cause
any obvious adverse effects in mouse retina.13 In the present
study, Li-pshRNA did not affect cell viability ­(Figure 1), and webelieve that the highly biocompatible nature of RNAi-loaded
lipid nanoparticles could be ideal for clinical application.
We used single-stranded RNAi (pshRNA) because double-stranded siRNAs have been associated with adverse off-­target effects due to activation of innate immunity, specifically
due to Toll-like receptor 3 activation.10 Further, annealing is
mandatory for generation of double-stranded siRNA, but notfor pshRNA, which is a single-stranded RNAi in which thelinker region has been replaced by amino acid derivatives.
This substitution and the short hairpin construct exhibit highstability in vivo and preliminary data (not shown) suggest that
pshRNA is less susceptible to degeneration due to RNase
activity. Because annealing is not necessary to generatewww.moleculartherapy.org/mtna-155-

Topical RNAi Suppresses Corneal Neovascularization
Taketani et al.

4a1 hour3 hours
200 µ m200 µ m

b

c
6

*

5

Relative mRNA expression of ANGPTL2

Relative mRNA expression of ANGPTL2

6

*
4

3

2

1

0

5

4

3

2

1

0
Control

Negative
Li-pshRNA

ANGPTL2
Li-pshRNA

Control

Negative
Li-pshRNA

ANGPTL2
Li-pshRNA

Figure 4 Inhibition of ANGPTL2 mRNA by ANGPTL2 Li-pshRNA eye drops after chemical injury. (a) Fluorescence-labeled topical
ANGPTL2 Li-pshRNA permeated the entire thickness of the injured cornea at 1 and 3 hours after injury. (b) ANGPTL2 mRNA expression was
reduced after topical administration of ANGPTL2 Li-pshRNA eye drops 36 hours after injury (P = 0.017 and 0.032 for saline as control andnegative Li-pshRNA, respectively; n = 6). ANGPTL2 mRNA expression was not inhibited by negative Li-pshRNA. *P < 0.05. (c) At 48 hours
after injury, levels of ANGPTL2 expression were the same among all three groups. ANGPTL2, angiopoietin-like protein 2; Li-pshRNA, prolinemodified short hairpin RNA wrapped with liposome.

the pshRNA, the cost can be also reduced. Moreover, previous research showed that pshRNA did not have off-target
effects and it was more stable against nuclease than doublestranded RNAi constructs.14 Thus, pshRNA overcomes many
disadvantages hitherto encountered with double-stranded
siRNAs.
The corneal epithelium contains tight junctions that forma barrier to paracellular drug permeation of the inner layerof the cornea in vivo. The corneal epithelium is lipoidal innature, which poses considerable resistance to permeation
by topically administered hydrophilic agents,15 as demonstrated by the failure of unmodified pshRNA, which is hydrophilic, to penetrate the epithelial surface in our experiments(Figure 2). The charge of a drug also affects permeation.
Positively charged molecules exhibit poor permeation, presumably because they bind to the negatively charged corneal and scleral proteoglycan matrix.16 To overcome this, thepshRNA was loaded into lipid nanoparticles (Li-pshRNA) thatwere specifically designed to facilitate drug delivery to theretina.17 In this study, we showed that ANGPTL2 Li-pshRNA
could reduce in vivo corneal ANGPTL2 mRNA expression(Figures 3 and 4). This result and others, for example, thatthe topical administration of edaravone-loaded lipid nanoparticles can protect against light-induced retinal dysfunction,18
support the potential usefulness of lipid nanoparticle-loaded
topical agents in the treatment of corneal diseases.
We found that the marked reduction in the level of ANGPTL2 mRNA was not sustained beyond 48 hours after alkali
injury. Even in the control group, ANGPTL2 expression
dropped to the same level as that of GAPDH, suggesting that
ANGPTL2 is produced in the very early stage of inflammation. As alkali damage progresses, the cells are not able toproduce as much ANGPTL2, and it becomes more difficult to
detect the effects of ANGPTL2 RNAi.
In conclusion, extensive corneal injuries such as chemicalburns cause conjunctivalization of the corneal surface andmassive neovascularization, leading to corneal opacity andsevere reduction in and visual acuity. We have successfully
generated a topical RNAi agent (Li-pshRNA) that inhibits
corneal inflammation and CNV by inhibiting expression of
ANGPTL2. Li-pshRNA eye drops show promise for preventing CNV and subsequent visual impairment without the side
effects commonly experienced with steroids or immunosuppressive agents.

Molecular Therapy—Nucleic Acids-156-

Topical RNAi Suppresses Corneal Neovascularization
Taketani et al.

5

a

d

b

Control

Negative Li-pshRNAe Negative Li-pshRNA

Control

2.0 mmg

Statement for the Use of Animals in Ophthalmic and Vision
Research. All surgical procedures were performed underketamine hydrochloride (35 mg/kg) and xylazine chloride(5 mg/kg) anesthesia, and all efforts were made to minimize
suffering. C57BL/6 mice aged 8–12 weeks were used in all
experiments.

ANGPTL2 Li-pshRNAf ANGPTL2 Li-pshRNA

2.0 mm

2.0 mm**45%
Percent are of angiogenesis in cornea

c

**

40%
35%
30%
25%
20%
15%
10%
5%
0%
Control

Negative
Li-pshRNA

ANGPTL2
Li-pshRNA

Figure 5 Inhibition of corneal neovascularization by ANGPTL2
Li-psh RNA eye drops. Corneal neovascularization after alkali injury wasmarkedly reduced after 10 days of once-daily treatment with ANGPTL2
Li-pshRNA eye drops (c) compared to eyes treated with control (a) or
negative Li-pshRNA (b). Corneas treated with ANGPTL2 Li-pshRNA
showed smaller areas of neovascularization (f) compared to control (d)and negative Li-pshRNA (e) corneas. The samples were stained with
CD31 to visualize the neovascularization. (g) Corneal neovascularization(CNV) was quantified by area (vascularized area/total cornea area) using
image J software. The extent of CNV after ANGPTL2 treatment wassignificantly smaller compared to the other groups (P < 0.01, n = 9). **P <0.01. ANGPTL2, angiopoietin-like protein 2; Li-pshRNA, proline-modified
short hairpin RNA wrapped with liposome.

Figure 6 ANGPTL2 pshRNA platform. PshRNA was synthesized
in solid phase as single-stranded RNAs that self-anneal after
synthesis into a helical structure containing a single stem loop. Red
characters indicated guide sequence, and green characters showed
nucleotide cassette sequence of pshRNA. ANGPTL2, angiopoietinlike protein 2; pshRNA, proline-modified short hairpin RNA.
A, adenine; C, cytosine; G, guanine; U, uracil; P, proline derivative.

Materials and methods
Animals. All animal experiments were approved by the University of Tokyo Hospital Animal care committee and conformed
to the Association for Research in Vision and Ophthalmology

Reagents. The ANGPTL2-specific single-stranded RNAisegment was contained in a short hairpin structure (ANGPTL2 pshRNA) with a stem loop, which was synthesized in
the solid phase.14 The stem loop contained proline derivatives similar to PnkRNA, as previously described.19 The basesequence of the ANGPTL2 pshRNA was 5′-CCA GAA AGC
GAG UAC UAU AUU CC proline modified-3′, 5′-GGA AUAUAF UAC UCG CUU UCU GGU U-3′ (structure shown in
Figure 6), and the negative control was 5′-UAC UAU UCGACA CGC GAA GUU CC proline modified-3′, 5′-GGA ACUUCG CGU GUC GAA UAG UAU U-3′. The reagents were
supplied by BONAC (Fukuoka, Japan). The linker region of
the pshRNA was replaced by a fluorescent pigment for drugdelivery experiments.
The lipid nanoparticle was prepared as described previously.17
1,2-Distearoyl-sn-glycero-3-phosphocholine,
cholesterol,and stearylamine (7:3:1 molar ratio, lipid concentrations: 20
mmol/l) were dissolved in a small amount of chloroform anddried in a rotary evaporator under reduced pressure at 40°C to form a thin lipid film. The film was dried in a vacuum
oven overnight to ensure complete removal of the solvent.
After hydration of the film with a suitable amount of Tris–­
ethylenediaminetetraacetic acid buffer containing RNAi, theresultant lipid suspension was incubated in a water bath at
70 °C. The multilamellar vesicle suspension was incubated atroom temperature for 15 minutes. Submicron-sized liposomes
were prepared using an extruder (LipoFast-­Pneumatic, Avestin, Ottawa, Ontario, Canada) equipped with a polycarbonate membrane (0.1-µm membrane filter pore size; Whatman
Japan KK, Tokyo, Japan). Extrusion was performed 41 times
with nitrogen pressure (200 psi). The final phospholipid andRNAi concentrations in liposomal suspensions were 20
mmol/l and 0.5 or 1 mol/l, respectively.
Cell toxicity of Li-pshRNA in vitro. Cellular viability wasevaluated using a nonradioactive colorimetric assay (WST1; TAKARA Bio, Shiga, Japan) according to the manufacturer’s protocol. Simian virus 40-immortalized human corneal
epithelial cells were cultured in Dulbecco’s modified Eagle
medium (Wako Pure Chemical Industry, Osaka, Japan)/F12
containing 10% fetal bovine serum supplemented with cholera toxin (100 ng/ml), human recombinant epidermal growth
factor (10 µg/ml), and insulin (5 µg/ml). After the culture
reached confluence, ANGPTL2 Li-pshRNA, negative control
Li-pshRNA, Amphotericin B (0.1% Fungizone, Bristol-Myers,
Tokyo, Japan), or saline was applied for 30 minutes followed
by WST-1 reagent in phenol red-free Dulbecco’s modified
Eagle medium/F12 medium for 1 hour at 37 °C, and the dye
was measured at 450 nm by a Victor 3V Multilabel Countermodel 1420 plate reader (Perkin Elmer, Waltham, MA). The
results were expressed as the mean ± SD of the percentage of cell viability compared with that of the control-treatedcontrols.
www.moleculartherapy.org/mtna-157-

Topical RNAi Suppresses Corneal Neovascularization
Taketani et al.

6
Table 1 Quantitative reverse transcriptase PCR primersGenesymbolForward 5′–3′Reverse 5′–3′

MusAACCATGGAGGACTGGTCTG
ANGPTL2TGAGGTTGGAGTGAGCACAG

MusGAPDH

AACTTTGGCATTGTGGAAGGCACATTGGGGGTAGGAACAC

Drug delivery to the cornea. Drops of Li-pshRNA labeledwith fluorescent pigment (5 µl) were instilled on the corneal
surface of anesthetized mice, after which the eyes wereenucleated. Specimens were embedded in optimal cutting
temperature compound (Sakura Finetechnical, Tokyo, Japan)
after 1, 3, and 6 hours. The samples were immediately frozen
in liquid nitrogen and cut into 10-µm sections. Corneal distribution of the labeled pshRNA was examined by fluorescencemicroscopy (BZ-9000; Keyence, Osaka, Japan). The sameprocedure was performed in the alkali-burn models withdrops instilled 1, 3, and 6 hours after alkali burn.
To examine changes in mRNA expression after administration of the Li-pshRNA RNAi agent, 5 µl of ANGPTL2 LipshRNA was instilled on the corneal surface in anesthetized
mice. Eyes were enucleated at 12, 24, 48, and 72 hours afteradministration, and the corneal epithelium was separated
from the corneal stroma using trypsin-ethylenediaminetetraacetic acid. The total RNA of each sample was extracted
and ANGPTL2 expression was quantified by quantitative
real-time reverse transcriptase PCR.
Real-time reverse transcriptase PCR. cDNA was synthesizedusing PrimerScript RT Master Mix (TAKARA Bio). The PCR
amplifications were performed in an instrument using Takara
PCR Thermal Cycler Dice (TAKARA Bio) as described previously.9 The primer sets are shown in Table 1.
Mouse model of alkali-induced inflammatory CNV. The
mouse alkali-burn models were produced as previously
described20–22 with modifications to suit our experiment.
Chemical injury was induced by placing 5 µl of 0.5 N sodiumhydroxide (NaOH) on the cornea of anesthetized mice for
30 seconds. The eyes were then washed with 20 ml saline(day 0). On day 1, the injured eyes were treated with 5 µl of
ANGPTL2 Li-pshRNA or negative Li-pshRNA for 5 minutes,
after which reagents were absorbed (KimWipe: Nikkei Products, Osaka, Japan) and eyes were subsequently treatedwith ofloxacin ophthalmic ointment (Talibit, Santen, Osaka,
Japan). The RNAi reagent and antibiotic treatments wererepeated daily until day 10, then the mice were euthanized
and the cornea with limbus was excised.
CNV was quantified using CD31 staining as previously
described.9 Briefly, corneas were fixed in acetone for 10 minutes at −20 °C, washed three times in phosphate-bufferedsaline, stained with rat anti-mouse CD31 antibody (BD Biosciences, Franklin Lakes, NJ), and incubated at 4 °C overnight.
The samples were washed again with phosphate-bufferedsaline and stained with the secondary antibody (Alexa Fluor
594 donkey anti-rat IgG, Invitrogen, San Diego, CA) for 5
hours at room temperature. The stained samples were evaluated by fluorescence microscopy (BZ-9000, Keyence), andthe CD31-positive blood vessels were quantified by area
using Image J software (http://rsb.info.nih. gov/ij/). The results
are expressed as vessel area/total field.
To examine changes in mRNA expression in our model,
the corneas were extracted 36 and 48 hours after injury, andquantitative real-time reverse transcriptase PCR of ANGPTL2 was performed for each group (control, negative LipshRNA, and ANGPTL2 Li-pshRNA) using the conditions
described above.
Statistical analysis. All of the data were analyzed by Mann–
Whitney U-test using statistical software R (Ver. 3.1.1), andvalues of P < 0.05 were considered statistically significant
after Holm’s correction.
Acknowledgments This work has been supported by agrant-in-aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology.
We would like to specially thank Azusa Kamikawa, assistantresearcher, for her assistance in conducting our experiments.
This work was done in Tokyo, Japan.
1. Bachmann, B, Taylor, RS and Cursiefen, C (2010). Corneal neovascularization as a risk
factor for graft failure and rejection after keratoplasty: an evidence-based meta-analysis.
Ophthalmology 117: 1300–5.
2. Epstein, RJ, Stulting, RD, Hendricks, RL and Harris, DM (1987). Corneal
neovascularization. Pathogenesis and inhibition. Cornea 6: 250–257.
3. Petsoglou, C, Balaggan, KS, Dart, JK, Bunce, C, Xing, W, Ali, RR et al. (2013).
Subconjunctival bevacizumab induces regression of corneal neovascularisation: a pilot
randomised placebo-controlled double-masked trial. Br J Ophthalmol 97: 28–32.
4. Chang, JH, Garg, NK, Lunde, E, Han, KY, Jain, S and Azar, DT (2012). Corneal
neovascularization: an anti-VEGF therapy review. Surv Ophthalmol 57: 415–429.
5. Kim, TI, Chung, JL, Hong, JP, Min, K, Seo, KY and Kim, EK (2009). Bevacizumab
application delays epithelial healing in rabbit cornea. Invest Ophthalmol Vis Sci 50:
4653–4659.
6. Kadomatsu, T, Endo, M, Miyata, K and Oike, Y (2014). Diverse roles of ANGPTL2 inphysiology and pathophysiology. Trends Endocrinol Metab 25: 245–254.
7. Thorin-Trescases, N and Thorin, E (2014). Angiopoietin-like-2: a multifaceted protein withphysiological and pathophysiological properties. Expert Rev Mol Med 16: e17.
8. Kanda, A, Noda, K, Oike, Y and Ishida, S (2012). Angiopoietin-like protein 2 mediates
endotoxin-induced acute inflammation in the eye. Lab Invest 92: 1553–1563.
9. Toyono, T, Usui, T, Yokoo, S, Kimakura, M, Nakagawa, S, Yamagami, S et al. (2013).
Angiopoietin-like protein 2 is a potent hemangiogenic and lymphangiogenic factor incorneal inflammation. Invest Ophthalmol Vis Sci 54: 4278–4285.
10. Kleinman, ME, Yamada, K, Takeda, A, Chandrasekaran, V, Nozaki, M, Baffi, JZ et al.
(2008). Sequence- and target-independent angiogenesis suppression by siRNA via TLR3.
Nature 452: 591–597.
11. Kim, B, Tang, Q, Biswas, PS, Xu, J, Schiffelers, RM, Xie, FY et al. (2004). Inhibition ofocular angiogenesis by siRNA targeting vascular endothelial growth factor pathway genes:
therapeutic strategy for herpetic stromal keratitis. Am J Pathol 165: 2177–2185.
12. Qazi, Y, Stagg, B, Singh, N, Singh, S, Zhang, X, Luo, L et al. (2012). Nanoparticlemediated delivery of shRNA.VEGF-a plasmids regresses corneal neovascularization.
Invest Ophthalmol Vis Sci 53: 2837–2844.
13. Nakama, T, Yoshida, S, Ishikawa, K, Kobayashi, Y, Zhou, Y, Nakao, S et al. (2015).
Inhibition of choroidal fibrovascular membrane formation by new class of RNA interference
therapeutic agent targeting periostin. Gene Ther 22: 127–137.
14. Takanashi, M, Sudo, K, Ueda, S, Ohno, S, Yamada, Y, Osakabe, Y et al. (2015). Noveltypes of small RNA exhibit sequence- and target-dependent angiogenesis suppression
without activation of Toll-like receptor 3 in an age-related macular degeneration (AMD)mouse model. Mol Ther Nucleic Acids 4: e258.
15. Gaudana, R, Ananthula, HK, Parenky, A and Mitra, AK (2010). Ocular drug delivery. AAPS
J 12: 348–360.
16. Kim, SH, Lutz, RJ, Wang, NS and Robinson, MR (2007). Transport barriers in transscleraldrug delivery for retinal diseases. Ophthalmic Res 39: 244–254.
17. Murata, M, Yonamine, T, Tanaka, S, Tahara, K, Tozuka, Y and Takeuchi, H (2013). Surface
modification of liposomes using polymer-wheat germ agglutinin conjugates to improve theabsorption of peptide drugs by pulmonary administration. J Pharm Sci 102: 1281–1289.
18. Shimazaki, H, Hironaka, K, Fujisawa, T, Tsuruma, K, Tozuka, Y, Shimazawa, M et al.
(2011). Edaravone-loaded liposome eyedrops protect against light-induced retinal damage
in mice. Invest Ophthalmol Vis Sci 52: 7289–7297.

Molecular Therapy—Nucleic Acids-158-

Topical RNAi Suppresses Corneal Neovascularization
Taketani et al.

7
19. Hamasaki, T, Suzuki, H, Shirohzu, H, Matsumoto, T, D’Alessandro-Gabazza, CN, GilBernabe, P et al. (2012). Efficacy of a novel class of RNA interference therapeutic agents.
PLoS One 7: e42655.
20. Sotozono, C, He, J, Matsumoto, Y, Kita, M, Imanishi, J and Kinoshita, S (1997). Cytokine
expression in the alkali-burned cornea. Curr Eye Res 16: 670–676.
21. Zhou, SY, Xie, ZL, Xiao, O, Yang, XR, Heng, BC and Sato, Y (2010). Inhibition of mousealkali burn induced-corneal neovascularization by recombinant adenovirus encoding
human vasohibin-1. Mol Vis 16: 1389–1398.
22. Anderson, C, Zhou, Q and Wang, S (2014). An alkali-burn injury model of corneal
neovascularization in the mouse. J Vis Exp (86), e51159, doi:10.3791/51159 (2014).

This work is licensed under a Creative Commons
Attribution-NonCommercial-NoDerivs 4.0 International
License. The images or other third party material in this article areincluded in the article’s Creative Commons license, unless indicatedotherwise in the credit line; if the material is not included under theCreative Commons license, users will need to obtain permission
from the license holder to reproduce the material. To view a copy ofthis license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/

www.moleculartherapy.org/mtna-159-

RESEARCH ARTICLE

Promotion of Expansion and Differentiationof Hematopoietic Stem Cells by Interleukin27 into Myeloid Progenitors to Control
Infection in Emergency Myelopoiesis
Jun-ichi Furusawa1, Izuru Mizoguchi1, Yukino Chiba1, Masayuki Hisada2,
Fumie Kobayashi3, Hiroki Yoshida4, Susumu Nakae5, Akihiko Tsuchida2,
Tetsuya Matsumoto6, Hideo Ema7, Junichiro Mizuguchi8, Takayuki Yoshimoto1*
1 Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan,
2 Department of Gastrointestinal and Pediatric Surgery, Tokyo Medical University, Tokyo, Japan,
3 Department of Infectious Diseases, Kyorin University of Medicine, Tokyo, Japan, 4 Department of
Biomolecular Sciences, Faculty of Medicine, Saga University, Saga, Japan, 5 Laboratory of Systems
Biology, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University ofTokyo, Tokyo, Japan, 6 Department of Microbiology, Tokyo Medical University, Tokyo, Japan, 7 Department
of Cell Differentiation, The Sakaguchi Laboratory of Developmental Biology, Keio University School of
Medicine, Tokyo, Japan, 8 Department of Immunology, Tokyo Medical University, Tokyo, Japan
OPEN ACCESS
Citation: Furusawa J-i, Mizoguchi I, Chiba Y, Hisada
M, Kobayashi F, Yoshida H, et al. (2016) Promotion of
Expansion and Differentiation of Hematopoietic StemCells by Interleukin-27 into Myeloid Progenitors toControl Infection in Emergency Myelopoiesis. PLoS
Pathog 12(3): e1005507. doi:10.1371/journal.
ppat.1005507
Editor: Mary M. Stevenson, McGill University,
CANADA
Received: August 17, 2015
Accepted: February 24, 2016
Published: March 18, 2016
Copyright: © 2016 Furusawa et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source arecredited.
Data Availability Statement: All relevant data arewithin the paper and its Supporting Information files.
Funding: This study was supported in part by
Grants-in-Aid for Scientific Research (B) (TY),
Scientific Research on innovative areas (TY), andYoung Scientists (Start-up) (JF) from the Ministry of
Education, Culture, Sports, Science, and Technology,
Japan. The funders had no role in study design, datacollection and analysis, decision to publish, or
preparation of the manuscript.

* yoshimot@tokyo-med.ac.jp

Abstract
Emergency myelopoiesis is inflammation-induced hematopoiesis to replenish myeloid cells
in the periphery, which is critical to control the infection with pathogens. Previously, proinflammatory cytokines such as interferon (IFN)-α and IFN-γ were demonstrated to play a
critical role in the expansion of hematopoietic stem cells (HSCs) and myeloid progenitors,
leading to production of mature myeloid cells, although their inhibitory effects on hematopoiesis were also reported. Therefore, the molecular mechanism of emergency myelopoiesis
during infection remains incompletely understood. Here, we clarify that one of the interleukin(IL)-6/IL-12 family cytokines, IL-27, plays an important role in the emergency myelopoiesis.
Among various types of hematopoietic cells in bone marrow, IL-27 predominantly and continuously promoted the expansion of only Lineage−Sca-1+c-Kit+ (LSK) cells, especiallylong-term repopulating HSCs and myeloid-restricted progenitor cells with long-term repopulating activity, and the differentiation into myeloid progenitors in synergy with stem cell factor. These progenitors expressed myeloid transcription factors such as Spi1, Gfi1, andCebpa/b through activation of signal transducer and activator of transcription 1 and 3, andhad enhanced potential to differentiate into migratory dendritic cells (DCs), neutrophils, andmast cells, and less so into macrophages, and basophils, but not into plasmacytoid DCs,
conventional DCs, T cells, and B cells. Among various cytokines, IL-27 in synergy with thestem cell factor had the strongest ability to augment the expansion of LSK cells and their differentiation into myeloid progenitors retaining the LSK phenotype over a long period of time.
The experiments using mice deficient for one of IL-27 receptor subunits, WSX-1, and IFN-γ
revealed that the blood stage of malaria infection enhanced IL-27 expression through IFN-γ

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 2016

1 / 25-160-

Role of IL-27 in Emergency Myelopoiesis

Competing Interests: The authors have declaredthat no competing interests exist.

production, and the IL-27 then promoted the expansion of LSK cells, differentiating andmobilizing them into spleen, resulting in enhanced production of neutrophils to control theinfection. Thus, IL-27 is one of the limited unique cytokines directly acting on HSCs to promote differentiation into myeloid progenitors during emergency myelopoiesis.

Author Summary
Emergency myelopoiesis is inflammation-induced hematopoiesis that is critical for controlling infection with pathogens, but the molecular mechanism remains incompletely
understood. Here, we clarify that one of the interleukin (IL)-6/IL-12 family cytokines, IL27, plays an important role in emergency myelopoiesis. Among various types of hematopoietic cells in bone marrow, IL-27 predominantly and continuously promoted expansionof only Lineage−Sca-1+c-Kit+ (LSK) cells, especially long-term repopulating hematopoietic
stem cells, and differentiation into myeloid progenitors in synergy with stem cell factor.
These progenitors expressed myeloid transcription factors such as Spi1, Gfi1, and Cebpa/bthrough activation of signal transducer and activator of transcription 1 and 3, and hadenhanced potential to differentiate into neutrophils, but not into plasmacytoid dendritic
cells. Among various cytokines, IL-27 in synergy with stem cell factor had the strongest
ability to augment the expansion of LSK cells and their differentiation into myeloid progenitors. The blood stage of malaria infection was revealed to enhance IL-27 expression
through interferon-γ production, and IL-27 then promoted the expansion of LSK cells, differentiating and mobilizing them into the spleen, resulting in enhanced production of neutrophils to control the infection. Thus, IL-27 is one of the limited unique cytokines directly
acting on hematopoietic stem cells to promote differentiation into myeloid progenitors
during emergency myelopoiesis.

Introduction
Emergency myelopoiesis is inflammation-induced hematopoiesis, which is critical for controlling systemic infection with pathogens such as a virus, bacteria, or parasite [1,2]. In contrast to
adaptive immune cells such as T cells and B cells, which can vigorously proliferate in responseto their specific antigens, innate immune cells need to be replenished from hematopoietic stem
cells (HSCs) and progenitors in bone marrow (BM) because of their low proliferative activity.
However, the molecular mechanism of emergency myelopoiesis during infection remains
incompletely understood. HSCs and hematopoietic progenitors can directly sense the presenceof pathogens via pattern recognition receptors (Rs) such as Toll-like receptors (TLRs), andthey can also respond to pro-inflammatory cytokines such as interferon (IFN)-α, IFN-γ, interleukin (IL)-1, tumor necrosis factor (TNF)-α, and granulocyte colony-stimulating factor(G-CSF) [1]. IFN-α and IFN-γ have pleiotropic effects on many cell types, including HSCs andhematopoietic progenitors [1]. Recently, these cytokines were demonstrated to induce an
expansion of HSCs and myeloid progenitors, leading to the production of mature myeloid cells[3–6], although their inhibitory effects on hematopoiesis were previously reported [7–9]. Currently, thus, there are several conflicting positive and negative effects of IFN-α and IFN-γ inhematopoiesis [10,11]. However, these discrepancies may be explained by compensatory mechanisms, including IFN-γ-mediated secretion of other cytokines such as IL-6 [12] and fmsrelated tyrosine kinase 3 ligand (Flt3L) [13].

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 2016

2 / 25-161-

Role of IL-27 in Emergency Myelopoiesis

IL-27 is one of the IL-6/IL-12 family cytokines; it plays important roles in immune regulation with both pro-inflammatory and anti-inflammatory properties [14–16]. IL-27 consists of
p28 and Epstein-Barr virus-induced gene 3 (EBI3), and its receptor is composed of WSX-1 andglycoprotein (gp)130, which is a common receptor subunit in many of the IL-6 family cytokines. We previously demonstrated that IL-27 plays a role in HSC regulation, and that IL-27
expands HSCs and promotes their differentiation in vitro [17]. Moreover, transgenic (Tg) miceexpressing IL-27 showed enhanced myelopoiesis in BM and extramedullary hematopoiesis in
the spleen [17].
In the present study, we further examined the effects of IL-27 on hematopoiesis, the molecular mechanisms, and the physiological role of IL-27 in the control of malaria infection. IL-27
acted on and expanded Lineage (Lin)−Sca-1+c-Kit+ (LSK) cells, which are highly enriched in
HSCs together with very primitive hematopoietic progenitors [18,19], in BM cells in synergy
with stem cell factor (SCF, c-Kit ligand) and differentiated HSCs into myeloid progenitors
through activation of signal transducer and activator of transcription 1 (STAT1) and STAT3.
Moreover, malaria infection induced IFN-γ production, which augmented IL-27 expression,and the IL-27 then promoted the expansion and mobilization of LSK cells into the spleen,
resulting in enhanced myelopoiesis to resolve the infection. Our results revealed that IL-27 is
one of the limited unique cytokines directly acting on long-term HSCs (LT-HSC), which represent the true stem cells capable of self-renewing, and promotes the expansion and differentiation of them into myeloid progenitors.

Results
IL-27 and SCF expand only LSK cells among various kinds of BM
progenitors
Previously, we demonstrated that stimulation of LSK cells with IL-27 and SCF induces an
expansion of HSCs and hematopoietic progenitors, including short-term repopulating cells[17]. Moreover, we found that only the combination of IL-27 and SCF, but not either alone,
vigorously and continuously expands BM cells to produce LSK cells and CD11b+c-Kit− cells[17]. To examine which cell populations in BM cells respond to IL-27 and SCF in more detail,BM cells were divided into two populations positive or negative for Lin markers except CD11b,
and the Lin− population was further divided into four populations positive for either c-Kit or
CD11b, or both positive, or both negative. Each population purified by sorting was then stimulated with IL-27 and SCF. Among these five populations, only the Lin−c-Kit+ population
greatly expanded (Fig 1A). Next, the BM cells were divided into respective hematopoietic progenitors according to the expression of cell surface markers, as reported previously [20–22],
and stimulated with IL-27 and SCF. Only the LSK cell population vigorously and continuously
expanded over more than 6 weeks, although transient and slight expansion was seen in the cell
populations of granulocyte/macrophage progenitor (GMP), common myeloid progenitor(CMP), and megakaryocyte/erythrocyte progenitor (MEP) (Fig 1B–1E). The expanding cells in
the LSK cell population were further analyzed for the expression of cell surface markers. In linewith the preliminary results, there seemed to be two populations, the phenotypical LSK population and the Lin+c-Kit− population (Fig 1E).
We previously demonstrated that IL-27 Tg mice, which express high amounts of IL-27 inblood, show an increased number of LSK cells in the BM and spleen [17]. To further examine
the in vivo effects of IL-27 on the expansion of LSK cells, LSK cells were purified by sortingfrom BM cells of GFP Tg mice and transferred into wild-type (WT) and IL-27 Tg mice. The
transferred GFP+ LSK cells vigorously expanded in the BM and spleen of IL-27 Tg mice, but
not in those of WT mice, and approximately half of the expanding cells retained the cell surface

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 2016

3 / 25-162-

Role of IL-27 in Emergency Myelopoiesis

Fig 1. IL-27 and SCF most strongly expand only LSK cells among various kinds of BM progenitors and differentiate them into myeloid progenitors
retaining the LSK phenotype. (A) Expansion of only the c-Kit+CD11b−Lin− population by IL-27 and SCF in BM cells. Total BM cells were divided into two
populations positive or negative for Lin markers, and the Lin− population was further divided into four populations positive for either c-Kit or CD11b, both
positive, or both negative. Each population (5 × 103) purified by sorting was stimulated by either IL-27 or SCF alone, or both, in a round 96-well plate andphotographed 2 weeks later. (B-E) Expansion of only the LSK cell population by IL-27 and SCF among various BM progenitors. BM progenitors (4–5 × 103)
purified by sorting were stimulated with IL-27 and SCF. Cell expansion was photographed at indicated periods (B) and cell number of the expanded LSK cell
population retaining the LSK phenotype was counted at 1 week (C). Time kinetic analysis of cell numbers in the LSK population retaining the LSK phenotype

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 2016

4 / 25-163-

Role of IL-27 in Emergency Myelopoiesis(D). Representative flow cytometry dot plot analysis of c-Kit and Lin (upper) and of c-Kit and Sca-1 in the Lin−c-Kit+ population (lower) of expanded LSK cells
and progenitor cells at 1 week (E). (F) Expansion of LSK populations in vivo by IL-27 in IL-27 Tg mice. LSK cells were purified by sorting from BM cells of GFP
Tg mice and transferred into non-lethally irradiated WT and IL-27 Tg mice. Twenty days later, BM and spleen in these recipient mice were analyzed for GFP+LSK populations. Data are shown as mean ± SEM (n = 2–3) and are representative of at least two independent experiments. *P < 0.05. (G-H) Augmented
and prolonged expansion of the LSK cell population by only IL-27 and SCF in vitro. LSK cells (1 × 103) from WT mice were stimulated by various cytokines
together with SCF for 1 to 4 weeks, the stimulated cells were analyzed by flow cytometry (G), and cell number was counted with time course (H). Data arerepresentative of at least two independent experiments.
doi:10.1371/journal.ppat.1005507.g001

markers for LSK phenotype (Fig 1F). These results suggest that IL-27 vigorously and continuously augments the expansion of LSK cells both in vitro and in vivo.

IL-27 has the strongest ability to augment the expansion of LSK cells
and their differentiation into myeloid progenitors retaining the LSK
phenotypeBecause it was previously reported that IFN-α and IFN-γ induce proliferation of HSCs in vivo
[3–5], we next explored the effects of various cytokines in collaboration with SCF on the expansion of LSK cells in vitro. However, IFN-α and IFN-γ augmented the expansion of LSK cells
very little, and only IL-27 enhanced it vigorously over 4 weeks (Fig 1G and 1H). Moreover,although there are several cytokines, such as IL-3, IL-11, G-CSF, and TPO, that are known totransiently expand and differentiate HSCs [1], none showed an ability superior to that of IL-27
in expanding LSK cells retaining the LSK phenotype over a long period of time (S1 Fig). Thus,
IL-27 has the strongest ability to augment the expansion of LSK cells.

LSK cells expanded by IL-27 and SCF are multipotent myeloid
progenitors
The LSK cells expanded by IL-27 and SCF were further analyzed for the cell surface expressionof various markers, and the expression levels were compared with those of primary LSK cells
freshly prepared from BM of WT mice. The expression levels of macrophage colony-stimulating factor receptor (M-CSFR), CD16/32, and MHC class II in the LSK cells expanded by IL-27
and SCF were much higher than those in primary LSK cells (Fig 2A). The expression levels of
CD34 and CD150 in the expanded LSK cells were slightly less than those in primary LSK cells(Fig 2A). In contrast, the expanded LSK cells were almost completely negative for Flt3 expression, whereas primary LSK cells were positive for Flt3 (Fig 2A). Thus, IL-27 and SCF expand
and differentiate primary LSK cells into M-CSFR+Flt3−CD16/32+ LSK cells (myeloid progenitor cells).
Next, multipotency of the LSK cells expanded by IL-27 and SCF were examined under various differentiation conditions for migratory dendritic cells (mDCs) by granulocyte/macrophage (GM)-CSF, plasmacytoid DCs (pDCs) and conventional DCs (cDCs; lymphoid-residentDCs) were examined using Flt3L and thrombopoietin (TPO) [23], myeloid cells were examinedusing IL-3 and SCF, and T cells and B cells were examined by using thymic stromal cells (TSt4)
with and without expressing Notch ligand Delta-like 1 (DLL1), respectively [24]. The expanded
LSK cells much more rapidly proliferated and differentiated into MHC class II+CD11c+ mDCs
than primary LSK cells in response to GM-CSF, although the total number of mDCs achieved
seemed to be similar for both (Fig 2B and 2C). However, LSK cells stimulated with IL-27 and
SCF rapidly lost the ability to differentiate into pDC and cDC (Fig 2D and 2E). These phenomena are highly consistent with the almost complete abolishment of Flt3 expression on the
expanded LSK cells (Fig 2A). Under myeloid differentiation conditions, the expanded LSK cells
differentiated much more greatly into neutrophils and slightly into mast cells, but less so into

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 2016

5 / 25-164-

Role of IL-27 in Emergency Myelopoiesis

Fig 2. LSK cells expanded by IL-27 and SCF are multipotent myeloid Progenitors. (A) Flow cytometry histogram analysis of cell surface markers in the
LSK cells expanded by IL-27 and SCF for 4 weeks and primary BM cells using antibodies as indicated (solid line) and their control antibodies (plane line withshading). (B-C) Augmented potential of the LSK cells expanded by IL-27 and SCF to differentiate into mDCs. LSK populations (3 × 103) purified from the LSK
cells expanded by IL-27 and SCF for 2 and 4 weeks and primary BM cells were stimulated with GM-CSF. After the indicated time, these stimulated cells wereanalyzed for the expression of MHC class II and CD11c (B), and the cell numbers of mDC (MHC class II+CD11c+) were counted (C). (D-E) Decreased

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 2016

6 / 25-165-

Role of IL-27 in Emergency Myelopoiesis

potential of the LSK cells expanded by IL-27 and SCF to differentiate into pDCs (Siglec H+PDCA1+CD11c+) and cDCs (Siglec H−PDCA1−CD11c+). (F-G)
Enhanced potential of the LSK cells expanded by IL-27 and SCF for 2 weeks to differentiate into neutrophils. Neutrophil; Ly6G+CD11b+, macrophage; F4/80+CD11b+, mast cell; c-Kit+FcεR1α+CD11b−, basophil; CD49b+FcεR1α+CD11b−. (H) Abrogated potential of the LSK cells expanded by IL-27 and SCF for 2
weeks to differentiate into T (day 18) and B (day 15) cells. (I) Enhanced potential of the LSK cells expanded by IL-27 and SCF to differentiate into neutrophils
in vivo. LSK populations purified from the LSK cells from CD45.1 congenic mice expanded by IL-27 and SCF for 2 weeks were transferred into sublethally
irradiated CD45.2 recipient mice with the same congenic BM cells. Development of Gr-1+CD11b+ neutrophils in the BM and spleen were analyzed by flow
cytometry after 9 days, and the percentage of neutrophils in each CD45.1+ and CD45.2+ cells was compared. (J) Increased expression of transcription
factors critical for the differentiation into myeloid cells in the LSK cells expanded by IL-27 and SCF. RNA was prepared from the LSK population purified from
LSK cells expanded for 2 weeks together with other progenitors and subjected to real-time RT-PCR. Data are shown as mean ± SEM (n = 2–4) and are
representative of two to three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005.
doi:10.1371/journal.ppat.1005507.g002

macrophages and basophils (Fig 2F and 2G). Similar enhanced differentiation into myeloid
cells was observed using the LSK cells obtained from WT and IL-27 Tg mice (S2 Fig). In contrast, the ability to differentiate into B cells and T cells was almost completely abrogated in the
expanded LSK cells (Fig 2H).
Moreover, the ability of the LSK cells expanded by IL-27 and SCF to differentiate into myeloid cells in vivo was explored by using mixed BM chimeras. The equal cell numbers of the LSK
cell population expanded from CD45.1 congenic mice and BM cells from CD45.2 congenic
mice were mixed and transferred into sublethally irradiated CD45.2 congenic mice. After 9days, cell populations of neutrophils in the BM and spleen were analyzed. In agreement with
the in vitro results described, the percentages of neutrophils derived from the expanded LSK
cells were markedly higher than those from primary LSK cells in both BM and spleen (Fig 2I).
To explore the molecular mechanisms whereby IL-27 and SCF expand LSK cells, total RNA
was prepared from the expanded LSK cells and respective hematopoietic progenitors and analyzed with real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The expanded
cells highly expressed the transcription factors critical for differentiation into myeloid cells
such as Spi1, Gfi1, and Cebpa, but expression was much less for those important for the othertypes of cells such as Irf8, Tcf4, and Ikzf1 [25–27] (Fig 2J). No expression of transcription factors important for B cells and erythrocytes, Pax5 [28] and Gata1 [29], respectively, wasobserved. In addition, the expression of Cbepb, which is a transcription factor recently demonstrated to be regulated by cytokines and control emergency granulopoiesis [30–32], was also
increased (S3 Fig).
These results suggest that LSK cells expanded by IL-27 and SCF are multipotent myeloid
progenitors that have unique potential to differentiate into mDCs, neutrophils, and mast cells,
and less so into macrophages, and basophils, but not into pDCs, cDCs, T cells, and B cells.

STAT1 and STAT3 are important for expansion and differentiation of
LSK cells by IL-27 and SCF
We and others previously demonstrated that IL-27 activates both STAT1 and STAT3 throughWSX-1 and gp130, respectively [14,15]. Consistent with these reports, real-time RT-PCR analysis revealed that LSK populations purified from LSK cells expanded by IL-27 and SCF and primary BM cells were positive for mRNA expression of STAT1 and STAT3 (Fig 3A).
Phosphorylation of STAT1 and STAT3 was also detected in primary WT LSK cells, but not
WSX-1-deficient LSK cells (Fig 3B), in response to IL-27 and SCF, which were analyzed byflow cytometry. Furthermore, IL-27 alone induced phosphorylation of both STAT1 and
STAT3, whereas SCF alone failed to induce phosphorylation of either one, as discussed previously [33] (S4 Fig).
To further investigate the roles of STAT1 and STAT3 in the expansion of LSK cells and theability to differentiate into myeloid progenitors by IL-27 and SCF, we used STAT1-deficient
LSK cells and conditional STAT3-knockout (STAT3 cKO) LSK cells. LSK cells from WT (129)

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 2016

7 / 25-166-

Role of IL-27 in Emergency Myelopoiesis

Fig 3. STAT1 and STAT3 are important for expansion and differentiation of LSK cells by IL-27 and SCF. (A) mRNA expression of STAT1 and STAT3 in
the LSK cells expanded by IL-27 and SCF for 2 weeks and in primary LSK cells. (B) Flow cytometry histogram analysis of primary LSK cells after stimulation
with IL-27 and SCF for 60 min using anti-pY-STAT1 or anti-pY-STAT3 (solid line) and control antibody (plain line with shading). (C-D) Dispensable role for
STAT1 in the expansion of LSK cells in response to IL-27 and SCF. LSK cells (1 × 103) purified from BM cells of WT (129) mice and STAT1-deficient mice
were expanded by IL-27 and SCF for 2 weeks and analyzed for expression of LSK phenotype (C). The cell numbers of LSK and LS−K cell populations werecounted. Purified cells of the STAT1-deficient LSK and LS−K cells (1 × 103) were further stimulated by IL-27 and SCF for more 1 week and the cell number of
expanded cells was counted (D). (E-G) Contribution of STAT1 to the differentiation into mDC (E) and myeloid cells (F) of LSK cells expanded by IL-27 and

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 2016

8 / 25-167-

Role of IL-27 in Emergency Myelopoiesis

SCF for 2 weeks together with their mRNA expression of transcription factors (G). (H) Indispensable role for STAT3 in the expansion of LSK cells in response
to IL-27 and SCF. Purified GFP− STAT3flox/flox LSK cells and GFP+ STAT3 cKO LSK cells (1 × 102) were expanded by IL-27 and SCF for 10 days andanalyzed for expression of LSK phenotype and their cell numbers. (I-K) Critical role for STAT3 in the differentiation into mDC (I) and myeloid cells (J) of LSK
cells expanded by IL-27 and SCF for 10 days together with their mRNA expression of transcription factors (K). Data are shown as mean ± SEM (n = 3–4) andrepresentative of two to three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005.
doi:10.1371/journal.ppat.1005507.g003

and STAT1-deficient mice were stimulated with IL-27 and SCF. Although WT LSK cells comprised more than 90% of cells with the LSK phenotype after 7 days, STAT1-deficient LSK cells
comprised half LSK phenotype cells and half Sca-1−LK (LS−K) (Fig 3C). Nevertheless, thenumber of expanded cells was comparable among them, probably due to the anti-proliferative
effects by STAT1 signaling [34]. However, the purified STAT1-deficient LS−K cells failed tosurvive thereafter, even in the presence of IL-27 and SCF (Fig 3D), although the purified
STAT1-deficient LSK cells expanded well (Fig 3D), as did WT 129 LSK cells (Fig 3C). Moreover, WT and STAT1-deficient LSK cells had similar abilities to differentiate into MHC class
II+CD11c+ mDC cells (Fig 3E) and macrophages (Fig 3F). STAT1-deficient LSK cells showed
reduced ability to differentiate into neutrophils and mast cells, but increased ability to differentiate into basophils (Fig 3F). In line with the reduced ability to differentiate into neutrophils,mRNA expression of the critical transcription factor Gfi1 was significantly reduced in STAT1deficient LSK cells compared with that in WT LSK cells (Fig 3G). In contrast, STAT3 cKO LSK
cells expanded very little in response to IL-27 and SCF (Fig 3H). Moreover, the residual surviving LSK cells almost completely lost the ability to differentiate into mDCs (Fig 3I) and myeloid
cells (Fig 3J). Consistent with the abrogated abilities, these cells showed reduced expression of
the critical transcription factors such as Spi1, Gfi1, and Cebpa, but not Irf8 (Fig 3K). These
results suggest that both STAT1 and STAT3 are necessary for LSK cells to fully expand and differentiate into myeloid progenitor cells in response to IL-27 and SCF.

IL-27 and SCF expand CD34−CD150+ LSK cells into myeloid progenitor
cells
To more precisely define which cell population responds to stimulation with IL-27 and SCF, LSK
cells were further divided into two populations according to CD34 expression. Although the percentage of the more primitive population of CD34− LSK cells was much less than that of CD34+
LSK cells, the CD34− LSK cells responded much better to stimulation with IL-27 and SCF andexpanded more vigorously than CD34+ LSK cells (Fig 4A and 4B). Then, the LSK cells were further divided into eight populations, F1 to F8, including LT-HSCs (CD34−CD150+CD41− LSK,
F1) and myeloid-restricted progenitor cells with long-term repopulating activity (MyRPs,
CD34−CD150+CD41+ LSK, F4) according to the recently revised criteria [19]. Respective populations purified by sorting (Fig 4C) were stimulated with IL-27 and SCF. Only two populations, F1
and F4, vigorously expanded. F5, which corresponds to populations more differentiated toward
myeloid cells such as macrophages, slightly expanded (Fig 4D and 4E). The F1 and F4 populations expanded by IL-27 and SCF had great abilities to differentiate into myeloid cells, particularly neutrophils (Fig 4E). Thus, IL-27 and SCF expand CD34−CD150+ LSK cells, including
LT-HSCs and MyRPs, and differentiate them into myeloid progenitor cells, which have great
potential to differentiate mainly into neutrophils.

IL-27 plays an important role in expansion, differentiation, andmobilization of LSK cells to control malaria infection
We previously demonstrated that in the blood stage of malaria infection with the attenuatedvariant Plasmodium (P) berghei XAT derived from the lethal strain P. berghei NK65 IFN-γ

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 2016

9 / 25-168-

Role of IL-27 in Emergency Myelopoiesis

Fig 4. IL-27 and SCF expand CD34−CD150+ LSK cells into multipotent myeloid progenitor cells. (A-B) Enhanced expansion of CD34− LSK cells by IL27 and SCF. LSK cells from WT mice were divided into two populations according to the expression of CD34, CD34− LSK, and CD34+ LSK cells, and each
population (1 × 102) was stimulated with IL-27 and SCF. One to 4 weeks later, these stimulated cells were analyzed by flow cytometry; representative dot
plots of c-Kit and Sca-1 in the Lin− population at 2 weeks are shown (A). Cell numbers of these stimulated cells were counted with time course (B). (C-E)
Augmented expansion of CD34−CD150+ LSK cells by IL-27 and SCF. LSK cells were further divided into eight populations (F1-F8) according to theexpression of CD34, CD150, and CD41 (C), and each population (50 cells) purified by sorting was stimulated with IL-27 and SCF. One to 4 weeks later, thesestimulated cells were analyzed by flow cytometry. Representative dot plots of c-Kit and Sca-1 in the Lin− population at 4 weeks are shown, and the cellnumber of the LSK cell population in these stimulated cells was counted with time course (D). LSK populations (1 × 103) purified from primary or IL-27/SCF-

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 201610 / 25-169-

Role of IL-27 in Emergency Myelopoiesis

expanded F1, F4, and F5 LSK cells were differentiated into myeloid cells by IL-3 and SCF, and cell number was counted (E). Data are shown as mean ± SEM(n = 3–4) and are representative of two to three independent experiments. *P < 0.05, ***P < 0.005.
doi:10.1371/journal.ppat.1005507.g004

production induced by IL-12 and phagocytic cells in the spleen are critical for controlling parasitemia [35,36]. Recently, it was reported that the blood stage of P. chabaudi infection induces
mobilization of early myeloid progenitor cells out of BM, thereby transiently establishing myelopoiesis in the spleen through IFN-γ to resolve the infection [6,37]. In line with these results,
WSX-1-deficient mice showed more increased parasitemia than WT mice at 7 days (Fig 5A),
just prior to when the parasitemia reaches its peak after infection with P. berghei XAT (S5A
Fig). In contrast, no significant difference was observed in the serum IFN-γ levels in WT and
WSX-1-deficient mice (Fig 5B). The infection markedly induced the enhanced percentage andnumber of LSK cells in the BM and spleen of WT mice (Fig 5C and 5D). The LSK cells in the
BM showed greatly augmented abilities to differentiate in vitro into neutrophils and mast cells,
but had slightly reduced abilities to differentiate into macrophages and basophils after infection(Fig 5E). Moreover, the LSK cells in the spleen exhibited a much more enhanced ability to differentiate into neutrophils, macrophages, and mast cells (Fig 5E). In contrast, of note, WSX-1deficient mice showed significantly reduced percentage and number of LSK cells in the BM
and spleen compared with WT mice after infection (Fig 5C and 5D). In particular, the cellnumber of the neutrophils in the spleen was increased very little in WSX-1-deficient mice (Fig
5F and 5G). Consistent with this, LSK cells from BM of WSX-1-deficient mice showed reduced
abilities to differentiate in vitro into neutrophils after infection compared with those of WT
mice (S6 Fig). In addition, more greatly reduced abilities to differentiate in vitro into various
myeloid cells were observed when LSK cells from spleen were used (S6 Fig). Moreover, mixedbone marrow chimera experiments using bone marrow cells from WT and WSX-1-deficient
mice revealed that the effect of IL-27 on the expansion of LSK cells and neutrophils is actually acell-autonomous direct effect (S7 Fig).
To further elucidate the protective role of LSK cells in malaria infection, LSK cells purified
from BM cells of infected WT CD45.1 mice were injected into the infected WSX-1-deficientCD45.2 mice. Consistent with the increased percentage of neutrophils differentiated from theWT LSK cells in the BM and spleen of transferred WSX-1-deficient mice (Fig 5H), parasitemia
was significantly decreased in the WSX-1-deficient mice by the transfer of WT LSK cells compared with that in non-transferred WSX-1-deficient mice (Fig 5I). Thus, the blood stage ofmalaria infection induces expansion, differentiation, and mobilization of LSK cells into thespleen to produce myeloid cells such as neutrophils in an IL-27-dependent manner.

Malaria infection enhances IL-27 expression through IFN-γ production topromote the expansion, differentiation, and mobilization of LSK cells
Although it was previously reported that IFN-α and IFN-γ induce proliferation of HSCs invivo [3–5], IFN-α and IFN-γ augmented the expansion of LSK cells very little in vitro, and only
IL-27 enhanced it vigorously over 4 weeks (Fig 1G and 1H). However, similar to the work previously reported [37,38], IFN-γ-deficient mice showed increased parasitemia with almost no
increase in the number of LSK cells in BM and spleen (Fig 6A and 6B). To clarify the molecularmechanism whereby IFN-γ induces the expansion of LSK cells, the expression of IL-27 subunits EBI3 and p28 was examined. Although the infection did not increase EBI3 mRNA expression in the BM and spleen of both WT and IFN-γ-deficient mice (S8 Fig), intriguingly, theinfection greatly enhanced p28 mRNA expression in WT mice but failed to enhance it in IFNγ-deficient mice (Fig 6C). In agreement with this, p28 protein levels in the serum were greatly

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 201611 / 25-170-

Role of IL-27 in Emergency Myelopoiesis

Fig 5. IL-27 plays an important role in expansion, differentiation, and mobilization of LSK cells to control malaria infection. (A-D) Reduced inductionof LSK cell population in WSX-1-deficient mice after malaria infection, accompanied by increased parasitemia and comparable production of IFN-γ. WT or
WSX-1-deficient mice were infected with the blood stage of P. berghei XAT. Seven days later, parasitemia (A) and serum IFN-γ level (B) were determined,and LSK populations in the BM and spleen were analyzed by flow cytometry, and representative dot plots of c-Kit and Sca-1 in the Lin− population are shown(C). Cell number of the LSK cell population was counted (D). (E) Augmented potential of LSK cells to differentiate into myeloid cells by malaria infection. LSK
cells (1 × 103) in the BM and spleen of the malaria-infected or non-infected WT mice were purified and differentiated into myeloid cells in vitro by IL-3 and
SCF, and cell number of differentiated cells was counted. (F-G) Reduced cell number of neutrophils in WSX-1-deficient mice after malaria infection. The BM
and spleen cells were analyzed for expression of Gr-1 and CD11b at 7 days after the infection (F), and cell number of neutrophils (Gr-1+CD11b+) was

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 201612 / 25-171-

Role of IL-27 in Emergency Myelopoiesis

counted (G). (H-I) Decreased parasitemia in the WSX-1-deficient mice transferred with LSK cells purified from BM cells of the malaria-infected WT mice. LSK
cells purified from BM cells of the infected WT CD45.1 mice were transferred into non-lethally irradiated WSX-1-deficient CD45.2 mice 7 days before
infection. Neutrophil population in the BM and spleen was analyzed by flow cytometry, and representative dot plots of CD11b and Gr-1 in the CD45.1+
population are shown (H). Parasitemia was measured 4 and 7 days after the infection (I). Data are shown as mean ± SEM (n = 3–9) and are representative ofat least two independent experiments. *P < 0.05, ***P < 0.005.
doi:10.1371/journal.ppat.1005507.g005

increased by the infection in WT mice but not in IFN-γ-deficient mice (Fig 6D). Consistent
with the in vivo role of IFN-γ, we also observed the augmentation of mRNA expression of p28,
but not EBI3, and p28 protein production in the culture supernatants of WT BM cells stimulated with IFN-γ in vitro (S9 Fig).
To further clarify the role of IL-27 downstream of IFN-γ, we next performed the experiment
to see the effects of forced expression of IL-27 on the susceptibility to malaria infection in IFNγ-deficient mice. The hydrodynamic injection of IL-27 expression vector into the infected IFNγ-deficient mice showed significantly decreased parasitemia compared with that of control vector (Fig 6E). This phenomenon was accompanied by the enhanced percentage of LSK cells andaugmented numbers of LSK cells and neutrophils in both BM and spleen (Fig 6F and 6G).
Thus, the blood stage of malaria infection augments the expression of IL-27 through IFN-γ,
and IL-27 then promotes the expansion, differentiation, and mobilization of LSK cells into thespleen to control parasitemia.

Discussion
Previously, we found that IL-27, which is in the IL-6/IL-12 family of cytokines, plays a role in
the regulation of HSCs in vitro and in vivo [17]. Here, we have further elucidated that IL-27 is aunique cytokine that directly acts on LSK cells to promote their differentiation into myeloid
progenitor cells called M-CSFR+Flt3−CD16/32+ LSK cells, which still retain the LSK phenotype(Fig 2A–2I). These progenitors have great potential to give rise to neutrophils, mDCs, andmast cells, but not to pDCs, cDCs, T cells, and B cells. Interestingly, among various BM progenitor cells, IL-27 and SCF vigorously and continuously expand only HSCs and primitive
myeloid progenitor cells with long-term repopulating activity, such as LT-HSCs and MyRPs,
respectively [19], for more than 4 weeks (Fig 4). Consistent with the ability to differentiate into
myeloid progenitor cells, the LSK cells expanded by IL-27 and SCF expressed transcription factors such as Spi1, Gfi1, Cebpa, and Cebpb, which are critical for myeloid differentiation [25–27,30–32] (Fig 2J and S3 Fig). Although Cebpb was reported to be an important transcription
factor for emergency granulopoiesis [30–32], STAT3 signaling was revealed to be importantfor its upregulation, whereas STAT1 signaling unexpectedly suppressed its expression (S10Fig). This phenomenon seems to correlate to the expression level of the anti-apoptotic gene
Bcl-2 [39,40], but not the transcription factor E2-2, which is critical for pDC differentiation[41]. Further studies are necessary to elucidate the precise roles of each STAT in the regulationof Cebpb expression. Thus, IL-27 is one of the limited unique cytokines that directly acts on themost primitive LT-HSCs; it promotes their expansion and differentiation into myeloid progenitor cells, presumably through MyRPs [19], to replenish myeloid cells such as neutrophils in
the periphery during emergency myelopoiesis.
Sca-1 is an IFN-responsive molecule that is highly upregulated in many hematopoietic cells
following exposure to IFNs [10,11,37]. Consequently, myeloid-restricted progenitor cells normally identified as Lin−Sca-1−c-Kit+ (LS−K) become positive for Sca-1 and can no longer bedistinguished from the real multipotent LSK cells, resulting in overestimation of the latter population, and this is a problem. IL-27 was previously reported to enhance the expression of Sca1 on T cells [42]. However, to alleviate the problem, we initially identified the cell population

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 201613 / 25-172-

Role of IL-27 in Emergency Myelopoiesis

Fig 6. Malaria infection enhances IL-27 expression through IFN-γ production to promote the expansion, differentiation, and mobilization of LSK
cells. (A-B) Indispensable role for IFN-γ in the expansion of LSK cells after malaria infection. WT and IFN-γ-deficient mice were infected with the blood stageof P. berghei XAT. Seven days later, parasitemia was determined (A) and LSK populations were analyzed by flow cytometry (B). (C-D) IFN-γ-dependent
induction of IL-27 p28 subunit expression by malaria infection. RNA was prepared 7 days after the infection and analyzed for expression of p28 by real-time
RT-PCR (C), and serum p28 levels were determined by ELISA (D). (E-G) Decreased parasitemia and augmented expansion of LSK cell population in IFN-γdeficient mice by IL-27. IFN-γ-deficient mice were hydrodynamically injected with IL-27-expression vector or control vector at days 0 and 4 after infection; atday 7, parasitemia was measured (E), LSK population was analyzed by flow cytometry (F), and cell numbers of LSK cells and neutrophils were counted (G).
Data are shown as mean ± SEM (n = 3–5) and are representative of at least two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005.
doi:10.1371/journal.ppat.1005507.g006

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 201614 / 25-173-

Role of IL-27 in Emergency Myelopoiesis

responsive to IL-27 and SCF among BM cells by using LSK cells and various hematopoietic
progenitor cells purified by sorting. Intriguingly, it turned out that the SCF and LSK cell populations expanded vigorously and continuously in response to IL-27, and that the LS−K cell populations including GMP, CMP, and MEP only transiently and slightly responded during thefirst week and then disappeared thereafter (Fig 1A–1E). Moreover, in almost all in vitro experiments, we used primary LSK cells that were purified by sorting. The high responsiveness of the
LSK cells to IL-27 seems to be partially due to the higher mRNA expression of WSX-1 in the
LSK cells compared to that of other hematopoietic progenitor cells (S3 Fig) [17].
We previously demonstrated that during the blood stage of malaria infection with attenuated P. berghei XAT, IL-12-mediated IFN-γ production and phagocytic cells (including neutrophils) in the spleen are critical for controlling parasitemia [35,36,43]. Previous studies
demonstrated that neutrophils play an important role in killing malaria parasites in mice, rats,and humans [43–45]. In marked contrast, regarding infection with lethal P. berghei NK65, IL12-mediated IFN-γ production was shown to contribute to T-cell-dependent immunopathology [46]. However, a major role of IL-27 in infection is its suppression of excess immune
responses against infection by controlling the production of pro-inflammatory cytokines [14–16]. Consistent with this, WSX-1/IL-27 was recently demonstrated to have a critical role in limiting the effector CD4+ T-cell-mediated immunopathology caused by IL-12-dependent IFN-γ
production during infection with lethal P. berghei NK65 [47–49]. The present study clearly
revealed that WSX-1/IL-27 contributes to clearance of parasites due to enhanced myelopoiesis
during the early phase of infection with attenuated P. berghei XAT (Fig 5 and S5A Fig). However, during the late phase of infection, WSX-1/IL-27 seems to play a role in limiting the production of pro-inflammatory cytokines such as IFN-γ (S5B Fig), leading to augmented
reduction of parasitemia (S5A Fig), as in the case of infection with lethal P. berghei NK65 [47–49]. Moreover, our preliminary data revealed that there were no apparent differences observedin parasitemia or expansion of LSK cells and neutrophils in the BM and spleen of WT and
WSX-1-deficient mice 7 days after infection with lethal P. berghei NK65 (S11 Fig). It is conceivable that pro-inflammatory cytokines other than IL-27 were abundantly produced in theabsence of IL-27 during infection with lethal P. berghei NK65 and that late-phase infection
with attenuated P. berghei XAT may have redundantly compensated for the loss of IL-27 topromote myelopoiesis. However, other studies have shown that IL-27 limits migration of neutrophils from the BM to the site of inflammation by reducing production of cytokines and chemokines during influenza infection [50] and septic peritonitis [51]. IL-27 was also reported to
be a negative regulator of neutrophil function [52]. Although IL-27 directly promotes myelopoiesis to produce myeloid progenitors in BM, as shown in the present study, IL-27 may indirectly regulate migration of these progenitors and neutrophils to the site of inflammation andlimit neutrophil function. Thus, IL-27 has both positive and negative effects on neutrophils;
therefore, the overall outcome of the effects of IL-27 is likely to be governed by the balance
between these effects, depending on the disease model.
It was recently demonstrated that P. chabaudi infection induces mobilization of early myeloid progenitor cells out of BM, thereby transiently establishing myelopoiesis in the spleenthrough IFN-γ [37]. However, the expression of IFN-γR in the hematopoietic compartment
was dispensable, whereas its expression in the irradiation-insensitive cellular compartment,
including endothelial cells and stromal cells, was important [37]. Secretion of IFN-γ-induced
chemokines such as CCL2 and CCL7 by non-hematopoietic cells plays a critical role in themobilization of CCR2-expressing HSCs [37]. In this study, however, there is no experimental
evidence regarding how IFN-γ regulates the activation of HSCs. In the present study, WSX-1deficient mice showed significantly reduced numbers of LSK cells and neutrophils comparedwith WT mice after P. berghei XAT infection, resulting in increased parasitemia (Fig 5A–5F

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 201615 / 25-174-

Role of IL-27 in Emergency Myelopoiesis

and S5A Fig). These results suggest that endogenous IL-27 greatly contributes to the clearanceof parasitemia through augmentation of myelopoiesis. Moreover, similar to P. chabaudi infection, P. berghei XAT infection could not increase the number of LSK cells in IFN-γ-deficient
mice with increased parasitemia (Fig 6A and 6B). Of note, after P. berghei XAT infection, p28mRNA expression and its serum protein level were markedly upregulated in an IFN-γ-dependent manner (Fig 6C and 6D and S9 Fig). This is consistent with previous reports indicatingthat p28 gene transcription in macrophages is induced by IFN-γ and TLR ligands [53], andthat IFN-γ limits Th17-mediated and Th9-mediated autoimmune inflammation through IL-27
production [54,55]. In addition, the hydrodynamic injection of the IL-27 expression vector
into infected IFN-γ-deficient mice greatly recovered the number of LSK cells and neutrophils
in the BM and spleen and eventually reduced parasitemia (Fig 6E–6G). Thus, during malaria
infection, it is highly conceivable that the proliferative effects on LSK cells by IFN-γ are indirectly mediated by IL-27. In our study, we could not observe any direct proliferative effect of
IFN-α and IFN-γ on LSK cells in vitro, as recently pointed out by others [10,11], and only IL27 augmented the proliferative effect for more than 4 weeks (Fig 1G and 1H).
Recently, IL-27 was reported to have a polyglutamic acid domain in the p28 subunit, whichis unique among cytokines, and to confer hydroxyapatite-binding and bone-binding properties
and bone tropism to bone sialoprotein and the endosteal bone surface [56]. This location in the
BM has been identified as a niche for HSCs [57], and these properties support the idea that IL27 plays a critical role in the regulation of HSCs in that niche. We detected much higher
expressions of IL-27 subunits (both p28 and EBI3) at mRNA levels in BM than in the spleen
during the steady state, and P. berghei XAT infection greatly augmented the expression of p28
mRNA in both BM and spleen, and also its serum protein level (Fig 6C and 6D). Further studies are necessary to clarify which BM cells produce IL-27 during malaria infection; mesenchymal stromal cells might be a candidate because of their reported IL-6 production during viral
infection [12], as described in the next section.
It was recently demonstrated that specific cytotoxic CD8+ T cells during an acute viral infection with lymphocytic choriomeningitis virus secrete IFN-γ, thus enhancing the production of
IL-6 in BM mesenchymal stromal cells and resulting in an increased number of early multipotent progenitors and committed myeloid precursors in the BM and accumulation of myeloid
cells in the periphery [12]. The IL-6Rα chain is only expressed at the stage of early multipotent
progenitors and downstream myeloid precursors, and it is lacking HSCs [12]. In contrast, IL27 most predominantly acts on only HSCs, as shown in the present study. Both IL-6 and IL-27
share gp130, which is ubiquitously expressed as a common receptor subunit. Therefore, downstream of IFN-γ, both IL-27 and IL-6 may be necessary to induce the maximum myelopoiesis
to control infection. However, the mode of IL-6 action is complex and there are two majormechanisms: IL-6 classic signaling through membrane IL-6Rα and IL-6 trans-signaling
through soluble IL-6Rα [58,59]. It was recently reported that IL-6Rα-deficient mice show
increased resistance to P. chabaudi infection and that IL-6 trans-signaling, but not IL-6 classic
signaling, contributes to a lethal outcome of infection [60]. In contrast to the viral infection, we
could not detect any increased mRNA expression of IL-6 in the BM or spleen of WT and IFNγ-deficient mice with P. berghei XAT infection (S8 Fig). A similar inability of IFN-γ to enhanceIL-6 mRNA expression in WT BM cells in vitro was also observed (S9A Fig). In addition, IL-6deficient mice showed little increased susceptibility to the P. berghei XAT infection, reduced
cell numbers in the LSK cell population, and reduced neutrophils in the BM and spleen (S12Fig). Thus, individual pathogens may utilize different mechanisms to induce emergency myelopoiesis through IL-27, IL-6, and others.

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 201616 / 25-175-

Role of IL-27 in Emergency Myelopoiesis

In conclusion, the present results provide a novel role and mechanism for the action of IL27 downstream of IFN-γ in the efficient expansion of myeloid progenitor cells from LT-HSCs
and MyRP cells and their mobilization into the spleen during acute malaria infection.

Materials and Methods
Ethics statement
The animal study was approved by the Animal Care and Use Committee of Tokyo Medical
University (S-230043, S-24012, S-25059, S-26003, and S-27009) and was performed in accordance with our institutional guidelines and the Fundamental Guidelines for Proper Conduct ofAnimal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, 2006.

Mice
C57BL/6 (CD45.2) mice and C57BL/6 (CD45.1) mice were purchased from Sankyo Lab(Tokyo, Japan). The 129/Sv mice and STAT1-deficient mice (129/Sv background) were purchased from Taconic Farms (Germantown, NY, USA). IL-6-deficient mice (C57BL/6 background) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). IFN-γ-deficient
mice (C57BL/6 background), STAT3flox/flox mice (a mixed background of 129/Sv and C57BL/6), and GFP Tg mice (C57BL/6 background) [61] were provided by Dr. Iwakura (Tokyo University of Science), Dr. Takeda (Osaka University), Dr. Okabe (Osaka University) and Dr. Ito(Tokyo Medical University), respectively. In addition to these mice, IL-27 Tg mice (C57BL/6
background) [17], and WSX-1-deficient mice (C57BL/6 background) [62] were maintained in
specific pathogen-free conditions under the care of the Laboratory Animal Center of Tokyo
Medical University. STAT3 cKO cells were obtained by infecting STAT3flox/flox cells with Creexpressing retrovirus in vitro.

Antibodies and reagents
Monoclonal antibodies (mAbs) for mouse c-Kit (2B8), Sca-1 (D7), CD3ε (145-2C11), CD4(GK1.5), CD8α (53–6.7), CD19 (6D5), CD49b (DX5), Gr-1 (RB6-8C5), TER119/erythroid cell
(TER-119), CD11c (N418), CD11b (M1/70), F4/80 (BM8), NK1.1 (PK136), B220 (RA3-6B2),
FcεRIα (MAR-1), M-CSFR (AFS98), Flt3 (A2F10), CD16/32 (2.4G2), CD150 (TC15-12F12.2),
CD41 (MWReg30), IL-7Rα (A7R34), MHC class II I-A/I-E (M5/114.15.2), Siglec H (551),
Ly6G (1A8), CD45.1 (A20), and CD45.2 (104) were purchased from BioLegend (San Diego,
CA). mAbs against mouse pY701-STAT1 (4a) and pY705-STAT3 (4/P-STAT3) were purchased from BD Pharmingen (San Diego, CA). mAbs against mouse CD34 (RAM34) was purchased from eBioscience (La Jolla, CA). mAbs against mouse PDCA1 (JF05-1C2.4.1) was
purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). APC-Cy7-conjugated streptavidin, PerCP/Cy5.5-conjugated streptavidin, and Brilliant Violet 510-conjugated streptavidin
were purchased from BioLegend and used to reveal staining with biotinylated Abs. Mouserecombinant IL-27 and hyper-IL-6 were prepared as a tagged single-chain fusion protein byflexibly linking EBI3 to p28 and soluble IL-6Rα to IL-6, respectively, using HEK293-F cells(Life Technologies, Carlsbad, CA) as described previously [63,64]. Mouse recombinant SCF,
IL-1β, IL-3, IL-6, IL-7, IL-11, IL-12, thymic stromal lymphopoietin (TSLP), G-CSF, M-CSF,
GM-CSF, TNF-α, and human recombinant TPO were purchased from PeproTech (Rocky Hill,
NJ). Human recombinant Flt3L was purchased from Miltenyi Biotec. Mouse recombinant IL23, IL-25, and IL-33 were purchased from R&D Systems (Minneapolis, MN). Mouse IFN-α

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 201617 / 25-176-

Role of IL-27 in Emergency Myelopoiesis

was purchased from PBL Biomedical Laboratories (Piscataway, NJ). Mouse recombinant IFNγ was provided from Shionogi Pharmaceutical Co., Ltd. (Osaka, Japan),

Preparation of cells
Spleen and BM Lin− cells were enriched by negative selection using an autoMACS Pro (Miltenyi Biotec) with a combination of magnetic beads conjugated with mAbs against CD3ε, CD4,
CD8α, Gr-1, TER119, CD11b, CD11c, NK1.1, B220, and FcεRIα. Subsequently, cells were
stained with mAbs against c-Kit, and Sca-1 was used for LSK. For multiple fractions of HSC,
cells were stained with CD34, c-Kit, Sca-1, CD150, and CD41 mAbs [19]. In the case of common lymphoid progenitor (CLP), macrophage-DC progenitor (MDP), and common DC progenitor (CDP), cells were stained with c-Kit, Sca-1, IL-7Rα, M-CSFR, and Flt3 mAbs [20–22].
For GMP, CMP, and MEP, cells were stained with CD34, c-Kit, Sca-1, and CD16/32 mAbs
[20–22]. Sorting was performed on FACS Aria or FACS Aria III (BD Bioscience).

Culture of cells
Cells were cultured at 37°C under 5% CO2/95% air in RPMI-1640 (SIGMA, St. Louis, MO)
containing 10% fetal calf serum, 50 μM 2-mercaptoethanol (GIBCO, Grand Island, NY), and100 μg/ml kanamycin (Meiji Seika, Tokyo, Japan). To proliferate progenitors, sorted cells were
cultured with 10 ng/ml IL-27 and/or 10 ng/ml SCF. To examine the effect of various cytokines
on proliferation of LSK, IL-1β, IL-3, IL-6, hyper-IL-6, IL-11, IL-12, IL-23, IL-25, IL-33, TSLP,
G-CSF, TPO, TNF-α, and IFN-α were used as a final concentration of 10 ng/ml. IFN-γ wasused as a final concentration of 100 U/ml. Half of the medium was changed every 3 days, withcytokines added.

In vitro differentiation assay
For evaluation of mDC potential, sorted cells (1–5 × 103) in 96-well plates were cultured with
20 ng/ml GM-CSF for 3 to 10 days. For pDC and cDC potential, sorted cells (5 × 103) were cultured with 100 ng/ml Flt3L and 20 ng/ml TPO for 10 days. For analysis of multipotent myeloid
potential, sorted cells (1 × 102–5 × 103) in 96-well plates were cultured with 10 ng/ml SCF and10 ng/ml IL-3 for 6 days. Half of the medium was replaced every 3 days, with cytokines added.
For detection of B-cell potential, sorted cells (2 × 102) were cultured with a monolayer of thymic stromal cells (TSt4) containing 2 ng/ml IL-7 for 15 days [24]. For detection of T-cell potential, sorted cells (2 × 102) were cultured with TSt4 cells expressing DLL1, TSt4/DLL1 cells,
containing 2 ng/ml IL-7 and 2 ng/ml Flt3L for 18 days [24]. Half of the medium was replaced
every 7 days, with cytokines added. The following cell surface markers were used to identify
respective cells: mDC; MHC class II+CD11c+, pDC; Siglec H+PDCA1+CD11c+, cDC; Siglec
H−PDCA1−CD11c+, neutrophil; Ly6G+CD11b+ or Gr-1+CD11b+, macrophage; F4/80+CD11b+, mast cell; c-Kit+FcεR1α+CD11b−, basophil; CD49b+FcεR1α+CD11b−, B cell;
B220+CD19+, double positive T cell; and CD4+CD8+.

Flow cytometry
Flow cytometry was performed on a FACS Canto II (BD Bioscience, San Jose, CA) and data
were analyzed using FlowJo Software (Tree Star, Ashland, OR). Cell number was counted
using flow cytometry unless otherwise indicated. For intracellular cytokine staining, cells were
fixed with Fixation Buffer (BD Bioscience) for 30 min and permeabilized with Perm Buffer II(BD Bioscience) for 30 min. Then, samples were stained with antibodies for pY701-STAT1 andpY705-STAT3.

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 201618 / 25-177-

Role of IL-27 in Emergency Myelopoiesis

Quantitative real-time RT-PCR
Total RNA was prepared using RNeasy Mini Kit (QIAGEN, Hilden, Germany), and cDNA wasprepared using oligo(dT) primer and SuperScript III RT (Invitrogen, Carlsbad, CA, USA).
Real-time quantitative PCR was performed using SYBR Premix Ex Taq II and a Thermal cycler
Dice real-time system according to the manufacturer’s instructions (TAKARA, Otsu, Shiga,
Japan). Glyceraldehyde-3-phosphate (GAPDH) was used as housekeeping gene to normalize
mRNA. Relative expression of real-time PCR products was determined by using the ΔΔCt
method to compare target gene and GAPDH mRNA expression. Primers used in this study arelisted in S1 Table.

Adoptive cell transfer
For in vivo proliferation analysis, BM LSK cells (8 × 103) purified from GFP Tg mice wereintravenously transferred into irradiated (4 Gy) WT and IL-27 Tg mice. To evaluate in vivo
development, IL-27/SCF-cultured BM LSK cells (2 × 104) from CD45.1 congenic mice weretransplanted into sublethally irradiated (6 Gy) CD45.2 recipient mice with the same congenic
BM cells (2 × 106). As in the case of malaria infection, BM LSK cells (5 × 104) were sorted frommalaria-infected CD45.1 mice and intravenously transferred into irradiated (4 Gy) WSX1-deficient mice 7 days before infection.

Retroviral transduction
The retroviral vector pMX-Cre-GFP (from Dr. M. Kubo) was transfected into Platinum-E
packaging cells [65] using FuGENE 6 (Promega, Madison, WI), and supernatants of these cultures were used as the source of viral particles. LSK cells sorted from BM cells of STAT3flox/flox
mice were stimulated with IL-27 and SCF (10 ng/ml each) and transduced with viral particles
by spin infection (2,000 rpm, 90 min, 25°C) using 8 μg/ml Polybrene at 24 hr and 48 hr later.
The next day, GFP+ LSK cells were sorted and used as STAT3 cKO LSK cells.

Malaria infection
Mice were injected intravenously with a red blood cell (RBC) suspension containing parasitized
RBC (1 × 104) with the nonlethal strain P. berghei XAT [35], which is an irradiation-induced
attenuated variant of the lethal strain P. berghei NK65, or the P. berghei NK65 [46]. Parasitemia
was assessed by the microscopic examination of Giemsa-stained smears of tail blood after
infection. The percentage of parasitemia was calculated as follows: parasitemia (%) = [(numberof infected RBC) / (total number of RBC counted)] × 100.

Hydrodynamic tail-vein injection
IFN-γ-deficient mice were intravenously injected with 25 μg of p3xFLAG-CMV (Sigma Chemical Co., St. Louis, MO), or p3xFLAG-IL-27 plasmids at days 0 and 4 after malaria infection.

ELISA
Amounts of IL-27 p28 in culture supernatants or serum were determined by using Quantikine
kits (R&D) according to the manufacturer’s instruction.

Statistical analysis
Data are represented as mean ± SEM. Statistical analyses were performed by two-tailed Student’s t test for two groups, and by one-way ANOVA and Bonferroni’s multiple comparison

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 201619 / 25-178-

Role of IL-27 in Emergency Myelopoiesis

tests for multiple groups. P < 0.05 was considered to indicate a statistically significant
difference.

Supporting Information
S1 Table. Primers used in this study.
(TIF)
S1 Fig. IL-27 possesses the strongest ability to augment the expansion of LSK cells retaining
the LSK phenotype among various cytokines. LSK cells (5 × 102) from WT mice were stimulated by various cytokines together with SCF. The stimulated and expanded cells were analyzed
for expression of c-Kit and Sca-1 in the Lin− population by flow cytometry 1 week (A) and 3
weeks (B) later, and cell number of the LSK cell population was counted. Data are shown asmean ± SEM (n = 3–4) and are representative of two independent experiments.
(TIF)
S2 Fig. LSK cells in the BM cells of IL-27 Tg mice have enhanced ability to differentiate
into neutrophils. (A) An increased number of LSK cells in IL-27 Tg mice. BM and spleen cellsof WT and IL-27 Tg mice were analyzed for the expression of c-Kit and Sca-1 in the Lin− population by flow cytometry, and cell number of the LSK cell population was counted. (B-D) Augmented potential of LSK cells in IL-27 Tg mice to differentiate into neutrophils, but markedly
reduced differentiation into pDCs and cDCs. LSK populations (3 × 103) purified from BM cellsof WT and IL-27 Tg mice were stimulated with GM-CSF. After 10 days, stimulated cells wereanalyzed for the expression of MHC class II and CD11c, and cell number of mDC (MHC class
II+CD11c+) was counted (B). LSK populations (5 × 103) were also stimulated with Flt3L and
TPO. Ten days later, stimulated cells were analyzed for the expression of Siglec H and PDCA1in the CD11c+ population, and the cell numbers of pDC and cDC were counted (C). The LSK
populations were also stimulated with IL-3 and SCF. Six days later, these stimulated cells wereanalyzed regarding their multipotency in differentiating to Ly6G+CD11b+ neutrophils, F4/80+CD11b+ macrophages, c-Kit+FcεR1α+CD11b− mast cells, and CD49b+FcεR1α+CD11b−basophils, and the cell numbers of respective cells were counted (D). Data are shown asmean ± SEM (n = 3) and are representative of two to four independent experiments.  P < 0.05,

P < 0.01,  P < 0.005.
(TIF)
S3 Fig. Increased expression of Cebpb in the LSK cells expanded by IL-27 and SCF, and thehighest expression of WSX-1 in primary LSK cells among hematopoietic progenitors. RNA
was prepared from the LSK cells expanded by IL-27 and SCF for 2 weeks together with primary
LSK cells and other progenitors, and subjected to real-time RT-PCR. Data are shown asmean ± SEM (n = 2–4) and are representative of two independent experiments.
(TIF)
S4 Fig. IL-27 alone, but not SCF alone, induces phosphorylation of STAT1 and STAT3.
Flow cytometry histogram analysis of primary LSK cells after stimulation with the combination
with IL-27 (10 ng/ml) and SCF (10 ng/ml), IFN-γ (100 U/ml) and SCF (10 ng/ml), or eachalone for 60 min using anti-pY-STAT1 or anti-pY-STAT3 (solid line) and control antibody(plain line with shading). Data are shown as mean ± SEM (n = 3).
(TIF)
S5 Fig. Time course of parasitemia and serum IFN-γ level in WT and WSX-1-deficient
mice after infection with the blood stage of P. berghei XAT. WT mice and WSX-1-deficient
mice were infected with the blood stage of P. berghei XAT and parasitemia was measured with

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 2016

20 / 25-179-

Role of IL-27 in Emergency Myelopoiesis

time course after infection (A). Serum IFN-γ level was determined 14 days later (B). Data areshown as mean ± SEM (n = 3–5) and are representative of at least two independent experiments.  P < 0.05,  P < 0.01.
(TIF)
S6 Fig. Reduced potential of LSK cells from WSX-1-deficient mice to differentiate into
myeloid cells compared with those from WT mice after malaria infection. LSK cells in the
BM and spleen of WT and WSX-1-deficient mice infected with malaria for 7 days were purified
and differentiated into myeloid cells in vitro by IL-3 and SCF, and cell number of differentiatedcells was measured. Data are shown as mean ± SEM (n = 3) and are representative of at least
two independent experiments.  P < 0.05,  P < 0.01,  P < 0.005.
(TIF)
S7 Fig. Promotion of expansion of LSK cells by IL-27 in a cell-autonomous direct manner.
BM cells (1 × 106) from CD45.1 congenic mice and BM cells (1 × 106) from WSX-1-deficient
mice (CD45.2) were equally mixed and transferred into lethally (9 Gy) irradiated CD45.2 recipient mice. After 7 days, these mice were infected with the blood stage of P. berghei XAT; an
additional 7 days later, and populations of LSK cells (A) and neutrophils (B) in the BM andspleen were analyzed by flow cytometry. Representative dot plots of CD45.1+ and CD45.2+
cells in these populations are shown and percentages of these CD45.1+ and CD45.2+ cells ineach population were compared. Data are shown as mean ± SEM (n = 3–4).  P < 0.05,

P < 0.005.
(TIF)
S8 Fig. Expression of cytokine and chemokine mRNA in the BM and spleen after malaria
infection. WT and IFN-γ-deficient mice were infected with the blood stage of P. berghei XAT,
and RNA was prepared from BM and spleen of WT and IFN-γ-deficient mice 7 days after the
infection, and the mRNA expression levels of cytokines were analyzed as indicated by real-time
RT-PCR. Data are shown as mean ± SEM (n = 3) and are representative of two independent
experiments.  P < 0.05,  P < 0.005.
(TIF)
S9 Fig. Augmentation of mRNA expression of IL-27 p28, but not IL-6, by IFN-γ in WT BM
cells in vitro. Total BM cells of WT mice were stimulated with IFN-γ (100 U/ml) for 48 hr andthe mRNA expression of IL-27 p28, EBI3, and IL-6 was analyzed by real-time RT-PCR (A). IL27 p28 levels in culture supernatants were also determined by ELISA (B). Data are shown asmean ± SEM (n = 3) and are representative of two independent experiments.  P < 0.05,

P < 0.005.
(TIF)
S10 Fig. mRNA expression of transcription factors and molecule critical for cell differentiation and proliferation in STAT1 KO and STAT3 cKO LSK cells. (A) LSK cells purified fromBM cells of WT (129) mice and STAT1-deficient mice were expanded by IL-27 and SCF for 2
weeks, and the LSK population was then purified by sorting and subjected to real-time
RT-PCR. (B) Purified GFP− STAT3flox/flox LSK cells and GFP+ STAT3 cKO LSK cells wereexpanded by IL-27 and SCF for 10 days, and the LSK population was then purified by sortingand subjected to real-time RT-PCR. Data are shown as mean ± SEM (n = 3–4) and representative of two to three independent experiments.  P < 0.05,  P < 0.005.
(TIF)
S11 Fig. There was no apparent difference in the parasitemia and expansion of LSK cells
and neutrophils between WT mice and WSX-1-deficient mice 7 days after lethal P. berghei

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 2016

21 / 25-180-

Role of IL-27 in Emergency Myelopoiesis

NK65 infection. WT or WSX-1-deficient mice were infected with the blood stage of P. berghei
NK65. Seven days later, parasitemia was determined (A), and populations of LSK cells (B) andneutrophils (C) in the BM and spleen were analyzed by flow cytometry, and representative dot
plots of c-Kit and Sca-1 in the Lin− population and CD11b and Gr-1 are shown. Cell numberof these populations in the BM and spleen was also counted. Data are shown as mean ± SEM(n = 2–4).  P < 0.05.
(TIF)
S12 Fig. Dispensable role of IL-6 in infection with the blood stage of P. berghei XAT. (A)Comparable susceptibility of WT and IL-6-deficient mice to malaria. WT and IL-6-deficient
mice were infected with the blood stage of P. berghei XAT and parasitemia was counted at 7days after infection. Data are shown as mean ± SEM (n = 5) and are representative of at least
two independent experiments.  P < 0.05,  P < 0.01,  P < 0.005. (B-C) Similar cell numbersof the LSK cell population and neutrophils in the BM and spleen of WT and IL-6-deficient
mice compared with mice infected with malaria. BM and spleen cells were analyzed by flow
cytometry 7 days after malaria infection; representative dot plots of c-Kit and Sca-1 in the Lin−
population (B) and Gr-1 and CD11b (C) are shown. The cell numbers of the LSK cell population and neutrophils were counted (B-C). Data are shown as mean ± SEM (n = 3) and are representative of two independent experiments.  P < 0.05,  P < 0.01,  P < 0.005.
(TIF)

Acknowledgments
The authors thank Dr. K. Takeda (Osaka University), Dr. Y. Iwakura (Tokyo University of Science), Dr. M. Kubo (Tokyo University of Science), Dr. K. Ikawa (RIKEN) and Dr. H. Kawamoto (University of Kyoto), Dr. M. Okabe (Osaka University) and Dr. M. Ito (Tokyo Medical
University), and the Animal Research Center of Tokyo Medical University for STAT3flox/flox
mice, IFN-γ-deficient mice, pMX-Cre-GFP, TSt-4 and TSt-4/DLL1, GFP Tg mice, and animalcare.

Author Contributions
Conceived and designed the experiments: TY JF MH AT. Performed the experiments: JF IMYC TM HE JM. Analyzed the data: JF IM YC. Contributed reagents/materials/analysis tools:
FK HY SN. Wrote the paper: TY JF. Directed the study: TY. Provided technical support anddiscussed results: MH HE TM AT JM.

References
1.

Takizawa H, Boettcher S, Manz MG (2012) Demand-adapted regulation of early hematopoiesis ininfection and inflammation. Blood 119: 2991–3002. doi: 10.1182/blood-2011-12-380113 PMID:
22246037

2.

King KY, Goodell MA (2011) Inflammatory modulation of HSCs: viewing the HSC as a foundation for
the immune response. Nat Rev Immunol 11: 685–692. doi: 10.1038/nri3062 PMID: 21904387

3.

Sato T, Onai N, Yoshihara H, Arai F, Suda T, et al. (2009) Interferon regulatory factor-2 protects quiescent hematopoietic stem cells from type I interferon-dependent exhaustion. Nat Med 15: 696–700. doi:
10.1038/nm.1973 PMID: 19483695

4.

Essers MA, Offner S, Blanco-Bose WE, Waibler Z, Kalinke U, et al. (2009) IFNalpha activates dormant
haematopoietic stem cells in vivo. Nature 458: 904–908. doi: 10.1038/nature07815 PMID: 19212321

5.

Baldridge MT, King KY, Boles NC, Weksberg DC, Goodell MA (2010) Quiescent haematopoietic stem
cells are activated by IFN-gamma in response to chronic infection. Nature 465: 793–797. doi: 10.1038/
nature09135 PMID: 20535209

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 201622 / 25-181-

Role of IL-27 in Emergency Myelopoiesis

6.

Belyaev NN, Brown DE, Diaz AI, Rae A, Jarra W, et al. (2010) Induction of an IL7-R(+)c-Kit(hi) myelolymphoid progenitor critically dependent on IFN-gamma signaling during acute malaria. Nat Immunol
11: 477–485. doi: 10.1038/ni.1869 PMID: 20431620

7.

Broxmeyer HE, Cooper S, Rubin BY, Taylor MW (1985) The synergistic influence of human interferongamma and interferon-alpha on suppression of hematopoietic progenitor cells is additive with theenhanced sensitivity of these cells to inhibition by interferons at low oxygen tension in vitro. J Immunol
135: 2502–2506. PMID: 3928752

8.

