᣿එ‫ܖٻ‬ⅻ⇂‒ᡶ‫ޒ‬Сࣂ‒ᄂᆮ৑‒ᏽၨСࣂ≋଒≝ᢡˡ‫܇‬ᚮૺ≌ᄂᆮЎ᣼‒◊✲ࡢ࠶ࡺࡳ࡜ᴗ⦼ 2021

㸨㸨㸨㸨㸨㸨㸨㸨㸨㸨㸨㸨㸨㸨㸨㸨㸨㸨㸨㸨㸨
Annual Report 2021
Division of Translational and Clinical Oncology(previously: Division of Diagnostic Molecular Oncology)

Cancer Research Institute, Kanazawa University
Kanazawa, Japan

2021 ᖺ 12 ᭶
December 2021

研究のあゆみと業績 2021

まえがき
今年６月末、オリンピック東京2020がまさに強行されようとしていた頃に、私たちの研究室が開設満20年を迎えようとしていました。
本誌のまえに編集した「研究のあゆみと業績 Vicennial Report
2016-2020」にも書いたように、今年7月に生まれた2nd grandkidがやがて成人するくらいの年月に相当します。これがはたして長かったのか、短かったのか？私にはちょうどよかったようです。ただ、昨夏から楽しみにして、７月に予定していた共同研究セミナーと記念会を11月末に延長しても、開催がかないませんでした【附記１】。
毎年、日本漢字能力検定協会（京都市東山区）が全国から募っ 出典：https://jp.quora.com/
た、その年の世相を漢字1文字で表現する「今年の漢字」を選定するのが年末の恒例です。
2021年は「金」が選ばれ、清水寺の森 清範貫主の揮毫により発表されました。私は、もしウィ
ルスあるいはウイルス感染症を一文字で現わ
ウィルス
せる漢字があれば選ばれたのではないかと変なことを想像し、ネットをみてみました。多くのサイトでは、病毒や濾過性病毒とか書かれています。これではピンとこないと思っていたとこ
ろ、冒頭のような造字をみかけました。20年の節目に当たる今年はまたしても、隣国が発生 出典：エンタメRBB（https://www.rbbtoday.com/）
源といわれる新たな疫病に振りまわされてしまい、この造字が「金」に重くのしかかる一年でした。
記念会ができなかったことは悔やまれるとはいえ、出不精の私にはわりと快適な１年でした。
今春は、２名の大学院が学位を獲り【第５頁、附記３】、学位を目指す第一線外科医が９月から研究生に在籍しています。４月には２名のポスドクが採用され、少し活気がでてきました。あと、がん研News Letterの「共同研究者の紹介」欄に、もう長年（2013年～）になる香川大学病理学：
松田陽子さんとの交流を交換日記のようなかたちで紹介したことも、殺伐としたこの１年ではほっとできることです【附記２】。私自身は年末になって、日本消化器内視鏡学会から次回（2022年度）の北陸支部セミナーを担当するように指名されました。2008年には消化器・腫瘍外科から身を引いたものの、消化器内視鏡専門医、指導医としてなんとか持ちこたえている自身に課せられた任務と受けとめ、承諾しました。また仲間に助けてもらって、なんとか役目をはたすよう努めます。
この年末には、妙な変異が現れたようで、この先が案じられます。それでも、2020年末の「まえがき」にも云いました。さあ皆さん、元気を出してすばらしい2022年を迎えましょう。それでは、これからもどうぞよろしくお願いします。
2021年１2月 年末〒920-0934 石川県金沢市宝町 13 番 1 号
進展
金沢大学がん 制御 研究所・腫瘍制御
金沢大学附属病院がんセンター（併任）金沢医科大学病院内視鏡科（非常勤）☎ 076-265-2792（直通），2798（事務，研究室）
📠📠 076-234-4529
📨📨 minamoto@staff.kanazawa-u.ac.jp

Division of Translational and Clinical Oncology
Cancer Research Institute and Cancer Center
Kanazawa University and Hospital
13-1 Takara-machi
Kanazawa 920-0934, Japan
Phone 81-76-265-2792 (office), 2798 (secretary)
Fax 81-76-234-4523
e-mail: minamoto@staff.kanazawa-u.ac.jp

HP（更新中）: http://www.kanazawa-u.ac.jp/~ganken/shuyoseigyo/index.html—

1- 1—-

金沢大学がん進展制御研究所 腫瘍制御 (旧：遺伝子診断) 研究分野

【目次】

研究スタッフ ………………………………………………………………………

共同研究者 ………………………………………………………………………… 年のあゆみとできごと ………………………………………………………

研究分野と活動の概要 ……………………………………………………………

研究費 ………………………………………………………………………………

研究業績 ……………………………………………………………………………
 論文発表 学会発表その他（地域・社会貢献など）

知的財産，関連活動など …………………………………………………………
メディア・新聞報道など …………………………………………………………

【附記１】腫瘍制御共同研究セミナーと  周年記念会（誌上開催） ……………
【誌上開催：七夕の会】酒呑もおよ！………………………………………………
【附記２】金沢大がん研 News Letter（抜粋）：共同研究者紹介…………………