Chen J, Lipovsky K, Ellison FM, Calado RT, Young NS (2004) Bystander destruction of hematopoietic
progenitor and stem cells in a mouse model of infusion-induced bone marrow failure. Blood 104: 1671–
1678. PMID: 15166031

9.

Murphy M, Loudon R, Kobayashi M, Trinchieri G (1986) Gamma interferon and lymphotoxin, releasedby activated T cells, synergize to inhibit granulocyte/monocyte colony formation. J Exp Med 164: 263–
279. PMID: 3088195

10.

de Bruin AM, Demirel O, Hooibrink B, Brandts CH, Nolte MA (2013) Interferon-gamma impairs proliferation of hematopoietic stem cells in mice. Blood 121: 3578–3585. doi: 10.1182/blood-2012-05-432906
PMID: 23487025

11.

Pietras EM, Lakshminarasimhan R, Techner JM, Fong S, Flach J, et al. (2014) Re-entry into quiescence protects hematopoietic stem cells from the killing effect of chronic exposure to type I interferons.
J Exp Med 211: 245–262. doi: 10.1084/jem.20131043 PMID: 24493802

12.

Schurch CM, Riether C, Ochsenbein AF (2014) Cytotoxic CD8+ T cells stimulate hematopoietic progenitors by promoting cytokine release from bone marrow mesenchymal stromal cells. Cell Stem Cell
14: 460–472. doi: 10.1016/j.stem.2014.01.002 PMID: 24561082

13.

Guermonprez P, Helft J, Claser C, Deroubaix S, Karanje H, et al. (2013) Inflammatory Flt3l is essential
to mobilize dendritic cells and for T cell responses during Plasmodium infection. Nat Med 19: 730–738.
doi: 10.1038/nm.3197 PMID: 23685841

14.

Hall AO, Silver JS, Hunter CA (2012) The Immunobiology of IL-27. Adv Immunol 115: 1–44. doi: 10.
1016/B978-0-12-394299-9.00001-1 PMID: 22608254

15.

Mizoguchi I, Higuchi K, Mitobe K, Tsunoda R, Mizuguchi J, et al. (2013) Interleukin-27: Regulation of
immune responses and disease development by a pleiotropic cytokine with pro- and anti-inflammatory
properties. In: Yoshimoto T, Yoshimoto T, editors. Cytokine frontiers: Regulation of immune responses
in health and disease Tokyo: Springer. pp. 353–375.

16.

Yoshida H, Hunter CA (2015) The immunobiology of interleukin-27. Annu Rev Immunol 33: 417–443.
doi: 10.1146/annurev-immunol-032414-112134 PMID: 25861977

17.

Seita J, Asakawa M, Ooehara J, Takayanagi S, Morita Y, et al. (2008) Interleukin-27 directly induces
differentiation in hematopoietic stem cells. Blood 111: 1903–1912. PMID: 18042804

18.

Osawa M, Hanada K, Hamada H, Nakauchi H (1996) Long-term lymphohematopoietic reconstitution bya single CD34-low/negative hematopoietic stem cell. Science 273: 242–245. PMID: 8662508

19.

Yamamoto R, Morita Y, Ooehara J, Hamanaka S, Onodera M, et al. (2013) Clonal analysis unveils selfrenewing lineage-restricted progenitors generated directly from hematopoietic stem cells. Cell 154:
1112–1126. doi: 10.1016/j.cell.2013.08.007 PMID: 23993099

20.

Kondo M, Weissman IL, Akashi K (1997) Identification of clonogenic common lymphoid progenitors inmouse bone marrow. Cell 91: 661–672. PMID: 9393859

21.

Akashi K, Traver D, Miyamoto T, Weissman IL (2000) A clonogenic common myeloid progenitor thatgives rise to all myeloid lineages. Nature 404: 193–197. PMID: 10724173

22.

Onai N, Kurabayashi K, Hosoi-Amaike M, Toyama-Sorimachi N, Matsushima K, et al. (2013) A clonogenic progenitor with prominent plasmacytoid dendritic cell developmental potential. Immunity 38:
943–957. doi: 10.1016/j.immuni.2013.04.006 PMID: 23623382

23.

van de Laar L, Coffer PJ, Woltman AM (2012) Regulation of dendritic cell development by GM-CSF:
molecular control and implications for immune homeostasis and therapy. Blood 119: 3383–3393. doi:
10.1182/blood-2011-11-370130 PMID: 22323450

24.

Masuda K, Kubagawa H, Ikawa T, Chen CC, Kakugawa K, et al. (2005) Prethymic T-cell development
defined by the expression of paired immunoglobulin-like receptors. EMBO J 24: 4052–4060. PMID:
16292344

25.

Rosenbauer F, Tenen DG (2007) Transcription factors in myeloid development: balancing differentiation with transformation. Nat Rev Immunol 7: 105–117. PMID: 17259967

26.

Geissmann F, Manz MG, Jung S, Sieweke MH, Merad M, et al. (2010) Development of monocytes,
macrophages, and dendritic cells. Science 327: 656–661. doi: 10.1126/science.1178331 PMID:
20133564

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 201623 / 25-182-

Role of IL-27 in Emergency Myelopoiesis

27.

Belz GT, Nutt SL (2012) Transcriptional programming of the dendritic cell network. Nat Rev Immunol
12: 101–113. doi: 10.1038/nri3149 PMID: 22273772

28.

Medvedovic J, Ebert A, Tagoh H, Busslinger M (2011) Pax5: a master regulator of B cell development
and leukemogenesis. Adv Immunol 111: 179–206. doi: 10.1016/B978-0-12-385991-4.00005-2 PMID:
21970955

29.

Kaneko H, Shimizu R, Yamamoto M (2010) GATA factor switching during erythroid differentiation. Curr
Opin Hematol 17: 163–168. doi: 10.1097/MOH.0b013e32833800b8 PMID: 20216212

30.

Hirai H, Zhang P, Dayaram T, Hetherington CJ, Mizuno S, et al. (2006) C/EBPbeta is required for 'emergency' granulopoiesis. Nat Immunol 7: 732–739. PMID: 16751774

31.

Hirai H, Yokota A, Tamura A, Sato A, Maekawa T (2015) Non-steady-state hematopoiesis regulated by
the C/EBPbeta transcription factor. Cancer Sci.

32.

Manz MG, Boettcher S (2014) Emergency granulopoiesis. Nat Rev Immunol 14: 302–314. doi: 10.
1038/nri3660 PMID: 24751955

33.

Ronnstrand L (2004) Signal transduction via the stem cell factor receptor/c-Kit. Cell Mol Life Sci 61:
2535–2548. PMID: 15526160

34.

Lee CK, Smith E, Gimeno R, Gertner R, Levy DE (2000) STAT1 affects lymphocyte survival and proliferation partially independent of its role downstream of IFN-gamma. J Immunol 164: 1286–1292. PMID:
10640742

35.

Yoshimoto T, Yoneto T, Waki S, Nariuchi H (1998) Interleukin-12-dependent mechanisms in the clearance of blood-stage murine malaria parasite Plasmodium berghei XAT, an attenuated variant of P. berghei NK65. J Infect Dis 177: 1674–1681. PMID: 9607848

36.

Yoneto T, Waki S, Takai T, Tagawa Y, Iwakura Y, et al. (2001) A critical role of Fc receptor-mediatedantibody-dependent phagocytosis in the host resistance to blood-stage Plasmodium berghei XAT
infection. J Immunol 166: 6236–6241. PMID: 11342646

37.

Belyaev NN, Biro J, Langhorne J, Potocnik AJ (2013) Extramedullary myelopoiesis in malaria depends
on mobilization of myeloid-restricted progenitors by IFN-gamma induced chemokines. PLoS Pathog 9:e1003406. doi: 10.1371/journal.ppat.1003406 PMID: 23762028

38.

Yoneto T, Yoshimoto T, Wang CR, Takahama Y, Tsuji M, et al. (1999) Gamma interferon production is
critical for protective immunity to infection with blood-stage Plasmodium berghei XAT but neither NO
production nor NK cell activation is critical. Infect Immun 67: 2349–2356. PMID: 10225894

39.

Borner C (2003) The Bcl-2 protein family: sensors and checkpoints for life-or-death decisions. Mol
Immunol 39: 615–647. PMID: 12493639

40.

Chipuk JE, Moldoveanu T, Llambi F, Parsons MJ, Green DR (2010) The BCL-2 family reunion. Mol Cell
37: 299–310. doi: 10.1016/j.molcel.2010.01.025 PMID: 20159550

41.

Cisse B, Caton ML, Lehner M, Maeda T, Scheu S, et al. (2008) Transcription factor E2-2 is an essential
and specific regulator of plasmacytoid dendritic cell development. Cell 135: 37–48. doi: 10.1016/j.cell.
2008.09.016 PMID: 18854153

42.

Liu Z, Liu JQ, Talebian F, Wu LC, Li S, et al. (2013) IL-27 enhances the survival of tumor antigen-specific CD8+ T cells and programs them into IL-10-producing, memory precursor-like effector cells. Eur J
Immunol 43: 468–479. doi: 10.1002/eji.201242930 PMID: 23225163

43.

Waki S (1994) Antibody-dependent neutrophil-mediated parasite killing in non-lethal rodent malaria.
Parasite Immunol 16: 587–591. PMID: 7532294

44.

Nnalue NA, Friedman MJ (1988) Evidence for a neutrophil-mediated protective response in malaria.
Parasite Immunol 10: 47–58. PMID: 3281102

45.

Pierrot C, Adam E, Hot D, Lafitte S, Capron M, et al. (2007) Contribution of T cells and neutrophils inprotection of young susceptible rats from fatal experimental malaria. J Immunol 178: 1713–1722.
PMID: 17237421

46.

Yoshimoto T, Takahama Y, Wang CR, Yoneto T, Waki S, et al. (1998) A pathogenic role of IL-12 inblood-stage murine malaria lethal strain Plasmodium berghei NK65 infection. J Immunol 160: 5500–
5505. PMID: 9605153

47.

Findlay EG, Greig R, Stumhofer JS, Hafalla JC, de Souza JB, et al. (2010) Essential role for IL-27
receptor signaling in prevention of Th1-mediated immunopathology during malaria infection. J Immunol
185: 2482–2492. doi: 10.4049/jimmunol.0904019 PMID: 20631310

48.

Villegas-Mendez A, de Souza JB, Lavelle SW, Gwyer Findlay E, Shaw TN, et al. (2013) IL-27 receptor
signalling restricts the formation of pathogenic, terminally differentiated Th1 cells during malaria infection by repressing IL-12 dependent signals. PLoS Pathog 9: e1003293. doi: 10.1371/journal.ppat.
1003293 PMID: 23593003

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 201624 / 25-183-

Role of IL-27 in Emergency Myelopoiesis

49.

Gwyer Findlay E, Villegas-Mendez A, de Souza JB, Inkson CA, Shaw TN, et al. (2013) IL-27 receptor
signaling regulates CD4+ T cell chemotactic responses during infection. J Immunol 190: 4553–4561.
doi: 10.4049/jimmunol.1202916 PMID: 23536628

50.

Liu FD, Kenngott EE, Schroter MF, Kuhl A, Jennrich S, et al. (2014) Timed action of IL-27 protects from
immunopathology while preserving defense in influenza. PLoS Pathog 10: e1004110. doi: 10.1371/
journal.ppat.1004110 PMID: 24809349

51.

Wirtz S, Tubbe I, Galle PR, Schild HJ, Birkenbach M, et al. (2006) Protection from lethal septic peritonitis by neutralizing the biological function of interleukin 27. J Exp Med 203: 1875–1881. PMID:
1688026052.

Li JP, Wu H, Xing W, Yang SG, Lu SH, et al. (2010) Interleukin-27 as a negative regulator of humanneutrophil function. Scand J Immunol 72: 284–292. doi: 10.1111/j.1365-3083.2010.02422.x PMID:
20883313

53.

Liu J, Guan X, Ma X (2007) Regulation of IL-27 p28 gene expression in macrophages through MyD88and interferon-gamma-mediated pathways. J Exp Med 204: 141–152. PMID: 17227910

54.

Murugaiyan G, Mittal A, Weiner HL (2010) Identification of an IL-27/osteopontin axis in dendritic cells
and its modulation by IFN-gamma limits IL-17-mediated autoimmune inflammation. Proc Natl Acad Sci
U S A 107: 11495–11500. doi: 10.1073/pnas.1002099107 PMID: 20534530

55.

Murugaiyan G, Beynon V, Pires Da Cunha A, Joller N, Weiner HL (2012) IFN-gamma limits Th9-mediated autoimmune inflammation through dendritic cell modulation of IL-27. J Immunol 189: 5277–5283.
doi: 10.4049/jimmunol.1200808 PMID: 23125412

56.

Tormo AJ, Beaupre LA, Elson G, Crabe S, Gauchat JF (2013) A polyglutamic acid motif confers IL-27
hydroxyapatite and bone-binding properties. J Immunol 190: 2931–2937. doi: 10.4049/jimmunol.
1201460 PMID: 23390294

57.

Morrison SJ, Scadden DT (2014) The bone marrow niche for haematopoietic stem cells. Nature 505:
327–334. doi: 10.1038/nature12984 PMID: 24429631

58.

Heinrich PC, Behrmann I, Haan S, Hermanns HM, Muller-Newen G, et al. (2003) Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J 374: 1–20. PMID: 12773095

59.

Drucker C, Gewiese J, Malchow S, Scheller J, Rose-John S (2010) Impact of interleukin-6 classic- andtrans-signaling on liver damage and regeneration. J Autoimmun 34: 29–37. doi: 10.1016/j.jaut.2009.
08.003 PMID: 19717281

60.

Wunderlich CM, Delic D, Behnke K, Meryk A, Strohle P, et al. (2012) Cutting edge: Inhibition of IL-6trans-signaling protects from malaria-induced lethality in mice. J Immunol 188: 4141–4144. doi: 10.
4049/jimmunol.1102137 PMID: 22467660

61.

Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y (1997) 'Green mice' as a source of ubiquitous green cells. FEBS Lett 407: 313–319. PMID: 9175875

62.

Yoshida H, Hamano S, Senaldi G, Covey T, Faggioni R, et al. (2001) WSX-1 is required for the initiationof Th1 responses and resistance to L. major infection. Immunity 15: 569–578. PMID: 11672539

63.

Morishima N, Mizoguchi I, Takeda K, Mizuguchi J, Yoshimoto T (2009) TGF-beta is necessary for
induction of IL-23R and Th17 differentiation by IL-6 and IL-23. Biochem Biophys Res Commun 386:
105–110. doi: 10.1016/j.bbrc.2009.05.140 PMID: 19501566

64.

Yoshimoto T, Okada K, Morishima N, Kamiya S, Owaki T, et al. (2004) Induction of IgG2a class switching in B cells by IL-27. J Immunol 173: 2479–2485. PMID: 15294962

65.

Morita S, Kojima T, Kitamura T (2000) Plat-E: an efficient and stable system for transient packaging ofretroviruses. Gene Ther 7: 1063–1066. PMID: 10871756

PLOS Pathogens | DOI:10.1371/journal.ppat.1005507 March 18, 201625 / 25-184-

ONCOLOGY REPORTS 35: 992-998, 2016

992

Hypoxia inducible factor-1 mediates upregulation of
urokinase-type plasminogen activator receptor genetranscription during hypoxia in cervical cancer cells
Hirotaka Nishi1, Toru Sasaki1, Yuzo Nagamitsu1, Fumitoshi Terauchi1,
Takeshi Nagai2, Toshitaka Nagao2 and Keiichi Isaka1
1

Department of Obstetrics and Gynecology, and 2Department of Anatomic Pathology,
Tokyo Medical University, Shinjuku-ku, Tokyo 160-0023, Japan
Received September 18, 2015; Accepted October 26, 2015
DOI: 10.3892/or.2015.4449

Abstract. Hypoxia occurs during development of cervicalcancer and is considered to correlate with its invasion. Hypoxia
mediates tumor cells to have more invasive property in avariety of cancers. Urokinase plasminogen activator receptor(uPAR) which mediates invasion is considered to be inducedby hypoxia. We sought to determine the regulators of uPAR
expression during hypoxia in cervical cancer. We showed thatcervical cancer cell lines, CaSki and CA, were more invasive
under hypoxic condition (1% O2) than under normoxic condition (20% O2) by invasion assays. Using western blot analysis,
hypoxia enhanced the endogenous hypoxia-inducible factor(HIF)-1α and uPAR protein expression. uPAR mRNA level
was also upregulated by hypoxia using real-time RT-PCR.
Overexpression of HIF-1α which is induced by hypoxia
activated the transcriptional activity of the uPAR promoter
by luciferase assays. HIF-1 protein bound the putative HIF-1
response element on the uPAR promoter using electrophoretic
mobility shift analysis, and additional luciferase assays showthat this is essential for uPAR transactivation by HIF-1. HIF-1
overexpression enhanced the endogenous uPAR expression
and introduction of siRNA for HIF-1α diminishes uPAR
expression during hypoxia. These results indicate the upregulation of uPAR by hypoxia in cervical cancer cells is mediatedthrough HIF-1. In cervical cancer tissues, we also demonstrated that uPAR protein expression was detected in cervicalcancer but not in normal cervix or cervical intraepithelial
neoplasia (CIN) by immunohistopathological staining. Our
results provide evidence that regulation of uPAR expression
by HIF-1 represents a mechanism for cervical cancer invasion
during hypoxia.

Correspondence to: Dr Hirotaka Nishi, Department of Obstetrics
and Gynecology, Tokyo Medical University, 6-7-1 Nishishinjuku,
Shinjuku-ku, Tokyo 160-0023, Japan
E-mail: nishih@tokyo-med.ac.jp

Key words: hypoxia, cervical cancer, uPAR promoter, invasion

Introduction
Hypoxia within solid tumors may have a profound impact
in malignant progression. Hypoxia within the tumor mass isan independent marker of poor prognosis for patients witha variety of cancers including cervical cancers, soft tissue
sarcoma and head neck cancers (1-3). Hypoxic stress underlies
a number of biologically important processes in which cellularmigration and invasion occur (4). Hypoxia may play an important role in promoting tumor metastasis and invasion into theextracellular matrix (5). Hypoxia upregulates the expression of
the urokinase plasminogen activator receptor (uPAR) which is
a component of a protease system implicated in tumor invasion and metastasis (6). The uPAR is activated by binding with
its ligand, urokinase plasminogen activator (uPA), allowing theconversion of inactive plasminogen into plasmin that in turncan degrade extracellular matrix proteins, facilitating invasion
by tumor cells (7).
Hypoxia-inducible factor (HIF)-1 is a heterodimeric transcriptional complex that plays a pivotal role in the regulationof cellular utilization of oxygen and is an essential regulator ofangiogenesis in solid tumor and ischemic disorders. HIF-1 iscomposed of the bHLH-PAS proteins HIF-1α and aryl hydrocarbon receptor nuclear translocator (ARNT). HIF-1 mediates
the transcriptional response to oxygen deprivation by binding
to HIF-1 response elements (HRE) within the promoters orenhancers of genes involved in glycolysis, glucose transport,
erythropoiesis and angiogenesis (8,9). The HIF-1 activity
is critical for cancer development and HIF-1 is essential for
proliferation, survival or differentiation of multiple tumor
tissues (9). In light of the above considerations, we investigated
how uPAR expression is induced by hypoxia in cervical cancer
cell lines. We also sought to determine the regulator of uPAR
expression during hypoxia. HIF-1 is a possible candidate forthe regulator of uPAR expression.
Materials and methods
Samples. The samples were obtained from patients undergoing
surgery or biopsy at Tokyo Medical University Hospital withinformed consent. We used ten normal cervices and 35 cervical-185-

nishi et al: HIF-1 activates uPAR in cervical cancer

cancers. To extract total RNA, the tissues were finely minced
into small pieces with scissors, washed in phosphate-bufferedsaline (PBS), snap-frozen and stored at -80˚C. For immunohistochemistry analysis, the tissues were formalin‑fixed andembedded in paraffin.
Cell culture. The human cervical cancer cell lines, CaSki andCA, were maintained in Modified Eagle Medium (MEM;
Invitrogen, Carlsbad, CA, USA) supplemented with 10%
fetal bovine serum (FBS). For hypoxic exposure, these cells(1.0x107 cells) were plated in 10 ml of medium in 100-mm
dishes and incubated overnight in 1% O2 in a water-jacketed
CO2 incubator (NAPCO, Winchester, VA, USA) at 37˚C in ahumidified atmosphere with 5% CO2 for indicated times. In
experiments using transfected cells, each transfection was
performed before exposure to hypoxia.
DNA plasmids. Sequence analysis of the human uPAR
promoter revealed one putative binding site for HIF-1, theHRE motifs 5'-BACGTSSK-3'. The putative HIF binding siteat position -34 to -39 was identified on the sense strand (10).
Luciferase reporter constructs, puPAR-141-luc, containing theuPAR promoter which is produced by PCR were preparedby ligation into pGL3-basic vector (Fig. 3A). We prepared
the puPAR-141mt-luc, where the putative HRE is mutated byreplacement of ACG with TTT using PCR-based site-specificmutagenesis. HIF-1α and ARNT constructs were kindly
provided by Dr L. Eric Huang (Departments of Neurosurgery
and Oncological Sciences, University of Utah, USA).
Invasion assays. For invasion assays, 12-mm-diameterTranswell polycarbonate filters (12-mm pore size; Costar) in a
modified Boyden chamber were coated with Matrigel (100 ml;
Sigma, St. Louis, MO, USA) at 1:20 dilution in serum-free
medium and air-dried for 24 h. Cells (5x104) in the complete
medium (200 ml) were seeded into the inner chamber. A totalof 600 ml of the medium was added to the lower chamber,and the plate was incubated at 37˚C in a 5% CO2/95% air
incubator (20% O2). For hypoxic treatment, plates containing
Boyden chambers were placed in 1% O2 in a water-jacketed
CO2 incubator (NAPCO) at 37˚C in a humidified atmospherewith 5% CO2 overnight. Cells on the lower surface of thefilter were scraped with a rubber scraper into the medium
from the lower chamber, pelleted, resuspended in the medium(50 ml), and counted using a hemocytometer. Each condition
was performed in quadruplicates, and the experiments werecarried out twice.
Real-time RT-PCR analysis. Total RNA was isolated using
Isogen reagent (Nippon Gene, Tokyo, Japan) and quantified by A260/A280 measurement using an Ultraspec 3000
(Amersham Biosciences, Piscataway, NJ, USA). Total RNA(50 mg) was reverse-transcribed into cDNA using an RT-PCR
kit (Stratagene, La Jolla, CA, USA) according to the manufacture's recommendations.
Real-time PCR was performed for the quantitative estimation of the DNA level. PCR reactions (20 ml) were set-up withthe final concentrations of 5 mM MgCl2, SYBR-Green MasterMix (2 ml; Roche Applied Science, Mannheim, Germany),cDNA (1:10 dilution; 5 ml), and forward and reverse primers

993(0.3 mM each). The primer sequences were as follows: 5'-CAA
CACCACCAAATGCAACG-3' (forward), and 5'-GGTTTTT
CGGTTCGTGAGTG-3' (reverse). The reactions were thencycled in a LightCycler (Roche Applied Science) with thefollowing parameters: denaturation for 1 cycle at 95˚C for
10 sec, 45 cycles (temperature transition of 20˚C/sec) of 95˚C
for 10 sec, 62˚C for 10 sec, and 72˚C for 6 sec. Fluorescencereadings were taken at 72˚C and, melting curve analysis was
performed with continuous fluorescence reading. The
LightCycler software generated a standard curve (frommeasurements taken during the exponential phase of theamplification) that enabled the amount of each gene in eachtest sample to be determined.
Western blot analysis. CaSki and CA cells were seeded
at 2.5x10 6 cells/150 mm dish, and incubated overnight at
37˚C. Subsequently, cells were exposed to hypoxic conditions. Cells were harvested and lysed on ice for 30 min. In
lysis buffer [10 mM Tris (pH 8.0), 1 mM EDTA, 400 mM
NaCl, 10% glycerol, 0.5% NP40, 5 mM sodium fluoride,
0.1 mM phenylmetylsulphonyl fluoride and 1 mM DTT],
containing complete protease inhibitor cocktail (Boehringer
Mannheim, Indianapolis, IN, USA). The lysate was centrifuged at 14,000 rpm for 15 min and the soluble fractions
were collected. Protein concentrations were measured using
Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules,
CA, USA). Equal amounts of protein (40 mg) were loaded
onto a 4-12% SDS-polyacrylamide gel and subjected to electrophoresis at 200 V for 50 min. The protein was transferred
onto a polyviniylidene difluoride membrane and probed withan anti‑uPAR antibody (FL-290; Santa Cruz Biotechnology,
Santa Cruz, CA, USA), anti-HIF-1α antibody (NB100-105;
Novus Biologicals, Littleton, CO, USA), and anti-actin antibody (C4; Boehringer Mannheim). The same blot was probedwith different antibodies after stripping the membrane. Each
protein was detected by horseradish peroxidase-conjugatedsecondary antibody coupled with enhanced chemiluminescence western blotting detection reagents (Amersham
Biosciences). Each band intensity was normalized to the intensity of the actin band.
Transfection and luciferase assays. Each cell line was seeded
at 5.0x105 cells/35-mm dish and incubated overnight at 37˚Cin a 5% CO2 incubator. For each transfection, empty or expression vectors (1.0 mg) along with the promoter-luciferase DNA(0.3 mg) were mixed in Opti-MEM (0.2 ml) and a precipitate
was formed using Lipofectamine 2000 (both from Invitrogen)according to the manufacturer's instructions. Cells were washedwith Opti-MEM, and complexes were applied to the cells.
After transfection for 24 h, cells were harvested and extracts
were prepared with the Glo Lysis Buffer (Promega, Madison,
WI, USA). Luciferase activity was measured in extracts from
triplicate samples using the Bright-Glo Luciferase Assay
system (Promega).
Electrophoretic mobility shift assays. The HIF-1 protein was
synthesized in vitro in the presence of unlabeled amino acids
using the pcDNA3-HIF-1α and pcDNA3-ARNT expression
constructs with the coupled transcription/translation system(TNT) from Promega (11). Translated products were analyzed

-186-

994

ONCOLOGY REPORTS 35: 992-998, 2016

by western blotting using anti-HIF-1α antibody (NB100-105;
Novus Biologicals).
Oligonucleotides containing the HIF-1 consensus
DNA-binding site from uPAR promoter (HRE/uPAR) andthe HIF-1 binding site from the erythropoietin promoter(HRE) were purchased as single-stranded DNAs from
Genosys Biotechnologies (Woodlands, TX, USA). Doublestranded oligonucleotides were prepared by annealing
complementary oligonucleotides, in a buffer containing
10 mM Tris (pH 8.0), 500 mM NaCl and 1 mM EDTA.
The sequences of the complementary pairs were as
follows: 5'-AAGGAGAGAAGACGTGCAGGGACCCC-3'
a nd 5'- G G G GT CCC T G CACGT C T T C T C T CC T T-3'
(HRE/uPAR); 5'-TCTGTACGTGACCACACTCACCTC-3'
and 5'-GAGGTGAGTGTGGTCACGTACAGA-3' (HRE).
Equimolar amounts of the complementary oligonucleotides
were mixed in a 1.5-ml microcentrifuge tube and placed ina heat block at 95˚C. The heat block was allowed to cool toroom temperature, and the samples were desalted on a G-25
Microspin column (Amersham). The double-stranded oligonucleotides were end-labeled with 32P using T4 polynucleitide
kinase and [γ-32P]-ATP. For electrophoretic mobility shift
analysis, end-labeled double stranded oligonucleotides,
5,000 cpm, were incubated with HIF-1 protein (2 ml) preparedby in vitro transcription/translation at room temperature (22˚C)
for 30 min in the presence of a binding buffer containing 10%
glycerol, 20 mM HEPES (pH 7.5), 25 mM KCl, 2 mM DTT,
2 mM MgCl2, 0.4 % NP-40 and 1 mg sheared salmon sperm
DNA. When competition assays were performed, an unlabeled
HIF-1 consensus sequence oligonucleotide was incubatedwith proteins and buffer for 5 min before the addition ofeach labeled oligonucleotide. For supershift assays, 0.5 mg of
HIF-1α antibody (OZ15; Lab Vision Corporation, Fremont,
CA, USA) was incubated with the binding mixtures for 5 min
before the addition of the labeled oligonucleotide. Samples(20 ml) were loaded onto a 5% non-denaturing polyacrylamide
gel and subjected to electrophoresis at 150 V for 1 h using
0.5X Tris‑borate EDTA [1X Tris-borate EDTA: 89 mM Tris,
8 mM boric asid and 2 mM EDTA (pH 8.3)] as running buffer.
After electrophoresis, gels were transferred to Whatman 3
MM paper and exposed to Kodak XAR film with intensifying
screens at -80˚C.
siRNA transfection. Gene silencing was achieved by transienttransfection of siRNA oligonucleotides encoding specificsequences for HIF-1α according to the manufacturer's instructions (Santa Cruz Biotechnology). In brief, CaSki and CA
were plated overnight in antibiotic-free medium to reach 50%
confluency. Cells were transfected the following morning
using Lipofectamine 2000 with 100 pmol/ml of siRNAoligonucleotides. Control siRNA was transfected to exclude
non-specific effects. After exposure to 1% O2 for 24 h, cells
were harvested for protein.
Immunohistopathological analysis. Immunohistochemistry
was performed on representative formalin-fixed, paraffin
embedded sections. Each tissue block was sectioned at
4-mm thickness, deparaffinized in xylene and rehydrated in
graded alcohols. Primary antibodies against uPAR (CD87,
clone HD-uPAR-13.1; 1:50 dilution; American Diagnostica,

Figure 1. Hypoxia induces invasion in cervical cancer cells. Error bars indicate standard deviation from quadruplicate assays.

Figure 2. Hypoxia induces uPAR protein expression. CaSki and CA cells
were cultured at 20% O2 or 1% O2 for 24 h. Whole cell extracts (40 mg) wereprepared and subjected to western blot analysis.

Stamford, CT, USA) and HIF-1α (NB100-105, clone
H1alpha67; 1:500 dilution; Novus Biologicals) were used.
Pretreatment for unmasking of antigens was carried out either
via protease XXIV (Sigma Chemicals, Perth, WA, USA) for
10 min at room temperature for uPAR, or by autoclaving for
10 min at 110˚C for HIF-1α.
Results
Hypoxia mediates invasion. To determine the functionalcorrelates of hypoxia-induced uPAR expression and invasion,
we first compared the invasion of cervical cancer cells througha reconstituted basement membrane (Matrigel). The cervicalcancer cell line, CaSki and CA, were incubated in hypoxic (1%O2) and normoxic conditions (20% O2), respectively. Invasion
assays showed that these cells were 2-fold more invasive underhypoxic conditions than under normoxic conditions (Fig. 1).
Hypoxia induces endogenous uPAR expression. To analyze
whether there is a correlation between HIF-1α and uPAR, we
examined the effect of hypoxia on HIF-1α and uPAR expression in cervical cancer cell line, CaSki and CA. Whole cell
extracts prepared from controls and from cells exposed to
1% O2 for 24 h were subjected to western blot analysis. As
shown in Fig. 2, both HIF-1α and uPAR protein expressions
were increased significantly under hypoxic conditions. These
results indicate that hypoxia induces the endogenous expressions of uPAR.
HIF-1 transactivates the uPAR promoter. To examine whether
the induction of uPAR expression by hypoxia is mediated-187-

nishi et al: HIF-1 activates uPAR in cervical cancer

995

HIF-1 overexpression increased the activity of this mutatedpromoter, indicating that this putative HRE (HRE/uPAR)
is essential for the regulation of uPAR by hypoxia andHIF-1 (Fig. 3B and C).
HIF-1 interacts with the putative HRE in the uPAR promoter. To
determine whether HIF-1 has direct interaction with the HRE on
the uPAR promoter, electrophoretic mobility shift assays were
performed. Oligonucleotide corresponding to nucleotides -50
to -25 (HRE/uPAR) of the uPAR promoter was incubated with
HIF-1α and ARNT prepared by in vitro transcription/translation and subjected to electrophoresis. A DNA-protein complex
was formed when HRE/uPAR was incubated with the HIF-1programmed rabbit reticulocyte lysate (Fig. 4A), but not withthe unprogrammed lysate (data not shown). This complex was
specifically retarded by anti-HIF-1α antibody (Fig. 4). The
addition of a 30-fold molar excess of cold HIF-1 consensus
oligonucleotide (HRE) markedly reduced binding (Fig. 4A).
To additionally substantiate a HIF-1 binding to this oligonucleotide, we performed competition assays using end-labeled
HIF-1 consensus oligonucleotide and cold HRE/uPAR as acompetitor. The unlabeled HRE/uPAR competed with HIF-1
binding to the wild-type probe when present in the reaction at
50- and 200-fold molar excess (Fig. 4B). These results confirm
that HIF-1 binds to the HRE/uPAR and indicate that theaffinity of HIF-1 binding to the HRE/uPAR is similar to that of
the consensus HIF-1 sequence.
These data indicate that hypoxia-induced uPAR expression
is induced by the enhanced activity of HIF-1 on the HRE/uPAR
promoter. Taken together, the putative HIF-1 binding site in
the proximal region of the uPAR promoter is essential for the
regulation of uPAR.

Figure 3. Transactivation of uPAR promoter activity by hypoxia exposure andHIF-1 overexpression. (A) The schematics of the HIF-1 response elements
and the puPAR141-luc and puPAR141mt-luc reporters are shown. (B) CaSki
and CA cells were transfected with the puPAR141-luc/puPAR141mt-luc(0.3 mg) in hypoxic conditions (1% O2) and activity reported as relative luminescence units (RLU). Error bars indicate standard deviation in triplicate
assays. (C) CaSki and CA cells were co-transfected with the puPAR141luc/puPAR141mt-luc (0.3 mg) and 1.0 mg of HIF-1 expression constructs(HIF-1α and ARNT) or empty vector, pcDNA3 in hypoxic conditions (1%O2) and activity reported as relative luminescence units (RLU). Error bars
indicate standard deviation in triplicate assays.

via a transcriptional mechanism, CaSki and CA cells weretransfected with puPAR-141-luc (Fig. 3A) and then exposed to
1% O2 for 24 h. Hypoxia transactivated the uPAR promoter
by 2-fold (Fig. 3B). Additional cotransfection experiments
were performed with puPAR-141-luc, HIF-1α and ARNT or
empty vectors. HIF-1 overexpression transactivated the uPAR
promoter by 5-6 fold (Fig. 3C).
The uPAR promoter contains a putative HRE. To confirm
whether the uPAR promoter regions are responsible fortransactivation by HIF-1, we prepared a reporter construct,
puPAR-141mt-luc, where the putative HRE is mutated byreplacement of AGG with TTT (Fig. 3A). Neither hypoxia nor

Disruption of the uPAR expression by HIF-1 siRNA. HIF-1αsiRNA was previously used to inhibit HIF-1α expression (12).
In the present study, we used this HIF-1α siRNA to examine
whether hypoxia-induced uPAR expression is mediated by
HIF-1 upregulation. When CaSki cells were transfected withsiRNA and exposed to hypoxic conditions, the HIF-1α anduPAR expressions were downregulated compared with thecontrol siRNA transfected cells (Fig. 5A). The level of actin
was monitored as a control and was found to be unalteredby siRNA (Fig. 5A). Similar results were obtained using CA
cells (Fig. 5A). These results indicate that HIF-1α is requiredfor hypoxia induced uPAR expression.
To examine the effect on invasiveness by HIF-1α knockdown, we performed invasion assay in CaSki and CA cells
after HIF-1α siRNA tranfection. Downregulation of HIF-1
expression significantly diminishes invasion in cervical cancer
cells (Fig. 5).
uPAR mRNA expression in cervical cancer tissue. The expression of uPAR mRNA was examined in cervical cancer from
35 patients and normal cervix from 10 patients who sufferedfrom uterine myoma or prolapse of the uterus. Triplicate
measurements were carried out. uPAR mRNA levels weremuch higher in cervical cancer than in normal tissues (Fig. 6).
Localization of the uPAR expression. Immunohistochemical
analysis of uPAR may lead to the understanding of neoplastic

-188-

996

ONCOLOGY REPORTS 35: 992-998, 2016

Figure 4. Analysis of HIF-1 interaction with the putative HRE in the uPAR promoter. (A) 32P end-labeled HRE/uPAR oligonucleotide was used as a probe.
For the competition assays, 30- or 100-fold molar excess of the HRE was used. For the supershift assay, anti-HIF-1α antibody (0.5 mg) was added into thebinding reaction. The thin arrow indicates HIF-1/DNA complexes and the thick arrow indicates supershifted bands. (B) The HIF-1 consensus oligonucleotide
was end-labeled with 32P. For the competition assays, 50- or 200-fold molar excess of HRE/uPAR was added to the binding reaction, respectively. For thesupershift assay, anti-HIF-1α antibody (0.5 mg) was added into binding reaction. The thin arrow indicates HIF-1/DNA complexes and the thick arrow indicatessupershifted bands. NS, non-specific.

Figure 6. uPAR mRNA level in cervical cancer.

Figure 5. (A) Disruption of uPAR expression by the HIF-1α siRNA. CaSki
and CA cells were transfected with the HIF-1α siRNA or control siRNAusing Lipofectamine 2000. Cells were then exposed to hypoxic conditions(1% O2). A cell lysate was prepared after 24 h and analyzed by western blotting for protein level using antibodies to HIF-1α, uPAR and actin. (B) HIF-1αsiRNA diminishes invasion in cervical cancer cells.

progression of invasion in cervical carcinogenesis. Increased
nuclear staining of HIF-1α was observed in invasive cervicalcancer tissue samples (Fig. 7C). However, no or faint staining
was observed in CIN3 (Fig. 7A). High-level cytoplasmic
staining of uPAR was detected in invasive cervical cancer,
whereas not in CIN3 (Fig. 7B and D).
Discussion
Tumor cells need to migrate through the extracellular matrix
and invade blood vessels into systemic circulation to bedisseminated to metastatic organs (13,14). Firstly, tumor cells
detach from the primary tumor, invade blood vessels and thenmetastasize in organs where they form the secondary lesion. In
spite of significant progress in cancer biology, it is not known-189-nishi et al: HIF-1 activates uPAR in cervical cancer

997

Figure 7. Immunohistochemical staining of HIF-1α and uPAR in cervical cancer. (A) CIN3 shows no staining with HIF-1α antibody. (B) CIN3 shows nostaining with uPAR antibody. (C) Increased nuclear staining was observed in invasive cervical cancer. (D) Strong cytoplasmic staining was detected in invasive
cervical cancer. (A-D) Magnification, x100.

why only some but not all cells of a clonal tumor populationacquire the ability to migrate through the tissue barriers (14).
One hypothesis is that specific transient regional tumor microenvironment of low oxygen mediates a distinctive epigenetic
gene expression profile in a subset of cells, which likely causes
selection of more invasive cell clones (15,16). Tumor hypoxia
is one of the most critical situations during development oftumor invasion.
Our data show that HIF-1 transactivates the uPAR
promoter and then mediates the induction of uPAR andinvasion in cervical cancer cells during hypoxia. We havealso shown that the protein level of uPAR in cervical cancer
samples is higher than that in normal cervix. The level ofuPAR was increased under hypoxic conditions, as determinedby real-time RT-PCR analysis and western blotting in cervicalcancer cell lines. When siRNA was used to reduce HIF-1α
expression, not only uPAR expression but also invasivenesswas significantly diminished during hypoxia. According toelectrophoretic mobility shift assay, we identified the active
HRE in the uPAR core promoter. These findings may be
relevant to several biological processes in which cell invasion occurs. For example, increased expression of uPAR
in cancer may facilitate their invasion through hypoxic
regions of the stroma (17). In addition, increased expressionof uPAR is enhanced in migrating endothelial cells in vitro,and the fact that neovascularization of transplanted tumors
in vivo is inhibited by uPAR antagonists suggests a role forhypoxia‑stimulated uPAR expression in tumor angiogenesis (18-20). uPAR antagonists inhibit tumor metastasis, aswell as local invasion, perhaps explaining the association ofelevated uPAR levels in cervical, breast and other types ofcarcinomas with a poor clinical outcome (21-23).
Correlation between HIF-1 and regulation of uPAR has
rarely been shown. The protease system of uPA/uPAR andmetalloproteinases participate in the metastatic diseaseprogression, which depends on hypoxia (13). A previous
study showed that exposure of MDA-MB231 human breast
carcinoma cells to hypoxia upregulated uPAR expression andincreased invasion (17). It has also shown that these effects
were prevented by incubating the cells with a blocking antibody to uPAR (17). It was hypothesized in that previous study
that HIF-1 may be involved in the transcriptional activationof the uPAR gene (17). Also, Büchler et al has shown thatthe effects of hypoxia on uPAR and invasion were mediateddirectly by HIF-1 in pancreatic cancer cells (24).
The HRE (5'-RCGTG-3') overlaps the E-box (CACGTG),
which is known to bind to several nuclear factors, such as
c-Myc, Max and Mad (25-28). It is unclear whether HIF-1
competes with these factors for binding to these sites, whereas
recent findings showed that hypoxia downregulated thec-Myc expression (29). Under hypoxic conditions, HIF-1α
may play a predominant role in regulating promoter activity
of uPAR. Taken together, these data strongly indicate thathypoxia‑induced uPAR expression is due to the enhanced
activity of HIF-1 on the uPAR promoter. Furthermore,
the proximal HRE in the uPAR promoter is essential forthe upregulation of uPAR during hypoxia since the uPAR
promoter was no longer transactivated by hypoxia when we

-190-

998

ONCOLOGY REPORTS 35: 992-998, 2016used the puPAR-141mt-luc which had the mutated putative
HRE by replacement of AGG with TTT.
These data may be important for future therapeutics sincelocal tumor invasion and early metastatic progression are themost challenging clinical features of cervical cancer. The
coordinated activation by HIF-1 of a large battery of target
genes, the protein products of which perform diverse but
related functions contributing to tumor invasion, suggests that
inhibitors of HIF-1 activity may have therapeutic utility as
anticancer agents (30). In conclusion, our results indicate thathypoxia-induced upregulation of uPAR is mediated by HIF-1
in cervical cancer cells, and provide evidence that regulationof uPAR expression by HIF-1 represents a mechanism forinvasive cervical cancer during hypoxia.
Acknowledgements
The present study was supported by Grants-in-Aids from
the Ministry of Education, Culture, Sports, Science andTechnology of Japan (MEXT).
References
1. Brizel DM, Scully SP, Harrelson JM, Layfield LJ, Bean JM,
Prosnitz LR and Dewhirst MW: Tumor oxygenation predicts forthe likelihood of distant metastases in human soft tissue sarcoma.
Cancer Res 56: 941-943, 1996.
2. Brizel DM, Sibley GS, Prosnitz LR, Scher RL and Dewhirst MW:
Tumor hypoxia adversely affects the prognosis of carcinoma of
the head and neck. Int J Radiat Oncol Biol Phys 38: 285-289,
1997.
3.	Vaupel P, Thews O, Mayer A, Höckel S and Höckel M:
Oxygenation status of gynecologic tumors: What is the optimal
hemoglobin level? Strahlenther Onkol 178: 727-731, 2002.
4. Duong HS, Le AD, Zhang Q and Messadi DV: A novel 3-dimensional culture system as an in vitro model for studying oral
cancer cell invasion. Int J Exp Pathol 86: 365-374, 2005.
5. Petrella BL, Lohi J and Brinckerhoff CE: Identification of
membrane type-1 matrix metalloproteinase as a target ofhypoxia-inducible factor-2 alpha in von Hippel-Lindau renal cell
carcinoma. Oncogene 24: 1043-1052, 2005.
6. Maity A and Solomon D: Both increased stability and transcription contribute to the induction of the urokinase plasminogenactivator receptor (uPAR) message by hypoxia. Exp Cell Res 255:
250-257, 2000.
7. Montuori N, Mattiello A, Mancini A, Santoli M, Taglialatela P,
Caputi M, Rossi G and Ragno P: Urokinase-type plasminogenactivator up-regulates the expression of its cellular receptor
through a post-transcriptional mechanism. FEBS Lett 508:
379-384, 2001.
8. Giatromanolaki A, Koukourakis MI, Sivridis E, Turley H,
Talks K, Pezzella F, Gatter KC and Harris AL: Relation of
hypoxia inducible factor 1 alpha and 2 alpha in operable
non-small cell lung cancer to angiogenic/molecular profile oftumours and survival. Br J Cancer 85: 881-890, 2001.
9. Semenza GL: Hypoxia-inducible factors in physiology andmedicine. Cell 148: 399-408, 2012.
10. Fukuda R, Zhang H, Kim JW, Shimoda L, Dang CV and
Semenza GL: HIF-1 regulates cytochrome oxidase subunits tooptimize efficiency of respiration in hypoxic cells. Cell 129:111-122, 2007.
11. Nishi H, Nakada T, Kyo S, Inoue M, Shay JW and Isaka K:
Hypoxia-inducible factor 1 mediates upregulation of telomerase(hTERT). Mol Cell Biol 24: 6076-6083, 2004.
12. McNally SJ, Harrison EM, Ross JA, Garden OJ and Wigmore SJ:
Curcumin induces heme oxygenase 1 through generation ofreactive oxygen species, p38 activation and phosphatase inhibition. Int J Mol Med 19: 165-172, 2007.

13.	Kim J, Yu W, Kovalski K and Ossowski L: Requirement for
specific proteases in cancer cell intravasation as revealed by anovel semiquantitative PCR-based assay. Cell 94: 353-362, 1998.
14. Yamano M, Fujii H, Takagaki T, Kadowaki N, Watanabe H
and Shirai T: Genetic progression and divergence in pancreatic
carcinoma. Am J Pathol 156: 2123-2133, 2000.
15. Lal A, Peters H, St Croix B, Haroon ZA, Dewhirst MW,
Strausberg RL, Kaanders JH, van der Kogel AJ and Riggins GJ:
Transcriptional response to hypoxia in human tumors. J NatlCancer Inst 93: 1337-1343, 2001.
16.	Koong AC, Denko NC, Hudson KM, Schindler C, Swiersz L,
Koch C, Evans S, Ibrahim H, Le QT, Terris DJ, et al: Candidate
genes for the hypoxic tumor phenotype. Cancer Res 60: 883-887,
2000.
17. Graham CH, Forsdike J, Fitzgerald CJ and MacdonaldGoodfellow S: Hypoxia-mediated stimulation of carcinoma cell
invasiveness via upregulation of urokinase receptor expression.
Int J Cancer 80: 617-623, 1999.
18. Mazar AP, Henkin J and Goldfarb RH: The urokinase plasminogen activator system in cancer: Implications for tumor
angiogenesis and metastasis. Angiogenesis 3: 15-32, 1999.
19. Bauer TW, Liu W, Fan F, Camp ER, Yang A, Somcio RJ,
Bucana CD, Callahan J, Parry GC, Evans DB, et al: Targeting ofurokinase plasminogen activator receptor in human pancreatic
carcinoma cells inhibits c-Met- and insulin-like growth factor-I
receptor-mediated migration and invasion and orthotopic tumor
growth in mice. Cancer Res 65: 7775-7781, 2005.
20. Reuning U, Sperl S, Kopitz C, Kessler H, Krüger A, Schmitt M
and Magdolen V: Urokinase-type plasminogen activator(uPA) and its receptor (uPAR): Development of antagonists ofuPA/uPAR interaction and their effects in vitro and in vivo. Curr
Pharm Des 9: 1529-1543, 2003.
21. Degryse B, Sier CF, Resnati M, Conese M and Blasi F: PAI-1
inhibits urokinase-induced chemotaxis by internalizing theurokinase receptor. FEBS Lett 505: 249-254, 2001.
22. Ramont L, Pasco S, Hornebeck W, Maquart FX andMonboisse JC: Transforming growth factor-beta1 inhibits tumor
growth in a mouse melanoma model by down-regulating theplasminogen activation system. Exp Cell Res 291: 1-10, 2003.
23. Fox SB, Taylor M, Grøndahl-Hansen J, Kakolyris S, Gatter KC
and Harris AL: Plasminogen activator inhibitor-1 as a measureof vascular remodelling in breast cancer. J Pathol 195: 236-243,
2001.
24. Büchler P, Reber HA, Tomlinson JS, Hankinson O, Kallifatidis G,
Friess H, Herr I and Hines OJ: Transcriptional regulation of
urokinase-type plasminogen activator receptor by hypoxiainducible factor 1 is crucial for invasion of pancreatic and livercancer. Neoplasia 11: 196-206, 2009.
25. Montagne M, Naud JF, McDuff FO and Lavigne P: Toward theelucidation of the structural determinants responsible for themolecular recognition between Mad1 and Max. Biochemistry 44:
12860-12869, 2005.
26. Hu J, Banerjee A and Goss DJ: Assembly of b/HLH/z proteinsc-Myc, Max, and Mad1 with cognate DNA: Importance ofprotein-protein and protein-DNA interactions. Biochemistry 44:11855-11863, 2005.
27. Semov A, Marcotte R, Semova N, Ye X and Wang E: Microarray
analysis of E-box binding-related gene expression in young andreplicatively senescent human fibroblasts. Anal Biochem 302:
38-51, 2002.
28.	Vervoorts J and Lüscher B: DNA binding of Myc/Max/Mad
network complexes to oligonucleotides containing two E box
elements: c-Myc/Max heterodimers do not bind DNA cooperatively. Biol Chem 380: 1121-1126, 1999.
29.	Koshiji M, Kageyama Y, Pete EA, Horikawa I, Barrett JC and
Huang LE: HIF-1alpha induces cell cycle arrest by functionallycounteracting Myc. EMBO J 23: 1949-1956, 2004.
30. Powis G and Kirkpatrick L: Hypoxia inducible factor-1alpha as acancer drug target. Mol Cancer Ther 3: 647-654, 2004.

-191-

Okabe et al. Journal of Hematology & Oncology (2015) 8:97
DOI 10.1186/s13045-015-0190-9

JOURNAL OF HEMATOLOGY& ONCOLOGYLETTER TO THE EDITOR

Open Access

Anti-leukemic activity of axitinib against cells
harboring the BCR-ABL T315I point mutation
Seiichi Okabe*, Tetsuzo Tauchi, Yuko Tanaka, Juri Sakuta and Kazuma Ohyashiki

Abstract
The BCR-ABL; breakpoint cluster region-Abelson point mutation T315I is resistant to ABL tyrosine kinase inhibitors.
However, axitinib, a vascular endothelial growth factor receptor inhibitor, is effective against this mutation. In this study,
we investigated axitinib activity against ponatinib-resistant cells and found that axitinib inhibited cellular growth andapoptosis in Ba/F3 T315I-mutant cells and T315I-mutant primary samples, but not in ponatinib-resistant Ba/F3 cells andprimary samples. Thus, an alternative strategy may be required to improve the prognosis of Philadelphia-chromosomepositive leukemia patients harboring BCR-ABL point mutations.
Keywords: Axitinib, Resistant cell, Ponatinib, T315I, Compound mutation

Letters to the editor
The BCR-ABL1 fusion gene is a causative oncogene inchronic myeloid leukemia (CML) and 30–50 % of acutelymphoblastic leukemia cases [1, 2]. Although ABL tyrosine kinase inhibitors (ABL TKIs) such as imatinib, nilotinib, dasatinib, and bosutinib have improved CML
treatment [3], such therapies cannot cure patients with
Philadelphia chromosome (Ph)-positive leukemia becauseof leukemia stem cells [4]. Moreover, some patients develop
BCR-ABL point mutations and become resistant to ABL
TKI therapy [5]. In particular, the ABL kinase domain mutation T315I is resistant to imatinib and second-generation
ABL TKIs (e.g., nilotinib, dasatinib, and bosutinib). Accordingly, this mutation is often found in patients with TKIresistant disease [6]. A third-generation ABL TKI, ponatinib, and omacetaxine which is a semisynthetic form ofhomoharringtonine, was recently developed [7]. Ponatinib
is a potent oral tyrosine kinase inhibitor that affects both
unmutated and mutated BCR-ABL [8]; it is effective
against T315I-mutant cells and has been approved for
TKI-resistant or intolerant CML and Ph-positive ALL patients. Omacetaxine is approved for the treatment ofchronic or accelerated-phase CML refractory to TKIs [7].
Recently, the vascular endothelial growth factor receptor (VEGFR) inhibitor axitinib was found to exhibit antileukemic activity against T315I-mutant disease. In the* Correspondence: okabe@tokyo-med.ac.jp
Department of Hematology, Tokyo Medical University, 6-7-1 Nishi-shinjuku,
Shinjuku-ku, Tokyo 160-0023, Japan

comparative effectiveness of axitinib versus sorafenib in
advanced renal cell carcinoma (AXIS) trial [9], axitinib
improved progression-free survival (PFS) compared tosorafenib, which is an all-multikinase inhibitor thatblocks angiogenesis targets [10], in patients with advanced renal cell carcinoma (RCC). Axitinib was approved for the treatment of advanced RCC. Axitinib isan orally active and potent TKI of VEGFRs 1, 2, and 3
and inhibits BCR-ABL1, especially the T315I variant, viaa distinct binding conformation [11]. In this study, weinvestigated whether axitinib could suppress ponatinibresistant compound-mutant cells harboring the T315I
mutation. A 72-h axitinib treatment inhibited the growthof Ba/F3 T315I cells (Fig. 1a). Immunoblot analysis of
axitinib-treated cells revealed dose-dependent decreases
in BCR-ABL, the downstream molecule Crk-L, and ribosomal S6 protein phosphorylation and increases in caspase
3 and Poly (ADP-ribose) polymerase (PARP) activity(Fig. 1b, c, e). Ponatinib and axitinib also induced apoptosis, significantly increased caspase activity (Fig. 1d), andreduced Akt activity (Fig. 1f).
In contrast, clinically available concentrations of axitinib did not inhibit the growth of ponatinib-resistant
Ba/F3 cells (Fig. 1a). Immunoblot analysis revealed that
BCR-ABL, Crk-L, and S6 kinase phosphorylation were
not inhibited by axitinib or ponatinib (Fig. 1b, e). Similarly, no increase in caspase activity or decrease in Akt
activity was observed following axitinib treatment (Fig. 1c,© 2015 Okabe et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium,provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://
creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

-192-

Okabe et al. Journal of Hematology & Oncology (2015) 8:97

A

B

D

E

F

G

H

I

Page 2 of 4

C

Fig. 1 (See legend on next page.)-193-

Okabe et al. Journal of Hematology & Oncology (2015) 8:97

Page 3 of 4

(See figure on previous page.)
Fig. 1 Growth inhibition and cellular signaling following axitinib/ponatinib treatment in T315I-mutant and compound-mutant cells. a Ba/F3 T315I
or Ba/F3 ponatinib-resistant (Ba/F3 ponatinib-R) cells were exposed to axitinib or ponatinib for 72 h at the indicated concentrations and subjected
to quantitative cell proliferation analysis. Each result is presented as the mean percentage of proliferation relative to unexposed control cultures.
*P < 0.05 compared to T315I cells. b Ba/F3 T315I or Ba/F3 ponatinib-R cells were treated with ponatinib (10 nM) or axitinib (500 nM) for 24 h. Total
cell extracts were examined via immunoblot analysis with anti-phospho ABL, phospho-Crk-L, phospho-S6, cleaved caspase 3, cleaved PARP, ABL,
Crk-L, and β-actin antibodies. c Caspase activity was analyzed using an ApoAlert® Caspase-3 Colorimetric Assay Kit (Takara Bio Inc. Otsu, Shiga,
Japan) according to the manufacturer’s protocol. Data represent three independent sets of experiments. *P < 0.05 compared to Ba/F3 T315I andBa/F3 ponatinib-R cells. d Apoptosis in Ph-positive cell lines was assayed using a FITC Annexin V Apoptosis Detection Kit I™ (BD Pharmingen, San
Jose, CA, USA). The experiments were performed in triplicate. e Ba/F3 T315I or Ba/F3 ponatinib-R cells were treated with axitinib at the indicatedconcentrations for 24 h. Total cell extracts were examined via immunoblot analysis with anti-phospho ABL, phospho-Crk-L, phospho-S6, cleaved
caspase 3, cleaved PARP, ABL, Crk-L, and β-actin antibodies. f Akt activity was analyzed using a phospho-AKT 1/2/3 (Ser473) InstantOne™ enzyme-linked
immunosorbent assay kit (Affymetrix, Cleveland, OH, USA) according to the manufacturer’s protocol. Data represent three independent sets of experiments.
*P < 0.05 compared to Ba/F3 T315I and Ba/F3 ponatinib-R cells. g, h T315I-positive or compound-mutant primary cells were subjected to quantitative cellproliferation analysis after a 72-h exposure to axitinib or ponatinib. Each result is presented as the mean percentage of proliferation relative to unexposed
control cultures. *P < 0.05 compared to control. i T315I-positive or compound-mutant primary cells were treated with axitinib at the indicatedconcentrations for 24 h. Total cell extracts were examined via immunoblot analysis with anti-phospho-Crk-L, phospho-S6, cleaved PARP, andβ-actin antibodiesf), and neither ponatinib nor axitinib affected apoptosis inthese cells (Fig. 1d).
We next evaluated primary T315I-mutant and ponatinibresistant compound-mutant samples. Axitinib potently
inhibited the growth of T315I-mutant primary cells in adose-dependent manner (Fig. 1g). Immunoblot analysis further revealed reduced Crk-L and S6 kinase phosphorylation
after axitinib or ponatinib treatment (Fig. 1i). In contrast,
the growth of ponatinib-resistant primary cells was not affected by ponatinib or axitinib (Fig. 1h). Immunoblotting
revealed that neither ponatinib nor axitinib affected thephosphorylation of Crk-L and S6 kinase in ponatinibresistant cells (Fig. 1i).
In CML, ABL TKI resistance is frequently caused by
ABL kinase domain mutations. The T315I mutation isresistant to all ABL TKIs except ponatinib. However, wepreviously described ponatinib-resistant cells resulting
from a BCR-ABL compound mutation [12]. Althoughaxitinib, which is currently being investigated for efficacy
in patients with Ph-positive T315I-mutant leukemia, induced apoptosis in T315I-mutant cells, it was ineffective
against cells with a compound mutation including T315I.
An alternative strategy will be required to improve theprognosis of patients with Ph-positive, BCR-ABL-mutant
leukemia. Current evidence to direct the managementof ABL TKI-resistant disease, particularly those harboring T315I and compound mutations, is limited. New
molecular-targeted drugs and an understanding of ABL
TKI resistance mechanisms are required to apply an appropriate therapeutic approach.
Abbreviations
CML: chronic myeloid leukemia; Ph: Philadelphia chromosome; TKI: tyrosine
kinase inhibitor; VEGFR: vascular endothelial growth factor receptor.
Competing interests
Kazuma Ohyashiki received research support from Novartis and BMS.

Authors’ contributions
SO performed the experimental procedures; TT, YT, JS, and KO designed andcoordinated the study and interpreted data. All authors have read andapproved the final manuscript.
Acknowledgements
This work was supported by a High-Tech Research Center Project for private
universities, a matching fund subsidy from the Ministry of Education, Culture,
Sports, Science, and Technology (MEXT), and the University-Industry Joint
Research Project for private universities, a matching fund subsidy from
MEXT. This work was also supported by Grants-in-Aid for Scientific Researchfrom MEXT and Supporting Positive Activities for Female Researchers. We also
thank the Tokyo Medical University Research Center for technical support.
Received: 7 July 2015 Accepted: 17 July 2015

References
1. Rowley JD. Letter: a new consistent chromosomal abnormality in chronic
myelogenous leukemia identified by quinacrine fluorescence and Giemsa
staining. Nature. 1973;243:290–3.
2. Kurzrock R, Shtalrid M, Talpaz M, Kloetzer WS, Gutterman JU. Expression ofc-abl in Philadelphia-positive acute myelogenous leukemia. Blood.
1987;70:1584–8.
3. Sweet K, Zhang L, Pinilla-Ibarz J. Biomarkers for determining the prognosis
in chronic myelogenous leukemia. J Hematol Oncol. 2013;6:54.
4. Ahmed W, Van Etten RA. Alternative approaches to eradicating themalignant clone in chronic myeloid leukemia: tyrosine-kinase inhibitor
combinations and beyond. Hematology Am Soc Hematol Educ Program.
2013;2013:189–200.
5. Kantarjian HM, Talpaz M, Giles F, O’Brien S, Cortes J. New insights into thepathophysiology of chronic myeloid leukemia and imatinib resistance. Ann
Intern Med. 2006;145:913–23.
6. Ravandi F. Managing Philadelphia chromosome-positive acutelymphoblastic leukemia: role of tyrosine kinase inhibitors. Clin Lymphoma
Myeloma Leuk. 2011;11:198–203.
7. Lü S, Wang J. Homoharringtonine and omacetaxine for myeloid
hematological malignancies. J Hematol Oncol. 2014;7:2.
8. Cortes JE, Kantarjian H, Shah NP, Bixby D, Mauro MJ, Flinn I, et al. Ponatinibin refractory Philadelphia chromosome-positive leukemias. N Engl J Med.
2012;367:2075–88.
9. Rini BI, Escudier B, Tomczak P, Kaprin A, Szczylik C, Hutson TE, et al.
Comparative effectiveness of axitinib versus sorafenib in advanced renal cell
carcinoma (AXIS): a randomised phase 3 trial. Lancet. 2011;378:1931–9.
10. Smith AD, Roda D, Yap TA. Strategies for modern biomarker and drug
development in oncology. J Hematol Oncol. 2014;7:70.

-194-

Okabe et al. Journal of Hematology & Oncology (2015) 8:97

Page 4 of 4

11. Pemovska T, Johnson E, Kontro M, Repasky GA, Chen J, Wells P, et al.
Axitinib effectively inhibits BCR-ABL1(T315I) with a distinct binding
conformation. Nature. 2015;519:102–5.
12. Okabe S, Tauchi T, Tanaka Y, Katagiri S, Kitahara T, Ohyashiki K. Activity ofomacetaxine mepesuccinate against ponatinib-resistant BCR-ABL-positive
cells. Blood. 2013;122:3086–8.

Submit your next manuscript to BioMed Central
and take full advantage of:• Convenient online submission• Thorough peer review• No space constraints or color ﬁgure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit-195-Oncotarget, Vol. 6, No. 24

www.impactjournals.com/oncotarget/

Efficacy of the polo-like kinase inhibitor rigosertib, alone or in
combination with Abelson tyrosine kinase inhibitors, against
break point cluster region-c-Abelson-positive leukemia cells
Seiichi Okabe1, Tetsuzo Tauchi1, Yuko Tanaka1, Juri Sakuta1 and Kazuma Ohyashiki1
1

Department of Hematology, Tokyo Medical University, Tokyo, Japan

Correspondence to: Seiichi Okabe, email: okabe@tokyo-med.ac.jp
Keywords: chronic myeloid leukemia, ABL tyrosine kinase inhibitor, resistant cell, polo-like kinase
Received: January 21, 2015

Accepted: April 22, 2015

Published: May 08, 2015

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract
The potency of Abelson (ABL) tyrosine kinase inhibitors (TKIs) against chronic
myeloid leukemia (CML) has been demonstrated. However, ABL TKI resistance candevelop. In this study, we investigated the efficacy of a combination therapy including
rigosertib (ON 01910.Na), a polo-like kinase (PLK) and phosphoinositide 3-kinase(PI3K) inhibitor, and ABL TKIs. A 72-h rigosertib treatment was found to inhibit cell
growth, induce apoptosis, reduce phosphorylation of the breakpoint cluster region-c(BCR)-ABL and its substrate Crk-L, and increase the activities of caspase 3 and poly
(ADP-ribose) polymerase (PARP). This combination therapy also exerted a synergistic
inhibitory effect on Philadelphia chromosome (Ph)-positive cell proliferation andreduced the phosphorylation of BCR-ABL and Crk-L while increasing that of cleaved
PARP and the H2A.X histone. Rigosertib also potently inhibited the growth of ABL
TKI-resistant cells, and cotreatment with ABL TKIs and rigosertib induced higher
cytotoxicity. These results indicate that rigosertib treatment may be a powerfulstrategy against ABL TKI-resistant cells and could enhance the cytotoxic effects of
ABL TKIs.

Introduction

Although the majority of patients with CML-CP
achieve impressive clinical responses, increasing evidenceof acquired resistance to TKIs has been documented [8].
In particular, the T315I mutation seems to be the mostfrequently observed resistance mechanism after dasatinib
treatment [9]. The TKI ponatinib is also effective forpatients with heavily pretreated resistant CML and for
one third of patients with accelerated phase (AP) or blastic
phase (BP) CML [10]. In the Ph-positive ALL and CML
Evaluation (PACE) trial, ponatinib inhibited the growth ofleukemia cells, including those harboring T315I mutants[11]. Although ponatinib was developed as a BCR-ABL
inhibitor, recent studies have identified novel compoundmutations that cause resistance to ponatinib [12, 13],indicating that mutations in the BCR-ABL kinase domain
may confer resistance to ABL TKIs. Moreover, ABL TKIs
cannot eradicate leukemia stem cells [14]. Therefore,
alternative strategies are needed to improve the outcomesof patients with CML.
Mitosis is the key event of cell cycle, and polo-likekinase (PLK) is a pivotal regulator of mitosis as well as

Chronic myeloid leukemia (CML) is associatedwith the Philadelphia chromosome (Ph), which is atranslocation of the Abelson (ABL)1 oncogene on
chromosome 9 with a breakpoint cluster region (BCR) on
chromosome 22 and is designated t(9;22) [1]. This fusion
gene encodes the chimeric oncogenic fusion protein BCRABL, a constitutively active tyrosine kinase. Imatinib,
an orally available ABL tyrosine kinase inhibitor (TKI),
effectively induces cytogenic remission in patients with
CML [2]. Despite the efficacy of imatinib, some patientsdevelop resistance to ABL TKI therapy. Primary or
secondary resistance to imatinib has been reported tooccur in approximately 40% of patients with CML in thechronic phase (CML-CP) [3]. Second-generation ABL
TKIs such as nilotinib or dasatinib have been shown to
induce major cytogenetic responses in approximately 50%
of patients with imatinib-resistant or intolerant CML [4, 5]
and are currently used clinically as first-line therapies for
newly diagnosed CML [6, 7].
www.impactjournals.com/oncotarget

20231

-196-

Oncotarget

cytokinesis [15]. Five members of the PLK family havebeen discovered in humans. PLK1, which is mainly
expressed during the late G2 and M phase, regulates
various stage of mitosis. This protein contains a highly
conserved N-terminal protein kinase domain and a
C-terminal polo box domain (PBD), which is required for
docking to other targets. PLK1 is also highly expressedin a broad spectrum of tumors, and its expression level
correlates with poor prognosis in patients. Therefore,high PLK1 levels are a marker of cellular proliferation incancers such as leukemia [16, 17].
Rigosertib, also known as ON0190.Na, is a benzyl
sulfone analogue and an adenosine triphosphate (ATP)noncompetitive, multitargeted inhibitor [18]. Rigosertib
inhibits both PLK1 and phosphatidylinositol 3-kinase(PI3K) and has been investigated in clinical trials against
solid tumors and hematological malignancies such as

myelodysplastic syndrome (MDS) [19, 20]. Herein, we
have investigated the effects of rigosertib against Phpositive leukemia cells. We have also investigated whether
co-treatment with ABL TKIs and rigosertib would increasecytotoxicity against Ph-positive primary leukemia cells.

Results
Rigosertib induces cytotoxicity and inhibits theproliferation of Ph-positive cell lines
PLK1 is up-regulated in many human tumors,
including hematological malignancies [17]. Therefore,
we first examined PLK1 expression in Ph-positive
leukemia cells and observed expression in Ph-positive

Figure 1: Analysis of polo-like kinase 1 expression and rigosertib activity against Philadelphia (Ph)-positive leukemia
cells. A. Polo-like kinase 1 (PLK1) expression was examined via immunoblotting, with actin as the loading control. B., E. K562 or Ba/

F3 T315I cells were treated with the indicated concentrations of rigosertib for 72 h. The percentages of cell growth were examined; *P <0.05 compared with the control. Results represent three separate experiments. C. K562 cells were treated with the indicated concentrationsof rigosertib for 48 h. Percentages of apoptotic cells were examined. These results represent three independent experiments; *P < 0.05
compared with the control. D., F. K562 or Ba/F3 T315I cells were treated with rigosertib at the indicated concentrations for 24 h. Total
extracts were examined via immunoblotting with anti-phospho ABL, phospho-Crk-L, phosphohistone H2A.X, cleaved caspase 3, cleavedPARP, ABL, Crk-L, and β-actin antibodies (abs). ABL, Abelson; PARP, poly (ADP-ribose) polymerase; abs, antibodies.

www.impactjournals.com/oncotarget

20232

-197-

Oncotarget

Rigosertib overcomesleukemia cells

leukemia cell lines and primary samples as well ascluster of differentiation (CD) 34-positive CML samples(Figure 1A). As rigosertib is a potent PLK1 and PI3K
inhibitor, we examined its efficacy against Ph-positive
leukemia cells. Incubation for 72 h with the indicatedconcentrations of rigosertib decreased growth (Figure1B) and increased apoptosis in K562 cells in a dosedependent manner (Figure 1C). Immunoblotting revealed
reduced phosphorylation of BCR-ABL and its downstream
substrate Crk-L but increased caspase 3 and PARP activity
after rigosertib treatment (Figure 1D). We also observed
increased H2A.X histone phosphorylation after treatment
with rigosertib. Patients with resistance to imatinib
therapy harbor the T315I mutation [8], and we, therefore,
evaluated the efficacy of rigosertib against T315I-mutant
Ba/F3 cells. Cell growth was inhibited in a dose-dependent
manner after a 72-h treatment with rigosertib (Figure1E). In an immunoblot analysis, rigosertib treatment
reduced the phosphorylation of BCR-ABL and Crk-L andincreased both the caspase and PARP activity levels, aswell as H2A.X phosphorylation (Figure 1F).

TKI-resistant

To investigate whether rigosertib could sensitize
ABL TKI-resistant cells to ABL TKIs, a cell growth assay
was performed using Ba/F3 ponatinib-resistant (Ba/F3
ponatinib-R) cells and nilotinib-resistant K562 (K562
nilotinib-R) cells. We found that treatment with rigosertib
reduced the growth of these cells in a dose-dependent
manner (Figure 3A). We also observed increased apoptosis(Figure 3B) and activation of caspase 3 and PARP (Figure3C) in Ba/F3 ponatinib-R cells following rigosertib
treatment; in contrast, rigosertib reduced ABL and Crk-L
phosphorylation in a dose-dependent manner. Further,
we investigated the efficacy of ponatinib and rigosertib
against ponatinib-resistant Ba/F3 cells. Although ponatinib
treatment did not increase the activity of PARP, cotreatment with ponatinib and rigosertib inhibited cell
growth (Figure 3D) and increased PARP cleavage, caspase3 activity, and H2A.X phosphorylation (Figure 3E). We
further examined the efficacy of rigosertib against K562
nilotinib-R cells and found that this agent decreased
cell growth in a dose-dependent manner (Figure 3F).
Rigosertib treatment also reduced the phosphorylationof BCR-ABL and Crk-L while increasing the activities
of caspase 3 and PARP (Figure 3G). We further observed
increased cytotoxicity and reduced phosphorylationof BCR-ABL and Crk-L following co-treatment withnilotinib and rigosertib (Figures 3H, 3I). These results
indicate that rigosertib increases the cytotoxicity of ABL
TKIs against ABL TKI-resistant cells.

Co-treatment with ABL TKIs and rigosertib
increased cytotoxicity against Ph-positive cell lines
We tested the effect of a combination of rigosertib(0, 10, 20, 50, 100, and 200 nM) and imatinib (0, 100,
500 nM, and 1 µM) on Ph-positive K562 cells. The
combination of the two drugs decreased the proliferationof K562 cells more than each drug alone (Figure 2A).
Additionally, combined treatment with imatinib andrigosertib led to apoptosis at a higher rate than with eachdrug alone (Figure 2B). Immunoblot analysis revealed
that phosphorylation of BCR-ABL and Crk-L decreased
whereas caspase 3 and PARP activities increased following
co-treatment with imatinib and rigosertib (Figure 2C) andthat H2A.X phosphorylation also increased. Furthermore,
the combination of imatinib and rigosertib down-regulated
the expression of the anti-apoptotic protein myeloid cellleukemia (Mcl)-1. T315I mutant cells are resistant tosecond-generation ABL TKIs. We found that co-treatment
with ponatinib and rigosertib decreased the proliferationof T315I mutant cells (Figure 2D). A two-way analysis ofvariance (ANOVA) revealed that these drugs acted in asynergistic manner (Figure 2A, 2D). Combined treatment
with ponatinib and rigosertib also increased apoptosis
in T315I mutant cells (Figure 2E), and immunoblotting
revealed decreased phosphorylation of BCR-ABL and
Crk-L and increased caspase 3 and PARP activities (Figure2F) along with increased H2A.X phosphorylation. These
results suggest that rigosertib enhances ABL TKI-induced
cytotoxicity against Ph-positive leukemia cells.

www.impactjournals.com/oncotarget

ABL

PLK1 knockdown induces cytotoxicity against
Ph-positive leukemia cells
We investigated the inhibitory effects of PLK1 on
Ph-positive leukemia cells by examining the effects of
PLK1 siRNA knockdown in K562 cells. Immunoblot
analysis revealed that PLK1 expression was completely
inhibited (Figure 4A). Subsequently, we observed inhibited
growth inhibition in these PLK1 siRNA-transfected cells(Figure 4B) and greater imatinib-mediated inhibition in the
PLK1 siRNA-transfected K562 cells relative to the control
siRNA-transfected cells. We also observed increased
apoptosis in the PLK1 siRNA-transfected cells following
treatment with imatinib (Figure 4C). Immunoblot analysis
revealed increased PARP activity following imatinib
treatment in the PLK1 siRNA-transfected K562 cells(Figure 4D). These results indicate that PLK1 knockdown
increased the sensitivity of these cells to ABL TKI.

20233

-198-

Oncotarget

Rigosertib is effective against Ph-positive primarytumor samples

examined the activity of rigosertib against Ph-positive
primary tumor samples. Co-treatment with ABL TKIs
and rigosertib strongly inhibited cell proliferation relative
to treatment with each drug alone (Figure 5A). Next, we
examined intracellular signaling mechanisms and foundthat rigosertib treatment increased PARP activity (Figure

The activity of rigosertib has been investigated innumerous types of cancer cells [20]. Accordingly, weFigure 2: Co-treatment with Abelson tyrosine kinase inhibitors and rigosertib inhibits the proliferation of Philadelphia(Ph)-positive leukemia cells. A., D. K562 or Ba/F3 T315I cells were treated with the indicated concentrations of rigosertib or imatinib,

both, or ponatinib for 72 h. The percentages of cell growth were determined as described in the Methods. Data are representative of three
independent sets of experiments. B., E. K562 or Ba/F3 T315I cells were treated with the indicated concentrations of ponatinib or rigosertib,
both, or imatinib for 48 h. Percentages of apoptotic cells was determined; P < 0.05 compared with imatinib or ponatinib treatment. C., F.
K562 or Ba/F3 T315I cells were treated with ponatinib or rigosertib, both, or imatinib for 24 h. Total cellular lysates were analyzed via
immunoblotting with anti-phospho ABL, phospho-Crk-L, phosphohistone H2A.X, cleaved caspase 3, cleaved-PARP, ABL, Crk-L, Mcl-1
and β-actin antibodies. ABL, Abelson; PARP, poly (ADP-ribose) polymerase; Mcl-1, myeloid cell leukemia.
www.impactjournals.com/oncotarget

20234

-199-

Oncotarget

Figure 3: Rigosertib activity against Abelson tyrosine kinase inhibitor -resistant cells. A., F. Cells were cultured in thepresence or absence of rigosertib for 72 h. Viable cell numbers were counted; *P < 0.05 compared with the control. Results represent three
independent experiments. B. Cells were treated with the indicated concentrations of rigosertib for 48 h. Percentages of apoptotic cells
were determined; *P < 0.05 compared with the control. C., G. Cells were treated with rigosertib at the indicated concentrations for 24 h.
Total extracts were examined via immunoblotting with anti-phospho ABL, phospho-Crk-L, phosphohistone H2A.X, cleaved caspase 3,
cleaved-PARP, ABL, Crk-L, and β-actin antibodies. D., H. Cells were treated with the indicated concentrations of rigosertib or ponatinib,
both, or nilotinib for 72 h. Percentages of cell growth were determined; *P < 0.05 compared with rigosertib treatment. E., I. Cells weretreated with nilotinib or rigosertib, both, or ponatinib for 24 h. Total extracts were examined via immunoblotting with anti-phospho ABL,
phospho-Crk-L, phosphohistone H2A.X, cleaved caspase 3, cleaved-PARP, ABL, Crk-L, and β-actin antibodies. ABL, Abelson; PARP,
poly (ADP-ribose) polymerase.
www.impactjournals.com/oncotarget

20235

-200-

Oncotarget

5B) while inhibiting the phosphorylation of BCR-ABL
and Crk-L. Co-treatment with ponatinib and rigosertib also
increased PARP activity in primary T315I-positive cells
while decreasing the phosphorylation of BCR-ABL and
Crk-L (Figure 5C). These results indicate that co-treatment
with ABL TKI and rigosertib enhanced the cytotoxicity
against Ph-positive primary samples.

most well studied member of the PLK family and a key
regulator of mitotic progression [15]. In this study, wedemonstrated that PLK1 protein was abundantly expressedin Ph-positive leukemia and CD34-positive primary CML
cells. These observations support the use of PLK1 as atherapeutic target.
Because PLK1 expression is increased in CD34positive CML stem cells (Figure 1A), PLK inhibition may
eradicate these cells. We demonstrated that one PLK1
inhibitor, rigosertib, could induce H2A.X phosphorylationin Ph-positive leukemia cells (Figures 1D, 1F). H2A.X
phosphorylation is a sensitive cellular response to various

Discussion
Recent studies have suggested PLK1 as a usefultarget in the treatment of various tumors. PLK1 is the

Figure 4: Transfection of polo-like kinase 1-specific siRNA inhibits proliferation and promotes apoptosis in Philadelphia(Ph)-positive leukemia cells. A. PLK1 protein expression was measured via immunoblotting with a PLK1-specific antibody. β-actin

was used as a loading control. B. PLK1 and control siRNA transfected cells were incubated with imatinib for 72 h. Viable cell numbers werecounted; *P < 0.05 compared with control siRNA transfected cells. These results represent three separate experiments. C. Cells were treatedwith imatinib for 48 h. Percentages of apoptotic cells were determined; P < 0.05 compared with control siRNA transfected cells. Results
represent three independent experiments. D. Cells were treated with imatinib for 24 h. Whole extracts were examined via immunoblottingwith anti-phospho ABL, phospho-Crk-L, cleaved-PARP, and β-actin antibodies. ABL, Abelson; PARP, poly (ADP-ribose) polymerase.
www.impactjournals.com/oncotarget

20236

-201-

Oncotargetevents that results in DNA damage [21]. Therefore,rigosertib may induce an early chromatin modification
event following DNA damage and fragmentation. Our dataalso demonstrated that PLK1 inhibition synergisticallyenhanced sensitivity to ABL TKIs in Ph-positive cells. In
cancer cells, PLK1 depletion has been shown to inhibit
cell proliferation [22], perturb spindle assembly, prolong
mitotic arrest, and induce apoptosis. In this study, we also
confirmed that siRNA-induced PLK1 depletion inhibited
cell growth, increased apoptosis, and enhanced theefficacy of ABL TKI (Figure 4C).
Rigosertib is a small molecule that induces mitotic
arrest, and we showed that rigosertib alone could induce
dose-dependent cytotoxicity in ABL TKI-resistant celllines. In contrast, no response was observed in ABL TKIresistant cells even after 72 h of treatment with ABL
TKIs such as nilotinib and ponatinib, when administeredalone. Other studies have reported that rigosertib alone
induces apoptosis in hematological cancer cells [23].
We demonstrated that co-treatment with ABL TKIs andrigosertib inhibited cell proliferation, induced apoptosis,
activated caspase-3, and altered the expression of Mcl-1(Figure 2C).
ABL TKI-resistant patients have a poor prognosis.
Accordingly, we investigated the efficacy of rigosertib
against ABL TKI-resistant cell lines. Rigosertib iscurrently being investigated in clinical trials and has
shown promising results when administered alone or as
part of a combination therapy [24]. Other PLK inhibitors
have also been investigated [25]. PLK1 inhibition by a
combination of rigosertib and ABL TKIs offers a novel
targeted anti-tumor therapy for patients with Ph-positive
leukemia.
In summary, this pre-clinical study provides

Figure 5: Rigosertib suppresses growth and alters signaling in Philadelphia (Ph)-positive primary cells. A. CML or Ph-

positive ALL cells were treated with ponatinib or rigosertib, both, or imatinib for 72 h. Cell viability was determined as described in the
Methods; *P < 0.05 compared with ponatinib or imatinib treatment. B. Lysates from primary cells treated with or without rigosertib for 24
h were immunoblotted using the indicated antibodies. C. Cells were treated with ponatinib, rigosertib, or both for 24 h. Whole extracts were
examined via immunoblotting with anti-phospho ABL, phospho-Crk-L, cleaved caspase 3, cleaved-PARP and β-actin antibodies. CML,
chronic myeloid leukemia; ALL, acute lymphoblastic leukemia; PARP, poly (ADP-ribose) polymerase.

www.impactjournals.com/oncotarget

20237

-202-

Oncotarget

siRNA transfection

scientific rationale for the continued investigation of
PLK inhibition as a therapeutic strategy for patients with
Ph-positive leukemia. We also provide new information
regarding the efficacy of combined treatment with the
PLK inhibitor rigosertib and a potent ABL TKI against
ABL TKI-resistant leukemia cells.

PLK1-targeting siRNA was purchased fromSanta Cruz Biotechnology (sc-36277). K562 cells
were transfected with PLK1 or control siRNA via
electroporation as described previously [29].

Materials and Methods

Cell viability assays
The viability of Ph-positive cells treated withrigosertib alone or in combination with imatinib,
nilotinib, or ponatinib was measured via trypan blue
exclusion or staining with a cell counting kit solution(Dojin, Kumamoto, Japan) followed by photometricmeasurements at A450 nm to determine cell viability. The
experiments were performed in triplicate.

Ethics statement
Peripheral blood samples were collected frompatients with CML and Ph-positive leukemia after
obtaining written informed consent. The Institutional
Review Board of the Tokyo Medical University approved
this study in adherence to the Declaration of Helsinki.

Apoptosis assay

Ph-positive cell lines and patient cells

Apoptosis of the Ph-positive cell lines was assayedusing previously described methods [30]. The experiments
were performed in triplicate.

The Ph-positive leukemia cell line (K562) was
obtained from the American Type Culture Collection(ATCC, Manassas, VA, USA). K562 nilotinib-R [26], aT315I mutant Ph-positive ALL cell line (SK-9) [27], Ba/
F3 ponatinib-R [13], and T315I mutant Ba/F3 cells [28]were established previously. All cell lines were cultured
in Roswell Park Memorial Institute (RPMI) 1640 medium
containing 10% fetal bovine serum (FBS) and maintained
at 37°C in a 5% CO2 humidified atmosphere. Freshperipheral blood samples were collected in heparinizedtubes from patients after obtaining informed consent.
Mononuclear cells were separated from blood using
LymphoSepare(Immuno-Biological
Laboratories,
Minneapolis, MN, USA). These cells were used
immediately or cryopreserved in liquid nitrogen until used.

Immunoblotting
Immunoblot analysis was performed according
to previously described methods [31]. Ph-positive
cell lines or patient cells were incubated with theindicated concentrations of ABL TKI or rigosertib forthe indicated time periods. Following the indicatedtreatments, cells were harvested and lysed by sonicationin radioimmunoprecipitation assay (RIPA) lysis buffer.
Protein lysate concentrations were measured using aprotein assay kit (Bio-Rad Laboratories, Hercules, CA,
USA). Equal amounts of proteins were loaded on 4%–
20% polyacrylamide gel and transferred to polyvinylidene
difluoride (PVDF) membranes. The membranes wereprobed with the primary antibodies of interest at theappropriate dilutions for 2 h at room temperature.
Blots were developed using an Amersham enhanced
chemiluminescence (ECL) plus kit (GE Healthcare, Little
Chalfont, UK). The experiments with Ph-positive celllines were performed thrice independently.

Reagents
Rigosertib and ponatinib were purchased from
MedKoo Biosciences (Chapel Hill, NC, USA), andimatinib and nilotinib were provided by Novartis Pharma
AG (Basel, Switzerland). Stock solutions of rigosertib,
nilotinib, and ponatinib were prepared in dimethyl
sulfoxide (DMSO). Imatinib was dissolved in distilledwater, aliquoted, and stored at -20°C. Anti-phospho ABL,
phospho Crk-L, cleaved caspase 3, and PARP antibodies(Abs) were purchased from Cell Signaling (Danvers, MA,
USA). Anti-phosphohistone H2A.X and Crk-L Abs wereobtained from Millipore (Billerica, MA, USA). ABL,
PLK1, and Mcl-1 Abs were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). Other reagentswere obtained from Sigma chemical company (St. Louis,
MO, USA).

www.impactjournals.com/oncotarget

Statistical analysis
Differences in dose responses and apoptosis between
treatment groups were determined using Student’s t test.
P values of <0.05 were considered significant. In someexperiments, results of multiple-group comparisons wereanalyzed using a two-way ANOVA and presented asmeans ± standard deviations.

20238

-203-

Oncotarget

Acknowledgments

myeloid leukemia: dasatinib, nilotinib, or imatinib. J
Hematol Oncol. 2010; 3: 47.

This work was supported by a High-Tech ResearchCenter Project for Private Universities, a matching fundsubsidy from the Ministry of Education, Culture, Sports,
Science, and Technology (MEXT), and the UniversityIndustry Joint Research Project for Private Universities, amatching fund subsidy from MEXT. This work was also
supported by Grants-in-Aid for Scientific Research from
MEXT and Supporting Positive Activities for Female
Researchers.

The authors declare that they have no conflicts ofinterest.

Rowley JD. Letter: A new consistent chromosomal
abnormality in chronic myelogenous leukaemia identified
by quinacrine fluorescence and Giemsa staining. Nature.
1973; 243: 290-293.
Kantarjian H, Sawyers C, Hochhaus A, Guilhot F, Schiffer
C, Gambacorti-Passerini C, Niederwieser D, Resta D,
Capdeville R, Zoellner U, Talpaz M, Druker B, Goldman
J, et al. Hematologic and cytogenetic responses to imatinib
mesylate in chronic myelogenous leukemia. N Engl J Med.
2002; 346: 645-652.

3.

Hochhaus A, O’Brien SG, Guilhot F, Druker BJ, Branford
S, Foroni L, Goldman JM, Müller MC, Radich JP, Rudoltz
M, Mone M, Gathmann I, Hughes TP, et al. Six-year
follow-up of patients receiving imatinib for the first-line
treatment of chronic myeloid leukemia. Leukemia. 2009;23:1054-1061.

4.

5.

6.

7.

Soverini S, Colarossi S, Gnani A, Castagnetti F, Rosti G,
Bosi C, Paolini S, Rondoni M, Piccaluga PP, Palandri F,
Giannoulia P, Marzocchi G, Luatti S, et al. Resistance todasatinib in patients with Philadelphia-positive leukemia
is mainly mediated by the presence or the selection of
mutations at residues 315 and 317 in the Bcr-Abl kinasedomain. Haematologica. 2007; 92:401-404.

12. Zabriskie MS, Eide CA, Tantravahi SK, Vellore NA,
Estrada J, Nicolini FE, Khoury HJ, Larson RA, Konopleva
M, Cortes JE, Kantarjian H, Jabbour EJ, Kornblau SM, etal. BCR-ABL1 compound mutations combining key kinasedomain positions confer clinical resistance to ponatinib in
Ph chromosome-positive leukemia. Cancer Cell. 2014; 26:428-442.
13. Okabe S, Tauchi T, Tanaka Y, Katagiri S, Kitahara T,
Ohyashiki K. Activity of omacetaxine mepesuccinate
against ponatinib-resistant BCR-ABL-positive cells. Blood.
2013; 122: 3086-3088.
14. Ahmed W, Van Etten RA. Alternative approaches toeradicating the malignant clone in chronic myeloid
leukemia: tyrosine-kinase inhibitor combinations andbeyond. Hematology Am Soc Hematol Educ Program.
2013; 2013: 189-200.

Kantarjian HM, Giles F, Gattermann N, Bhalla K, Alimena
G, Palandri F, Ossenkoppele GJ, Nicolini FE, O’Brien SG,
Litzow M, Bhatia R, Cervantes F, Haque A, et al. Nilotinib(formerly AMN107), a highly selective BCR-ABL tyrosine
kinase inhibitor, is effective in patients with Philadelphia
chromosome-positive chronic myelogenous leukemia inchronic phase following imatinib resistance and intolerance.
Blood. 2007; 110: 3540-3546.

15. Zitouni S, Nabais C, Jana SC, Guerrero A, Bettencourt-Dias
M. Polo-like kinases: structural variations lead to multiple
functions. Nat Rev Mol Cell Biol. 2014; 15: 433-452.
16. Degenhardt Y, Lampkin T. Targeting Polo-like kinase incancer therapy. Clin Cancer Res. 2010; 16: 384-389.
17. Renner AG, Dos Santos C, Recher C, Bailly C, Créancier L,
Kruczynski A, Payrastre B, Manenti S. Polo-like kinase 1 isoverexpressed in acute myeloid leukemia and its inhibition
preferentially targets the proliferation of leukemic cells.
Blood. 2009; 114: 659-662.

Shah NP, Kantarjian HM, Kim DW, Réa D, DorlhiacLlacer PE, Milone JH, Vela-Ojeda J, Silver RT, Khoury
HJ, Charbonnier A, Khoroshko N, Paquette RL, Deininger
M, et al. Intermittent target inhibition with dasatinib 100
mg once daily preserves efficacy and improves tolerability
in imatinib-resistant and -intolerant chronic-phase chronic
myeloid leukemia. J Clin Oncol. 2008; 26: 3204-3212.

18. Gumireddy K, Reddy MV, Cosenza SC, Boominathan
R, Baker SJ, Papathi N, Jiang J, Holland J, Reddy EP.
ON01910, a non-ATP-competitive small molecule inhibitorof Plk1, is a potent anticancer agent. Cancer Cell. 2005; 7:
275-286.

Kantarjian HM, Baccarani M, Jabbour E, Saglio G, Cortes
JE. Second-generation tyrosine kinase inhibitors: the futureof frontline CML therapy. Clin Cancer Res. 2011; 17: 16741683.

19. Bowles DW, Diamond JR, Lam ET, Weekes CD, Astling
DP, Anderson RT, Leong S, Gore L, Varella-Garcia M,

Wei G, Rafiyath S, Liu D. First-line treatment for chronicwww.impactjournals.com/oncotarget

9.

11. Cortes JE, Kim DW, Pinilla-Ibarz J, le Coutre P, Paquette
R, Chuah C, Nicolini FE, Apperley JF, Khoury HJ, Talpaz
M, DiPersio J, DeAngelo DJ, Abruzzese E, et al. A phase2 trial of ponatinib in Philadelphia chromosome-positive
leukemias. N Engl J Med. 2013; 369: 1783-1796.

References2.

Diamond JM, Melo JV. Mechanisms of resistance to BCRABL kinase inhibitors. Leuk Lymphoma. 2011; 52: 12-22.

10. Cortes JE, Kantarjian H, Shah NP, Bixby D, Mauro MJ,
Flinn I, O’Hare T, Hu S, Narasimhan NI, Rivera VM,
Clackson T, Turner CD, Haluska FG, et al. Ponatinib in
refractory Philadelphia chromosome-positive leukemias. N
Engl J Med. 2012; 367: 2075-88.

Conflicts of interest

1.

8.

20239

-204-

Oncotarget

Vogler BW, Keysar SB, Freas E, Aisner DL, et al. Phase
I study of oral rigosertib (ON 01910.Na), a dual inhibitorof the PI3K and Plk1 pathways, in adult patients withadvanced solid malignancies. Clin Cancer Res. 2014; 20:1656-1665.

Ohyashiki K. Activity of histone deacetylase inhibitors
and an Aurora kinase inhibitor in BCR-ABL-expressing
leukemia cells: Combination of HDAC and Aurora
inhibitors in BCR-ABL-expressing cells. Cancer Cell Int.
2013; 13: 32.

20. Komrokji RS, Raza A, Lancet JE, Ren C, Taft D, Maniar M,
Wilhelm F, List AF. Phase I clinical trial of oral rigosertib
in patients with myelodysplastic syndromes. Br J Haematol.
2013; 162: 517-524.

31. Okabe S, Tauchi T, Tanaka Y, Ohyashiki K. Efficacy of
ponatinib against ABL tyrosine kinase inhibitor-resistantleukemia cells. Biochem Biophys Res Commun. 2013; 435:506-511.

21. Mariotti LG, Pirovano G, Savage KI, Ghita M, Ottolenghi
A, Prise KM, Schettino G. Use of the γ-H2AX assay toinvestigate DNA repair dynamics following multiple
radiation exposures. PLoS One. 2013; 8: e79541.
22. Liu X, Erikson RL. Polo-like kinase (Plk)1 depletioninduces apoptosis in cancer cells. Proc Natl Acad Sci. 2003;
100: 5789-5794.
23. Bachegowda L, Gligich O, Mantzaris I, Schinke C, WyvilleD, Carrillo T, Braunschweig I, Steidl U, Verma A. Signaltransduction inhibitors in treatment of myelodysplastic
syndromes. J Hematol Oncol. 2013; 6: 50.
24. Ma WW, Messersmith WA, Dy GK, Weekes CD,
Whitworth A, Ren C, Maniar M, Wilhelm F, Eckhardt
SG, Adjei AA, Jimeno A. Phase I study of Rigosertib, an
inhibitor of the phosphatidylinositol 3-kinase and Polo-likekinase 1 pathways, combined with gemcitabine in patientswith solid tumors and pancreatic cancer. Clin Cancer Res.
2012; 18: 2048-2055.
25. Mross K, Dittrich C, Aulitzky WE, Strumberg D, Schutte J,
Schmid RM, Hollerbach S, Merger M, Munzert G, Fleischer
F, Scheulen ME. A randomised phase II trial of the Pololike kinase inhibitor BI 2536 in chemo-naïve patients withunresectable exocrine adenocarcinoma of the pancreas - a
study within the Central European Society Anticancer Drug
Research (CESAR) collaborative network. Br J Cancer.
2012; 107: 280-286.
26. Okabe S, Tauchi T, Tanaka Y, Ohyashiki K. Dasatinib
preferentially induces apoptosis by inhibiting Lyn kinasein nilotinib-resistant chronic myeloid leukemia cell line. J
Hematol Oncol. 2011; 4: 32.
27. Okabe S, Tauchi T, Ohyashiki K. Establishment of a new
Philadelphia chromosome-positive acute lymphoblastic
leukemia cell line (SK-9) with T315I mutation. Exp
Hematol. 2010; 38: 765-772.
28. Kimura S, Naito H, Segawa H, Kuroda J, Yuasa T, Sato
K, Yokota A, Kamitsuji Y, Kawata E, Ashihara E, Nakaya
Y, Naruoka H, Wakayama T, et al. NS-187, a potent andselective dual Bcr-Abl/Lyn tyrosine kinase inhibitor, is a
novel agent for imatinib-resistant leukemia. Blood. 2005;
106: 3948-3954.
29. Okabe S, Tauchi T, Tanaka Y, Katagiri S, Ohyashiki K.
Effects of the hedgehog inhibitor GDC-0449, alone orin combination with dasatinib, on BCR-ABL-positive
leukemia cells. Stem Cells Dev. 2012; 21: 2939-2948.
30. Okabe S, Tauchi T, Tanaka Y, Kimura S, Maekawa T,
www.impactjournals.com/oncotarget

20240

-205-

Oncotarget

Review Article

Potential clinical application of interleukin-27 as anantitumor agent
Takayuki Yoshimoto, Yukino Chiba, Jun-Ichi Furusawa, Mingli Xu, Ren Tsunoda, Kaname Higuchi and
Izuru Mizoguchi
Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan

Key words
IL-27, antitumor effects, protumor effects, IL-10, Treg
Correspondence
Takayuki Yoshimoto, Department of Immunoregulation,
Institute of Medical Science, Tokyo Medical University,
6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan.
Tel: +81-3-3351-6141 Ext. 431; Fax: +81-3-3351-6250;
E-mail: yoshimot@tokyo-med.ac.jp
Funding Information
This work was supported in parts by grants from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan, and from the MEXT-Supported
Program for the Strategic Research Foundation at Private
Universities, 2013-2017.
Received April 16, 2015; Revised June 17, 2015; Accepted
June 25, 2015
Cancer Sci 106 (2015) 1103–1110
doi: 10.1111/cas.12731

Cancer immunotherapies such as sipuleucel-T and ipilimumab are promising newtreatments that harness the power of the immune system to fight cancer andachieve long-lasting remission. Interleukin (IL)-27, a member of the IL-12 heterodimeric cytokine family, has pleiotropic functions in the regulation of immune
responses with both pro-inflammatory and anti-inflammatory properties. Evidence obtained using a variety of preclinical mouse models indicates that IL-27
possesses potent antitumor activity against various types of tumors through multiple mechanisms without apparent adverse effects. These mechanisms include
those mediated not only by CD8+ T cells, natural killer cells and macrophages,
but also by antibody-dependent cell-mediated cytotoxicity, antiangiogenesis,
direct antiproliferative effects, inhibition of expression of cyclooxygenase-2 andprostaglandin E2, and suppression of epithelial–mesenchymal transition, depending on the characteristics of individual tumors. However, the endogenous role ofIL-27 subunits and one of its receptor subunits, WSX-1, in the susceptibility totumor development after transplantation of tumor cell lines or endogenously
arising tumors seems to be more complicated. IL-27 functions as a double-edged
sword: IL-27 increases IL-10 production and the expression of programmed deathligand 1 and T-cell immunoglobulin and mucin domain-3, and promotes the generation of regulatory T cells, and IL-27 receptor a singling enhances transformation; IL-27 may augment protumor effects as well. Here, we review both facets of
IL-27, antitumor effects and protumor effects, and discuss the potential clinicalapplication of IL-27 as an antitumor agent.

Cancer ImmunotherapyC

ancer immunotherapy has recently come into the spotlight
as a promising new treatment that harnesses the power ofthe immune system; sipuleucel-T and ipilimumab wereapproved by the US Food and Drug Administration for patientswith cancer in 2010 and 2011, respectively.(1,2) Sipuleucel-T isa therapeutic vaccine using dendritic cells (DC) stimulated bya tumor-specific antigen combined with granulocyte-macrophage colony-stimulating factor.(1) Ipilimumab is an inhibitory
monoclonal antibody specific to one of the immune checkpoints, cytotoxic T-lymphocyte antigen 4.(2) Currently, various
therapeutic strategies to overcome tumor immune evasion andenhance antitumor immunity are also undergoing investigation.
Interleukin-12 Cytokine Family

The interleukin (IL)-12 cytokine family is unique in that it is aheterodimeric cytokine composed of two different subunits,

and one subunit is often shared by another cytokine in thesame family.(3,4) These member cytokines are mainly produced
by antigen-presenting cells such as DC and play critical rolesin the regulation of differentiation into respective helper T(Th) cells and their functions. IL-12 is composed of p35 andp40 subunits and induces the differentiation of naive CD4+ T
cells into Th1 cells to enhance the cellular immunity by augmentation of interferon (IFN)-c production from natural killer(NK) cells and T cells and generation of cytotoxic T lymphocytes (CTL).(3) The p40 subunit is also covalently bound with
an IL-12 p35-related protein, p19, to form IL-23, which plays
an important role in developing tissue-specific autoimmune
diseases and inflammatory diseases by enhancing IL-17 production and maintaining pathogenic Th17 cells.(4) IL-27 consists of an IL-12 p35-related protein, p28, which is also called
IL-30, and an IL-12 p40-related protein, Epstein–Barr virus-induced gene 3 (EBI3)(4–7) (Fig. 1). IL-27 receptor (R) is composed of IL-27Ra (WSX-1 ⁄ TCCR) and glycoprotein (gp) 130,
a common receptor subunit for the IL-6 family of cytokines.
EBI3 was previously reported to associate with p35 to form

© 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd
on behalf of Japanese Cancer Association.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and distribution
in any medium, provided the original work is properly cited, the use is noncommercial and no modifications or adaptations are made.

-206-

Cancer Sci | September 2015 | vol. 106 | no. 9 | 1103–1110

Review
Potent antitumor effects of IL-27

www.wileyonlinelibrary.com/journal/cas

Fig. 1. Antitumor and protumor effects of IL-27 depending on thetype of cells that IL-27 stimulates and the tumor context. CTL, cytotoxic T lymphocyte; DC, dendritic cell; EBI3, Epstein–Barr virus-induced
gene 3; gp, glycoprotein; IFN, interferon; IL, interleukin; NK, naturalkiller; PD-L1, programmed death ligand 1; R. receptor; STAT, signaltransducer and activator of transcription; Th, helper T; Tim-3, T-cell
immunoglobulin and mucin domain-3; Tr1, IL-10-producing regulatory
T; Treg, regulatory T.

heterodimeric molecule EBI3 ⁄ p35, and EBI3 ⁄ p35 was recently
found to be produced by regulatory T (Treg) cells and to playa suppressive role in the Treg cells; therefore, it is named
IL-35.
Pro-Inflammatory and Anti-Inflammatory Properties ofInterleukin-27

Interleukin-27 is one of the pleiotropic cytokines with bothpro-inflammatory and anti-inflammatory properties acting on
various types of cells, such as T cells, B cells, macrophages
and DC, depending on the context (Fig. 1).(4–6) IL-27 efficiently activates both signal transducer and activator of transcription (STAT) 1 and STAT3, which bind to distinct IL-27R
subunits WSX-1 and gp130, respectively. IL-27 promotes theearly induction of Th1 differentiation and generation ofCTL,(8,9) but it inhibits the differentiation of naive CD4+ T
cells into Th2 cells, Th9 cells and Th17 cells, and suppresses
the production of pro-inflammatory cytokines, leading to amelioration of allergic diseases and autoimmune diseases. In addition, IL-27 augments IL-10 production from these Th cells andgeneration of IL-10-producing regulatory (Tr1) T cells,(10–12)
whereas the role of IL-27 in induction of Treg cells is still controversial. IL-27 was initially shown to suppress the differentiation of inducible Treg cells from naive CD4+ T cells,(13–15) andtransfer of TCCR-deficient CD4+CD45RBhi cells in a colitis
model was demonstrated to reduce development of colitis withan increased percentage of Treg cells in the gut.(16) Contrary
to the antagonistic effects of IL-27 on Treg cells, recent
reports have paradoxically revealed that IL-27 does not inhibit
the generation of Treg cells, but rather promotes it and produces a distinct Treg cell population expressing T-cell-specific
T-box transcription factor (T-bet) and CXCR3 to control
Th1-mediated immunity at the local sites of inflammation.(17)Moreover, IL-27 was recently demonstrated to exert immunosuppressive functions via induction of the programmed
death ligand 1 (PD-L1) on CD4+ T cells and DC.(18–20) IL-27mediated induction of PD-L1 inhibits Th17 cell differentiation
and reduces pathology in experimental autoimmune
encephalomyelitis.(20)Fig. 2. Potent antitumor activity of IL-27 through multiple mechanisms that are mediated by CD8+ T cells, NK cells, macrophages,
macrophages, ADCC, anti-angiogenesis, direct anti-proliferative effect,
inhibition of COX-2 and PGE2 expression, and suppression of EMT,
depending on the characteristics of individual tumors. ADCC, antibody-dependent cell-mediated cytotoxicity; COX-2, cyclooxygenase-2;
CTL, cytotoxic T lymphocyte; DC, dendritic cell; EBI3, Epstein-Barrvirus–induced gene 3; EMT, epithelial–mesenchymal transition; IRF,
interferon regulatory factor; IL, interleukin; MHC, major histocompatibility complex; MMP, matrix metalloproteinase; NK, naturalkiller; PGE2, prostaglandin E2; PD-L1, programmed death ligand 1;
Tim-3, T-cell immunoglobulin and mucin domain-3; TLR, Toll-likereceptor; Tr1, IL-10-producing regulatory T; TRAIL, tumor necrosis
factor–related apoptosis-inducing ligand; Treg, regulatory T; VEGF,
vascular endothelial growth factor.

Therapeutic Potential of Interleukin-27 as an Antitumor
Agent
Antitumor effects of interleukin-27 against transplanted
tumors genetically engineered to secrete interleukin-27. The

antitumor efficacy of IL-27 was first demonstrated in 2004
using a transplanted mouse tumor genetically engineered tosecrete IL-27 before transplantation as a preclinical tumor
model.(21) Since then, accumulating evidence has revealed that
IL-27 possesses potent antitumor activity against a variety oftumor models through multiple mechanisms without apparent
adverse effects.(21,22) The mechanisms are those mediatedby CD8+ T cells,(21,23–30) NK cells(22,24,25,31) and macrophages,(32) as well as antibody-dependent cell-mediated cytotoxicity(ADCC),(33)anti-angiogenesis,(34–40)
directantiproliferative effects,(36,41–43) inhibition of expression of
cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2),(44)and suppression of epithelial–mesenchymal transition(EMT),(32,39) depending on the characteristics of individual
tumors (Fig. 2 and Table 1). Transfection of highly immunogenic tumors such as mouse colon carcinoma (colon 26) with
IL-27 expression vector greatly reduced tumor growth, which
was mainly mediated by CD8+ T cells.(21) IL-27 plays a particularly important role in the generation of CTL by enhancing
the survival of tumor-specific CD8+ T cells and the differentiation into effector and memory cells.(8,9) In contrast, the antitumor effects of IL-27 against poorly immunogenic tumors suchas mouse melanoma B16F10 were mediated by multiple mechanisms via angiogenesis,(34) NK cells,(22,25,31) and its direct
antiproliferative effects on tumors.(32,39,41,44) IL-27 stimulated
human umbilical vein endothelial cells and induced productionof anti-angiogenic chemokines such as CXCR9 and CXCR10
to elicit strong anti-angiogenic activity against melanomas,
which contributed to its antitumor and antimetastatic activi-© 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd
on behalf of Japanese Cancer Association.

-207-

Cancer Sci | September 2015 | vol. 106 | no. 9 | 1104

Review
Yoshimoto et al.

www.wileyonlinelibrary.com/journal/cas

Table 1. Susceptibility of mice transplanted with tumors expressing IL-27 and mice injected with IL-27 to development of tumors
Treatment

Susceptibility

IL-27
transfectant
IL-27
transfectant

Reduced tumor growth in vivo by augmented antitumor CTL

Mouse colon carcinoma (Colon 26)

Reduced tumor growth in vivo by augmented antitumor CTL

Mouse orthotopic primary and metastatic
neuroblastoma (TBJ)

IL-27
transfectant
IL-27
transfectant

Reduced tumor growth in vivo by enhanced T-celldependent and NK-cell-dependent antitumor immunity
Reduced tumor growth in vivo by upregulation of CXCL9and CXCL10 and inhibition of angiogenesis

Mouse colon carcinoma (Colon 26)

IL-27
transfectant
IL-27
transfectant
In vitro
treatment

Reduced tumor growth in vivo by NK-cell-mediatedantitumor immunity independent of IFN-c
Reduced tumor growth in nude mice by NK cells throughFas ⁄ FasL pathway
Reduced tumor growth in vitro by direct antiproliferative
activity through WSX-1 ⁄ STAT1 ⁄ IRF-1 signaling

Mouse melanoma (B16F10)IL-27transfectant
IL-27
transfectant
IL-27
transfectant
IL-27 plasmid
injection

Reduced tumor growth in nude mice by augmented IFN-c
production and NK activity
Reduced tumor growth in vivo by NK cell–mediated ADCC

Human esophageal carcinoma (Eca109)

IL-27
transfectant
IL-27 plasmid
injection
IL-27 protein
injection
IL-27 protein
injection
IL-27 genetherapy
IL-27 protein
injection
IL-27
transfectant byretroviral
infection
IL-27 protein
injection
IL-27
transfectant

IL-27
transfectant
vaccineIL-27 protein
injection

Tumor

Reduced tumor growth in vivo by CD8+ and CD4+ T cells,
IFN-c and NK cells
Therapeutic synergy of hydrodynamic injection of IL-27
plasmid and IL-2 by augmented generation of tumorspecific CTLs but suppressed expansion of Treg cells
Restraint of tumorigenicity in vivo by suppressing theexpression of COX-2 and PGE2
Elimination of distal aggressive tumor by IL-12 and IL-27
sequential gene therapy via intramuscular electroporation
through T and NK cells
Damped tumorigenicity of human multiple myeloma celllines in NOD ⁄ SCID mice through inhibition of angiogenesis
Inhibition of leukemia cell spreading in NOD ⁄ SCID ⁄ IL-2Rc ⁄mice through suppression of proliferation, angiogenesis,
miR-155 expression and induction of apoptosis
Inhibition of tumor growth by sonoporation delivery of IL-27
gene therapy through enhanced accumulation of effector
cells
Inhibition of tumor growth in NOD ⁄ SCID mice throughsuppressed angiogenesis
Inhibition of tumor growth in nude mice through inductionof apoptosis and cell-cycle arrest

Inhibition of tumor growth in NOD ⁄ SCID ⁄ IL-2Rc ⁄ micethrough suppression of angiogenesis and spreading related
genes
Enhanced antitumor CTL responses in vivo via programmingtumor antigen-specific CD8+ T cells into IL-10-producing
memory precursor-like effector cells characterized by agreater survival advantage
T-cell-mediated antitumor effects by tumor vaccine
engineered to secrete IL-27 by means of cationic liposome
Inhibition of tumor growth in NOD ⁄ SCID mice by
upregulation of TRAIL and TLR3 in cooperation with a TLR3
agonist poly(I:C) in a partly TRAIL-dependent manner

Cancer Sci | September 2015 | vol. 106 | no. 9 | 1105

Mouse melanoma (B16F10) lung metastasis

Human esophageal carcinoma (Eca109)
Mouse melanoma (B16F10)
Human melanoma (SK-MEL-13, 28, 37)

Mouse head and neck squamous cell
carcinoma (SCCVII)
Mouse hepatocellular carcinoma (MM45T.Li)
Mouse disseminated metastatic neuroblastoma(TBJ) in liver
Mouse Lewis lung carcinoma (LLC)
Mouse colon carcinoma (Colon 26)
Mouse adenocarcinoma (4T1)

Reference
Hisada et al.
2004(21)
Salcedoet al.
2004(23)
Chiyo et al.
2004(24)
Shimizu
et al.
2006(34)
Oniki et al.
2006(22)
Liu et al.
2008(45)
Yoshimotoet al.
2008(41)
Liu et al.
2008(31)
Matsui et al.
2009(33)
Hu et al.
2009(25)
Salcedoet al.
2009(26)
Ho et al.
2009(44)
Zhu et al.
2010(27)

Human multiple myeloma (NCI-H929 andU266)
Human B-ALL

Cocco et al.
2010(35)
Canale et al.
2011(36)

Mouse prostate cancer (TCR2)

Zolochevska
et al.
2011(28)
Cocco et al.
2012(37)
Liu et al.
2012(42)

Human follicular lymphoma and diffuse large
B-cell lymphoma (SU-DHL-4)
Human pancreatic carcinoma (AsPC1)

Human AML

Zorzoli et al.
2012(38)

Mouse plasmacytoma (J558)

Liu et al.
2013(29)

Mouse Lewis lung carcinoma (LLC)

Zhang et al.
2013(30)

Human melanoma (SK-ME-13, 28, 37)

Chiba et al.
2013(43)© 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd
on behalf of Japanese Cancer Association.

-208-

Review
Potent antitumor effects of IL-27

www.wileyonlinelibrary.com/journal/cas

Table 1 (continued)
Treatment

Susceptibility

Tumor

In vitro
treatment

Inhibition of EMT and angiogenic factor production in a
STAT1-dominant pathway

Human non-small cell lung cancer

IL-27 protein
injection

Inhibition of tumor growth in athymic nude mice throughreduced proliferation and vascularization by
downregulation of pro-angiogenesis-related genes (FLT1,
PTGS1 ⁄ COX-1, FGFR3) and upregulation of antiangiogenesis-related genes (CXCL10, TIMP3)
Inhibition of tumor growth in athymic nude and NOD ⁄ SCID
mice through granulocyte-driven and macrophage-driven
colliquative necrosis, CXCL3 production, and reduced
pluripotency-related and EMT-related gene expression

Human prostate cancer (PC3 and DU145)

IL-27 protein
injection

Human non-small cell lung cancer, particularly
adenocarcinoma (Calu-6) and squamous cell
carcinoma (SK-MES)

Reference
Kachroo
et al.
2013(39)
Di Carlo
et al.
2014(40)

Airoldi et al.
2015(32)

ADCC, antibody dependent cell-mediated cytotoxicity; AML, acute myeloid leukemia; B-ALL, B-acute lymphoblastic leukemia; COX-2, cyclooxygenase-2; CTL, cytotoxic T lymphocyte; EBI3, Epstein–Barr virus-induced gene 3; EMT, epithelial–mesenchymal transition; FGFR, fibroblast growth
factor receptor; FLT, fms-related tyrosine kinase; IFN, interferon; IRF, interferon regulatory factor; IL, interleukin; NK, natural killer; LLC, Lewis
lung carcinoma; NOD ⁄ SCID, nonobese diabetic ⁄ severe combined immunodeficient; PGE2, prostaglandin E2; poly(I:C), polyinosinic-polycytidylicacid; PTGS1, prostaglandin G ⁄ H synthase 1; STAT, signal transducer and activator of transcription; TLR, Toll-like receptor; TRAIL, tumor necrosis
factor–related apoptosis-inducing ligand; Treg, regulatory T; TIMP3, tissue inhibitor of metalloproteinase 3.

ties.(34) In addition, IL-27 has potent direct antiproliferative
activity on melanomas through WSX-1 ⁄ STAT1 and, in part,through interferon regulatory factor (IRF)-1 and tumor necrosis
factor (TNF)-related apoptosis-inducing ligand (TRAIL).(41,43)
It also directly restrained tumorigenicity of mouse Lewis lungcarcinoma LLC by suppressing expression of COX-2 andPGE2.(44) Moreover, IL-27 activated NK cells and augmented
tumor-specific immunoglobulin production, which cooperatively exerted ADCC activity against mouse head and necksquamous cell carcinoma SCCVII,(33) and inhibited EMT andangiogenic factor production in a STAT1-dominant pathway inhuman non-small cell lung cancer.(32,39) Similar suppression oftumor growth was observed with human tumors such as esophageal carcinoma(45) and pancreatic carcinoma.(42)
Antitumor effects of interleukin-27 protein injection against
human tumors transplanted in immunodeficient mice. Moreover,

IL-27 has demonstrated potent antitumor activity in a varietyof human therapeutic models through injection of IL-27 protein into immunodeficient mice such as nude mice, nonobese
diabetic ⁄ severe combined immunodeficient (NOD ⁄ SCID) miceand NOD ⁄ SCID ⁄ IL-2Rc / mice after transplantation ofhuman tumors. The human tumors reported on so far are melanoma,(43) multiple myeloma,(35) B-acute lymphoblastic leukemia (B-ALL),(36) follicular lymphoma,(37) diffuse large B-cell
lymphoma,(37) acute myeloid leukemia (AML),(38) prostatecancer(40) and non-small cell lung cancer(32,39) (Table 1). IL27 strongly inhibited in vitro tumor growth of human melanoma, multiple myeloma, follicular lymphoma and diffuse
large B-cell lymphoma through suppression of angiogenesis
and induction of apoptosis, and the tumorigenicity of thesetumors transplanted in NOD ⁄ SCID mice was greatly hampered
by IL-27.(35,37,43) Similarly, IL-27 suppressed leukemic spreading of B-ALL cells and leukemia dissemination of AML cells
transplanted in NOD ⁄ SCID ⁄ IL-2Rc / because of significant
reduction of angiogenic and spreading-related genes, including
vascular endothelial growth factors, angiopoietins and matrix
metalloproteinases (MMP), and also because of upregulationof angiostatic molecules, such as tissue inhibitor of
MMP.(36,38) Direct antiproliferative effects of IL-27 in collaboration with polyinosinic-polycytidylic acid [poly(I:C)], one of
the Toll-like receptor 3 (TLR3) ligands whose expression was

revealed to be upregulated by IL-27, was also observed in
NOD ⁄ SCID mice transplanted with human melanoma.(43) In
addition, IL-27 was recently shown to inhibit tumor growth ofhuman prostate cancers in athymic nude mice throughreduced proliferation and vascularization by downregulation of
pro-angiogenesis-related genes and upregulation of anti-angiogenesis-related genes.(40) Inhibition of tumor growth of humannon-small cell lung cancers was also demonstrated to be mediated by granulocyte-driven and macrophage-driven colliquative
necrosis, CXCL3 production, and reduced pluripotency-related
and EMT-related gene expression.(32,39)
Endogenous Role of IL-27 in the Susceptibility to
Development of Tumors
Antitumor and protumor effects of endogenous WSX-1. To
gain further insight into the antitumor effects of IL-27, it isimportant to clarify its endogenous role in the exertion of antitumor effects or protumor effects. Therefore, mice deficient in
IL-27 subunits and receptor subunits were analyzed for susceptibility to tumor development (Table 2). WSX-1-deficient miceoverall showed more excessive tumor growth of melanoma
B16 injected subcutaneously than did WT mice.(46) However,this phenotype appears to be the sum of the effects of lacking
WSX-1 in different immune responses, such as generation of
CTL and antigen-presenting capacity of DC after maturation.
Tumor-specific CTL generation was lower in WSX-1-deficient
mice than in WT mice, and CTL induction in WSX-1-deficient
mice was not restored by transfer of WT DC pulsed withtumor antigen, indicating that IL-27 is directly required forgeneration of tumor-specific CTL.(46) In contrast, when transferred into tumor-bearing mice, WSX-1-deficient DC pulsedwith tumor antigen were more potent than WT DC in the inhibition of tumor growth and generation of CTL, indicating the
suppressive effects of IL-27 on DC function.(46) It is also
reported that WSX-1-deficient mice had reduced resistance toendogenously arising mouse tumor models, 3-methylcholanthrene (MCA)-induced fibrosarcoma and polyoma middle T
antigen (PyMT)-induced mammary carcinoma.(47) This reduced
resistance was accompanied by decreased IFN-c production
from CD4+ and CD8+ T cells and an increased number of Treg

© 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd
on behalf of Japanese Cancer Association.

-209-

Cancer Sci | September 2015 | vol. 106 | no. 9 | 1106

Review
Yoshimoto et al.

www.wileyonlinelibrary.com/journal/cas
Table 2. Susceptibility of mice deficient in WSX-1, EBI3 and p28 to development of tumors
Deficient mice
WSX-1

Susceptibility

Tumor

Overall reduced resistance to tumor growth due to impaired antitumor CTL
generation but accompanied with augmented antitumor immunity by DC

Melanoma (B16)

Reduced resistance to endogenously arising tumor growth due to decreased IFN-c
production by CD4+ and CD8+ T cells and increased numbers of Treg cells

MCA-induced
fibrosarcoma
PyMT-induced
mammary carcinoma
Melanoma (B16F10)
Lewis lungcarcinoma (LLC)
Melanoma (B16F10)
lung metastasis
MCA-induced
fibrosarcoma
Melanoma (B16)
Melanoma (B16F10)
Lymphoma (EL-4)
MCA-induced
fibrosarcoma

Augmented resistance to tumor growth due to reduced number of the most
exhausted Tim-3+PD-1+CD8+ T cells among TILs
EBI3
DC-specificconditional
p28

Augmented resistance to tumor growth due to increased numbers of IFN-c-producing
killer DC with T-bet-mediated antitumor CD8+ T-cell responses in the lung
Reduced resistance to tumor growth due to impairment of IL-27-mediated CXCL10
expression in MDSCs accompanied by reduced recruitment and activation of NK and
NKT cells
Enhanced antitumor immune responses due to impairment of IL-27-mediated CCL22
expression in DC with reduced infiltration of Treg cells into tumors

Reference
Shinozaki
et al.
2008(46)
Natividad
et al.
2013(47)
Zhu et al.
2015(48)
Sauer et al.
2008(49)
Wei et al.
2013(53)
Xia et al.
2014(54)

CTL, cytotoxic T lymphocyte; DC, dendritic cells; EBI3, Epstein–Barr virus-induced gene 3; IFN, interferon; IL, interleukin; MCA, 3-methylcholanthrene; MDSC, myeloid-derived suppressor cell; NK, natural killer; PD-1, programmed death-1; PyMT, polyoma middle T antigen; T-bet, T-cellspecific T-box transcription factor; TIL, tumor-infiltrating lymphocyte; Tim-3, T-cell immunoglobulin and mucin domain-3; Treg, regulatory T.

cells. In marked contrast, however, it was recently demonstrated that WSX-1-deficient mice showed more attenuatedtumor growth of B16F10 and LLC than did WT mice.(48) Thisaugmented antitumor effect was explained by the reduced
number of T-cell immunoglobulin and mucin domain-3 (Tim3)+ programmed death-1 (PD-1)+ CD8+ T cells, which are themost exhausted T-cell population among tumor-infiltrating
lymphocytes, indicating IL-27 signaling as a key regulator ofeffector T-cell responses via induction of Tim-3.(48) Taken
together, these results suggest that IL-27 ⁄ WSX-1 signaling
plays critical roles in both generation and exhaustion of CTL,
together with suppression of DC function.
Protumor effects of endogenous Epstein–Barr virus-induced
gene 3. In contrast to both the antitumor and protumor effectsof WSX-1, whose deficiency seems to highly agree with lossof representative IL-27 functions, EBI3-deficient mice showed
augmented antitumor activity against lung metastasis of
B16F10 (Table 2).(49) This effect was induced by expansion ofa newly described cell subset called IFN-c-producing killer
DC in the lung of EBI3-deficient mice, and these DC thenactivated CD8+ T cells to produce IFN-c and TNF-a, resulting
in tumor apoptosis in a T-bet-dependent manner. This phenotype is consistent with increased EBI3 expression in serum andtumor tissue correlating with higher risk of cancer progression.(50,51) Although EBI3-deficient mice showed abrogatedIL-35-mediated Treg activity(52) and also may be devoid of
IL-27-mediated regulation of Treg cells, if any, the endogenous role of EBI3 likely contributes more strongly to the
protumor activity mediated by EBI3 alone than the antitumor
activity by IL-27.
Both antitumor and protumor effects of endogenous p28. So
far, there have been two seemingly opposite articles regarding
the endogenous role of p28 in the induction of antitumor
immune responses(53,54) (Table 2). DC-specific conditional
p28-deficient mice were initially demonstrated to have reduced
resistance to progression of tumors such as MCA-induced
fibrosarcoma and transplanted melanoma B16.(53) These effects
were due to impairment of IL-27-mediated CXCL10 expresCancer Sci | September 2015 | vol. 106 | no. 9 | 1107

sion in myeloid-derived suppressor cells (MDSC) and IL-12
production from DC, which led to recruitment and activationof NK and NKT cells, resulting in immunological control oftumors.(53) In contrast, using the same p28-deficient mice, thesame group also reported that IL-27 plays a critical role intumor infiltration of Treg cells.(54) In tumor-associated DC, IL27 promoted the expression of CCL22, which is critical for
recruitment of peripheral Treg cells into tumors, and the p28deficient mice showed reduced tumor infiltration of Treg
cells.(54) Because IL-27, but not p28, restored the respective
phenomena in the p28-deficient mice, the endogenous role of
p28 seems to strongly agree with IL-27 functions. However,
the role of IL-27 in the induction of Treg cells still appears to
be controversial in the tumor microenvironment, as describedlater.
Potential Protumor Effects of Interleukin-27
Contribution of interleukin-27-mediated upregulation of interleukin-10 to its antitumor effects. Because IL-27 possesses bothpro-inflammatory and anti-inflammatory activities, some suchas IL-27-mediated augmentation of IL-10 production(10–12) and
upregulation of PD-L1(18–20) and Tim-3(48) may function
against its antitumor effects. However, the role of IL-10 in the
induction of antitumor immune responses is often controversial, and increasing evidence supports a positive role of IL-10
for induction of antitumor CTL responses, which were
demonstrated in IL-10-deficient mice and IL-10 transgenic
mice.(55–57) IL-27 was recently demonstrated to significantly
enhance the survival of activated tumor-specific CD8+ T cells
in vitro and in vivo and to induce a unique memory precursor
cell phenotype in tumor antigen-specific CD8+ T cells,
characterized by, for instance, upregulation of IL-10.(29) The
IL-27-mediated IL-10 production from CTL contributed to the
induction of memory precursor cell phenotype, CTL memory
and, consequently, tumor rejection.(29)Contribution of interleukin-27-mediated upregulation of programmed death ligand 1 and T-cell immunoglobulin and mucin© 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd
on behalf of Japanese Cancer Association.

-210-

Review
Potent antitumor effects of IL-27

www.wileyonlinelibrary.com/journal/cas

domain-3 to its protumor effects. Exhausted T cells are charac-terized by their sustained expression of inhibitory receptors
such as PD-1 and Tim-3.(58–60) The PD1 ⁄ PD-L1 pathway has
emerged as a central player in immune regulation, and cancer
cells that express PD-L1 promote tumor progression throughinhibition of PD-1 expressing effector cells.(61) Co-expressionof PD-1 and other inhibitory receptors such as Tim-3 contributes to the induction of T-cell exhaustion and defines T
cells with more deeply exhausted phenotype.(60) IL-27 wasrecently shown to induce expression of PD-L1 in not only T
cells and DC(18–20) but also tumors.(62) Histological analyses
revealed that when IL-27 was detected in primary cutaneous
melanomas, its expression was maintained in metastatic
lesions.(62) In addition, IL-27 induced PD-L1 expression inmelanoma cell lines and the in situ expression of IL-27 in melanoma correlated with those of PD-L1 and IL-10.(62) Thus, it
is highly conceivable that IL-27-induced PD-L1 expression in
T cells and tumors could contribute to the protumor effects of
IL-27, whose possibility remains to be directly clarified. Moreover, IL-27 was recently demonstrated to induce the expressionof the transcription factor nuclear factor, IL-3 regulated(NFIL3), which cooperates with T-bet, to induce the expression of Tim-3.(48) IL-27 signaling was required for the induction of Tim-3+ exhausted T cells and promotion of tumor
growth.(48) Thus, IL-27-mediated upregulation of inhibitory
molecules such as PD-L1 and Tim-3 likely contributes totumor progression.
Positive and negative effects of interleukin-27 on Treg cells.

The role of IL-27 in the induction of Treg cells in the context of tumorigenesis is also controversial. The induction ofTreg cells(54,63) and the suppression of them(47) by IL-27
have both been reported. Initially, apoptotic tumor cells were
demonstrated to induce IL-27 release from human DC toactivate Treg cells that express CD69 and CD39 to generateadenosine, thereby suppressing cytotoxicity of CD73-expressing CD8+ T cells and limiting antitumor immunity.(63) Moreover, the effect of endogenous IL-27 on Treg cells in thetumor microenvironment was investigated using IL-27p28
conditional knockout mice. IL-27 induced the expression of
CCL22 on tumor-associated DC, which mediates tumor infiltration of Treg cells, resulting in tumor progression.(54) In
contrast, it was also demonstrated that tumor development
and growth are accelerated in WSX-1-deficient mice.(47) The
enhanced tumor growth in both carcinogen-induced fibrosarcoma and oncogene-driven mammary carcinoma was associated with increased number of Treg cells and decreased
IFN-c-production by CD4+ and CD8+ T cells.(47) Collectively, if IL-27 were to promote the generation of Treg
cells, the antitumor effects of IL-27 could be weakened. In
contrast, if IL-27 were to inhibit it, the antitumor effects of
IL-27 could be augmented, but side effects such as development of autoimmune diseases might be expected. Further

References
1 Kantoff PW, Higano CS, Shore ND et al. Sipuleucel-T immunotherapy
for castration-resistant prostate cancer. N Engl J Med 2010; 363: 411–
22.
2 Hodi FS, O’Day SJ, McDermott DF et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 2010; 363:
711–23.
3 Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 2003; 3: 133–46.

studies are necessary to elucidate the precise effect of IL-27
on Treg cells in the tumor microenvironment.
Potential transforming activity of interleukin-27Ra. A point
mutation in Janus kinase (JAK)2 (JAK2-V617F), which has anincreased inherent kinase activity, was identified in a numberof neoplastic myeloproliferative disorders.(64) From a patientwith acute myeloid leukemia, IL-27Ra was recently identified
as a gene that can induce ligand-independent transformation ofhematopoietic cells via JAK-dependent pathways.(65) In addition, it was demonstrated that IL-27Ra induces the tyrosine
phosphorylation of JAK2 and its downstream target STAT5,
and transforms cytokine-dependent hematopoietic cells tocytokine independence when co-expressed with mutated
JAK2-V617F but not wild-type JAK2.(65,66) This activating
effect on JAK2-V617F by IL-27Ra was dependent on a functional IL-27Ra Box 1 motif but was independent of the gp130
co-receptor, suggesting that IL-27Ra may be functioning as a
homodimer.(65) Intriguingly, we have previously shown that IL27 stimulates and expands hematopoietic stem cells to differentiate into myeloid cells in IL-27 transgenic mice.(67) Taken
together, aberrant IL-27Ra signaling may enhance myeloid cell
growth, potentially leading to myeloproliferative neoplasms,
although higher incidence of development of spontaneoustumors was not observed in the IL-27 transgenic mice.(67)
Future Prospects and Concluding Remarks

Cytokine-based cancer immunotherapies currently used include
systemic administration, local delivery, vaccination with tumor
cells engineered to secrete cytokine, adjuvants for cancer vaccine and adoptive therapy of immune cells expanded by cytokines. Because IL-27 is a multifunctional cytokine acting witha double-edged sword, both antitumor and protumor effects of
IL-27 are conceivably expected depending on the type of cells
that IL-27 stimulates and the tumor context. Currently, antibody drugs have become very popular and are one of the mostpromising cancer immunotherapies. Therefore, to overcome
the protumor effects of IL-27, it is highly possible that IL-27mediated antitumor effects could be further augmented by antibodies against immune checkpoints, such as PD-L1 and Tim-3,
and also by antibodies against Treg cells, such as cytotoxic
T-lymphocyte-associated protein 4. These possibilities remain
to be investigated in the near future. In addition, IL-27 wasshown to have lower toxicity in mouse models, probably
because of low induction of IFN-c;(21,22) therefore, the combination of IL-27 treatment with other cancer immunotherapies
to overcome the protumor effects of IL-27 could be a promising therapeutic strategy against cancer.
Disclosure Statement

The authors have no conflict of interest to declare.

4 Kastelein RA, Hunter CA, Cua DJ. Discovery and biology of IL-23 and IL27: related but functionally distinct regulators of inflammation. Annu Rev
Immunol 2007; 25: 221–42.
5 Villarino AV, Huang E, Hunter CA. Understanding the pro- and anti-inflammatory properties of IL-27. J Immunol 2004; 173: 715–20.
6 Mizoguchi I, Higuchi K, Mitobe K, Tsunoda R, Mizuguchi J, Yoshimoto T.
Interleukin-27: Regulation of immune responses and disease development by
a pleiotropic cytokine with pro- and anti-inflammatory properties. In: Yoshimoto T, Yoshimoto T, eds. Cytokine Frontiers: Regulation of Immune
Responses in Health and Disease. Tokyo: Springer, 2013; 353–75.

© 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd
on behalf of Japanese Cancer Association.

-211-

Cancer Sci | September 2015 | vol. 106 | no. 9 | 1108

Review
Yoshimoto et al.

www.wileyonlinelibrary.com/journal/cas
7 Yoshida H, Hunter CA. The immunobiology of interleukin-27. Annu Rev
Immunol 2015; 33: 417–43.
8 Morishima N, Owaki T, Asakawa M, Kamiya S, Mizuguchi J, Yoshimoto T.
Augmentation of effector CD8+ T cell generation with enhanced granzyme
B expression by IL-27. J Immunol 2005; 175: 1686–93.
9 Schneider R, Yaneva T, Beauseigle D, El-Khoury L, Arbour N. IL-27
increases the proliferation and effector functions of human naive CD8+ T
lymphocytes and promotes their development into Tc1 cells. Eur J Immunol
2011; 41: 47–59.
10 Awasthi A, Carrier Y, Peron JP et al. A dominant function for interleukin
27 in generating interleukin 10-producing anti-inflammatory T cells. Nat
Immunol 2007; 8: 1380–9.
11 Fitzgerald DC, Zhang GX, El-Behi M et al. Suppression of autoimmune
inflammation of the central nervous system by interleukin 10 secreted byinterleukin 27-stimulated T cells. Nat Immunol 2007; 8: 1372–9.
12 Stumhofer JS, Silver JS, Laurence A et al. Interleukins 27 and 6 induce
STAT3-mediated T cell production of interleukin 10. Nat Immunol 2007; 8:
1363–71.
13 Neufert C, Becker C, Wirtz S et al. IL-27 controls the development of inducible regulatory T cells and Th17 cells via differential effects on STAT1.
Eur J Immunol 2007; 37: 1809–16.
14 Huber M, Steinwald V, Guralnik A et al. IL-27 inhibits the development ofregulatory T cells via STAT3. Int Immunol 2008; 20: 223–34.
15 Wojno ED, Hosken N, Stumhofer JS et al. A role for IL-27 in limiting T
regulatory cell populations. J Immunol 2011; 187: 266–73.
16 Cox JH, Kljavin NM, Ramamoorthi N, Diehl L, Batten M, Ghilardi N. IL27 promotes T cell-dependent colitis through multiple mechanisms. J Exp
Med 2011; 208: 115–23.
17 Hall AO, Beiting DP, Tato C et al. The cytokines interleukin 27 and interferon-gamma promote distinct Treg cell populations required to limit infection-induced pathology. Immunity 2012; 37: 511–23.
18 Karakhanova S, Bedke T, Enk AH, Mahnke K. IL-27 renders DC immunosuppressive by induction of B7-H1. J Leukoc Biol 2011; 89: 837–45.
19 Matta BM, Raimondi G, Rosborough BR, Sumpter TL, Thomson AW. IL-27
production and STAT3-dependent upregulation of B7-H1 mediate immune
regulatory functions of liver plasmacytoid dendritic cells. J Immunol 2012;
188: 5227–37.
20 Hirahara K, Ghoreschi K, Yang XP et al. Interleukin-27 priming of T cells
controls IL-17 production in trans via induction of the ligand PD-L1. Immunity 2012; 36: 1017–30.
21 Hisada M, Kamiya S, Fujita K et al. Potent antitumor activity of interleukin27. Cancer Res 2004; 64: 1152–6.
22 Oniki S, Nagai H, Horikawa T et al. Interleukin-23 and interleukin-27 exert
quite different antitumor and vaccine effects on poorly immunogenic melanoma. Cancer Res 2006; 66: 6395–404.
23 Salcedo R, Stauffer JK, Lincoln E et al. IL-27 mediates complete regressionof orthotopic primary and metastatic murine neuroblastoma tumors: role forCD8+ T cells. J Immunol 2004; 173: 7170–82.
24 Chiyo M, Shimozato O, Yu L et al. Expression of IL-27 in murine carcinoma cells produces antitumor effects and induces protective immunity ininoculated host animals. Int J Cancer 2005; 115: 437–42.
25 Hu P, Hu HD, Chen M et al. Expression of interleukins-23 and 27 leads tosuccessful gene therapy of hepatocellular carcinoma. Mol Immunol 2009; 46:
1654–62.
26 Salcedo R, Hixon JA, Stauffer JK et al. Immunologic and therapeutic
synergy of IL-27 and IL-2: enhancement of T cell sensitization, tumorspecific CTL reactivity and complete regression of disseminated neuroblastoma metastases in the liver and bone marrow. J Immunol 2009; 182:
4328–38.
27 Zhu S, Lee DA, Li S. IL-12 and IL-27 sequential gene therapy via intramuscular electroporation delivery for eliminating distal aggressive tumors. J
Immunol 2010; 184: 2348–54.
28 Zolochevska O, Xia X, Williams BJ, Ramsay A, Li S, Figueiredo ML. Sonoporation delivery of interleukin-27 gene therapy efficiently reduces prostate
tumor cell growth in vivo. Hum Gene Ther 2011; 22: 1537–50.
29 Liu Z, Liu JQ, Talebian F, Wu LC, Li S, Bai XF. IL-27 enhances the survival of tumor antigen-specific CD8+ T cells and programs them into IL-10producing, memory precursor-like effector cells. Eur J Immunol 2013; 43:
468–79.
30 Zhang J, Tian H, Li C et al. Antitumor effects obtained by autologous Lewis
lung cancer cell vaccine engineered to secrete mouse interleukin 27 bymeans of cationic liposome. Mol Immunol 2013; 55: 264–74.
31 Liu L, Wang S, Shan B et al. IL-27-mediated activation of natural killer
cells and inflammation produced antitumour effects for human oesophageal
carcinoma cells. Scand J Immunol 2008; 68: 22–9.
32 Airoldi I, Tupone MG, Esposito S et al. Interleukin-27 re-educates intratumoral myeloid cells and down-regulates stemness genes in non-small celllung cancer. Oncotarget 2015; 6: 3694–708.
33 Matsui M, Kishida T, Nakano H et al. Interleukin-27 activates natural killer
cells and suppresses NK-resistant head and neck squamous cell carcinoma

through inducing antibody-dependent cellular cytotoxicity. Cancer Res 2009;69: 2523–30.
34 Shimizu M, Shimamura M, Owaki T et al. Antiangiogenic and antitumor
activities of IL-27. J Immunol 2006; 176: 7317–24.
35 Cocco C, Giuliani N, Di Carlo E et al. Interleukin-27 acts as multifunctional
antitumor agent in multiple myeloma. Clin Cancer Res 2010; 16: 4188–97.
36 Canale S, Cocco C, Frasson C et al. Interleukin-27 inhibits pediatric B-acutelymphoblastic leukemia cell spreading in a preclinical model. Leukemia
2011; 25: 1815–24.
37 Cocco C, Di Carlo E, Zupo S et al. Complementary IL-23 and IL-27 anti-tumor activities cause strong inhibition of human follicular and diffuse large
B-cell lymphoma growth in vivo. Leukemia 2012; 26: 1365–74.
38 Zorzoli A, Di Carlo E, Cocco C et al. Interleukin-27 inhibits the growth ofpediatric acute myeloid leukemia in NOD ⁄ SCID ⁄ Il2rg- ⁄ - mice. Clin Cancer
Res 2012; 18: 1630–40.
39 Kachroo P, Lee MH, Zhang L et al. IL-27 inhibits epithelial-mesenchymaltransition and angiogenic factor production in a STAT1-dominant pathway
in human non-small cell lung cancer. J Exp Clin Cancer Res 2013; 32: 97.
40 Di Carlo E, Sorrentino C, Zorzoli A et al. The antitumor potential of Interleukin-27 in prostate cancer. Oncotarget 2014; 5: 10332–41.
41 Yoshimoto T, Morishima N, Mizoguchi I et al. Antiproliferative activity of
IL-27 on melanoma. J Immunol 2008; 180: 6527–35.
42 Liu L, Meng J, Zhang C et al. Effects on apoptosis and cell cycle arrest contribute to the antitumor responses of interleukin-27 mediated by retrovirus inhuman pancreatic carcinoma cells. Oncol Rep 2012; 27: 1497–503.
43 Chiba Y, Mizoguchi I, Mitobe K et al. IL-27 enhances the expression of
TRAIL and TLR3 in human melanomas and inhibits their tumor growth incooperation with a TLR3 agonist poly(I:C) partly in a TRAIL-dependent
manner. PLoS ONE 2013; 8: e76159.
44 Ho MY, Leu SJ, Sun GH, Tao MH, Tang SJ, Sun KH. IL-27 directly
restrains lung tumorigenicity by suppressing cyclooxygenase-2-mediatedactivities. J Immunol 2009; 183: 6217–26.
45 Liu LH, Shan BE, Shao LL, Wang SJ. Inhibitory effect of IL-27 gene on
tumor formation activity of Eca109 cells in nude mice and its mechanisms.
Ai Zheng 2008; 27: 12–7.
46 Shinozaki Y, Wang S, Miyazaki Y et al. Tumor-specific cytotoxic T cell
generation and dendritic cell function are differentially regulated by interleukin 27 during development of anti-tumor immunity. Int J Cancer 2009;
124: 1372–8.
47 Natividad KD, Junankar SR, Mohd Redzwan N et al. Interleukin-27 signaling promotes immunity against endogenously arising murine tumors. PLoS
ONE 2013; 8: e57469.
48 Zhu C, Sakuishi K, Xiao S et al. An IL-27 ⁄ NFIL3 signalling axis drives
Tim-3 and IL-10 expression and T-cell dysfunction. Nat Commun 2015; 6:6072.
49 Sauer KA, Maxeiner JH, Karwot R et al. Immunosurveillance of lung melanoma metastasis in EBI-3-deficient mice mediated by CD8+ T cells. J Immunol 2008; 181: 6148–57.
50 Nishino R, Takano A, Oshita H et al. Identification of Epstein–Barr virus-induced gene 3 as a novel serum and tissue biomarker and a therapeutic target
for lung cancer. Clin Cancer Res 2011; 17: 6272–86.
51 Gonin J, Larousserie F, Bastard C et al. Epstein-Barr virus-induced gene 3(EBI3): a novel diagnosis marker in Burkitt lymphoma and diffuse large Bcell lymphoma. PLoS ONE 2011; 6: e24617.
52 Collison LW, Workman CJ, Kuo TT et al. The inhibitory cytokine IL-35
contributes to regulatory T-cell function. Nature 2007; 450: 566–9.
53 Wei J, Xia S, Sun H et al. Critical role of dendritic cell-derived IL-27 inantitumor immunity through regulating the recruitment and activation of NK
and NKT cells. J Immunol 2013; 191: 500–8.
54 Xia S, Wei J, Wang J et al. A requirement of dendritic cell-derived interleukin-27 for the tumor infiltration of regulatory T cells. J Leukoc Biol
2014; 95: 733–42.
55 Mumm JB, Emmerich J, Zhang X et al. IL-10 elicits IFNgamma-dependent
tumor immune surveillance. Cancer Cell 2011; 20: 781–96.
56 Tanikawa T, Wilke CM, Kryczek I et al. Interleukin-10 ablation promotes
tumor development, growth, and metastasis. Cancer Res 2012; 72: 420–9.
57 Groux H, Cottrez F, Rouleau M et al. A transgenic model to analyze theimmunoregulatory role of IL-10 secreted by antigen-presenting cells. J
Immunol 1999; 162: 1723–9.
58 Trautmann L, Janbazian L, Chomont N et al. Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction.
Nat Med 2006; 12: 1198–202.
59 Jones RB, Ndhlovu LC, Barbour JD et al. Tim-3 expression defines a novel
population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection. J Exp Med 2008; 205: 2763–79.
60 Jin HT, Anderson AC, Tan WG et al. Cooperation of Tim-3 and PD-1 in
CD8 T-cell exhaustion during chronic viral infection. Proc Natl Acad Sci
USA 2010; 107: 14733–8.
61 Jin HT, Ahmed R, Okazaki T. Role of PD-1 in regulating T-cell immunity.
Curr Top Microbiol Immunol 2011; 350: 17–37.

Cancer Sci | September 2015 | vol. 106 | no. 9 | 1109

© 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd
on behalf of Japanese Cancer Association.

-212-

Review
Potent antitumor effects of IL-27

www.wileyonlinelibrary.com/journal/cas

62 Gonin J, Carlotti A, Dietrich C et al. Expression of IL-27 by tumor cells ininvasive cutaneous and metastatic melanomas [corrected]. PLoS ONE 2013;
8: e75694.
63 Sekar D, Hahn C, Brune B, Roberts E, Weigert A. Apoptotic tumor cells
induce IL-27 release from human DCs to activate Treg cells that express
CD69 and attenuate cytotoxicity. Eur J Immunol 2012; 42: 1585–98.
64 Reuther GW. JAK2 activation in myeloproliferative neoplasms: a potential
role for heterodimeric receptors. Cell Cycle 2008; 7: 714–9.

65 Pradhan A, Lambert QT, Reuther GW. Transformation of hematopoietic cells
and activation of JAK2-V617F by IL-27R, a component of a heterodimeric
type I cytokine receptor. Proc Natl Acad Sci USA 2007; 104: 18502–7.
66 Pradhan A, Lambert QT, Griner LN, Reuther GW. Activation of JAK2V617F by components of heterodimeric cytokine receptors. J Biol Chem
2010; 285: 16651–63.
67 Seita J, Asakawa M, Ooehara J et al. Interleukin-27 directly induces differentiation in hematopoietic stem cells. Blood 2008; 111: 1903–12.

© 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd
on behalf of Japanese Cancer Association.

Cancer Sci | September 2015 | vol. 106 | no. 9 | 1110

-213-

OncoImmunology

ISSN: (Print) 2162-402X (Online) Journal homepage: http://www.tandfonline.com/loi/koni20

Therapeutic potential of interleukin-27 against
cancers in preclinical mouse models
Izuru Mizoguchi, Yukino Chiba, Jun-ichi Furusawa, Mingli Xu, Ren Tsunoda,
Kaname Higuchi & Takayuki Yoshimoto
To cite this article: Izuru Mizoguchi, Yukino Chiba, Jun-ichi Furusawa, Mingli Xu, Ren
Tsunoda, Kaname Higuchi & Takayuki Yoshimoto (2015) Therapeutic potential of interleukin-27
against cancers in preclinical mouse models, OncoImmunology, 4:10, e1042200, DOI:
10.1080/2162402X.2015.1042200
To link to this article: https://doi.org/10.1080/2162402X.2015.1042200

Accepted author version posted online: 27
May 2015.
Published online: 27 May 2015.
Submit your article to this journal

Article views: 163

View related articles

View Crossmark data

Citing articles: 3 View citing articles

Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=koni20-214-

AUTHOR'S VIEW
OncoImmunology 4:10, e1042200; October 2015; © 2015 Taylor & Francis Group, LLC

Therapeutic potential of interleukin-27 against
cancers in preclinical mouse models
Izuru Mizoguchi, Yukino Chiba, Jun-ichi Furusawa, Mingli Xu, Ren Tsunoda, Kaname Higuchi, and Takayuki Yoshimoto*Department of Immunoregulation; Institute of Medical Science; Tokyo Medical University; Tokyo, Japan

Keywords: antitumor, IL-27, poly(I:C), TLR3, TRAIL
Abbreviations: ADCC, antibody-dependent cell-mediated cytotoxicity; COX-2, cyclooxygenase-2; DC, dendritic cell; EBI3,
Epstein–Barr virus-induced gene 3; EMT, epithelial–mesenchymal transition; gp, glycoprotein; IL, interleukin; IRF, interferon regulatory factor; MHC, major histocompatibility complex; MMP, matrix metalloproteinase; NK, natural killer; PD-L1, programmed
death-ligand 1; PGE2, prostaglandin E2; poly(I:C), polyinosinic-polycytidylic acid; STAT, signal transducer and activator of transcription; Tim-3, T-cell immunoglobulin and mucin domain-3; TLR, Toll-like receptor; Tr1, IL-10-producing regulatory T; TRAIL,
tumor necrosis factor-related apoptosis-inducing ligand; Treg, regulatory T; VEGF, vascular endothelial growth factor.

Since we ﬁrst reported the antitumor efﬁcacy of IL-27 in 2004, accumulating evidence obtained by several groups
using a variety of preclinical mouse models indicates that IL-27 possesses potent antitumor activity against various
types of tumors through multiple mechanisms depending on the characteristics of individual tumors without apparent
adverse effects.

Cancer immunotherapy has become
one of the most promising new treatmentsthat harnesses powers of the immune system to control cancer and achieve longterm cure. This strategy includes therapeutic vaccines, T-cell checkpoint inhibitors, adoptive therapy of geneticallymodified immune cells, elimination of
immunosuppressive cells such as regulatory T (Treg) cells, and new cytokines.
Interleukin (IL)-27 is one of the IL-12
family cytokines, which play critical rolesin the regulation of differentiation into
respective helper T (Th) cells and theirfunctions.1 IL-27 consists of p28, which isalso called IL-30, and Epstein–Barr virusinduced gene 3 (EBI3), and its receptor(R) is composed of IL-27Ra (WSX-1)
and glycoprotein (gp)130, a common
receptor subunit for the IL-6 family cytokines.1 IL-27 possesses both pro-inflammatory and anti-inflammatory properties;
it promotes the early induction of Th1
differentiation and generation of cytotoxic
T lymphocytes (CTLs), but it inhibits thedifferentiation of naive CD4C T cells into
Th2, Th9, and Th17 cells and suppresses
the production of pro-inflammatory cytokines through activation of both signaltransducer and activator of transcription(STAT)1 and STAT3.1
Antitumor effect of IL-27 was first
demonstrated in 2004 using mouse colon
carcinoma Colon 26 transfected with IL27 expression vector, which greatly
reduced tumor growth through mainly
CD8C T cells.2 Since then accumulating
evidence by several groups has revealed
that IL-27 possesses potent antitumor
activity against a variety of tumor models,
ranging from a transplanted mouse tumor
genetically engineered to secret IL-27
before transplantation to a human therapeutic model by injection of IL-27 protein
into immunodeficient mice after transplantation of human tumor as preclinicaltumor models.2-7 Molecular mechanismsof the antitumor activity were demonstrated to be mediated in multiple ways by
CD8C T cells,2 natural killer (NK) cells,3and macrophages,4 antibody-dependentcell-mediated cytotoxicity (ADCC), antiangiogenesis,5,6 direct antiproliferative
effects,7 inhibition of expression of cyclooxygenase-2 (COX-2) and prostaglandin
E2 (PGE2), and suppression of epithelial–mesenchymal transition (EMT),4 depending on the characteristics of individual

tumors (Fig. 1). Since several tumors wererevealed to express both IL-27R subunits
on the cell surface, IL-27 elicited direct
antiproliferative effects via WSX-1/
STAT1 signaling, presumably, by its cytostatic effects rather than by cytotoxic
effects.1 Recently, we have further elucidated a novel antitumor mechanism that
IL-27 augments the expression of tumor
necrosis factor-related apoptosis-inducing
ligand (TRAIL) and Toll-like receptor 3(TLR3) in human melanoma cell lines,
SK-MEL-13, 28, and 37, and that IL-27
inhibits the tumor growth in cooperation
with a TRL3 agonist, polyinosinic-polycytidylic acid [poly(I:C)], partly in a
TRAIL-dependent manner.7 Moreover,repeated injections of IL-27 protein andpoly(I:C) cooperatively suppressed in vivo
tumor growth of human melanoma in
immunodeficient non-obese diabetic/severe combined mice.7 Similar therapeutic effects by repeated injections of IL-27
protein were also demonstrated against
various types of human tumors transplanted in immunodeficient mice.4,6 The
human tumors so far revealed to be susceptible to the treatment by IL-27 are
not only melanoma7 but also multiple*Correspondence to: Takayuki Yoshimoto; Email: yoshimot@tokyo-med.ac.jp
Submitted: 04/10/2015; Accepted: 04/10/2015
http://dx.doi.org/10.1080/2162402X.2015.1042200

www.tandfonline.com

OncoImmunology-215-

e1042200-1

Figure 1. Potent antitumor activity of IL-27 through multiple mechanisms, which are mediated by
CD8C T cells, NK cells, macrophages, macrophages, ADCC, anti-angiogenesis, direct anti-proliferative effect, inhibition of COX-2 and PGE2 expression, and suppression of EMT, depending on thecharacteristics of individual tumors. Abbreviations: ADCC, antibody-dependent cell-mediated cytotoxicity; COX-2, cyclooxygenase-2; DC, dendritic cell; EBI3, Epstein–Barr virus-induced gene 3; EMT,
epithelial–mesenchymal transition; gp, glycoprotein; IL, interleukin; IRF, interferon regulatory factor;
MHC, major histocompatibility complex; MMP, matrix metalloproteinase; NK, natural killer; PD-L1,
programmed death-ligand 1; PGE2, prostaglandin E2; poly(I:C), polyinosinic-polycytidylic acid; STAT,
signal transducer and activator of transcription; Tim-3, T-cell immunoglobulin and mucin domain-3;
TLR, Toll-like receptor; Tr1, IL-10-producing regulatory T; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; Treg, regulatory T; VEGF, vascular endothelial growth factor.

myeloma,6 B-acute lymphoblastic leukemia, follicular lymphoma, diffuse large Bcell lymphoma, acute myeloid leukemia,
prostate cancer, and non-small cell lungcancer.4 IL-27 strongly inhibited theirtumor growth in vivo mainly due to suppression of pro-angiogenesis-related molecules such as vascular endothelial growth
factors, angiopoietins, and matrix metalloproteinases, and also due to inverse augmentation of anti-angiogenesis-related
molecules such as tissue inhibitor of
metalloproteinase.5,6
Since IL-27 possesses both pro-inflammatory and anti-inflammatory activities,1
some of them such as IL-27-mediatedaugmentation of IL-10 production and

up-regulation of programmed deathligand 1 (PD-L1) may function against its
antitumor effects, although the role of IL27 in the induction of Treg cells is still
controversial.8,9 Therefore, it is reasonable
to consider that the potent antitumor
activity of IL-27 is the sum of these positive and negative effects on the immune
responses to tumors. Although the role of
IL-10 in induction of antitumor immune
responses is often controversial, recent evidence supports a positive role for IL-10 in
the induction of antitumor CTL
responses, which were demonstrated in
IL-10-deficeient mice and IL-10-transgenic mice. Indeed, IL-10 production by
IL-27-induced CTLs was recently

References

Cytokine Frontiers: Regulation of Immune Responses
in Health and Disease. Tokyo: Springer. pp. 353-75
2. Hisada M, Kamiya S, Fujita K, Belladonna ML, Aoki
T, Koyanagi Y, Mizuguchi J, Yoshimoto T. Potent
antitumor activity of interleukin-27. Cancer Res 2004;
64:1152-6;
PMID:14871851;
http://dx.doi.org/
10.1158/0008-5472.CAN-03-2084

1. Mizoguchi I, Higuchi K, Mitobe K, Tsunoda R, Mizuguchi J, Yoshimoto T (2013) Interleukin-27: regulationof immune responses and disease development by apleiotropic cytokine with pro- and anti-inflammatory
properties. In: Yoshimoto T, Yoshimoto T, editors.

e1042200-2

OncoImmunology-216-

demonstrated to contribute to the generation of memory precursor-like effector
CTLs to augment antitumor immunity.10
Moreover, IL-27 was recently shown to
induce the expression of PD-L1 in T cells,a major mediator of T-cell exhaustion,
which hampers the induction of antitumor immune responses.1 However, since
antibody drugs have currently become
very popular as one of the most promisingcancer immunotherapies, IL-27 injection
could augment antitumor immune
responses in synergy with antibodies
against immune checkpoints such as PDL1, whose possibility remains to be clarified. On the other hand, IL-27 was initially demonstrated to inhibit thedifferentiation of naive CD4C T cells to
Treg cells,1 whereas recent reports oppositely revealed that IL-27 does not inhibit
the generation of Treg cells but rather promotes it; IL-27 produced a distinct Treg
cell population which expresses T-cell-specific T-box transcription factor T-bet andCXCR3, to control Th1-mediated immunity.1 Similar paradoxical roles of IL-27 in
the tumor microenvironment were also
reported.8,9 Thus, the effects of IL-27 on
Treg cells may depend on the context.
However, so far there is no report showingthat injection of IL-27 protein or tumor
cells transfected with IL-27 expression
vector would augment the number ofTreg cells in mice bearing tumors,although transgenic mice over-expressing
IL-27 were previously demonstrated todecrease the number of Treg cells,1 which
could presumably result in enhancementof antitumor immune responses. Besides,
since IL-27 was shown to have lower toxicity in mouse models,2,3 collectively, IL27 may have great potential as one of themost promising therapeutic cytokines
against cancer.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest weredisclosed.
3. Oniki S, Nagai H, Horikawa T, Furukawa J, Belladonna ML, Yoshimoto T, Hara I, Nishigori C. Interleukin-23 and interleukin-27 exert quite different
antitumor and vaccine effects on poorly immunogenic
melanoma. Cancer Res 2006; 66:6395-404;
PMID:16778218;
http://dx.doi.org/10.1158/00085472.CAN-05-4087

Volume 4 Issue 10

4. Airoldi I, Tupone MG, Esposito S, Russo MV, Barbarito G, Cipollone G, Di Carlo E. Interleukin-27 re-educates intratumoral myeloid cells and down-regulates
stemness genes in non-small cell lung cancer. Oncotarget 2015; 6:3694-708; PMID: 25638163
5. Shimizu M, Shimamura M, Owaki T, Asakawa M,
Fujita K, Kudo M, Iwakura Y, Takeda Y, Luster AD,
Mizuguchi J et al. Antiangiogenic and antitumor activities of IL-27. J Immunol 2006; 176:7317-24;
PMID:16751375; http://dx.doi.org/10.4049/
jimmunol.176.12.7317
6. Cocco C, Giuliani N, Di Carlo E, Ognio E, Storti P,
Abeltino M, Sorrentino C, Ponzoni M, Ribatti D, Airoldi I. Interleukin-27 acts as multifunctional antitumor
agent in multiple myeloma. Clin Cancer Res 2010;www.tandfonline.com

16:4188-97; PMID:20587591; http://dx.doi.org/
10.1158/1078-0432.CCR-10-0173
7. Chiba Y, Mizoguchi I, Mitobe K, Higuchi K, Nagai H,
Nishigori C, Mizuguchi J, Yoshimoto T. IL-27 enhances the expression of TRAIL and TLR3 in human melanomas and inhibits their tumor growth in cooperation
with a TLR3 agonist poly(I:C) partly in a TRAILdependent manner. PLoS One 2013; 8:e76159;
PMID:24155891; http://dx.doi.org/10.1371/journal.
pone.0076159
8. Natividad KD, Junankar SR, Mohd Redzwan N, Nair
R, Wirasinha RC, King C, Brink R, Swarbrick A, Batten M. Interleukin-27 signaling promotes immunity
against endogenously arising murine tumors. PLoS

OncoImmunology-217-

One 2013; 8:e57469; PMID:23554861; http://dx.doi.
org/10.1371/journal.pone.0057469
9. Xia S, Wei J, Wang J, Sun H, Zheng W, Li Y, Sun Y,
Zhao H, Zhang S, Wen T et al. A requirement ofdendritic cell-derived interleukin-27 for the tumor
infiltration of regulatory T cells. J Leukoc Biol 2014;
PMID: 24443555; http://www.jleukbio.org/content/
95/5/733.long
10. Liu Z, Liu JQ, Talebian F, Wu LC, Li S, Bai XF. IL-27
enhances the survival of tumor antigen-specific CD8C
T cells and programs them into IL-10-producing,memory precursor-like effector cells. Eur J Immunol
2013; 43:468-79; PMID:23225163; http://dx.doi.org/
10.1002/eji.201242930

e1042200-3

ONCOLOGY REPORTS 33: 292-296, 2015

292

Vaccination with OVA-bound nanoparticles encapsulating IL-7
inhibits the growth of OVA-expressing E.G7 tumor cells in vivo
HIROKO TOYOTA1, NORIKO YANASE1, TAKAYUKI YOSHIMOTO2, MITSUNORI HARADA3,YASUKI KATO3 and JUNICHIRO MIZUGUCHI1
1

Department of Immunology, Tokyo Medical University, Shinjuku-ku, Tokyo 160-8402;
Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University,
Shinjuku‑ku, Tokyo 160‑8402; 3NanoCarrier Co. Ltd., Kashiwa-shi, Chiba 277-0871, Japan

2

Received July 22, 2014; Accepted September 19, 2014
DOI: 10.3892/or.2014.3603
Abstract. Immunotherapy has gained special attention due
to its specific effects on tumor cells and systemic action toblock metastasis. We recently demonstrated that ovalbumin(OVA) conjugated to the surface of nanoparticles (NPs)(OVA‑NPs) can manipulate humoral immune responses. In
the present study, we aimed to ascertain whether vaccination
with OVA-NPs entrapping IL-7 (OVA-NPs-IL-7) are able toinduce antitumor immune responses in vivo. Pretreatment witha subcutaneous inoculation of OVA-NPs delayed the growthof thymic lymphoma cells expressing a model tumor antigen
OVA (E.G7-OVA), and OVA-NPs-IL-7 substantially blocked
the growth of E.G7-OVA tumor cells, although NPs-IL-7 alone
had a meager effect, as assessed by the mean tumor size andthe percentage of tumor-free mice. However, pretreatment
with OVA-NPs-IL-7 failed to reduce the growth of parental
thymic tumor cells, suggesting that the antitumor effect was
antigen-specific. A tetramer assay revealed that vaccination
with OVA-NPs-IL-7 tended to enhance the proportion ofcytotoxic T cells (CTLs) specific for OVA. When the tumorfree mice inoculated with OVA-NPs-IL-7 plus EG.7 cells were
rechallenged with E.G7-OVA cells, they demonstrated reduced
growth compared with that in the control mice. Thus, a single
subcutaneous injection of OVA-NPs-IL-7 into mice induced
tumor-specific and also memory-like immune responses,
resulting in regression of tumor cells. Antigens on NPs entrapping IL-7 would be a promising carrier to develop and enhance
immune responses, including humoral and cellular immunity

Correspondence to: Professor Junichiro Mizuguchi, Department of
Immunology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku,
Tokyo 160-8402, Japan
E-mail: mizu@tokyo-med.ac.jp

Abbreviations: TAA, tumor-associated antigen; DCs, dendritic cells;
CTLs, cyototoxic T cells; NPs, nanoparticles; OVA, ovalbumin

Key words: vaccination, nanoparticles, tumor antigen, E.G7-OVA
tumor cells

as well as a method of drug delivery to a specific target ofinterest.
Introduction
Although currently well-established treatments, including
chemotherapy, radiotherapy and surgery, are initially effective,
some tumors have inherent or acquired resistance to current
treatments. Alternative strategies such as cancer immunotherapy have attracted special attention (1-3). Immunotherapy
could specifically target tumor cells through de novo expressionof tumor-associated antigens (TAAs). TAA-expressing tumor
cells are efficiently phagocytosed by immature dendritic cells(DCs) (4), which mature to generate co-stimulatory molecules
and/or cytokines under inflammatory conditions. The mature
DCs present antigens along with costimulatory signals to
T cells, resulting in activation of T cells. CD8-positive (CD8+)
T cells specifically recognize and respond to TAA-expressing
tumor cells in combination with CD4 + T cells or theirderived cytokines, resulting in generation of cytotoxic T cells(CTLs) that specifically kill tumor cells systemically (2).
CD8-mediated immunotherapy has had considerable successin selected patients (2), and development of novel therapeutic
approaches and optimization of current treatments hold great
promise in the field of antitumor immunotherapy capable ofcontrolling tumor growth, while minimizing damage to hosttissues.
To evaluate CD8-mediated control of tumor growth, we
employed the ovalbumin (OVA)-expressing thymoma cell line
E.G7-OVA as a model for immunotherapy where OVA is amodel TAA (5). Cytokines such as IL-2, IL-12, IFN-γ and IL-7
have been used for modulating antitumor immune responses
with limited success (6,7). IL-7, originally known as a functional molecule sharing a receptor component (γc chain), plays
a crucial role in the maintenance of memory T cells as wellas the survival of naïve T cells (8). Moreover, a recent study
demonstrated that IL-7 enhances immune responses against
tumors (9,10).
Nanoparticles (NPs) have been recently used as carriers
to manipulate immune responses as well as a specific system
to deliver drugs to a target of interest (3,11-13). The physicalcharacteristics of NPs, including size, hydrophobicity, stability,-218-

toyota et al: OVA-BOUND-NPs-IL-7-MEDIATED INHIBITION OF OVA-EXPRESSING THYMIC TUMOR CELLS

antigen loading and antigen-release kinetics, modulate immune
responses. Moreover, immune responses are further modified
by the antigen dose and method of conjugation of antigens on
the surface of or inside NPs (11). Our preparation of biodegradable NPs consisting of poly(ethylene glycol) (PEG)-poly(glutamic acid) block copolymers carrying chemotherapeutic
agents was effective for targeting tumors in vivo (14). We
recently demonstrated that a single subcutaneous injection of
OVA conjugated on the surface of NPs (OVA-NPs) induced
OVA-specific IgG responses with poor IgE synthesis (15). The
present study examined whether OVA-NPs loaded with IL-7
(OVA-NPs-IL-7) elicited antitumor immune responses in vivo.
Vaccination with a single injection of OVA-NPs-IL-7 prophylactically prevented the growth of E.G7-OVA tumors but notparental thymic lymphoma cells in vivo, probably throughinduction of CD8+ CTLs.
Materials and methods
Mice. Female C57BL/6 mice were purchased from the JacksonLaboratory. The mice were maintained at the animal facilityof Tokyo Medical University and used for experiments at theage of 8 weeks. The Ethics Committee of Animal Experimentsof the Tokyo Medical University approved the experiments.

293

Figure 1. Preparation of OVA-NPs encapsulating IL-7. OVA-NPs were loadedwith various concentrations of IL-7, which showed a size range of 80-100 nm
in diameter. OVA, ovalbumin; NPs, nanoparticles.

Flow cytometric analysis. Single spleen cell suspensions
from C57BL/6 mice primed with OVA-NPs-IL-7 and control
mice were stained with anti-CD4-FITC, anti-CD62L-APC(BioLegend, San Diego, CA, USA), anti-CD8-FITC (MBL),
and anti-CD44-PE/Cy7 (eBioscience, San Diego, CA, USA).
Relative fluorescence intensities were measured using the
FACSCanto II and analyzed with FACSDiva software(BD Biosciences).

Cell lines. EL4 thymoma cells (TIB-39) and E.G7-OVA
cells (a derivative of EL4 cells expressing OVA, CRL-2113)
were obtained from the American Type Culture Collection(Manassas, VA, USA). Cells were maintained in RPMI-1640
supplemented with 10% (v/v) fetal bovine serum, 50 µM
2-mercaptoethanol, and 100 µg/ml kanamycin at 37˚C in ahumidified atmosphere of 5% CO2.

Statistical analysis. Statistical analysis was performed with
the Student's t-test. A p-value of <0.05 was considered toindicate a statistically significant result. Regression analysis
was carried out using MS Excel software to determine therelationship between the two methods.

Preparation of OVA-NPs loaded with IL-7. Two types of
PEG-pGlu with a carbonyl carbon of an amino acid partiallyreplaced by a benzyl or octyl group through an ester linkage
were synthesized at NanoCarrier Co., Ltd. (Kashiwa, Japan).
One had a maleimide group at the PEG terminus and benzyl
groups in pGlu, and the other had a methoxy group at the PEG
terminus and octyl groups in pGlu. OVA-bound polymeric
micelles were prepared as previously described (16). The
micelles were separated from unbound OVA by ultrafiltration.
IL-7 was encapsulated into the micelles at different weightpercentages of protein to polymer of 5, 2 and 0.7% at pH 6.0.
The final solvent contained 10% (w/v) sucrose and 20 mM
sodium phosphate buffer (pH 6.0). The micelles were stored
at -80˚C prior to use.

Preparation of OVA-NPs loaded with IL-7. OVA was conjugated on the surface of NPs, and then IL-7 was loaded into theNPs (Fig. 1). The average diameter of the OVA-NPs-IL-7 was
100 nm.

Prophylactic protocol for tumor growth. Mice were subcutaneously immunized with OVA-NPs-IL-7 in the back. Seven days
later, the mice were challenged with E.G7-OVA or parental
EL-4 tumor cells (1x106 cells in 100 µl PBS/mouse), followedby assays for tumor growth.
Tetramer assay for splenocytes. Detection of OVA-specific CTLs
was performed using an H-2Kb/OVA Tetramer-SIINFEKL kit(MBL, Nagoya, Japan). Briefly, splenocytes from immunized
mice were stained with anti-mouse CD8-FITC (KT15) (MBL),
followed by an assay using a FACSCanto II flow cytometer(BD Biosciences) according to the manufacturer's instructions.

Results

Pretreatment with OVA-NPs-IL-7 reduces the growth of
E.G7-OVA tumor cells in vivo. Whether OVA on NPs in combination with IL-7 exert antitumor effects was examined in thepresent study. Pretreatment of C57BL/6 mice with OVA-NPs
7 days before E.G7-OVA tumor inoculation delayed tumor
growth compared with the NPs alone (Fig. 2A). Vaccination ofmice with OVA-NPs-IL-7 completely prevented the growth of
E.G7-OVA tumor cells in 4 of the 5 mice for at least 28 days,
although NPs-IL-7 had only a meager effect on tumor growth.
However, the growth of parental EL4 tumor cells was almost
comparable in the OVA-NPs-IL-7-treated and control NPs
alone groups (Fig. 2B), suggesting that vaccination with
OVA-NPs-IL-7 induced OVA-specific antitumor immunity inthe mice.
OVA-NPs encapsulating various concentrations of IL-7
were examined for adjuvant action against tumor growth.
OVA-NPs containing 5 µg IL-7 completely blocked the growthof E.G7-OVA tumor cells in 4 of the 5 mice, whereas partial
protection from tumor growth was obtained by 1.8 or 12.5 µg
IL-7 incorporated into the OVA-NPs (Fig. 3). Thus, 5 µg IL-7
incorporated into the OVA-NPs was employed for the subsequent study.

-219-

294

ONCOLOGY REPORTS 33: 292-296, 2015

Figure 2. OVA-NPs-IL-7 blocks the growth of E.G7-OVA tumor cells in vivo. C57BL/6 mice were pretreated with OVA-NPs-IL-7, OVA-NPs, NPs-IL-7 and
NPs alone, and then inoculated with 1x106 (A) E.G7-OVA or (B) EL4 tumor cells 7 days later, followed by assay every 3-4 days for tumor size up to (A) 28 and(B) 15 days, respectively. Data are means ± SD for 5 mice/group. Data are from one of two independent experiments with essentially similar results. *p<0.05.
OVA, ovalbumin; NPs, nanoparticles.

Figure 3. Dose response of IL-7 incorporated into OVA-NPs efficiently blocks the growth of E.G7-OVA tumor cells. Mice were primed with OVA-NPs
containing various concentrations of IL-7, and then challenged with 1x106 E.G7-OVA tumor cells, followed by assay for tumor size. Data are means ± SD for5 mice/group. *p<0.05. OVA, ovalbumin; NPs, nanoparticles.

Generation of CTLs after vaccination with OVA-NPs-IL-7.
Vaccination of tumor antigen with a suitable adjuvant induces
tumor-specific CTLs, inhibiting tumor growth in vivo (11).
Vaccination with OVA-NPs-IL-7 tended to enhance a portionof splenic CTLs specific for OVA compared with the control
NPs alone (Fig. 4A). To examine the relationship between
CTLs and tumor size, tumor size was measured (Fig. 4B). A
regression plot of CTLs and tumor size is shown in Fig. 4C.
The coefficient of determination, R2=0.988, suggested a goodcorrelation between CTLs and tumor size. Thus, OVA-NPsIL-7-induced CTLs may have contributed to prevention oftumor growth in vivo.
Generation of memory-like T cells. Whether memory-likecells were generated by the immunization with OVA-NPs-IL-7
together with E.G7-OVA was examined. The tumor-free micereceiving OVA-NPs-IL-7 together with E.G7-OVA tumor
cells showed partial resistance to rechallenge with E.G7-OVA
cells (Fig. 5A), suggesting that this immunization generated amemory-like cell. Then, we analyzed CD4+ and CD8+ T cells
from mice that had been immunized with OVA-NPs-IL-7 incombination with E.G7-OVA. The CD4+ T cells from the rechallenged mice expressed a memory phenotype (CD44hiCD62L+)
relative to the controls (39.5 vs. 29.9) (Fig. 5B). The percentage
of memory CD8+ T cells from the primed mice tended to behigher than that from the control mice, although statisticallynot significant. Thus, priming with OVA-NPs-IL-7 along with
E.G7-OVA generated memory-like T cells in vivo, which may
contribute to prevention of the growth of subsequently inoculated E.G7-OVA cells.
Discussion
Growth of tumors is controlled by the immune system (17). To
induce a strong antitumor immune response to suppress tumor
growth in vivo, a vaccine comprising a model tumor-associatedantigen OVA on NPs loaded with IL-7 was prepared. A single
immunization with OVA-NPs-IL-7 suppressed the growth of-220-

toyota et al: OVA-BOUND-NPs-IL-7-MEDIATED INHIBITION OF OVA-EXPRESSING THYMIC TUMOR CELLS

295

Figure 4. OVA-NPs-IL-7 generates OVA-specific CTLs in vivo. (A) Spleen T cells were prepared from mice primed with OVA-NPs-IL-7 or control NPs alone
or in combination with E.G7 tumor inoculation 21 days previously, followed by assessment with tetramer assay (3 independent examples), as described inMaterials and methods. (B) Tumor size of the mice was obtained when the spleen was removed. *p<0.05. (C) Regression analysis was carried out to determine
the relationship between tumor size and CTLs. OVA, ovalbumin; NPs, nanoparticles; CTL, cytotoxic T cell.

Figure 5. Single injection of OVA-NPs-IL-7 generates memory-like cells in vivo. (A) Tumor-free mice (n=5) receiving OVA-NPs-IL-7 together with E.G7-OVA
tumor cells or control (n=3) mice were then rechallenged with the same number of E.G7-OVA cells 53 days after the 1st inoculation, followed by assay fortumor size. Data are means ± SD. *p<0.05. (B) Spleen cells (n=3) from the E.G7-OVA rechallenged and control mice receiving E.G7 tumor cells alone were
stained with anti-CD4‑FITC, anti-CD8-FITC, anti-CD44-PE/Cy7 and anti-CD62L-APC 33 days after rechallenge. The percentage of T cells with a memory(CD44hiCD62L+) phenotype was calculated. *p<0.05. OVA, ovalbumin; NPs, nanoparticles.

OVA-expressing E.G7-OVA tumor cells. Memory-like CD4+
cells were generated after immunization with OVA-NPs-IL-7

in vivo, which may have contributed to the resistance to therechallenge with E.G7-OVA cells. Thus, tumor antigen on NPs

-221-

296

ONCOLOGY REPORTS 33: 292-296, 2015

in the presence of cytokine IL-7 would function as a powerfulprophylactic vaccine to prevent the growth of subsequent
inoculations of tumor cells in vivo.
Synthetic particles mimicking the structure of microbes
function as powerful immune adjuvant (3,18). The adjuvant
activity can be modified by physical characteristics of NPs
including size, antigen loading and antigen-release kinetics.
Our preparation of OVA-NPs-IL-7 (~100 nm) prevented thegrowth of EG.7-OVA but not parental EL4 tumors (Fig. 2B).
OVA-NPs may be endocytosed by DCs or macrophages
to cross-prime CD8+ T cells in regional lymph nodes (19),since small particles in a range of 20-200 nm freely migrate
to draining lymph nodes from the injection site (20). A slow
release of IL-7 from OVA-NPs (13) would facilitate to activateand maintain CD4+-memory T cells.
Cytokines produced by a variety of cells enhance orinhibit immune responses against pathogens and tumors. A
clinical cancer trial demonstrated that IL-7 has efficacy as atherapeutic agent (21). IL-7 promotes the survival of effector
T cells as well as memory T cells (22-24). Consistent withthese findings, a single injection of OVA-NPs-IL-7 tended
to enhance the proportion of CTLs specific for OVA relative
to the control. The proportion of CD8+ CTLs appeared tocorrelate with poor tumor growth (Fig. 4B) (17,25). Moreover,
OVA-NPs-IL-7 in combination with E.G7-OVA cells enhanced
the proportion of a CD4+ memory phenotype (Fig. 5B), which
may have contributed to the resistance to the rechallenge with
E.G7-OVA cells (Fig. 5A). However, the proportion of memory
B cell phenotype was comparable between OVA-NPs-IL-7 andthe control group (Toyota et al, unpublished data). Thus, IL-7
appeared to shift the balance toward an antitumor effect byrepressing regulatory networks (24), which are predominant in
the tumor-bearing host (26,27).
Collectively, we demonstrated that a single subcutaneous
injection of OVA-NPs-IL-7 suppressed the growth in vivo ofsubsequently inoculated E.G7-OVA tumor cells expressing
OVA, yet not parental EL4 cells. In addition to antitumor
responses, OVA-NPs strongly generated humoral immunity
including IgG1 and IgG2a antibody production in mice (15).
Thus, antigens conjugated on NPs would be a novel adjuvant
enhancing cellular as well as humoral immune responses since
they are stable, biodegradable and non-toxic to humans.
References
1. Sanlorenzo M, Vujic I, Posch C, et al: Melanoma immunotherapy.
Cancer Biol Ther 15: 665-674, 2014.
2. Disis ML, Bernhard H and Jaffee EM: Use of tumour-responsive
T cells as cancer treatment. Lancet 373: 673-683, 2009.
3. Yoo JW, Irvine DJ, Discher DE, et al: Bio-inspired, bioengineeredand biomimetic drug delivery carriers. Nat Rev Drug Discov 10:
521-535, 2011.
4. Radford KJ, Tullett KM and Lahoud MH: Dendritic cells andcancer immunotherapy. Curr Opin Immunol 27: 26-32, 2014.
5. Moore MW, Carbone FR and Bevan MJ: Introduction of soluble
protein into the class I pathway of antigen processing and presentation. Cell 54: 777-785, 1988.

6. Oppenheim JJ: Cytokines: Past, present, and future. Int J
Hematol 74: 3-8, 2001.
7. Christian DA and Hunter CA: Particle-mediated delivery ofcytokines for immunotherapy. Immunotherapy 4: 425-441,
2012.
8. Katzman SD, Hoyer KK, Dooms H, et al: Opposing functionsof IL-2 and IL-7 in the regulation of immune responses.
Cytokine 56: 116-121, 2011.
9. Liu S, Lizée G, Lou Y, et al: IL-21 synergizes with IL-7 toaugment expansion and anti-tumor function of cytotoxic T cells.
Int Immunol 19: 1213-1221, 2007.
10. Gou HF, Huang J, Shi HS, et al: Chemo-immunotherapy withoxaliplatin and interleukin-7 inhibits colon cancer metastasis inmice. PLoS One 9: e85789, 2014.
11. Oyewumi MO, Kumar A and Cui Z: Nano-microparticles as
immune adjuvants: Correlating particle sizes and the resultant
immune responses. Expert Rev Vaccines 9: 1095-1107, 2010.
12. Krishnamachari Y, Geary SM, Lemke CD, et al: Nanoparticle
delivery systems in cancer vaccines. Pharm Res 28: 215-236,
2011.
13. Osada K, Christie RJ and Kataoka K: Polymeric micelles frompoly(ethylene glycol)-poly(amino acid) block copolymer for drug
and gene delivery. J R Soc Interface 6 (Suppl 3): S325-S339,
2009.
14. Matsumura Y and Kataoka K: Preclinical and clinical studiesof anticancer agent-incorporating polymer micelles. Cancer
Sci 100: 572-579, 2009.
15. Yanase N, Toyota H, Hata K, et al: OVA-bound nanoparticles
induce OVA-specific IgG1, IgG2a, and IgG2b responses with low
IgE synthesis. Vaccine 32: 5918-5924, 2014.
16. Harada M, Ohuchi M, Hayashi T and Kato Y: Prolonged circulation and in vivo efficacy of recombinant human granulocytecolony-stimulating factor encapsulated in polymeric micelles. J
Control Release 156: 101-108, 2011.
17. DeNardo DG, Andreu P and Coussens LM: Interactions between
lymphocytes and myeloid cells regulate pro- versus anti-tumor
immunity. Cancer Metastasis Rev 29: 309-316, 2010.
18. Mahapatro A and Singh DK: Biodegradable nanoparticles are
excellent vehicle for site directed in-vivo delivery of drugs andvaccines. J Nanobiotechnology 9: 55, 2011.
19. Andersen BM and Ohlfest JR: Increasing the efficacy of tumor
cell vaccines by enhancing cross priming. Cancer Lett 325:
155-164, 2012.
20. Manolova V, Flace A, Bauer M, et al: Nanoparticles target
distinct dendritic cell populations according to their size. Eur J
Immunol 38: 1404-1413, 2008.
21. Möller P, Sun Y, Dorbic T, et al: Vaccination with IL-7
gene-modified autologous melanoma cells can enhance the antimelanoma lytic activity in peripheral blood of patients with agood clinical performance status: a clinical phase I study. Br J
Cancer 77: 1907-1916, 1998.
22. Li J, Huston G and Swain SL: IL-7 promotes the transition of CD4
effectors to persistent memory cells. J Exp Med 198: 1807‑1815,
2003.
23. Schluns KS, Kieper WC, Jameson SC and Lefrançois L:
Interleukin-7 mediates the homeostasis of naïve and memory
CD8 T cells in vivo. Nat Immunol 1: 426-432, 2000.
24. Rochman Y, Spolski R and Leonard WJ: New insights into theregulation of T cells by γc family cytokines. Nat Rev Immunol 9:480-490, 2009.
25. Hussein MR: Tumour-infiltrating lymphocytes and melanoma
tumorigenesis: an insight. Br J Dermatol 153: 18-21, 2005.
26. Egeblad M, Nakasone ES and Werb Z: Tumors as organs:
Complex tissues that interface with the entire organism. Dev
Cell 18: 884-901, 2010.
27. de Visser KE, Eichten A and Coussens LM: Paradoxical rolesof the immune system during cancer development. Nat Rev
Cancer 6: 24-37, 2006.

-222-

Okabe et al. Journal of Hematology & Oncology 2014, 7:37
http://www.jhoonline.org/content/7/1/37

JOURNAL OF HEMATOLOGY& ONCOLOGYRESEARCH

Open Access

Combination of the ABL kinase inhibitor imatinibwith the Janus kinase 2 inhibitor TG101348 fortargeting residual BCR-ABL-positive cells
Seiichi Okabe*, Tetsuzo Tauchi, Seiichiro Katagiri, Yuko Tanaka and Kazuma Ohyashiki

Abstract
Background: The ABL kinase inhibitor imatinib is highly effective in treating most, but not all, patients with chronic
myeloid leukemia (CML). This is because residual CML cells are generally present in the bone marrow
microenvironment and are refractory to imatinib. Hematopoietic cytokine receptor signaling is mediated by Janus
kinases (JAKs) and their downstream transcription factor, signal transducer and activator of transcription (STAT).
TG101348 (SAR302503) is an oral inhibitor of JAK2.
Methods: We investigated the efficacy of imatinib and TG101348 using the break point cluster region-c-Abelson(BCR-ABL)-positive cell line and primary CML samples wherein leukemia cells were protected by a feeder cell line (HS-5).
Results: Imatinib treatment resulted in partial inhibition of cell growth in HS-5-conditioned medium. Furthermore,combined treatment with imatinib and TG101348 abrogated the protective effects of HS-5-conditioned medium on
K562 cells. Phosphorylation of Crk-L, a BCR-ABL substrate, decreased considerably, while apoptosis increased. In addition,
the combined treatment of CD34-positive primary samples resulted in considerably increased cytotoxicity, decreased
Crk-L phosphorylation, and increased apoptosis. We also investigated TG101348 activity against feeder cells andobserved that STAT5 phosphorylation, granulocyte macrophage colony-stimulating factor, and interleukin 6 levels
decreased, indicating reduced cytokine production in HS-5 cells treated with TG101348.
Conclusions: These results showed that JAK inhibitors may enhance the cytotoxic effect of imatinib against residual
CML cells and that a combined approach may be a powerful strategy against the stroma-associated drug resistance ofPhiladelphia chromosome-positive cells.
Keywords: Chronic myeloid leukemia, JAK2 inhibitor, Imatinib, Cytokine, Feeder cell

Background
Chronic myeloid leukemia (CML) is a malignant myeloproliferative clonal disorder associated with the presenceof the Philadelphia chromosome (Ph) [1]. Ph results froma translocation that yields the break point cluster regionc-Abelson (BCR-ABL) fusion protein, which is a constitutively active tyrosine kinase [2]. BCR-ABL activation is
responsible for modulating different signaling pathways,
and consequently, hematopoietic stem and progenitor cellproliferation, as well as abnormal interactions with theextracellular matrix and stroma [3]. The BCR-ABL fusion
protein activates several downstream signaling molecules,* Correspondence: okabe@tokyo-med.ac.jp
First Department of Internal Medicine, Tokyo Medical University, Shinjuku-ku,
Tokyo 160-0023, Japan

including those of the Ras/Raf/MAPK, PI3K/AKT/mTOR,
and JAK/STAT pathways [4-6].
Imatinib, a first generation ABL tyrosine kinase inhibitor(ABL TKI), is a competitive inhibitor that binds to theATP-binding catalytic site of ABL and decreases kinasedomain activity by stabilizing the protein in an active conformation [7]. Imatinib is highly for treating patients in thechronic phase of CML. As observed in the International
Randomized Study of Interferon and STI571 study, 87% of
patients treated with imatinib achieved a complete cytogenetic response, with many patients achieving major molecular responses [8]. However, discontinuing imatinib isusually associated with disease relapse. In particular, theFrench Stop Imatinib trial reported that approximately
60% of patients who had maintained a complete molecular© 2014 Okabe et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.

-223-

Okabe et al. Journal of Hematology & Oncology 2014, 7:37
http://www.jhoonline.org/content/7/1/37

Page 2 of 11

response for 2 years on imatinib relapsed after terminationof treatment [9]. Thus, it is currently recommended thatpatients with CML stop TKI therapy only in the context ofa clinical trial. The major cause of relapse in patients with
CML is considered to be the existence of leukemia stem
cells as imatinib is believed to be unable to kill quiescent
CML stem cells. It is possible that these quiescentleukemia stem cells do not depend on BCR-ABL signaling[10]. Therefore, new strategies are required to specificallytarget CML stem cells and improve outcomes in patientswith CML.
Janus kinases (JAKs) are a family of intracellular, nonreceptor tyrosine kinases including four family members such as JAK1, JAK2, JAK3, and Tyk2. JAK signaling
pathways are required for cytokine and growth factor
signaling [11]. The downstream molecules, which belong to the signal transducer and activator of transcription (STAT) family, are activated by JAKs. The JAK/STAT pathway is responsible for various functions.
JAK2 plays an important role in the signaling pathways
induced by hematopoietic growth factors such as granulocyte macrophage colony-stimulating factor (GM-CSF) andinterleukin (IL)-3 [12,13]. Although JAK2 is phosphorylated in cytokine-independent BCR-ABL-positive CML
cells, aberrant JAK/STAT signaling has been implicated invarious tumors [14]. For example, a point mutation in
JAK2 that results in non-synonymous amino acid substitution, V617F, was discovered in hematological malignancies. In fact, the V617F variant is common in patients withmyeloproliferative neoplasms (MPNs) such as polycythemiavera, essential thrombocythemia, and primary myelofibrosis [15]. Several JAK2 inhibitors have been developed forpatients with MPNs. These inhibitors are currently in clinical trials. One of the JAK2 inhibitors, TG101348 (alsoknown as SAR302503), is a small-molecule JAK2 antagonist. TG101348 inhibits the growth of hematopoietic cells
derived from patients with MPNs who have the V617F
mutation [16].
JAK2 is part of the BCR-ABL signaling network pathway and is activated in CML cells [17]. JAK2 including
the point mutation is also involved in CML maintenance[18-20]. Thus, JAK2 inhibitors may become a therapeutic target for CML cells. Although several reports
have demonstrated that BCR-ABL/JAK2 inhibits CML
cells including ABL TKI-resistant cells [21,22], it is notcompletely known whether JAK2 is involved in CMLstem cell survival mediated by cytokines in the presenceof ABL TKI.
Here, we investigated the effect of TG101348 on residual CML cells. We demonstrated that co-treatment
with imatinib and TG101348 increased the cytotoxic effect in CD34-positive CML samples. We also found thatcytokine production, which supported growth of CML
cells, was reduced by TG101348.

Results
Effects of imatinib on BCR-ABL-expressing cells in thepresence of human stromal cells

We investigated the cell proliferation effects of imatinib
on K562 cells when cultured in the presence or absenceof HS-5 conditioned medium, which was collected andpooled from a HS-5 stromal cell culture. We found that
K562 cell proliferation was inhibited by imatinib in adose-dependent manner when cultured in the absence ofHS-5 conditioned medium (Figure 1A). In contrast, weobserved that anti-leukemic activity of imatinib was partially reduced in the presence of HS-5 conditioned
medium (Figure 1A). The HS-5 stromal cell line secretes
several cytokines [23]. As JAK2 is essential for signalingof several of these cytokines, we used the JAK2 inhibitor
TG101348 to investigate the role of JAK2 in the observed protection of K562 cells by HS-5 conditioned
medium. We found that co-treatment with imatinib andTG101348 inhibited K562 cell proliferation in the presence of the HS-5 conditioned medium (Figure 1B). We
also found that another JAK inhibitor, AG490, also
inhibited K562 cell growth in the presence of HS-5 conditioned medium (Figure 1B). We next investigated theeffect of TG101348 alone on K562 cells. We found thathigh TG101348 concentration partially inhibited K562
cell proliferation in the absence of the HS-5 conditioned
medium (Figure 1C). The IC50 value for TG101348 was
up to 2 μM in BCR-ABL-positive cells. The concentration of TG101348 used in a clinical trial was >1 μM[16]. It has been reported that a high TG101348 concentration is associated with severe adverse events in patients with MF [16], thus, we investigated concentrations
below <1 μM in this study. Next, we investigated the effectsof this inhibitor on intracellular signaling. We observed adecrease in BCR-ABL and STAT5 phosphorylation in thepresence of a high TG101348 concentration (Figure 1D).
Treatment of Ph-positive (Ph+) leukemia cells withimatinib and TG101348

We next investigated whether imatinib and TG101348
could inhibit Ph + cell growth in the presence of HS-5
cells. K562 cells were exposed to imatinib alone or in
combination with TG101348 at different concentrationsin the presence or absence of HS-5 cells. Growth ofK562 cells treated with imatinib alone in the absence ofHS-5 was considerably inhibited in a dose-dependent
manner. However, when K562 cells were treated in thepresence of HS-5 cells, the effects of imatinib decreased
considerably (Figure 2A). Thus, HS-5 cells supported
K562 cell proliferation even in the presence of a highimatinib concentration. Next, we investigated the effectof TG101348 treatment. We observed that cell contact
between K652 and HS-5 cells was required for protection from imatinib. We also found that treatment with-224-

Okabe et al. Journal of Hematology & Oncology 2014, 7:37
http://www.jhoonline.org/content/7/1/37

Page 3 of 11

Figure 1 Cytotoxic effects of imatinib in the presence of HS-5 conditioned medium. (A) K562 cells were cultured at a concentration of 8 ×
104/mL in the presence of varying concentrations of imatinib in the presence or absence of HS-5 conditioned medium for 72 h. Viable cellnumbers were calculated. Results are representative of three separate experiments. (B) K562 cells were treated with 2 μM imatinib alone or in
combination with either 1 μM TG101348 or 5 μM AG490 in the presence or absence of HS-5 conditioned medium for 72 h. Viable cell numbers
were calculated. Results are representative of three separate experiments. (C) K562 cells were cultured with the indicated concentrations of
TG101348 for 72 h. Viable cell numbers were calculated. Results are representative of three separate experiments. (D) K562 cells were treated with
TG101348 for 24 h, and total extracts were examined by immunoblotting using anti-phospho ABL, ABL, phospho-STAT5, STAT5, and β-actin Abs.
Blots were scanned and optical densities were determined using ImageJ software.

TG101348 overcame the HS-5-mediated protection of
K562 cells (Figure 2A). Using immunoblotting, we determined that phosphorylation of Crk-L, a BCR-ABL substrate, STAT5, and mitogen activated protein kinase(MAPK) decreased following co-treatment with imatinib
and TG101348. In contrast, we observed that poly(ADPribose) polymerase (PARP) activity increased during cotreatment (Figure 2B). Next, we used the pan-caspase
inhibitor Z-VAD-fmk to inhibit caspase pathways. We
found that Z-VAD-fmk treatment inhibited imatinib- andTG101348-induced apoptosis, suggesting that a caspasedependent mechanism is involved in TG101348- andimatinib-mediated cell death in K562 cells (Figure 2C).

Effects of TG101348 in combination with imatinib on
CD34-positive CML samples

To determine whether co-treatment with TG101348
and imatinib affects primary chronic phase CML cell
viability, we isolated untreated peripheral blood samples
from patients at the time of diagnosis or healthy donors
and cultured the cells in the presence of HS-5 cells for
72 h with either imatinib alone or in combination with
TG101348 at the indicated concentrations (Figure 3A).
Cell viability was assessed by trypan blue exclusion. Viability of CD34-positive primary cells was not affected byimatinib treatment alone or in the presence of HS-5
stromal cells (Figure 3A). In contrast, viability of the-225-

Okabe et al. Journal of Hematology & Oncology 2014, 7:37
http://www.jhoonline.org/content/7/1/37

Page 4 of 11

Figure 2 (See legend on next page.)-226-

Okabe et al. Journal of Hematology & Oncology 2014, 7:37
http://www.jhoonline.org/content/7/1/37

Page 5 of 11(See figure on previous page.)
Figure 2 Co-treatment with imatinib and TG101348 increased inhibition of cell growth and induced apoptosis of BCR-ABL-expressing
cells in the presence of HS-5 cells. (A) K562 cells were cultured at a concentration of 8 × 104/mL in the presence or absence of HS-5 cells. Cells
were then treated with varying concentrations of imatinib alone or in combination with TG101348 for 72 h. Viable cell numbers were calculated.
Results are representative of three separate experiments. (B) K562 cells were treated with imatinib alone or in combination with TG101348 for
24 h, and total extracts were examined by immunoblotting using anti-phospho ABL, ABL, phospho-Crk-L, Crk-L, phospho-STAT5, STAT5,
phospho-MAPK, Erk-1, cleaved PARP, and β-actin Abs. Blots were scanned and optical densities were determined using ImageJ software. (C) K562
cells were cultured with Z-VAD-fmk (20 μM) and/or TG101348 and imatinib for 24 h. Total extracts were analyzed by immunoblotting using
anti-cleaved caspase-3 and cleaved PARP Abs. Actin was used as the loading control. Data are representative of two separate experiments. Blots
were scanned and optical densities were determined using ImageJ software.

primary cells decreased significantly by imatinib in theabsence of HS-5 cells (data not shown). However, cotreatment with TG101348 and imatinib resulted in a
progressive reduction in viable cell number comparedwith treatment with imatinib alone (Figure 3A). Furthermore, viable cell numbers of normal samples decreased partially after treatment with imatinib andTG101348 suggests that these treatments were selectively, and more effectively influence against leukemia(Figure 3A). The efficacy of the co-treatment was different in each group of primary samples, which may indicate different chemical sensitivities of these compounds.
We next investigated the effect of the inhibitors on
intracellular signaling in CD34-positive primary samples. We found that BCR-ABL, Crk-L, and STAT5 phosphorylation decreased, while PARP activity increased
after the co-treatment (Figure 3B). We investigated theefficacy of co-treatment with TG101348 and imatinib ina xenograft model. Growth of tumors decreased considerably in TG101348- and imatinib-treated mice compared with that in control mice. These results indicatethat co-treatment with imatinib and TG101348 is aneffective strategy to reduce expansion of BCR-ABLpositive cells including CD34-positive primary cells.

signaling in these cells. We found that BCR-ABL phosphorylation in the JAK2 siRNA-transfected K562 cells
was not reduced after imatinib treatment compared tocontrol cells (Figure 4C). In contrast, we observed that
Crk-L and MAPK phosphorylation decreased after imatinib treatment in JAK2-siRNA transfected K562 cells(Figure 4C). Thus, these results indicate that JAK2 is involved in the downstream signaling of BCR-ABL.

Knockdown of JAK2 increased sensitivity to imatinib

Discussion
Previous studies revealed that BCR-ABL-positive progenitor cells persist in patients with CML despite TKI
therapy. This may be attributable, in part, to quiescent
CML stem cells residing in protected hematopoietic
niches [24]. Hematopoietic growth factors, including cytokines, are secreted by endothelial cells and fibroblasts.
JAK2 phosphorylates STAT5 in normal hematopoietic
stem cells following activation of cytokine receptors.
However, BCR-ABL phosphorylates STAT5 at the same
critical tyrosine residue close to the SH2 domain in
CML cells, and activated STAT5 translocates to the nucleus [14,25].
Residual leukemia cells in bone marrow niches are exposed to many cytokines in situ [26]. Our results indicatethat cytokine-enriched HS-5 conditioned medium protected CML cells from imatinib. Furthermore, we demonstrated that the imatinib inhibitory effect decreased when
CML cells were co-cultured with HS-5 cells. This stromal

We next knocked down JAK2 by transfecting JAK2 smallinterfering RNA (siRNA) into K562 cells. Cell lysates
were analyzed by immunoblotting 72 h after transfection
using an anti-JAK2 Ab. We confirmed that Jak2 expression decreased considerably when compared to that incontrol cells (Figure 4A). To test the effect of JAK2
knockdown on K562 cells, we examined the effect ofimatinib on them when cultured in the presence of HS5. We observed that the viability of JAK2 siRNA transfected cells was not reduced when compared with thatof control cells (Figure 4B). In contrast, viability of JAK2
siRNA-transfected cells was partially reduced in thepresence of HS-5 cells when compared to control cells
cultured under the same conditions (Figure 4B). Importantly, we found that viability of JAK2 siRNA-transfected
K562 cells decreased considerably after imatinib treatment in the presence of HS-5 cells (Figure 4B). Next,
we investigated the effect of imatinib on intracellular

Detection of cytokines in HS-5 conditioned medium

The HS-5 cell line was treated with TG101348 at different
concentrations for 24 h (Figure 5). We found that STAT5
phosphorylation was partially reduced after TG101348
treatment using immunoblotting analysis (Figure 5A). We
next investigated cytokine levels by assaying HS-5 conditioned medium after 1 day of culture in the presence orabsence of TG101348 using the human cytokine array according to the manufacturer’s instructions. We analyzed
36 cytokines simultaneously and evaluated their relative
expression levels. We found that GM-CSF, and IL-6
levels decreased considerably after TG101348 treatmentcompared to that in the untreated conditioned medium(Figure 5B). These results indicate that TG101348 regulates cytokine production in HS-5 cells.

-227-

Okabe et al. Journal of Hematology & Oncology 2014, 7:37
http://www.jhoonline.org/content/7/1/37

Page 6 of 11

Figure 3 (See legend on next page.)-228-

Okabe et al. Journal of Hematology & Oncology 2014, 7:37
http://www.jhoonline.org/content/7/1/37

Page 7 of 11(See figure on previous page.)
Figure 3 Effects of imatinib and TG101348 on CD34-positive primary cells. (A) CD34-positive CML cells or healthy donor samples were
cultured in the presence of HS-5 cells. Cells were then treated with varying concentrations of imatinib alone or in combination with TG101348 for
72 h. Viable cell numbers were calculated. Results are representative of three separate experiments. (B) CD34-positive primary cells were treatedwith the indicated concentrations of imatinib alone or in combination with TG101348 for 24 h. Total cell extracts were analyzed by immunoblotting
using phospho-specific ABL, STAT5, Crk-L, and cleaved PARP Abs. β-actin was used as the loading control. Results are representative of at least three
reproducible experiments. Blots were scanned and optical densities were determined using ImageJ software. (C) In vivo studies were performed as
described in Methods. Tumors were treated with or without imatinib and TG101348 5 days per week, and growth was measured. *p < 0.05, comparedwith control mice.

cell line secretes multiple cytokines that support hematopoietic progenitor proliferation. Treatment of K562 cellswith imatinib in the presence of HS-5 conditioned
medium resulted in only partial inhibition of cell growth,
indicating that imatinib alone may not be sufficient toeradicate residual CML cells that reside within a cytokinerich microenvironment. Thus, it is important to probe thecytokine-mediated leukemia stem cell signaling pathways
as targets for combined treatments with TKIs to develop
more efficient therapies for patients with CML.
In this study, we analyzed the ability of cytokine signaling to protect CML cells. A previous study revealed
GM-CSF-mediated imatinib and nilotinib resistancethrough BCR-ABL independent activation of the JAK2/
STAT5 pathway [27]. We demonstrated that inhibiting
JAK2 by either JAK2 siRNA transfection or the JAK2 inhibitor TG101348 downregulated STAT5 activity. Moreover, STAT5 phosphorylation decreased considerably and
PARP activity increased following co-treatment with imatinib and TG101348 in Ph+ leukemia cells including primary CD34-positive cells. Inhibiting STAT5 in leukemia
was sufficient to prevent leukemia cell proliferation. JAK2
functions downstream of BCR-ABL. Although we did nottried to combine the two drugs at their IC50s in this cellline, we showed that inhibiting JAK2 resulted in reduced
STAT5 activity and enhanced imatinib efficacy even whenleukemia cells were protected by hematopoietic stromalcells-mediating cytokine/chemokine production.
We also examined the effect of TG101348 on cytokine
expression in HS-5 cells using a cytokine array containing
36 different cytokines. We observed that expression levelsof several cytokines, such as GM-CSF, G-CSF, and IL-6,
decreased considerably after treatment. Because GM-CSF
and IL-6 are involved in maintenance of hematopoietic
stem cells, these cytokines suppress apoptosis and regulate
cell viability, growth, and differentiation [28]. Thus, activating cytokine-mediated JAK2/STAT5 signaling may circumvent the need for BCR-ABL signaling and cell survival[29]. Here, we demonstrated that blocking cytokine signaling using a JAK2 inhibitor in combination with imatinib
might be an effective strategy to eradicate residual CML
cells in vitro and in vivo. Cytokine-mediated signaling may
not be critical for cell survival when BCR-ABL signaling is

activated; however, it is possible that residual CML cells
treated with imatinib can be rescued through a cytokinetriggered JAK2/STAT5 signaling pathway.

Conclusions
Our results indicate that co-treatment with imatinib and
JAK2 inhibitors may have the potential for targeting residual CML cells by enhancing imatinib efficiency andreducing cytokine production by stromal cells. Therefore, this approach may represent an important therapeutic strategy for patients with CML.
Methods
Cell culture and reagentsThe BCR-ABL-positive leukemia cell line K562 and humanbone marrow stromal cell line HS-5 were obtained fromthe American Type Culture Collection (Rockville, MD,
USA). Cells were cultured in the Roswell Park Memorial
Institute 1640 medium containing 10% fetal bovine serum
and maintained at 37°C in a 5% CO2 humidified atmosphere. The JAK2 inhibitor TG101348 was purchased from
Shanghai APIs Chemical (Shanghai, China) and the ABL
kinase inhibitor imatinib was provided by Novartis Pharma
AG (Basel, Switzerland). Stock solutions of TG101348
were prepared in dimethyl sulfoxide, while that of imatinib
was prepared in distilled water. These stock solutions were
further diluted in growth medium to achieve the desiredconcentrations. Antibodies (Abs) against phosphor-Abl,phospho Crk-L, STAT5, MAPK, cleaved caspase-3, and
PARP were purchased from Cell Signaling Technology(Danvers, MA, USA). Erk-1, Crk-L, and STAT5 Abs werepurchased from BD Biosciences (San Jose, CA, USA). The
anti-Abl Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other reagents were obtained
from Sigma-Aldrich (St. Louis, MO, USA).
Primary BCR-ABL-positive and normal control cells

This study protocol was approved by the Institutional
Review Board of Tokyo Medical University, and written
informed consent was provided by all patients in accordance with the Declaration of Helsinki. Primary samples
were collected from untreated peripheral blood of patients with CML or from the bone marrow of normal-229-

Okabe et al. Journal of Hematology & Oncology 2014, 7:37
http://www.jhoonline.org/content/7/1/37

Page 8 of 11

Figure 4 JAK2 siRNA transfected K562 cells had increased sensitivity to imatinib. (A) K562 cells were transfected with JAK2 siRNA or acontrol siRNA. Cell lysates were analyzed by immunoblotting 72 h after transfection using Jak2 and β-actin Abs. Blots were scanned and opticaldensities were determined using the ImageJ system. (B) K562 and siRNA-transfected K562 cells were cultured at a concentration of 8 × 104/mL in
the presence or absence of feeder cells. The cells were treated with 1 μM imatinib for 72 h. Viable cell numbers were calculated. Results are
representative of three separate experiments. (C) K562 and siRNA-transfected K562 cells were treated with imatinib for 24 h, and total extracts
were examined by immunoblotting using anti-phospho ABL, ABL, phospho-Crk-L, Crk-L, phospho-MAPK, Erk-1 and β-actin Abs. Blots were
scanned and optical densities were determined using ImageJ software.

healthy donor after obtaining written informed consent.
The peripheral blood was collected in heparinized tubes,
and mononuclear cells were separated using LymphoSepare (Immuno-Biological Laboratories, Minneapolis,
MN, USA). These cells were either used immediately or
cryopreserved in liquid nitrogen until use.

Cell proliferation assay

Cells were seeded in 24- or 96-well plates at a density of
8 × 104 or 8 × 103 cells/well. Next, cells were treated with
the inhibitors at the indicated concentrations: imatinib,
100 nM to 2 μM; and TG101348, 100 nM to 1 μM. Viable cells were either counted using trypan blue-230-

Okabe et al. Journal of Hematology & Oncology 2014, 7:37
http://www.jhoonline.org/content/7/1/37

Page 9 of 11

Figure 5 Effect of TG101348 on HS-5 cells. (A) HS-5 cells were cultured in the presence of different concentrations of TG101348 for 24 h, andtotal extracts were examined by immunoblotting using anti-phospho STAT5, STAT5, and β-actin Abs. Blots were scanned and optical densities
were determined using ImageJ software. (B) HS-5 cells were treated with TG101348 for 24 h and conditioned medium was collected. The relative
levels of 36 different cytokines were analyzed using the Human Cytokine Array Kit. The expression of cytokines was determined using ImageJsoftware. Results are representative of three separate experiments. *p < 0.05, TG101348 treatment vs. control.

exclusion or were stained with a cell counting kit solution(Dojin, Kumamoto, Japan) and measured spectrophotometrically (A450) to determine cell viability. In some experiments, cells were co-cultured with human stromal cells.
After cultivating the HS-5 cells at a density of 1 × 105
cells/well in 24-well plates overnight, the K562 cells wereplated onto the HS-5 cells, cultured for 3 days, and analyzed for cell proliferation. To analyze the activity of thesoluble factors produced by HS-5 cells, K562 cells were
cultured for 72 h in the presence of HS-5 conditioned
medium from cultures that that were either not treatedwith imatinib or treated with the drug at the indicatedconcentrations. Cell proliferation was then analyzed.

siRNA transfection

siRNAs targeting the JAK family members were purchased from Santa Cruz Biotechnology (sc-39099). K562
cells were transfected with a siRNA targeting JAK2 byelectroporation, as described previously [30].
Immunoblotting

Immunoblotting was performed as described previously[31]. After different treatments were performed as indicated above, cells were collected by centrifugation andlysed by sonication using the radioimmunoprecipitation
assay lysis buffer. Protein content in the lysates was determined using a protein assay kit (Bio-Rad Laboratories,-231-

Okabe et al. Journal of Hematology & Oncology 2014, 7:37
http://www.jhoonline.org/content/7/1/37

Page 10 of 11

Hercules, CA, USA). Forty micrograms of total cellular proteins were separated on 4–20% polyacrylamide gels andtransferred to polyvinylidene difluoride membranes. Next,
the membranes were probed using primary Abs of interestat the appropriate dilutions for 2 h at room temperature.
The blots were visualized by the chemiluminescent method
using the Amersham ECL chemiluminescence kit (GEHealthcare, Tokyo, Japan). Immunoblots were quantified
using ImageJ software (NIH, Bethesda, MD, USA).
Cytokine array

After culturing HS-5 cells in the presence or absence ofTG101348, the culture medium was collected. Cytokine
levels in the medium were measured using the humancytokine panel A (Proteome Profiler™) (R&D Systems,
Minneapolis, MN, USA). Horseradish peroxidase substrate was used to detect protein expression, and data
were visualized using Kodak Biomax Light film. Arrays
were scanned and optical densities were determinedusing ImageJ software.
In vivo studies

Six-week-old female BALB/c nude mice were purchasedfrom CLEA Japan Inc. (Tokyo, Japan). Mice were injectedsubcutaneously with 1 × 107 Ba/F3 wild-type BCR-ABL
cells. Mice were treated after 5 days with 40 mg/kg imatinib intraperitoneally or 30 mg/kg TG101348 orally or a
combination of the two agents for 5 days per week. Control mice were treated with phosphate-buffered saline.
Tumor sizes were analyzed every day. The average tumor
weight per mouse (n = 6) was calculated.
Statistical analysis

We used Student’s t-test to determine differences in response to the various treatments. p < 0.05 was considered statistically significant.
Abbreviations
Ph: Philadelphia chromosome; TKI: Tyrosine kinase inhibitor; CML: Chronic
myeloid leukemia; JAK: Janus kinase; GM-CSF: Granulocyte macrophagecolony-stimulating factor; STAT: Signal transducer and activator oftranscription; IL: Interleukin.
Competing interests
The authors declare no competing interests.
Authors’ contributions
SO, TT, SK, YT, and KO conceived and designed the experiments; SO
performed the experiments; and SO, TT, SK, YT, and KO contributed reagentsand wrote the manuscript. All authors read and approved the final
manuscript.
Acknowledgments
This study was supported by a “High-Tech Research Center” Project for private
universities, a matching fund subsidy from the Ministry of Education, Culture,
Sports, Science and Technology (MEXT); and by the “University-Industry Joint
Research Project” for private universities, a matching fund subsidy from MEXT.
This study was also supported by Grants-in-Aid for Scientific Research from
MEXT.

Received: 7 February 2014 Accepted: 22 April 2014
Published: 28 April 2014

References
1. Sattler M, Griffin JD: Molecular mechanisms of transformation by the BCRABL oncogene. Semin Hematol 2003, 40(suppl 1):4–10.
2. Kantarjian HM, Talpaz M, Giles F, O’Brien S, Cortes J: New insights into thepathophysiology of chronic myeloid leukemia and imatinib resistance.
Ann Intern Med 2006, 145:913–923.
3. Helgason GV, Karvela M, Holyoake TL: Kill one bird with two stones:
potential efficacy of BCR-ABL and autophagy inhibition in CML.
Blood 2011, 118:2035–2043.
4. Sawyers CL, McLaughlin J, Witte ON: Genetic requirement for Ras in thetransformation of fibroblasts and hematopoietic cells by the Bcr-Abl
oncogene. J Exp Med 1995, 181:307–313.
5. Skorski T, Bellacosa A, Nieborowska-Skorska M, Majewski M, Martinez R, Choi JK,
Trotta R, Wlodarski P, Perrotti D, Chan TO, Wasik MA, Tsichlis PN, Calabretta B:
Transformation of hematopoietic cells by BCR/ABL requires activation of a
PI-3k/Akt-dependent pathway. EMBO J 1997, 16:6151–6161.
6. Chai SK, Nichols GL, Rothman P: Constitutive activation of JAKs and STATs
in BCR-Abl-expressing cell lines and peripheral blood cells derived fromleukemic patients. J Immunol 1997, 159:4720–4728.
7. Roskoski R Jr: STI-571: an anticancer protein-tyrosine kinase inhibitor.
Biochem Biophys Res Commun 2003, 309:709–717.
8. Druker BJ, Guilhot F, O’Brien SG, Gathmann I, Kantarjian H, Gattermann N,
Deininger MW, Silver RT, Goldman JM, Stone RM, Cervantes F, Hochhaus A,
Powell BL, Gabrilove JL, Rousselot P, Reiffers J, Cornelissen JJ, Hughes T, Agis
H, Fischer T, Verhoef G, Shepherd J, Saglio G, Gratwohl A, Nielsen JL, Radich
JP, Simonsson B, Taylor K, Baccarani M, So C, et al: Five-year follow-up ofpatients receiving imatinib for chronic myeloid leukemia. N Engl J Med
2006, 355:2408–2417.
9. Mahon FX, Réa D, Guilhot J, Guilhot F, Huguet F, Nicolini F, Legros L,
Charbonnier A, Guerci A, Varet B, Etienne G, Reiffers J, Rousselot P,
Intergroupe Français des Leucémies Myéloïdes Chroniques: Discontinuationof imatinib in patients with chronic myeloid leukemia who havemaintained complete molecular remission for at least 2 years: theprospective, multicentre Stop Imatinib (STIM) trial. Lancet Oncol 2010,
11:1029–1035.
10. Sloma I, Jiang X, Eaves AC, Eaves CJ: Insights into the stem cells of chronic
myeloid leukemia. Leukemia 2010, 24:1823–1833.
11. Murray PJ: The JAK-STAT signaling pathway: input and output
integration. J Immunol 2007, 178:2623–2629.
12. Wheadon H, Roberts PJ, Watts MJ, Linch DC: Changes in signaltransduction downstream from the granulocyte-macrophagecolony-stimulating factor receptor during differentiation of primary
hemopoietic cells. Exp Hematol 1999, 27:1077–1086.
13. Nagata Y, Todokoro K: Interleukin 3 activates not only JAK2 and STAT5,
but also Tyk2, STAT1, and STAT3. Biochem Biophys Res Commun 1996,
221:785–789.
14. Fabbro D: BCR-ABL signaling: A new STATus in CML. Nat Chem Biol 2012,
8:228–229.
15. Vainchenker W, Constantinescu SN: JAK/STAT signaling in hematologicalmalignancies. Oncogene 2013, 32:2601–2613.
16. Pardanani A, Gotlib JR, Jamieson C, Cortes JE, Talpaz M, Stone RM, Silverman
MH, Gilliland DG, Shorr J, Tefferi A: Safety and efficacy of TG101348, a
selective JAK2 inhibitor, in myelofibrosis. J Clin Oncol 2011, 29:789–796.
17. Warsch W, Walz C, Sexl V: JAK of all trades: JAK2-STAT5 as novel
therapeutic targets in BCR-ABL1+ chronic myeloid leukemia.
Blood 2013, 122:2167–2175.
18. Xu W, Chen B, Tong X: Chronic myeloid leukemia patient withco-occurrence of BCR-ABL junction and JAK2 V617F mutation. Int J
Hematol 2014, 99:87–90.
19. Lee YJ, Moon JH, Shin HC, Seo JW, Han SA, Seo SK, Sohn SK: Two CML
patients who subsequently developed features of essential
thrombocythemia with JAK2-V617F mutation while in complete
cytogenetic remission after treatment with imatinib mesylate. Int J
Hematol 2013, 97:804–807.
20. Park SH, Chi HS, Cho YU, Jang S, Park CJ, Kim DY, Lee JH, Lee KH: Two casesof myeloproliferative neoplasm with a concurrent JAK2 (V617F)
mutation and BCR/ABL translocation without chronic myelogenous-232-

Okabe et al. Journal of Hematology & Oncology 2014, 7:37
http://www.jhoonline.org/content/7/1/37

21.

22.
23.

24.

25.

26.

27.

28.

29.

30.

31.

Page 11 of 11

leukemia phenotype acquisition during hydroxyurea treatment. Ann Lab
Med 2013, 33:229–232.
Samanta AK, Chakraborty SN, Wang Y, Schlette E, Reddy EP, Arlinghaus RB:
Destabilization of Bcr-Abl/Jak2 network by a Jak2/Abl kinase inhibitor
ON044580 overcomes drug resistance in blast crisis chronic
myelogenous leukemia (CML). Genes Cancer 2010, 1:346–359.
Cogle CR: Overcoming chronic myeloid leukemia stem cell resistance toimatinib by also targeting JAK2. J Natl Cancer Inst 2013, 105:378–379.
Nakao T, Kim S, Ohta K, Kawano H, Hino M, Miura K, Tatsumi N, Iwao H:
Role of mitogen-activated protein kinase family in serum-induced
leukemia inhibitory factor and interleukin-6 secretion by bone marrowstromal cells. Br J Pharmacol 2002, 136:975–984.
Ernst T, Hochhaus A: Chronic myeloid leukemia: clinical impact of BCRABL1 mutations and other lesions associated with disease progression.
Semin Oncol 2012, 39:58–66.
Warsch W, Kollmann K, Eckelhart E, Fajmann S, Cerny-Reiterer S, Hölbl A,
Gleixner KV, Dworzak M, Mayerhofer M, Hoermann G, Herrmann H, SillaberC, Egger G, Valent P, Moriggl R, Sexl V: High STAT5 levels mediate imatinib
resistance and indicate disease progression in chronic myeloid leukemia.
Blood 2011, 117:3409–3420.
Corbin AS, Agarwal A, Loriaux M, Cortes J, Deininger MW, Druker BJ: Human
chronic myeloid leukemia stem cells are insensitive to imatinib despite
inhibition of BCR-ABL activity. J Clin Invest 2011, 121:396–409.
Wang Y, Cai D, Brendel C, Barett C, Erben P, Manley PW, Hochhaus A,
Neubauer A, Burchert A: Adaptive secretion of granulocyte-macrophagecolony-stimulating factor (GM-CSF) mediates imatinib and nilotinib
resistance in BCR/ABL + progenitors via JAK-2/STAT-5 pathway
activation. Blood 2007, 109:2147–2155.
Mohamed AA, Ibrahim AM, El-Masry MW, Mansour IM, Khroshied MA, Gouda
HM, Riad RM: Ex vivo expansion of stem cells: defining optimum
conditions using various cytokines. Lab Hematol 2006, 12:86–93.
McCubrey JA, Steelman LS, Abrams SL, Bertrand FE, Ludwig DE, Bäsecke J,
Libra M, Stivala F, Milella M, Tafuri A, Lunghi P, Bonati A, Martelli AM:
Targeting survival cascades induced by activation of Ras/Raf/MEK/ERK,
PI3K/PTEN/Akt/mTOR and Jak/STAT pathways for effective leukemia
therapy. Leukemia 2008, 22:708–722.
Okabe S, Tauchi T, Tanaka Y, Katagiri S, Ohyashiki K: Effects of thehedgehog inhibitor GDC-0449, alone or in combination with dasatinib,
on BCR-ABL-positive leukemia cells. Stem Cells Dev 2012, 21:2939–2948.
Okabe S, Tauchi T, Tanaka Y, Ohyashiki K: Efficacy of ponatinib against ABL
tyrosine kinase inhibitor-resistant leukemia cells. Biochem Biophys Res
Commun 2013, 435:506–511.

doi:10.1186/1756-8722-7-37
Cite this article as: Okabe et al.: Combination of the ABL kinase inhibitor
imatinib with the Janus kinase 2 inhibitor TG101348 for targeting
residual BCR-ABL-positive cells. Journal of Hematology & Oncology
2014 7:37.

Submit your next manuscript to BioMed Central
and take full advantage of:• Convenient online submission• Thorough peer review• No space constraints or color ﬁgure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit-233-Cancer Biology & Therapy

ISSN: 1538-4047 (Print) 1555-8576 (Online) Journal homepage: http://www.tandfonline.com/loi/kcbt20

Efficacy of the dual PI3K and mTOR inhibitor NVPBEZ235 in combination with nilotinib against
BCR-ABL-positive leukemia cells involves the ABL
kinase domain mutation
Seiichi Okabe, Tetsuzo Tauchi, Yuko Tanaka, Toshihiko Kitahara, Shinya
Kimura, Taira Maekawa & Kazuma Ohyashiki
To cite this article: Seiichi Okabe, Tetsuzo Tauchi, Yuko Tanaka, Toshihiko Kitahara, Shinya
Kimura, Taira Maekawa & Kazuma Ohyashiki (2014) Efficacy of the dual PI3K and mTOR inhibitor
NVP-BEZ235 in combination with nilotinib against BCR-ABL-positive leukemia cells involves the
ABL kinase domain mutation, Cancer Biology & Therapy, 15:2, 207-215, DOI: 10.4161/cbt.26725
To link to this article: https://doi.org/10.4161/cbt.26725

Published online: 08 Nov 2013.

Submit your article to this journal

Article views: 365

View related articles

View Crossmark data

Citing articles: 16 View citing articles

Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=kcbt20-234-

Research Paper

Research Paper

Cancer Biology & Therapy 15:2, 207–215; February 2014; © 2014 Landes Bioscience

Efficacy of the dual PI3K and mTOR inhibitor
NVP-BEZ235 in combination with nilotinib
against BCR-ABL-positive leukemia cells
involves the ABL kinase domain mutation
First Department of Internal Medicine; Tokyo Medical University; Tokyo, Japan; 2Division of Hematology, Respiratory Medicine and Oncology;
Department of Internal Medicine; Faculty of Medicine; Saga University; Saga, Japan; 3Department of Transfusion Medicine and Cell Therapy;
Kyoto University Hospital; Kyoto, Japan

1

Keywords: chronic myeloid leukemia, PI3K, mTOR, NVP-BEZ235, nilotinib, BCR-ABL mutation, ponatinib resistance

Imatinib, an ABL tyrosine kinase inhibitor (TKI), has shown clinical efficacy against chronic myeloid leukemia (CML).
However, a substantial number of patients develop resistance to imatinib treatment due to the emergence of clones
carrying mutations in the protein BCR-ABL. The phosphoinositide 3 kinase (PI3K)/Akt/mammalian target of rapamycin(mTOR) pathway regulates various processes, including cell proliferation, cell survival, and antiapoptosis activity. In thisstudy, we investigated the efficacy of NVP-BEZ235, a dual PI3K and mTOR inhibitor, using BCR-ABL-positive cell lines.
Treatment with NVP-BEZ235 for 48 h inhibited cell growth and induced apoptosis. The phosphorylation of the AKT kinase,eukaryotic initiation factor 4-binding protein 1 (4E-BP1), and p70 S6 kinase were decreased after NVP-BEZ235 treatment.
The combination of NVP-BEZ235 with a BCR-ABL kinase inhibitor, imatinib, or nilotinib, induced a more pronounced
colony growth inhibition, whereas the combination of NVP-BEZ235 and nilotinib was more effective in inducing apoptosis
and reducing the phosphorylation of AKT, 4E-BP1, and S6 kinase. NVP-BEZ235 in combination with nilotinib also inhibitedtumor growth in a xenograft model and inhibited the growth of primary T315I mutant cells and ponatinib-resistant cells.
Taken together, these results suggest that administration of the dual PI3K and mTOR inhibitor NVP-BEZ235 may be aneffective strategy against BCR-ABL mutant cells and may enhance the cytotoxic effects of nilotinib in ABL TKI-resistant
BCR-ABL mutant cells.

Introduction
Chronic myeloid leukemia (CML) is a myeloproliferative
disorder of hematopoietic stem cells, characterized by the presence of the Philadelphia chromosome (Ph).1,2 BCR-ABL kinaseis largely responsible for the pathogenesis of CML. BCR-ABL
activates the phosphatidylinositol-3 kinase (PI3K)/AKT, Ras,and Janus kinase (JAK)/signal transducers and activator of transcription (STAT) downstream signaling pathways; therefore,
these molecules may have leukemogenic activity.3-5 Althoughimatinib is used for the treatment of CML and Ph-positive
acute lymphoblastic leukemia (ALL),6,7 some patients develop
secondary resistance to imatinib therapy. Several imatinib resistance mechanisms have been reported in vitro, which involve
reactivation of the BCR-ABL signaling pathway. These include
BCR-ABL gene amplification, BCR-ABL point mutation, andoverexpression of LYN, a SRC-family kinase protein.8,9 Secondline BCR-ABL kinase inhibitors such as nilotinib and dasatinib
are clinically available. The majority of imatinib-resistant or

imatinib-intolerant patients responded to treatment with thesesecond-generation BCR-ABL kinase inhibitors.10 However,therapy with second-generation BCR-ABL kinase inhibitors ortransplantation is less successful in patients with advanced or
blast-phase CML than in those with chronic phase.11 Furthermore, if quiescent leukemia stem cells are not eradicated, current
BCR-ABL tyrosine kinase inhibitor will not cure the disease.12
Alternative treatment modalities, such as BCR-ABL targeting tyrosine kinase inhibitors (TKIs) combined with tolerated
agents that target pathways downstream of BCR-ABL could prevent the emergence of resistant clones.
PI3K is involved in the phosphorylation of membrane inositol
lipids. AKT, also referred as to as protein kinase B, is a 57-kDa
serine/threonine kinase that is the cellular homolog of thev-AKT oncogene, and it has 3 isoforms that share a high degreeof sequence homology. All the 3 members (AKT1, AKT2, andAKT3) belong to the AGC family of protein kinases.13 AKT1 andAKT2 are ubiquitously expressed and AKT3 is found in nervous
tissue.14 The PI3K/AKT signaling pathways play an important*Correspondence to: Seiichi Okabe; Email: okabe@tokyo-med.ac.jp
Submitted: 08/17/2013; Revised: 10/02/2013; Accepted: 10/06/2013
http://dx.doi.org/10.4161/cbt.26725
www.landesbioscience.com	Cancer Biology & Therapy

-235-

207

©2014 Landes Bioscience. Do not distribute.

Seiichi Okabe1,*, Tetsuzo Tauchi1, Yuko Tanaka1, Toshihiko Kitahara1, Shinya Kimura2, Taira Maekawa3, and Kazuma Ohyashiki1

Figure 1. Effects of NVP-BEZ235 and imatinib or nilotinib on Ba/F3 BCRABL random mutagenesis cells. Ba/F3 BCR-ABL random mutagenesis
cells were treated with the indicated concentration of NVP-BEZ235 and/or nilotinib (A) or imatinib (B) for 14 d. The colony-formation assay was
performed as described in Materials and Methods. The data are representative of 3 independent sets of experiments. Statistical analysis was
also performed by using two-way ANOVA. Two-way ANOVA analysis
revealed combined effects of two drugs were additive.

role in normal cellular processes such as proliferation, survival,and differentiation.15 Mammalian target of rapamycin (mTOR)
is a 289-kDa serine/threonine protein kinase belonging to the
PI3K-related kinase family. It is established that the upregulation
and activation of PI3K/AKT/mTOR signaling is important forconferring a growth advantage to leukemia cells, including CML
cells. Moreover, activation of the PI3K/AKT/mTOR pathway
negatively influences the tumor response to conventional antileukemic treatments.16
In this study, we investigated the effects of NVP-BEZ235 on
Ph-positive leukemia cells. NVP-BEZ235 is a dual inhibitor of
PI3K and mTOR.17 We clarified the mechanisms of action of
NVP-BEZ235 in BCR-ABL–expressing cells and determined its
efficacy alone and in combination with ABL kinase inhibitors
such as nilotinib or imatinib. We found that the co-treatment
with NVP-BEZ235 and BCR-ABL kinase inhibitors is effective
against imatinib-resistant BCR-ABL-positive leukemia cells.

Effect of NVP-BEZ235 and imatinib or nilotinib on Ba/F3
BCR-ABL random mutagenesis cells
BCR-ABL point mutations are major mechanism of imatinib
resistance.8,9 In many leukemia cases, patient samples containmultiple genetically distinct leukemia-initiating cell subclones
and reconstitution with the predominant diagnosis clone is associated with more aggressive growth properties.18 In particular,
compound mutations are common in patients with sequencing evidence for double BCR-ABL1 mutations and frequently
reflect a highly complex clonal network in Ph-positive leukemia
patients.19 Therefore, we used the in vitro screen of randomly
mutagenized BCR-ABL Ba/F3 cells that confer resistance to
ABL tyrosine kinase inhibitor in this study. We evaluated thevarious NVP-BEZ235 combinations employing random mutagenesis of Ba/F3 BCR-ABL cells and analyzed the antiproliferative activity by long-term culture colony assay. After exposure
to indicated concentrations of NVP-BEZ235, the percentage ofcolony formation was lower than that in the control (Fig. 1). The
inhibition rate calculated by colony assay was 62% with 50 nM
NVP-BEZ235 and 84% with 75 nM. In this study, we examinedthe efficacy of the combination treatment of nilotinib or imatinib with NVP-BEZ235. We found that the proliferation of Ba/
F3 BCR-ABL random mutagenesis cells was partially reduced bytreatment with imatinib or nilotinib alone compared with thecontrol. We found that the percentage of colony formation was
significantly inhibited by the co-treatment with NVP-BEZ235
and nilotinib or imatinib. We also found that colony growth wascompletely inhibited by treatment with 50 nM NVP-BEZ235
and 500 nM nilotinib. Statistical analysis was also performed.
Two-way ANOVA analysis revealed these drugs combined effects
were additive.
NVP-BEZ235 inhibits growth and induces apoptosis in
BCR-ABL-positive cells
We next evaluated the effect of NVP-BEZ235 on BCR-ABLpositive cell line K562 using the cell proliferation assay. K562 cells
were incubated for 48 h with increasing concentrations of NVPBEZ235. As shown in Figure 2A, the cell growth of K562 wasinhibited in a dose-dependent manner. Effective inhibitory concentrations for this cell line were found in the nanomolar range.
We next examined whether NVP-BEZ235 induced apoptosis in
K562 cells. As indicated in Figure 2B, the percentage of Annexin
V-positive cells increased after 48 h NVP-BEZ235 treatment ina dose-dependent manner. We also evaluated the intracellularsignaling after NVP-BEZ235 treatment. 4E-BP1 and S6 kinases
are the downstream targets of PI3K/AKT and mTOR pathways,
and these are correlated with protein transcription.20 In immunoblot analysis, we found that the phosphorylation level of AKT,
4E-BP1, and S6 kinase decreased after 24 h NVP-BEZ235 treatment in a dose-dependent manner. However, the phosphorylation of MAPK was not found to be reduced (Fig. 2C). We next
examined the effect on Ph-positive primary cells. We found thatthe cellular growth of Ph-positive primary cells was inhibited by
NVP-BEZ235 in a dose dependent manner (Fig. 2D).

208	Cancer Biology & Therapy-236-

Volume 15 Issue 2©2014 Landes Bioscience. Do not distribute.

Results©2014 Landes Bioscience. Do not distribute.
Figure 2. NVP-BEZ235 inhibits cell growth and induces apoptosis in Ph-positive cells. (A) Cells were cultured at a concentration of 8 × 104/mL in the presence or absence of NVP-BEZ235 for 48 h. Viable cells were evaluated as described in Materials and Methods. (B) K562 cells were treated with NVP-BEZ235
at the indicated concentration for 48 h. The percentage of apoptotic cells was examined as described in Materials and Methods. (C) K562 cells weretreated with NVP-BEZ235 for 24 h; total extracts were analyzed by immunoblot analysis with anti-phospho Akt, 4E-BP1, S6 kinase, MAPK, and anti-actin
antibodies. The data presented here are representative of 3 separate experiments. (D) Ph-positive primary cells were cultured at a concentration of 2 ×
105/mL in the presence or absence of NVP-BEZ235 for 48 h. Viable cells were evaluated as indicated in Materials and Methods.

www.landesbioscience.com	Cancer Biology & Therapy

-237-

209

Co-treatment with NVP-BEZ235 and nilotinib inhibits
growth and induces apoptosis of K562 and primary cells
To examine the inhibitory effect of NVP-BEZ235 and nilotinib on the proliferation of CML cells, we evaluated the growthof K562 cells before and after NVP-BEZ235 and/or nilotinib
treatment. The cell growth of K562 was inhibited by NVPBEZ235 and nilotinib treatment (Fig. 3A). As indicated in Figure 3B, the percentage of Annexin V-positive cells increased after
NVP-BEZ235 and nilotinib treatment. We next examined theintracellular signaling before and after NVP-BEZ235 and/or
nilotinib treatment in K562 cells. The results indicated that thephosphorylation of BCR-ABL was reduced after 24 h treatment
with nilotinib. The phosphorylation of AKT, 4E-BP1, and S6
kinase reduced after co-treatment with NVP-BEZ235 and nilotinib. Moreover, BCL-XL was downregulated and cleaved PARP
was upregulated after treatment with NVP-BEZ235 and nilotinib (Fig. 3C).

Effects of NVP-BEZ235 and nilotinib on Ba/F3 BCR-ABL
random mutagenesis cells in a xenograft model
To assess the activity of NVP-BEZ235, we tested CML tumor
formation in mice. Therefore, we injected nude mice subcutaneously with 1 × 107 Ba/F3 BCR-ABL random mutagenesis
cells. The next day, mice were separated into 4 groups (control,
nilotinib, NVP-BEZ235, and nilotinib + NVP-BEZ235). Control mice were treated with 0.9% NaCl daily. Tumor size was
evaluated every 3 d. An orally administered dose of 30 mg/kg/
day of nilotinib or NVP-BEZ235 inhibited tumor growth andreduced tumor volume compared with control mice. Moreover,it was observed that the tumor volume in the nilotinib + NVPBEZ235 group decreased significantly (P < 0.001) (Fig. 4A). The
tumor from mice treated with nilotinib and NVP-BEZ235 displayed higher necrosis levels compared with that from vehicletreated mice. We also performed immunohistochemical analysis.
TdT-mediated dUTP nick-end labeling (TUNEL) assay showed

210	Cancer Biology & Therapy-238-

Volume 15 Issue 2©2014 Landes Bioscience. Do not distribute.

Figure 3. Effect of NVP-BEZ235 and nilotinib treatment on K562 cells. (A and B) Cells were cultured in the presence or absence of NVP-BEZ235 or nilotinib for 48 h. Viable cells and apoptotic cells were evaluated as described in Materials and Methods. *P < 0.05 for nilotinib and NVP-BEZ235 treatment
vs. treatment with 50 nM nilotinib alone in the same cell line. (C) K562 cells were treated with NVP-BEZ235 and/or nilotinib for 24 h; total cellular lysates
were immunoblotted with anti-phospho Abl, Akt, 4E-BP1, S6 kinase, BCL-XL, PARP, and actin Abs.

that the number of apoptotic cells was higher and the expression
level of the proliferation maker Ki-67 was lower in the nilotinib
and NVP-BEZ235 treatment group than in the other groups(Fig. 4B). Furthermore, we found that the phosphorylation of S6
kinase was significantly lower in the nilotinib and NVP-BEZ235
combination treatment group compared with that in the control
mice. These results suggest that nilotinib and NVP-BEZ235
treatment effectively suppress tumor growth in vivo and that thetumor inhibition achieved by the combinatorial treatment wassuperior to that achieved by nilotinib or NVP-BEZ235 alone.
Co-treatment with NVP-BEZ235 and nilotinib inhibits thegrowth of wild type and mutant BCR-ABL-positive cells
Because co-treatment with NVP-BEZ235 and nilotinib
inhibited colony formation, we investigated whether NVPBEZ235 and nilotinib treatment could inhibit Ph-positive primary cells as well. The results showed that 48 h NVP-BEZ235
and nilotinib co-treatment suppressed the growth of Ph-positive
primary cells (Fig. 5A). We next investigated the effect of thetreatment on T315I point mutant primary cells. We found that
NVP-BEZ235 and nilotinib inhibited cell growth and induced

apoptosis of T315I-positive cells (Fig. 5B and C). In addition,
we found that NVP-BEZ235 and nilotinib combination treatment inhibited the growth of ponatinib (AP24534)-resistantprimary cells (Fig. 5D). These results indicated that the combination of NVP-BEZ235 and nilotinib treatment is effective
against Ph-positive primary cells, including ABL TKI-resistantcells.

Discussion
Imatinib resistance caused by ABL kinase domain mutation
activates the downstream targets of BCR-ABL, such as the PI3K/
Akt/mTOR pathways, which have recently been implicated in
the survival and expansion of leukemic cells. Therefore, the targeting of the PI3K/Akt/mTOR signaling pathway by specifickinase inhibitors have been the focus of extensive treatment for
Ph-positive leukemia cells including T315I mutation.
A number of PI3K/Akt/mTOR signaling inhibitors are underinvestigation. Among these, NVP-BEZ235 has shown antitumor activity against various tumor types,21-23 and it is now

www.landesbioscience.com	Cancer Biology & Therapy

-239-

211

©2014 Landes Bioscience. Do not distribute.

Figure 4. Effect of NVP-BEZ235 and nilotinib on Ba/F3 BCR-ABL random mutagenesis cells in xenograft model. (A) In vivo studies were performed as
described in Materials and Methods. (B) Tumor cells treated with or without NVP-BEZ235 and nilotinib for 24 d were examined by immunohistochemical
analysis as described in Materials and Methods. Original magnification: 400×. H&E, hematoxylin and eosin; TUNEL, TdT-mediated dUTP nick-end labeling. *P < 0.01, **P < 0.001 compared with control.

©2014 Landes Bioscience. Do not distribute.
Figure 5. For figure legend, see page 213.

212	Cancer Biology & Therapy-240-

Volume 15 Issue 2

Figure 5 (See opposite page). Co-treatment with NVP-BEZ235 and nilotinib inhibits cell growth and induces apoptosis of wild-type and T315I mutant
BCR-ABL-positive cells. (A) Wild-type primary cells were cultured at a concentration of 2 × 105/mL in the presence or absence of NVP-BEZ235 or nilotinibfor 48 h. Viable cells were evaluated. (B) Ba/F3 T315I cells were cultured in the presence or absence of NVP-BEZ235 or nilotinib for 48 h. Viable cells andpercentage of apoptotic cells was examined. (C) T315I mutant primary cells were cultured in the presence or absence of NVP-BEZ235 or nilotinib for 48
h. Viable cells and percentage of apoptotic cells was examined. (D) Ponatinib-resistant primary cells were treated with NVP-BEZ235 and/or nilotinib at
the indicated concentration for 48 h. Viable cells were evaluated as described in Materials and Methods. *P < 0.05, nilotinib and NVP-BEZ235 treatment
vs. treatment with 1 μM nilotinib alone in the same cell line.

Materials and Methods
Cell, reagents, and animals
A BCR-ABL-positive cell line K562 was obtained from theAmerican Type Culture Collection. Ba/F3 BCR-ABL, BCRABL point mutant cells, and BCR-ABL random mutagenesis
cells were described previously.31,32 A full-length BCR-ABL cDNA
was cloned into pMSCV-IRES-GFP retroviral vector, generating MSCV-IRES-GFP-B/A. XL-1 Red competent Escherichia
coli cells (Stratagene) deficient in DNA repair mechanisms wereused for transformation. The plasmid libraries were transfected
onto HEK 293T cells and the viral supernatant was collected.
Murine Ba/F3 cells were then transfected with the mutated vector. Ba/F3 cells expressing the different BCR-ABL constructs
became interleukin-3 (IL-3)-independent and were grown in
Roswell Park Memorial Institute (RPMI) without IL-3. Thesecells were cultured in the RPMI1640 medium containing 10%
fetal bovine serum (FBS). Nude mice were obtained from Clea
Japan and maintained under standard conditions according to
the institutional guidelines. NVP-BEZ235, nilotinib and imatinib were obtained from Novartis Pharma AG. Stock solutionsof NVP-BEZ235 and nilotinib were prepared in dimethyl sulfoxide (DMSO), and imatinib was dissolved in distilled water prior
to dilution to the desired concentration in the growth medium.
Anti-phospho Abl, anti-phospho CRK-L, S6 kinase, eukaryotic
initiation factor 4-binding protein 1 (4E-BP1), mitogen-activatedProtein kinase (MAPK), anti-cleaved caspase 3, and poly (ADPribose) polymerase (PARP) antibodies (Abs) were purchasedfrom Cell Signaling. Other reagents were obtained from Sigma.
Cell proliferation assay
Cell proliferation analysis was performed as previously
described.33,34 Briefly, the cells were seeded in 24- or 96-wellplates at a density of 1 × 105 or 1 × 104 cells/well. The cells weretreated with the inhibitors at the following concentrations: NVPBEZ235, 1–100 nM; nilotinib, from 10 nM to 1 μM. Viable
cells were counted by trypan blue exclusion or fluorescence-activated cell sorting (FACS).
FACS analysis
The cells were treated with NVP-BEZ235 and/or nilotinib at
the concentrations described above for 48 h. Annexin V/propidium iodide apoptosis assay was performed according to the
manufacturer’s protocol (Becton, Dickinson and Company). The
cells were mixed gently and were analyzed immediately by flow
cytometry.
Western blotting
The cells were treated with NVP-BEZ235 at indicated times
and concentrations, and washed once with ice-cold phosphatebuffered saline (PBS). The cells were then lysed in lysis bufferwww.landesbioscience.com	Cancer Biology & Therapy

-241-

213

©2014 Landes Bioscience. Do not distribute.

being evaluated in clinical trials. NVP-BEZ235 has shown invivo anticancer efficacy in models of lung cancer, breast cancer,
glioma, and myeloma.23-26 Another dual PI3K/mTOR inhibitor,
PI-103 and Mdm2 inhibitor, Nutlin-3 enhances the antileukemic activity-mediated p53 pathway in acute myeloid leukemia(AML) cells.27 The simultaneous inhibition of PI3K/Akt/mTOR
and MAPK signaling is effective against epidermal growth factor receptor (EGFR) mutant lung cancer.28 Although it has beenreported that the co-treatment of the PI3K inhibitors LY294002
or wortmannin with imatinib enhances the antileukemic activity in CML,29 these compounds cannot be used in clinical setting due to their severe toxicity. Rapamycin has been shown tobe effective in vitro against imatinib-resistant CML cells30 anda phase I–II study of everolimus (RAD001), a derivative of
rapamycin, in combination with imatinib in imatinib-resistant
CML patients.
In the present study, we evaluated the potential antileukemic effect and the action mechanism of NVP-BEZ235 against
BCR-ABL-positive cells. NVP-BEZ235 displayed a statisticallysignificant growth inhibition of BCR-ABL mutant cell (Fig. 5)
and the combined use of NVP-BEZ235 and nilotinib induced
apoptosis in BCR-ABL-positive cells, Ph-positive primary cells
including T315I mutation, and ponatinib-resistant cells (Fig. 5C
and D). In this study, we could not demonstrate the synergistic effect when the two agents were combined in colony assay.
Our screen links mutations in these residues to drug resistance.
Because the number of clones isolated from this type of screen,
many of the novel ABL TKI-resistant variants recovered. These
TKI-resistant variants suggest these might be weak cooperating
mutations whose modest effects might not be resolved in shortterm cell growth assays. We also demonstrated that co-treatment
with nilotinib and NVP-BEZ235 enhanced antitumor activity
in vivo and was less toxic in a xenograft model. The challenge forthe development of an effective Ph-positive leukemia therapy is todevelop an alternative treatment strategy that does not rely solely
on kinase domain inhibition. In this study, we showed that thetargeting of PI3K/Akt/mTOR signaling network with pharmacological inhibitors in combination with ABL TKIs is an efficacious treatment of Ph-positive leukemia.
In summary, the experimental data presented in this reportsuggests that NVP-BEZ235, a PI3K/AKT-mTOR inhibitor,
potentiates nilotinib-induced apoptosis of BCR-ABL-positive
leukemia cells through S6 kinase inhibition. Combination regimens involving novel and conventional anti-BCR-ABL agents
may be a key strategy to improve clinical benefits and reduce thedevelopment of imatinib-resistant clones. This molecular model
will provide useful information for development of therapeuticstrategies against Ph-positive cells.

References
1.

2.

3.

4.

5.

6.

Nowell PC, Hungerford DA. A minute chromosome
in human granulocytic leukemia. Science 1960;
142:1497
Sawyers CL. Chronic myeloid leukemia. N Engl J
Med 1999; 340:1330-40; PMID:10219069; http://dx.doi.org/10.1056/NEJM199904293401706
Tauchi T, Okabe S, Miyazawa K, Ohyashiki K. The
tetramerization domain-independent Ras activation
by BCR-ABL oncoprotein in hematopoietic cells. Int
J Oncol 1998; 12:1269-76; PMID:9592185
Skorski T, Kanakaraj P, Nieborowska-Skorska M,
Ratajczak MZ, Wen SC, Zon G, Gewirtz AM, Perussia
B, Calabretta B. Phosphatidylinositol-3 kinase
activity is regulated by BCR/ABL and is required
for the growth of Philadelphia chromosome-positive
cells. Blood 1995; 86:726-36; PMID:7606002
Chai SK, Nichols GL, Rothman P. Constitutive
activation of JAKs and STATs in BCR-Abl-expressing
cell lines and peripheral blood cells derived fromleukemic patients. J Immunol 1997; 159:4720-8;
PMID:9366395
Druker BJ, Sawyers CL, Kantarjian H, Resta DJ,
Reese SF, Ford JM, Capdeville R, Talpaz M. Activityof a specific inhibitor of the BCR-ABL tyrosine kinase
in the blast crisis of chronic myeloid leukemia andacute lymphoblastic leukemia with the Philadelphia
chromosome. N Engl J Med 2001; 344:103842; PMID:11287973; http://dx.doi.org/10.1056/
NEJM200104053441402

Peripheral blood was collected in heparinized tubes and mononuclear cells were separated using LymphoSepare (ImmunoBiological Laboratories). These cells were cultured in RPMI1640
medium containing 10% FBS.
Colony-formation assay
Ba/F3 BCR-ABL random mutagenesis cells were seeded in
6-well culture plates in MethoCult GFH4434 medium (StemCell Technology) containing the indicated concentration of nilotinib and/or NVP-BEZ235. Cells were incubated for more than
14 d in a humidified atmosphere with 5% CO2 at 37 °C, and thecolonies were counted with a reversed microscope. Cell aggregates containing 40 or more cells were considered colonies. Eachexperiment was performed in triplicate.
Statistical analysis
Differences between treatments in terms of responses were
determined using the Student t test. P < 0.05 was considered statistically significant. Data for comparison of multiple groups arepresented as mean ± SD and were analyzed by ANOVA.
Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.
Acknowledgments

We thank Novartis for providing the drugs (NVP-BEZ235,
nilotinib, and imatinib). This work was supported by a HighTech Research Center Project for private universities—a matching fund subsidy from the Ministry of Education, Culture,
Sports, Science and Technology (MEXT)—and by the University-Industry Joint Research Project for private universities—amatching fund subsidy from the MEXT. This work was also
supported by Grants-in-Aid for Scientific Research from the
MEXT.

7.

Druker BJ, Guilhot F, O’Brien SG, Gathmann I,
Kantarjian H, Gattermann N, Deininger MW,
Silver RT, Goldman JM, Stone RM, et al.; IRIS
Investigators. Five-year follow-up of patients receiving
imatinib for chronic myeloid leukemia. N Engl J Med
2006; 355:2408-17; PMID:17151364; http://dx.doi.
org/10.1056/NEJMoa062867
8. Kantarjian HM, Talpaz M, Giles F, O’Brien S,
Cortes J. New insights into the pathophysiology
of chronic myeloid leukemia and imatinib
resistance. Ann Intern Med 2006; 145:91323;
PMID:17179059;
http://dx.doi.
org/10.7326/0003-4819-145-12-200612190-00008
9. Hochhaus A, Kreil S, Corbin AS, La Rosée P,
Müller MC, Lahaye T, Hanfstein B, SchochC, Cross NC, Berger U, et al. Molecular andchromosomal mechanisms of resistance to imatinib(STI571) therapy. Leukemia 2002; 16:2190-6;
PMID:12399961;
http://dx.doi.org/10.1038/
sj.leu.2402741
10. Baccarani M, Cortes J, Pane F, Niederwieser D, Saglio
G, Apperley J, Cervantes F, Deininger M, Gratwohl
A, Guilhot F, et al.; European LeukemiaNet.
Chronic myeloid leukemia: an update of concepts
and management recommendations of European
LeukemiaNet. J Clin Oncol 2009; 27:6041-51;
PMID:19884523;
http://dx.doi.org/10.1200/
JCO.2009.25.0779

214	Cancer Biology & Therapy-242-

11. Garg RJ, Kantarjian H, O’Brien S, Quintás-Cardama
A, Faderl S, Estrov Z, Cortes J. The use of nilotinib
or dasatinib after failure to 2 prior tyrosine kinase
inhibitors: long-term follow-up. Blood 2009;
114:4361-8;
PMID:19729517;
http://dx.doi.
org/10.1182/blood-2009-05-221531
12. Pavlovsky C, Kantarjian H, Cortes JE. Firstline therapy for chronic myeloid leukemia: Past,
present, and future. Am J Hematol 2009; 84:28793; PMID:19306355; http://dx.doi.org/10.1002/
ajh.21380
13. Vasudevan KM, Garraway LA. AKT signaling inphysiology and disease. Curr Top Microbiol Immunol
2010; 347:105-33; PMID:20549472; http://dx.doi.
org/10.1007/82_2010_66
14. Dummler B, Hemmings BA. Physiological rolesof PKB/Akt isoforms in development and disease.
Biochem Soc Trans 2007; 35:231-5; PMID:17371246;
http://dx.doi.org/10.1042/BST0350231
15. Brazil DP, Yang ZZ, Hemmings BA. Advances inprotein kinase B signalling: AKTion on multiple
fronts. Trends Biochem Sci 2004; 29:233-42;
PMID:15130559;
http://dx.doi.org/10.1016/j.
tibs.2004.03.006
16. LoPiccolo J, Blumenthal GM, Bernstein WB, Dennis
PA. Targeting the PI3K/Akt/mTOR pathway:
effective combinations and clinical considerations.
Drug Resist Updat 2008; 11:32-50; PMID:18166498;
http://dx.doi.org/10.1016/j.drup.2007.11.003

Volume 15 Issue 2©2014 Landes Bioscience. Do not distribute.

for 20 min on ice. The lysates were centrifuged at 12 000 rpm
for 20 min at 4 °C. The protein content of the lysates wasdetermined using a protein assay kit (Bio-Rad Laboratories).
Equivalent amounts of proteins of the cell lysates were boiled
with 2× SDS sample buffer for 5 min. The proteins were loaded
onto polyacrylamide gels and then transferred to polyvinylidine
difluoride (PVDF) membranes (Millipore). The membranes
were blocked by 1% bovine serum albumin (BSA) in PBS-Tween
20 (PBST) and probed with the indicated primary antibodies atappropriated dilution for 2 h at room temperature (RT). Blots
were probed with secondary antibodies conjugated with horseradish peroxidase and developed using the enhanced chemiluminescence (ECL, Amersham Phamacia Biotech) system with
ECL-film according to the manufacture’s specifications.
In vivo studies
The mice were injected subcutaneously with 1 × 107 Ba/F3
BCR-ABL mutagenesis cells. The mice were treated orally with30 mg/kg NVP-BEZ235 or 30 mg/kg nilotinib, or a combination of the 2 agents daily. Control mice were treated with 0.9%
NaCl of the vehicle. Tumor sizes were observed at various time
points. The average tumor weight per mouse (n = 5) was calculated and was used to analyze the group mean tumor weight ±standard error (s.e.).
Immunohistochemical analysis
Tumor cells were collected at indicated time points and fixedin paraformaldehyde. Paraffin-embedded tissues were stainedwith hematoxylin and eosin (H&E) and TdT-mediated dUTP
nick-end labeling (TUNEL), and treated with phospho S6 kinase
and Ki-67 antibodies.
Primary BCR-ABL-positive cells
This study was performed according to approved laboratory
protocols and in accordance with the Declaration of Helsinki.

18.

19.

20.

21.

22.

23.

Maira SM, Stauffer F, Brueggen J, Furet P,
Schnell C, Fritsch C, Brachmann S, Chène P, De
Pover A, Schoemaker K, et al. Identification andcharacterization of NVP-BEZ235, a new orally
available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor withpotent in vivo antitumor activity. Mol Cancer Ther
2008; 7:1851-63; PMID:18606717; http://dx.doi.
org/10.1158/1535-7163.MCT-08-0017
Notta F, Mullighan CG, Wang JC, Poeppl A,
Doulatov S, Phillips LA, Ma J, Minden MD,
Downing JR, Dick JE. Evolution of human BCRABL1 lymphoblastic leukaemia-initiating cells.
Nature 2011; 469:362-7; PMID:21248843; http://dx.doi.org/10.1038/nature09733
Khorashad JS, Kelley TW, Szankasi P, Mason CC,
Soverini S, Adrian LT, Eide CA, Zabriskie MS, Lange
T, Estrada JC, et al. BCR-ABL1 compound mutations
in tyrosine kinase inhibitor-resistant CML: frequency
and clonal relationships. Blood 2013; 121:48998; PMID:23223358; http://dx.doi.org/10.1182/
blood-2012-05-431379
Hidalgo M, Rowinsky EK. The rapamycinsensitive signal transduction pathway as a target
for cancer therapy. Oncogene 2000; 19:66806; PMID:11426655; http://dx.doi.org/10.1038/
sj.onc.1204091
Schnell CR, Stauffer F, Allegrini PR, O’Reilly T,
McSheehy PM, Dartois C, Stumm M, Cozens R,
Littlewood-Evans A, García-Echeverría C, et al.
Effects of the dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVPBEZ235 on the tumor vasculature: implications forclinical imaging. Cancer Res 2008; 68:6598-607;
PMID:18701483; http://dx.doi.org/10.1158/00085472.CAN-08-1044
Serra V, Markman B, Scaltriti M, Eichhorn PJ, Valero
V, Guzman M, Botero ML, Llonch E, Atzori F, Di
Cosimo S, et al. NVP-BEZ235, a dual PI3K/mTOR
inhibitor, prevents PI3K signaling and inhibits thegrowth of cancer cells with activating PI3K mutations.
Cancer Res 2008; 68:8022-30; PMID:18829560;
http://dx.doi.org/10.1158/0008-5472.CAN-08-1385
Engelman JA, Chen L, Tan X, Crosby K, Guimaraes
AR, Upadhyay R, Maira M, McNamara K,
Perera SA, Song Y, et al. Effective use of PI3K and
MEK inhibitors to treat mutant Kras G12D andPIK3CA H1047R murine lung cancers. Nat Med
2008; 14:1351-6; PMID:19029981; http://dx.doi.
org/10.1038/nm.1890

24. Liu TJ, Koul D, LaFortune T, Tiao N, Shen RJ, Maira
SM, Garcia-Echevrria C, Yung WK. NVP-BEZ235, anovel dual phosphatidylinositol 3-kinase/mammaliantarget of rapamycin inhibitor, elicits multifaceted
antitumor activities in human gliomas. Mol Cancer
Ther 2009; 8:2204-10; PMID:19671762; http://dx.doi.org/10.1158/1535-7163.MCT-09-0160
25. Crowder RJ, Phommaly C, Tao Y, Hoog J, Luo
J, Perou CM, Parker JS, Miller MA, Huntsman
DG, Lin L, et al. PIK3CA and PIK3CB inhibition
produce synthetic lethality when combined withestrogen deprivation in estrogen receptor-positive
breast cancer. Cancer Res 2009; 69:3955-62;
PMID:19366795; http://dx.doi.org/10.1158/00085472.CAN-08-4450
26. McMillin DW, Ooi M, Delmore J, Negri J,
Hayden P, Mitsiades N, Jakubikova J, Maira
SM, Garcia-Echeverria C, Schlossman R, et al.
Antimyeloma activity of the orally bioavailable dual
phosphatidylinositol 3-kinase/mammalian targetof rapamycin inhibitor NVP-BEZ235. Cancer Res
2009; 69:5835-42; PMID:19584292; http://dx.doi.
org/10.1158/0008-5472.CAN-08-4285
27. Kojima K, Shimanuki M, Shikami M, Samudio
IJ, Ruvolo V, Corn P, Hanaoka N, Konopleva M,
Andreeff M, Nakakuma H. The dual PI3 kinase/mTOR inhibitor PI-103 prevents p53 induction
by Mdm2 inhibition but enhances p53-mediatedmitochondrial apoptosis in p53 wild-type AML.
Leukemia 2008; 22:1728-36; PMID:18548093;
http://dx.doi.org/10.1038/leu.2008.158
28. Faber AC, Li D, Song Y, Liang MC, Yeap BY,
Bronson RT, Lifshits E, Chen Z, Maira SM,
García-Echeverría C, et al. Differential inductionof apoptosis in HER2 and EGFR addicted cancers
following PI3K inhibition. Proc Natl Acad Sci U S A
2009; 106:19503-8; PMID:19850869; http://dx.doi.
org/10.1073/pnas.0905056106
29. Klejman A, Rushen L, Morrione A, Slupianek A,
Skorski T. Phosphatidylinositol-3 kinase inhibitors
enhance the anti-leukemia effect of STI571.
Oncogene 2002; 21:5868-76; PMID:12185586;
http://dx.doi.org/10.1038/sj.onc.1205724

www.landesbioscience.com	Cancer Biology & Therapy-243-

30. Sillaber C, Mayerhofer M, Böhm A, Vales A, Gruze
A, Aichberger KJ, Esterbauer H, Pfeilstöcker M, Sperr
WR, Pickl WF, et al. Evaluation of antileukaemic
effects of rapamycin in patients with imatinibresistant chronic myeloid leukaemia. Eur J Clin
Invest 2008; 38:43-52; PMID:18173550; http://dx.doi.org/10.1111/j.1365-2362.2007.01892.x
31. Kimura S, Naito H, Segawa H, Kuroda J, Yuasa T,
Sato K, Yokota A, Kamitsuji Y, Kawata E, Ashihara
E, et al. NS-187, a potent and selective dual Bcr-Abl/
Lyn tyrosine kinase inhibitor, is a novel agent for
imatinib-resistant leukemia. Blood 2005; 106:394854; PMID:16105974; http://dx.doi.org/10.1182/
blood-2005-06-2209
32. Ray A, Cowan-Jacob SW, Manley PW, Mestan
J, Griffin JD. Identification of BCR-ABL point
mutations conferring resistance to the Abl kinase
inhibitor AMN107 (nilotinib) by a randommutagenesis study. Blood 2007; 109:5011-5;
PMID:17303698;
http://dx.doi.org/10.1182/
blood-2006-01-015347
33. Okabe S, Tauchi T, Nakajima A, Sashida G, Gotoh
A, Broxmeyer HE, Ohyashiki JH, Ohyashiki
K. Depsipeptide (FK228) preferentially induces
apoptosis in BCR/ABL-expressing cell lines and cells
from patients with chronic myelogenous leukemia
in blast crisis. Stem Cells Dev 2007; 16:503-14;
PMID:17610380;
http://dx.doi.org/10.1089/
scd.2007.9994
34. Okabe S, Tauchi T, Tanaka Y, Katagiri S, Ohyashiki
K. Effects of the hedgehog inhibitor GDC-0449,
alone or in combination with dasatinib, on BCRABL-positive leukemia cells. Stem Cells Dev 2012;
21:2939-48;
PMID:22642671;
http://dx.doi.
org/10.1089/scd.2012.0016

215©2014 Landes Bioscience. Do not distribute.

17.

Drug Design, Development and Therapy

Dovepress
open access to scientific and medical research

Original Research

Drug Design, Development and Therapy downloaded from https://www.dovepress.com/ by 210.137.139.190 on 29-Apr-2018
For personal use only.

Open Access Full Text Article

Downregulated microRNA-148b in circulating
PBMCs in chronic myeloid leukemia patients with
undetectable minimal residual disease: a possible
biomarker to discontinue imatinib safely
This article was published in the following Dove Press journal:
Drug Design, Development and Therapy
25 August 2014
Number of times this article has been viewed

Junko H Ohyashiki 1
Kazushige Ohtsuki 1
Izuru Mizoguchi 2
Takayuki Yoshimoto 2
Seiichiro Katagiri 3
Tomohiro Umezu 1,4
Kazuma Ohyashiki 3,4
Department of Molecular Oncology,
Institute of Medical Science,2
Department of Immunoregulation,
Institute of Medical Science,3
Department of Hematology,
4
Department of Molecular Science,
Tokyo Medical University, Tokyo, Japan
1

Background: A subset of patients with chronic myeloid leukemia (CML) can sustain a complete
molecular response after discontinuing imatinib mesylate (IM). We focused on microRNAs(miRNAs), with the aim of finding a molecular biomarker to discriminate which patients cansafely and successfully discontinue IM use.
Methods: To identify miRNAs that showed altered expression in patients who had discontinued
IM (STOP-IM group), we first screened miRNA expression of peripheral blood mononuclear
cells by using a TaqMan miRNA array on samples from five unselected patients from the STOPIM group, seven CML patients receiving IM (IM group), and five healthy volunteers. We thenperformed miRNA quantification in 49 CML patients with deep molecular response. Mann–
Whitney U and chi-square tests were used to determine statistical significance for comparisons
between the control (healthy volunteers) and test groups (STOP-IM and IM groups). Multiple
groups were compared by one-way analysis of variance.
Results: Downregulation of miR-148b was noted in patients in the STOP-IM group andin a ­subset of the IM group. We then subdivided the IM patients into two groups: one withdownregulated miR-148b expression (IM-1; less than the cut-off value) and the other without
downregulated miR-148b expression (IM-2; greater than the cut-off value). The number ofpatients who had a sustained stable molecular response was significantly lower in IM-2 group.
This group also had a significantly lower percentage of natural killer cells.
Conclusion: Downregulated miR-148 may contribute to immune surveillance in STOP-IM
patients and may therefore have potential as additive information in managing CML patients
undergoing treatment with IM.
Keywords: chronic myeloid leukemia, imatinib mesylate, discontinuation, miR-148b

Background

Correspondence: Junko H Ohyashiki
Department of Molecular Oncology,
Institute of Medical Science, Tokyo
Medical University, 671 Nishishinjuku,
Shinjuku, Tokyo 160 0023, Japan
Tel +81 3 3342 6111 Ext 5171
Fax +81 3 3348 0713
Email junko@tokyo-med.ac.jp1151

submit your manuscript | www.dovepress.com

Drug Design, Development and Therapy 2014:8 1151–1159

Dovepress© 2014 Ohyashiki et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0)
License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further
permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on
how to request permission may be found at: http://www.dovepress.com/permissions.php

http://dx.doi.org/10.2147/DDDT.S66812
Powered by TCPDF (www.tcpdf.org)

The treatment of chronic myeloid leukemia (CML) has been greatly impacted by thedevelopment of imatinib mesylate (IM), the first specific inhibitor of BCR-ABL1.1
In newly diagnosed patients with chronic-phase CML, IM induces a complete hematological response in more than 90% of cases and a complete cytogenetic response
in more than 80% of cases,1 and IM has been established as the standard therapy
for CML. Recent reports have demonstrated that a subset of CML patients with acomplete molecular response (currently referred to as undetectable minimal residual
disease, UMRD) can sustain this state after IM discontinuation (STOP-IM).2–4 We and­others have postulated that immunological surveillance may play an important role inDovepress

Drug Design, Development and Therapy downloaded from https://www.dovepress.com/ by 210.137.139.190 on 29-Apr-2018
For personal use only.

Ohyashiki et al

these patients.5–8 More recently, Ng et al demonstrated that
East Asian CML patients with a common intronic deletion
polymorphism in the gene coding BIM (at intron 2) had aninferior response to tyrosine kinase inhibitors compared tothose without the genetic variation.9,10 Taken together, immunological diversity as well as genetic background should
be considered in discussions of the efficacy and safety of
IM cessation in CML patients. Currently there is no definitive classifier to discriminate CML patients with UMRD who
could safely stop IM and those who should not.
MicroRNAs (miRNAs), which are noncoding singlestranded RNAs of 21–25 nucleotides, have recently been
implicated in the regulation of cellular processes such as
development, differentiation, apoptosis, proliferation, andhematopoiesis. MiRNAs have also been implicated in thedevelopment of human cancers, either as tumor suppressors
or as oncogenes.11,12 Approximately 100 miRNAs are known
to be expressed in cells of the immune system and to play key
roles in the development of innate and acquired immunity,13,14
therefore, aberrant expression of miRNAs that target theimmune system may be related to the immunological background of cancer patients. In CML cells, abnormal expressionof several miRNAs (miR-15a, miR-16, miR-31, miR-142,
miR-155, miR-181, miR-221, miR-564, let7a, and the­polycistronic miR-17–92 cluster) has been described.15–18
Although the miRNA expression profile in CML cells
has been extensively studied, little is known about miRNA
expression in CML patients with UMRD whose CML cells
cannot be detected. We therefore determined the miRNAprofiles of peripheral blood mononuclear cells (PBMCs) in
CML patients with UMRD who were receiving IM or had
discontinued IM, with the goal of clarifying whether altered
miRNA expression is linked to pathophysiology in CML
patients who safely discontinued IM. Here, we provide a
distinct miRNA expression profile of circulating PBMCs in
CML patients with successful IM cessation. We additionallydescribe a subset of CML patients receiving IM that showedsimilar miRNA expression profiles.

Materials and methods
Patients
Forty-nine consecutive patients with CML who had UMRD
were enrolled in this study. In 16 patients who had discontinued IM and had sustained UMRD for more than 6 months(STOP-IM group), samples were collected when IM wasstopped. In contrast, 33 patients who were receiving IM andhad UMRD at the time of sample collection were ­categorized
as the IM group. Fifteen healthy volunteers served1152

as ­controls. CML patients before treatment and those who
did not respond to IM were not included in this study. There
was no significant difference in sex or age between the CML
groups and the healthy volunteers. UMRD was defined as theabsence of a BCR-ABL transcript as determined by nested
reverse transcription-polymerase chain reaction ­(RT-PCR)
or a highly sensitive transcription-mediated amplification(TMA) method. These PCR methods can detect at least a4-log reduction in the BCR-ABL transcript (international
scale 0.01%). The study was approved by the institutional
review board of Tokyo Medical University (no 930, approved
24 June 2008). Written informed consent was obtained fromall participants prior to specimen collection, in accordancewith the Declaration of Helsinki.

RNA isolation from peripheral blood
PBMCs were obtained from 10 mL of whole blood by the Ficoll
density gradient method (GE Healthcare ­Bio-Sciences AB,
Uppsala, Sweden). Cell pellets were quickly ­frozen at −80°C
until use. Total RNA was isolated from cell pellets with a
mirVana PARIS kit (Ambion, ­Austin, TX, USA), as reported
previously.19 Briefly, 600 µL of cell lysate was mixed withan equal volume of denaturing solution, and the subsequent
phenol extraction and cartridge filtration were carried out
according to the manufacturer’s instructions.

TaqMan low-density array screening
The miRNA expression profiles were determined with a
TaqMan Array Human microRNA Card A (Thermo Fisher
Scientific, Waltham, MA, USA). Ninety nanograms of total
RNA was used as an input in each RT reaction. The RT
reaction and pre-amplification step was set up according to
the manufacturer’s recommendations. miRNAs were reverse
transcribed with the Megaplex Primer Pools (Human Pools
A version 2.1; Thermo Fisher Scientific). RT reaction products from the PBMC samples were further amplified with
Megaplex PreAmp primers (Primers A version 2.1; Thermo
Fisher Scientific). ­Quantitative RT-PCR was performed onan Applied ­Biosystems 7900HT thermocycler according to
the manufacturer’s recommended program. With the use of
SDS 2.4 software and DataAssist (Thermo Fisher Scientific),the expression of miRNA was calculated based on cycle
threshold (Ct) values normalized by those of RNU6B. Data
analysis was done using GeneSifter® software (Perkin Elmer,
Waltham, MA, USA). P-values of less than 0.05 were considered to indicate a statistically significant difference, and theBenjamini–Hochberg algorithm was used for estimation offalse discovery rates, as we have reported previously.19

Drug Design, Development and Therapy 2014:8

submit your manuscript | www.dovepress.com

Dovepress-245Powered by TCPDF (www.tcpdf.org)

Dovepress

A possible biomarker to discontinue imatinib safely

IM

To identify candidate miRNAs that showed altered expression in patients in the STOP-IM group, we first screened
miRNA expression using a TaqMan miRNA array (Thermo
Fisher Scientific) on five unselected patients from the
STOP-IM group, seven from the IM group, and five healthy
volunteers. Among these three groups, we observed differential expression of 22 miRNAs as identified by using­GeneSifter software (Figure 1; Gene Expression Omnibus[GEO] accession no GSE47652). Although 22 miRNAs were
STOP-IMWe determined the amounts of the individual miRNAs by realtime quantitative RT-PCR with a TaqMan MicroRNA Assay(Thermo Fisher Scientific) and the following ­miRNA-specificstem-loop primers: hsa-miR-148b (000471), hsa-miR-326(000542), hsa-let7b (000378), and RNU6B (001093).
­Subsequently, quantitative real-time PCR was performed
with an ABI Prism 7000 sequence detection system (Thermo
Fisher Scientific). The reaction was initiated by incubation
at 95°C for 2 minutes, followed by 50 cycles of 95°C for
15 seconds and then 60°C for 1 minute. All reactions were runin duplicate. Mean (Ct) values for all miRNAs were quantified with sequence detection system software (SDS version
1.02; Thermo Fisher Scientific). All miRNA expression was
normalized to RNU6B expression, yielding a −∆Ct value, asreported elsewhere.20 The −∆∆Ct value was then calculated
by subtracting the −∆Ct value of the normal sample from therespective −∆Ct values of patient samples. Expression of all
miRNAs was normalized by using the 2−∆∆Ct method.

Results
The miRNA expression profilingby Taqman miRNA array

ControlDrug Design, Development and Therapy downloaded from https://www.dovepress.com/ by 210.137.139.190 on 29-Apr-2018
For personal use only.

Real-time quantitative RT-PCR

hsa-miR-96-4373372
hsa-miR-338-3p-4395363
hsa-miR-362-3p-4395228
hsa-miR-326-4373050
hsa-miR-323-3p-4395338

Flow cytometry
Freshly obtained PBMCs were separated using a Ficoll density gradient (GE Healthcare Bio-Sciences) and resuspendedin Roswell Park Memorial Institute (RPMI) 1640 medium
supplemented with 10% fetal calf serum. ­Immunophenotyping
was performed on a FACSCanto II (BD Biosciences, San Jose,
CA, USA) flow cytometer using ­CellQuest software (BD
Biosciences) with a 5-color flow cytometry panel including
antibodies against the following cell ­surface antigens andeffector molecules: CCR7, CD3, CD8, CD45RO, and CD56­(eBioscience, San Diego, CA, USA, and ­BioLegend, San
Diego, CA, USA). Cells were analyzed with the FACSCanto II(BD Biosciences) flow cytometer using CellQuest software.8

hsa-miR-210-4373089
hsa-miR-329-4373191
hsa-miR-454-4395434
hsa-let-7b-4395446
hsa-miR-148b-4373129
hsa-miR-449a-4373207
hsa-miR-487b-4378102
hsa-let-7c-4373167
hsa-miR-127-3p-4373147
hsa-miR-625-4395542
hsa-miR-107-4373154
hsa-miR-551b-4380945
hsa-miR-495-4381078

Statistical analysis and pathway analysis

hsa-miR-539-4378103

Data are expressed as means ± standard deviation (SD).
Mann–Whitney U and chi-square tests were used to determine statistical significance for comparisons between thecontrol and test groups. Multiple groups were comparedby one-way analysis of variance. GraphPad Prism software(version 5c for Macintosh; GraphPad Software Inc., La Jolla,
CA, USA) was used for statistical analyses. Results wereconsidered statistically significant when P,0.05.
Following identification of differentially expressing­miRNAs, the predicted targets for these altered miRNAs were
subjected to microRNA Target Filter in Ingenuity Pathways
Analysis (IPA) software (Ingenuity System, Redwood City,
CA, USA).

Drug Design, Development and Therapy 2014:8

hsa-miR-23a-4373074
hsa-miR-503-4373228
hsa-miR-18a-4395533
Legend:0.5

1Figure 1 miRNA profiling by TaqMan (Thermo Fisher Scientific, Waltham, MA,
USA) miRNA array.
Notes: A distinct expression pattern was found in the STOP-IM group (middle lane).
Using SDS (version 2.4) and DataAssist software (Thermo Fisher), we calculated theexpression of miRNAs based on their Ct values normalized by the Ct value of RNU6B.
Data were analyzed with GeneSifter software (Perkin Elmer, Waltham, MA, USA).
P-values of ,0.05 were considered to indicate a statistically significant difference, andthe Benjamini–Hochberg algorithm was used for estimation of false discovery rates.
Abbreviations: miRNA, microRNA; Ct, cycle threshold; IM, imatinib mesylate;
STOP-IM, patients who had discon­tinued IM and had sustained UMRD for morethan 6 months; UMRD, undetectable minimal residual disease.

submit your manuscript | www.dovepress.com

Dovepress-246Powered by TCPDF (www.tcpdf.org)

2

1153

Dovepress

selected based on fold change, the expression profile was
highly variable among individuals. We therefore selected
three of these miRNAs (let-7b, ­miR-148b, and miR-326)
for further validation on the basis of their averaged foldchange in expression (log2 variation .0.6), significant
P-value, quality control value, and their predicted target
genes (Table S1).

miR-362 being upregulated in the STOP-IM group basedon miRNA array screening, the validation analysis using alarge number of samples revealed that miR-326 tended to be
downregulated in the STOP-IM group in comparison to the
IM group and the control group. We observed no statisticallysignificant difference in the expression of miR-326 between
each group (Figure 2C).

Quantification of individual miRNAby real-time quantitative RT-PCR

Clinical and biological relevanceof miR-148b expression

We then validated the miRNA array results by real-timequantitative RT-PCR using samples from 16 patients of
the STOP-IM group, 33 of the IM group, and 15 healthy
volunteers. Validation of miR-148b, miR-362, and let-7b
revealed that expression of miR-148b was significantly
lower in the STOP-IM group than in the healthy volunteers(Figure 2A). The miR-148b expression tended to be lower
in the STOP-IM group than in the IM group, although thedifference was not statistically significant. We observed nostatistically significant difference in the expression of let-7b
between the STOP-IM and IM groups (Figure 2B). Despite

Based on the statistical significant results from quantifying
individual miRNAs, we particularly focused on miR-148b.
To estimate a cut-off value for miR-148b expression inmononuclear cells for distinguishing between the STOP-IM
and healthy control groups, we performed receiver operating characteristic (ROC) curve analysis (Figure 2D). The
area under the ROC curve was 0.8792 with 75% sensitivity (95% confidence interval [CI], 47.62%–90%) and 80%
specificity (95% CI, 51.91%–95%). The cut-off value wasthus ­determined to be 0.5880, which was used in subsequent
analyses of miR-148b expression. We next subdivided the IM

C

P<0.0001*
P=0.055

P=0.064
3
2
1
0

STOP-IMIM

miR-326a expression

miR-148b expressionA

P=0.088B

4
2
0Control

STOP-IM

100% sensitivity

2.0
1.5
1.0
0.5

80
60
40
20
0

0.0
STOP-IM

IM

Control

D 100

P=0.1751

P=0.7253

IM

CML-UMRD

P=0.21552.5

P=0.6883

P=0.057

6

CML-UMRD

let-7b expression

Drug Design, Development and Therapy downloaded from https://www.dovepress.com/ by 210.137.139.190 on 29-Apr-2018
For personal use only.

Ohyashiki et al

0

20

40

60

80 100100% – specificity%

Control

CML-UMRD
Figure 2 Expression of miR-148b (A), let-7b (B), and miR-326b (C) in the STOP-IM, IM, and control groups. (D) Cut-off level for miR-148b expression between the STOPIM and control groups.
Notes: The asterisk indicates a statistically significant difference. The area under the ROC curve was 0.8792 with 75% sensitivity (95% CI, 47.62%–90%) and 80% specificity(95% CI, 51.91%–95%). The cut-off value was thus determined to be 0.5880, which was used in the following analyses of miR-148b expression.
Abbreviations: IM, imatinib mesylate; ROC, receiver operating characteristic; CI, confidence interval; CML, chronic myeloid leukemia; STOP-IM, patients who had
discontinued IM and had sustained UMRD for more than 6 months; UMRD, undetectable minimal residual disease.

1154

Drug Design, Development and Therapy 2014:8

submit your manuscript | www.dovepress.com

Dovepress-247Powered by TCPDF (www.tcpdf.org)

Drug Design, Development and Therapy downloaded from https://www.dovepress.com/ by 210.137.139.190 on 29-Apr-2018
For personal use only.

Dovepress

A possible biomarker to discontinue imatinib safely

patients into two groups: one with downregulated ­miR-148b
expression (IM-1; less than the cut-off value) and the otherwithout downregulated miR-148b expression (IM-2; greaterthan the cut-off value) (Table 1).
Comparison of the STOP-IM, IM-1, and IM-2 groups
revealed no significant differences in sex, age, Sokal score,
duration of IM therapy, months to achieve UMRD fromdiagnosis, or prior interferon (IFN)-α therapy (Table 1). The
total IM dose was significantly higher (P=0.0289) in the IM-2
group (893±294 g) than in the STOP-IM group (636±369 g),
while there was no statistically significant difference between
the IM-1 group and STOP-IM group. Similarly, the numberof patients who sustained stable molecular response wassignificantly lower (P=0.0232) in the IM-2 group than in the
STOP-IM group. Notably, the percentage of natural killer(NK) cells was significantly lower (P=0.03) in the IM-2 groupthan in the STOP-IM group. We could not find any statisticalsignificance of BIM deletion among the 3 groups.

Network analysis of miRNA-148b
ROC analysis of the miR-148b suggests that the miRNAsignature in PBMCs may predict whether IM discontinuation
Table 1 Clinico-hematological, immunological, and molecularcharacteristics of patients with and without downregulation of
miR-148b expression
Downregulationof miR-148b
expression

STOP-IM

IM-1

IM-2

Yes

Yes

No

Number of patients

16

18

15

Age (years)
Sex (male/female)
Sokal score
Total imatinib dose(grams)Duration of imatinib
therapy (months)
Months to achieve
UMRD from diagnosis
Stable MR* (yes/no)

57.5±11.6
7/9
0.74±0.22637±36054.5±15.4
11/4
0.73±0.24954±2450.404
0.201§
0.849
0.057967.4±34.9

60.4±9.1
12/6
0.70±0.09
848±3240.0185¶75.8±29.3

88.9±76.0

0.226459.3±68.0

48.2±39.8

67.5±44.9

0.547314/2

7/8

0.0454§

Prior IFN-α (yes/no)
NK cells (%)

4/1213.2±6.9

5/10
8.1±0.96

0.3888§0.141

CCR7−CR45RO−
CD8+ cells (%)
BIM deletion (yes/no)8.4±3.9

13/5
0.0232¶3/15
12.2±9.1
0.03¶
10.5±5.4

7.6±6.1

0.1733

1/10

2/16

1/13

0.9288§P-value

Notes: *If BCR-ABL1 remained undetectable for a continuous period of 24 monthsof imatinib treatment, the response was termed stable molecular response (MR).
Patients who received IM and did not have a sustained UMRD but maintained amajor molecular response for more than 24 months (fluctuating UMRD) were
not referred as “stable MR”. P values are calculated by one-way ANOVA unlessotherwise indicated; ¶Student’s t-test; §chi-square test.
Abbreviations: IM, imatinib mesylate; UMRD, undetectable minimal residual disease;
ANOVA, analysis of variance; IFN, interferon; NK, natural killer; STOP-IM, patients
who had discon­tinued IM and had sustained UMRD for more than 6 months.

Drug Design, Development and Therapy 2014:8

is appropriate. Therefore, using in silico network analysis we
assessed whether predicted targets of miR-148b signature could
provide insights into analogous changes enriched in the PBMC
signaling pathways in CML-UMRD patients. Network analysis
on predicted targets of miRNA-148 was carried out using IPA(Ingenuity System), which provides an independent methodof assessing the role of predicted ­miR-148b targets. The top
network has ten nodal molecules that are predicted targets of
miR-148b like DNMT3B, NR1I2, and TLR3 (Figure 3).

Discussion
One of the major questions in research on STOP-IM centers
is the genetic and epigenetic markers for discriminating
patients who can safely stop IM. Several empirical clinical studies have shown that durable stable UMRD before
discontinuation, prior IFN therapy, and early deep molecularresponse are important to avoid molecular relapse.2,7,21,22 In
this context, we particularly focused on miRNA expression
profiling of PBMCs in CML patients who could safely stop
IM. Since all patients in this study exhibited deep molecularresponse, we did not do miRNA profiling of CML cells; therefore, we could compare CML patients with or without IM andhealthy volunteers. We found a distinct miRNA profile thatwas specific for the STOP-IM group, and we concluded thatthe miR-148b expression level might be a possible diagnostic
marker for safely stopping IM.
miR-148a, miR-148b, and miR-152 are the three members of the miR148/152 family.23 Aberrant expression of themiR148/152 family has been reported in tumor as well as
non-tumor disease. The present study was performed using
non-tumor PBMCs; target genes regulated by miR-148b
might be different from those in tumor tissue. For this reason,
we tried to identify possible targets through use of the IPA
software (Ingenuity System). There were several candidate
targets for miR-148b as shown in Figure 3, such as cholecystokinin B receptor (CCBR), DNA methyltransferase 1(DNMT1), DNMT3, nuclear receptor subfamily 1, group 1,
member 2 (NR112), peroxisome proliferator-activated receptor alpha (PPARA), ribosomal protein S6 kinase, and polypeptide 5 (RPS6KA). Notably, DNMT1 and DNMT3 wereamong the candidates. The CpG island in the first intron of
Foxp3 DNA is demethylated in naturally occurring humanand murine T-regulatory (Treg) cells, and the methylation
status is linked with Foxp3 expression.24 A recent report byWang et al demonstrated Foxp3+ Treg cells require Dnmt1
expression to prevent development of lethal autoimmunity.25
Therefore, we could not rule out the possibility that downregulation of miR-148b induces DNMT1 expression andsubmit your manuscript | www.dovepress.com

Dovepress-248Powered by TCPDF (www.tcpdf.org)

1155

Dovepress

Ohyashiki et al

TMEM8B
miR-148

Drug Design, Development and Therapy downloaded from https://www.dovepress.com/ by 210.137.139.190 on 29-Apr-2018
For personal use only.

RPS6KA5

EnzymeG-protein coupled receptor

InsulinGroup/complex/other
KinaseLigand-dependent nuclear receptor
DNMT3B

miR-148b-3p

Transmembrane receptor

PPARA

Unknown
MicroRNA
Mature MicroRNA
Relationship
Relationship

NR1I2
DNMT1
CCKBR

TLR3

Figure 3 Network analysis of miR-148b by Ingenuity Pathways Analysis.
Notes: The miR-148b-3p is located in the center. Solid lines indicate a direct relation, and broken lines indicates an indirect relation.

promotes Treg suppression regulated by Foxp3 expression. In the present study, we were not able to analyze Treg
­function; however, Rea et al recently reported that NK cells
rather than Treg cells are associated with clinical outcome
after IM discontinuation.26 Since imatinib itself is known to
inhibit CD4+CD25+ Treg activity,27 regulation of Treg after
IM discontinuation might be partly compensated by downregulation of miR-148b in a subset of STOP-IM patients.
In addition to DNMT1, we noted that miR-148b is indirectly linked to toll-like receptor 3 (TLR3) in the network
presented by IPA (Ingenuity System). Liu et al recently
reported that miRNA-148 and miRNA-152 fine-tune theregulation of the innate immune response and the antigenpresenting capacity of dendritic cells, and this fine-tuning
may contribute to immune homeostasis and immune regulation.28 The expression of miR-148a, 148b, and miR152
was upregulated in mouse dendritic cells upon maturation,
and activation was induced by Tlr3, Tlr4, and Tlr9 agonists.
In the clinical context, we subdivided CML patients basedon miR-148b expression and found that CML patients in
the STOP-IM group and the IM-2 group showed different
immunological features. Additionally, the percentage of NK
cells was significantly lower in the IM-2 group. Although
we did not intend to focus on immunological surveillancein STOP-IM patients at the beginning of this miRNA study,
the present data may support previous reports regarding a
possible role of NK cells in STOP-IM patients.8,10,26
In line with this, CML patients in the IM-1 group could stopIM, and miR-148 expression in PBMCs might be an additive
biomarker for IM cessation. The correlation between miR-148b
1156

levels and treatment-free remission is not clear at this moment(Figure S1A); therefore, follow-up study is needed to clarify theclinical significance of miR-148b. We only had two patients who
relapsed shortly after IM discontinuation (Figure S1B), and all
STOP-IM patients enrolled in the current study had sustained
UMRD for more than 12 months. Consequently, we could not
determine whether miR-148b levels are correlated with relapse
after stopping IM. Since the number of patients in the present
study was rather small, an analysis of a larger number of patientsin an independent cohort may clarify this point.
Furthermore, it is a matter for discussion whether PBMCs
are suitable for miRNA analysis in clinical specimens. ­Analysisof the lymphocyte fractions (eg, NK cells and T cells) would be
useful, but using fractionated samples from clinical specimens
is difficult in practice. For this reason, we used circulating
lymphocytes rather than purified NK or T cells; however, we
could not completely rule out the possibility that the difference in miRNA expression was due to differences in cellularcomposition. Another limitation in the current study is thatthe miRNA profile was not from cancer cells; therefore, we
could not perform a functional analysis of the target genes.
Unfortunately, we could not perform biological experiments
to demonstrate a possible association between downregulation of miR-148b and the immune surveillance in CML. We
alternatively applied an IPA (Ingenuity ­System) bioinformatics tool to estimate the network between ­miR-148b and its
target genes (Figure 3). Although our bioinformatics analysis
is mainly based on previous knowledge base, it may help tounderstand the ­biological relevance of miR-148b in the current study. Finally, we could not find any relationship between
Drug Design, Development and Therapy 2014:8

submit your manuscript | www.dovepress.com

Dovepress-249Powered by TCPDF (www.tcpdf.org)

Dovepress

A possible biomarker to discontinue imatinib safely

BIM deletion and the level of miR-148b expression. This pointshould be clarified in a larger cohort in the near future.

Drug Design, Development and Therapy downloaded from https://www.dovepress.com/ by 210.137.139.190 on 29-Apr-2018
For personal use only.

Conclusion
Our observation of downregulated expression of miR-148b
supports the idea that miRNA may contribute to immune surveillance in STOP-IM patients and may therefore have potential
as an additive information marker in managing CML patients
being treated with IM. Whether miR-148b may be used as abiomarker among other factors (eg, NK cells) to predict disease
relapse after IM discontinuation needs to be investigated.

Authors’ contributions
Junko H Ohyashiki designed and performed the research;
Kazushige Ohtsuki performed PCR analysis; Seiichiro
Katagiri and Kazuma Ohyashiki contributed essential reagentsor tools; Izuru Mizoguchi, Takayuki Yoshimoto performed theFACS analysis; Tomohiro Umezu performed the bioinformatic
analysis; Junko H Ohyashiki wrote the paper; and Kazuma
Ohyashiki performed critical reading. All authors contributed
toward data analysis, drafting, and revising the manuscript.

Acknowledgment
This work was supported by the Private University ­Strategic
Research Based Support Project: Epigenetics Research­Project Aimed at General Cancer Cure Using Epigenetic
Targets from MEXT (Ministry of Education, Culture, Sports,
Science and Technology), Tokyo, Japan.

Disclosure
Kazuma Ohyashiki received research support from Novartis
and BMS. Junko H Ohyashiki received research support from
Novartis. This work was supported by the Private University
Strategic Research Based Support Project: Epigenetics
Research Project Aimed at General Cancer Cure Using­Epigenetic Targets from MEXT (Ministry of Education,
Culture, Sports, Science and Technology), Tokyo, Japan. The
authors report no other conflicts of interest in this work.

References

1. Druker BJ, Guilhot F, O’Brien SG, et al. Five-year follow-up of patients
receiving imatinib for chronic myeloid leukemia. N Engl J Med.
2006;355(23):2408–2417.
2. Branford S, Yeung DT, Ross DM, et al. Early molecular response
and female sex strongly predict stable undetectable BCR-ABL1, thecriteria for imatinib discontinuation in patients with CML. Blood.
2013;121(19):3818–3824.
3. Mahon FX, Rea D, Guilhot J, et al. Discontinuation of imatinibin patients with chronic myeloid leukaemia who have maintainedcomplete molecular remission for at least 2 years: the prospective,
multicentre Stop Imatinib (STIM) trial. Lancet Oncol. 2010;11(11):1029–1035.
Drug Design, Development and Therapy 2014:8

4. Ross DM, Branford S, Seymour JF, et al. Safety and efficacy of­imatinib cessation for CML patients with stable undetectable minimal
residual disease: results from the TWISTER Study. Blood. 2013;122(4):515–522.
5. Melo JV, Ross DM. Minimal residual disease and discontinuation oftherapy in chronic myeloid leukemia: can we aim at a cure? Hematology
Am Soc Hematol Educ Program Book. 2011:136–142.
6. Ohyashiki K, Katagiri S, Tauchi T, et al. Increased natural killer cells
and decreased CD3(+)CD8(+)CD62L(+) T cells in CML patients
who sustained complete molecular remission after discontinuation of
imatinib. Br J Haematol. 2012;157(2):254–256.
7. Takahashi N, Kyo T, Maeda Y, et al. Discontinuation of imatinib inJapanese patients with chronic myeloid leukemia. Haematologica.
2012;97(6):903–906.
8. Mizoguchi I, Yoshimoto T, Katagiri S, et al. Sustained upregulationof effector natural killer cells in chronic myeloid leukemia after­discontinuation of imatinib. Cancer Sci. 2013;104(9):1146–1153.
9. Ng KP, Hillmer AM, Chuah CT, et al. A common BIM deletion
polymorphism mediates intrinsic resistance and inferior responses
to tyrosine kinase inhibitors in cancer. Nat Med. 2012;18(4):521–528.
10. Katagiri S, Umezu T, Ohyashiki JH, Ohyashiki K. The BCL2L11(BIM) deletion polymorphism is a possible criterion for ­discontinuationof imatinib in chronic myeloid leukaemia patients. Br J Haematol.
2013;160(2):269–271.
11. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, andfunction. Cell. 2004;116(2):281–297.
12. He L, Thomson JM, Hemann MT, et al. A microRNA polycistron as apotential human oncogene. Nature. 2005;435(7043):828–833.
13. Baltimore D, Boldin MP, O’Connell RM, Rao DS, Taganov KD.
MicroRNAs: new regulators of immune cell development and function.
Nature Immunol. 2008;9(8):839–845.
14. O’Connell RM, Rao DS, Chaudhuri AA, Baltimore D. Physiologicaland pathological roles for microRNAs in the immune system. Nat Rev
Immunol. 2010;10(2):111–122.
15. Yu Y, Yang L, Zhao M, et al. Targeting microRNA-30a-mediatedautophagy enhances imatinib activity against human chronic myeloid
leukemia cells. Leukemia. 2012;26(8):1752–1760.
16. Venturini L, Battmer K, Castoldi M, et al. Expression of the miR-17–92
polycistron in chronic myeloid leukemia (CML) CD34+ cells. Blood.
2007;109(10):4399–4405.
17. San Jose-Eneriz E, Roman-Gomez J, Jimenez-Velasco A, et al.
MicroRNA expression profiling in Imatinib-resistant Chronic Myeloid
Leukemia patients without clinically significant ABL1-mutations. Mol
Cancer. 2009;8:69.
18. Rokah OH, Granot G, Ovcharenko A, et al. Downregulation of miR-31,
miR-155, and miR-564 in chronic myeloid leukemia cells. PLoS One.
2012;7(4):e35501.
19. Umezu T, Ohyashiki K, Ohyashiki JH. Detection method for quantifying
global DNA methylation by fluorescence correlation spectroscopy. Anal
Biochem. 2011;415(2):145–150.
20. Ohyashiki K, Umezu T, Yoshizawa S, et al. Clinical impact of downregulated plasma miR-92a levels in non-Hodgkin’s lymphoma. PLoS
One. 2011;6(2):e16408.
21. Etienne G, Dulucq S, Nicolini FE, et al. Achieving deeper molecularresponse is associated with a better clinical outcome in chronic myeloid
leukemia patients on imatinib front-line therapy. Haematologica.
2014;99(3):458–464.
22. Rousselot P, Charbonnier A, Cony-Makhoul P, et al. Loss of major
molecular response as a trigger for restarting tyrosine kinase inhibitor
therapy in patients with chronic-phase chronic myelogenous leukemia
who have stopped imatinib after durable undetectable disease. J Clin
Oncol. 2014;32(5):424–430.
23. Chen Y, Song YX, Wang ZN. The microRNA-148/152 family:
multi-faceted players. Mol Cancer. 2013;12:43.
24. Kim HP, Leonard WJ. CREB/ATF-dependent T cell receptor-induced
FoxP3 gene expression: a role for DNA methylation. J Exp Med.
2007;204(7):1543–1551.
submit your manuscript | www.dovepress.com

Dovepress-250Powered by TCPDF (www.tcpdf.org)

1157

Dovepress

Drug Design, Development and Therapy downloaded from https://www.dovepress.com/ by 210.137.139.190 on 29-Apr-2018
For personal use only.

Ohyashiki et al
25. Wang L, Liu Y, Beier UH, et al. Foxp3+ T-regulatory cells require
DNA methyltransferase 1 expression to prevent development of lethal
autoimmunity. Blood. 20132;121(18):3631–3639.
26. Rea D, Dulphy N, Henry G, et al. Low Natural Killer (NK) Cell counts
and functionality are associated with molecular relapse after imatinib
discontinuation in patients (pts) with chronic phase (CP)-chronic
myeloid leukemia (CML) with undetectable BCR-ABL transcripts forat least 2 years: preliminary results rrom immunostim, on behalf of
STIM investigators. Blood. 2013;122(21). Abstract #856.

1158

27. Larmonier N, Janikashvili N, LaCasse CJ, et al. Imatinib
mesylate inhibits CD4+ CD25+ regulatory T cell activity and enhances
active immunotherapy against BCR-ABL- tumors. J Immunol. 2008;
181(10):6955–6963.
28. Liu X, Zhan Z, Xu L, et al. MicroRNA-148/152 impair innate response
and antigen presentation of TLR-triggered dendritic cells by targeting
CaMKIIalpha. J Immunol. 2010;185(12):7244–7251.

Drug Design, Development and Therapy 2014:8

submit your manuscript | www.dovepress.com

Dovepress-251Powered by TCPDF (www.tcpdf.org)

Dovepress

A possible biomarker to discontinue imatinib safely

Supplementary materials
B
Stop
1.5

miR-148b expression

miR-148b expression

Drug Design, Development and Therapy downloaded from https://www.dovepress.com/ by 210.137.139.190 on 29-Apr-2018
For personal use only.

A

1.0
0.5
0.0

0

10

20

30

40

50

1.5
Relapse
1.0
0.5
0.0
0

TFS (months)

36MonthsFigure S1 (A) miR-148b expression and treatment-free survival (TFS) in STOP-IM patients. Two patients who relapsed shortly after IM discontinuation are shown in open
circles. (B) Sequential analysis of miR-148b expression in two patients who relapsed shortly after IM discontinuation.
Note: Patients who had discontinued IM and had sustained UMRD for more than 6 months (STOP-IM group). In contrast, patients who were receiving IM and had UMRD
at the time of sample collection were categorized as the IM group.
Abbreviation: IM, imatinib mesylate; UMRD, undetectable minimal residual disease.
ID

ANOVA

Mean 1

Mean 2

Mean 3

SEM 1

SEM 2

SEM 3

Quality 1

Quality 2

Quality 3

hsa-miR-96-4373372

0.027347

0.055553

0.310587

0.205137

0.017893

0.081936

0.063318000hsa-miR-338-3p-4395363

0.010837

0.725438

1.79030.988980.178231

0.194392

0.52424800.2

0.142857

hsa-miR-362-3p-4395228

0.009886

1.79764.791131.02369

0.588755

0.7632210.200925

00.6

0.142857

hsa-miR-326-4373050

0.01577

0.697104

1.67510.306825

0.384774

0.295916

0.130009

0

0.2

0

hsa-miR-323-3p-4395338

0.019971

1.58278

0.694484

2.709940.238736

0.118801

1.0002000.142857

hsa-miR-210-4373089

0.036942

99.0538

41.6222151.605

43.0505

9.48987

32.31821

1

1

hsa-miR-329-4373191

0.044299

0.227578

0.112847

0.263752

0.074624

0.011992

0.048371000hsa-miR-454-4395434

0.0394732.624

287.192738.77190.59962.7271154.128111

hsa-let-7b-4395446

0.040507

924.122304.3091128.06

333.482

84.3708265.7461

1

1

hsa-miR-148b-4373129

0.016993

70.180919.1018

122.726

24.9349

5.41085

27.55641

11

hsa-miR-449a-4373207

0.04547

0.268017

0.073807

0.406233

0.101705

0.022575

0.116278000hsa-miR-487b-4378102

0.014862

5.60136

1.505816.744180.96490.323029

4.30608

0.6

0.4

0.142857

hsa-let-7c-4373167

0.042081

6.04992

1.277318.23423

2.83505

0.284943

2.290540.4

00.428571

hsa-miR-127-3p-4373147

0.000099

16.1789

7.28123

13.24540.098157

0.826206

4.045161

0.6

0.714286

hsa-miR-625-4395542

0.02117127.76825.90378

30.5204

10.5496

1.21956.9852410.8

1

hsa-miR-107-4373154

0.013937

12.7226

4.78675

8.64006

1.880170.770132

2.135261

0.6

0.571429

hsa-miR-551b-4380945

0.02058

0.349719

0.118071

0.293659

0.059812

0.036671

0.063495000hsa-miR-495-4381078

0.024363

19.4487.30647

12.0573.20282

0.834396

4.050081

0.6

0.571429

hsa-miR-539-4378103

0.043747

5.212121.310433.7235

1.19105

0.313569

2.093320.6

0.4

0.142857

hsa-miR-23a-4373074

0.004595

100.70931.682917.4238

14.1188

14.08819.889961

0.6

0.428571

hsa-miR-503-4373228

0.007081

1.17271

0.064389

2.963140.460532

0.025522

0.643415000.142857

hsa-miR-18a-4395533

0.006033

80.68284.7681585.37229.64220.762338

17.47611

0.6

1

Table S1 Fold changes, P-values, and quality control values of 22 genes differentially expressed among 3 groups (GEO accession number GSE47652).
Abbreviations: ANOVA, analysis of variance ID, identification document; SEM, standard error of measurement; GEO, Gene Expression Omnibus.

Dovepress

Drug Design, Development and Therapy

Publish your work in this journal
Drug Design, Development and Therapy is an international, peerreviewed open-access journal that spans the spectrum of drug designand development through to clinical applications. Clinical outcomes,
patient safety, and programs for the development and effective, safe,
and sustained use of medicines are a feature of the journal, which

has also been accepted for indexing on PubMed Central. The manuscript management system is completely online and includes a very
quick and fair peer-review system, which is all easy to use. Visit
http://www.dovepress.com/testimonials.php to read real quotes frompublished authors.

Submit your manuscript here: http://www.dovepress.com/drug-design-development-and-therapy-journal

Drug Design, Development and Therapy 2014:8

submit your manuscript | www.dovepress.com

Dovepress-252Powered by TCPDF (www.tcpdf.org)

1159

Biochemical Pharmacology 89 (2014) 361–369

Contents lists available at ScienceDirect

Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharmConstitutive activation of the ATM/BRCA1 pathway prevents DNA
damage-induced apoptosis in 5-azacytidine-resistant cell lines
Satoshi Imanishi a,*, Tomohiro Umezu a,b, Kazushige Ohtsuki a,c, Chiaki Kobayashi c,
Kazuma Ohyashiki c, Junko H. Ohyashiki aabcInstitute for Medical Science, Tokyo Medical University, 6-7-1 Nishi-shinjuku, Shinjuku, Tokyo 160-0023, Japan
Department of Molecular Science, Tokyo Medical University, 6-7-1 Nishi-shinjuku, Shinjuku, Tokyo 160-0023, JapanFirst Department of Internal Medicine, Tokyo Medical University, 6-7-1 Nishi-shinjuku, Shinjuku, Tokyo 160-0023, JapanA R T I C L E I N F O

A B S T R A C T

Article history:
Received 27 January 2014
Accepted 17 March 2014
Available online 26 March 2014

5-Azacytidine (AZA) exerts its anti-tumor effects by exerting cytotoxicity via its incorporation into RNA
and DNA, which causes the reactivation of aberrantly silenced growth-regulatory genes by promoter
demethylation, as well as DNA damage. AZA is used for patients with myelodysplastic syndrome andacute myeloid leukemia. However, some patients demonstrate resistance to AZA, the mechanisms ofwhich are not fully elucidated. We therefore sought to better characterize the molecular mechanism of
AZA resistance using an in vitro model of AZA resistance. We established AZA-resistant cell lines by
exposing the human leukemia cell lines U937 and HL-60 to clinical concentrations of AZA, andcharacterized these cells. AZA-resistant cells showed a down-regulation of the DNMT3A protein, in
correlation with their marked genome-wide DNA hypomethylation. Furthermore, genes involved inpyrimidine metabolism were down-regulated in both AZA-resistant cell lines; AZA sensitivity was
restored by inhibition of CTP synthase. Of note is that the DNA damage response pathway isconstitutively activated in the AZA-resistant cell lines, but not in the parental cell lines. Inhibition of the
DNA damage response pathway canceled the AZA resistance, in association with an increase in apoptotic
cells. We found that the molecular mechanism underlying AZA resistance involves pyrimidine
metabolism and the DNA damage response through ATM kinase. This study therefore sheds light on themechanisms underlying AZA resistance, and will enable better understanding of AZA resistance inpatients undergoing AZA treatment.
ß 2014 Elsevier Inc. All rights reserved.

Chemical compounds studied in this article:
5-Azacytidine (PubChem CID: 9444)
2-desoxy-5-azacytidine (PubChem CID:
451668)3-deazauridine (PubChem CID: 3000824)
RG108 (PubChem CID: 702558)
caffeine (PubChem CID: 2519)
KU55933 (PubChem CID: 5278396)
mitomycin C (PubChem CID: 5746)
etoposide (PubChem CID: 36462)
cisplatin (PubChem CID: 441203)
Keywords:
5-Azacytidine
DNA demethylation
DNA damage

1. Introduction
5-Azacytidine (AZA) is one of the most effective DNA
demethylating agents used in cancer treatment. It is used for

Abbreviations: AZA, 5-azacytidine; MDS, myelodysplastic syndromes; AML, acute
myeloid leukemia; azaCMP, azacytidine monophosphate; azaCTP, azacytidine
triphosphate; DNMT, DNA methyltransferase; DAC, 2-desoxy-AZA; CDKN, cyclin
dependent kinase inhibitor; TP53, tumor protein p53; PTEN, phosphatase andtensin homolog; WT1, Wilms tumor 1; RB1, retinoblastoma 1; POLR2B, polymerase(RNA) II (DNA directed) polypeptide B; UCK2, uridine–cytidine kinase 2; CDA,
cytidine deaminase; AK3, adenylate kinase 3; POL II, RNA polymerase II; CTP,
cytidine triphosphate; UTP, uridine triphosphate; 3-DU, 3-deazauridine; MMC,
mitomycin C; phosphorylated proteins were indicated as, p-protein symbol.
* Corresponding author at: 6-7-1 Nishi-shinjuku, Shinjuku, Tokyo 160-0023,
Japan. Tel.: +81 3 3342 6111; fax: +81 3 3345 0185.
E-mail address: s-ima@tokyo-med.ac.jp (S. Imanishi).
http://dx.doi.org/10.1016/j.bcp.2014.03.008
0006-2952/ß 2014 Elsevier Inc. All rights reserved.

-253-

the treatment of patients with myelodysplastic syndromes (MDS)
and acute myeloid leukemia (AML) in the USA, Europe, and Japan,
among others, and yields a 40–60% response rate in these diseases[1]. However, some patients treated with AZA develop resistanceagainst AZA after various treatment durations [2,3]. The prognosis
of MDS patients after AZA treatment failure is poor, with a medianoverall survival time of 5.6 months [4].
AZA exerts its anti-tumor effects via its incorporation into RNA
and DNA [5]. Induction of the differentiation of malignant cells
might also be involved in the anti-tumor effects of AZA [6]. AZA
taken up by cells is phosphorylated to azacytidine monophosphate(azaCMP) by uridine–cytidine kinase 2 (UCK2), and approximately
80–90% of azaCMP is subsequently phosphorylated to azacytidine
triphosphate (azaCTP), which is incorporated into RNA. AzaCTP
incorporated into RNA then promotes the degradation of RNA andinhibits protein synthesis [5].

362

S. Imanishi et al. / Biochemical Pharmacology 89 (2014) 361–369

The remaining 10–20% of azaCMP is further mono-phosphorylated and converted to 20 -deoxy-azaCTP (aza-dCTP) by ribonucleotide reductase. Aza-dCTP is subsequently incorporated into DNA
during replication [5]. When incorporated into a hemimethylated
CpG site, the 20 -deoxy-azaCTP entraps DNA methyltransferase(DNMT) 1, whereas when it is incorporated into an unmethylated
CpG site, it captures DNMT3A or DNMT3B [7], and forms covalent
DNMT–DNA adducts. The formation of DNMT–DNA adducts
induces the DNA damage response [8], subsequently resulting incytotoxicity, and also causes the depletion of soluble DNMT
protein levels, leading to replication-dependent global demethylation [9] and gene reactivation [10]. The DNA damage response
induced by DNA damaging stimuli, as well as AZA [11], involves theactivation of the Ataxia telangiectasia mutated (ATM) protein [12].
This is followed by phosphorylation and activation of checkpoint
kinase 1 and 2 (CHK1 and CHK2) and breast cancer 1, early onset(BRCA1) proteins. Activated CHK1 and CHK2 phosphorylate p53
resulting in cell cycle arrest and apoptosis, whereas activated of
BRCA1 promotes DNA damage repair [12].
The recent attention given to the activity of AZA or 2-desoxyAZA (DAC) has focused on their demethylating activity rather thanon their induction of DNA damage. Attention has particularly been
given to demethylation in the promoter region of tumorsuppressor genes, because MDS and AML involve the abnormal
regulation of epigenetic modiﬁcations [13,14]. The major obstacle
toward the understanding of AZA sensitivity and resistance is thatthe degrees of involvement of each mechanism, namely, theincorporation of AZA into RNA, DNA, or both, in exerting theclinical and hematological improvements in MDS and AML are notyet fully elucidated.
To overcome this obstacle, we established cell lines that areresistant to clinical doses of AZA, from AZA-sensitive humanleukemia cell lines, and compared their molecular and cellular
properties to clarify the mechanism underlying AZA activity andresistance.

2. Materials and methods
2.1. Cell culture and reagent treatment
A human histiocytic leukemia cell line U937 [15], a humanpromyelocytic leukemia cell line HL-60 [16], and their AZAresistant derivatives R-U937 and R-HL-60 cells were incubated in
RPMI 1640 medium (Life Technologies Inc., Carlsbad, CA, USA)
including 10% inactivated fetal bovine serum and 1% penicillin/
streptomycin (Life Technologies). For the maintenance of R-U937
and R-HL-60 cells, AZA was added to the medium at a ﬁnal
concentration of 3 mM. For treatment with the reagents, cells werecollected by centrifugation and resuspended at 3  105 cells/ml in
fresh medium with the agents or the vehicle. For immunostaining
and western blotting of proteins of the DNA damage response
pathway, R-U937 and R-HL-60 cells were incubated in the absenceof AZA for one week before the experiment. Cell viability wasmeasured by the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan),as reported previously [17]. Apoptotic cells were quantiﬁed using
the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences,Franklin Lakes, NJ, USA) as follows. Cells were treated with theindicated reagents for 36 h. After washing in ice-cold phosphatebuffered saline (PBS), the cells were incubated with FITC-labeledannexin V and propidium iodide (PI) for 15 min at room
temperature in the dark. Flow cytometric measurements were
performed on a BD Accuri C6 Flow Cytometer (BD Biosciences). A
488 nm blue laser was used for the excitation, and signals weredetected by the FL1 channel (533 nm) for FITC and were detectedby the FL2 channel (585 nm) for PI. The signals of 30,000 eventswere obtained. Analyses of the obtained data were performed
using the C6 software Ver. 1.0 (BD Biosciences).
2.2. Chemical reagents and antibodies
The reagents used in this study were from the following sources.
AZA, DAC, RG108, mitomycin C (MMC), etoposide (ETP) and cisplatin(CDDP) were purchased from Wako Pure Chemical Industries Ltd.
(Osaka, Japan), and 3-deazauridine (3-DU) and caffeine werepurchased from Sigma Aldrich Inc. (St. Louis, MO, USA). The ATM
kinase inhibitor KU55933, the PARP inhibitor olaparib, the antiDNMT1 antibody 4H80, the anti-DNMT3A antibody H-295, the antiDNMT3B antibody Q-25, the anti-ATM antibody 2C1 and the anti-bACTIN antibody C4 were purchased from Santa Cruz Biotechnology
Inc. (Dallas, TX, USA). The anti-phosphorylated H2AX (p-H2AX)
antibody was purchased from Trevigen Inc. (Gaithersburg, MD,
USA). The anti-phosphorylated ATM (Ser1981) (p-ATM) antibody
10H11.E12 was purchased from Upstate (Temecula, CA, USA).
Antibodies for the phosphorylated form of p53 (Ser15) (p-p53),
BRCA1 (Ser1524) (p-BRCA1), CHK1 (Ser345) (p-CHK1), CHK2
(Thr68) (p-CHK2), and ATR (Ser428) (p-ATR) were contained in
the DNA Damage Antibody Sampler Kit purchased from Cell
Signaling Technology Inc. (Danvers, MA, USA) and the anti-ATR
antibody was purchased from Rockland Immunochemicals Inc.
(Gilbertsville, PA, USA). Anti-P-glycoprotein antibody JSB-1 waspurchased from Abcam Plc. (Cambridge, UK). The secondary
antibodies, namely, horseradish peroxidase (HRP)-labeled antimouse IgG antibody and HRP-labeled anti-rabbit IgG antibody werepurchased from GE Healthcare (Buckinghamshire, UK). Alexa 546labeled anti-rabbit IgG antibody and Alexa 488-labeled anti-mouse
IgG antibody were purchased from Life Technologies Inc.
2.3. Western blotting
Western blotting was performed as previously described [17].
Brieﬂy, the membranes were probed with antibodies directedagainst DNMT1 (1:200), DNMT3A (1:200), DNMT3B (1:200), pATM (1:500), p-ATR (1:500), p-p53 (1:500), p-BRCA1 (1:500), pCHK1 (1:500), p-CHK2 (1:500), ATM (1:200), ATR (1:500) or bACTIN (1:200) and then treated with the appropriate secondary
antibodies. The amount of each protein was determined using theImage J software.
2.4. Methylation analysis
To determine the global DNA methylation levels, we performed
the single-molecule methylation assay (SMMA) as previously
described [18]. Brieﬂy, TAMRA-labeled methyl-CpG-binding domain protein 2 (MBD2), unlabeled MBD2, 0% methylated DNA(negative control) and 100% methylated DNA (positive control) wereused. Comprehensive analyses using the Inﬁnium HumanMethylation450 BeadChip Kit (Illumina, San Diego, CA, USA) were also
performed according to the manufacturer’s instructions. Brieﬂy,
DNA samples were treated with bisulﬁte using the EZ DNA
Methylation Kit (Zymo Research, CA, USA). The bisulﬁte-treated
DNA was fragmented and suspended in hybridization buffer. The
fragmented DNA was then dispensed onto a HumanMethylation450
BeadChip and hybridization was performed for 20 h. The methylation level of each CpG locus was calculated using the GenomeStudio
Methylation Module (Illumina) as a methylation b-value (b = intensity of the methylated allele (M)/(intensity of the unmethylated
allele (U) + intensity of the methylated allele (M) + 100). The heatmaps were created using the GeneSpring 12.5 GX software (Agilent
Technologies, Santa Clara, CA, USA) using the b-value without any
modiﬁcation. Analyses of the methylation levels in the promoter
regions of speciﬁc genes were performed using the Methylated DNA-254-

S. Imanishi et al. / Biochemical Pharmacology 89 (2014) 361–369

immunoprecipitation (MeDIP) assay kit (Active Motif, Carlsbad, CA,
USA) following the manufacturer’s instructions.

363

3. Results
3.1. Growth inhibitory effect of AZA in AZA-resistant cell lines

2.5. Quantitative RT-PCR
Quantitative RT-PCR was performed as previously described[19]. Taqman gene expression assays were used for AK3
CDA(Hs00156401_m1),
UCK2(Hs00750254_s1),(Hs00367072_m1) and POLR2B (Hs00265358_m1). The TaqMan
Pre-Developed Assay Reagent (Life Technologies) was used for
ACTB. The expression level of each gene relative to the expression
level of ACTB was determined by the DCT method.
2.6. Immunocytochemistry
Cells were ﬁxed in 4% formalin in PBS at 4 8C overnight. Afterwashing and permeabilization in 0.1% TritonX-100 in PBS, cells
were treated with an anti-p-H2AX antibody (1:500) or an anti-pATM antibody (1:200) at room temperature for 1 h, and thenwashed in PBS. After treatment with the appropriate secondary
antibody, nuclei were stained with Hoechst 33342 (Dojindo,
Kumamoto, Japan). The cells were then observed and photographed using the Biozero BZ-8100 ﬂuorescence microscope
system (Keyence, Osaka, Japan). For the analysis of p-H2AXpositive cells, 3 photographs of each sample, with each photographincluding >200 cells, were analyzed using the Image J software.
The ratio of p-H2AX-positive cells was determined as Alexa 546positive cells/Hoechst positive nuclei.
2.7. Statistical analysis
For statistical analyses, one-way or two-way ANOVA followedby the t-test was performed using the GraphPad PRISM 6 software(GraphPad Software Inc., La Jolla, CA, USA). The means  SD are
shown in the ﬁgures.

To establish AZA-resistant human leukemia cell lines, U937 and
HL-60 cells were incubated with gradually increasing doses of AZA,
from 0.1 mM to 3 mM for 9 months and 3 months respectively. The
surviving cells were strongly resistant to AZA (Fig. 1A), and were
designated as AZA-resistant U937 (R-U937) cells and AZA-resistant
HL-60 (R-HL-60) cells. These cells were also resistant to DAC(Fig. 1B), indicating that they acquired cross-resistance to DAC. The
AZA-resistant phenotype of the R-U937 and R-HL-60 cells weremaintained even after incubation in medium without AZA for 6
months (Fig. 1C). Both AZA-resistant cell lines demonstrated alower proliferation rate than their parental cell lines (Fig. 1D). AZA
treatment increased the ratio of annexin V-positive and PI-positive
cells in U937 and HL-60 cells, but not in R-U937 and R-HL-60 cells(Fig. 2A and B), suggesting that AZA-treated U937 and HL-60 cells
die from apoptosis. FACS analysis for the multi-drug transporter Pgp detected no signiﬁcant difference in expression between the
AZA-resistant cells and the parental cells (data not shown),
indicating that the resistant phenotype of the AZA-resistant cells
does not involve multi-drug resistance.
3.2. DNMT protein expression and DNA methylation in AZA-resistantcell lines
We next examined the protein expression levels of DNMT1,
DNMT3A and DNMT3B in the AZA-resistant cell lines. Comparedwith their parental cells, the AZA-resistant cells had equivalent
expression levels of DNMT1, whereas their DNMT3A levels weresigniﬁcantly reduced (Fig. 3A). DNMT3B was not detected in any of
the cell lines (data not shown). The SMMA demonstrated that
global DNA methylation levels were signiﬁcantly reduced in the
AZA-resistant cell lines compared with their parental cells (Fig. 3B).

Fig. 1. Establishment of the AZA-resistant cell lines. (A, B) Viability of the cell lines in the presence of AZA (A) or DAC (B). R-U937 and R-HL-60 cells showed resistance to AZA
and DAC. Each triangle indicates increasing doses, from left to right, of AZA (0, 1, 5 and 10 mM) or DAC (0, 0.2, 1 and 2 mM). *: P < 0.01 compared with cells in the absence ofeach reagent. (C) Comparison of AZA resistance of R-U937 and R-HL-60 cells after incubation with (+) or without () AZA for 6 months. The R-U937 and R-HL-60 cells wereresistant to AZA even after incubation in medium without AZA for 6 months. Each triangle indicates increasing doses, from left to right, of AZA (0, 1, 5 and 10 mM). (D)Comparison of the cell growth between AZA-resistant cells and the parental cells. The AZA-resistant cell lines proliferated more slowly than their parental cells. The resultsfrom 3 independent experiments were analyzed.

-255-

364

S. Imanishi et al. / Biochemical Pharmacology 89 (2014) 361–369

Fig. 2. FACS analyses of apoptosis of the cells in the presence of 10 mM AZA. (A, B) FACS analyses of U937 and R-U937 cells (A) and HL-60 and R-HL-60 cells (B) for annexin V
and PI staining. The annexin V-positive and PI-positive fraction was increased in the parental cells but not in the AZA-resistant cells after 72 h of treatment with 10 mM of AZA.
Typical plots from a representative experiment are shown. The experiments were repeated 3 times.

Global DNA demethylation was also conﬁrmed by the methylationarray (Fig. 3C). Furthermore, the DNA methylation array revealed alarge variety of demethylation patterns among the tumor
suppressor genes (Fig. 3D). For example, we found that most of
the promoter regions of cyclin dependent kinase inhibitor 1A(CDKN1A) were demethylated in AZA-resistant cell lines comparedwith those in their parental cells, whereas the demethylation
patterns of CDKN1B, CDKN2A and CDKN2B did not show a constanttendency. The promoter region of tumor protein p53 (TP53) was
demethylated in both AZA-sensitive and AZA-resistant cells,

Fig. 3. DNMT protein expression and DNA methylation in AZA-resistant cells. (A) Protein levels of DNMT1 and DNMT3A. A decrease in the level of DNMT3A was evident inboth AZA-resistant cell lines. The experiment was repeated 3 times. A representative blot is shown. The quantitative analyses are provided in the histogram. *: P < 0.01
compared with the parental cells. (B, C) Global DNA methylation was measured by the SMMA assay (B) and methylation array (C). Experiments were repeated 3 times for (B),and 2 times for (C). Methylation was signiﬁcantly decreased in R-U937 and R-HL-60 cells compared with U937 and HL-60 cells. (D) DNA methylation patterns of various
tumor suppressor genes.

-256-

S. Imanishi et al. / Biochemical Pharmacology 89 (2014) 361–369

whereas a part of the promoter regions of phosphatase and tensin
homolog (PTEN) and Wilms tumor 1 (WT1) were demethylated in
AZA-resistant cells. Demethylation in retinoblastoma 1 (RB1) was
not prominent in the AZA-resistant cell lines.
3.3. Down-regulation of genes involved in the pyrimidine metabolism
pathway in AZA-resistant cell lines
To investigate the biological relevance of the RNA-dependent
pathway in AZA resistance, we ﬁrst examined the RNA expressionof genes involved in pyrimidine metabolism, such as polymerase(RNA) II (DNA directed) polypeptide B (POLR2B), uridine–cytidine
kinase 2 (UCK2), cytidine deaminase (CDA), and adenylate kinase 3(AK3), which were identiﬁed from the KEGG PATHWAY database(http://www.genome.jp/dbget-bin/www_bget?pathway:map00240) [20,21]. We found that the RNA expression of UCK2 andPOLR2B was signiﬁcantly decreased in both R-U937 and R-HL-60
cells compared with their parental cells (Fig. 4A and B), whereas
the RNA expression of AK3 and CDA was variable between the 2 celllines. POLR2B encodes a rate-limiting subunit of RNA polymerase II(POL II) and UCK2 encodes an enzyme speciﬁc for pyrimidine
metabolism. These results indicate the biological relevance of
UCK2 and POLR2B in the AZA-resistant phenotype.
To determine whether altered pyrimidine metabolism results
in a reduction of cytidine salvaging and acceleration of cytidine
triphosphate (CTP) synthesis from uridine triphosphate (UTP), we
incubated the AZA-resistant cell lines in 3-DU, a CTP synthasespeciﬁc inhibitor [22], with increasing doses of AZA. The viability of
R-U937 and R-HL-60 cells was not affected by 40 mM and 5 mM 3DU, respectively. AZA induced growth inhibition in a dosedependent manner in the presence of 3-DU (Fig. 4C). Therefore,

365

the accelerated conversion of UTP to CTP by CTP synthase plays animportant role in AZA resistance.
To clarify the possible association between the down-regulation of genes involved in pyrimidine metabolism and thedemethylation of their promoter region, we performed the MeDIP
assay for the promoter regions of UCK2 and POLR2B. Against our
expectations, the promoter regions of these genes were highly
demethylated in both the AZA-resistant cell lines and theirparental cells, even when AZA was not added (Fig. 4D). We
therefore could not ﬁnd any AZA-induced demethylation in the
AZA-resistant cell lines and their parental cells. These ﬁndings
indicate that the down-regulation of genes involved in the RNAdependent pathway is not due to demethylation of gene promoters
by AZA.
3.4. Involvement of the DNA damage response pathway in AZA
resistance
AZA is incorporated not only into RNA, but also into DNA, andinhibits DNMT activity. To clarify whether DNMT inhibition plays acentral role in AZA sensitivity, we next compared the viability of
the AZA-resistant cells and their parental cells in the presence orabsence of RG108, which is a non-nucleoside analog DNMT
inhibitor that does not damage DNA [23]. Since cell growth was notaffected by RG108, we concluded that the growth-inhibitory effectof AZA might be due to DNA damage rather than DNMT inhibition(Fig. 5A). We then performed immunocytochemistry for phosphorylated H2AX (p-H2AX), which is a hallmark of the DNA
damage response (Fig. 5B). U937 and HL-60 cells incubated with
10 mM AZA or 2 mM DAC showed a signiﬁcantly higher ratio of pH2AX-positive cells, whereas no such increase was detected inFig. 4. Altered expression of genes involved in pyrimidine metabolism in AZA-resistant cells. (A, B) mRNA expression levels of pyrimidine metabolism genes in R-U937 (A) and
R-HL-60 cells (B). The results from 3 independent experiments were analyzed. Means  SD of relative mRNA expression levels normalized to ACTB are shown. The mRNA level inthe parental cells was regarded as 1 for each gene. *: P < 0.01 compared with the mRNA expression level of each gene in the parental cells. (C) AZA reduced the viability of R-U937
and R-HL-60 cells in the presence of 3-DU. The results from 3 independent experiments were analyzed. *: P < 0.01 compared with the 3-DU-untreated control at each indicated dose.
(D) Results of the MeDIP assay. The recovery rates for the transcription start site (TSS) and the area 500 bp upstream from the TSS (US) of UCK2 and POLR2B are shown. Even in theabsence of AZA, the AZA-resistant cells as well as their parental cells showed very low recovery rates in these regions. The results from 2 independent experiments were analyzed. A
primer pair for ZC3H13 was used as a positive control. IgG was used as a negative control for the anti-5-methylcytosine antibody. N.D., not detected.

-257-

366

S. Imanishi et al. / Biochemical Pharmacology 89 (2014) 361–369

Fig. 5. DNA damage response in AZA-resistant cell lines. (A) Treatment with RG108 at the indicated doses did not reduce the viability of U937 and HL-60 cells. The results from
3 independent experiments were analyzed. (B) 10 mM AZA and 2 mM DAC increased the ratio of p-H2AX-positive cells in all the cell lines, whereas RG108 did not. *: P < 0.01
compared with the untreated control of each cell line. #: P < 0.01 compared with the parental cells treated with AZA or DAC. The results are shown as means  SD.

U937 and HL-60 cells incubated with RG108. These results indicatethat DNA damage plays a major role in exerting the effects of AZA
and DAC on human leukemia cells. We also found that the ratio of
p-H2AX-positive cells was increased in R-U937 and R-HL-60 cells
exposed to AZA or DAC, although the increase was signiﬁcantly
lower in the AZA-resistant cells than in the parental cells.
To further elucidate the machinery associated with DNA
damage, we stained phosphorylated ATM (p-ATM) in the AZAresistant cells and their parental cells. In U937 and HL-60 cells, pATM-positive nuclear foci were not detected before AZA treatment,
whereas p-ATM-positive nuclear foci were detected in the cells
after AZA treatment (Fig. 6A). Unlike the parental cells, R-U937 and
R-HL-60 cells already had a few p-ATM positive foci before AZA
treatment, and the number of foci increased after AZA treatment.
To clarify the role of the DNA damage response in AZA resistance,
we compared the phosphorylation levels of the proteins involvedin the DNA damage response, that is, ATM, Ataxia telangiectasia
and Rad3 related (ATR), BRCA1, CHK1, CHK2 and p53, in U937 and

R-U937 cells at 6, 24 and 48 h in the presence of 10 mM AZA, as well
as before the treatment (Mock) (Figs. 6B and 7). In U937 cells in thepresence of AZA, a signiﬁcant increase in the phosphorylation of
ATM and BRCA1 was detected at 6 h, and phosphorylation of CHK1,
CHK2 and p53 was increased at 24 and 48 h. In R-U937 cells, ATM
and BRCA1 were highly phosphorylated even in the absence ofAZA, and their phosphorylation levels were further increased at 6 h
and 24 h, respectively, in the presence of AZA. Phosphorylation of
p53 and CHK2 was not increased even in the presence of AZA in RU937 cells. Although phosphorylated CHK1 was signiﬁcantly
increased in R-U937 cells at 6 and 24 h, it was decreased to thebasal level at 48 h. We were unable to detect the phosphorylated
ATR protein (data not shown).
We then evaluated cell viability with or without caffeine, which
is known to inhibit the DNA damage response pathway, withincreasing doses of AZA or DAC. Eventually, we found that 1 mM
caffeine reduced the viability of R-U937 and R-HL-60 cells in
10 mM of AZA or 2 mM of DAC (Fig. 8A). Although caffeine canFig. 6. ATM signaling plays roles in the acquisition of AZA resistance. (A) Immunostaining for p-ATM (Ser1981). Positive foci were observed in the nuclei of AZA-treated cells(right column). R-U937 and R-HL-60 possessed a few positive foci even in the absence of AZA (left column). Green: p-ATM (Ser1981). Blue: DAPI. *: Non-speciﬁc staining ofdead cells. (B) Results of Western blotting for the phosphorylated proteins involved in the DNA damage response, p-ATM (Ser1981), p-p53 (Ser15), p-BRCA1 (Ser1524), pCHK1 (Ser345), and p-CHK2 (Thr68), at the indicated times in the presence of 10 mM AZA. Typical photographs obtained from 3 independent experiments for each protein are
shown. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)-258-

S. Imanishi et al. / Biochemical Pharmacology 89 (2014) 361–369

367

Fig. 7. Relative amounts of phosphorylated proteins of the DNA damage response pathway in U937 cells and R-U937 cells. Relative amount of p-ATM (Ser1981) (A), p-BRCA1(Ser1524) (B), p-p53 (Ser15) (C), p-CHK1 (Ser345) and p-CHK2 (Thr68) (D) was quantiﬁed based on photographs obtained from 3 independent Western blotting experiments.
The amount of p-ATM (Ser1981) was normalized to the amount of total ATM, and the others were normalized to the amount of b-ACTIN. *: P < 0.05 compared with the mockcontrol. #: P < 0.05 compared with U937 cells of the corresponding hours.

affect multiple pathways, 10 mM of the ATM kinase activity
speciﬁc inhibitor KU55933 also canceled the AZA resistance in
R-U937 and R-HL-60 cells (Fig. 8A), demonstrating the criticalrole of the ATM-dependent DNA damage response pathway in
the AZA resistance. Finally, to conﬁrm the resistance to DNA
damage of the AZA-resistant cell lines, we treated the cells with

the DNA synthesis inhibitor MMC, the topoisomerase II inhibitor
ETP, and the cross-linking agent CDDP. Although the susceptibility to these drugs greatly differed between the two parental
cell lines, the AZA-resistant cell lines demonstrated higher
resistance to the drugs than their corresponding parental celllines (Fig. 8B and C).

Fig. 8. Inhibition of the DNA damage response restores AZA sensitivity in AZA-resistant cells. (A) Caffeine and KU55933 canceled the AZA resistance of R-U937 and R-HL-60
cells, and caffeine canceled the DAC resistance of R-U937 and R-HL-60 cells. (B, C) Higher viabilities were detected in R-U937 (B) and R-HL-60 cells (C) than in their parental
cells in the presence of mitomycin C (MMC), etoposide (ETP) and cisplatin (CDDP). *: The results from 3 independent experiments were analyzed for each graph. P < 0.01
compared with the parental cell line treated with the indicated dose of MMC, ETP or CDDP. The statistical analysis for each agent was performed independently. (D) Annexin
V-positive cells were signiﬁcantly increased in AZA-treated R-U937 cells in the presence of KU55933 compared with those in AZA-treated R-U937 cells in the absence of
KU66933. In contrast, olaparib did not affect the percentage of annexin V-positive cells in the AZA-treated R-U937 cells.

-259-

368

S. Imanishi et al. / Biochemical Pharmacology 89 (2014) 361–369

3.5. Inhibition of ATM kinase activity induces apoptosis in AZAresistant cell lines
To determine whether reversal of AZA resistance by KU55933 isdue to the induction of apoptosis via the DNA damage response, wequantiﬁed the annexin V-positive cells in AZA-treated U937 and RU937 cells (Fig. 8D). Annexin V-positive cells were signiﬁcantly
increased in AZA-treated U937 cells, whereas such an increase was
not evident in AZA-treated R-U937 cells, as shown in Fig. 1C.
Annexin V-positive cells were signiﬁcantly increased in thepresence of KU55933 in AZA-treated R-U937 cells. This indicatesthat the DNA damage response, via the activity of ATM kinase, isindeed involved in the AZA-resistant phenotype. In contrast, PARP
inhibition by 1 mM olaparib did not cancel the AZA resistance in RU937 cells. Taken together, these results show that the DNA
damage response pathway involving ATM, rather than PARP, plays
a key role in the AZA-resistant phenotypes.
4. Discussion
The demethylating agent AZA has received FDA approval for thetreatment of MDS. However, the reason why a subset of patients is
resistant to AZA remains unclear. In terms of clinical practice, there
are two types of AZA resistance. The ﬁrst type is primary treatment
failure, namely, non-responders. The second type is acquired AZA
resistance during the course of AZA-treatment. The molecularmechanism involved might be different between the two types of
AZA resistance. In the current study, we demonstrated that the
suppression of UCK2, which is involved in pyrimidine metabolism,
was detected in the AZA-resistant cell lines, which is consistent
with previous studies [20]. Several studies reported that thedifferences in AZA sensitivity among cell lines correlate with thedifferences in the expression levels of UCK2 [24,25]. Furthermore,
recent studies demonstrated that genetic variations in UCK2
determine the sensitivity of cells to a cytidine analog [26,27].
Taken together, it is likely that the mechanism underlying acquired
AZA resistance partially overlaps with that of intrinsic resistance.
Further research using clinical samples will promote the understanding of the role of UCK2 expression, polymorphisms, andmutations in AZA resistance. We also identiﬁed POLR2B as anothergene involved in AZA resistance. To the best of our knowledge, therole of POLR2B in AZA resistance has not been reported previously.
The roles of POLR2B or the other subunits of POL II in AZA
resistance should be clariﬁed by further research.
Contrary to the detailed knowledge about the decreased
incorporation of AZA into RNA in AZA-resistant cells, it is still
unclear whether or not AZA is incorporated into DNA in AZAresistant cells. DAC resistance and the DNA damage response in
AZA-resistant cells used in the present study strongly suggest theincorporation of AZA into DNA in the resistant cells. The mostimportant ﬁnding of this study is the involvement of ATM signaling
in the AZA resistance of cells in which AZA has been incorporated
into DNA. Indeed, the constitutive activation of ATM/BRCA1dependent DNA repair and the inhibition of ATM/p53-dependent
apoptosis prevented R-U937 cells from undergoing AZA-induced
apoptosis. Although HL-60 cells do not possess the TP53 gene [28],the ATM inhibitors canceled the AZA-resistant phenotype of R-HL60 cells, suggesting that AZA resistance involves the inhibition of
p53-independent apoptosis. Previous studies have reported theconstitutive phosphorylation of ATM in radiation-resistant celllines and the radio-sensitization of these cells by ATM inhibitors[29,30], whereas the constitutive phosphorylation of ATM has
never been reported in cells resistant to DNA-damaging agents.
Since the AZA-resistant cell lines were exposed to clinical doses of
AZA for several months for their establishment, the constitutive
phosphorylation of ATM is likely to be linked to the mechanism

active in patients with acquired AZA resistance, whereas the role of
the DNA damage response in AZA non-responders is still unclear.
It is widely thought that AZA and DAC exert their anti-tumor
activities via the demethylation and reactivation of tumor
suppressor genes [31]. If resistance to DNA demethylation is
involved in AZA resistance, epigenetic silencing by the restorationof methylation in tumor suppressor genes is a candidate
mechanism for gaining resistance to AZA. However, we did notﬁnd any increased methylation in the promoters of tumor
suppressor genes in the AZA-resistant cell lines. Taken together
with the results from the region-speciﬁc analysis of the promotersof UCK2 and POLR2B and global analysis of the DNA in AZAresistant cells, our results indicate that restoration of DNA
methylation is not involved in AZA resistance.
Unlike AZA, DAC is metabolized by DNA metabolism. Although
AZA and DAC are considered to rarely induce cross-resistance [32],the AZA-resistant cell lines in this study were resistant to DAC, andthis resistance was canceled by treatment with caffeine. Our
results strongly suggest the possibility that DAC resistance in the
AZA-resistant cell lines is dependent on the ATM/BRCA1 pathway.
We observed that the viability of the AZA-resistant cell lines washigher than that of their parental cell lines upon MMC, ETP or CDDP
treatment. This indicates that the constitutive activation of ATM
may allow the cells to gain resistance to a wide range of DNAdamaging drugs. The development of cross-resistance to otherdrugs should be an important consideration when AZA is usedclinically.
Treatment with the CTP synthase inhibitor 3-DU canceled theresistance of R-U937 and R-HL-60 cells to AZA, as well as to ATM
inhibitors. Recently, Raynal et al. [33] demonstrated that 3-DU
enhances the cytotoxic activity of DAC by enhancing theincorporation of DAC into DNA. 3-DU causes a reduction in the
CTP pool, resulting in a reduction in the deoxy-CTP (dCTP) pool,which leads to enhanced incorporation of aza-dCTP into DNA due
to less competition with dCTP. The metabolites of AZA might beincorporated into not only DNA but also RNA, but nevertheless, thismechanism is plausible for the observed restoration of AZA
sensitivity by 3-DU in this study. Although the utility of CTP
synthase inhibitors for leukemia therapy should be evaluated in afurther study, our results indicate that targeting CTP synthase canbe beneﬁcial for the treatment of leukemia.
Another important issue that should be addressed is thepossible role of DNMT3A in AZA resistance. DNMT3A methylates
the non-methylated CpGs and plays important roles in the decisionof cell fates of hematopoietic stem cells [34,35]. In the AZAresistant cell lines with highly demethylated DNA, DNMT3A might
function to re-methylate the CpGs and might be entrapped by AZA.
Various studies showed that CD34+ cells from patients withmyeloid malignancy demonstrate lower DNA methylation levels
than the healthy CD34+ cells [36,37]. In the patients with MDS, theprogenitor cells that differentiate from pathogenic CD34+ cells
cannot proliferate or differentiate normally and die from apoptosis.
The patients then suffer from severe ineffective hematopoiesis,
which can be improved by treatment with AZA or DAC. On theother hand, a previous study showed the persistent proliferation ofpathogenic CD34+ cells in patients treated with AZA [38]. Further
characterization of our AZA-resistant cell lines will promote theunderstanding of the mechanism of action of demethylating agentsin leukemia cells with hypomethylated DNA.
On the other hand, some limitations exist in our approach. Since
R-U937 and R-HL-60 cells are not of clonal origin, they may beheterogeneous populations. Hence, we cannot determine whether
multiple changes are needed to acquire resistance to AZA, orwhether a single change can be responsible for AZA resistance. It is
probable that the slow proliferation rate of AZA-resistant cell lines
contributes to the AZA-resistant phenotype. However, we failed to-260-

S. Imanishi et al. / Biochemical Pharmacology 89 (2014) 361–369

ﬁnd any evidence, for example phase-speciﬁc prolongation of cell
cycle, in the present study indicating that the slow proliferation
rate of AZA-resistant cell lines is an active mechanism underlying
AZA resistance. Finally, although we proposed a molecularmechanism underlying acquired AZA resistance, our results are
not necessarily able to explain the AZA resistance mechanism in
non-responders. Since approximately half of the patients with
MDS show no response to AZA treatment, it is very important todetermine whether or not the same mechanisms are involved in
the acquired and primary resistance to AZA. These problems
should be clariﬁed by further studies both in vitro and in vivo.
In the present study, AZA-resistant cell lines were establishedand characterized at the molecular level. We propose that themolecular mechanism underlying resistance to AZA involves
pyrimidine metabolism and sustained DNA damage response
involving ATM as well as BRCA1. These ﬁndings provide new
insights into the diagnostic and therapeutic strategies for thetreatment of AZA-resistant MDS patients.
Competing interests
The authors declare no competing interests.
Acknowledgements
This work was supported by the Private University Strategic
Research-Based Support Project: Epigenetics Research Project
Aimed at General Cancer Cure Using Epigenetic Targets S0801020
from MEXT (Ministry of Education, Culture, Sports, Science andTechnology), Japan.
The authors thank Dr. Helena A. Popiel (PhD, Lecturer) and Dr.
Edward Barroga (PhD, Associate Professor) of the Department ofInternational Medical Communications of Tokyo Medical University for reviewing and editing the manuscript.
References[1] Tefferi A, Vardiman JW. Myelodysplastic syndromes. N Engl J Med
2009;361:1872–85.
[2] Fenaux P, Mufti GJ, Hellstrom-Lindberg E, Santini V, Finelli C, Giagounidis A,
et al. Efﬁcacy of azacitidine compared with that of conventional care regimens
in the treatment of higher-risk myelodysplastic syndromes: a randomised,open-label, phase III study. Lancet Oncol 2009;10:223–32.
[3] Fenaux P, Mufti GJ, Hellstrom-Lindberg E, Santini V, Gattermann N, Germing U,
et al. Azacitidine prolongs overall survival compared with conventional careregimens in elderly patients with low bone marrow blast count acute myeloid
leukemia. J Clin Oncol 2010;28:562–9.
[4] Prebet T, Gore SD, Esterni B, Gardin C, Itzykson R, Thepot S, et al. Outcome ofhigh-risk myelodysplastic syndrome after azacitidine treatment failure. J Clin
Oncol 2011;29:3322–7.
[5] Stresemann C, Lyko F. Modes of action of the DNA methyltransferase inhibitors
azacytidine and decitabine. Int J Cancer 2008;123:8–13.
[6] Curik N, Burda P, Vargova K, Pospisil V, Belickova M, Vlckova P, et al. 5-Azacitidine
in aggressive myelodysplastic syndromes regulates chromatin structure at PU.1
gene and cell differentiation capacity. Leukemia 2012;26:1804–11.
[7] Schneider-Stock R, Diab-Assef M, Rohrbeck A, Foltzer-Jourdainne C, Boltze C,
Hartig R, et al. 5-Aza-cytidine is a potent inhibitor of DNA methyltransferase 3aand induces apoptosis in HCT-116 colon cancer cells via Gadd45- and p53dependent mechanisms. J Pharmacol Exp Ther 2005;312:525–36.
[8] Nakano T, Katafuchi A, Matsubara M, Terato H, Tsuboi T, Masuda T, et al.
Homologous recombination but not nucleotide excision repair plays a pivotal
role in tolerance of DNA–protein cross-links in mammalian cells. J Biol Chem
2009;284:27065–76.
[9] Yamagata Y, Szabo P, Szuts D, Bacquet C, Aranyi T, Paldi A. Rapid turnover of
DNA methylation in human cells. Epigenetics 2012;7:141–5.
[10] Claus R, Lubbert M. Epigenetic targets in hematopoietic malignancies. Oncogene 2003;22:6489–96.
[11] Jiemjit A, Fandy TE, Carraway H, Bailey KA, Baylin S, Herman JG, et al.
p21(WAF1/CIP1) induction by 5-azacytosine nucleosides requires DNA damage. Oncogene 2008;27:3615–23.

-261-

369

[12] Khanna KK, Jackson SP. DNA double-strand breaks: signaling, repair and thecancer connection. Nat Genet 2001;27:247–54.
[13] Itzykson R, Fenaux P. Epigenetics of myelodysplastic syndromes. Leukemia
2013;28:497–506.
[14] Abdel-Wahab O, Levine RL. Mutations in epigenetic modiﬁers in thepathogenesis and therapy of acute myeloid leukemia. Blood
2013;121:3563–72.
[15] Shibanuma M, Kuroki T, Nose K. Effects of the protein kinase C inhibitor H-7
and calmodulin antagonist W-7 on superoxide production in growing andresting human histiocytic leukemia cells (U937). Biochem Biophys Res Commun 1987;144:1317–23.
[16] Collins SJ. The HL-60 promyelocytic leukemia cell line: proliferation, differentiation, and cellular oncogene expression. Blood 1987;70:1233–44.
[17] Umezu T, Ohyashiki K, Kuroda M, Ohyashiki JH. Leukemia cell to endothelial
cell communication via exosomal miRNAs. Oncogene 2012;32:2747–55.
[18] Umezu T, Ohyashiki K, Ohyashiki JH. Detection method for quantifying global
DNA methylation by ﬂuorescence correlation spectroscopy. Anal Biochem
2011;415:145–50.
[19] Ohyashiki JH, Hisatomi H, Nagao K, Honda S, Takaku T, Zhang Y, et al.
Quantitative relationship between functionally active telomerase and major
telomerase components (hTERT and hTR) in acute leukaemia cells. Br J Cancer
2005;92:1942–7.
[20] Vesely J, Dvorak M, Cihak A, Sorm F. Biochemical mechanisms of drug resistance. XII. Uridine kinases from mouse leukemic cells sensitive and resistant to5-azacytidine. Int J Cancer 1971;8:310–9.
[21] Mahfouz RZ, Jankowska A, Ebrahem Q, Gu X, Visconte V, Tabarroki A, et al.
Increased CDA expression/activity in males contributes to decreased cytidine
analog half-life and likely contributes to worse outcomes with 5-azacytidine
or decitabine therapy. Clin Cancer Res 2013;19:938–48.
[22] Balzarini J, Gago F, Kulik W, van Kuilenburg AB, Karlsson A, Peterson MA, et al.
Introduction of a ﬂuorine atom at C3 of 3-deazauridine shifts its antimetabolic
activity from inhibition of CTP synthetase to inhibition of orotidylate decarboxylase, an early event in the de novo pyrimidine nucleotide biosynthesis
pathway. J Biol Chem 2012;287:30444–54.
[23] Brueckner B, Garcia Boy R, Siedlecki P, Musch T, Kliem HC, Zielenkiewicz P,
et al. Epigenetic reactivation of tumor suppressor genes by a novel smallmolecule inhibitor of human DNA methyltransferases. Cancer Res
2005;65:6305–11.
[24] Qin T, Jelinek J, Si J, Shu J, Issa JP. Mechanisms of resistance to 5-aza-20 deoxycytidine in human cancer cell lines. Blood 2009;113:659–67.
[25] Saunthararajah Y. Key clinical observations after 5-azacytidine and decitabine treatment of myelodysplastic syndromes suggest practical solutions forbetter outcomes. Hematology Am Soc Hematol Educ Program
2013;2013:511–21.
[26] Murata D, Endo Y, Obata T, Sakamoto K, Syouji Y, Kadohira M, et al. A crucialrole of uridine/cytidine kinase 2 in antitumor activity of 30 -ethynyl nucleosides. Drug Metab Dispos 2004;32:1178–82.
[27] Hasegawa T, Futagami M, Kim HS, Matsuda A, Wataya Y. Analysis of single
nucleotide polymorphisms in uridine/cytidine kinase gene encoding metabolic enzyme of 30 -ethynylcytidine. Nucleic Acids Res Suppl 2002;2:237–8.
[28] Wolf D, Rotter V. Major deletions in the gene encoding the p53 tumor antigen
cause lack of p53 expression in HL-60 cells. Proc Nati Acad Sci U S A
1985;82:790–4.
[29] Yin H, Glass J. The phenotypic radiation resistance of CD44+/CD24(-or low)breast cancer cells is mediated through the enhanced activation of ATM
signaling. PLoS ONE 2011;6:e24080.
[30] Hazawa M, Hosokawa Y, Monzen S, Yoshino H, Kashiwakura I. Regulation of
DNA damage response and cell cycle in radiation-resistant HL60 myeloid
leukemia cells. Oncol Rep 2012;28:55–61.
[31] Baylin SB. DNA methylation and gene silencing in cancer. Nat Clin Prac Oncol
2005;2(Suppl. 1):S4–11.
[32] Uchida T, Ogawa Y, Kobayashi Y, Ishikawa T, Ohashi H, Hata T, et al. Phase I and
II study of azacitidine in Japanese patients with myelodysplastic syndromes.
Cancer Sci 2011;102:1680–6.
[33] Raynal NJ, Momparler LF, Rivard GE, Momparler RL. 3-Deazauridine enhances
the antileukemic action of 5-aza-20 -deoxycytidine and targets drug-resistancedue to deﬁciency in deoxycytidine kinase. Leuk Res 2011;35:110–8.
[34] Challen GA, Sun D, Jeong M, Luo M, Jelinek J, Berg JS, et al. Dnmt3a is essential
for hematopoietic stem cell differentiation. Nat Genet 2012;44:23–31.
[35] Trowbridge JJ, Orkin SH. Dnmt3a silences hematopoietic stem cell self-renewal. Nat Genet 2012;44:13–4.
[36] Martin-Subero JI, Ammerpohl O, Bibikova M, Wickham-Garcia E, Agirre X,
Alvarez S, et al. A comprehensive microarray-based DNA methylation study of
367 hematological neoplasms. PLoS ONE 2009;4:e6986.
[37] Will B, Zhou L, Vogler TO, Ben-Neriah S, Schinke C, Tamari R, et al. Stem andprogenitor cells in myelodysplastic syndromes show aberrant stage-speciﬁc
expansion and harbor genetic and epigenetic alterations. Blood
2012;120:2076–86.
[38] Craddock C, Quek L, Goardon N, Freeman S, Siddique S, Raghavan M, et al.
Azacitidine fails to eradicate leukemic stem/progenitor cell populations inpatients with acute myeloid leukemia and myelodysplasia. Leukemia
2013;27:1028–36.

From www.bloodjournal.org by guest on April 28, 2018. For personal use only.

Regular Article
LYMPHOID NEOPLASIA

Exosomal miR-135b shed from hypoxic multiple myeloma cells enhances
angiogenesis by targeting factor-inhibiting HIF-1
Tomohiro Umezu,1,4 Hiroko Tadokoro,2 Kenko Azuma,3,4 Seiichiro Yoshizawa,3 Kazuma Ohyashiki,1,3 and Junko H. Ohyashiki4
1

Department of Molecular Science, Tokyo Medical University, Tokyo, Japan; 2Department of Pharmaceutical Health Care and Sciences, Tokyo University of
Pharmacy and Life Sciences, Tokyo, Japan; 3Department of Hematology and 4Institute of Medical Science, Tokyo Medical University, Tokyo, Japan

Exosomes are small endosome-derived vesicles containing a wide range of functional
proteins, mRNA, and miRNA. Exosomal miRNA from cancer cells helps modulate themicroenvironment. In multiple myeloma (MM), the massive proliferation of malignant• We established hypoxiaresistant cells that can mimic plasma cells causes hypoxia. To date, the majority of in vitro hypoxia studies of cancer
cells have used acute hypoxic exposure (3-24 hours). Thus, we attempted to clarify thein vivo conditions of hypoxicrole of MM-derived exosomes in hypoxic bone marrow by using MM cells grownbone marrow.
continuously in vitro under chronic hypoxia (hypoxia-resistant MM [HR-MM] cells). The• Exosomal miR-135b derived
HR-MM cells produced more exosomes than the parental cells under normoxia or acutefrom these cell lines
hypoxia conditions, and miR-135b was significantly upregulated in exosomes from HRenhanced endothelial tube
MM cells. Exosomal miR-135b directly suppressed its target factor–inhibiting hypoxiaformation under hypoxia via
inducible factor 1 (FIH-1) in endothelial cells. Finally, exosomal miR-135b from HR-MM
the HIF-FIH signaling
cells enhanced endothelial tube formation under hypoxia via the HIF-FIH signaling
pathway.
pathway. This in vitro HR myeloma cell model will be useful for investigating MM
cell–endothelial cell interactions under hypoxic conditions, which may mimic the in vivobone marrow microenvironment. Although tumor angiogenesis is regulated by various factors, exosomal miR-135b may be a target
for controlling MM angiogenesis. (Blood. 2014;124(25):3748-3757)

Key Points

Introduction
Multiple myeloma (MM) is a unique B-cell neoplasm characterized
by the accumulation of clonal malignant plasma cells in the bonemarrow (BM).1,2 The massive number of plasma cells usually
disseminates into multiple bone lesions that are located far fromthe primary lesion, much like cancer metastasis. The molecularmechanism by which a primary myeloma lesion progresses to multiple
lesions has not been fully elucidated. Although autologous stem celltransplantation combined with chemotherapeutic agents such as
thalidomide, lenalidomide, and bortezomib can signiﬁcantly improve
response rates and the prognosis of MM patients,3-5 MM remains
incurable for the majority of patients because of relapse.6,7
Hypoxia is an important element of the cancer microenvironment
and is known to be associated with metastasis. Under hypoxia, cancer
cells secrete substances that modulate their hostile microenvironment to promote tumor angiogenesis.8-10 Aberrant angiogenesis has
been reported in MM-inﬁltrated BM,11-13 and increased angiogenic
activity is associated with endothelial activation, increased capillarypermeability, and hyperperfusion.14-16 Evidence suggests that MM
cells promote angiogenic activity via hypoxia-inducible factor(HIF)-1a, a key transcription factor of hypoxia, leading to theoverproduction of angiogenic cytokines such as vascular endothelial
growth factor (VEGF),17 angiopoietin-1,18 and osteopontin.19

In addition to conventional signaling pathways responding tohypoxia (ie, direct cell-cell contact or VEGF signaling),10 ourgroup and others have shown that exosomes, small endosome-derived
vesicles containing a wide range of functional proteins, mRNA, andmiRNA, from hypoxic cancer cells help to modulate the microenvironment without contacting the surrounding noncancer cells.20
Previous studies demonstrated that oxygen tension in MM-inﬁltrated
BM was lower than in normal BM, which is already hypoxic innature.21 The massive proliferation of MM cells produces hypoxic
conditions in the tumor, which may lead to more rapid cell proliferation, drug resistance, and angiogenesis.11,22 However, little is
known about how hypoxia affects the biological properties of
MM cells in vivo. Previous studies using a human tumor syngeneic
mouse model (the 5T33M mouse MM model) demonstrated thatmyelomatous BM is more hypoxic than normal BM.21,23,24 In contrast
to those in vivo models, the majority of in vitro hypoxia studies ofcancer cells have used acute hypoxic exposure (3-24 hours).
To clarify the role of MM-derived exosomes in hypoxic BM,
we established an in vitro chronic hypoxia model using MM cells
that show continuous growth in vitro under hypoxic conditions
lasting more than 6 months (hypoxia-resistant [HR] cells). Here,
we investigated the MM cell–endothelial cell interaction via miR-135b

Submitted May 16, 2014; accepted October 2, 2014. Prepublished online as
Blood First Edition paper, October 15, 2014; DOI 10.1182/blood-2014-05576116.

The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.

The online version of this article contains a data supplement.
There is an Inside Blood Commentary on this article in this issue.

© 2014 by The American Society of Hematology

BLOOD, 11 DECEMBER 2014 x VOLUME 124, NUMBER 25

3748

-262-

From www.bloodjournal.org by guest on April 28, 2018. For personal use only.
BLOOD, 11 DECEMBER 2014 x VOLUME 124, NUMBER 25

miR-135b ENHANCES ANGIOGENESIS

3749

Figure 1. The response of HR-MM cells to hypoxia.
(A) The growth of parental cells (RPMI8226, KMS-11,
U266) and HR-MM cells (RPMI8226-HR, KMS-11-HR,
U266-HR) were measured after 24, 48, and 72 hours
of normoxic (20% O2) or hypoxic (1% O2) culture conditions. (B) The expression level of HIF-1a protein inparental cells and HR-MM cells cultured under 20% or
1% O 2 conditions for 24 hours. Numbers below thepanels represent the normalized HIF-1a expression
signal by b-actin (ratio).

shed from MM cells under hypoxia, which may promote MM diseaseprogression without directly contacting adjacent tissue.

mean pixel density obtained from image analysis of 5 random microscopicﬁelds using ImageJ software (http://rsb.info.nih.gov/nih-image/).
miRNA expression profiles

Materials and methods
Cell lines and culture conditions
Human MM cell lines (RPMI8226, KMS-11, U266) and human umbilicalvein endothelial cells (HUVECs) were purchased from the Human Science
Research Resource Bank (Osaka, Japan) and Lonza Inc. (Allendale, NJ),
respectively. See the supplemental Methods on the Blood Web site for details.
Establishment of HR-MM cell lines
Cell lines RPMI8226, KMS-11, and U266 were incubated under hypoxic
conditions (1% O2) for 6 to 7 months. The sublines that survived well underlong-term hypoxia were designated HR-MM cells RPMI8226-HR, KMS-11HR, and U266-HR, respectively.
Preparation of exosomal fraction
MM cell lines were seeded at a density of 5 3 105 cells/mL and cultured for
24 hours (unless otherwise indicated) under hypoxic (1% O2) or normoxic(20% O2) conditions in serum-free AIM-V medium (Invitrogen, Carlsbad,
CA). The exosomes derived from MM cells were puriﬁed by Exoquick
Exosome Precipitation Solution (System Biosciences, Mountain View,
CA) as described previously.25
Transmission electron microscopy
Exosomes were prepared and ﬁxed as described previously.20 The samples
were observed with a transmission electron microscope (JEM-1200EX;
JEOL Ltd., Tokyo, Japan) at an acceleration voltage of 80 kV.
Nanoparticle tracking analysis of exosomes
Measurements for nanoparticle tracking analysis were performed using the
Nanosight LM10 system (Nanosight, Amesbury, United Kingdom), ﬁxed
as described previously.20 The capture settings and analysis settings were
performed manually according to the manufacturer’s instructions.
Tube formation assay
The formation of capillarylike structures was assessed as described previously.20 HUVECs (2 3 104 cells/well) were plated on top of Matrigel(280 mL/well) and treated with exosomes (500 mL of exosome fraction/well)derived from MM cells (2.5 3 106 cells) cultured for 24 hours under hypoxic
conditions or under normoxic conditions. The total tube area was quantiﬁed as

Isolation of cellular and exosomal miRNAs was performed using themiRNeasy kit (Qiagen, Hiden, Germany) as described previously.20 See
the supplemental Methods for details. MiRNA proﬁling in both cells andexosomes was performed using a TaqMan low-density miRNA array(Applied Biosystems, Foster City, CA) according to the manufacturer’s
recommendations. The relative expression of each gene was calculated
by using the comparative threshold cycle (Ct) method, as described previously.25
RNU6B was used as an invariant control for the cellular miRNA. The
synthetic spike control (ath-miR-159) was used as an invariant control for the
exosomal miRNA.
Transfection of MM cells with Cy3-labeled pre-mir miRNA
precursor and PKH67-labeled exosome transfer
Pre-mir miRNA precursor (has-miR-210 or has-miR-135b; Ambion, Austin,TX) was labeled with Label IT siRNA Tracker Cy3 kit (Mirus, Madison,
WI) according to the manufacturer’s instructions. The PKH67-labeled
exosomes including Cy3-miR-210 or Cy3-miR-135b were collected as described previously.20
miRNA target reporter luciferase assay
Synthetic oligonucleotides bearing the miR-135b binding sequence (59TCACATAGGAATGAAAAGCCATA-39) or factor-inhibiting hypoxiainducible factor 1 (FIH-1) 39-UTR with the miR-135b complementarybinding site (59-TTAGATAGGGTTCCAACTGGGCCTACAAGCTCAAGC
CATACATAAAAGGACCTTGGG-39) were cloned into the ﬁreﬂy luciferase
reporter plasmid pMIR-Report (Ambion) according to the manufacturer’s
protocol (the seed sequence of miR-135b is shown in bold italics). We also
generated the mutated sensor vector in which the seed sequence of miR-135b
was changed to CCTAACGC. For measuring luciferase activity, see the supplemental Methods for details.
Knockdown and overexpression experiment of exosomal
miR-135b
RPMI8226 cells (1 3 105) or RPMI8226-HR cells (1 3 105) were transfectedwith anti–miR-135b miRNA inhibitors (Ambion), miR-135b mimic (Ambion),or scrambled control (Negative Control 1; Ambion) or by using HiPerFect(Qiagen). The next day, after a change to fresh media, cells were cultured
under hypoxic conditions for 24 hours. The exosomes derived from
RPMI8226-HR1%O2/anti–miR-135b cells or RPMI8226-HR1%O2/control
were puriﬁed as described.

-263-

From www.bloodjournal.org by guest on April 28, 2018. For personal use only.
3750

BLOOD, 11 DECEMBER 2014 x VOLUME 124, NUMBER 25

UMEZU et al

Hypoxia response-element reporter assay
HUVECs were maintained at 50% to 60% conﬂuence and were transducedby either 0.1 mg of pGL4.42(luc2P/HRE/Hygrp) transcription reporter orpGL4.15(luc2P/Hygro) control (Promega, Mannheim, Germany). The luciferase plasmid was cotransfected with 0.01 mg of pGL4.75(hRluc/CMV)
control plasmid (Promega) as a control for the transfection efﬁciency.
HUVECs were then treated with RPMI822620%O2-exosome, RPMI82261%O2exosome, or RPMI8226-HR1%O2-exosome, and the luciferase activity wasassessed after 48 hours with the Dual-Glo Luciferase System (Promega)according to the manufacturer’s protocol.
Immunoblotting
The cells were lysed in lysis buffer (Roche, Penzberg, Germany), and equalamounts of protein were separated on sodium dodecyl sulfate–polyacrylamidegels. The exosome pellets isolated from the same amount of culture medium(5 mL) were lysed in 200 mL of lysis buffer (Roche), and the same amounts
of lysate (30 mL) were loaded in each lane of the gels. See the supplemental
Methods for details of antibodies used for the immunoblots.
In vivo Matrigel plug assay
The exosome derived from RPMI8226/miR135b mimic (RPMI8226-transfected
miR-135b mimic) or RPMI8226-HR/anti-miR135b (RPMI8226-HR–transfectedanti–miR-135b inhibitor) were mixed with Matrigel (200 mL), and then the
mixture was subcutaneously injected into nude mice (female, 8-week-old
BALB/c-n/n; CLEA Japan, Tokyo, Japan). After 1 week, the Matrigel plugs
were harvested and processed for analysis. See the supplemental Methods fordetails of analysis of capillary density.
Statistical analyses
Data are expressed as mean 6 standard deviation (SD). Two treatment groups
were compared by Mann-Whitney U test or Student t test. Multiple groupcomparisons were performed by analysis of variance. GraphPad Prism
version 5c for Macintosh (GraphPad Inc., La Jolla, CA) was used for statisticalanalyses. Results were considered statistically signiﬁcant when P , .05.

Results
Establishment and characterization of HR-MM cells

The cell growth of parental cells and cell growth of HR-MM cells
were measured at 24, 48, and 72 hours (Figure 1A). Growth of theparental cells was signiﬁcantly reduced by incubation under hypoxic
conditions for 48 to 72 hours, but the HR-MM cells continued togrow, although the proliferation rate was reduced. The expression of
HIF-1a was upregulated in parental cells under acute hypoxia for
24 hours, and upregulation of HIF-1a expression was more evident
in HR-MM cells under hypoxic conditions (1% O2) compared withparental cells (Figure 1B). Furthermore, we compared the MM surface
markers (CD19–, CD1381) between the parental cells and HR-MM cellsby ﬂow cytometry (supplemental Figure 1A). We could not ﬁnd aremarkable change in the CD138 expression pattern, but there was achange in the KMS-11 cells. In RPMI8226 and U266 cells, there wasno difference in the expression of 2 genes between parental cells andHR-MM cells (supplemental Figure 1B), and they have maintained theexpression of MM cell-surface markers and transcription factors.

Figure 2. Characterization of exosomes derived from HR-MM cells. (A) Transmission electron micrographs of exosomes derived from parental cells (RPMI8226 cells
cultured under 20% or 1% O2 conditions) and HR-MM cells (RPMI8226-HR cells
cultured under 1% O2 condition). The scale bar represents 50 nm. (B) CD63 andCD81 (common exosomal markers) immunoblots of exosomes derived from RPMI8226
and RPMI8226-HR cells. These exosomes were isolated from 5 mL of culture media of
RPMI8226 (5 3 10 5 cells/mL) or RPMI8226-HR cells (5 3 10 5 cells/mL) cultured
under 20% O2 or 1% O2 conditions for 24 hours, respectively, and the same amountsof exosome lysate (30 mL) were loaded in each lane of the gels. The ratio of CD63
and CD81 expression is indicated below the panels. (C) The nanoparticle concentrationand size distribution of the exosomes derived from RPMI8226 cells cultured under 20%
or 1% O2 conditions for 24 hours (dotted lines) and RPMI8226-HR cells cultured under
1% O2 conditions for 24 hours (solid lines).

shaped appearance with the common exosomal markers (CD63 andCD81) (Figure 2A-B). There was no difference between the cell lines
in the nanoparticle size distribution of exosomes, but the amount of
exosomes secreted from the HR-MM cells was twofold that of theparental cells (Figure 2C). Similar results were observed in the other2 cell lines, KMS-11-HR and U266-HR (supplemental Figure 2A-C).

Characterization of exosomes derived from HR-MM cells

We next compared the size, ultrastructures, and quantity of exosomes
between parental and HR-MM cells. Transmission electron microscopy revealed that the size of exosomes was similar between theparental cells (RPMI822620%O2 and RPMI82261%O2) and HR-MM
cells (RPMI8226-HR1%O2), and each vesicle showed the classic cup-

Remote effect of exosomes: exosomes derived from HR-MM
cells induce tube formation in normoxic HUVECs in adose-dependent manner

To clarify how exosomes derived from HR-MM cells affect endothelial
cells that are remote from tumor tissue, we performed an endothelial-264-

From www.bloodjournal.org by guest on April 28, 2018. For personal use only.
BLOOD, 11 DECEMBER 2014 x VOLUME 124, NUMBER 25

miR-135b ENHANCES ANGIOGENESIS

3751

Figure 3. Endothelial tube formation assay innormoxic or hypoxic HUVECs. The formation of
tubelike structures was observed under dark fieldusing a cell-permeable dye, Calcein AM (green).
Endothelial tube formation of HUVECs cultured
under normoxic conditions (20% O 2 ) with (A)RPMI8226 20%O2 -exosomes, (B) RPMI8226 1%O2 exosomes, (C) RPMI8226-HR 1%O2 -exosomes, (D)half the amount of RPMI8226-HR1%O2-exosomes, or(E) without exosomes (control; HUVECs20%O2 ). (F)Quantitative data for the tubelike structures determinedby pixel density. RPMI8226 1%O2 -exosomes and
RPMI8226-HR1%O2-exosomes significantly enhanced
tube formation of HUVECs20%O2 compared with control(HUVECs 20%O2) (*P , .01, **P , .001). Because
nanoparticle tracking analysis indicated that the HR
cells secreted double the amount of exosomes compared with parental cells (Figure 2B), half the amountof RPMI8226-HR1%O2-exosomes was also assessed(RPMI8226-HR1%O2-exosomes vs RPMI8226-HR1%O2exosome (1/2): #P , .01, Student t test). Endothelial tube
formation of HUVECs cultured under hypoxic conditions(1% O2 ) with (G) RPMI8226 20%O2-exosomes, (H)RPMI82261%O2 -exosomes, (I) RPMI8226-HR1%O2 exosomes, (J) half the amount of RPMI8226-HR1%O2exosomes, or (K) without exosomes (control;
HUVECs1%O2). Because HUVECs were exposed tohypoxic conditions, the induction of tube formation wasenhanced in all treatments. (L) Quantitative data forthe tubelike structures determined by pixel density.
RPMI82261%O2-exosomes and RPMI8226HR1%O2exosomes enhanced tube formation of HUVECs1%O2(*P , .01, **P , .001). Even when RPMI8226-HR1%O2exosomes were reduced to half the dose, induction of tube
formation did not decrease (RPMI8226-HR1%O2-exosomes vs RPMI8226-HR1%O2-exosome [1/2]; P 5 .38,
Student t test). Values are mean 6 SD. The scale bar
indicates 500 mm. exo, exosome.

tube formation assay. HUVECs cultured under normoxic conditions(HUVEC20%O2) were treated with RPMI822620%O2-exosomes,
RPMI8226 1%O2 -exosomes, or RPMI8226-HR 1%O2 -exosomes.
RPMI82261%O2-exosomes enhanced the tube formation of HUVECs,
whereas RPMI8226 20%O2-exosomes did not (Figure 3A-B,E-F).
Exosomes from HR-MM cells signiﬁcantly enhanced tube formationof HUVECs compared with the control (Figure 3C,E-F). Because
exosome release from HR-MM cells was twofold that of parental
cells, we also tested the effect of adding half the amount of
RPMI8226-HR1%O2-exosomes; we found that tube formation
signiﬁcantly decreased in an exosome dose–dependent manner(Figure 3C-D,F and supplemental Table 1). These ﬁndings indicatethat the amount of exosomes affects tube formation in HUVECs
under normoxic conditions.
Local effect of exosome: increased tube formation by
exosomes from HR-MM cells is dose independent in
hypoxic HUVECs

In the majority of cases, both MM and endothelial cells might beunder hypoxic conditions, especially when endothelial cells are
adjacent to MM cells. Therefore, we investigated the local effectof exosomes and whether exosomes derived from HR-MM cells
could affect tube formation of HUVECs cultured under hypoxic-265-

From www.bloodjournal.org by guest on April 28, 2018. For personal use only.
3752

BLOOD, 11 DECEMBER 2014 x VOLUME 124, NUMBER 25

UMEZU et al

Table 1. Cellular and exosomal miRNA profiles of RPMI8226-HR
cells(1) Acute hypoxia2,
RPMI8226-HR↑(2) Acute hypoxia↑,
RPMI8226-HR↑

Fold change (vsparental cell)

Fold change (vsparental cell)Ct valuemiRNA

Exosome

Cell

miRNA

Exosome

Cell

20-25

miR-425

2.15582.4911

miR-21017.48623.9463

miR-27a1.76222.7311

miR-27b2.06824.4697miR-223

2.671

6.33

miR-135a16.99118.1049

miR-324-3p

1.57671.8605miR-361-5p

1.71

3.448925-30

miR-383

4.160910.8108

miR-551b3.65552.5412

miR-140-3p

2.19082.5292

miR-500

2.21114.8255miR-135b

7.955121.8038

miR-483-5p

2.50791.6511

miR-362-5p

1.747

2.7095whereas miR-210 expression was upregulated soon after hypoxic
exposure (1 hour) and then increased gradually until 72 hours(Figure 4A-B). In HR-MM cells, high miR-210 expression wasmaintained in hypoxic culture, but the expression gradually disappeared over the course of 1 week in culture under normoxic
conditions (Figure 4A-B). These ﬁndings indicate that upregulated miR-210 expression is reversible and depends on the oxygen
concentration. In contrast, miR-135b was barely detected in cells
and exosomes of the parental line. miR-135b upregulation only
occurred in HR-MM cells, and the expression level was maintained
when cells were cultured under normoxic conditions for 1 week
(Figure 4C-D). These results indicate that miR-210 is a universal
hypoxia-responsive miRNA with transient expression, whereas miR135b is an HR-MM cell–speciﬁc miRNA with continuous expression.
Exosomal miR-135b derived from HR-MM cells regulates thetarget gene in HUVECs

After the cellular and exosomal miRNAs were extracted by a fold change of ,1.5
and Ct value, they were subdivided into 2 groups: (1) miRNAs upregulated only in
HR-MM cells (Acute hypoxia2, RPMI8226-HR↑), and (2) miRNAs upregulated inboth acute hypoxia and HR-MM cells (Acute hypoxia↑, RPMI8226-HR↑).

conditions (HUVECs1%O2). Similar to the results using HUVECs20%O2,
RPMI8226 1%O2 -exosomes enhanced the tube formation of
HUVECs1%O2 compared with the control (Figure 3H,K-L). Exosomesfrom HR-MM cells enhanced tube formation of HUVECs1%O2 to aneven greater degree (Figure 3I,K-L and supplemental Table 1). It
would be logical to assume that tube formation might decrease byhalf with half the amount of exosomes, but the decrease of tube
formation of HUVECs was not so evident at this lower dose. This
indicates that the accelerated tube formation does not simply dependon the amount of exosomes.
Cellular and exosomal miRNA profiling of HR-MM cells

We then compared miRNA proﬁles of cells and exosomes using the
3 parental cells and 3 HR-MM sublines (NCBI, gene expression
omnibus; GSE48983). We extracted the cellular and exosomal miRNAs
with a fold change of ,1.5 and on the basis of Ct value (20-25,
25-30, 30-35). We arbitrarily subdivided miRNA as follows: (1)miRNAs upregulated in acute hypoxia and HR cells, (2) miRNAs
upregulated in only acute hypoxia, and (3) miRNAs upregulated
in only HR cells (Tables 1-3). The upregulation of miR-210 was
found in acute hypoxia and HR cells in all cell lines. In contrast,
we could not ﬁnd any HR-speciﬁc miRNA universally expressedin the 3 cell lines. Several miRNAs were HR speciﬁc in at least 2cell lines: miR-425 and miR-135b (RPMI8226-HR and KMS-11-HR
cells); miR-335, miR-328, and miR-200c (KMS-11-HR and U266HR cells); and miR-223 (RPMI8226-HR and U266-HR cells)(Tables 1-3). Among them, the expression level of miR-135b was ashigh as that of miR-210. Therefore, we investigated miR-135b further.

We visualized the transport of exosomal miR-135b derived from
RPMI8226-HR cells into HUVECs transfected with b-gal control
plasmid (HUVECs/b-gal) using the method modiﬁed from ourprevious report.25 After incubation with PKH67-labeled exosomes
derived from RPMI8226-HR/Cy3-miR-135b cells, the Cy3-miR135b signals and PKH67 signals were colocalized in the cytoplasm
of HUVECs/b-gal (Figure 5A-F). We performed a luciferase
reporter assay to assess whether the exogenous miR-135b via
exosomal transport could function like endogenous miRNA in
endothelial cells. When HUVECs/b-gal transduced with the reporter
plasmid–containing complementary miR-135b binding site (sensor
vector, HUVECs/LUC/b-gal) were incubated with RPMI8226HR1%O2-exosomes, ﬁreﬂy luciferase activity was signiﬁcantly
reduced compared with the control (HUVECs only; **P , .01)
(Figure 5G). In contrast, the RPMI8226-HR1%O2-exosomes did
not reduce the luciferase activity using a mutated sensor vector of
miR-135b (Figure 5G).
To investigate whether RPMI8226-HR exosomes mediatedangiogenesis is directly dependent on miR-135b, we performed
knockdown experiments of exosomal miR-135b. The anti–miR135b inhibitor disrupted the activity of exosomal miR-135b;
RPMI8226-HR/anti–miR135b-exosome and could not reduce theluciferase activity in HUVECs/LUC/b-gal (RPMI8226-HR/anti–
miR135b-exo vs RPMI8226-HR/con-exo; #P , .01) (Figure 5G).
Furthermore, the knockdown of miR-135b led to a decrease in
Table 2. Cellular and exosomal miRNA profiles of KMS-11-HR cells(2) Acute hypoxia↑,
KMS-11-HR↑

Fold change (vsparental cell)

Fold change (vsparental cell)Ct valuemiRNA

Exosome

Cell

miRNA

Exosome

Cell

25-30

miR-425

1.7072

2.196

miR-21013.9761

16.5659

miR-335

6.5755

7.066830-35

miR-132

1.96415.6163Kinetics of miR-210 and miR-135b in hypoxia

We measured the intracellular or exosomal miR-210 and miR-135b
expressions of RPMI8226 and RPMI8226-HR with real-timereverse-transcription polymerase chain reaction. We found different
miRNA kinetics of miR-210 and miR-135b during culture underhypoxic conditions. In parental cells, the expression of intracellularand exosomal miR-210 was not found under normoxic conditions,(1) Acute
hypoxia2,
KMS-11-HR↑

miR-518b1.6252

3.0374miR-328

1.94084.8109miR-200c

2.39883.1656miR-331-5p

1.79192.0219miR-135b

5.94242.0893After the cellular and exosomal miRNAs were extracted by a fold change of ,1.5
and Ct value, they were subdivided into 2 groups: (1) miRNAs upregulated only in
HR-MM cells (Acute hypoxia2, KMS-11-HR↑), and (2) miRNAs upregulated in bothacute hypoxia and HR-MM cells (Acute hypoxia↑, KMS-11-HR↑).

-266-

From www.bloodjournal.org by guest on April 28, 2018. For personal use only.
BLOOD, 11 DECEMBER 2014 x VOLUME 124, NUMBER 25

miR-135b ENHANCES ANGIOGENESIS

Table 3. Cellular and exosomal miRNA profiles of U266-HR cells

Exosomal miR-135b enhances neovascularization in vivo

(1) Acute
hypoxia2,
U266-HR↑(2) Acute hypoxia↑,
U266-HR↑

Fold change(vs parental cell)

Fold change(vs parental cell)Ct valuemiRNA

Exosome

Cell

miRNA

Exosome

Cell

20-25

miR-193b

2.01122.6867miR-21017.48633.3568

miR-138

2.1346

3.1043miR-328

2.11381.9223

let-7e1.6542

1.8644miR-204

16.01215.2747miR-223

2.45

12.9772

miR-335

11.6635

21.1083

miR-23a2.41245.4919miR-200c

3.18722.8421

miR-185

2.41972.7435

25-30

30-35

3753

After the cellular and exosomal miRNAs were extracted by a fold change of ,1.5
and Ct value, they were subdivided into 2 groups: (1) miRNAs upregulated only in
HR-MM cells (Acute hypoxia2, U266-HR↑), and (2) miRNAs upregulated in bothacute hypoxia and HR-MM cells (Acute hypoxia↑, U266-HR↑).

endothelial tube formation via exosomes (RPMI8226-HR/con-exo
vs RPMI8226-HR/anti–miR135b-exo; *P , .05) (Figure 5H-J).
These results suggest that the RPMI8226-HR exosome–mediatedangiogenesis is dependent on exosomal miR-135b.

To analyze angiogenic responses to exosomes (exosomal miR-135b)
derived from HR-MM cells, we performed an in vivo Matrigel plugangiogenesis assay to detect the newly-formed blood vessels in
the transplanted gel plugs in nude mice (Figure 6A). The densityof the neovessels line in Matrigel plugs was quantiﬁed by immunohistochemical staining with anti-mouse CD31 (Figure 6B). The plugs
containing the exosomes derived from RPMI8226-HR/anti-miR135b
signiﬁcantly reduced the density of CD311 neovesssels comparedwith control (the exosomes derived from RPMI8226-HR/con)(Figure 6C). These additional experiments provide evidence ofimportant roles for exosomal miR-135b derived from HR-MM
cells on angiogenesis in vivo.
Exosomal miR-135b derived from HR-MM cells regulated the
HIF-1 signaling in HUVECs

We performed in silico analysis to determine possible miR-135b
targets that may be responsible for its angiogenic function, speciﬁcallyunder hypoxia, using database resources including Targetscan
and MiRanda. Seven-hundred eighteen transcripts with conserved
miR-135b binding sites were predicted. Among them, we selected
2 candidates with high predicted efﬁcacy calculated by the context
scores. One is angiopoietin-2 (ANGPT2), a key regulator of angiogenesis that exerts context-dependent effects on endothelial cells,

Figure 4. Time course of miR-210 and miR-135b
expression under hypoxic conditions. Parental cells(RPMI8226) and HR-MM cells (RPMI8226-HR) were
cultured under normoxic conditions (20% O2) or hypoxic
conditions (1% O2) for the indicated times (0, 24, 48, or
72 hours). Cellular and exosomal miRNA expression
levels were quantified by quantitative reverse-transcription
polymerase chain reaction: (A) cellular miR-210, (B)exosomal miR-210, (C) cellular miR-135b, and (D)exosomal miR-135b. RNU6B and ath-miR-159 wereused as the invariant control for cell and exosome,respectively. Values are means 6 SD of 3 independent
experiments, with each performed on different days.

-267-

From www.bloodjournal.org by guest on April 28, 2018. For personal use only.
3754

BLOOD, 11 DECEMBER 2014 x VOLUME 124, NUMBER 25

UMEZU et al

Figure 5. Transfer of miR-135b derived from HRMM cells to HUVECs via exosomes. HUVECs/b-gal(HUVECs transfected with pMIR-reporter b-gal control
vector) were cultured with PKH67-labeled exosomes
derived from the parental cells (RPMI8226) transfectedwith Cy3-pre-miR-135b. The Cy3-miR-135b signals
were detected in (A) the cytoplasm of HUVECs (red),and (B) green signals indicate PKH67-labeled exosomes. (C) Cy3-miR-135b signals are colocalized with
PKH67 in HUVECs (yellow). Parts of areas in (A-C) are
enlarged in (D-F), respectively. Nuclear counterstaining was performed using 49,6-diamidino-2-phenylindole(DAPI) (blue). The scale bar indicates 10 mm. (G)Luciferase reporter vector for assessing miR-135b–
specific activity contained complementary miR-135b
sequences in its 39-UTR. The normalized firefly luciferase
activity was obtained by firefly luciferase activity/b-gal
activity. Sensor vector: luciferase activity of HUVECs/
Luc/b-gal (HUVECs cotransfected with luciferase reporter vector and b-gal control vector) treated with
RPMI8226-HR1%O2-exosomes was significantly reduced
compared with control (HUVECs/Luc/b-gal without
exosomes) (**P , .01, n 5 3). RPMI8226-HR/antimiR135b exosomes (miR-135b–deactivated exosomesof RPMI8226-HR cells) could not reduce the luciferase
activity in HUVECs (RPMI8226-HR/anti–miR135b-exo
vs RPMI8226-HR/con-exo: #P , .01, Student t test, n 5 3).
In the mutated sensor vector, there was no difference inluciferase activity with or without exosomes. (H-I)
Tube formation assay of HUVECs cultured under
1% O 2 with RPMI8226-HR1%O2/control exosomes(H) and with RPMI8226-HR 1%O2 /anti-miR135b exosomes (I). The scale bar indicates 500 mm. (J) The
tubelike structures determined by pixel density are
reduced by the addition of RPMI8226-HR 1%O2 /antimiR135b exosomes compared with control (*P , .05
vs control, Student t test, n 5 3). Values are means 6 SDof 3 independent experiments, with each performed ona different day. exo, exosome; con, control.

and the other is FIH-1, which is also known as hypoxic-inducible
factor-1a subunit inhibitor. However, we found that only FIH-1
showed direct binding with miR-135b by luciferase reporter assay(supplemental Figure 3). To validate the direct binding between
miR-135b and the FIH-1 39-UTR region, we performed a luciferase
reporter assay using the reporter plasmid containing FIH-1 39-UTR
with the miR-135b binding site. Reduction of luciferase activity fromthe pLuc-FIH1-39-UTR plasmid was observed in HUVECs cultured
with RPMI822620%O2/miR-135b exosome (exosomes derived from
RPMI822620%O2 transfected with miR-135b mimics; 70% reduction) or in HUVECs cultured with RPMI8226-HR1%O2-exosome(30% reduction) compared with control (HUVECs only) (Figure 7A,
left panel). As expected, deletion of the FIH-1 39-UTR sequence
from the construct abolished the inhibitory effects of miR-135b
on luciferase activity (Figure 7A, right panel). To clarify whether
FIH-1 expression levels are reduced in RPMI8226-HR cells,
we compared FIH-1 between the parental cells (RPMI8226) and
HR-MM cells (RPMI8226-HR) using immunoblot analysis. FIH-1

expression was reduced in RPMI8226-HR cells (supplemental
Figure 4A). In contrast, FIH-1 protein was not detected in exosomes
derived from RPMI8226-HR cells (supplemental Figure 4B). The
reduction of FIH-1 expression levels in RPMI8226-HR cells
may be induced by endogenous miR-135b, and the FIH-1 proteinof RPMI8226-HR cells did not transfer to the other cells via
exosomes. These results indicate that exosomal (exogenous) miR-135b
interacts with the FIH-1 39-UTR to exert translational repression in
HUVECs.
To determine whether FIH-1 expression is suppressed by adding
HR-MM–derived exosomes (exogenous-miR-135b), and whether
the suppression depends on the oxygen levels of HUVECs in culture,
we compared FIH-1 protein expression levels in normoxic HUVECs(Figure 7B, left panel) and hypoxic HUVECs (Figure 7B, right panel).
A reduction of approximately 70% of FIH-1 protein was observedin HUVECs cultured with RPMI8226-HR1%O2-exosomes comparedwith the control (Figure 7B). There was no signiﬁcant difference between HUVECs 20%O2 and HUVECs1%O2. These ﬁndings-268-

From www.bloodjournal.org by guest on April 28, 2018. For personal use only.
BLOOD, 11 DECEMBER 2014 x VOLUME 124, NUMBER 25

miR-135b ENHANCES ANGIOGENESIS

3755

indicate that the suppression of FIH-1 by HR-MM–derived exosomes
was independent of the oxygen levels of HUVECs.
Finally, we investigated the HIF transcriptional activity toclarify the involvement of the HIF-1 and FIH-1 pathways in exosomal
miR-135b–mediated tube formation in HUVECs. We transfected
HUVECs with a reporter plasmid containing an SV40 promoterluciferase transcription unit downstream of a 68-bp hypoxiaresponse element that mediates HIF-1–dependent gene transcription.
The reporter-gene expression was markedly increased in HUVECs1%O2
relative to HUVECs20%O2 (Figure 7C, right panel). In HUVECs20%O2,
HIF-1 activity was barely detected before and after adding exosomes(Figure 7C, left panel). These ﬁndings strongly indicate that
exosomal miR-135b accelerated HIF-1 transcriptional activity
via inhibition of FIH-1.

Discussion

Figure 6. Exosomal miR-135b enhances neovascularization in vivo. (A) Representative light microscopic photographs of Matrigel plugs harvested 1 week after
subcutaneous injection into nude mice. The exosomes derived from RPMI8226,
RPMI8226-HR/control, RPMI8226-HR/anti-miR135b, or phosphate-buffered saline(as a vehicle control) were mixed with growth factor–reduced Matrigel. (B) The
neovasculature induced by the exosome of RPMI8226-HR cells in Matrigel was
visualized by immunohistochemical staining of frozen sections with anti-mouse CD31
antibody. Representative photographs reveal abundant vasculature positively stained
for CD31 (red). Nuclear counterstaining was performed using DAPI (blue). The
scale bar indicates 200 mm. (C) Quantitative data for the neovessels lined in Matrigel
plugs determined by pixel density. The exosome derived from RPMI8226-HR cells
increased the density of vasculature positive for CD31 in Matrigel plugs comparedwith vehicle control (*P , .01 vs control, Student t test). The HR exosome–induced
increase of neoangiogenesis was canceled by anti–miR-135b disrupting the activityof exosomal miR-135b (RPMI8226-HR/con-exo vs RPMI8226-HR/anti-miR135bexo; #P , .05, Student t test). Values are mean 6 SD. exo, exosome; con, control.

Hypoxic MM cells release various diffusible factors (eg, VEGF) that
promote the angiogenic switch in MM.26,27 Although the hypoxic
signaling pathway via VEGF by cell-cell contact has been studied in
MM angiogenesis,11,17 the complexity of the hypoxic response in the
MM microenvironment needs to be deﬁned more clearly. Here, we
provide evidence of exosome-mediated angiogenesis in MM cells
under prolonged hypoxia.
One major barrier to investigating the effects of hypoxia on MM
cells in vitro was that cells could grow only for several days underhypoxic conditions. Therefore, most studies were performed using
MM cells exposed to hypoxia for 24 to 72 hours. In such cases, MM
cells might respond to hypoxic stress in a manner similar to thatof an ischemic attack rather than long-lasting intratumor hypoxia.
Therefore, we established HR-MM cells, which can serve as asurrogate of therapy-resistant MM cells, because the biologicaleffects of hypoxia on tumor cells are known to be resistant toanticancer chemotherapy and are known to increase the risk oftumor metastasis.28-30
In the current study, we investigated the characteristics of
exosome release in HR-MM cells. We ﬁrst noted that the amountof exosomes from HR-MM cells was signiﬁcantly greater thanthat of parental cells, and a subset of exosomal miRNA mightwork in a dose-dependent manner. Exosomal miR-210 induced
tube formation in a dose-dependent manner when HUVECs were
cultured under normoxic conditions.20 One important ﬁnding isthat upregulation of miR-210 is not speciﬁc to HR-MM cells. When
HUVECs were cultured under hypoxic conditions, miR-210 expression in the HUVECs themselves increased. Under such conditions,certain miRNAs, other than miR-210, were likely to play more
important roles, especially in local cell-cell communication in MM.
We then focused on exosomal miR-135b as an miRNA speciﬁc to
HR-MM cells. miR-135b was speciﬁcally upregulated in both exosomesand HR-MM cells, whereas the expression was barely detected in
the parental cells, even when they were cultured under short-term
hypoxia. miR-135b is an oncogenic microRNA that has been linked
to the progression of several types of cancers, including coloncancer,31,32 osteosarcoma,33 and non–small-cell lung cancer.34 Sometargets already reported the direct binding of miR-135b (such as
SMAD5).35 To the best of our knowledge, there is no reportdealing with an association between those targets and angiogenesis.
Zhang et al demonstrated a parallel correlation between miR-135b
and HIF-1a; miR-135b is more involved with mechanisms ofhypoxic response.36 We noted that exosomal miR-135b derived-269-

From www.bloodjournal.org by guest on April 28, 2018. For personal use only.
3756

BLOOD, 11 DECEMBER 2014 x VOLUME 124, NUMBER 25

UMEZU et al

Figure 7. Exosomal miR-135b targets FIH-1 in hypoxic HUVECs. (A) miR-135b
binding sites in the FIH-1 39-UTR were cloned into pMIR luciferase reporter vector(left, sensor vector). Identical construct mutation was generated (right, mutated vector). RPMI82261%O2/miR-135b exosomes (exosomes derived from RPMI82261%O2
transfected with miR-135b mimics), RPMI82261%O2/control exosomes (derived from
RPMI82261%O2 transfected with scramble control miR), or RPMI8226HR1%O2 exosomes were treated with HUVEC/Luc/b-gal (HUVECs cotransfected with pMIRFIH1-39-UTR vector and b-gal control vector). Luciferase activity of the sensor vector
displayed a significant decrease by treatment with RPMI82261%O2/miR-135b exosome(**P , .01, Student t test, n 5 3) and RPMI8226-HR1%O2-exosome (*P , .05, Student
t test, n 5 3) compared with the control (HUVECs/Luc/b-gal without exosomes). Theseexperiments were performed in triplicate, and the results are shown as mean 6 SD.
(B) FIH-1 protein expression levels measured by immunoblot after treatment with
RPMI8226 20%O2 -exosome, RPMI8226 1%O2 -exosome, or RPMI8226-HR 1%O2 exosome of normoxic HUVECs (solid bars) and hypoxic HUVECs (open bars). The
intensity of each band was quantified and normalized to FIH-1 expression signals
by b-actin (**P , .01 vs control, Student t test, n 5 3). (C) Effect of exosomes derived
from HR-MM cells (RPMI8226-HR) on HIF-1–dependent transactivation of luciferase
activity in normoxic HUVECs (solid bars) and hypoxic HUVECs (open bars) transfectedwith luciferase reporter genes linked to hypoxia response element (HRE). Luciferase
activity was measured using the Dual-Glo Luciferase System (Promega). All assays
were performed in triplicate. Means 6 SD are shown (**P , .01 vs control; HUVECs1%
O2
without exosomes, Student t test, n 5 3). exo, exosome.

from chronic HR cells functioned as a signaling factor to transmit
a hypoxic response. miR-135b may also contribute to angiogenesis
in anaplastic large-cell lymphoma, implying that it plays a central
role in regulating several signaling pathways. 36-38 In HR-MM
cells, we identiﬁed the direct target of miR-135b, FIH-1, which is an
asparaginyl hydroxylase enzyme binding to HIF-1a that inhibits
its transactivation function.39 Exosomal miR-135b may have theability to improve the surrounding BM microenvironment by HR-MM
cells via the HIF-FIH signaling pathway.

This study has several limitations. Tumor angiogenesis is known
to be regulated by various kinds of factors, including cytokines,
chemokines, and growth factors derived from the BM microenvironment such that a complex mechanism may support MM diseaseprogression and resistance to chemotherapy.40,41 Exosomes carryvarious molecules, including proteins, lipids, mRNAs, and miRNA.
Thus, further studies are required to understand how soluble factors
and/or exosomal contents affect miR-135b in the BM microenvironment in MM. There is another issue that needs to be addressed.
A recent study by King et al has clearly demonstrated that hypoxia
promotes the release of exosomes in breast cancer cells: the amountof exosomes increased when cells were incubated under hypoxia.42
Therefore, it is important to take into consideration the amount of
exosomes released in hypoxia. In the current study, we demonstrated
both the quality and quantity of exosomes from HR-MM cells;
however, it is still unknown whether known members of the HIF
pathway affect exosome pathways or processing. Most importantly,
the clinical relevance of miR-135b expression in MM patients is still
uncertain. We and others have reported miRNA expression proﬁlesin MM, but miR-135b expression was barely detected in MM cells
and plasma,43 which is consistent with our results using parental MM
cell lines. In addition, we have determined the exosomal miR-135b
expression level in plasma of 15 MM patients and 5 healthy
volunteers. However, the expression levels of exosomal miR-135b
in MM patients were signiﬁcantly lower compared with those ofhealthy volunteers (supplemental Figure 5). The most plausible
explanation for this phenomenon is that exosomal miR-135b
might play a major role in local area rather than circulatingplasma. For this reason, plasma miR-135b expression is not asuitable prognostic factor at present, but exosomal miR-135b might
be a potent molecular target related to local tumor angiogenesis. We
also investigated exosomes from the primary myeloma cells of 2
MM patients (supplemental Figure 6), and the ﬁndings indicatethat at least a subset of MM patients exhibits high miR-135b
expression, whereas MM patients are a highly heterogeneousgroup. Even when miR-135b was elevated in exosomes fromMM cells of patient 2 (supplemental Figure 5), plasma miR135b expression level was low. Furthermore, the Matrigel plug
assay may provide evidence of an important role for exosomal
miR-135b derived from HR-MM cells on angiogenesis in vivo
(Figure 6).
In conclusion, we constructed an in vitro HR-MM cell modelthat reﬂects prolonged intratumor hypoxia. The majority of previous
studies refer to acute hypoxia, which may differ somewhat fromin vivo conditions. To our knowledge, this is the ﬁrst report to deal
with cell-cell communication via exosomes under chronic hypoxia.
Using this model, we provide new evidence that hypoxia-driven
accelerated tube formation is attributable to exosomal miR-135b
shed from HR-MM cells by targeting the HIF-1/FIH signaling
pathway.

Acknowledgments
The authors thank C. Kobayashi and Y. Yamamoto for their technicalassistance.
This study was supported in part by the Private University
Strategic Research-Based Support Project (S1311016) from the
Ministry of Education, Culture, Sports, Science, and Technology(Tokyo, Japan); and the Kobayashi Foundation for Cancer research(Tokyo, Japan).

-270-

From www.bloodjournal.org by guest on April 28, 2018. For personal use only.
BLOOD, 11 DECEMBER 2014 x VOLUME 124, NUMBER 25

miR-135b ENHANCES ANGIOGENESIS

Authorship
Contribution: T.U., K.O., and J.H.O. designed the research and wrote
the paper; T.U., H.T., and K.A. performed research and analyzed
data; and S.Y. provided patient samples.

3757

Conﬂict-of-interest disclosure: K.O. received research support from Celgene KK, Chugai Pharmaceutical KK, and Janssen
Pharma KK.
Correspondence: Tomohiro Umezu, Tokyo Medical University,
6-7-1 Nishi-shinjuku, Shinjuku, Tokyo 160-0023, Japan; e-mail:t_umezu@tokyo-med.ac.jp.

References
1. Palumbo A, Anderson K. Multiple myeloma.
N Engl J Med. 2011;364(11):1046-1060.
2. Kyle RA, Rajkumar SV. Multiple myeloma. Blood.
2008;111(6):2962-2972.
3. Kumar SK, Rajkumar SV, Dispenzieri A, et al.
Improved survival in multiple myeloma and theimpact of novel therapies. Blood. 2008;111(5):2516-2520.
4. Agarwal A, Mahadevan D. Novel targeted
therapies and combinations for the treatment ofmultiple myeloma. Cardiovasc Hematol Disord
Drug Targets. 2013;13(1):2-15.

paracrine tumor-stromal cell interactions in
multiple myeloma. Blood. 2000;95(8):2630-2636.
18. Giuliani N, Colla S, Lazzaretti M, et al.
Proangiogenic properties of human myeloma
cells: production of angiopoietin-1 and its potential
relationship to myeloma-induced angiogenesis.
Blood. 2003;102(2):638-645.
19. Colla S, Morandi F, Lazzaretti M, et al. Human
myeloma cells express the bone regulating geneRunx2/Cbfa1 and produce osteopontin that is
involved in angiogenesis in multiple myeloma
patients. Leukemia. 2005;19(12):2166-2176.

5. Fu C, Wang J, Xin X, et al. Therapeutic effects
of autologous hematopoietic stem celltransplantation in multiple myeloma patients. Exp
Ther Med. 2013;6(4):977-982.

20. Tadokoro H, Umezu T, Ohyashiki K, Hirano T,
Ohyashiki JH. Exosomes derived from hypoxic
leukemia cells enhance tube formation inendothelial cells. J Biol Chem. 2013;288(48):34343-34351.

6. Laubach JP, Mahindra A, Mitsiades CS, et al. The
use of novel agents in the treatment of relapsed
and refractory multiple myeloma. Leukemia. 2009;23(12):2222-2232.

21. Asosingh K, De Raeve H, de Ridder M, et al. Roleof the hypoxic bone marrow microenvironment in
5T2MM murine myeloma tumor progression.
Haematologica. 2005;90(6):810-817.

7. van de Donk NW, Lokhorst HM, Dimopoulos M,
et al. Treatment of relapsed and refractory
multiple myeloma in the era of novel agents.
Cancer Treat Rev. 2011;37(4):266-283.

22. Martin SK, Diamond P, Gronthos S, Peet DJ,
Zannettino AC. The emerging role of hypoxia,
HIF-1 and HIF-2 in multiple myeloma. Leukemia.
2011;25(10):1533-1542.

8. Quail DF, Joyce JA. Microenvironmental
regulation of tumor progression and metastasis.
Nat Med. 2013;19(11):1423-1437.

23. Hu J, Van Valckenborgh E, Menu E, De Bruyne E,
Vanderkerken K. Understanding the hypoxic
niche of multiple myeloma: therapeutic
implications and contributions of mouse models.
Dis Model Mech. 2012;5(6):763-771.

9. Mizukami Y, Kohgo Y, Chung DC. Hypoxia
inducible factor-1 independent pathways in tumor
angiogenesis. Clin Cancer Res. 2007;13(19):5670-5674.
10. Tonini T, Rossi F, Claudio PP. Molecular basis ofangiogenesis and cancer. Oncogene. 2003;
22(42):6549-6556.
11. Kocemba KA, van Andel H, de Haan-Kramer A,
et al. The hypoxia target adrenomedullin isaberrantly expressed in multiple myeloma andpromotes angiogenesis. Leukemia. 2013;27(8):1729-1737.
12. Rajkumar SV, Leong T, Roche PC, et al.
Prognostic value of bone marrow angiogenesis in
multiple myeloma. Clin Cancer Res. 2000;6(8):3111-3116.
13. Vacca A, Ribatti D, Roncali L, et al. Bone marrow
angiogenesis and progression in multiple
myeloma. Br J Haematol. 1994;87(3):503-508.
14. Otjacques E, Binsfeld M, Noel A, Beguin Y,
Cataldo D, Caers J. Biological aspects ofangiogenesis in multiple myeloma. Int J Hematol.
2011;94(6):505-518.
15. Giuliani N, Storti P, Bolzoni M, Palma BD,
Bonomini S. Angiogenesis and multiple myeloma.
Cancer Microenviron. 2011;4(3):325-337.
16. Munshi NC, Wilson C. Increased bone marrow
microvessel density in newly diagnosed multiple
myeloma carries a poor prognosis. Semin Oncol.
2001;28(6):565-569.
17. Dankbar B, Padró T, Leo R, et al. Vascularendothelial growth factor and interleukin-6 in

24. Azab AK, Hu J, Quang P, et al. Hypoxia promotes
dissemination of multiple myeloma throughacquisition of epithelial to mesenchymaltransition-like features. Blood. 2012;119(24):5782-5794.
25. Umezu T, Ohyashiki K, Kuroda M, Ohyashiki JH.
Leukemia cell to endothelial cell communication
via exosomal miRNAs. Oncogene. 2013;32(22):2747-2755.
26. Storti P, Bolzoni M, Donofrio G, et al. Hypoxiainducible factor (HIF)-1a suppression in myeloma
cells blocks tumoral growth in vivo inhibiting
angiogenesis and bone destruction. Leukemia.
2013;27(8):1697-1706.
27. Podar K, Anderson KC. The pathophysiologic roleof VEGF in hematologic malignancies: therapeutic
implications. Blood. 2005;105(4):1383-1395.
28. Bache M, Kappler M, Said HM, Staab A,
Vordermark D. Detection and specific targeting of
hypoxic regions within solid tumors: current
preclinical and clinical strategies. Curr Med Chem.
2008;15(4):322-338.
29. Burroughs SK, Kaluz S, Wang D, Wang K,
Van Meir EG, Wang B. Hypoxia inducible factor
pathway inhibitors as anticancer therapeutics.
Future Med Chem. 2013;5(5):553-572.
30. Chaidos A, Barnes CP, Cowan G, et al. Clinicaldrug resistance linked to interconvertible
phenotypic and functional states of tumorpropagating cells in multiple myeloma. Blood.
2013;121(2):318-328.

-271-

31. Wu W, Wang Z, Yang P, et al. MicroRNA-135b
regulates metastasis suppressor 1 expression
and promotes migration and invasion in colorectal
cancer. Mol Cell Biochem. 2014;388(1-2):
249-259.
32. Xu XM, Qian JC, Deng ZL, et al. Expression of
miR-21, miR-31, miR-96 and miR-135b iscorrelated with the clinical parameters ofcolorectal cancer. Oncol Lett. 2012;4(2):339-345.
33. Lulla RR, Costa FF, Bischof JM, et al.
Identification of differentially expressed
microRNAs in osteosarcoma. Sarcoma. 2011;2011:732690.
34. Lin CW, Chang YL, Chang YC, et al. MicroRNA135b promotes lung cancer metastasis byregulating multiple targets in the Hippo pathway
and LZTS1. Nat Commun. 2013;4:1877.
35. Xu S, Cecilia Santini G, De Veirman K, et al.
Upregulation of miR-135b is involved in the
impaired osteogenic differentiation ofmesenchymal stem cells derived from multiple
myeloma patients. PLoS ONE. 2013;8(11):e79752.
36. Zhang L, Sun ZJ, Bian Y, Kulkarni AB. MicroRNA135b acts as a tumor promoter by targeting thehypoxia-inducible factor pathway in geneticallydefined mouse model of head and necksquamous cell carcinoma. Cancer Lett. 2013;
331(2):230-238.
37. Khatri R, Subramanian S. MicroRNA-135b and its
circuitry networks as potential therapeutic targets
in colon cancer. Front Oncol. 2013;3:268.
38. Matsuyama H, Suzuki HI, Nishimori H, et al.
miR-135b mediates NPM-ALK-driven
oncogenicity and renders IL-17-producing
immunophenotype to anaplastic large cell
lymphoma. Blood. 2011;118(26):6881-6892.
39. Mahon PC, Hirota K, Semenza GL. FIH-1: a novel
protein that interacts with HIF-1alpha and VHL tomediate repression of HIF-1 transcriptional
activity. Genes Dev. 2001;15(20):2675-2686.
40. Caers J, Van Valckenborgh E, Menu E, Van
Camp B, Vanderkerken K. Unraveling the biologyof multiple myeloma disease: cancer stem cells,
acquired intracellular changes and interactions
with the surrounding micro-environment. Bull
Cancer. 2008;95(3):301-313.
41. Hideshima T, Mitsiades C, Tonon G, Richardson
PG, Anderson KC. Understanding multiple
myeloma pathogenesis in the bone marrow to
identify new therapeutic targets. Nat Rev Cancer.
2007;7(8):585-598.
42. King HW, Michael MZ, Gleadle JM. Hypoxic
enhancement of exosome release by breast
cancer cells. BMC Cancer. 2012;12:421.
43. Yoshizawa S, Ohyashiki JH, Ohyashiki M, et al.
Downregulated plasma miR-92a levels haveclinical impact on multiple myeloma and relateddisorders. Blood Cancer J. 2012;2(1):e53.

From www.bloodjournal.org by guest on April 28, 2018. For personal use only.

2014 124: 3748-3757
doi:10.1182/blood-2014-05-576116 originally published
online October 15, 2014

Exosomal miR-135b shed from hypoxic multiple myeloma cells
enhances angiogenesis by targeting factor-inhibiting HIF-1
Tomohiro Umezu, Hiroko Tadokoro, Kenko Azuma, Seiichiro Yoshizawa, Kazuma Ohyashiki and
Junko H. Ohyashiki

Updated information and services can be found at:
http://www.bloodjournal.org/content/124/25/3748.full.html
Articles on similar topics can be found in the following Blood collections
Lymphoid Neoplasia (2773 articles)
Multiple Myeloma (395 articles)
Vascular Biology (509 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml

Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Societyof Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.

-272-

Journal of Hematology & Transfusion

CentralResearch Article

Detection of BIM (BCL2L11)
Polymorphic Variants in Chronic
Myeloid Leukemia by Q-Invader
Assay and Their ClinicalSignificance
1,3

2

Kazuma Ohyashiki *, Kenichi Tadokoro , Toshikazu
Yamaguchi2, Seiichiro Katagiri1, Tomohiro Umezu3, Kazushige
Ohtsuki4, Tetsuzo Tauchi1, Toshinori Yamamoto2 and Junko H.
Ohyashiki4*Corresponding author
Kazuma Ohyashiki, Department of Hematology, Tokyo
Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo
160-0023, Japan, Tel: 81-3-3342-1510; Fax: 81-5381-6651,
E-mail:Submitted: 09 August 2014
Accepted: 01 November 2014
Published: 03 November 2014
ISSN: 2333-6684
Copyright© 2014 Ohyashiki et al.
OPEN ACCESS

Keywords• Chronic myeloid leukemia• Imatinib• BIM polymorphism

Department of Hematology, Tokyo Medical University, Japan
Development of Clinical Genomics, BML. Inc., Japan3
Department of Molecular Science, Tokyo Medical University, Japan
4
Division of Molecular Oncology, Institute of Medical Science, Tokyo Medical
University, Japan
1
2

Abstract
Background: Accumulating evidence suggests that genetic variants, including
deletion and single nucleotide polymorphism (SNP) have role in the genesis andprogression of various cancers. BIM (also known as BCL2L11) is a proapoptotic proteinthat is essential in the tyrosine kinase inhibitor (TKI)-induced apoptosis in chronic myeloid
leukemia (CML) cells. We therefore attempted to develop new detection assay systemof the BIM genetic variants in CML patients and clinical relevance.
Subjects and methods: We assessed knownBIM polymorphic variants (BIM
deletion polymorphism in intron 2 and silent SNP in BIM exon 5) by using the Q-Invader
method with molecular response by TKIs in 47 Japanese chronic myeloid leukemia(CML) patients who achieved 4-log reduction of molecular response (MR4.0) or more.
Results: The Q-Invader assay was able to detect BIM deletion polymorphism
expanding approximately 2900 bp and BIMSNP at exon 5 (c465C>T). Six of 47(12.8%) showed the BIM intron 2 deletion polymorphism and 11 of 47 (23.4%) CML
patients did the SNP. In healthy volunteers, 4 of 20 (20%) had the BIM deletion
polymorphism and 4 of 20 (20%) did the BIM SNP: none of our subjects had the
BIM deletion polymorphism and SNP (c465C>T) concurrently. CML patients with BIM
polymorphic variants showed high frequencies of reduction of imatinib dose andswitching to second-line
Conclusion: TKIs. BIM is an essential protein for the apoptotic process in cancer
cells with TKI therapy, the detection assay of polymorphic variants, such as Q-Invader
assay, could be useful in clinical practice.

BACKGROUND
Tyrosine kinase inhibitors (TKIs) are now used as the central
therapeutic approach for treatment of several tumors, including
chronic myeloid leukemia (CML), and the introduction of TKIs asa first-line treatment for CML has introduced new possibilities forcuring CML [1,2], for example, the STOP Imatinib (IM) study by

Mahon et al. [3]. It is well-known that TKIs, including IM, activate
proapoptotic BCL-2 homology domain 3–only proteins, such as
BCL2-like 11 (BCL2L11, also known as BIM) [4]. Therefore, BIM
plays a major role in TKI-induced apoptosis of BCR-ABL1−positive
CML cells [5]. Recently, Ng et al. [6] demonstrated that East Asian
CML patients with a common intronic deletion polymorphism in
the gene coding BIM (at intron 2) had inferior responses to TKIs

Cite this article: Ohyashiki K, Tadokoro K, Yamaguchi T, Katagiri S, Umezu T, et al. (2014) Detection of BIM (BCL2L11) Polymorphic Variants in Chronic
Myeloid Leukemia by Q-Invader Assay and Their Clinical Significance. J Hematol Transfus 2(3): 1032.

-273-

Ohyashiki et al. (2014)
Email:

CentralA

Prim-

Q-BIM-F1

AC096670

107801:AACTCAACAAACCCATCAGAACAGACACTGGAACAAAATGACATTTCTAAATACCATCCAGCTCTGTCTTCATAGGCTTCAGTGAGGTAAATCAGGCAGG 107900

Invader oligo
Q-BIM-WR1

ary probe 1

AC096670

107901:CCTTTGCCCATGTTATAGAATTGGAAAGAACCTCAGAGTGGTGGTCACTTGTCAGAGGTTGGGCACACCTGTGAGGTGGTGGGGAGAAATGACAGACATC 108000

AC096670

108001:CCAGCAGCTACACATGCTGGCTGCACGTCTCTTGCCAAATGCCAGGAGGTAATTTTTTAGGGTCCCTCCTTAGGGAAAGGGGCTGGAAGTTTTATTATTG 108100

AC096670

110601:TCATTTATCCATTCATCCATCAGGAGATACTTGGGTTGCTTCCACTTTTTGGCTATTGTGAGTAGTGCTGCTATGAACATGGGTATGCAAATATCTTTTG 110700

AC096670

110701:GGGGATTCTGCTTTGAATTTTTTTGGATATATACTTGGAAGTGGAATTGCTGGATCATATGGTAATTCTATTTTTAATTTTTTGGGGAACCATCATGCTG 110800

AC096670

110801:TTCTCCATAGAGGCTGTGCCATTTTACATTCCCACCAACAGGGCACAAGGGTTCCAGTTTCTCCACATACTTACCAACACTTTTTTTTTTTTTTTTTTAA 110900

Primary probe 2

Q-BIM-dR1:Deletion region
Invader probes
Primary probe 1: CGCGCCGAGGGGCAGCCTTTGCCCATG
Primary probe 2（for deletion）: ACGGACGCGGAGCTGTTCTCCATAGAGGCTGTG
Invader oligo: TCTTCATAGGCTTCAGTGAGGTAAATCAA
Primers
Q-BIM-F1: CAGAACAGACACTGGAACAA
Q-BIM-WR1: CTGAGGTTCTTTCCAATTCT
Q-BIM-dR1: CCCTGTTGGTGGGAATGTAA

B

rs724710-F2

ATTTTTGTTTTGTTTTGTTCTGATGCAGCTTCCATGAGGCAGGCTGAACCTGCAGATATGCGCCCAG

Primary probe
AGATATGGATCGCCCAAGAGTTGCGGCGTATYGGAGACGAGTTTAACGCTTACTATGCAAGGAGGGT
Invader oligo
rs724710-R2

AATGATGTTTTCTTTACCCGCTTTTCTGCTCACACCCTCCCCTTCCACACTATATTTTTTTAAAAAG

Invader probes
Primary probe 1: CGCGCCGAGGCGGAGACGAGTTTAACGC
Primary probe 2: ACGGACGCGGAGTGGAGACGAGTTTAACGC
Invader oligo: GGATCGCCCAAGAGTTGCGGCGTATC
Primers
rs724710-F2 : AGGCAGGCTGAACCTGCAGA
rs724710-R2 : TGTGAGCAGAAAAGCGGGTA

Figure 1 Design used to detect BIM genetic variants. A: Cleavage point for the Q-Invader assay at BIM deletion polymorphism. Cleavage points inpolymerase chain reaction amplicon (white boxes). Deletion area at intron 2 (shaded area). B: Cleavage point for the Q-Invader assay at rs724710.
Cleavage point in polymerase chain reaction amplicon (white box).

compared with those without the genetic variation. Augis et al.
[7] also found a single nucleotide polymorphism (SNP) c465C>Tat BIM exon 5, without amino acid change, in French CML patients
who did not respond to IM treatment. The detection of BIM
genetic variants is critical for TKI therapy, not only for CML but
also for other cancers [6].
Q-Invader assay is widely used to detect SNP in clinicalpractice [8,9]; however, this technique is not generally
available to detect deletion polymorphism, such as BIM deletion
polymorphism, because its deletion expands approximately 3 kb.
In the current study, we ascertained that Q-Invader assay could

detect BIM deletion polymorphism at intron 2 and SNP c465C>Tat BIM exon 5. We used the Q-Invader assay and determinedthe clinical implications in CML patients with maintained major
molecular response.

PATIENTS AND METHODSPatients

To investigate genetic variations of BIM, we obtained DNA
from 47 Japanese CML patients referred to the Tokyo Medical
University Hospital who had achieved molecular remission (MR)of 4.0 or more [10]. Since all CML patients studied achieved MR4.0,

J Hematol Transfus 2(3): 1032 (2014)

-274-

2/6

Ohyashiki et al. (2014)
Email:

CentralDenature step

Invader reaction in duplex format
Primary
probe

Invaderoligo

Primary
probe

Invaderoligo

Cleavase

Cleavase

Non-deletion amplicon

Deletion amplicon

Released
Flap at5’-endReleased
Flap at5’-endF

Fluorescence
Probe F

FluorescenceR

Q

Q Probe R

Q

Q

Cleavase

Cleavase

Identify non-deletion allele

Identify deletion allele

Figure 2 Schematic illustration of the Q-Invader assay in a duplex format for detecting BIM polymorphisms. Human DNA is amplified by two-step
PCR. The denatured PCR products formed an invasive complex with the primary probe and Invader oligo. The released 5’-flap probe, a product of
the first reaction, promotes cleavage of the fluorescence probe in the second Invader reaction, creating a detectable, amplified signal for detecting
BIM polymorphisms. Finally, the PCR products were made into duplex DNA with DNA polymerase.

DNAs obtained from peripheral blood from CML patients are of
representative germ-line change rather than somatic CML cells.
There were 10 patients who maintained MR for more than 12
months after discontinuation of IM, 3 patients who experienced
relapse within 4 months after cessation of IM, 16 patients who
maintained MR for 24 consecutive months while using TKIs,
and 18 patients treated with TKIs who showed fluctuating MR(but maintained major molecular response) over the courseof 24 months. We also studied DNA obtained from 20 healthy
volunteers used as the Japanese control group. This study wasapproved by the Institutional Review Board of Tokyo Medical
University (no. 1655; approved on January 28, 2011). KCL22
was used as a positive control and K562 was used as a negative
control for the BIM deletion polymorphism of intron 2 [6,10].

Detection of BIM deletion polymorphism and SNP by
Q-Invader techniquePreparation of genomic DNA was performed with a robotic
workstation (Magtraction System, 6GC; Precision System
Science, Chiba, Japan) with the EZ1 DNA Blood 350 μl kit (Qiagen,
Valencia, CA, USA) according to the manufacturer’s instructions.
The genomic DNA was subjected to polymerase chain reaction
amplification using primer designed to detect a deletion site(2903 bp) in intron 2 (deletion polymorphism) (Figure 1-A)

and a silent SNP in exon5 (c465C>T) (Figure 1-B), separately.
Thus, Two fluorescence signals (carboxyfluorescein or FAM fornon-deletion polymorphism in intron 2 and C allele in exon 5;
REDmond RED or RED for deletion polymorphism in intron 2 andT allele in exon5) could be detected in each single reaction with a
Universal General Purpose Reagent (Hologic, Bedford, MA, USA),
including Cleavase and FRET mix with two common fluorescenceprobes (Figure 2). Template DNA was added to a 15-μl reaction
mixture containing 500 nM primers (Sigma Aldrich Japan, Tokyo,
Japan)for amplification BIM gene, 600 nM of each primary probe(Sigma Aldrich Japan), 70 nM Invader oligo (Sigma Aldrich
Japan), Universal General Purpose Reagent (Hologic) and FRET
mix (Table 1). The reaction mixture was preheated in a 384-PCR
plate (Roche, Basel, Switzerland) at 95°C for 2 min, and a twostep PCR reaction was carried out for 40 cycles (95°C for 15 sec,
65°C for 60 sec) in a LightCycler 480 (Roche) [8,9]. Fluorescencevalues of FAM (wavelength/bandwidth: excitation, 465 nm;
emission, 510 nm) and RED (excitation, 533 nm; emission, 610nm) were measured at end of the incubation/extension step at
65°C for each cycle and by standard real-time PCR. By analyzing
the results, a crossing point (Cp) can be obtained by an endpoint
genotyping method in the Light Cycler 480 software.

Statistical analysis

The results were statistically analyzed with GraphPad Prism

3/6

J Hematol Transfus 2(3): 1032 (2014)-275-

Ohyashiki et al. (2014)
Email:

CentralTable 1: Polymerase−chain−reaction primer for detection of BIM deletion
polymorphism and BIM single−nucleotide−polymorphism.
Polymerase−chain−reaction primer

Q-BIM-F1 (polymorphism): 5’-CCTCGTGGAAGGCGACAACCTAT-3’
Q-BIM-WR1 (polymorphism): 5’-GGCCADGGRTACCCRGGCTG-3’
Q-BIM-dR1 (polymorphism): 5’-CCTGGGCTCAGCCYGGGTA-3’
rs724710-F2 (SNP): 5’-AGGCAGGCTGAACCTGCAGA-3’

rs724710-R2 (SNP): 5’-TGTGAGCAGAAAAGCGGGTA-3’
Invader probes

asBIMdel-p1: 5’-CGCGCCGAGGGGCAGCCTTTGCCCATG-3’

asBIMdel-p2: 5’-ACGGACGCGGAGCTGTTCTCCATAGAGGCTGTG-3’
asBIMdel-io: 5’-TCTTCATAGGCTTCAGTGAGGTAAATCAA-3’

as_rs724710-p1: 5’-CGCGCCGAGGCGGAGACGAGTTTAACGC-3’

as_rs724710-p2: 5’-ACGGACGCGGAGTGGAGACGAGTTTAACGC-3’
as_rs724710-io: 5’-GGATCGCCCAAGAGTTGCGGCGTATC-3’

p1: primary probe (FAM)
p2: primary probe (RED)
io: Inavaderoligo
5’−prime of the p1 probe (CGGCCGAGG) and 5’−prime of the p2 probe (ACGGACGCGGAG) are FLAP sequence.
3’-prime of P1 and P2 probes are amino modification
io: Invader oligo, p1: primary probe (FAM), p2: primary probe (Red),
SNP: single nucleotide polymorphism.

5 (GraphPad Software, La Jolla, CA, USA). The profiles of twogroups were analyzed by chi-square test. P< 0.05 was considered
to indicate a statistically significant difference.

RESULTS

Using the Q-Invader assay, the fluorescent intensity of FAM
increases when the sample has no deletion polymorphism orno SNP (c465), whereas the intensity of Red increases when
the sample has the intron 2BIM deletion polymorphism or SNP(c465C>T). These two fluorescent intensities plot the x-axis andthe y-axis, respectively, and the polymorphism is identified by thescatter plot (Figure 3). Data for the BIM deletion polymorphism
were confirmed by standard polymerase chain reaction assay
and gel electrophoresis, as described previously [10]. Q-Invader
assay was used for assessment of BIM polymorphic variants in
the Japanese population.

None of the CML patients or healthy subjects had the BIM
deletion polymorphism and SNP (c465C>T) concurrently; thus,
we analyzed BIM polymorphism variants separately and then
combined as a group of BIM genetic variants. None of our subjects
showed T/T genotype of the c465C>T SNP. Six of 47 (12.8%)
Japanese CML patients and 4 of 20 (20%) Japanese volunteers
showed the BIM intron 2 deletion polymorphism. Regarding the
SNP (c465C>T), using the Q-Invader technique, 11 of 47 (23.4%)
CML patients and 4 of 20 (20%) volunteers showed the SNP. As
reported previously, 3 of 13 CML patients who stopped IM had
the BIM deletion polymorphism, and all 3 patients with BIM
deletion had molecular relapse within 4 months, whereas none of
the remaining 10 patients without the BIM deletion maintained
molecular response after stopping IM [10]. Although we foundthree CML patients with BIM SNP at c465C>T in the group that
stopped using IM, none of them had molecular relapse. Of the 34

CML patients who experienced MR of 4.0 while still taking TKIs, 3
of 34 (8.8%) had BIM deletion polymorphism and 8 of 34 (23.5%)
had BIM SNP (c465C>T).

We next assessed clinical features of 44 CML patients
showing the BIM deletion polymorphism or BIM SNP (c465C>T;BIM polymorphism variants). Of the 47 CML patients with BIM
status, three were excluded from the clinical analysis because
of inappropriate IM intake (one with previous chemotherapy
and two with low adherence). Thirty patients were treated with
first-line IM without previous alpha-interferon therapy. Noneof the 28 CML patients without BIM genetic variants switched
to second-line TKIs, whereas CML patients with either BIM SNP(2/10 versus 0/34; P = 0.1722) or BIM deletion polymorphism(2/6 versus 0/38; P = 0.0262) had a history of switching tosecond-line TKIs. When we combined genetic variants of the BIM
gene (intron 2 deletion polymorphism and SNP [c465C>T]), asignificantly high frequency of switching to second-line TKIs in
CML patients with BIM genetic variants was again noted (4/16
versus 0/28; P = 0.0055) (Table 2). Switching to second-line
TKIs in our CML patients was attributable to hematologic or
non-hematologic adverse events, but not to IM resistance. We
next assessed IM dose for these CML patients. The frequency ofcontinuing the IM dose of 400 mg was significantly higher in CML
patients without BIM genetic variants than in those with genetic
variants (25/28 versus 6/16; P = 0.0003). These observations
indicate that Japanese CML patients without BIM genetic variants(deletion polymorphism at intron 2 and SNP [c465C>T]) could
benefit from the standard IM dose without switching to secondline TKIs. This tendency was also evident in 30 CML patientsadministered first-line IM therapy (data not shown).

DISCUSSION

In the current study, we determined BIM genetic variants
in Japanese CML patients using the Q-Invader assay. The
current Q-Invader assay could clarify which subjects have
BIM SNP as well as the long-distance deletion, such as BIM
deletion polymorphism at intron 2, separately. Therefore inclinical setting, the Q-invader assay should be performed twiceon the same patient sample, since none of our sample showed

Table 2: Correlation between BIM genetic variants and response totyrosine kinase inhibitors.
CML
patients

BIM SNP (c465c>T)
P value

BIM deletion
polymorphism
P value

BIM SNP or deletion
Polymorphism
P value

No genetic variants

10/44
2/44
16/44
28/44

Maintainimatinib

Imatinibdose

400 mgreduction
4/10

2/10

2/6

2/6
0.4

2/6

0.02620.00030.03410.0055

4/10

0.032

25/28

3/28

Switch tosecondTKIs

0/28

P values were calculated by chi-square test.
CML: chronic myeloid leukemia, SNP: single nucleotide polymorphism,
TKI: tyrosine kinase inhibitor.

4/6

J Hematol Transfus 2(3): 1032 (2014)-276-

Ohyashiki et al. (2014)
Email:

Central

B

A

RED

RED

C/T
Deletion Heterozygote

C/C

Figure 3 Scatter plot of the fluorescent intensity of FAM and Red. The fluorescent intensity of FAM increases when the sample has no deletion
polymorphism or no SNP (c465), whereas the intensity of Red increases when the sample has BIM deletion polymorphism (A) or BIM SNP (c465C>T)(B). These two fluorescent intensities plot the x-axis and y-axis, respectively, and the polymorphism is identified by the scatter plot. A: BIM deletion
heterozygote (circle) and non-BIM deletion homozygote. B: Scatter plot of rs724710 polymorphism for the patients. C/T heterozygote (circle) andC/C homozygote.

concomitant positivity for these two genetic variants. The BIM
genetic variants were detected in 40% of the Japanese control
population; 20% had intron 2 deletion polymorphism and 20%
had exon 5 SNP of the BIM gene. Ng et al. [6] reported that 12.3%
of East Asian healthy individuals had BIM intron 2 deletion
polymorphism. They reported that CML patients without the BIM
deletion polymorphism had a significantly high optimal responsecompared with those with the BIM genetic variant because ofswitched BIM splicing from exon 4 to exon 3, resulting in lack of
the proapoptotic BCL-2 homology domain 3 [6]. Augis et al. [7]also reported another BIM genetic variant at exon 5 in French
CML patients. This genetic variant was significantly associatedwith a late achievement of major molecular response thereby
leading frequent BCR-ABL1 tyrosine kinase domain mutations[7]. Our previous study also demonstrated that CML patients with
BIM deletion polymorphism are IM-dependent [10], suggestingthat BIM dysfunction attributable to polymorphism variants isessential in TKI treatment for CML patients. Since approximately
45% of CML patients who showed resistant to IM treatment had
no BCR-ABL1 domain mutation, BIM genetic variants, including
BIM-SNP, may have some role in such patients who resist to TKIs.
It has also been demonstrated that intron 2BIM deletion
polymorphism in epidermal growth factor receptor (EGFR)
mutation-positive nonsmall cell lung cancer showed intrinsic
resistance to EGFR TKIs [6,11], and the resistance wasovercome by the combination of TKIs and histone deacetylase
inhibitor because of restoration of the aberrant splicing of BIM
and upregulation of BCL-2 homology domain 3–containing
BIM isoforms to resensitize to EGFR TKIs [11]. However, thecorrelation between BIM SNP at exon 5 (c465C>T) and theinferior response to TKIs in non-small cell lung cancer has notbeen reported. Furthermore, biological implication of BIM-SNP
at exon 5 (c465C>T) should be clarified in various neoplasias.
The BIM deletion polymorphism is uncommon in Caucasians

and African Americans, whereas BIM SNP at exon 5 (c465C>T)
has been found in the Caucasian cohort [7]. Moreover, BIM haplo
insufficiency may affect the immune system [12-14]; for example,
BIM dysfunction affects interleukin-15–mediated survival ofnatural killer cells [13]. Immune surveillance is now considered to
be a key element for prevention of cancer proliferation, including
CML. Since Augis et al. demonstrated a reduction of Bim mRNAlevels in the circulating mononuclear cells of healthy controls
with c465C>T genotype [7], further information regarding BIM
genetic variants and immune function should be accumulated.

CONCLUSIONS

Our results show that a high-throughput assay like thecurrent Q-Invader assay could be a powerful tool to delineate theclinical relevance of BIM dysfunction in various cancers. Finally,
the Q-Invader assay has clearly identified individuals with either
BIM deletion polymorphism or BIM SNP.

ACKNOWLEDGEMENTS

Kazuma Ohyashiki received research support and honoraria
for lecture fees from Bristol-Meyers Squibb KK and Novartis KK.

Author’s contributions

K.O.: designed and wrote the manuscript; K.T. and T.Y.:
developed and performed Q-Invader assay; S.K. and T.T.: collectedpatient samples; T.U. and K.O.: performed the polymerase
chain reaction technique; Y.Y.: supported this study; and J.H.O.:
reviewed the manuscript.

REFERENCES

1. Baccarani M, Deininger MW, Rosti G, Hochhaus A, Soverini S,
Apperley JF, et al. European LeukemiaNet recommendations for the
management of chronic myeloid leukemia. Blood. 2013; 122: 872-884.
2. Mauro

J Hematol Transfus 2(3): 1032 (2014)

-277-

M.

Striving

toachievesafe,

permanent

treatment

5/6

Ohyashiki et al. (2014)
Email:

Centraldiscontinuation in chronic myeloid leukemia. Leuk Res. 2013; 37:1395−1403.

3. Mahon FX, Réa D, Guilhot J,Guilhot F, Huguet F, Nicolini F, et al.
Discontinuation of imatinib in patients with chronic myeloid
leukaemia who have maintained complete molecular remission for atleast 2 years: the prospective. Multicenter Stop Imatinib (STIM) trial.
Lancet Oncol. 2010; 11: 1029-1035.

4. Kuroda J, Puthalakah H, Cragg MS,Kelly PN, Bouillet P, Huang DC, et al.
Bim and Bad mediate imatinib-induced killing of Bcr/Abl+ leukemic
cells and resistance due to their loss is overcome by a BH3 mimetic.
ProcNatlAcadSci USA. 2006; 103:14907-14912.
5. Kuribara R, Honda H, Matsui H, Shinjyo T, Inukai T, Sugita K, et al. Rolesof Bim in apoptosis of normal and Bcr-Abl-expressing hematopoietic
progenitors. Mol Cell Biol. 2004; 24: 61724-61783.
6. Ng KP, Hillmer AM, Chuah CT, Juan WC, Ko TK,Teo AS, et al. A common
BIM deletion polymorphism mediates intrinsic resistance and inferior
responses to tyrosine kinase inhibitors in cancer. Nat Med. 2012; 18:521-528.
7. Augis V, Airiau K,Josselin M, Turcq,Mahon FX, Belloc F. A single
nucleotide polymorphism in the cBIM is associated with a slow
achievement of major molecular response in chronic myeloid
leukaemia treated with Imatinib. Plos ONE. 2013; 8: e78582.
8. Tadokoro K, Kobayashi M, Suzuki F,Tanaka C, Yamaguchi T, Nagano
M,et al. Comparative quantitative analysis of hepatitis C mutations atamino acids 70 and 91 in the core region by the Q-Invader assay. J
Virol Meth. 2013; 189: 221-227.

9. Tadokoro K, Yamaguchi T, Egashira T, and Hara T. Quantitation ofviral load by real-time PCR-monitoring Invader reaction. J Virol Meth.
2009; 155: 182-186.

10. Katagiri S, Umezu T, Ohyashiki JH, Ohyashiki K. The BCL2L11 (BIM)
deletion polymorphism is a possible criterion for discontinuation of
imatinib in chronic myeloid leukaemia patients. Br J Haematol. 2013;
160: 269-271.
11. Nakagawa T, Takeuchi S, Yamada T, Ebi H, Sano T, Nanjo S, et al.
EGFR-TKI resistance due to BIM polymorphism can be circumvented
in communication with HDAC inhibition. Cancer Res. 2013; 73: 24282434.
12. Bouillet P, Purton JF, Godfrey DI,Zhang LC, Coultas L, Puthalakath
H, et al. BH3-only family member Bim is required for apoptosis of
autoreactive thymocytes. Nature. 2002; 415: 922-926.

13. Huntington ND, Puthalakath H, Gunn P, Naik E, Michalak EM, Smyth
MJ, et al. Interleukin-15 mediated survival of natural killer cells isdetermined by interacting among Bim, Noxa, and Ncl-1. Nat Immunol.
2007; 8: 856-863.
14. Sandalova E, Wei C-H, Masucci MG, Levitsky V. Regulation of
expression of Bcl-2 protein family Bim by T cell receptor triggering.
Proc Natl Acad Sci USA. 2004; 101: 3011-3016.

Cite this article
Ohyashiki K, Tadokoro K, Yamaguchi T, Katagiri S, Umezu T, et al. (2014) Detection of BIM (BCL2L11) Polymorphic Variants in Chronic Myeloid Leukemia by QInvader Assay and Their Clinical Significance. J Hematol Transfus 2(3): 1032.

6/6

J Hematol Transfus 2(3): 1032 (2014)-278-

Author's View

Author's View

OncoImmunology 3, e1; May 2014; © 2014 Landes Bioscience

Immunosurveillance markers may predict patients
who can discontinue imatinib therapy
without relapse
Takayuki Yoshimoto1,*, Izuru Mizoguchi1, Seiichiro Katagiri2, Tetsuzo Tauchi2, Jun-ichi Furusawa1, Yukino Chiba1,3,
Junichiro Mizuguchi3, Junko H Ohyashiki4, and Kazuma Ohyashiki2
Department of Immunoregulation; Institute of Medical Science; Tokyo Medical University; Tokyo, Japan; 2Department of Hematology; Tokyo Medical University; Tokyo, Japan;
3
Department of Immunology; Tokyo Medical University; Tokyo, Japan; 4Department of Molecular Oncology; Institute of Medical Science; Tokyo Medical University;
Tokyo, Japan

Keywords: chronic myeloid leukemia, imatinib, NK cells, cytotoxic T lymphocytes, predictive marker, immunosurvelliance
Abbreviations: CML, chronic myeloid leukemia; CMR, complete molecular response; CTL, cytotoxic lymphocyte; IFN,
interferon; NK, natural killer; TKI, tyrosine kinase inhibitor

Tyrosine kinase inhibitors have dramatically improved the treatment of chronic myeloid leukemia. Recent evidence
revealed that some patients with chronic myeloid leukemia can stop imatinib without relapse after achieving a complete
molecular response. This review discusses the possible predictive markers to identify these patients who can stop imatinib without relapse.

Imatinib, a tyrosine kinase inhibitor,
has dramatically improved the treatmentof chronic myeloid leukemia (CML).
Recent evidence has revealed that somepatients with CML can safely discontinue
imatinib therapy without relapse, particularly after achieving a complete molecularresponse. This review discusses the possible immunosurveillance predictive markers useful to discriminate patients who
may stop imatinib therapy without eliciting disease recurrence.
Chronic myeloid leukemia (CML) isa clonal myeloproliferative disorder ofhematopoietic stem cells caused by formation of the BCR-ABL1 chimeric geneencoding an aberrant tyrosine kinasewith oncogenic activity.1 Tyrosine kinase
inhibitors (TKIs) are the current standard-of-care treatment for patients with
CML. Imatinib (Glivec®) is the first TKI
used to treat chronic-phase CML, replacing conventional interferon α (IFNα)
administration.2 However, discontinuation of TKI therapy usually causes rapid
disease relapse, presumably due to thereactivation of dormant CML stem cells
that are resistant to TKI-induced leukemic cell ablation. TKI therapy is therefore
considered to be necessary throughout the
lifetime of the patient although an indefinite intake of TKI causes concerns about
long-term safety, tolerability, drug resistance, and costs. If CML can be cured
permitting safe cessation of an expensive
drug treatment, such as imatinib, thenboth personal and governmental medical
expenses could be expected to dramatically decrease without sacrificing patientcare. Of note, recent accumulating evidence indicates that some CML patients
can stop imatinib treatment without suffering disease relapse after achieving acomplete molecular response (CMR).3
Therefore, there is currently a strong needfor specific predictive markers that could
precisely determine which patients candiscontinue therapy without experiencing
relapse. To date, several markers have beenreported. Physiological variables associated with resistance to relapse include:
male sex, low Sokal risk score, shortertime to BCR-ABL1 negativity, longer
duration of CMR before discontinuation,and longer duration of imatinib therapy.3,4However, further investigation of thisissue in larger clinical studies encompassing more patients is necessary to provereliability.
It has been previously reported that
41% of imatinib-treated CML patientswith CMR lasting more than 2 y can safely
discontinue treatment without relapse.3 In
another study, a unique subset of CML
patients also demonstrated maintenanceof CMR after imatinib discontinuationand yet, intriguingly, high sensitivity
quantitative polymerase chain reaction
assay revealed that these patients harboredpersistent BCR-ABL1 translocated DNA.5
Thus, it may not be necessary to continue
imatinib therapy indefinitely, and some
CML patients can stop imatinib without
apparent disease relapse, despite the presence of persistent residual CML cells. Thisevidence strongly suggests that although
TKI therapy plays a central role in minimizing BCR-ABL1–positive CML cells,*Correspondence to: Takayuki Yoshimoto; Email: yoshimot@tokyo-med.ac.jp
Submitted: 04/08/2014; Accepted: 04/11/2014; Published Online: 05/14/2014
Citation: Yoshimoto T, Mizoguchi I, Katagiri S, Tauchi T, Furusawa Ji, Chiba Y, Mizuguchi J, Ohyashiki JH, Ohyashiki K. Immunosurveillance markers may predictpatients who can discontinue imatinib therapy without relapse. OncoImmunology 2014; 3:e28861; http://dx.doi.org/10.4161/onci.28861
www.landesbioscience.com	OncoImmunology

-279-

e1-1

© 2014 Landes Bioscience. Do not distribute.

1other endogenous factors could also bevital for restraining CML cells even in theabsence of TKIs. Among such native anticancer effectors are immune cells mediating immunosurveillance. Increasing
evidence suggests that natural killer (NK)cells play an important role in controlling growth of CML cells and sustaining
CMR.6-9 Recently, CML patients who sustained a CMR after imatinib discontinuation were shown to exhibit higher levelsof functional NK cells than either normal(non-diseased) subjects or CML patients
who did not sustain a CMR but did
maintain a major molecular response formore than 2 y with continuing imitinib
therapy (Fig. 1A).7 In accordance withthis report, increased counts of NK cells
have also been reported for IFNα-treated
CML patients who were able to discontinue treatment without relapse.8 The
essential role of NK cells in constraining
CML relapse has also been demonstrated
by implantation of NK cells into thebone marrow of irradiated recipient mice,
revealing that NK cells are able to control
the growth of CML cells in vivo throughmissing self-recognition.9 The effect ofNK cells was considered to be mediated,
at least in part, by targeting leukemiainitiating stem cells.9 Although off-target
effects secondarily induced by imatinib

therapy may be involved in triggering activation of NK cells as has been previously
reported in gastrointestinal stromal tumor
patients, the molecular mechanisms bywhich NK cells are activated in CML
patients undergoing imatinib treatment
remain to be clarified.
Cytotoxic T lymphocyte (CTL)
responses are also attractive candidates forpredictive markers of relapse risk following
TKI discontinuation, but there have been
few reports of this occurrence so far. Thisis presumably due to the attenuation of
CTL responses that may be more sensitive
than NK cells to TKI-mediated inhibitionof off-target kinases. For instance, onesuch off-target kinase, lymphocyte-specific protein tyrosine kinase (LCK), binds
to CD4 + and CD8 + T cells and plays an
indispensable signaling role in the selection and maturation of stimulated T cells.
CML patients have lower numbers of total
CD8 + T cells while undergoing imatinib
treatment. However, after imatinib discontinuation this lymphocyte number
returns to the homeostatic level of normal
healthy controls (Fig. 1A).7 However, alloreactive CTLs have been demonstrated tomediate curative anti-leukemic effects inallogeneic hematopoietic stem cell transplantation. Moreover, one of several mechanisms by which IFNα controls CML ise1-2	OncoImmunology-280-

considered to be via the enhancement of
CTL responses against CML antigens,such as the leukemia-associated antigen
serine protease, proteinase-3.10 Therefore,taken together, these results indicate thatfurther detailed analyses of CML antigenspecific CTL responses, for example using
several sensitive human leukocyte antigen
tetramer–peptide complexes, could help
to predict those patients who can safely
stop imatinib therapy.
In conclusion, in addition to thedepth of CMR achieved and the patients
genetic background, the presence of relatively abundant and functional NK cells
is a good prognostic marker for safe discontinuation of imatinib.6,7 Moreover,
CTLs specific for CML antigens such as
BCR-ABL1 or proteinase-3 would also
be good candidates for predictive markers. However, to further confirm reliability, the development and availability ofhighly sensitive human leukocyte antigen
tetramer–peptide complexes that recognize unique CML antigen-specific CTLs
are necessary tools for such analyses.
Moreover, longitudinal studies before andafter discontinuation of imatinib involving a larger patient cohort are warranted,
kinetic analyses that may reveal a combination of these multiple markers offeringa more reliable and attractive strategy to

Volume 3© 2014 Landes Bioscience. Do not distribute.

Figure 1. Predictive immune cell markers for identifying patients who can stop imatinib without relapse. (A) Hypothetical kinetics regarding the activation level of natural killer (NK) cells, total CD8+ T cells, and chronic myeloid leukemia (CML) antigen-specific cytotoxic T lymphocyte (CTLs). Total CD8+
T cells appear to be more susceptible to imatinib than NK cells. It is predicted that patients who have sustained and higher levels of activated NK cells
and/or CML antigen–specific CTLs can safely stop imatinib without relapse. (B) Combined prediction using multiple markers, such as the presence of
IFNγ+ NK cells and CML antigen–specific CTLs, could be a more reliable strategy.

3.

Disclosure of Potential Conflicts of Interest

K.O. received research support from
Bristol–Myers Squibb KK and Novartis
KK, served as consultant and advisor
for Novartis KK, Bristol-Myers Squibb
KK, and Ariad, and received honoraria
for lecture fees from Novartis KK and
Bristol–Myers Squibb KK. This study was
supported in part by the Private University
Strategic Research-Based Support Project:
Epigenetics Research Project Aimed atGeneral Cancer Cure using Epigenetic
Targets and by a Grant-in-Aid for
Scientific Research from the Ministry of
Education, Culture, Sports, Science, andTechnology, Japan.

4.

5.

6.

References1.

2.

Deininger MW, Goldman JM, Melo JV. The molecular biology of chronic myeloid leukemia. Blood 2000;
96:3343-56; PMID:11071626
Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal
GM, Fanning S, Zimmermann J, Lydon NB. Effects
of a selective inhibitor of the Abl tyrosine kinase on
the growth of Bcr-Abl positive cells. Nat Med 1996;
2:561-6; PMID:8616716; http://dx.doi.org/10.1038/
nm0596-561

7.

Mahon FX, Réa D, Guilhot J, Guilhot F, Huguet
F, Nicolini F, Legros L, Charbonnier A, Guerci A,
Varet B, et al.; Intergroupe Français des Leucémies
Myéloïdes Chroniques. Discontinuation of imatinibin patients with chronic myeloid leukaemia whohave maintained complete molecular remission for
at least 2 years: the prospective, multicentre Stop
Imatinib (STIM) trial. Lancet Oncol 2010; 11:102935; PMID:20965785; http://dx.doi.org/10.1016/
S1470-2045(10)70233-3
Takahashi N, Kyo T, Maeda Y, Sugihara T, Usuki K,
Kawaguchi T, Usui N, Okamoto S, Ohe Y, Ohtake S,
et al. Discontinuation of imatinib in Japanese patientswith chronic myeloid leukemia. Haematologica
2012; 97:903-6; PMID:22180435; http://dx.doi.
org/10.3324/haematol.2011.056853
Ross DM, Branford S, Seymour JF, Schwarer AP,
Arthur C, Bartley PA, Slader C, Field C, Dang P,
Filshie RJ, et al. Patients with chronic myeloid leukemia who maintain a complete molecular response
after stopping imatinib treatment have evidenceof persistent leukemia by DNA PCR. Leukemia
2010; 24:1719-24; PMID:20811403; http://dx.doi.
org/10.1038/leu.2010.185
Ohyashiki K, Katagiri S, Tauchi T, Ohyashiki JH,
Maeda Y, Matsumura I, Kyo T. Increased naturalkiller cells and decreased CD3(+)CD8(+)CD62L(+)
T cells in CML patients who sustained complete
molecular remission after discontinuation of imatinib.
Br J Haematol 2012; 157:254-6; PMID:22077498;
http://dx.doi.org/10.1111/j.1365-2141.2011.08939.x
Mizoguchi I, Yoshimoto T, Katagiri S, Mizuguchi
J, Tauchi T, Kimura Y, Inokuchi K, Ohyashiki
JH, Ohyashiki K. Sustained upregulation of effector natural killer cells in chronic myeloid leukemia
after discontinuation of imatinib. Cancer Sci 2013;
104:1146-53;
PMID:23758044;
http://dx.doi.
org/10.1111/cas.12216

www.landesbioscience.com	OncoImmunology-281-

Kreutzman A, Rohon P, Faber E, Indrak K, Juvonen
V, Kairisto V, Voglová J, Sinisalo M, Flochová E,
Vakkila J, et al. Chronic myeloid leukemia patientsin prolonged remission following interferon-α
monotherapy have distinct cytokine and oligoclonal lymphocyte profile. PLoS One 2011; 6:e23022;
PMID:21857985; http://dx.doi.org/10.1371/journal.
pone.0023022
9. Kijima M, Gardiol N, Held W. Natural killer cell
mediated missing-self recognition can protect micefrom primary chronic myeloid leukemia in vivo.
PLoS One 2011; 6:e27639; PMID:22132120; http://dx.doi.org/10.1371/journal.pone.0027639
10. Burchert A, Müller MC, Kostrewa P, Erben P, Bostel
T, Liebler S, Hehlmann R, Neubauer A, Hochhaus
A. Sustained molecular response with interferonalfa maintenance after induction therapy with imatinib plus interferon alfa in patients with chronic
myeloid leukemia. J Clin Oncol 2010; 28:142935; PMID:20142590; http://dx.doi.org/10.1200/
JCO.2009.25.5075
8.

e1-3

© 2014 Landes Bioscience. Do not distribute.

stratify CML patients according to predicted outcome (Fig. 1B).

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 48, pp. 34343–34351, November 29, 2013
© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

Exosomes Derived from Hypoxic Leukemia Cells Enhance
Tube Formation in Endothelial Cells*□
S

Received for publication, April 27, 2013, and in revised form, October 15, 2013 Published, JBC Papers in Press, October 16, 2013, DOI 10.1074/jbc.M113.480822

Hiroko Tadokoro‡§, Tomohiro Umezu§¶, Kazuma Ohyashiki¶储, Toshihiko Hirano‡, and Junko H. Ohyashiki§1
From the ‡Department of Clinical Pharmacology, Tokyo University of Pharmacy and Life Science, Tokyo 192-0392, Japan, the¶Department of Molecular Science, Tokyo Medical University, Tokyo 160-0023, Japan, the 储First Department of Internal Medicine,Tokyo Medical University, Tokyo 160-0023, Japan, and the §Institute of Medical Science, Tokyo Medical University,
Tokyo 160-0023, Japan

Hypoxia plays an important role during the evolution of cancer cells and their microenvironment. Emerging evidence suggests communication between cancer cells and their microenvironment occurs via exosomes. This study aimed to clarify
whether hypoxia affects angiogenic function through exosomes
secreted from leukemia cells. We used the human leukemia cellline K562 for exosome-generating cells and human umbilicalvein endothelial cells (HUVECs) for exosome target cells. Exosomes derived from K562 cells cultured under normoxic (20%)
or hypoxic (1%) conditions for 24 h were isolated and quantitated by nanoparticle tracking analysis. These exosomes werethen cocultured with HUVECs to evaluate angiogenic activity.
The exosomes secreted from K562 cells in hypoxic conditions
significantly enhanced tube formation by HUVECs comparedwith exosomes produced in normoxic conditions. Using a TaqMan low-density miRNA array, we found a subset of miRNAs,
including miR-210, were significantly increased in exosomes
secreted from hypoxic K562 cells. We demonstrated that cancer
cells and their exosomes have altered miRNA profiles underhypoxic conditions. Although exosomes contain various molecular constituents such as proteins and mRNAs, altered exosomal
compartments under hypoxic conditions, including miR-210,
affected the behavior of endothelial cells. Our results suggestthat exosomal miRNA derived from cancer cells under hypoxic
conditions may partly affect angiogenic activity in endothelial
cells.

The tumor microenvironment plays a crucial role in thepathophysiology of cancer (1, 2). Hypoxia is an important element of cancer microenvironment and is associated with* This work was supported by the Private University Strategic Research Based
Support Project Epigenetics Research Project Aimed at General Cancer
Cure Using Epigenetic Targets from MEXT (Ministry of Education, Culture,
Sports, Science, and Technology) (Tokyo, Japan).
□
S
This article contains supplemental Tables S1 and S2.
1
To whom correspondence should be addressed: Inst. of Medical Science,
Tokyo Medical University, 6-7-1 Nishi-shinjuku, Shinjuku, Tokyo 160-0023,
Japan. Tel.: 81-3-3342-6111; Fax: 81-3-3345-0185; E-mail: junko@hh.
iij4u.or.jp.

aggressive tumor phenotypes and poor patient outcomes (3, 4).
Hypoxia has been shown to help maintain normal stem cells aswell as cancer stem cell populations, but its roles in cancer stem
cells are largely unknown (5). Under hypoxia, cancer cells
secrete substances that modulate their hostile microenvironment to sustain growth and survival (1, 6, 7). In addition toconventional signaling pathways responding to hypoxia (i.e.
direct cell-to-cell contact or VEGF signaling) (8, 9), recent studies have shown the importance of communication between
tumor cells and microenvironmental factors, including angiogenesis via microvesicles or exosomes, secreted from hypoxic
tumor cells (10, 11).
Exosomes are small endosome-derived vesicles (30 –100 nm)
containing a wide range of functional proteins, mRNAs, andmiRNAs2 that are actively secreted via exocytosis from different cell types, such as dendritic cells, lymphocytes, and tumor
cells (12). Recent studies demonstrated that exosomes may act
as mediators of cell-to-cell communication (13–16). Exosomemediated signaling promotes tumor progression through communication between the tumor and surrounding stromal tissues (17), activation of proliferative and angiogenic pathways(18), and initiation of premetastatic sites (19). We have also
demonstrated that exosomal miRNA can be transported fromleukemia cells to endothelial cells and certain exogenous
miRNAs modulate endothelial migration and tube formation(20).
Evidence suggests a possible role for exosomes derived fromhypoxic tumor cells in modulating the tumor microenvironment (10, 11); however, the precise mechanism by which tumor
progression and angiogenesis are affected has not been fully
elucidated. Here we provide data showing that hypoxic tumor
cells secrete exosomal miRNA, which enhances tube formationin human umbilical vein endothelial cells (HUVECs) due toinhibition of the receptor tyrosine kinase ligand Ephrin-A3.

2

The abbreviations used are: miRNA, microRNA; HUVECs, human umbilicalvein endothelial cells; NTA, nanoparticle tracking analysis; EFNA3, EphrinA3; HIF, hypoxia-inducible factor.

NOVEMBER 29, 2013 • VOLUME 288 • NUMBER 48

JOURNAL OF BIOLOGICAL CHEMISTRY

-282-

34343

Downloaded from http://www.jbc.org/ at TOKYO MEDICAL UNIVERSITY on April 28, 2018

Background: We recently showed communication between leukemia and endothelial cells and induction of angiogenesis
via exosomes.
Results: Hypoxic leukemia cells secrete exosomal miRNA, which enhances tube formation in endothelial cells.
Conclusion: Exosomal miRNA from a tumor itself helps modulate the microenvironment of the tumor.
Significance: This study provides novel insight into the role of exosomes in cancer development.

Tumor Exosome under Hypoxic Conditions
EXPERIMENTAL PROCEDURES

34344 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 288 • NUMBER 48 • NOVEMBER 29, 2013

-283-

Downloaded from http://www.jbc.org/ at TOKYO MEDICAL UNIVERSITY on April 28, 2018

Cell Lines and Culture Conditions—We used human leukemia cell line K562 for exosome-generating cells and HUVECs as
exosome target cells. Cells were maintained as described previously (20). Pooled HUVECs were purchased from Lonza, Inc.
(Allendale, NJ) and cultured in endothelial basal medium(EBM-2; Lonza, Inc.) supplemented with EGM-2 singleQuotsand 5% FBS at 37 °C in a humidified atmosphere of 95% air and
5% CO2 until the third passage. K562 cells were cultured for
24 –72 h under hypoxic conditions (1% O2, 5% CO2, 94% N2) ina humidified gas-sorted hypoxic incubator (MCO-5M, Sanyo,
Osaka, Japan) using the degassed culture mediums (AIM-V
medium, Invitrogen). RPMI8226 (multiple myeloma) and
SUDHL4 (diffuse large B-cell lymphoma) cell lines were also
used for the tube formation assay and miRNA profiling.
Preparation of the Exosomal Fraction—K562, RPMI8226,
and SUDHL4 cells were seeded at a density of 5 ⫻ 105 cells/ml
and cultured for 24 h unless otherwise indicated under hypoxic
conditions (1% O2) or normoxic conditions (20% O2) in serumfree AIM-V medium (Invitrogen). The exosomes were purified
by Exoquick Exosome Precipitation Solution (System Biosciences, Mountain View, CA) as described previously (20). Exosome pellets were resuspended with 500 ␮l of the serum-free
AIM-V medium (Invitrogen).
Transmission Electron Microscopy—For electron microscopy analysis, exosomes were prepared, fixed with 4% paraformaldehyde and 4% glutaraldehyde in 0.1 M phosphate buffer,
pH 7.4, at incubation temperature, and placed in a refrigerator
to lower temperature to 4 °C. The samples were adsorbed to a
400-mesh carbon-coated grid and immersed in 2% phosphotungstic acid solution (pH 7.0) for 30 s. The samples wereobserved by transmission electron microscope (JEM-1200EX;
JEOL, Ltd., Japan) at an acceleration voltage of 80 kV.
Nanoparticle Tracking Analysis (NTAs) for Exosomes—NTA
measurements were performed using the Nanosight LM10 system (Nanosight, Amesbury, UK) equipped with a blue laser(405 nm). Exosomes were illuminated by the laser and their
movement under Brownian motion was recorded in 90-s sample videos, which were analyzed with NTA analytical software(version 2.0, Nanosight). We serially diluted all samples with
PBS to reach a particle concentration suitable for analysis withNTA (1 ⫻ 108 to 2.5 ⫻ 109 particles/ml). The capture settings(shutter and gain) and analysis settings were done manuallyaccording to the manufacturer’s instructions. All analysis settings were kept constant within each experiment. NTAs wereaveraged within each sample across the video replicates andthen averaged across samples to provide total nanoparticle concentrations. The nanoparticle concentration was normalized tocell numbers at the time of harvest.
Tube Formation Assay—The formation of capillary-likestructures was assessed in a 24-well plate using growth factorreduced Matrigel (BD Biosciences). For this procedure,
HUVECs (2 ⫻ 104 cells/well) were plated on top of Matrigel(280 ␮l/well) and treated with exosomes derived from K562
cells cultured under hypoxic conditions (K5621%O2-exosome)or under normoxic conditions (K56220%O2-exosome). After
24 h, cells were visualized under the bright-field microscope(BZ-8000, Keyence, Osaka, Japan). The total tube area wasquantified as the mean pixel density obtained from image analysis of five random microscopic fields using ImageJ software(http://rsb.info.nih.gov/nih-image/).
RNA Isolation—Isolation of cellular and exosomal miRNAs
was performed using the miRNeasy kit (Qiagen, Hilden, Germany). The cell or exosome pellets were dissolved with 700 ␮lof QIAzol lysis regent (Qiagen). After a 2-min incubation, 1 ␮lof 1 nM ath-miR-159 (Hokkaido System Science, Hokkaido,
Japan) was added to each aliquot as a spike control for losses in
preparation, followed by vortexing for 30 s, and incubation on
ice for 10 min. Subsequent phenol extraction and cartridge filtration were carried out according to the manufacturer’s
instructions.
miRNA Expression Profile—miRNA profiling in both cells
and exosomes was done using a TaqMan low-density miRNAarray (Applied Biosystems, Bedford, MA). To identify cellularand exosomal miRNAs universally responding to hypoxic conditions, we also performed miRNA profiles in two additional
cell lines derived from hematologic malignancies (RPMI8226,
multiple myeloma; SUDHL4, diffuse large B-cell lymphoma). A
fixed volume of 3 ␮l of RNA solution from 50 ␮l of the elution
was used as input in each RT reaction. An RT reaction andpreamplification step were set up according to the manufacturers’ recommendations. miRNAs were reverse transcribed using
the Megaplex Primer Pools (Human Pools A, version 2.1) from
Applied Biosystems. RT reaction products from the exosome
sample were further amplified using the Megaplex PreAmp
Primers (Primers A, version 2.1). The expression profile ofmiRNAs was determined using the Human TaqMan miRNA
Arrays A (Applied Biosystems). RNU6B and a spike control(ath-miR159) were used as an invariant control for the cell andculture medium, respectively. Quantitative RT-PCR was carried out on an Applied Biosystems 7900HT thermal cyclerusing the manufacturer’s recommended program. Finally, all of
the raw data from each array were retrieved from the 7900HT
and run on Data Assist Software (version 3.1, Applied
Biosystems).
Transfection of K562 Cells with Cy3-Labeled Pre-mir miRNA
Precursor and PKH67-labeled Exosome Transfer—Pre-mir
miRNA precursor (has-miR-210; Applied Biosystems) waslabeled with Label IT siRNA Tracker Cy3 Kit according to the
manufacturer’s instructions (Mirus, Madison, WI). K562 cells(1 ⫻ 105) were transfected with 10 nM of Cy3-labeled pre-mir
miRNA precursor using HiPerFect (Qiagen) (K562/Cy3-miR210). The day after transfection, cells were washed three times
with PBS, and the medium was switched to fresh serum-free
AIM-V medium (Invitrogen). After incubation for a day, theculture medium was collected and used for purification of exosome fraction by Exoquick (System Biosciences). The exosomes, including Cy3-miR-210, were incubated with 2 ␮M
PKH67 (Sigma Aldrich) for 5 min at 25 °C and washed fourtimes using Amicon Ultra-0.5 (100 kDa, Millipore, Billerica,
MA).
Knockdown Experiment of Exosomal miR-210—Donor cells(K562 cells; 1 ⫻ 105) or recipient cells (HUVECs; 3 ⫻ 104) weretransfected with scrambled control (negative control 1,
Ambion) or anti-miR-210 miRNA inhibitors (Ambion) using

Tumor Exosome under Hypoxic Conditions

HiPerFect (Qiagen). The following day, after a change to fresh
media, cells were cultured under hypoxic conditions for 24 h.
The exosomes derived from K5621%O2/anti-miR-210 orK5621%O2/control cells were purified as described previously.
Luciferase Assay—Synthetic oligonucleotides bearing theEphrin-A3 (EFNA3) 3⬘-UTR with the miR-210 complementarybinding site (5⬘-TTTGTCTTCTGTGAAGACAGGACCTATGCAACGCACAGACACTTTTGGAGACCGT-3⬘) were cloned
into the firefly luciferase reporter plasmid pMIR-Report (Applied
Biosystems) according to the manufacturer’s instructions. The
seed sequence of miR-210 (ACGCACA) is indicated by boldface
and underlined type. We also generated the mutated sensor vector
that replaced the seed sequence of miR-210 with CATACAC. For
measuring luciferase activity, HUVECs were grown in 24-wellplates until 50–60% confluence. The Luciferase plasmid (0.1 ␮g)
was co-transfected with 0.01 ␮g of pMIR-Report ␤-gal control
plasmid (Applied Biosystems) as control for the transfection efficiency. After incubation for a day, HUVECs were treated with theexosome derived from K562 cells cultured under hypoxic conditions (K5621%O2-exosome) or under normoxic conditions(K56220%O2-exosome), and the activity of luciferase and ␤-gal wasassessed after 48 h with the Dual-Light System according to the
manufacturer’s instructions (Applied Biosystems).
Immunoblotting—The cells were lysed in lysis buffer (Roche
Diagnostics) and equal amounts of protein were separated on
SDS-polyacrylamide gels. Immunoblots were probed with antibodies directed against hypoxia-inducible factor 1␣ (HIF-1␣;rabbit polyclonal anti-HIF-1␣; 1:500, NB100 –134, Novus Biologicals, Littleton, CO), CD63 (rabbit polyclonal anti-CD63;
1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), Ephrin-A3(sc-733954; Santa Cruz Biotechnology), or ␤-actin (mousemonoclonal anti-␤-actin, 1:2500, Chemicon International,

Temecula, CA). Secondary antibodies were purchased from GE
Healthcare.
Immunocytochemistry—HUVECs were seeded on glass coverslips (Matsunami Glass) and cultured in normoxic conditions(20% O2) with K5621%O2-exosomes or K56220%O2-exosomes.
Ephrin-A3 was detected by using polyclonal anti-Ephrin-A3
antibody (sc-1012; Santa Cruz Biotechnology). Alexa Fluor
488-conjugated anti-mouse IgG goat serum (Molecular Probes,
Invitrogen) were used as secondary antibodies. DAPI (Abbott,
Abbott Park, IL) was used for nuclear staining. Analyses were
performed with a fluorescent microscope (Biozero BZ-8000;
Keyence, Osaka, Japan).
Statistical Analyses—Data are expressed as mean ⫾ S.D. Twotreatment groups were compared by Mann-Whitney U test orStudent’s t test. Multiple group comparisons were done by analysis of variance. For statistical analysis, GraphPad Prism for
Macintosh (version 5c, GraphPad, Inc., La Jolla, CA) was used.
Results were considered statistically significant when p ⬍ 0.05.

RESULTS
Cell Growth of K562 Cells Cultured in Hypoxic Conditions—
We first examined the response to hypoxia in K562 cells. The
cell growth was measured following 24, 48, and 72 h of normoxic (20% O2) or hypoxic (1% O2) culture. K562 cell growth
was not significantly different between normoxia and hypoxia
for 24 h, but it was reduced by incubation under hypoxic conditions for 48 –72 h (Fig. 1A). The expression of a master regulator of hypoxic response, HIF-1␣ was remarkably up-regulated
in hypoxic conditions after 24 h (Fig. 1B).
Amount of Exosomes Released from K562 Cells—K562 cells
were cultured under hypoxic or normoxic conditions, and theexosome fraction was isolated by Exoquick after 24 h, after

NOVEMBER 29, 2013 • VOLUME 288 • NUMBER 48

JOURNAL OF BIOLOGICAL CHEMISTRY

-284-

34345

Downloaded from http://www.jbc.org/ at TOKYO MEDICAL UNIVERSITY on April 28, 2018

FIGURE 1. The response to hypoxia in K562 cells. A, 5 ⫻ 104 cells/ml of K562 cells were seeded, and cell growth was measured following 24, 48, and 72 h ofnormoxic (20% O2) or hypoxic (1% O2) cultivate conditions. B, the expression level of HIF-1␣ protein in K562 cells cultured under 20% O2 or 1% O2 conditionsfor 24 h. C, photomicrograph of an exosome derived from hypoxic K562 cells. Scale bar, 100 nm. D, photomicrograph of an exosome derived from normoxic
K562 cells. Scale bar, 100 nm. E, CD63 (exosomal marker) immunoblot of exosomes derived from K562 cells cultured under 20% O2 or 1% O2 conditions for 24 h.
Equal amount of exosomes (300 ng) were used for the assay. F and G, the nanoparticle size distribution for the exosomes derived from K562 cells cultured under
20% O2 or 1% O2 conditions for 24 h were obtained by NTA (F), and the nanoparticle concentrations were normalized to final cell counts for 24 h under normoxic
or hypoxic conditions. Each bar is presented as the mean ⫾ S.D. (n ⫽ 3) (G).

Tumor Exosome under Hypoxic Conditions

which we observed the ultrastructure of exosomes using transmission electron microscopy. The size of exosomes derived
from hypoxic K562 cells was similar to the exosomes derived
from normoxic K562 cells (50 –100 nm in diameter), and eachof those vesicles showed the classical cup-shaped appearance(Fig. 1, C and D). We further checked the expression of theexosome marker CD63 by immunoblotting. The expression
level of CD63 was not changed when K562 cells were cultured
under hypoxic conditions for 24 h (Fig. 1E). To further investigate the size distribution profile of exosomes derived from
K562 cells under normoxic conditions (K56220%O2-exosome)and hypoxic conditions (K5621%O2-exosome), we performed
NTA using the Nanosight LM10 system. The nanoparticle size
distribution of K56220%O2-exosomes was similar to K5621%O2exosomes when cells were cultured for 24 h; the peaks of particle size were ⬃100 nm within the expected size of exosomes(Fig. 1F). When nanoparticle concentrations were normalized
to cell numbers at the time of harvest, there were no significant
differences observed between K56220%O2-exosomes andK5621%O2-exosomes (Fig. 1G). These results indicate that thesize and concentration of exosomes secreted from K562 cells
were not affected when cells were exposed to hypoxia for 24 h.
In contrast, the nanoparticle size distribution of K56220%O2exosomes (control) was different from that of K5621%O2-exosomes when cells were exposed to hypoxia for 72 h. We foundnanoparticles of various sizes in the control sample; therefore,
we could not evaluate the amount of exosome release whencells were cultured for 72 h (data not shown). Consequently, weused the exosomes isolated from culture medium following
24 h of normoxic or hypoxic conditions because culture
hypoxic conditions for 24 h affected neither the cancer cell
growth nor the amount of exosomes secreted into the culture
medium.

The Exosomes Derived from K562 Cells in Hypoxic Conditions
Enhanced Tube Formation of HUVECs—We next performed
endothelial tube formation assays with the exosomes derived
from K562 cells following 24 h of normoxic (K5621%O2exosomes) or hypoxic (K56220%O2-exosomes) conditions.
K5621%O2-exosomes remarkably enhanced the tube formationof HUVECs under normoxic conditions (Fig. 2, A and D; **, p ⬍0.001; *, p ⬍ 0.01, respectively) compared with K56220%O2-exosomes (Fig. 2, B and D) or control (HUVECs20%O2; Fig. 2, C and
D). We also investigated whether or not K5621%O2-exosomes
could affect growth and cell viability of HUVECs; however, we
could not find any significant difference in the growth of
HUVECs between K5621%O2-exosomes and K56220%O2-exosomes (data not shown). Enhanced tube formation of HUVECs
also occurred with exosomes derived from hypoxic RPMI8226
cells (RPMI82261%O2-exosomes) (Fig. 2, E–H). These results
indicate that hypoxic cancer exosomes may affect tube formation rather than endothelial cell proliferation.
Cellular and Exosomal miRNA Profiling of K562 Cells Cultured in Hypoxic Conditions—To identify hypoxia-induced
exosomal miRNAs, we performed miRNA profiling using the
TaqMan low-density array (NCBI, gene expression omnibus(GEO) accession no. GSE45289). Based on the results obtained
from the characterization of hypoxic K562 cells and the endothelial tube formation assay, we used the exosomes derived
from K562 cells cultured under hypoxic or normoxic conditions for 24 h. The 2(⫺⌬Ct) values of cellular and exosomal
miRNAs were ranked with a fold change ⬎ 1.5 by hypoxic conditions compared with normoxic conditions (Table 1). TwomiRNAs (miR-18b and miR-210) were found among the top 10
up-regulated miRNAs under hypoxic conditions in both cells
and exosomes. To clarify whether up-regulation of miR-18b
and miR-210 was specific for K562 cells, we performed the34346 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 288 • NUMBER 48 • NOVEMBER 29, 2013

-285-

Downloaded from http://www.jbc.org/ at TOKYO MEDICAL UNIVERSITY on April 28, 2018

FIGURE 2. The exosomes derived from K562 cells in hypoxic conditions enhance tube formation of HUVECs. A–C, the formation of tube-like structures wasobserved under bright field. Endothelial tube formation of HUVECs cultured on Matrigel with K5621%O2-exosomes (A), with K56220%O2-exosomes (B), or without
K562-exosomes (control) (C). The scale bar indicates 500 ␮m. D, the tube-like structures determined by pixel density are significantly enhanced by the additionof K56220%O2-exosomes (*, p ⬍ 0.01) or K5621%O2-exosomes (**, p ⬍ 0.001) as compared with control (HUVECs20%O2). E–G, endothelial tube formation of
HUVECs with RPMI82261%O2-exosomes (E) and with RPMI822620%O2-exosomes (F) or without RPMI8226-exosomes (control) (G). The scale bar indicates 500 ␮m.
H, the tube-like structures were significantly enhanced by the addition of RPMI822620%O2-exosomes (*, p ⬍ 0.01) or RPMI82261%O2-exosomes (**, p ⬍ 0.001) ascompared with control (HUVECs20%O2). Values are mean ⫾ S.D.

Tumor Exosome under Hypoxic Conditions
TABLE 1
Top 10 cellular and exosomal miRNAs elevated under hypoxic conditions in K562 cells
Cellular and exosomal miRNAs, which were up-regulated ⬎1.5-fold under hypoxic
conditions, are ranked by expression level.
Rank

Cellular miRNA

Exosomal miRNA

1
2
3
4
5
6
7
8
9
10

hsa-miR-19a
hsa-miR-146–5p
hsa-miR-454
hsa-miR-18b
hsa-miR-574–3p
hsa-miR-21
hsa-miR-431
hsa-miR-345
hsa-miR-210
hsa-miR-197

hsa-miR-20a
hsa-miR-24
hsa-miR-18b
hsa-miR-130b
hsa-miR-106b
hsa-miR-224
hsa-miR-210
hsa-miR-652
hsa-miR-379
hsa-miR-185

FIGURE 3. Transfer of miRNAs derived from K562 cells to HUVECs via exosomes. A–C, HUVECs were cultured with PKH67-labeled exosomes (exo.)
derived from K562 cells transfected Cy3-pre-miR-210 (K562/Cy3-miR-210
cells). The Cy3-miR-210 signals were detected in the cytoplasm of HUVECs(open arrowheads) (A), and closed arrowheads indicate PKH67 signals (B). Cy3miR-210 signals are co-localized with PKH67 in HUVECs (arrows) (C). Parts ofareas in A–C are enlarged in D–F, respectively. Nuclear counterstaining was
performed using DAPI (blue). The scale bar indicates 10 ␮m. G, luciferase
assay. The luciferase reporter vector for assessing miR-210 specific activity
expressed firefly luciferase containing complementary miR-210 sequences in
its 3⬘-untranslated region. The luciferase reporter vector and ␤-gal control
vector allowed simultaneous monitoring of miR-210 activity and transfection
efficiency, respectively. Sensor vector: Luciferase activity of HUVECs cultured
with exosomes from K562 cells cultured under hypoxic conditions (K5621%O2exosomes) was significantly reduced compared with HUVECs only (control) or
cultured with exosomes from K562 cells cultured under normoxic conditions(K56220%O2-exosomes) (*p ⬍ 0.05; n ⫽ 3). Mutated sensor vector: there was nodifference in luciferase activity between K5621%O2-exosomes and K56220%O2exosomes. Mean ⫾ S.D. of triplicates.

was no change in the amount of secreted exosomes between
K5621%O2/control (K5621%O2 cells transfected with negative
control miR) and K5621%O2/anti-miR-210 (K5621%O2 cells
transfected with miR-210 inhibitor) (Fig. 4B). In addition toaffecting intercellular miR-210, the miR-210 inhibitor disrupted the activity of exosomal miR-210; K5621%O2/anti-miR210-exosome could not reduce the luciferase activity in
HUVECs transduced with the reporter plasmids (with K562exosome versus control: *, p ⬍ 0.05; anti-miR-210 versus negative control: #, p ⬍ 0.05; Fig. 4C). Furthermore, the knockdownof miR-210 led to a decrease in endothelial tube formation via
exosomes (Fig. 4, D–F, K5621%O2/control-exosome versus
K5621%O2/anti-miR-210-exosome). These findings may indicate that exosomal miR-210 plays an important role in endothelial tube formation. However, we could not completely ruleout the possibility that the major effect on the HUVECs was

NOVEMBER 29, 2013 • VOLUME 288 • NUMBER 48

JOURNAL OF BIOLOGICAL CHEMISTRY

-286-

34347

Downloaded from http://www.jbc.org/ at TOKYO MEDICAL UNIVERSITY on April 28, 2018

miRNA profiles using two additional cell lines. The cellular andexosomal miRNAs with a fold change ⬎ 1.5 by hypoxic conditions compared with normoxic conditions were classified
according to Ct value (20 –25, 25–30, 30 –35 cycles) and areshown in supplemental Tables S1 and S2, respectively (GEO
accession nos. GSE45387 and GSE45388). Eventually, we foundthat miR-210 was elevated under hypoxic conditions in both
cells and exosomes, in all three cell lines analyzed. Thus, we
focused on miR-210 for further study.
Exosomal miR-210 Derived from K562 Cells Cultured in
Hypoxic Conditions Regulates the Target Gene in HUVECs—
Based on the endothelial tube formation assay, K5621%O2-exosomes containing miR-210 abundantly enhanced the capillarylike structure of HUVECs. To ascertain the association between
exosomal miR-210 from K562 cells and angiogenesis underhypoxic conditions, we further investigated whether miR-210
regulated the target factor in HUVECs via exosomes.
We first visualized the transport of exosomal miR-210
derived from K562 cells into HUVECs by using the modified
method that we reported previously (20). After incubation with
PKH67-labeled exosomes derived from K562/Cy3-miR-210
cells, the Cy3-miR-210 signals and PKH67 signals were co-localized in the cytoplasm of HUVECs (Fig. 3, A–F).
Next, we performed a luciferase reporter assay to test
whether exosomal miR-210 directly regulates the target gene,
EFNA3, anti-angiogenic factor, in HUVECs. When HUVECs
transduced with the reporter plasmids containing EFNA3
3⬘-UTR with the miR-210 complementary binding site (sensor
vector) were incubated with K5621%O2-exosomes, the firefly
luciferase activity was reduced as compared with K56220%O2exosomes (*, p ⬍ 0.05; Fig. 3G). In contrast, the K5621%O2exosomes did not reduce the luciferase activity using a mutatedsensor vector of the miR-210 (Fig. 3H).
To demonstrate that exosomal miR-210 is a major player inpromoting endothelial tube formation, we performed a knockdown experiment centered on it. We first validated the efficacyof the miR-210 inhibitor using a luciferase reporter assay.
When K562 cells were transduced with the sensor vector, miR210 inhibitor strongly inhibited miR-210 activity in K562 cells
cultured under hypoxic conditions (with K562-exosome versus
control: *, p ⬍ 0.05; anti-miR-210 versus control: #, p ⬍ 0.05;
Fig. 4A). In contrast, the miR-210 inhibitor did not affect theluciferase activity when a mutated vector of the miR-210 wasused (data not shown). The results of NTA suggested that thereTumor Exosome under Hypoxic Conditions

likely due to another agent because miR-210 inhibitor had onlya modest, albeit significant effect.
Additionally, to test whether recipient cells (HUVECs) rely
on miR-210 for their response to exosomes, HUVECs20%O2
were directly transfected with miR-210 inhibitor and plated on
Matrigel for 24 h. We could not find any significant differencein the tube formation between HUVECs20%O2/anti-miR-210
and HUVECs20%O2/control (Fig. 4, G–I).
Exosomal miR-210 Down-Regulated the Expression of EFNA3
in HUVECs—Finally, we confirmed expression of EFNA3 in
HUVECs incubated with K56220%O2-exosomes or K5621%O2exosomes. The expression of EFNA3 was suppressed by theaddition of K5621%O2-exosomes into HUVECs cultured under
normoxic conditions (Fig. 5, A–C; *, p ⬍ 0.001). We also demonstrated the suppression of EFNA3 expression via K5621%O2exosomes, as shown by immunoblot (Fig. 5D). The suppressed
EFNA3 expression of HUVECs was also found by exosomes
derived from hypoxic RPMI8226 cells (RPMI82261%O2-exosomes) (Fig. 5, E–H). We subsequently concluded that miR-210
might be transferred into HUVECs via exosomes as a signal of
hypoxic response and might induce the tubulogenesis of
HUVECs under normoxic conditions.

DISCUSSION
Tumor cell-derived exosomes and their roles in intercellularcommunication of the tumor microenvironment is an emerging concept in cancer research (17, 18, 21). Hypoxia is one of thecharacteristics of the tumor microenvironment, and hypoxic
tumor cells are known to acquire metastatic characteristics anddrug resistance (22, 23). We therefore sought to determine thecommunication between hypoxic tumor cells and endothelial
cells via exosomes derived from the tumor cells. This is the firstreport to circumstantiate the possible role of exosomal miRNAsecreted from hypoxic tumor cell in the tumor angiogenic
process.
Biological functions of exosomes released from cancer cells
have been extensively studied (12, 24, 25); however, the quantitative alternation of exosomes released from cancer cells
under a hypoxic state has not been fully elucidated. A recent
study by King et al. (26) has clearly demonstrated that hypoxia
promotes the release of exosomes in breast cancer cells; theamount of exosomes increased when cells were incubatedunder severe hypoxia (0.1% O2) for 24 h, or moderate hypoxia(1% O2) for more than 48 h. It is therefore important to take into
consideration the amount of exosomes released when discuss-

34348 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 288 • NUMBER 48 • NOVEMBER 29, 2013

-287-

Downloaded from http://www.jbc.org/ at TOKYO MEDICAL UNIVERSITY on April 28, 2018

FIGURE 4. Knockdown experiment of exosomal miR-210. A, the validation of the efficacy of miR-210 inhibitor in K562 cells transfected with the luciferase
reporter vector containing miR-210 sequences in EFNA3 3⬘-UTR. The transfection of anti-miR-210 to K5621%O2 cells inhibited the induction of luciferase activity(normoxia control versus hypoxia control: *, p ⬍ 0.05; anti-miR-210 versus negative control: #, p ⬍ 0.05; n ⫽ 3). B, the nanoparticle size distribution for the
exosomes derived from K5621%O2/control (K5621%O2 cells transfected with negative control miR) and K5621%O2/anti-miR-210 (K5621%O2 cells transfected with
miR-210 inhibitor) were obtained by NTA. C, HUVECs20%O2 were transfected with the luciferase reporter vector containing miR-210 sequences in EFNA3 3⬘-UTR.
Luciferase activity was compared with HUVECs20%O2 cultured with exosomes derived from K5621%O2/anti-miR-210 or K5621%O2/control (with K562-exosome
versus control: *, p ⬍ 0.05; anti-miR-210 versus negative control: #, p ⬍ 0.05; n ⫽ 3). D–F, endothelial tube formation of HUVECs20% cultured on Matrigel with
K5621%O2/control-exosomes (con-exo.; D) and with K5621%O2/anti-miR-210-exosomes (E). F, the tube-like structures determined by pixel density are reducedby the addition of K5621%O2/anti-miR-210-exosomes (*, p ⬍ 0.01) compared with K5621%O2/control-exosomes. G–I, endothelial tube formation of
HUVECs20%O2 transfected with scramble control miR (G) and with anti-miR-210 inhibitor (H). I, there was no difference in the tube-like structures with inhibitionof endogenous miR-210 in HUVECs20%O2 (ns; not significant; p ⬎ 0.1) compared with the control (con.). Values are mean ⫾ S.D.

Tumor Exosome under Hypoxic Conditions

ing functional transfer of exosomes from hypoxic cancer cells totarget cells. In agreement with the observation by King et al.
(26), we found that the amount of exosomes significantly
increased, and the size of exosomes measured by the Nanosight
LM10 system was highly variable, when cells were cultured at
1% O2 for more than 48 h. Although the size of exosomes fromhypoxic K562 cells at 24 h was almost similar to those of normoxic K562 cells, it is still uncertain how long-term exposure tohypoxia might affect release and function of tumor exosomes.
Therefore, we used exosomes derived from hypoxic tumor cells
at 1% O2 for 24 h in this study to determine the effect of exosome contents rather than the amount of exosomes.
Visualization of secreted exosome from K562 cells wasrecently reported by Mieno and colleagues (27). We could notvisualize the three-dimensional reconstruction of imported
exosomes; however, we were able to demonstrate the transfer of
PKH67-labeled exosomes and Cy3-labeled exosomal miRNA
from donor cells (leukemia cells) to the recipient cells (endothelial cells) by the modified method as previously reported(Fig. 3, A–F) (20).
Exosomes contain specific proteins, RNA, miRNA, and lipids. Recent reports demonstrated that the transfer of exosomederived unique miRNAs to recipient cells is an alternative
mechanism in addition to the classical mechanisms, including
direct cell-cell contact or chemical receptor-mediated events,
allowing gene-based communication between cells (14).
Hypoxia signaling pathways are known to be regulated by HIF
as a key transcriptional regulator (28). So far, several miRNAs
have been implicated in regulating both upstream and down-

stream signaling of the HIF pathways: miR-199a, miR-17–92
clusters, and miR-20b regulate HIF1␣ under hypoxia, and miR23, miR-24, miR-26, miR-107, miR-210, and miR-373 have beenshown to be induced by HIFs (29 –31). In the current study, themiRNA signature in hypoxic exosomes was variable among thethree different types of cancer cell lines analyzed. This indicatesthat hypoxic signaling pathways mediated by exosomal miRNAdepend on the types of cancer. In addition, some of the miRNAs
were expressed at higher levels in exosomes than in the cells,
implying that some miRNAs may be uniquely packed into exosomes. It was not unexpected that expression of exosomal miR210 was increased in all of the cell lines analyzed; therefore, we
particularly focused on miR-210 in the current study, as we
consider it to be the principal exosomal miRNA secreted fromhypoxic tumor cells.
Although several miR-210 target genes have been identified
to date (30), one that has been consistently reported is thereceptor tyrosine kinase ligand, Ephrin-A3 (EFNA3) (32). It is
known that EFNA1-EphA2 interaction plays an important rolein the regulation of angiogenesis and VEGF signaling (32).
Although the specific role of EFNA3 in the regulation of angiogenesis is still unknown, EphA2 has been shown to bind EFNA3
as well as EFNA1 (33). As a consequence, EFNA3 might act as anegative regulator for angiogenesis. Thus, we concluded that
exosomes containing a high expression level of miR-210 downregulate EFNA3 and thereby enhance tube formation of
HUVECs. A recent investigation by Day and colleagues (34)
demonstrated that the EphA3 receptor is highly expressed in asignificant proportion of gliomas, particularly by the undiffer-

NOVEMBER 29, 2013 • VOLUME 288 • NUMBER 48

JOURNAL OF BIOLOGICAL CHEMISTRY

-288-

34349

Downloaded from http://www.jbc.org/ at TOKYO MEDICAL UNIVERSITY on April 28, 2018

FIGURE 5. EFNA3 expression of HUVECs cultured with exosome derived from K562 cells. A and B, EFNA3 expression was inhibited by exosomes derived
from hypoxic K562 cells. HUVECs were cultured under normoxic conditions with the exosomes derived from K562 cells cultured under normoxic conditions(K56220%O2-exosomes) for 24 h (A) and with the exosomes derived from K562 cells cultured under hypoxic conditions (K5621%O2-exosomes) (B). The scale bar
indicates 10 ␮m. C, EFNA3 signal was quantified using ImageJ software. EFNA3 fluorescence signals were drastically inhibited by addition of K5621%O2exosomes (*, p ⬍ 0.001) as compared with K56220%O2-exosomes. D, expression of EFNA3 in HUVECs by immunoblot. E and F, HUVECs were cultured under
normoxic conditions with the RPMI822620%O2-exosomes for 24 h (E) and with the RPMI82261%O2-exosomes (F). The scale bar indicates 10 ␮m. G, EFNA3
fluorescence signals were drastically inhibited by addition of RPMI82261%O2-exosomes (*, p ⬍ 0.001) as compared with RPMI822620%O2-exosomes. H, expression of EFNA3 in HUVECs by immunoblot. Numbers below the panels represent the normalized EFNA3 expression signal by ␤-actin.

Tumor Exosome under Hypoxic Conditions

REFERENCES
1. Joyce, J. A., and Pollard, J. W. (2009) Microenvironmental regulation ofmetastasis. Nat. Rev. Cancer 9, 239 –252
2. Hu, M., and Polyak, K. (2008) Microenvironmental regulation of cancer
development. Curr. Opin. Genet. Dev. 18, 27–34
3. Harris, A. L. (2002) Hypoxia—a key regulatory factor in tumour growth.
Nat. Rev. Cancer 2, 38 – 47
4. Vaupel, P., and Mayer, A. (2007) Hypoxia in cancer: significance and impact on clinical outcome. Cancer Metastasis Rev. 26, 225–239
5. Heddleston, J. M., Li, Z., Lathia, J. D., Bao, S., Hjelmeland, A. B., and Rich,
J. N. (2010) Hypoxia inducible factors in cancer stem cells. Br. J. Cancer
102, 789 –795
6. Albini, A., and Sporn, M. B. (2007) The tumour microenvironment as atarget for chemoprevention. Nat. Rev. Cancer 7, 139 –147
7. Bennewith, K. L., and Dedhar, S. (2011) Targeting hypoxic tumour cells to
overcome metastasis. BMC Cancer 11, 504
8. Serini, G., Valdembri, D., and Bussolino, F. (2006) Integrins and angiogenesis: a sticky business. Exp. Cell Res. 312, 651– 658
9. Shibuya, M., and Claesson-Welsh, L. (2006) Signal transduction by VEGF
receptors in regulation of angiogenesis and lymphangiogenesis. Exp. Cell
Res. 312, 549 –560
10. Park, J. E., Tan, H. S., Datta, A., Lai, R. C., Zhang, H., Meng, W., Lim, S. K.,
and Sze, S. K. (2010) Hypoxic tumor cell modulates its microenvironment
to enhance angiogenic and metastatic potential by secretion of proteinsand exosomes. Mol. Cell Proteomics 9, 1085–1099
11. Svensson, K. J., Kucharzewska, P., Christianson, H. C., Sköld, S., Löfstedt,
T., Johansson, M. C., Mörgelin, M., Bengzon, J., Ruf, W., and Belting, M.
(2011) Hypoxia triggers a proangiogenic pathway involving cancer cell
microvesicles and PAR-2-mediated heparin-binding EGF signaling in endothelial cells. Proc. Natl. Acad. Sci. U.S.A. 108, 13147–13152
12. Vlassov, A. V., Magdaleno, S., Setterquist, R., and Conrad, R. (2012) Exosomes: current knowledge of their composition, biological functions, anddiagnostic and therapeutic potentials. Biochim. Biophys. Acta 1820,
940 –948
13. Valadi, H., Ekström, K., Bossios, A., Sjöstrand, M., Lee, J. J., and Lötvall,

J. O. (2007) Exosome-mediated transfer of mRNAs and microRNAs is a
novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9,654 – 659
14. Kosaka, N., Iguchi, H., Yoshioka, Y., Takeshita, F., Matsuki, Y., and Ochiya,
T. (2010) Secretory mechanisms and intercellular transfer of microRNAs
in living cells. J. Biol. Chem. 285, 17442–17452
15. Camussi, G., Deregibus, M. C., Bruno, S., Grange, C., Fonsato, V., andTetta, C. (2011) Exosome/microvesicle-mediated epigenetic reprogramming of cells. Am. J. Cancer Res. 1, 98 –110
16. Kosaka, N., Iguchi, H., Yoshioka, Y., Hagiwara, K., Takeshita, F., andOchiya, T. (2012) Competitive interactions of cancer cells and normal
cells via secretory microRNAs. J. Biol. Chem. 287, 1397–1405
17. Webber, J., Steadman, R., Mason, M. D., Tabi, Z., and Clayton, A. (2010)
Cancer exosomes trigger fibroblast to myofibroblast differentiation. Cancer Res. 70, 9621–9630
18. Skog, J., Würdinger, T., van Rijn, S., Meijer, D. H., Gainche, L., SenaEsteves, M., Curry, W. T., Jr., Carter, B. S., Krichevsky, A. M., and Breakefield, X. O. (2008) Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.
Nat. Cell Biol. 10, 1470 –1476
19. Hood, J. L., San, R. S., and Wickline, S. A. (2011) Exosomes released bymelanoma cells prepare sentinel lymph nodes for tumor metastasis. Cancer Res. 71, 3792–3801
20. Umezu, T., Ohyashiki, K., Kuroda, M., and Ohyashiki, J. H. (2013) Leukemia cell to endothelial cell communication via exosomal miRNAs. Oncogene 32, 2747–2755
21. Nazarenko, I., Rana, S., Baumann, A., McAlear, J., Hellwig, A., Trendelenburg, M., Lochnit, G., Preissner, K. T., and Zöller, M. (2010) Cell surface
tetraspanin Tspan8 contributes to molecular pathways of exosome-induced endothelial cell activation. Cancer Res. 70, 1668 –1678
22. Sullivan, R., Paré, G. C., Frederiksen, L. J., Semenza, G. L., and Graham,
C. H. (2008) Hypoxia-induced resistance to anticancer drugs is associatedwith decreased senescence and requires hypoxia-inducible factor-1 activity. Mol. Cancer Ther. 7, 1961–1973
23. Semenza, G. L. (2012) Molecular mechanisms mediating metastasis ofhypoxic breast cancer cells. Trends Mol. Med. 18, 534 –543
24. Corrado, C., Flugy, A. M., Taverna, S., Raimondo, S., Guggino, G., Karmali,R., De Leo, G., and Alessandro, R. (2012) Carboxyamidotriazole-orotate
inhibits the growth of imatinib-resistant chronic myeloid leukaemia cells
and modulates exosomes-stimulated angiogenesis. PLoS One 7, e42310
25. Kosaka, N., Iguchi, H., Hagiwara, K., Yoshioka, Y., Takeshita, F., andOchiya, T. (2013) Neutral sphingomyelinase 2 (nSMase2)-dependent exosomal transfer of angiogenic microRNAs regulate cancer cell metastasis.
J. Biol. Chem. 288, 10849 –10859
26. King, H. W., Michael, M. Z., and Gleadle, J. M. (2012) Hypoxic enhancement of exosome release by breast cancer cells. BMC Cancer 12, 421
27. Mineo, M., Garfield, S. H., Taverna, S., Flugy, A., De Leo, G., Alessandro,R., and Kohn, E. C. (2012) Exosomes released by K562 chronic myeloid
leukemia cells promote angiogenesis in a Src-dependent fashion. Angiogenesis 15, 33– 45
28. Pugh, C. W., and Ratcliffe, P. J. (2003) Regulation of angiogenesis by hypoxia: role of the HIF system. Nat. Med. 9, 677– 684
29. Kulshreshtha, R., Ferracin, M., Wojcik, S. E., Garzon, R., Alder, H., AgostoPerez, F. J., Davuluri, R., Liu, C. G., Croce, C. M., Negrini, M., Calin, G. A.,
and Ivan, M. (2007) A microRNA signature of hypoxia. Mol. Cell. Biol. 27,
1859 –1867
30. Huang, X., Le, Q. T., and Giaccia, A. J. (2010) MiR-210 —micromanager ofthe hypoxia pathway. Trends Mol. Med. 16, 230 –237
31. Hua, Z., Lv, Q., Ye, W., Wong, C. K., Cai, G., Gu, D., Ji, Y., Zhao, C., Wang,J., Yang, B. B., and Zhang, Y. (2006) MiRNA-directed regulation of VEGF
and other angiogenic factors under hypoxia. PLoS One 1, e116
32. Fasanaro, P., D’Alessandra, Y., Di Stefano, V., Melchionna, R., Romani, S.,
Pompilio, G., Capogrossi, M. C., and Martelli, F. (2008) MicroRNA-210
modulates endothelial cell response to hypoxia and inhibits the receptor
tyrosine kinase ligand Ephrin-A3. J. Biol. Chem. 283, 15878 –15883
33. Davis, S., Gale, N. W., Aldrich, T. H., Maisonpierre, P. C., Lhotak, V.,
Pawson, T., Goldfarb, M., and Yancopoulos, G. D. (1994) Ligands for
EPH-related receptor tyrosine kinases that require membrane attachment

34350 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 288 • NUMBER 48 • NOVEMBER 29, 2013

-289-

Downloaded from http://www.jbc.org/ at TOKYO MEDICAL UNIVERSITY on April 28, 2018

entiated, tumor-initiating cells. Because tumor-initiating cells
are known to exist in a hypoxic niche, targeting EFNA3 by miR210 might be beneficial not only in inhibiting angiogenesis but
also in eliminating tumor-initiating cells, although further
studies are needed.
Unlike solid tumors, little is known about the exosomes forthe angiogenic process in leukemia. The bone marrow ofchronic myeloid leukemia patients exhibits marked neovascularization and increased numbers of endothelial cells (35). Ourgroup and other investigators (20, 24, 27, 36) have shown that
exosomes released by CML cells have the potential to influencein vitro and/or in vivo angiogenesis by directly affecting endothelial cells properties. A salient feature of the present study
was that hypoxic CML cells secreted an exosomal miRNA,
which was different from those secreted in a normoxic state andable to modulate angiogenesis in vitro.
In conclusion, hypoxic K562 cells could release exosomes
containing distinct miRNAs. This has implications for how
tumor cells might convey signals to its microenvironment.
Hypoxic exosomes contained higher levels of miR-210, indicating a qualitative (not quantitative) difference between normoxic and hypoxic exosomes derived from hypoxic cancer
cells. Although tumor angiogenesis is known to be regulated byvarious factors, the current pathway, noncontact cell-to-cellcommunication via exosomes plays an important role, at leastin part, in the hypoxic tumor itself modulating the tumor
microenvironment.

Tumor Exosome under Hypoxic Conditions
or clustering for activity. Science 266, 816 – 819
34. Day, B. W., Stringer, B. W., Al-Ejeh, F., Ting, M. J., Wilson, J., Ensbey, K. S.,
Jamieson, P. R., Bruce, Z. C., Lim, Y. C., Offenhäuser, C., Charmsaz, S.,
Cooper, L. T., Ellacott, J. K., Harding, A., Leveque, L., Inglis, P., Allan, S.,
Walker, D. G., Lackmann, M., Osborne, G., Khanna, K. K., Reynolds, B. A.,
Lickliter, J. D., and Boyd, A. W. (2013) EphA3 maintains tumorigenicity
and is a therapeutic target in glioblastoma multiforme. Cancer Cell 23,
238 –248

35. Kvasnicka, H. M., Thiele, J., Staib, P., Schmitt-Graeff, A., Griesshammer,
M., Klose, J., Engels, K., and Kriener, S. (2004) Reversal of bone marrow
angiogenesis in chronic myeloid leukemia following imatinib mesylate(STI571) therapy. Blood 103, 3549 –3551
36. Taverna, S., Flugy, A., Saieva, L., Kohn, E. C., Santoro, A., Meraviglia,
S., De Leo, G., and Alessandro, R. (2012) Role of exosomes released bychronic myelogenous leukemia cells in angiogenesis. Int. J. Cancer 130,2033–2043

Downloaded from http://www.jbc.org/ at TOKYO MEDICAL UNIVERSITY on April 28, 2018

NOVEMBER 29, 2013 • VOLUME 288 • NUMBER 48

JOURNAL OF BIOLOGICAL CHEMISTRY

-290-

34351

Exosomes Derived from Hypoxic Leukemia Cells Enhance Tube Formation in
Endothelial Cells
Hiroko Tadokoro, Tomohiro Umezu, Kazuma Ohyashiki, Toshihiko Hirano and Junko
H. Ohyashiki
J. Biol. Chem. 2013, 288:34343-34351.
doi: 10.1074/jbc.M113.480822 originally published online October 16, 2013

Access the most updated version of this article at doi: 10.1074/jbc.M113.480822

Click here to choose from all of JBC's e-mail alerts

Supplemental material:
http://www.jbc.org/content/suppl/2013/10/16/M113.480822.DC1
This article cites 36 references, 13 of which can be accessed free at
http://www.jbc.org/content/288/48/34343.full.html#ref-list-1-291-

Downloaded from http://www.jbc.org/ at TOKYO MEDICAL UNIVERSITY on April 28, 2018

Alerts:• When this article is cited• When a correction for this article is posted

Correspondence
Calvi, L.M., Adams, G.B., Weibrecht, K.W., Weber,
J.M., Olson, D.P., Knight, M.C., Martin, R.P.,
Schipani, E., Divieti, P., Bringhurst, F.R., Milner,
L.A., Kronenberg, H.M. & Scadden, D.T. (2003)
Osteoblastic cells regulate the haematopoietic
stem cell niche. Nature, 425, 841–846.
Cheng, H., Qiu, L., Zhang, H., Cheng, M., Li, W.,
Zhao, X., Liu, K., Lei, L. & Ma, J. (2011) Arsenic
trioxide promotes senescence and regulates thebalance of adipogenic and osteogenic differentiation in human mesenchymal stem cells. Acta
Biochimica et Biophysica Sinica (Shanghai), 43,
204–209.
Corre, J., Barreau, C., Cousin, B., Chavoin, J.P.,
Caton, D., Fournial, G., Penicaud, L., Casteilla, L.
& Laharrague, P. (2006) Human subcutaneous
adipose cells support complete differentiation but

not self-renewal of hematopoietic progenitors.
Journal of Cellular Physiology, 208, 282–288.
Naveiras, O., Nardi, V., Wenzel, P.L., Hauschka, P.
V., Fahey, F. & Daley, G.Q. (2009) Bone-marrow adipocytes as negative regulators of the haematopoietic microenvironment. Nature, 460,
259–263.
Nishikawa, M., Ozawa, K., Tojo, A., Yoshikubo,T., Okano, A., Tani, K., Ikebuchi, K., Nakauchi,
H. & Asano, S. (1993) Changes in hematopoiesis-supporting ability of C3H10T1/2 mouseembryo fibroblasts during differentiation. Blood,81, 1184–1192.
Nuttall, M.E. & Gimble, J.M. (2004) Controllingthe balance between osteoblastogenesis and adipogenesis and the consequent therapeutic implications. Current Opinion in Pharmacology, 4,
290–294.

Rosenfeld, S., Follmann, D., Nunez, O. & Young,
N.S. (2003) Antithymocyte globulin and cyclosporine for severe aplastic anemia: association
between hematologic response and long-term
outcome. JAMA, 289, 1130–1135.
Tavassoli, M., Maniatis, A. & Crosby, W.H. (1974)
Induction of sustained hemopoiesis in fatty
marrow. Blood, 43, 33–38.
Zhang, Y., Harada, A., Bluethmann, H., Wang, J.
B., Nakao, S., Mukaida, N. & Matsushima, K.
(1995) Tumor necrosis factor (TNF) is a physiologic regulator of hematopoietic progenitor cells:
increase of early hematopoietic progenitor cells
in TNF receptor p55-deficient mice in vivo andpotent inhibition of progenitor cell proliferation
by TNF alpha in vitro. Blood, 86, 2930–2937.

The BCL2L11 (BIM) deletion polymorphism is a possible
criterion for discontinuation of imatinib in chronic myeloid
leukaemia patients

Treatment with tyrosine kinase inhibitors (TKIs) can beeffective in the majority of patients with chronic myeloid
leukaemia (CML) with the BCR-ABL1 fusiongene, and thestandard of care using imatinib has been established (Baccarani et al, 2009). Although 20% of CML patients showed primary resistance to first-line imatinib therapy, some patientsobtained an optimal response to imatinib and then could be
induced to complete molecular response (CMR), as assessedby the currently available quantitative polymerase chain reaction (PCR) assay (Mahon et al, 2010; Takahashi et al, 2012).
The reduction of the BCR-ABL1 transcript to the CMR levelis now considered to be the ideal target point of TKI treatment (Cortes et al, 2004).
Recent studies highlighted that some patients with sustained CMR are able to maintain this molecular condition
after the discontinuation of imatinib. In a nonrandomized
prospective study, among sustained CMR patients who had
stopped imatinib treatment for at least 2 years, 41% maintained CMR (Mahon et al, 2010). These studies demonstrated that some CML patients have long-lasting CMR after
the discontinuation of imatinib, although CML cells detectedby DNA PCR persisted even with CMR (Ross et al, 2010).
We recently demonstrated an increasing number of naturalkiller (NK) cells in sustained CMR patients after the discontinuation of imatinib (Ohyashiki et al, 2012), suggesting that
immunological surveillance could be important for maintaining CMR. However, we treated one CML patient in CMR

showing an increasing percentage of NK cells, but the patient
relapsed 3 months after discontinuation of imatinib. This
experience caused us to consider what factors underlie thesustained CMR status.
Ng et al (2012) demonstrated that CML patients witha common intronic deletion polymorphism in the geneencoding BCL2-like 11 protein (BCL2L11, also called
BIM), lacking the pro-apoptotic BCL2-homology domain
3 (BH3), show an inferior response to TKIs when compared to those without the polymorphism. It is wellknown that imatinib activates several pro-apoptotic BH3only proteins by a post-transcriptional mechanism, andtherefore BCL2L11 plays a major role in imatinib-induced
apoptosis of the BCR-ABL1 CML cells (Kuroda et al,2006). Thus, it remains unclear how the BCL2L11 deletion polymorphism affects imatinib therapy in patients
who have achieved CMR.
The aim of this study was to identify CML patients in
CMR who would be possible candidates for the safe discontinuation of imatinib.
We studied 50 CML patients referred to the Tokyo Medical University hospital who had achieved CMR. They
consisted of five patients who maintained CMR for>12 months after the discontinuation of imatinib, eightpatients who maintained CMR for <12 months after the discontinuation of imatinib, 20 patients who maintained CMR
for 24 consecutive months while taking TKIs, and 17 patientsª 2012 Blackwell Publishing Ltd
British Journal of Haematology, 2013, 160, 255–272

269

-292-

Correspondence

1·5 -Control
Cell lines

KCL22

K562

CMR under
TKI therapy

UPN 29UPN 67

UPN 49STOP imatinibfor < 12 months

UPN 22

UPN 47UPN 2

MarkerSize (kb)
5-

UPN 1

STOP imatinib
for > 12 months

treated with TKIs who showed fluctuating CMR over thecourse of the 24-month study. This study was approved by
the institutional review board of Tokyo Medical University(no. 1655: approved on January 28, 2011).
All five patients who maintained CMR for >12 months after
stopping imatinib showed only the 4226-bp band, whereas
three of the eight patients who had discontinued imatinib for<12 months had the BCL2L11 deletion polymorphism (Fig 1,
Table I). Notably, the three patients with the BCL2L11 deletion polymorphism (unique patient number [UPN] 22, UPN
47, and UPN 49) relapsed at 3–4 months after the discontinuation of imatinib, whereas the remaining five patients in thisgroup maintained CMR (median follow-up period after stopping imatinib was 7 months) (Table I). In the three relapsed
patients with the BCL2L11 deletion polymorphism, BCR-ABL1← 4226 bp← 1323 bp

Fig 1. BCL2L11 deletion polymorphism in chronic myeloid leukaemia patients. Genomic DNA was obtained from whole blood using a
DNA Extractor WB Kit (Wako, Osaka, Japan). The genomic DNA
was subjected to polymerase chain reaction (PCR) amplification
using primers designed to detect a deletion site (2903 bp) in intron
two of the BCL2L11 gene (forward: 5′-AATACCACAGAGGCCCACAG-3′; reverse: 5′-GCCTGAAGGTGCTGAGAAAG-3′) and JumpStart RedAccuTaq LA DNA polymerase (Sigma Aldrich, St. Louis,
MO, USA). The PCR conditions were 96°C for 30 s, 30 cycles ofdenaturation at 94°C for 15 s, annealing at 60°C for 60 s, and elongation at 68°C for 8 min. The resulting PCR products were fractionated in 12% agarose gels (Takara, Shiga, Japan), and gels stainedwith ethidium bromide were analysed using a Versa Doc Imaging
System version 3000 (Bio-Rad, Hercules, CA, USA). The 4226-bp
band indicates wild-type BCL2L11 (K562 cells; negative control),whereas the presence of the 1323-bp band in KCL22 cells (positive
control) in addition to the 4226-bp band indicates the BCL2L11
deletion polymorphism. All patients who maintained CMR for>12 months after stopping imatinib showed only the 4226-bp band
(UPN one and UPN two), whereas three of eight patients who had
discontinued imatinib for <12 months had the BCL2L11 deletion
polymorphism (UPN 22, UPN 47, and UPN 49). The BCL2L11 deletion polymorphism was also found in a CMR patient still taking
imatinib (UPN 67) and a fluctuating CMR patient (UPN 29). Fourof 20 (200%) DNA samples obtained from healthy Japanese volunteers showed the BCL2L11 deletion polymorphism (data not shown),
indicating no significant deviation of the BCL2L11 deletion polymorphism in the current CML patients compared with the normal
Japanese populationtranscript had completely disappeared for >24 months before
the discontinuation of imatinib. Among the 20 patients showing sustained CMR for >24 months while still taking TKIs,
one patient (UPN 67) had the BCL2L11 deletion polymorphism. In the group of 17 patients with fluctuating CMR
under TKI therapy, one patient (UPN 29, taking nilotinib)
had the BCL2L11 deletion polymorphism.
Three patients with the BCL2L11 deletion polymorphism
who discontinued imatinib relapsed within 3–4 months, suggesting that the BCL2L11 deletion polymorphism is strongly
linked to molecular relapse after discontinuation of imatinib.
In the current study, we identified five CML patients with
the BCL2L11 deletion polymorphism, and the incidence was
100% (5/50) in this population. We could not estimate theincidence of the deletion polymorphism in an eastern Asian
CML population, because CML cells with the deletion may
be more resistant to TKI therapy (Ng et al, 2012). It is possible that CML patients who do not achieve a major molecularresponse or CMR may show a much higher incidence of the
BCL2L11 deletion polymorphism. Ng et al (2012) reported ahigher incidence of the BCL2L11 deletion polymorphism inan eastern Asian population compared with that of Caucasians. However, molecular relapse after the discontinuationof imatinib is evident even in Caucasian CML patients, andthe resistance rate to first-line imatinib does not seem to behigh in the Japanese CML population, indicating that somefactors other than the BCL2L11 deletion polymorphism are
responsible for the resistance to TKI therapy in CML
patients. Our findings suggest that CML patients with the
BCL2L11 deletion polymorphism can achieve CMR under
TKI treatment (including imatinib), but they may not beable to discontinue TKI therapy. In other words, CML
patients with the BCL2L11 deletion polymorphism may be
TKI-dependent, even though they achieve long-lasting CMR.
Although this study did not reveal a difference in the incidence of BCL2L11 deletion polymorphism between thesustained and fluctuating CMR groups under TKI therapy(1/20 vs. 1/17), a large-scale study of CML populations ofvarious ethnicities may reveal the exact effect of the deletionon achieving sustained CMR under TKI therapy.
One possible factor underlying the maintenance of CMR
after the discontinuation of imatinib is immunologicalsurveillance, including cytotoxic T cells and/or NK cells.
Although it remains unclear whether the BCL2L11 deletion is
the only risk factor for relapse after stopping imatinib,
BCL2L11 status might serve as an additive marker when
deciding whether to discontinue TKI treatment in sustained
CMR patients. Moreover, the role of immunological surveillance in CML patients, especially those with the BCL2L11
deletion polymorphism, should be further researched.

Conflict of interest
The authors declare no conflict of interest.
ª 2012 Blackwell Publishing Ltd
British Journal of Haematology, 2013, 160, 255–272

270

-293-

Correspondence
Table I. BCL2L11 deletion polymorphism and clinical status in chronic myeloid leukaemia.
BCL2L11 deletion status

Patient groups
Maintained CMR after
discontinuation of
imatinib for >12 months
Maintained CMR
after discontinuationof imatinib for <12 months
Sustained CMR for >24
months under TKIs therapy
Fluctuating CMR for >24
months under TKIs therapy
Total number of patients

Clinical status

Cases
examined (n)Cases without
BCL2L11 deletion
polymorphism (n)Cases withBCL2L11 deletion
polymorphism (n)Molecular relapse
within 6 months

Sustained CMR
without imatinib

5

5

0

0

5 (1)

8

5

3

3

5 (1)

20

19

1

NA

NA

17

16

1

NA

NA

50

45

5

3

Parentheses indicate number of patients who maintained molecular remission with 4-log reduction of the BCR-ABL1 transcript.
Molecular relapse: escape from major molecular response.
CMR: complete molecular remission.
TKIs: tyrosine kinase inhibitors.
NA: not available.

Acknowledgements
This study was supported by the Private University Strategic
Research Based Support Project: Epigenetic Research Project
Aimed at General Cancer Cure Using Epigenetic Targets
from MEXT (Ministry of Education, Culture, Sports, Scienceand Technology), Tokyo, Japan. Thanks are due to Drs. T
Tauchi, Y Kimura, A Gotoh, K Ando, S Okabe, Y Tanaka,
and T Kitahara, Tokyo Medical University, for their participation in this study.

Seiichiro Katagiri1*
Tomohiro Umezu2*
Junko H. Ohyashiki3
Kazuma Ohyashiki1,2
1First Department of Internal Medicine, Tokyo Medical Univer-sityTokyo, Japan, 2Department of Molecular Science, Tokyo Medical
University,Tokyo, Japan and 3Institute of Medical Science, Tokyo Medical University, Tokyo, Japan
E-mail: ohyashik@rr.iij4u.or.jp*These two authors contributed equally to this work.

Keywords: chronic myeloid leukaemia, discontinuation of imatinib,

Author contributions

BCL2L11 deletion polymorphism, complete molecular remission.

SK and KO wrote the manuscript; TU performed the experiments; JHO analyzed the data and designed the study; KO
supervised the study.

References
Baccarani, M., Cortes, J., Pane, F., Niederwieser, D.,
Saglio, G., Apperley, J., Cervantes, F., Deininger,
M., Gratwohl, A., Guilhot, F., Hochhaus, A.,
Horowitz, M., Hughes, T., Kantarjian, H., Larson,
R., Radich, J., Simonsson, B., Silver, R.T.,
Goldman, J & Hehmann, R; European LeukemiaNet. (2009) Chronic myeloid leukemia: an updateof concepts and management recommendationsof European LeukemiaNet. Journal of ClinicalOncology, 27, 6041–6051.
Cortes, J., O’Brien, S. & Kantarjian, H. (2004) Discontinuation of imatinib therapy after achieving
a molecular response. Blood, 104, 2204–2205.
Kuroda, J., Puthalakath, H., Cragg, M.S., Kelly, P.N.,
Bouillet, P., Huang, D.C., Kimura, S., Ottmann, O.

First published online 1 November 2012
doi: 10.1111/bjh.12111

G., Druker, B.J., Villunger, A., Roberts, A.W. &Strasser, A. (2006) Bim and Bad mediate imatinibinduced killing of Bcr/Abl+ leukemic cells, and resistance due to their loss is overcome by a BH3 mimetic.
Proceeding of the National Academy of Science of theUnited States of America, 103, 14907–14912.
Mahon, F.X., Réa, D., Guilhot, J., Guilhot, F.,
Huguet, F., Nicolini, F., Legros, L., Charbonnier,A., Guerci, A., Varet, B., Etienne, G., Reiffers, J.
& Rousselot, P; Intergroupe Français des Leucémies Myéloı̈des Croniques. (2010) Discontinuation of imatinib in patients with chronic
myeloid leukaemia who have maintained complete molecular remission for at least 2 years:
the prospective, multicenter Stop Imatinib(STIM) trial. Lancet Oncology, 11, 1029–1035.

ª 2012 Blackwell Publishing Ltd
British Journal of Haematology, 2013, 160, 255–272

Ng, K.P., Hillmer, A.M., Chuah, C.T.H., Juan, W.
C., Ko, T.K., Teo, A.S., Ariyaratne, P.N.,
Takahashi, N., Sawada, K., Fei, Y., Soh, S., Lee,
W.H., Huang, J.W., Allen, Jr, J.C. ., Woo, X.Y.,
Nagarajan, N., Kumar, V., Thalamuthu, A., Poh,
W.T., Ang, A.L., Mya, H.T., How, G.F., Yang, L.
Y., Koh, L.P., Chowba, y B., Chang, C.T.,
Nadarajan, V.S., Chng, W.J., Than, H., Lim, L.
C., Goh, Y.T., Zhang, S., Poh, D., Tan, P., See, tJ.E., Ang, M.K., Chau, N.M., Ng, Q.S., Tan,
D.S., Soda, M., Isobe, K., Nöthen, M.M., Wong,
T.Y., Shahab, A., Ruan, X., Cacheux-Rataboul,V., Sung, W.K., Tan, E.H., Yatabe, Y., Mano,
H., Soo, R.A., Chin, T.M., Lim, W.T., Ruan,
Y. & Ong, S.T. (2012) A common BIM
deletion polymorphism mediates intrinsic resis-

271

-294-

Correspondencetance and inferior responses to tyrosine kinase
inhibitors in cancer. Nature Medicine, 18, 521–
528.
Ohyashiki, K., Katagiri, S., Tauchi, T., Ohyashiki,
J.H., Maeda, Y., Matsumura, I. & Kyo, T.
(2012) Increased natural killer cells anddecreased CD3+CD8+CD62L+ T cells in CML
patients who sustained complete molecularremission after discontinuation of imatinib. British Journal of Haematology, 157, 254–256.

Ross, D.M., Branford, S., Seymour, J.F., Schearer,
A.P., Arthur, C., Bartley, P.A., Slader, C., Field,
C., Dang, P., Filshie, R.J., Mills, A.K., Grigg, A.
P., Melo, J.V. & Hughes, T.P. (2010) Patientswith chronic myeloid leukemia who maintain acomplete molecular response after stopping
imatinib treatment have evidence of persistentleukemia by DNA PCR. Leukemia, 24, 1719–
1724.

Takahashi, N., Kyo, T., Maeda, Y., Sugihara, T.,
Usuki, K., Kawaguchi, T., Usui, N., Okamoto,S., Ohe, Y., Ohtake, S., Kitamura, K., Yamamoto,M., Teshima, H., Motoji, T., Tamaki, T., Sawada,
K. & Ohyashiki, K. (2012) Discontinuation of
imatinib in Japanese patients with chronic myeloid leukemia. Haematologica, 97, 903–906.

ª 2012 Blackwell Publishing Ltd
British Journal of Haematology, 2013, 160, 255–272

272

-295-

IL-27 Enhances the Expression of TRAIL and TLR3 in
Human Melanomas and Inhibits Their Tumor Growth in
Cooperation with a TLR3 Agonist Poly(I:C) Partly in a
TRAIL-Dependent Manner
Yukino Chiba1., Izuru Mizoguchi1., Kana Mitobe1, Kaname Higuchi1,2, Hiroshi Nagai3,
Chikako Nishigori3, Junichiro Mizuguchi2, Takayuki Yoshimoto1*
1 Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan, 2 Department of Immunology, Tokyo Medical University, Tokyo,
Japan, 3 Division of Dermatology, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan

Abstract
Interleukin (IL)-27 is a member of the IL-6/IL-12 cytokine family and possesses potent antitumor activity, which is mediatedby multiple mechanisms. Toll-like receptor (TLR)3 is the critical sensor of the innate immune system that serves to identify
viral double-stranded RNA. TLR3 is frequently expressed by various types of malignant cells, and recent studies reported
that a synthetic TLR3 agonist, polyinosinic-polycytidylic acid [poly(I:C)], induces antitumor effects on malignant cells. In thepresent study, we have explored the effect of IL-27 on human melanomas and uncovered a previously unknown
mechanism. We found that IL-27 inhibits in vitro tumor growth of human melanomas and greatly enhances the expressionof TNF-related apoptosis inducing ligand (TRAIL) in a dose-dependent manner. Neutralizing antibody against TRAIL partly
but significantly blocked the IL-27–mediated inhibition of tumor growth. In addition, IL-27 and poly(I:C) cooperatively
augmented TRAIL expression and inhibited tumor growth. The cooperative effect could be ascribed to the augmented
expression of TLR3, but not retinoic acid-inducible gene-I or anti-melanoma differentiation-associated gene 5, by IL-27. The
inhibition of tumor growth by the combination was also significantly abrogated by anti-TRAIL neutralizing antibody.
Moreover, IL-27 and poly(I:C) cooperatively suppressed in vivo tumor growth of human melanoma in immunodeficientmice. Taken together, these results suggest that IL-27 enhances the expression of TRAIL and TLR3 in human melanomas andinhibits their tumor growth in cooperation with poly(I:C), partly in a TRAIL-dependent manner. Thus, IL-27 and the
combination of IL-27 and poly(I:C) may be attractive candidates for cancer immunotherapy.
Citation: Chiba Y, Mizoguchi I, Mitobe K, Higuchi K, Nagai H, et al. (2013) IL-27 Enhances the Expression of TRAIL and TLR3 in Human Melanomas and Inhibits
Their Tumor Growth in Cooperation with a TLR3 Agonist Poly(I:C) Partly in a TRAIL-Dependent Manner. PLoS ONE 8(10): e76159. doi:10.1371/journal.pone.0076159
Editor: Hiroyasu Nakano, Juntendo University School of Medicine, Japan
Received June 24, 2013; Accepted August 20, 2013; Published October 14, 2013
Copyright: ß 2013 Chiba et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported in part by Grant-in-Aid for Scientific Research and ‘‘University-Industry Joint Research’’ Project from the Ministry of Education,
Culture, Sports, Science and Technology, Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of themanuscript. No additional external funding was received for this study.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: yoshimot@tokyo-med.ac.jp. These authors contributed equally to this work.

We and other groups previously reported that IL-27 has apotent ability to induce tumor-specific antitumor and protective
immunity through cytotoxic T lymphocyte (CTL) and naturalkiller (NK) cells in colon carcinoma colon 26 [7,8] andneuroblastoma TBJ [9] lines. IL-27 was further demonstrated toexert antitumor activity against poorly immunogenic B16F10
melanoma, which is mediated through NK cells but not CTL [10],and also against NK cell–resistant head and neck squamous cell
carcinoma SCCVII through NK cell–mediated antibody-dependent cellular cytotoxicity (ADCC) [11]. IL-27 was also shown tohave potent anti-angiogenic activity by inducing anti-angiogenic
chemokines, IFN-c–inducing protein (IP-10, CXCL10) andmonokine induced by IFN-c (MIG, CXCL9), as does IFN-c, but
in an IFN-c–independent manner [12]. In addition, we recently
demonstrated that IL-27 has anti-proliferative activity and acts
directly on melanomas through WSX-1/STAT1 signaling [13].
Thus, IL-27 exerts antitumor activities through multiple

Introduction
Interleukin (IL)-27 is a heterodimeric cytokine belonging to theIL-6/IL-12 cytokine family and consists of an IL-12 p40-related
protein, Epstein-Barr virus-induced gene 3, and an IL-12 p35related protein, p28 [1]. T-cell cytokine receptor/WSX-1, which ishomologous to the IL-12 receptor (R) b2 subunit, and gp130, a
common receptor chain for the IL-6 cytokine family, constitute afunctional signal-transducing receptor for IL-27 [1]. IL-27
activates signal transduction and activator of transcription(STAT)1 and STAT3 [2,3] and induces both pro- and antiinflammatory immune responses. IL-27 not only induces early
helper T (Th)1 differentiation, but also suppresses Th2 and Th17
differentiation and pro-inflammatory cytokine production [4].
Moreover, IL-27 induces the differentiation into IL-10–producing
regulatory T cells [5,6].

PLOS ONE | www.plosone.org

1

-296-

October 2013 | Volume 8 | Issue 10 | e76159

TRAIL-Mediated Inhibition of Tumor Growth by IL-27

mechanisms including CTL, NK cells, ADCC, anti-angiogenic
activity, and direct antiproliferative activity depending on thecharacteristics of individual tumors.
Moreover, it was recently demonstrated that IL-27 inhibits thegrowth of human tumors including melanoma, multiple myeloma,
B-acute lymphoblastic leukemia, follicular lymphoma, diffuse large
B-cell lymphoma, and acute myeloid leukemia [13–16]. IL-27
strongly inhibited tumor growth and in vivo tumorigenicity ofmultiple myeloma cells through suppression of angiogenesis [15].
IL-27 also severely hampered the leukemic spreading induced innonobese diabetic/severe combined immunodeficiency (NOD/
SCID)/IL-2Rc2/2 mice by injection with B-acute lymphoblastic
leukemia cells from pediatric patients [14]. Similarly, acute
myeloid leukemia cells injected into NOD/SCID/IL-2Rc2/2
mice gave rise to leukemia dissemination that was severely
inhibited by IL-27 [16]. These antitumor effects were mainly
attributed to significant reduction of angiogenic and spreadingrelated genes and also to up-regulation of angiostatic molecules[14–16].
Toll-like receptor (TLR) is a type of pattern recognition receptor
and recognizes molecules that are broadly shared by pathogens
but distinguishable from host molecules, collectively referred to as
pathogen-associated molecular patterns. It plays a key role in the
innate immune system, and once microbes have breached physicalbarriers such as the skin or intestinal tract mucosa, they arerecognized by TLRs, which activate immune cell responses. TLR3
is a major effector of the immune response against viral pathogens,and it is involved in the early activation of NK and dendritic cells[17]. It is also expressed in a wide range of nonimmune cells inwhich it plays a crirical role in the induction of interferon response.
Since TLR3 is frequently expressed by various types of malignant
cells and can directly trigger tumor cell apoptosis, TLR3 isconsidered to be a promising target for therapeutic purpose [18].
One of the TLR3 agonists, polyinosinic-polycytidylic acid[poly(I:C)], has been demonstrated to induce potent antitumor
activity against various tumors [19].
In the present study, we attempted to gain a better
understanding of the molecular events governing the antiproliferative effect of IL-27 on melanomas. We here show that
IL-27 enhances the expression of TNF-related apoptosis inducing
ligand (TRAIL) and TLR3 in human melanomas and therefore
inhibits their tumor growth in cooperation with poly(I:C) partly ina TRAIL-dependent manner. This is the first report to show a rolefor TRAIL in IL-27–mediated inhibition of tumor growth as well
as a cooperative effect between IL-27 and poly(I:C) on theinhibition of tumor growth through TLR3 up-regulation by IL-27.

Reagents
Human and mouse recombinant IL-27 was prepared as atagged single-chain fusion protein by flexibly linking EBI3 to p28
using HEK293-F cells (Life Technologies, Carlsbad, CA) as
described previously [12]. Anti-phosphotyrosine (pY)-STAT1,
anti-pY-STAT3, anti-TRAIL (C92B9), anti-retinoic acid-inducible gene-I (RIG-I, D14G6) and anti-melanoma differentiationassociated gene 5 (MDA5, D74E4) were purchased from Cell
Signaling Technology (Danvers, MA). Anti-STAT1 and antiSTAT3 were from Santa Cruz Biotechnology (Dallas, TX). AntiTRAIL and phycoerythrin (PE)-labeled anti-TRAIL (RIK-2) werefrom BioLegend (San Diego, CA). Anti-TLR3 (clone#512505)
was from R&D Systems (Minneapolis, MN). Anti-b-actin andpoly(I:C) were from Sigma-Aldrich (St. Louis, MO) and InvivoGen (San Diego, CA), respectively. Soluble TRAIL was purchasedfrom Alexis Biochemicals (San Diego, CA).

Western blotting
Cells were lysed in a lysis buffer containing protease inhibitors,
and the resultant cell lysates were separated by SDS-PAGE under
reducing conditions and transferred to polyvinylidene difluoride
membrane (Millipore, Billerica, MA) as described previously [21].
The membrane was then blocked, probed with a primary antibody
and then with an appropriate secondary antibody conjugated to
HRP, and visualized with the enhanced chemiluminescence
detection system (GE Healthcare, Uppsala, Sweden) according
to the manufacturer’s instructions. Immunoreactive bands weredetected with a ChemiDoc XRS (Bio-Rad, Hercules, CA), and theintensity of each band was quantified with the Image Lab (BioRad) or Image J (NIH, Bethesda, MD) program.

Proliferation assay
Cells (26103 to 86103 cells/200 ml/well) were stimulated with
IL-27 and/or poly(I:C) for 48–96 h and pulsed with 3H-thymidine
for the last 8–24 h. 3H-thymidine incorporation was counted using
TopCount (PerkinElmer, Waltham, MA).

Apoptosis assay
Apoptosis was assessed by flow cytometric analysis of cells
stained with Annexin V-fluorescein isothiocyanate (FITC) andpropidium iodide (PI) according to the manufacturer’s instruction(BD Biosciences, San Jose, CA).

RT-PCR
Total RNA was extracted using a guanidine thiocyanate
procedure, cDNA was prepared using oligo(dT) primer andSuperScript III RT (Life Technologies), and RT-PCR was
performed using Taq DNA polymerase. Cycle conditions were
94uC for 40 s, 60uC for 20 s, and 72uC for 40 s. Primers used for
gp130, WSX-1, and hypoxanthine phosphoribosyl transferase(HPRT) as an internal control were previously described [13,22].
The following primers were also used: TRAIL sense primer, 59GTGCTGATCGTGATCTT-39; TRAIL antisense primer, 59CCACTCCTTGATGATTCC-39; TRAIL-R1 sense primer, 59CTGAGCAACGCAGACTCGCTGTCCAC-39; TRAIL-R1 antisense primer, 59-TCCAAGGACACGGCAGAGCCTGTGCCAT-39; TRAIL-R2 sense primer, 59-GCCTCATGGACAATGAGATAAAGGTGGCT-39; TRAIL-R2 antisense primer,
59-CCAAATCTCAAAGTACGCACAAACGG-39; TRAIL-R3
sense primer, 59-GAAGAATTTGGTGCCAATGCCACTG-39;
TRAIL-R3 antisense primer, 59-CTCTTGGACTTGGCTGGGAGATGTG-39; TRAIL-R4 sense primer, 59-CTTTTCCGGCGGCGTTCATGTCCTTC-39; TRAIL-R4 antisense primer,

Materials and Methods
Ethics statement
The animal study was approved by the institutional review
board of Tokyo Medical University (S-22005, S-23044, S-24013
and S-25005).

Cell culture and mice
Human melanoma cell lines, SK-MEL-13, -28, and -37 [20],were kindly provided by Drs. L. J. Old and G. Ritter (Ludwig
Institute for Cancer Research, New York Branch, Memorial
Sloan-Kettering Cancer Center, New York, NY) and Dr. T.
Takahashi (Aichi Cancer Center, Nagoya, Japan), and cultured in
Iscoves’s modified Dulbecco’s medium supplemented with 10%
FBS. C57BL/6 mice and NOD/SCID mice were purchased fromSankyo Labo Service Corp. and Clea Japan, Inc., respectively.

PLOS ONE | www.plosone.org

2

-297-

October 2013 | Volume 8 | Issue 10 | e76159

TRAIL-Mediated Inhibition of Tumor Growth by IL-27

Figure 1. IL-27 induces TRAIL expression in human melanomas and inhibits their tumor growth partly in a TRAIL-dependent
manner. (A) Human melanoma cell lines (SK-MEL-13, -28, and -37) were stimulated with IL-27 (10 ng/ml) for 0, 20, and 60 min. Cell lysate was thenprepared and subjected to Western blotting with anti-pY-STAT1, anti-pY-STAT3, anti-total STAT1, and anti-total STAT3. Similar results were obtainedin two independent experiments. (B) These melanoma cell lines were stimulated with increasing doses of IL-27 (0–100 ng/ml) for 72–96 h in triplicate
and pulsed with 3H-thymidine for the last 24 h, and 3H-thymidine incorporation was measured. Data are shown as means 6 SD. *, {, and ` indicate
p,0.01, p,0.0001, and p,0.00001, respectively, compared with 0 ng/ml IL-27. Similar results were obtained in three independent experiments. (C)These melanoma cell lines were stimulated with increasing doses of IL-27 (0–100 ng/ml) for 24 h. Total RNA was then extracted and subjected to RTPCR analysis. Similar results were obtained in more than three independent experiments. (D) These melanoma cell lines were stimulated with IL-27

PLOS ONE | www.plosone.org

3

-298-

October 2013 | Volume 8 | Issue 10 | e76159

TRAIL-Mediated Inhibition of Tumor Growth by IL-27(100 ng/ml) for 48 h, and then analyzed for cell surface expression of TRAIL by FACS using PE-labeled anti-TRAIL (solid line) and its control Ab (plain
line with shading). Similar results were obtained in two independent experiments. (E) These melanoma cell lines were stimulated with IL-27 (10 and
100 ng/ml) in the presence of anti-TRAIL neutralizing Ab or its control Ab (10 mg/ml) for 72–96 h in triplicate and pulsed with 3H-thymidine for thelast 24 h, and 3H-thymidine incorporation was measured. Data are shown as means 6 SD. * indicates p , 0.05, compared with control Ab. Similar
results were obtained in three independent experiments.
doi:10.1371/journal.pone.0076159.g001

59-GTTTCTTCCAGGCTGCTTCCCTTTGTAG-39; TLR3
sense primer, 59-TTGCCTTGTATCTACTTTTGGGG-39; TLR3 antisense primer, 59-GCGGCTGGTAATCTTCTGAGTT39; RIG-I sense primer, 59-TGTGGGCAATGTCATCAAAA-39;
RIG-I antisense primer, 59-GAAGCACTTGCTACCTCTTGC39; MDA5 sense primer, 59-GGCACCATGGGAAGTGATT-39;
MDA5 antisense primer, 59-ATTTGGTAAGGCCTGAGCTG39; IFN regulatory factor (IRF)-1 sense primer, 59-GAAGTCCAGCCGAGATGC-39; IRF-1 antisense primer, 59-CGGCACAACTTCCACTG-39. Real-time quantitative PCR was carriedout using SYBR Premix Ex Taq II (TAKARA, Otsu, Shiga,
Japan) according to the manufacture’s instructions. Glyceraldehyde-3-phosphate (GAPDH) was used as house-keeping gene tonormalize mRNA. Relative expression of real-time PCR products
was determined by using the DDCt method to compare target
gene and GAPDH mRNA expression. Primers for TRAIL and
GAPDH were purchased from TAKARA: TRAIL sense primer,
59-CTTCACAGTGCTCCTGCAGTCTC-39; TRAIL antisense
primer, 59-AAGGTAGACTTCAAGATGGCAGCAA-39; GAPDH sense primer, 59-GCACCGTCAAGGCTGAGAAC-39; GAPDH antisense primer, 59-TGGTGAAGACGCCAGTGGA-39.

of the IL-27R subunits, WSX-1. However, B16F10 cells transfected with WSX-1-expression vector (B16F10-WSX-1) become as
responsive to IL-27 as several human melanomas [13]. In threedifferent human melanoma cell lines, SK-MEL-13, -28, and -37,
IL-27 induced tyrosine phosphorylation of STAT1 and STAT3,
indicating that these cells were responsive to IL-27 (Fig. 1A).
Moreover, IL-27 dose-dependently inhibited their tumor growth(Fig. 1B). Among them, SK-MEL-37 seemed to be mostsusceptible to stimulation by IL-27.

IL-27 induces TRAIL expression in human melanomas andinhibits their tumor growth partly in a TRAIL-dependent
mannerWe previously demonstrated that IL-27 inhibits the tumor
growth of B16F10-WSX-1 cells through the WSX-1/STAT1 andIRF-1 pathway [13]. To gain a better understanding of themolecular events governing the anti-proliferative effect of IL-27 on
melanomas, we first focused on TRAIL, which is an important
immune effector molecule in the surveillance and elimination ofdeveloping tumors [24]. TRAIL is a promising antineoplastic
agent because it induces apoptosis in cancer cells with only
negligible effects on normal cells. In addition, several previous
studies revealed that IFN-a induces anti-proliferative effects on
cancer cells by stimulating them to produce TRAIL [25,26]. Firstof all, we examined the sensitivity of three different cell lines ofhuman melanoma to soluble TRAIL. Cell growth of all three celllines was significantly inhibited in a dose-dependent manner,
although the sensitivity appeared to vary among them (Fig. S1).
Three human melanoma cell lines were then stimulated withincreasing doses of IL-27, and total RNA was extracted 24 h laterand subjected to RT-PCR analysis. The mRNA expression of
TRAIL was greatly enhanced by IL-27 in a dose-dependent
manner (Fig. 1C). Among the three melanoma cell lines, SKMEL-37 cells expressed the highest TRAIL mRNA amount after
stimulation by IL-27. At the protein level, IL-27 also enhanced theexpression of TRAIL, which was detected by Western blot analysis(see the later section), but these melanomas only slightly expressed
cell surface TRAIL in response to IL-27, which was detected byfluorescence-activated cell sorter (FACS) analysis (Fig. 1D).
We next examined whether or not these melanomas express
TRAIL-Rs (TRAIL-R1–R4) [27] to transduce the signal by
TRAIL. These melanomas expressed TRAIL-R1 and TRAIL-R2,
and the expression levels appeared not to be affected by IL-27 (Fig.
1C). TRAIL-R3 and TRAIL-R4, known as decoy receptors-1 and-2, cannot induce apoptosis but inhibit TRAIL-induced apoptosis
mediated by TRAIL-R1 and/or TRAIL-R2 when coexpressed[27]. TRAIL-R3 expression was detected in only SK-MEL-13
cells but not others, and its expression level was only minimallyaffected by IL-27 (Fig. 1C). Moreover, TRAIL-R4 expression was
hardly detected among all these cell lines even after stimulation by
IL-27 (data not shown). Thus, these melanomas would besusceptible to the stimulation by TRAIL, but the susceptibilities
may vary among the melanoma lines.
To explore the role of enhanced expression of TRAIL on tumor
growth, these melanomas were stimulated by IL-27 (10 and
100 ng/ml) in the presence of neutralizing antibody (Ab) against
TRAIL or its control Ab. IL-27 inhibited tumor growth of

Small interfering RNA (siRNA) transfection
Human TLR3 siRNA (ON-TARGETplus SMARTpool) andnegative control siRNA (siGLO RISC-free) were purchased from
Thermo Fisher Scientific (Lafayette, CO). Human RIG-I and
MDA-5 siRNAs, whose sequences were reported previously [23],were synthesized by Sigma-Aldrich. Human melanoma cells of
SK-MEL-37 cell line were plated in 12-well plates and transfectedwith these siRNAs using HiPerFect Transfection Reagent (Qiagen
Inc., Venlo, Netherlands) or Lipofectamine RNAiMAX Transfection Reagent (Life Technologies) according to the manufacturers’ protocols.

Human tumor xenograft model in immunodeficient mice
Human melanoma cells (56106 cells/mouse) of SK-MEL-37
cell line were injected subcutaneously (s.c.) into the right flank of
NOD/SCID mice (n = 4–5). The treatment was started 1 week
postengraftment and consisted of weekly intravenous (i.v.)
injections with PBS alone, mouse IL-27 (1 mg), poly(I:C) (30 mg),or mouse IL-27 (1 mg) plus poly(I:C) (30 mg) in 200 ml of PBS.
Mouse and human IL-27 were previously demonstrated to almost
equally inhibit human melanoma growth [13]. Tumor growth wasmonitored weekly with an electronic caliper and expressed asvolume (mm3) by calculating using the following volume equation:
0.5(ab2), where a is the long diameter and b is the short diameter.

Statistical analysis
Statistical analysis was performed by Student’s t test. A p valueof less than 0.05 was considered to indicate a statisticallysignificant difference.

Results
IL-27 inhibits tumor growth of human melanomas
We previously demonstrated that mouse melanoma B16F10
cells are not responsive to IL-27 due to a lack of expression of one
PLOS ONE | www.plosone.org

4

-299-

October 2013 | Volume 8 | Issue 10 | e76159

TRAIL-Mediated Inhibition of Tumor Growth by IL-27

Then, the involvement of up-regulated sensors for poly(I:C) ininhibiting SK-MEL-37 tumor growth by combining IL-27 andploy(I:C) was investigated by knock-down of their expression using
respective siRNAs. When up-regulated TLR3 expression was
reduced by 58% with 100 nM of its siRNA, which was determinedby the intensity of each band, suppressed tumor growth wassignificantly recovered (Fig. 3F and G). However, knock-down of
RIG-I or MDA5 failed to affect the suppressed tumor growth (Fig.
S2A-D).
We previously demonstrated that IL-27 induces IRF-1 expression, which is involved in the inhibition of tumor growth, in mousemelanoma B16F10 cells ectopically expressing WSX-1 [13]. In
addition, IFN-c and IFN-a were reported to enhance IRF-1mediated TRAIL expression in human tumor cell lines [29,30].
We then examined whether IL-27 induces IRF-1 expression inthese human melanomas. Consistent with the previous results[13,29,31], IL-27 induced IRF-1 expression at the mRNA level inall three human melanomas (Fig. 3A). Moreover, time course
analyses revealed that IRF-1 mRNA expression was rapidly
induced in response to IL-27 and poly(I:C), which was followed by
up-regulation of TLR3 and TRAIL (Fig. 3C).
These results suggest that IL-27 enhances the expression of
TLR3 and TRAIL probably through IRF-1 in human melanomas, which could account for the cooperative effect between IL-27
and poly(I:C).

melanomas, while anti-TRAIL Ab, but not control Ab, partly but
significantly abrogated the inhibitory effect by IL-27 on tumor
growth in all three melanoma cell lines (Fig. 1E).
These results suggest that IL-27 induces TRAIL expression at
the mRNA level in human melanomas and inhibits tumor growth
partly in TRAIL-dependent manner.

IL-27 and TLR3 agonist poly(I:C) cooperatively inhibit
tumor growth of human melanomas
TLR3 is a critical sensor of the innate immune system thatserves to identify viral double-stranded RNA [17]. Several recent
studies suggested that TLR3 activation by its synthetic agonist
poly(I:C) directly causes tumor cell apoptosis [18]. To enhance theanti-proliferative activity of IL-27 against melanomas, thecombined effect of IL-27 and poly(I:C) on tumor growth wasnext explored. Three human melanoma cell lines were treatedwith IL-27 and/or poly(I:C) for 72–96 h and their proliferative
responses were determined. Either IL-27 or poly(I:C) alone slightly
reduced tumor growth but their combination more strongly
inhibited tumor growth in the three melanoma cell lines (Fig. 2A).
Examination of morphology of SK-MEL-37 melanoma cells also
supported the enhanced inhibition of tumor growth by the
combination (Fig. 2B). Similar phenomena were also observed in
SK-MEL-13 and -28 melanomas (data not shown).
Annexin V binding to cell surface phosphatidylserine is one
hallmark of apoptotic cell death, and PI, which stains DNA, is amarker for permeabilized, necrotic cells. The FACS analysis of
SK-MEL-37 melanoma cells was then carried out after Annexin
V-PI staining. The IL-27 and poly(I:C) combination slightly, but
significantly, increased Annexin V-positive and PI-positive population containing apoptotic cells, but neither alone increased it
under the experimental conditions (Fig. 2C and D). These results
suggest that IL-27 mediates cytostatic activity against tumor cells
rather than cytotoxic activity as reported [13]. A similar tendency
was observed with the other melanomas, SK-MEL-13 and -28(data not shown).
These results suggest that IL-27 and poly(I:C) cooperatively
inhibit tumor growth of human melanomas.

IL-27 and poly(I:C) cooperatively induce TRAIL expression
in human melanomas and inhibit tumor growth partly ina TRAIL-dependent manner
To further demonstrate the cooperative effect between IL-27
and poly(I:C), we examined the effect of the combination on
TRAIL expression in human melanomas. Three human melanoma cell lines were treated with increasing doses of IL-27 in thepresence or absence of poly(I:C). Total RNA was extracted after
24 h and subjected to RT-PCR analysis. IL-27 enhanced theexpression of TRAIL mRNA, and the presence of poly(I:C)
further augmented it in all three melanomas (Fig. 4A). Then, total
cell lysates were prepared after 48 h and subjected to Western blot
analysis. Similar augmentation of TRAIL expression at protein
levels was observed in these melanomas, but the expression level
was highest in SK-MEL-37 and lowest in SK-MEL-13 (Fig. 4B).
Cell surface expression of TRAIL was also analyzed by FACS.
Synergistic induction of cell surface TRAIL expression by the
combination of IL-27 and poly(I:C) was clearly observed in SKMEL-28 and SK-MEL-37 cells 48 h after stimulation (Fig. 4C).
However, only slight augmentation was detected in SK-MEL-13
cells 48 h but not 24 and 72 h (data not shown) after stimulation.
Of note, regardless of the expression levels of TRAIL, thetreatment with anti-TRAIL neutralizing Ab, but not control Ab,
partly but significantly abrogated the inhibitory effect on tumor
growth of combined IL-27 and poly(I:C) in all three melanoma celllines (Fig. 4D).
These results suggest that IL-27 and poly(I:C) cooperatively
induce TRAIL expression in human melanomas and significantly
inhibit their tumor growth partly in a TRAIL-dependent manner.
However, the TRAIL expression level induced by IL-27 andpoly(I:C) seems to vary among three tumor cell lines; highest in
SK-MEL-37 and lowest in SK-MEL-12 cells.

IL-27 enhances TLR3 expression in human melanomas,
which could account for the cooperative effect between
IL-27 and poly(I:C)
To clarify the molecular mechanism whereby IL-27 andpoly(I:C) cooperatively inhibit tumor growth of melanomas, wenext investigated the effect of IL-27 on the expression of TLR3,
which is the receptor for poly(I:C), and also conversely the effect ofpoly(I:C) on the expression of IL-27R subunits, gp130 and WSX1. Human melanomas were treated with increasing concentrationsof IL-27 in the presence or absence of poly(I:C) for 24 h, andresultant cells were harvested. Total RNA and cell lysate were thenprepared and subjected to RT-PCR and Western blot analyses,
respectively. In all three cell lines, a slight amount of TLR3
expression was detected without any stimulation, while theexpression was enhanced by IL-27 at both mRNA and protein
levels (Fig. 3A and D). Moreover, the addition of poly(I:C) seemed
to further augment it (Fig. 3A and D). Besides TLR3, RIG-I andMDA5 are also known to be important sensors for poly(I:C) incytosols [28]. Therefore, we next examined the effect of IL-27 on
the expression of these molecules. IL-27 enhanced their expression
at both mRNA and protein levels (Fig. 3B and E). In contrast,
these human melanomas express mRNA of the IL-27R subunits
WSX-1 and gp130 even without any stimulation, and the additionof poly(I:C) hardly affected their expression (Fig. 3A).
PLOS ONE | www.plosone.org

IL-27 and poly(I:C) cooperatively inhibit in vivo tumor
growth of human melanoma in immunodeficient mice
Finally, in vivo antitumor effects of IL-27 or the combination of
IL-27 and poly(I:C) were examined using a tumor xenograft
5

-300-

October 2013 | Volume 8 | Issue 10 | e76159

TRAIL-Mediated Inhibition of Tumor Growth by IL-27

Figure 2. IL-27 and TLR3 agonist poly(I:C) cooperatively inhibits tumor growth of human melanomas. (A) Human melanoma cell lines(SK-MEL-13, -28 and -37) were stimulated with IL-27 (10 ng/ml) and/or poly(I:C) (10 mg/ml) for 72-96 h in triplicate and pulsed with 3H-thymidine forthe last 24 h, and 3H-thymidine incorporation was measured. Data are shown as means 6 SD. *, {, `, 1, ", and || indicate p,0.05, p,0.01, p,0.001,
p,0.00001, and p,0.000001, respectively, compared with 0 ng/ml IL-27 and 0 mg/ml poly(I:C). Similar results were obtained in thee independent
experiments. (B) Morphology was evaluated using light microscopy. A representative result of SK-MEL-27 from among the three human melanoma
cell lines is shown. Similar results were obtained in more than five independent experiments. (C) These melanoma cell lines were stimulated with IL27 (10 ng/ml) and/or poly(I:C) (10 mg/ml) for 48 h. Then, apoptosis was assessed by FACS analysis of cells stained with Annexin V-FITC and PI. A
representative result of SK-MEL-27 from among the three human melanoma cell lines is shown. Similar results were obtained in three independent
experiments. (D) Percentage of each population was calculated. Data are shown as means 6 SD of three independent experiments. * indicatesp , 0.05, compared to no stimulation.
doi:10.1371/journal.pone.0076159.g002

mouse model in immunodeficient NOD/SCID mice. Fromamong three human melanoma cell lines, SK-MEL-37 cells wereused, because this cell line appears to be most susceptible to thetreatment with IL-27 or combined IL-27 and poly(I:C) (Fig. 1B
and 2A). NOD/SCID mice were s.c. injected with SK-MEL-37
cells, and treated by weekly i.v. injections with PBS, IL-27,

PLOS ONE | www.plosone.org

poly(I:C), or IL-27 plus poly(I:C) from 1 week postengraftment. IL27 alone only slightly suppressed tumor growth in vivo (Fig. 5).
Poly(I:C) alone more greatly hampered in vivo tumor progression(Fig. 5), as reported previously [32]. Of note, the combination of
IL-27 and poly(I:C) significantly inhibited tumor progression
compared with PBS alone (Fig. 5). Then, the TRAIL expression at

6

-301-

October 2013 | Volume 8 | Issue 10 | e76159

TRAIL-Mediated Inhibition of Tumor Growth by IL-27

Figure 3. IL-27 enhances TLR3 expression in human melanomas, which could account for the cooperative effect between IL-27 andpoly(I:C). (A-C) Human melanoma cell lines (SK-MEL-13, -28, and -37) were stimulated with IL-27 (1, 3, 10 ng/ml) and/or poly(I:C) (1 mg/ml) for 24 h or
the indicated times. Total RNA was then extracted and subjected to RT-PCR analysis. Similar results were obtained in more than two independent
experiments. (D and E) Cell lysate was also prepared after the stimulation for 48 h and subjected to Western blotting with antibodies against TLR3,
RIG-I, MDA5 and b-actin. Similar results were obtained in more than two independent experiments. (F) SK-MEL-37 cells were transfected with siRNA
specific to TLR3 or control siRNA for 24 h. These cells were then stimulated with IL-27 (10 ng/ml) and poly(I:C) (1 mg/ml) for a further 24 h, and total
cell lysate was prepared and subjected to Western blot using anti-TLR3 and anti-b-actin. (G) The siRNA-transfected cells were also stimulated with IL27 (10 ng/ml) and poly(I:C) (1 mg/ml) for a further 48 h and pulsed with 3H-thymidine for the last 8 h in triplicate. 3H-thymidine incorporation wasmeasured, and relative proliferation (%) to that of respective unstimulated cells was calculated. Data are shown as means 6 SD. * indicates p , 0.05
compared with no siRNA and control siRNA. Similar results were obtained in two independent experiments.
doi:10.1371/journal.pone.0076159.g003

PLOS ONE | www.plosone.org

7

-302-

October 2013 | Volume 8 | Issue 10 | e76159

TRAIL-Mediated Inhibition of Tumor Growth by IL-27

Figure 4. IL-27 and poly(I:C) cooperatively induce TRAIL expression in human melanomas and inhibit their tumor growth partly in a
TRAIL-dependent manner. (A) Human melanoma cell lines (SK-MEL-13, -28, and -37) were stimulated with IL-27 (1, 3, 10 ng/ml) and/or poly(I:C) (0 or
1 mg/ml) for 24 h. Total RNA was then extracted and subjected to RT-PCR analysis. Similar results were obtained in three independent experiments. (B)These melanoma cell lines were stimulated with IL-27 (10 ng/ml) and/or poly(I:C) (10 mg/ml) for 48 h, and then total cell lysates were prepared andsubjected to Western blot analysis using anti-TRAIL and anti-b-actin. Relative expression level of TRAIL was determined by the intensity of each band of
TRAIL and b-actin. Similar results were obtained in two independent experiments. (C) These cells were also analyzed for cell surface expression of TRAIL
by FACS using PE-labeled anti-TRAIL (solid line) and its control Ab (plain line with shading). Similar results were obtained in more than two independent
experiments. (D) These melanoma cell lines were stimulated with IL-27 (10 ng/ml) and poly(I:C) (1 mg/ml) in the presence of anti-TRAIL neutralizing Ab or
its control Ab (10 mg/ml) for 72–96 h in triplicate and pulsed with 3H-thymidine for the last 24 h, and 3H-thymidine incorporation was measured. Data areshown as means 6 SD. * indicates p , 0.05, compared with control Ab. Similar results were obtained in three independent experiments.
doi:10.1371/journal.pone.0076159.g004

mRNA levels in the tumors was examined on day 63 after tumor
injection. The TRAIL mRNA expression levels tended to increaseby the IL-27 injections, although they were not significant (Fig.
S3). Thus, these results suggest that IL-27 and poly(I:C)
cooperatively inhibit in vivo tumor growth of human melanoma
in immunodeficient mice.
PLOS ONE | www.plosone.org

Discussion
Since antitumor efficacy of IL-27 was first evaluated in 2004 [7],accumulating evidence has revealed that IL-27 has potent
antitumor activity, which is mediated by multiple mechanisms
including CD8+ T cells [7–9,33], NK cells [10,34], ADCC [11],

8

-303-

October 2013 | Volume 8 | Issue 10 | e76159

TRAIL-Mediated Inhibition of Tumor Growth by IL-27

Figure 5. IL-27 and poly(I:C) cooperatively inhibit in vivo tumor
growth of human melanoma in immunodeficient mice. Immunodeficient NOD/SCID mice were s.c. injected with human melanoma
cells of the SK-MEL-37 cell line, and treated by weekly i.v. injections with
PBS alone, IL-27 (1 mg), poly(I:C) (30 mg), or IL-27 (1 mg) plus poly(I:C)(30 mg) from 1 week postengraftment. Tumor growth was monitored
weekly and expressed as volume (mm3). Data are shown as means 6
SD. * indicates p , 0.05, compared with PBS. Similar results wereobtained in two independent experiments.
doi:10.1371/journal.pone.0076159.g005

Figure 6. Hypothetical model of the pathway by which IL-27
and the combination of IL-27 and poly(I:C) induce TRAIL upregulation and inhibits tumor growth in human melanomas. IL27 induces IRF-1 expression through WSX-1/STAT1 signaling, resulting
in up-regulation of TRAIL expression. IL-27 also augments TLR3
expression in IRF-1-dependent and -independent manner. Thus, due
to the augmented TLR3 expression by IL-27, IL-27, and a synthetic TLR3
agonist, poly(I:C), cooperatively enhance TRAIL expression and greatly
inhibit tumor growth partly in a TRAIL-dependent manner.
doi:10.1371/journal.pone.0076159.g006

anti-angiogenesis [12,14–16,35,36], direct suppression of tumor
growth [13], and inhibition of cyclooxygenase-2 expression [37],depending on the characteristics of individual tumors. In thepresent study, we further explored the molecular mechanism
whereby IL-27 induces direct suppression of tumor growth ofhuman melanomas. We elucidated that IL-27 augments theexpression of TRAIL and TLR3 together with RIG-I and MDA5
in human melanomas, and that IL-27 and a TLR3 agonist,poly(I:C), cooperatively enhance TRAIL expression and inhibit
their tumor growth partly in a TRAIL-dependent manner as
illustrated in Fig. 6. The cooperative effect could be ascribed to theenhanced expression of TLR3, but not RIG-I or MDA5, by IL-27(Fig. 3A, B, D-G and Fig. S2A-D), which is consistent with facts
that RIG-I and MDA5 sense transfected poly(I:C), but not nakedpoly(I:C), in non-phagocytic cells [38,39]. Since IL-27 augmented
only marginal apoptosis even in collaboration with poly(I:C) (Fig.
2C and D), IL-27 is considered to mediate cytostatic activity rather
than cytotoxic activity as reported before [13]. Furthermore, thecombination of IL-27 and poly(I:C) significantly suppressed in vivo
tumor progression in the human melanoma xenograft model using
immunodeficient NOD/SCID mice (Fig. 5). When a higher dose(100 mg) of poly(I:C) was injected, tumor progression was moremarkedly inhibited regardless of the combination with IL-27 (datanot shown) as reported previously [32,40]. However, we noticed
that 3 out of 5 mice died even without any tumor after theexperiment was completed. In contrast, intriguingly, no mice diedwith treatment based on combined IL-27 and poly(I:C) (data notshown). Although further studies are necessary, the combination
treatment may be beneficial in reducing the potential toxicity
arising from a higher dose of poly(I:C) [41].
Among three human melanoma cell lines used in this study, SKMEL-13 showed slightly less susceptibility to the IL-27 andpoly(I:C)–mediated TRAIL-dependent growth inhibition and SKMEL-37 showed more susceptibility to it (Fig. 1B and Fig. 2A).
This tendency is consistent with the enhanced expression levels of
TRAIL (Fig. 4B and C) and also TLR3 (Fig. 3D and E) in
PLOS ONE | www.plosone.org

response to IL-27 and poly(I:C), although the expression levels of
the receptors gp130, WSX-1, RIG-I and MDA5 appeared to becomparable (Fig. 3A, B and E). This tendency might be also
affected by the constitutive expression of the decoy receptor for
TRAIL, TRAIL-R3 (Fig. 1C), which inhibits TRAIL signaling[27], or by the different sensitivity to soluble TRAIL (Fig. S1). Of
note, anti-TRAIL neutralizing Ab partly but significantly abrogated the IL-27 and poly(I:C)–mediated growth inhibition in allthese cell lines (Fig. 1E and 4D). This partial abrogation might bedue to the insufficient ability of the Ab to neutralize the TRAIL
activity, or alternatively, there could be TRAIL-independent
mechanisms as recently reported in the mechanism for IFNinduced apoptosis [42]. DNase II-deficient embryos die in utero
due to severe anemia caused by IFN-b produced in themacrophages carrying undigested DNA. Although a high level of
TRAIL mRNA was found in the fetal liver, a null mutation in
TRAIL failed to rescue the lethal anemia, indicating that TRAIL
is not necessary for inducing the apoptosis of erythroid cells in
DNase II-deficient embryos [42]. Further studies are necessary to
clarify the molecular mechanisms underlying IL-27 and poly(I:C)–
mediated inhibition of tumor growth in TRAIL-dependent and independent manners.
The IFNs, especially type I IFNs such as IFN-a and -b, are wellinvestigated cancer therapeutic agents [43]. IFN-a is used for thetreatment of renal cell carcinoma, leukemia, and malignant
melanoma. Several previous studies reported that IFN-a and
IFN-c induces anti-proliferative effects on a variety of cancer cells
by stimulating them to produce TRAIL [25,26]. IFN-a and IFN-cenhance TRAIL expression via a STAT1– and IRF-1–dependent
mechanism in cancer cells [29,30]. We previously demonstrated
that IL-27 induces IRF-1 expression in mouse B16F10 melanoma
cells ectopically expressing wild-type WSX-1, but not in thoseexpressing WSX-1 mutated in the tyrosine residue critical for
STAT1 binding [13]. Moreover, the induction of IRF-1 wasrevealed to contribute to the IL-27–mediated inhibition of tumor
9

-304-

October 2013 | Volume 8 | Issue 10 | e76159

TRAIL-Mediated Inhibition of Tumor Growth by IL-27

growth of B16F10 melanoma [13]. Taken together with thepresent results, it is highly conceivable that IL-27–induced upregulation of TRAIL in human melanomas could be also mediatedby STAT1/IRF-1 signaling (Fig. 6).
TLR agonists are currently being studied in many clinicalimmunotherapy trials as vaccine adjuvants. TLR3, a member
receptor of double-stranded RNAs, is a major effector of theimmune response against viral pathogens at the cellular andsystemic level [44]. It is involved in early activation of NK anddendritic cells and expressed in a wide range of nonimmune cells
in which it plays a key role in the induction of the IFN response[45]. TLR3 is also frequently expressed by various types ofmalignant cells, and several studies reported that the synthetic
TLR3 agonist, poly(I:C), induces proliferation blockade andapoptosis in malignant cells in vitro and in vivo
[18,19,32,40,46]. Moreover, it was reported that IFN-a upregulates the expression of TLR3 in lung carcinoma A549 cells
and human umbilical vein endothelial cells [47]. Although IFN-a
by itself did not induce massive apoptosis, it increased TLR3mRNA and enhanced the anti-proliferative effect of poly(I:C).
Inhibiting the JAK/STAT pathway diminished the induction of
TLR3 by IFN-a, which highlights the importance of JAK/STAT
signaling on IFN-a–induced TLR3 transcription. It was previously
noted that the TLR3 promoter contains an IFN-stimulated
response element and a STAT binding site, which were suggested
to be involved in regulating the response of TLR3 to IFN-a [48].
In addition, it was previously reported that IFN-b induces TLR3
up-regulation through IFN-a receptor 1, STAT1, and in part IRF1, but not Tyk2, in murine macrophages [49]. Similar to IFN-aand IFN-b, IL-27 activates STAT1 and induces IFN-inducible
genes including antiviral genes, resulting in inhibition of HIV
replication in CD4+ T cells and macrophages [50]. These reports
indicate similarity between IFN-a/b and IL-27 in the signaling
events and biological functions as previously suggested [51], which
could account for the IL-27–induced up-regulation of TLR3 andTRAIL in human melanomas.
TLR4 is important in mediating inflammatory cytokine
production in response to bacterial infection, and LPS is the main
ligand binding to TLR4 to induce inflammation. Interestingly,
although engagement of TLR3 expressed in human breast tumor
cells induces massive apoptosis [18], triggering TLR4 in murine
colon cancer MC26 cell line leads to immune evasion mediated by
the inhibition of T- and NK-cell activities [52]. Similar to thepresent results, it has very recently been reported that IL-27
enhances LPS-induced proinflammatory cytokine production
through up-regulation of TLR4 expression in human monocytes[53]. However, this up-regulation was mediated via JAK2, STAT3
and NF-kB, but not via STAT1. Thus, requirements of STAT1
and STAT3 in the signaling for up-regulation of distinct TLRs by
IL-27 appear to differ between TLR3 and TLR4. Further studies
are necessary to precisely elucidate the molecular mechanism
whereby IL-27 induces up-regulation of TLR3 and TLR4.
Taken together, the present results suggest that IL-27 exerts
anti-proliferative activity against human melanomas by mechanisms similar to those of IFN-a in a STAT1/IRF-1–dependent
manner and partly in a TRAIL-dependent manner. Moreover, IL27 increases TLR3 expression and therefore the combination of
IL-27 and poly(I:C) cooperatively enhances TRAIL expression
and inhibits tumor growth. IL-12 is one of the cytokines withstrong antitumor activity. The induction of the antitumor activity
and establishment of protective immunity by IL-12 are highly

PLOS ONE | www.plosone.org

dependent on production of IFN-c by NK and T cells [54].
However, this high IFN-c production also causes systemic
toxicities, which leads to limitation of the IL-12 therapy in clinical
trials [55]. In this regards, IL-27 has much less toxicities comparedwith IL-12, probably due to much lower ability of IL-27 toproduce IFN-c by NK and T cells [7,10]. Therefore, IL-27 andthe combination of IL-27 and poly(I:C) may be attractive
candidates as antitumor agents applicable to cancer immunotherapy.

Supporting Information
Figure S1 Human melanoma cell lines SK-MEL-13, 28
and 37 are sensitive to soluble TRAIL. Three melanoma celllines were stimulated with increasing doses of soluble TRAIL (0–100 ng/ml) for 48 h in triplicate and pulsed with 3H-thymidine forthe last 24 h, and 3H-thymidine incorporation was measured. Data
are shown as means 6 SD. *, p,0.01, compared with 0 ng/ml
soluble TRAIL. Similar results were obtained in two independent
experiments.
(TIF)
Figure S2 Knock-down of RIG-I or MDA5 hardly

affected the tumor growth suppressed by combining
IL-27 and ploy(I:C). SK-MEL-37 cells were transfected withsiRNAs specific to RIG-I (A and B), MDA5 (C and D) or control
siRNA for 24 h. These cells were then stimulated with IL-27(10 ng/ml) and poly(I:C) (1 mg/ml) for a further 24 h, and total
cell lysate was prepared and subjected to Western blot using antiRIG-I (A), anti-MDA5 (C) and anti-b-actin. The siRNAtransfected cells were also stimulated with IL-27 (10 ng/ml) andpoly(I:C) (1 mg/ml) for a further 48 h and pulsed with 3Hthymidine for the last 24 h in triplicate. 3H-thymidine incorporation was measured, and relative proliferation (%) to that ofrespective unstimulated cells was calculated (B and D). Data areshown as means 6 SD. * indicates p , 0.05 compared with nosiRNA and control siRNA. Similar results were obtained in two
independent experiments.
(TIF)
Figure S3 The TRAIL mRNA expression levels in thetumors tended to increase by the IL-27 injections into
tumor-bearing mice, although they were not significant.
Immunodeficient NOD/SCID mice were s.c. injected with humanmelanoma cells of the SK-MEL-37 cell line, and treated by weekly
i.v. injections with PBS alone, IL-27 (1 mg), poly(I:C) (30 mg), or
IL-27 (1 mg) plus poly(I:C) (30 mg) from 1 week postengraftment.
Tumor growth was monitored weekly, and on day 63 after tumor
injection TRAIL expression levels in the tumor sites werecompared by using real-time quantitative PCR.
(TIF)

Acknowledgments
The authors thank Drs. T. Takahashi, G. Ritter, and L. J. Old for humanmelanoma cell lines.

Author Contributions
Conceived and designed the experiments: TY JM. Performed theexperiments: YC IM KM KH. Analyzed the data: YC IM TY. Wrotethe paper: TY HN CN.

10

-305-

October 2013 | Volume 8 | Issue 10 | e76159

TRAIL-Mediated Inhibition of Tumor Growth by IL-27

References
1. Pflanz S, Timans JC, Cheung J, Rosales R, Kanzler H, et al. (2002) IL-27, aheterodimeric cytokine composed of EBI3 and p28 protein, induces proliferationof naive CD4+ T cells. Immunity 16: 779–790.
2. Takeda A, Hamano S, Yamanaka A, Hanada T, Ishibashi T, et al. (2003)
Cutting edge: Role of IL-27/WSX-1 signaling for induction of T-bet throughactivation of STAT1 during initial Th1 commitment. J Immunol 170: 4886–
4890.
3. Kamiya S, Owaki T, Morishima N, Fukai F, Mizuguchi J, et al. (2004) An
indispensable role for STAT1 in IL-27-induced T-bet expression but notproliferation of naive CD4+ T cells. J Immunol 173: 3871–3877.
4. Hunter CA (2005) New IL-12-family members: IL-23 and IL-27, cytokines withdivergent functions. Nat Rev Immunol 5: 521–531.
5. Stumhofer JS, Silver JS, Laurence A, Porrett PM, Harris TH, et al. (2007)
Interleukins 27 and 6 induce STAT3-mediated T cell production of interleukin
10. Nat Immunol 8: 1363–1371.
6. Awasthi A, Carrier Y, Peron JP, Bettelli E, Kamanaka M, et al. (2007) A
dominant function for interleukin 27 in generating interleukin 10-producing
anti-inflammatory T cells. Nat Immunol 8: 1380–1389.
7. Hisada M, Kamiya S, Fujita K, Belladonna ML, Aoki T, et al. (2004) Potent
antitumor activity of interleukin-27. Cancer Res 64: 1152–1156.
8. Chiyo M, Shimozato O, Yu L, Kawamura K, Iizasa T, et al. (2005) Expressionof IL-27 in murine carcinoma cells produces antitumor effects and induces
protective immunity in inoculated host animals. Int J Cancer 115: 437–442.
9. Salcedo R, Stauffer JK, Lincoln E, Back TC, Hixon JA, et al. (2004) IL-27
mediates complete regression of orthotopic primary and metastatic murine
neuroblastoma tumors: role for CD8+ T cells. J Immunol 173: 7170–7182.
10. Oniki S, Nagai H, Horikawa T, Furukawa J, Belladonna ML, et al. (2006)
Interleukin-23 and interleukin-27 exert quite different antitumor and vaccine
effects on poorly immunogenic melanoma. Cancer Res 66: 6395–6404.
11. Matsui M, Kishida T, Nakano H, Yoshimoto K, Shin-Ya M, et al. (2009)
Interleukin-27 activates natural killer cells and suppresses NK-resistant head andneck squamous cell carcinoma through inducing antibody-dependent cellularcytotoxicity. Cancer Res 69: 2523–2530.
12. Shimizu M, Shimamura M, Owaki T, Asakawa M, Fujita K, et al. (2006)
Antiangiogenic and antitumor activities of IL-27. J Immunol 176: 7317–7324.
13. Yoshimoto T, Morishima N, Mizoguchi I, Shimizu M, Nagai H, et al. (2008)
Antiproliferative activity of IL-27 on melanoma. J Immunol 180: 6527–6535.
14. Canale S, Cocco C, Frasson C, Seganfreddo E, Di Carlo E, et al. (2011)
Interleukin-27 inhibits pediatric B-acute lymphoblastic leukemia cell spreading
in a preclinical model. Leukemia 25: 1815–1824.
15. Cocco C, Giuliani N, Di Carlo E, Ognio E, Storti P, et al. (2010) Interleukin-27
acts as multifunctional antitumor agent in multiple myeloma. Clin Cancer Res
16: 4188–4197.
16. Zorzoli A, Di Carlo E, Cocco C, Ognio E, Ribatti D, et al. (2012) Interleukin-27
inhibits the growth of pediatric acute myeloid leukemia in NOD/SCID/Il2rc-/mice. Clin Cancer Res 18: 1630–1640.
17. Matsumoto M, Funami K, Oshiumi H, Seya T (2004) Toll-like receptor 3: a link
between toll-like receptor, interferon and viruses. Microbiol Immunol 48: 147–
154.
18. Salaun B, Coste I, Rissoan MC, Lebecque SJ, Renno T (2006) TLR3 candirectly trigger apoptosis in human cancer cells. J Immunol 176: 4894–4901.
19. Cheng YS, Xu F (2011) Anticancer function of polyinosinic-polycytidylic acid.
Cancer Biol Ther 10: 1219–1223.
20. Carey TE, Takahashi T, Resnick LA, Oettgen HF, Old LJ (1976) Cell surface
antigens of human malignant melanoma: mixed hemadsorption assays forhumoral immunity to cultured autologous melanoma cells. Proc Natl Acad Sci U
S A 73: 3278–3282.
21. Yoshimoto T, Furuhata M, Kamiya S, Hisada M, Miyaji H, et al. (2003) Positive
modulation of IL-12 signaling by sphingosine kinase 2 associating with the IL-12
receptor b 1 cytoplasmic region. J Immunol 171: 1352–1359.
22. Owaki T, Asakawa M, Kamiya S, Takeda K, Fukai F, et al. (2006) IL-27
suppresses CD28-medicated IL-2 production through suppressor of cytokine
signaling 3. J Immunol 176: 2773–2780.
23. Kuo RL, Kao LT, Lin SJ, Wang RY, Shih SR (2013) MDA5 plays a crucial rolein enterovirus 71 RNA-mediated IRF3 activation. PLoS One 8: e63431.
24. Johnstone RW, Frew AJ, Smyth MJ (2008) The TRAIL apoptotic pathway incancer onset, progression and therapy. Nat Rev Cancer 8: 782–798.
25. Kayagaki N, Yamaguchi N, Nakayama M, Eto H, Okumura K, et al. (1999)
Type I interferons (IFNs) regulate tumor necrosis factor-related apoptosisinducing ligand (TRAIL) expression on human T cells: A novel mechanism forthe antitumor effects of type I IFNs. J Exp Med 189: 1451–1460.
26. Chawla-Sarkar M, Leaman DW, Borden EC (2001) Preferential induction of
apoptosis by interferon (IFN)-b compared with IFN-a2: correlation with
TRAIL/Apo2L induction in melanoma cell lines. Clin Cancer Res 7: 1821–
1831.
27. Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, et al. (1997)
Control of TRAIL-induced apoptosis by a family of signaling and decoy
receptors. Science 277: 818–821.
28. Yoneyama M, Fujita T (2009) RNA recognition and signal transduction by
RIG-I-like receptors. Immunol Rev 227: 54–65.

PLOS ONE | www.plosone.org

29. Park SY, Seol JW, Lee YJ, Cho JH, Kang HS, et al. (2004) IFN-c enhances
TRAIL-induced apoptosis through IRF-1. Eur J Biochem 271: 4222–4228.
30. Papageorgiou A, Dinney CP, McConkey DJ (2007) Interferon-alpha induces
TRAIL expression and cell death via an IRF-1-dependent mechanism in human
bladder cancer cells. Cancer Biol Ther 6: 872–879.
31. Clarke N, Jimenez-Lara AM, Voltz E, Gronemeyer H (2004) Tumor suppressor
IRF-1 mediates retinoid and interferon anticancer signaling to death ligand
TRAIL. EMBO J 23: 3051–3060.
32. Salaun B, Zitvogel L, Asselin-Paturel C, Morel Y, Chemin K, et al. (2011) TLR3
as a biomarker for the therapeutic efficacy of double-stranded RNA in breast
cancer. Cancer Res 71: 1607–1614.
33. Salcedo R, Hixon JA, Stauffer JK, Jalah R, Brooks AD, et al. (2009)
Immunologic and therapeutic synergy of IL-27 and IL-2: enhancement of T
cell sensitization, tumor-specific CTL reactivity and complete regression ofdisseminated neuroblastoma metastases in the liver and bone marrow. J
Immunol 182: 4328–4338.
34. Liu L, Wang S, Shan B, Shao L, Sato A, et al. (2008) IL-27-mediated activationof natural killer cells and inflammation produced antitumour effects for humanoesophageal carcinoma cells. Scand J Immunol 68: 22–29.
35. Cocco C, Di Carlo E, Zupo S, Canale S, Zorzoli A, et al. (2012) Complementary
IL-23 and IL-27 anti-tumor activities cause strong inhibition of human follicularand diffuse large B-cell lymphoma growth in vivo. Leukemia 26: 1365–1374.
36. Airoldi I, Ribatti D (2011) Regulation of angiostatic chemokines driven by IL-12
and IL-27 in human tumors. J Leukoc Biol 90: 875–882.
37. Ho MY, Leu SJ, Sun GH, Tao MH, Tang SJ, et al. (2009) IL-27 directly
restrains lung tumorigenicity by suppressing cyclooxygenase-2-mediated activities. J Immunol 183: 6217–6226.
38. Gitlin L, Barchet W, Gilfillan S, Cella M, Beutler B, et al. (2006) Essential role of
mda-5 in type I IFN responses to polyriboinosinic:polyribocytidylic acid andencephalomyocarditis picornavirus. Proc Natl Acad Sci U S A 103: 8459–8464.
39. Besch R, Poeck H, Hohenauer T, Senft D, Hacker G, et al. (2009) Proapoptotic
signaling induced by RIG-I and MDA-5 results in type I interferon-independent
apoptosis in human melanoma cells. J Clin Invest 119: 2399–2411.
40. Inao T, Harashima N, Monma H, Okano S, Itakura M, et al. (2012) Antitumor
effects of cytoplasmic delivery of an innate adjuvant receptor ligand, poly(I:C),
on human breast cancer. Breast Cancer Res Treat 134: 89–100.
41. Krown SE, Kerr D, Stewart WE 2nd, Field AK, Oettgen HF (1985) Phase I
trials of poly(I,C) complexes in advanced cancer. J Biol Response Mod 4: 640–
649.
42. Kitahara Y, Kawane K, Nagata S (2010) Interferon-induced TRAILindependent cell death in DNase II-/- embryos. Eur J Immunol 40: 2590–2598.
43. Moschos S, Kirkwood JM (2007) Present role and future potential of type I
interferons in adjuvant therapy of high-risk operable melanoma. Cytokine
Growth Factor Rev 18: 451–458.
44. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA (2001) Recognition of
double-stranded RNA and activation of NF-kB by Toll-like receptor 3. Nature
413: 732–738.
45. Schulz O, Diebold SS, Chen M, Naslund TI, Nolte MA, et al. (2005) Toll-likereceptor 3 promotes cross-priming to virus-infected cells. Nature 433: 887–892.
46. Salaun B, Lebecque S, Matikainen S, Rimoldi D, Romero P (2007) Toll-likereceptor 3 expressed by melanoma cells as a target for therapy? Clin Cancer Res
13: 4565–4574.
47. Tissari J, Siren J, Meri S, Julkunen I, Matikainen S (2005) IFN-a enhances
TLR3-mediated antiviral cytokine expression in human endothelial andepithelial cells by up-regulating TLR3 expression. J Immunol 174: 4289–4294.
48. Taura M, Fukuda R, Suico MA, Eguma A, Koga T, et al. (2010) TLR3
induction by anticancer drugs potentiates poly I:C-induced tumor cell apoptosis.
Cancer Sci 101: 1610–1617.
49. Heinz S, Haehnel V, Karaghiosoff M, Schwarzfischer L, Muller M, et al. (2003)
Species-specific regulation of Toll-like receptor 3 genes in men and mice. J Biol
Chem 278: 21502–21509.
50. Imamichi T, Yang J, Huang DW, Brann TW, Fullmer BA, et al. (2008) IL-27, anovel anti-HIV cytokine, activates multiple interferon-inducible genes inmacrophages. AIDS 22: 39–45.
51. Owaki T, Asakawa M, Morishima N, Takeda K, Fukai F, et al. (2008) STAT3 is
indispensable to IL-27-mediated cell proliferation but not to IL-27-induced Th1
differentiation and suppression of proinflammatory cytokine production. J
Immunol 180: 2903–2911.
52. Huang B, Zhao J, Li H, He KL, Chen Y, et al. (2005) Toll-like receptors on
tumor cells facilitate evasion of immune surveillance. Cancer Res 65: 5009–
5014.
53. Guzzo C, Ayer A, Basta S, Banfield BW, Gee K (2012) IL-27 enhances LPSinduced proinflammatory cytokine production via upregulation of TLR4
expression and signaling in human monocytes. J Immunol 188: 864–873.
54. Colombo MP, Trinchieri G (2002) Interleukin-12 in anti-tumor immunity andimmunotherapy. Cytokine Growth Factor Rev 13: 155–168.
55. Ryffel B (1997) Interleukin-12: role of interferon-c in IL-12 adverse effects. Clin
Immunol Immunopathol 83: 18–20.

11

-306-

October 2013 | Volume 8 | Issue 10 | e76159

Sustained upregulation of effector natural killer cells
in chronic myeloid leukemia after discontinuation of
imatinibIzuru Mizoguchi,1 Takayuki Yoshimoto,1,6 Seiichiro Katagiri,2 Junichiro Mizuguchi,3 Tetsuzo Tauchi,2
Yukihiko Kimura,2,7 Koiti Inokuchi,4 Junko H. Ohyashiki5 and Kazuma Ohyashiki2
1

Department of Immunoregulation, Institute of Medical Science, Tokyo; Departments of 2Hematology; 3Immunology, Tokyo Medical University, Tokyo;
Division of Hematology, Department of Internal Medicine, Nippon Medical School, Tokyo; 5Department of Molecular Oncology, Institute of Medical
Science, Tokyo Medical University, Tokyo, Japan
4(Received April 25, 2013 ⁄ Revised June 3, 2013 ⁄ Accepted June 7, 2013 ⁄ Accepted manuscript online June 12, 2013 ⁄ Article first published online July 8, 2013)

A number of CML patients who achieve a sustained complete
molecular response (CMR) for at least 2 years during imatinib(IM) therapy can discontinue IM without relapse. With the longterm goal of developing immunological criteria for managing IM
therapy in CML patients, we compared the immunophenotypic
profiles of three groups of CML patients: those who received IM
and had a CMR for more than two consecutive years (CMR
group); patients who received IM and did not have a sustained
CMR but maintained a major molecular response for more than
2 years (fluctuating CMR group); and patients with a sustained
CMR for more than 6 months after IM discontinuation (STOP-IM
group), together with healthy controls. The percentages of effector populations of natural killer (NK) cells, such as interferon(IFN)-c+CD3 CD56+ cells, were significantly higher in the STOP-IM
and CMR groups than in the fluctuating CMR and control groups.
The elevated levels of these effector NK cells were sustained formore than 3 years after IM discontinuation. In contrast, the percentages of effector memory CD8+ T cells, such as IFN-c+
CCR7 CD45RO+CD8+ cells, were significantly higher in the STOPIM and control groups than in the CMR and fluctuating CMR
groups, possibly owing to IM intake. These results suggest thatthe immunological activation status of NK cells contributes to
CMR maintenance. Higher activation levels of effector NK cells in
CML patients being treated with IM might reflect minimization of
BCR-ABL1 transcript levels and therefore could be additive information for determining whether to stop IM. (Cancer Sci 2013;
104: 1146–1153)

T

he introduction of tyrosine kinase inhibitors (TKIs),
including imatinib (IM),(1) dramatically changed thetreatment of CML(2,3) and led to the establishment not onlyof new therapeutic guidelines but also of criteria for judging
the effectiveness of CML management.(4–6) Imatinib targets
the BCR-ABL oncoprotein, and the drug’s therapeutic effectiveness can be monitored by qualitatively and quantitatively
assessing BCR-ABL1 transcript levels,(7) although the current
techniques might not be sensitive enough to estimate theexact amount of minimal residual CML burden at the clinical level.(8) Moreover, CML stem cells, for example, CD34+
CML cells, carry much less BCR-ABL1 transcript than CML
non-stem cells, are insensitive to IM, and are considered tobe quiescent.(9–11) Nevertheless, a goal of TKI therapy for
CML is to obtain a major molecular response (MMR), but
hopefully a complete molecular response (CMR, also
referred to as undetectable molecular response), which isdefined as a ≥4.5-fold reduction in the amount of BCRABL1 transcript (CMR4.5) and is the optimal target point for
TKI treatment.(6)In a study of CML patients with a sustained CMR4.5 lasting
2 years before discontinuation of IM, Mahon et al.(12) foundthat approximately 40% of the patients maintained a CMR4.5
after stopping IM. Ross et al.(13) also reported that a subset of
CML patients maintained a CMR after IM cessation; however,these patients had persistent BCR-ABL DNA, as indicated bya highly sensitive patient-specific nested quantitative PCR
assay. These two studies clearly indicate that some patientswith persisting residual CML cells can stop IM without apparent molecular relapse. However, we do not know how long
CMR patients should take TKIs, including IM.(14) In the study
by Mahon et al.(12), most of the patients who experienced amolecular relapse did so within 6 months after discontinuationof IM, and the relapsed patients showed a molecular response
after restarting IM. This evidence strongly suggests that TKI
therapy may contribute to minimizing BCR-ABL-positive
CML cells but that eradicating CML cells is difficult. Although
the current techniques are not sensitive enough to detect the
BCR-ABL1 transcript,(13) other factors might also be involvedin maintaining the minimization of CML cells. One of these
could be immunological surveillance. We have previously
shown that CML patients who sustain a CMR after IM discontinuation have higher levels of natural killer (NK) cells than
do normal subjects or CMR patients under IM therapy.(15)In the current study, we characterized and compared thefunctional immunophenotypic profiles of CML-CMR patientsin detail with or without IM, along with the profiles of healthy
volunteers. Our results suggest that higher levels of functional
NK cells contribute to maintaining a CMR after stopping IM
and, therefore, that monitoring both the immune system andthe BCR-ABL1 transcript levels could be useful for identifying
CML patients who might be candidates for discontinuation ofIM.
Materials and Methods
Patient characteristics. We arbitrarily categorized 42 CML
patients into three groups as follows: 22 patients who had a
CMR for ≥2 consecutive years with IM (CMR group); 10
CML patients who had a CMR at least twice in the previous
2 years but did not have a sustained CMR under IM treatment(fluctuating CMR group; all patients maintained MMR: 3-log
reduction); and 10 patients who discontinued IM and had asustained CMR for ≥6 months (STOP-IM group).(14,15) We
also included 18 healthy volunteers as controls (control group).

6
To whom correspondence should be addressed.
E-mail: yoshimot@tokyo-med.ac.jp
7
Present address: Department of Center for Health Surveillance and Preventive
Medicine, Tokyo Medical University, Tokyo, Japan.

Cancer Sci | September 2013 | vol. 104 | no. 9 | 1146–1153

doi: 10.1111/cas.12216© 2013 Japanese Cancer Association-307-

The study was approved by the institutional review board ofTokyo Medical University (nos. 1655 and 2159) and Nippon
Medical School (no. 223064). Written informed consent was
obtained from all participants in accordance with the Declaration of Helsinki.
All patients studied were in the chronic phase of CML andhad no clinical signs of immunosuppression or apparent infection. In addition, none of the patients had been treated with
TKIs other than IM or had undergone allogeneic stem celltransplantation. Although some patients had previously beentreated with interferon (IFN)-a, the overall proportion of thesepatients was approximately 30%, and the proportions in eachgroup were similar (P = 0.981, Table 1). Overall, no patient
selection bias was apparent among these groups, except in thecase of the total IM dose (Table 1). The total IM dose, which
was calculated as the cumulative amount of IM taken prior toblood sample collection, was significantly higher in the CMR
group than in the STOP-IM group (P = 0.030). Because all
patients had a CMR, the lymphocyte fractions in the patientsanalyzed in this study were of non-leukemic origin.
Lymphocyte activation. Freshly obtained PBMCs were separated with a Ficoll density gradient (GE Healthcare, Uppsala,Sweden) and resuspended in RPMI-1640 medium supplemented with 10% FCS. PBMCs (5 9 106 cells ⁄ mL) were thenstimulated with PMA (10 ng ⁄ mL) and ionomycin (1 lg ⁄ mL;
Sigma-Aldrich, St. Louis, MO, USA) for 4 h in the presenceof monensin (GolgiStop, 2 lM; BD Biosciences, San Diego,
CA, USA) for intracellular staining of effector molecules.
Flow cytometry. Immunophenotyping was carried out with afive-color flow cytometry panel including antibodies against
the following cell surface antigens and effector molecules:
CD3, CD8, CD45RO, CD56, CCR7, IFN-c, granzyme B, andperforin (eBioscience, San Diego, CA, USA and BioLegend,
San Diego, CA, USA) (Fig. S1). We also analyzed NKG2Dpositive NK cells using anti-NKG2D antibody (eBioscience).
For intracellular staining of IFN-c, granzyme B, and perforin,cell surface antigens of PBMCs stimulated with PMA and ionomycin for 4 h in the presence of monensin were first stained,
fixed, and permeabilized with Cytofix ⁄ Cytoperm Fixation ⁄ Permeabilization Solution (BD Biosciences) according to the manufacturer’s instructions. These cells were then intracellularly
stained and analyzed with the FACSCanto II (BD Biosciences)flow cytometer and CellQuest software.
Real-time quantitative RT-PCR. Molecular genetic analysis of
BCR-ABL1 transcript was carried out by means of real-timequantitative RT-PCR. The molecular response was assessed atleast every 3 months. An MMR was defined as a 3-log reduction in the BCR-ABL1 transcript (international scale; <0.1%),
and a CMR was further confirmed as the disappearance of the

BCR-ABL1 transcript in a nested quantitative PCR assay, as
described previously.(14,15)
Statistical analysis. The immunophenotyping results were
statistically analyzed with GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). The profiles were analyzed by
one-way ANOVA or the v2-test for more than three groups andStudent’s t-test for two groups. Linear regression was used toassess the correlation between the percentages of IFN-c+ NK
cells and the duration times after stopping IM. The receiver–
operating characteristic (ROC) curve and the area under the
ROC curve were used to assess the cut-off level between two
groups. A P-value of less than 0.05 was considered to indicate a statistically significant difference.
Results
Percentages of effector populations of NK cells significantly
higher in STOP-IM and CMR groups than in fluctuating CMR andcontrol groups. Those CML patients receiving IM treatment(CMR and fluctuating CMR groups) had significantly lower
PBMC counts than the control or STOP-IM groups(P < 0.0001, Table 2, Fig. S2), although the STOP-IM grouptended to show slightly lower PBMC counts than the control
group; these results indicate that the low PBMC counts in the
CMR groups might have been due to IM intake and thus it ishard to find a marker in the cell counts for discrimination
between fluctuating CMR and STOP-IM groups (Figs S3,S4).
Therefore, we represent subsets of lymphocytes as percentages
rather than absolute cell counts.
Among the four groups, significant differences wereobserved in the percentages of the CD3 CD56+ NK cell population but not the CD3+CD56+ NKT cell population, whereas
no significant difference in NK cells between the STOP-IM
and CMR groups was observed, as reported previously(Fig. 1a).(15) To further examine the activation level of theselymphocytes, we carried out intracellular staining of effector
molecules, such as IFN-c, granzyme B, and perforin, after activation with PMA and ionomycin in the presence of monensin,
a protein transport inhibitor. This is the most commonly usedmethod to simply and efficiently activate many types of cells,
including NK cells and CD8+ T cells, to produce effector molecules such as cytokines. Natural killer cells play importantroles in innate immunity and also in linking the innate andadaptive immune responses. Their main functions are therelease of cytokines and the direct cell-mediated cytotoxicity.
Activated NK cells produce effector molecules such as IFN-c,
perforin, and granzyme B. IFN-c is critically important for
subsequent activation of macrophage and differentiation ofnaive CD4+ T cells into Th1 cells. Perforin and granzyme B

Table 1. Characteristics of CML patients who achieved a sustained complete molecular response (CMR) during imatinib (IM) therapy

No. of people
Age, years
Sex, male ⁄ female
Sokal Category, low ⁄ intermediate ⁄ highSokal score at CML diagnosis
Imatinib daily dose, ≥400 mg ⁄ <400 mg
Total IM dose, gDuration of IM therapy, months
Time to achieve CMR from diagnosis, months
Prior IFN-a, yes ⁄ noControl

Fluctuating CMR

CMR

STOP-IMP-value18
55.3  1.8
14 ⁄ 4
NA
NA
NA
NA
NA
NA
NA

10
56.7  4.5
8⁄2
7⁄2⁄1
0.785  0.078
9⁄1852.3  88.5
77.2  9.4
56.1  14.5
3⁄7

22
56.2  2.6
16 ⁄ 619 ⁄ 3 ⁄ 00.674  0.019
15 ⁄ 7960.0  62.1
89.5  5.5
58.9  8.8
6 ⁄ 16

10
56.5  4.0
4⁄6
6⁄2⁄1
0.740  0.068
6⁄4
636.9  113.9
67.4  11.0
59.3  21.5
3⁄7

0.987*
0.147**
0.542**
0.219*
0.295**
0.030*
0.133*
0.988*
0.981**

Data are presented as means  standard error. P-values were obtained by *one-way ANOVA or **v2 test. CMR group, patients who had a CMR
for ≥2 consecutive years with IM; Fluctuating CMR group, patients who had a CMR at least twice in the previous 2 years but did not have a sustained CMR under IM treatment; IFN, interferon; NA, not applicable; STOP-IM group, patients who discontinued IM and had a sustained CMRfor ≥6 months.

Mizoguchi et al.

Cancer Sci | September 2013 | vol. 104 | no. 9 | 1147
© 2013 Japanese Cancer Association-308-

Table 2. Immunophenotypic profiles of CML patients

6

Total no. of PBMCs (310 cells/mL)
CD56+ (%)
CD3+CD56+ (%)
CD3 CD56+ (%)
IFN-c+CD56+ (%)
Granzyme B+CD56+ (%)
Perforin+CD56+ (%)
IFN-c+ NKT (%)
Granzyme B+ NKT (%)
Perforin+ NKT (%)
IFN-c+ NK (%)
Granzyme B+ NK (%)
Perforin+ NK (%)
CD8+ (%)
IFN-c+CD8+ (%)
Granzyme B+CD8+ (%)
Perforin+CD8+ (%)
N CD8+ (%)
CM CD8+ (%)
EM CD8+ (%)
E CD8+ (%)
IFN-c+N CD8+ (%)
Granzyme B+N CD8+ (%)
Perforin+N CD8+ (%)
IFN-c+CM CD8+ (%)
Granzyme B+CM CD8+ (%)
Perforin+CM CD8+ (%)
IFN-c+EM CD8+ (%)
Granzyme B+EM CD8+ (%)
Perforin+EM CD8+ (%)
IFN-c+E CD8+ (%)
Granzyme B+E CD8+ (%)
Perforin+E CD8+ (%)

Control

Fluctuating CMR

CMR

STOP-IMP-value

2.038 0.179
13.0  1.2
4.1  1.0
8.7  1.1
10.3  1.2
10.6  0.9
10.9  1.2
3.6  1.0
3.4  0.9
3.3  1.0
6.7  1.0
7.0  0.9
7.7  1.1
28.7  2.4
21.8  2.0
14.7  1.5
10.5  1.5
5.0  0.6
2.6  0.3
7.6  0.9
13.2  1.4
1.3  0.2
0.3  0.1
0.2  0.0
1.5  0.3
0.7  0.1
0.5  0.1
7.2  0.9
3.4  0.5
1.6  0.4
11.9  1.4
10.2  1.3
8.2  1.3

1.180 0.103
11.0  2.2
3.2  1.0
7.8  1.3
7.6  1.6
8.7  1.9
7.9  1.3
2.2  0.9
2.3  0.9
1.1  0.2
5.4  0.9
6.3  1.1
6.8  1.2
22.1  2.4
13.5  2.2
10.1  1.8
6.1  0.9
5.3  1.1
2.4  0.3
5.1  1.0
9.4  1.8
0.8  0.1
0.4  0.1
0.2  0.0
0.8  0.1
0.7  0.1
0.5  0.1
4.2  1.0
2.2  0.6
0.6  0.2
7.7  1.7
6.8  1.5
4.7  0.8

1.106 0.103
17.9  2.4
3.7  0.5
14.3  2.3
13.8  2.2
13.0  1.6
15.6  2.4
2.8  0.4
2.5  0.5
2.2  0.4
11.0  2.0
10.3  1.5
13.2  2.2
27.8  1.8
17.8  1.3
12.3  1.0
9.3  1.1
6.0  0.8
3.4  0.4
6.6  0.6
11.6  1.2
1.0  0.2
0.4  0.1
0.2  0.0
1.3  0.2
0.7  0.1
0.5  0.1
5.4  0.5
2.5  0.3
1.0  0.2
10.1  0.9
8.7  1.0
7.5  1.0

1.579 0.160
18.9  3.0
4.3  0.9
14.7  2.1
17.1  3.0
15.9  2.8
16.8  3.0
3.8  0.9
3.2  0.9
2.9  0.9
13.5  2.2
12.9  2.1
13.9  2.2
28.6  3.1
22.2  3.1
15.7  2.3
10.7  1.4
4.4  0.3
1.9  0.3
8.4  1.2
13.9  2.4
0.6  0.1
0.2  0.1
0.1  0.0
0.8  0.1
0.5  0.2
0.4  0.2
7.6  1.2
3.6  0.6
1.6  0.4
13.4  2.5
11.6  2.1
7.9  1.2

<0.0001
0.085
0.863
0.036
0.043
0.077
0.043
0.554
0.718
0.276
0.021
0.022
0.030
0.265
0.031
0.117
0.153
0.635
0.090
0.117
0.318
0.102
0.375
0.337
0.071
0.673
0.825
0.034
0.151
0.067
0.168
0.264
0.264

Data are presented as means of percentage (%) of total lymphocytes  standard error. P-values were obtained by one-way ANOVA among fourgroups. Bold text indicates P < 0.05. CM, central memory (CCR7+CD45RO+); CMR, complete molecular response; CMR group, patients who had a
CMR for ≥2 consecutive years with imatinib (IM) therapy; Fluctuating CMR group, patients who had a CMR at least twice in the previous 2 years
but did not have a sustained CMR under IM treatment; E, effector (CCR7 CD45RO ); EM, effector memory (CCR7 CD45RO+); IFN, interferon; NK,natural killer (CD3 CD56+); NKT, natural killer T (CD3+CD56+); N, na€ıve (CCR7+CD45RO ); STOP-IM group, patients who discontinued IM and hada sustained CMR for ≥6 months.

are necessary for direct killing of target cells. Similar to thepercentages of the NK cell population, the percentages of the
IFN-c+, granzyme B+, and perforin+ NK cell populations weresignificantly higher in the STOP-IM and CMR groups than in
the fluctuating CMR and control groups (Fig. 1b). Amongthese distinct effector NK cell populations and total NK cell
population, the IFN-c+ NK cell population showed the lowest
P-value. No significant differences were observed in the percentages of IFN-c+, granzyme B+, or perforin+ NKT cells
among these four groups (Fig. 1c). Note that the percentagesof IFN-c+, granzyme B+, and perforin+ NK cells in the CMR
group were higher than the percentages in the fluctuating CMR
group (P = 0.039, 0.051, and 0.034, respectively), and approximately half of the patients in the CMR group showed highlevels of effector NK cells, which were similar to those in the
STOP-IM group.
Percentages of effector populations of CD8+ T cells were significantly higher in STOP-IM and control groups than in CMR and+fluctuating CMR groups. CD8 T cells are functionally divided

into four subpopulations: na€ıve (CCR7+CD45RO ); central memory (CCR7+CD45RO+); effector memory (CCR7 CD45RO+);and effector (CCR7 CD45RO ), according to the cell surface
expression of CCR7 and CD45RO.(16) We observed no significant
differences among the groups in the percentages of CD8+ T cells

or in the percentages of the four CD8+ T cell subpopulations,
including the effector memory population (Fig. 2a, Table 2). In
contrast, the percentages of effector populations, such as IFN-c+
CD8+ T cells, were significantly higher in the STOP-IM andcontrol groups than in the CMR and fluctuating CMR groups(Fig. 2b); differences that were more clearly apparent wereobserved in the IFN-c+ effector memory CD8+ T cell population
among the four subpopulations (Fig. 2c, Table 2).
Higher percentages of effector populations of NK cells and ⁄ oreffector memory CD8+ T cells clustered in patients with a CMR
after stopping IM. To estimate a cut-off value for the percent-age of IFN-c+ NK cells by which to distinguish between the
STOP-IM and fluctuating CMR groups, we carried out ROC
curve analysis (Fig. 3a). The area under the ROC curve was
0.9600 with 90% sensitivity (95% confidence interval [CI],55.50–99.75%) and 90% specificity (95% CI, 55.50–99.75%).
The cut-off value was thus determined to be 8.750. Furthermore, we also generated a ROC curve by using the percentages of IFN-c+ effector memory CD8+ T cells in the STOP-IM
group and in the fluctuating CMR group (Fig. 3b). The area
under the ROC curve was 0.7600 with 60% sensitivity(95% CI, 26.24–87.84%) and 90% specificity (95% CI,
55.50–99.75%). The cut-off value was determined to be 6.150.
Therefore, in the following analyses, we used 8.75% and1148

doi: 10.1111/cas.12216© 2013 Japanese Cancer Association

-309-

(a)

Fig. 1. Upregulation of functional natural killer(NK) cells in CML patients after stopping imatinib(IM). Immunophenotypic profiling was carried out
by five-color flow cytometry analysis of samples
from healthy controls and three patient groups:
those who received IM and had a complete
molecular response (CMR) for more than two
consecutive years (CMR group); patients who did
not have a sustained CMR but maintained a major
molecular response for more than 2 years(fluctuating CMR group); and patients with asustained CMR for more than 6 months after IM
discontinuation (STOP-IM group). The percentagesof cell populations of CD56+, CD3+CD56+ NKT, andCD3 CD56+ NK cells (a) effector (interferon [IFN]-c+,granzyme B+, and perforin+) cell populations of
CD3 CD56+ NK cells (b) and CD3+CD56+ NKT cells(c) were compared. Data are means of percentagesof total lymphocytes  standard error. *P < 0.05.
The percentage of IFN-c+ NK cells in the CMR groupis significantly higher than the percentages in thecontrol or fluctuating CMR groups, whereas there
was no difference between the STOP-IM and CMR
groups (P = 0.2388). This tendency was also noted
in granzyme B+ and perforin+ NK cells.

(b)

(c)

(a)

(b)

Fig. 2. Immunophenotypic profiles of CD8+ T cells
in CML patients. Immunophenotypic profiling was
carried out using five-color flow cytometry analysis
of samples from healthy controls and and threepatient groups: those who received imatinib (IM)and had a complete molecular response (CMR) for
more than two consecutive years (CMR group);
patients who did not have a sustained CMR but
maintained a major molecular response for morethan 2 years (fluctuating CMR group); and patientswith a sustained CMR for more than 6 months afterIM
discontinuation(STOP-IM
group).
The
percentages of cell populations of CD8+ T cells andCCR7 CD45RO+ effector memory CD8+ T cells(a) effector (interferon [IFN]-c+, granzyme B+, andperforin+) cell populations of CD8+ T cells (b) andCCR7 CD45RO+ effector memory CD8+ T cells(c) were compared. Data are means of percentagesof total lymphocytes  standard error. *P < 0.05.

(c)Mizoguchi et al.

Cancer Sci | September 2013 | vol. 104 | no. 9 | 1149
© 2013 Japanese Cancer Association

-310-

(a)

(b)

(a)

(c)

(b)

Fig. 3. Immunological characterization of CML patients after stopping imatinib (IM). (a) Differences in the percentage of interferon(IFN)-c+ natural killer (NK) cells between patients with a sustainedcomplete molecular response (CMR) for more than 6 months after IM
discontinuation (STOP-IM group) and patients who did not have a sustained CMR but maintained a major molecular response for more than
2 years (fluctuating CMR group) were compared by receiver–operating
characteristic (ROC) curve analysis. The analysis revealed that the cutoff value for IFN-c+ NK cells was 8.75%. (b) Differences in the percentage of IFN-c+ effector memory CD8+ T cells between the STOP-IM andfluctuating CMR groups were compared by ROC curve analysis. The
analysis revealed that the cut-off value for IFN-c+ effector memory
CD8+ T cells was 6.15%. (c) The correlation between the percentagesof the IFN-c+ NK cell population and the IFN-c+ effector memory CD8+
T cell population was analyzed for patients belonging to four different groups: healthy controls (n = 18); the fluctuating CMR group(n = 10); those who received IM and had a CMR for more than two
consecutive years (CMR group; n = 22); and the STOP-IM group(n = 10). The plot was then divided into four quadrants at the 6.15%
mark along the x-axis and at the 8.75% mark along the y-axis.
Patients in the quadrant below both cut-offs are considered to be athigh risk for relapse after stopping IM therapy. Patients in the CMR
group located outside of this high-risk area are considered to be
candidates for discontinuation of IM therapy.

6.15% as cut-off values for the IFN-c+ NK cells and the IFN-c+
effector memory CD8+ T cells, respectively.
The correlation between the percentages of the IFN-c+ NK
cell population and the IFN-c+ effector memory CD8+ T-cell
population was then analyzed (Fig. 3c). We used the cut-off
values (at the 6.15% mark along the x-axis and at the 8.75%
mark along the y-axis) to divide the plot into four quadrants:
one quadrant in which the percentages were higher than both
cut-offs; two quadrants in which the percentages were higher
than one of the cut-offs; and one quadrant in which the percentages were lower than both cut-offs. Almost all thepatients in the fluctuating CMR group fell into the quadrant
lower than both cut-offs. In marked contrast, almost all thepatients in the STOP-IM group fell outside the high-risk area,
especially into the area higher than the cut-off of the IFN-c+
NK cell population.

Fig. 4. Sustained upregulation of effector natural killer (NK) cell
population in CML patients who received imatinib (IM) and had acomplete molecular response (CMR) for more than two consecutive
years (CMR group) after IM discontinuation. The percentages of interferon (IFN)-c+ NK cells (a) and IFN-c+ effector memory CD8+ T cells (b)in patients with a sustained CMR for more than 6 months after IM discontinuation (STOP-IM group) plus patients who were in the CMR
group and stopped IM were plotted along the y-axis, and the numberof months after stopping IM was plotted along the x-axis for eachpatient. The correlation was examined by linear regression analysis;
the mean value is indicated by the solid line, and the 95% confidence
interval (CI) values are indicated by the dashed lines. One patient
retained an IFN-c+ NK cell level of 9.2% and an IFN-c+ effector
memory CD8+ T cell level of 5.6% 111 months after stopping IM.

Time-course analyses of immunophenotypic profiles of patientsin the CMR group after stopping IM. Finally, to examine howlong these higher activation levels of effector NK cells such as
IFN-c+ NK cells were sustained after stopping IM, we used
linear regression analysis to assess the correlation between thelevels of IFN-c+ NK cells in CMR patients who stopped IM
and the time after stopping IM (Fig. 4a). The mean percentage
of IFN-c+ NK cells more than 3 years after stopping IM wassustained at approximately 13%, which was much higher thanthe percentages for the control and fluctuating CMR groups(6.7  1.0% and 5.4  0.9%, respectively, Table 2) (95% CI,
9–17%). Intriguingly, one patient sustained an IFN-c+ NK celllevel of 9.2% without relapse 111 months after stopping IM.
Similar analysis of the levels of IFN-c+ effector memory CD8+
T cells was also carried out (Fig. 4b). The mean percentage
more than 3 years after stopping IM was sustained at approximately 7%, which is similar to the percentage for the control
group (7.2  0.9%, Table 2) (95% CI, 5–10%). These results
suggest that the elevated level of IFN-c+ NK cells was sustained for a long time in patients who did not relapse after
stopping IM, and the upregulation of effector NK cells may
have had some role in maintaining a CMR in CML patientswith minimized BCR-ABL1 transcript levels.
Discussion

In the present study, we showed that elevated levels of effector
NK cells were associated with minimization of CML cells and1150

doi: 10.1111/cas.12216© 2013 Japanese Cancer Association-311-

that sustained high levels of NK cells might be informative indetermining whether a CMR can be maintained after discontinuation of IM. The population of effector NK cells (such as
IFN-c+ NK) was significantly higher in patients who maintained a CMR under IM therapy than in the fluctuating CMR
group, and some of the CMR patients showed elevated effector
NK cell levels, similar to the elevated levels observed in the
STOP-IM patients. Recently, it has been reported that some
CML patients who achieve a sustained CMR on IM can stoptherapy and maintain a CMR.(12–14,17,18) However, even whenmolecular remission is sustained, CML stem cells may persist
after discontinuation of IM,(13) partly because the cells areinsensitive to IM.(9–11) Therefore, understanding the mechanism of drug-induced cure and determining which factors arenecessary for the maintenance of residual tumor cell dormancy
are important. To evaluate whether our immunological criteria
is a good predictive factor for sustained CMR even after IM
discontinuation, the time-course analyses of immunophenotypic
profiles of patients before and after stopping IM in the CMR
group are required.
Sustained elevation of effector NK cells after discontinuationof IM is considered to be linked to immunological surveillance.(19) As the elevation of effector NK cells appears topersist more than 3 years after IM discontinuation, these NK
cells might be memory-type cells. Although NK cells do notrearrange the genes encoding their activating receptors, recent
evidence has indicated that NK cells experience a selective
education process during development, undergo a clonal-like
expansion during virus infection, generate long-lived progeny,
and mediate more efficacious secondary responses against previously encountered pathogens like T and B cells.(20,21) At
diagnosis, CML cells are reported to show upregulated expression of NKG2D ligand, and expression of this ligand is associated with BCR-ABL by translational regulation involving thephosphoinositide 3-kinase ⁄ mammalian target of the rapamycin
pathway.(22) In addition, serum levels of MHC class I chainrelated molecules (for example, one of the partners of the
NKG2D activating receptor on NK and T cells) are reported tobe elevated in untreated CML patients, whereas NKG2D
expression on NK and CD8+ T cells is downregulated, owing
to immune escape; IM therapy may restore NKG2D-mediatedNK cytotoxic activity in CML patients.(22) This suggests that
upregulation of effector NK cells observed in the current study
may be the result of minimization of CML cells, and the maintained elevation of effector NK cells after stopping IM may belinked to a sustained CMR. Although we did not observe any
significant differences in the NKG2D+ NK population among
the fluctuating CMR, CMR, and STOP-IM groups(P = 0.8404, data not shown), the continuous upregulation ofeffector NK cells may have some immunological effects on
CML cells.
Imatinib is a specific inhibitor of tyrosine kinase receptors,
not only BCL-ABL but also KIT, that are required for themalignant transformation of stromal cells of the gut in gastrointestinal stromal tumors (GISTs). As IM enables disease control, including objective responses and stable disease in >80%
of GIST patients,(23) IM has also became the standard treatment of advanced GISTs. However, several lines of evidence
indicated that IM might mediate antitumor effects by an alternate mode of action instead of having a direct effect on tumoral c-kit mutations in GISTs.(24–26) Intriguingly, it waspreviously reported that IM acts on host dendritic cells to promote NK cell activation and NK cell-dependent antitumor
effects in mice.(25) In addition, most GIST-bearing patients thatwere treated with IM acquired NK cell activation, which positively correlated with clinical outcome,(25) and the IFN-c production level of NK cells after 2 months of treatment is
considered a possible independent predictor of long survival inadvanced GISTs treated with IM.(26) Whether such IM-mediated off-target effects trigger NK cell activation in CML
patients remains to be clarified.
It has also been shown that, in a small proportion ofpatients, IFN-a therapy can induce complete cytogenetic
remission with prolonged survival without disease relapse after
therapy is stopped.(27,28) Patients who discontinue IFN-a therapy but remain in remission for more than 2 years reportedly
show significantly higher NK cell levels than either patientsstill being treated with IFN-a or controls.(29) The immunophenotypic profiles of CML patients treated with IFN-a strongly
agree with our results for patients treated with IM. Although
IFN-a therapy has been suggested as a way to increase thelikelihood that IM therapy can be safely stopped,(12) weobserved no significant differences in the proportions ofpatients who previously used IFN-a among our three CML
patient groups (P = 0.981, Table 1).
Effector memory (CCR7 CD45RO ) CD8+ T cells mediate
protective memory by migrating to inflamed peripheral tissues
and showing immediate effector function. In contrast, central
memory CD8+ T cells mediate reactive memory by homing toT-cell areas of secondary lymphoid organs with little or noeffector function, and readily proliferate and differentiate toeffector cells in response to antigenic stimulation.(16) In CML,
CD8+ T cells reportedly recognize leukemic cells,(30,31) andautologous activated NK cells have been shown to efficiently
suppress the growth of malignant hematopoietic progenitors invitro.(32) Some patients with CML and Philadelphia-positive
acute lymphoblastic leukemia who were treated with the second-generation TKI dasatinib showed elevation of circulatinglarge granular lymphocytes and developed numerical expansion of NK cells, CD8+ T cells, or both, which is associatedwith superior therapeutic responses.(33,34) In contrast, dasatinib
has also been shown to suppress NK cell cytotoxicity.(35,36)
Therefore, the dissociation between the immune reactivationin vivo and immunosuppressive effects in vitro might exist in
dasatinib.(33,37) Salih et al.(38) showed that dasatinib impairs
expression of NKG2D ligand on CML cells and reduces the
production of IFN-c and the cytotoxicity of NK cells derived
from CML patients, whereas IM has no direct influence on NK
cell activity. These results indicate that understanding of theimmunological profiles of patients treated with TKIs is important and that caution should be exercised in stopping treatment
with these agents just after a CMR has been sustained for
2 years. Further studies are necessary to examine whether or
not the present criteria is applied to CML patients undergoing
dasatinib therapy.
Previously reported results indicate that NK cells ofpatients newly diagnosed with CML are reduced in numbers
or in proportion among lymphocytes and have limited cytolytic capacity at diagnosis of CML, and that these abnormalities persist during IM-induced remission.(39–41) The present
results suggest that NK cells are more sensitive to the residual BCR-ABL-positive cells than are NKT cells and CD8+
T cells. However, the patterns in CD8+ T cells were different from those in NK cells among the four groups, implyingthat these two types of immune cells play different roles in
CML tumor surveillance in different situations. It is possiblethat the effector population of CD8+ T cells, which were
suppressed by IM, was simply restored to the normal level
after stopping IM. Nevertheless, compared to patients in thefluctuating CMR group, some CMR patients treated with IM
show either upregulation of effector memory CD8+ T cells
or effector NK cells, or both (Fig. 3c), thus indicating a
possible immunological mechanism for the minimization of
BCR-ABL-positive cells that is linked to discontinuation ofIM, while other underlying factors for stopping IM may
exist.(42,43)

Mizoguchi et al.

Cancer Sci | September 2013 | vol. 104 | no. 9 | 1151
© 2013 Japanese Cancer Association-312-

In conclusion, our results suggest that monitoring of functional activation levels of NK cells, and perhaps of CD8+ T
cells as well, in CML patients could be important for indirectly
determining the level of residual CML cells, which contributeto immunological surveillance.

at General Cancer Cure using Epigenetic Targets (S0801020) and by aGrant-in-Aid for Scientific Research from the Ministry of Education,
Culture, Sports, Science and Technology, Japan, and Tokyo Medical
University Research Grant.

Disclosure Statement
Acknowledgments
We thank Ms C. Kobayashi and A. Hirota for their technical assistance.
This study was supported in part by the Private University Strategic
Research Based Support Project: Epigenetics Research Project Aimed

References
1 Druker BJ, Tamura S, Buchdunger E et al. Effects of a selective inhibitor of
the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med
1996; 2: 561–6.
2 Deininger MW, Goldman JM, Melo JV. The molecular biology of chronic
myeloid leukemia. Blood 2000; 96: 3343–56.
3 Deininger M, O’Brien SG, Guilhot F et al. International Randomized Studyof Interferon vs STI571 (IRIS) 8-year follow up: sustained survival and low
risk for progression or events in patients with newly diagnosed chronic myeloid leukemia in chronic phase (CML-CP) treated with imatinib. Blood(ASH Annual Meeting Abstracts) 2009; 114: 462. Abstract 1126.
4 Vardiman JW, Melo J, Baccarani M. Chronic myelogenous leukemia, BCR/ABL1 positive. In: Swerdlow SH, Campo E, Harris NL et al., eds. WHO
Classification of Tumours of Haematopoietic and Lymphoid Tissues, 4th edn.
Lyon, France: IARC Press, 2008; 32–7.
5 Baccarani M, Saglio G, Goldman J et al. Evolving concepts in the management of chronic myeloid leukemia: recommendations from an expert panel
on behalf of the European LeukemiaNet. Blood 2006; 108: 1809–20.
6 Baccarani M, Cortes J, Pane F et al. Chronic myeloid leukemia: an updateof concepts and management recommendations of European LeukemiaNet.
J Clin Oncol 2009; 27: 6041–51.
7 Muller MC, Cross NC, Erben P et al. Harmonization of molecular monitoring of CML therapy in Europe. Leukemia 2009; 23: 1957–63.
8 O’Hare T, Zabriskie MS, Eiring AM, Deininger MW. Pushing the limits oftargeted therapy in chronic myeloid leukaemia. Nat Rev Cancer 2012; 12:513–26.
9 Graham SM, Jorgensen HG, Allan E et al. Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia areinsensitive to STI571 in vitro. Blood 2002; 99: 319–25.
10 Corbin AS, Agarwal A, Loriaux M, Cortes J, Deininger MW, Druker BJ.
Human chronic myeloid leukemia stem cells are insensitive to imatinib
despite inhibition of BCR-ABL activity. J Clin Investig 2011; 121: 396–409.
11 Hamilton A, Helgason GV, Schemionek M et al. Chronic myeloid leukemia
stem cells are not dependent on Bcr-Abl kinase activity for their survival.
Blood 2012; 119: 1501–10.
12 Mahon FX, Rea D, Guilhot J et al. Discontinuation of imatinib in patientswith chronic myeloid leukaemia who have maintained complete molecularremission for at least 2 years: the prospective, multicentre Stop Imatinib(STIM) trial. Lancet Oncol 2010; 11: 1029–35.
13 Ross DM, Branford S, Seymour JF et al. Patients with chronic myeloid leukemia who maintain a complete molecular response after stopping imatinib
treatment have evidence of persistent leukemia by DNA PCR. Leukemia
2010; 24: 1719–24.
14 Takahashi N, Kyo T, Maeda Y et al. Discontinuation of imatinib in Japanesepatients with chronic myeloid leukemia. Haematologica 2012; 97: 903–6.
15 Ohyashiki K, Katagiri S, Tauchi T et al. Increased natural killer cells anddecreased CD3+CD8 +CD62L+ T cells in CML patients who sustained complete molecular remission after discontinuation of imatinib. Br J Haematol
2012; 157: 254–6.
16 Sallusto F, Geginat J, Lanzavecchia A. Central memory and effector memory
T cell subsets: function, generation, and maintenance. Annu Rev Immunol
2004; 22: 745–63.
17 Merante S, Orlandi E, Bernasconi P, Calatroni S, Boni M, Lazzarino M.
Outcome of four patients with chronic myeloid leukemia after imatinib mesylate discontinuation. Haematologica 2005; 90: 979–81.
18 Rousselot P, Huguet F, Rea D et al. Imatinib mesylate discontinuation inpatients with chronic myelogenous leukemia in complete molecular remission for more than 2 years. Blood 2007; 109: 58–60.
19 Nausch N, Cerwenka A. NKG2D ligands in tumor immunity. Oncogene
2008; 27: 5944–58.
20 Sun JC, Lopez-Verges S, Kim CC, DeRisi JL, Lanier LL. NK cells andimmune “memory”. J Immunol 2011; 186: 1891–7.

Kazuma Ohyashiki received research support from Bristol-Myers
Squibb KK and Novartis KK, and served as consultant and advisor ofNovartis KK, Bristol-Myers Squibb KK and Ariad, honoraria for
lecture fees from Novartis KK and Bristol-Myers Squibb KK.

21 Paust S, von AU. Natural killer cell memory. Nat Immunol 2011; 12: 500–8.
22 Boissel N, Rea D, Tieng V et al. BCR ⁄ ABL oncogene directly controls
MHC class I chain-related molecule A expression in chronic myelogenous
leukemia. J Immunol 2006; 176: 5108–16.
23 Dudley ME, Wunderlich JR, Robbins PF et al. Cancer regression andautoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science 2002; 298: 850–4.
24 Bauer S, Corless CL, Heinrich MC et al. Response to imatinib mesylate of agastrointestinal stromal tumor with very low expression of KIT. Cancer
Chemother Pharmacol 2003; 51: 261–5.
25 Borg C, Terme M, Taieb J et al. Novel mode of action of c-kit tyrosine
kinase inhibitors leading to NK cell-dependent antitumor effects. J Clin
Investig 2004; 114: 379–88.
26 Menard C, Blay JY, Borg C et al. Natural killer cell IFN-gamma levels predict long-term survival with imatinib mesylate therapy in gastrointestinal
stromal tumor-bearing patients. Cancer Res 2009; 69: 3563–9.
27 Talpaz M, Hehlmann R, Quintas-Cardama A, Mercer J, Cortes J. Re-emergence of interferon-a in the treatment of chronic myeloid leukemia. Leukemia 2013; 27: 803–12.
28 Veneri D, Tecchio C, De MG et al. Long-term persistence of molecularresponse after discontinuation of interferon-alpha in two patients withchronic myeloid leukaemia. Blood Transfus 2012; 10: 233–4.
29 Kreutzman A, Rohon P, Faber E et al. Chronic myeloid leukemia patients inprolonged remission following interferon-alpha monotherapy have distinct
cytokine and oligoclonal lymphocyte profile. PLoS ONE 2011; 6: e23022.
30 Rezvani K, Grube M, Brenchley JM et al. Functional leukemia-associatedantigen-specific memory CD8 + T cells exist in healthy individuals and inpatients with chronic myelogenous leukemia before and after stem cell transplantation. Blood 2003; 102: 2892–900.
31 Kanodia S, Wieder E, Lu S et al. PR1-specific T cells are associated withunmaintained cytogenetic remission of chronic myelogenous leukemia after
interferon withdrawal. PLoS ONE 2010; 5: e11770.
32 Cervantes F, Pierson BA, McGlave PB, Verfaillie CM, Miller JS. Autologous activated natural killer cells suppress primitive chronic myelogenous
leukemia progenitors in long-term culture. Blood 1996; 87: 2476–85.
33 Mustjoki S, Ekblom M, Arstila TP et al. Clonal expansion of T ⁄ NK-cells
during tyrosine kinase inhibitor dasatinib therapy. Leukemia 2009; 23: 1398–
405.
34 Kim DH, Kamel-Reid S, Chang H et al. Natural killer or natural killer ⁄ Tcell lineage large granular lymphocytosis associated with dasatinib therapy
for Philadelphia chromosome positive leukemia. Haematologica 2009; 94:
135–9.
35 Blake SJ, Bruce Lyons A, Fraser CK, Hayball JD, Hughes TP. Dasatinib
suppresses in vitro natural killer cell cytotoxicity. Blood 2008; 111: 4415–6.
36 Fraser CK, Blake SJ, Diener KR et al. Dasatinib inhibits recombinant viral
antigen-specific murine CD4 + and CD8 + T-cell responses and NK-cell
cytolytic activity in vitro and in vivo. Exp Hematol 2009; 37: 256–65.
37 Rohon P, Porkka K, Mustjoki S. Immunoprofiling of patients with chronic
myeloid leukemia at diagnosis and during tyrosine kinase inhibitor therapy.
Eur J Haematol 2010; 85: 387–98.
38 Salih J, Hilpert J, Placke T et al. The BCR ⁄ ABL-inhibitors imatinib, nilotinib and dasatinib differentially affect NK cell reactivity. Int J Cancer 2010;
127: 2119–28.
39 Chen CI, Koschmieder S, Kerstiens L et al. NK cells are dysfunctional in
human chronic myelogenous leukemia before and on imatinib treatment andin BCR-ABL-positive mice. Leukemia 2012; 26: 465–74.
40 Pierson BA, Miller JS. CD56 + bright and CD56 + dim natural killer cells
in patients with chronic myelogenous leukemia progressively decrease in
number, respond less to stimuli that recruit clonogenic natural killer cells,
and exhibit decreased proliferation on a per cell basis. Blood 1996;
88: 2279–87.
41 Fujimiya Y, Bakke A, Chang WC et al. Natural killer-cell immunodeficiency
in patients with chronic myelogenous leukemia I. Analysis of the defect1152

doi: 10.1111/cas.12216© 2013 Japanese Cancer Association-313-

using the monoclonal antibodies HNK-1 (LEU-7) and B73.1. Int J Cancer
1986; 37: 639–49.
42 Ng KP, Hillmer AM, Chuah CT et al. A common BIM deletion polymorphism mediates intrinsic resistance and inferior responses to tyrosine kinase
inhibitors in cancer. Nat Med 2012; 18: 521–8.

43 Katagiri S, Umezu T, Ohyashiki JH, Ohyashiki K. The BCL2L11 (BIM)
deletion polymorphism is a possible criterion for discontinuation of imatinibin chronic myeloid leukaemia patients. Br J Haematol 2013; 160: 269–71.

Supporting Information
Additional Supporting Information may be found in the online version of this article:
Fig. S1. Representative dot plot analyses of PBMCs in each group.
Fig. S2. Patients with CML taking imatinib (IM) showed low PBMC counts.
Fig. S3. Immunophenotypic profiles of natural killer (NK) cells in cell counts.
Fig. S4. Immunophenotypic profiles of CD8+ T cells in cell counts.

Mizoguchi et al.

Cancer Sci | September 2013 | vol. 104 | no. 9 | 1153
© 2013 Japanese Cancer Association-314-平成25年度～平成29年度≪文部科学省 私立大学戦略的研究基盤形成支援事業≫

東京医科大学

中間報告会『生体分子情報による次世代型がん個別最適化治療法の開発』

開催日時 ： 平成28年1月26日（火）18:00～20:00
会場 ： 東京医科大学病院
教育研究棟（自主自学館）3階 大教室
１．開 会

学 長

鈴

木

衞

２．プロジェクトの概要と今後の課題
プロジェクト長 医学総合研究所 教授 大屋敷 純子
３．研究進捗報告
I. 研究機器整備進捗状況：次世代シーケンサーの臨床応用(1) 稀少疾患におけるクリニカルシーケンス
小児科学分野 臨床研究医 鈴木 慎二(2) 眼内悪性黒色腫における体細胞変異解析
眼科学分野 助教 上田 俊一郎(3) 骨髄間質細胞の体細胞変異解析
医学総合研究所 ポスト・ドクター 東 剣虹(4) 肺癌におけるRNAシーケンスの有用性
分子病理学分野 助教 大野 慎一郎
Ⅱ. 研究基盤形成進捗状況(1) がん幹細胞を標的とした治療戦略:新たな分子標的の探索
血液内科学分野 准教授 田内 哲三(2) がん環境を標的とした治療戦略:エクソソームの役割
先端分子探索寄附講座 講師 梅津 知宏(3) 免疫学的側面から:サイトカインによる骨髄造血の制御と抗腫瘍効果
医学総合研究所 教授 善本 隆之(4) 核酸医薬の実現に向けて
分子病理学分野 主任教授 黒田 雅彦
４．総合討論５．閉 会
問合せ先：東京医科大学研究支援部研究支援課（E-mail） kenkyu-s@tokyo-med.ac.jp（TEL）03-3351-6141 内線412・385-315-

MEXT-Supported Program for the Strategic Research Foundation at Private Universities(2013-2017) ”Development of Personalized Cancer Therapy for the Next Generation
Based on Biomolecular Information”

Tokyo	
  Medical	
  University,	
  Ins5tute	
  of	
  Medical	
  Science	"Role of Aging and Cancer”
August 28, 2017 Tokyo
Venue:	
  Tokyo	
  Medical	
  University	
  Hospital	  Educa9on	
  and	
  Research	
  Building	  Mee9ng	
  Room	  (3rd	  ﬂoor)	
  	
  Organized	
  by	
  	
  
Masahiko	
  Kuroda,	
  Takayuki	
  Yoshimoto	
  and	
  Junko	  H	
  Ohyashiki	  Program	  Opening	
  Remarks:	
  14:00-­‐14:05	
  Prof.	
  Mamoru	
  Suzuki	
  (President	
  of	
  Tokyo	
  Medical	
  University)	
  Session	
  2:	
  16:10-­‐17:50	
  Session	
  1:	
  14:10-­‐15:50	
  (5)	
  Advances	
  in	
  cancer	
  immunotherapy:	
  Prof.	  Takayuki	
  Yoshimoto	
  (Tokyo	
  Medical	
  University)	
  (6)	
  Hypoxia	
  induced	
  paclitaxel	
  resistance	
  via	
  miR-­‐100	
  as	
  a	
  
poten9al	
  diagnos9c	
  biomarker	
  for	
  cervical	
  cancer:	
  Prof.	
  Hirotaka	
  Nishi	  (Tokyo	
  Medical	
  University） 	
  (7)	
  G-­‐quadruplexes	
  in	
  cancer	
  biology	
  and	  therapeu9cs:	
  Dr.	
  Hiroyuki	
  Seimiya	
  (Japanese	
  Founda5on	
  for	  Cancer	
  Research)	
  (8)	
  Recent	
  advance	
  of	
  telomere	
  biology	
  in	
  neuroblastoma:	
  Prof.	
  Eiso	  Hiyama	  (Hiroshima	
  University)	
  (1)	
  miRNA	
  in	
  aging	
  and	
  cancer:	
  Prof.	
  Hidetoshi	  	
  Tahara	  	  (Hiroshima	
  University)	
  (2)	
  Dendri9c	
  cell-­‐derived	
  exosomes	
  as	
  Immunotherapy:	
  Dr.	
  Masakatsu	
  Takanashi	
  (Tokyo	
  Medical	
  University)	
  (3)	
  Cross-­‐talk	
  mediated	
  by	
  bone	  marrow	  stromal	
  cell-­‐derived	
  extracellular	
  vesicles:	
  Dr.	
  Tomohiro	
  Umezu	
  (Tokyo	
  Medical	
  University)	
  (4)	
  Clinical	
  applica9on	
  of	
  mesenchymal	
  stromal	
  cells	
  (Kintaro	  cellsTM)	
  Mr.	
  Alexei	  Gladkov	
  (Kintaro	
  Cells	
  Power	  Corp.)	
  Special	
  Lecture	
  	
  18:00〜18:50	
  Chair:	
  Prof.	
  Kazuma	  Ohyashiki	  (Tokyo	
  Medical	
  University)	
  Title:	
  Recent	
  advances	
  in	
  telomeres	
  and	
  telomerase	  in	
  rela9on	
  to	
  aging	
  and	
  cancer:	
  TeSLA,	
  TPE-­‐OLD	
  and	
  6-­‐thio-­‐2’-­‐deoxyguanosine	
  
	
  
	
  	
  
Prof.	
  Jerry	
  W.	
  Shay	  	
  (Department	
  of	
  Cell	  Biology,	
  University	
  of	
  Texas	
  Southwestern	  Medical	
  Center,	
  Dallas,	
  U.S.A)	
  [Contact]	
  Chikako	
  Kato	  (Secretariat	
  of	
  the	
  1st	
  TMU-­‐IMS	  Interna5onal	
  Symposium	
  in	
  Tokyo)	
  E-­‐mail:	
  s-­‐idrc@tokyo-­‐med.ac.jp	
  TEL&FAX:	
  +81-­‐3-­‐3345-­‐0185	
  	  [URL]	
  hRp://team.tokyo-­‐med.ac.jp/ims_onc/symposium/index.html	
  
-316-

平成25年度～平成29年度≪文部科学省 私立大学戦略的研究基盤形成支援事業≫

東京医科大学最終成果報告会『生体分子情報による次世代型がん個別最適化治療法の開発』

日 時 ： 平成30年1月26日（金）17:20～19:20
会 場 ： 東京医科大学病院
教育研究棟（自主自学館）3階 大教室
１．開

会

学

２．本事業の研究成果総括
プロジェクト長 医学総合研究所長鈴木

分子腫瘍研究部門

衞

教授

大屋敷 純子

３．研究成果報告
I. 先端基礎研究の成果概要(1) 人工知能を用いた癌の予後システムの開発
分子病理学分野 主任教授 黒田 雅彦(2) IL-27による造血系の制御による抗腫瘍効果の増強
医学総合研究所 免疫制御研究部門 教授 善本 隆之
Ⅱ. 臨床への応用(1) 白血病のエクソーム解析：微小クローン推移による再発予測
血液内科学分野 助教 片桐 誠一朗(2) 次世代シークエンサーによる眼腫瘍の解析
眼科学分野 講師 臼井 嘉彦(3) 婦人科がんにおける腫瘍マーカーとしてのmiRNAの可能性
産科婦人科学分野 助教 高見澤 重篤
Ⅲ. 産学連携研究への発展
がん環境におけるMesenchymal Stromal Cellの役割
医学総合研究所 分子腫瘍研究部門・血液内科学分野
細胞治療研究開発講座 講師 梅津 知宏
４．総合討論５．外部評価委員のご意見
６．閉会

お問い合わせ：

病院長

土田

明彦

東京医科大学研究支援部研究支援課（E-mail） kenkyu-s@tokyo-med.ac.jp（TEL）03-3351-6141 内線457-317-