【附記３】学位論文： 2021 年３月 修了………………………………………………

—2 —研究のあゆみと業績 2021

【研究メンバー】 （2021 年 12 月現在）
教授

源 利成

金沢大学附属病院がんセンター（併任）

助教

堂本貴寛

博士研究員

上原将大

たか ひろ

ま さ ひろデ ィ リ レ バ

ボ

2021 年 4 月～
リドン

Diliraba Bolidong 2021 年 4 月～
さとし

本学大学院医学系研究科 消化器・腫瘍・再生外科学

大学院

竹中 哲

（博士課程）

中西宏佳

ひろよし

本学大学院医薬保健学総合研究科 腫瘍制御学（～3 月）

ま さ ひろ研究生

上原将大

本学大学院医薬保健学総合研究科 腫瘍制御学（～3 月）

太田亮介

本学大学院医薬保健学総合研究科 腫瘍制御学

まさのり

小竹優範

厚生連高岡病院 外科（9 月～）

も り た

医学類５年

守田周平

同４年

石田 岳

メディカルリサーチトレーニングプログラム（MRT）

研究支援員

浅香敦子

研究支援推進員

研究協力員

旭井亮一

（株）凸版印刷研究所

川島篤弘

独立行政法人国立病院機構金沢医療センター 臨床検査科

メディカルリサーチトレーニングプログラム（MRT）

たかし

あさ い

ひろ の り藤沢弘範

独立行政法人国立病院機構金沢医療センター 脳神経外科

横井健二

米国 ヒューストンメソジスト研究所ナノ医療部門

しまさき

島崎猛夫

金沢医科大学総合医学研究所，消化器内科

ひがし

東 朋美

金沢大学医薬保健研究域医学系 環境分子応答学（衛生学）

かさしま

笠島里美

金沢大学医薬保健研究域保健学系 検査技術科学

かつ よ し宮下勝吉

福井県立病院
脳神経外科
金沢大学医薬保健研究域医学系
脳神経外科学

なかじま

中島日出夫

上尾中央総合病院 腫瘍内科

下崎真吾

医療法人社団
下崎整形外科医院
公立南砺中央病院
整形外科

澤田 武

名古屋市立大学消化器・内分泌内科学

太田亮介

金沢大学大学院医薬保健学総合研究科 腫瘍制御学

デ ィ リ レ バ

ボ

リ

ド

ン

Diliraba Bolidong 腫瘍制御研究分野（2021 年 1 月～2021 年 3 月）
共同研究者

づか

塚 正彦

金沢大学医薬保健研究域医学系 法・社会医学（法医学）—

3- 3—-

金沢大学がん進展制御研究所 腫瘍制御 (旧：遺伝子診断) 研究分野

【共同研究者】 （2021 年現在で共同研究が稼動および予定しているもの．敬称略）
な か だ

教授

中田光俊

助教

け い し

中村慶史

金沢大学医薬保健研究域医学系 消化器・腫瘍・再生外科学

教授

土屋弘行

金沢大学医薬保健研究域医学系 機能再建学／整形外科学

特任教授 山本憲男

金沢大学医薬保健研究域医学系 機能再建学／整形外科学

教授

竹村博文

金沢大学医薬保健研究域医学系 先進総合外科学教授

Richard Wong

金沢大学理工学研究域自然システム学系／ナノ生命科学研究所

教授

滝野隆久

金沢大学国際基幹教育院

教授

元雄良治

金沢医科大学 腫瘍内科学（～3 月）

准教授

宮下知治

金沢医科大学 一般・消化器外科学／腫瘍外科学

教授

伊藤 透

金沢医科大学 消化器内視鏡学

教授

石垣靖人

金沢医科大学総合医学研究所分野長

よ し はるかつや

土原一哉

金沢大学医薬保健研究域医学系 脳機能制御学/脳神経外科学

国立がん研究センター 早期・探索臨床研究センター トランスレー
ショナルリサーチ分野

教授

曽我朋義

教授

竹田 扇

山梨大学大学院医学工学総合研究部
解剖学講座 細胞生物学
帝京大学医学部
解剖学

講師

吉村健太郎

山梨大学大学院医学工学総合研究部 解剖学講座 細胞生物学

せん

わた
いし
しわ

慶應義塾大学 先端生命科学研究所研究部長 石渡俊行

東京都健康長寿医療センター 老年病理学，高齢者がん研究

教授

松田陽子

香川大学医学部病理病態・生体防御講座 腫瘍病理学

教授

松下一之

千葉大学医学研究院 分子病態解析学，附属病院検査部

教授

や す し

佐々木泰史
え い じ

札幌医科大学 教養教育研究部門 医療人育成センター

准教授

久保田英嗣

名古屋市立大学消化器・内分泌内科学

助教

古田拓也

久留米大学医学部 病理学

研究員

小泉恵太

上尾中央総合病院
鶴見歯科大学かみ

紙 健次郎（株）ヒューマンメタボロームテクノロジーズ

教授

Ze’ev Ronai

Sanford Burnham Prebys Medical Discovery Institute, La Jolla, USA

教授

Andy Giraud

豪州オーストラリア王立小児病院

Louise M. Judd

豪州オーストラリア王立小児病院

Trevelyan R. Menheniott 豪州オーストラリア王立小児病院
Phil Sutton

豪州オーストラリア王立小児病院

准教授

Serge Y. Fuchs

ペンシルヴェニア大学 生物学

准教授

Barry Iacopetta

西オーストラリア大学 腫瘍学—

4 —研究のあゆみと業績 2021

【2021 年のあゆみとできごと】
2021年 1月 1日

・Dilireba Bolidong さんが研究協力員に採用された。

2021年 1月14日

・源 利成が第 29 回日本癌病態治療研究会 パネルディスカッション：癌治療
抵抗性の機序解明に基づく新たな治療法の開発で、基調講演した。

GSK3 のがん生物学とがん治療抵抗性の一隅
課題： GSK3β
2021年 3月 31日【附記３】

・中西宏佳君と上原将大君が大学院を修了し、医学博士の学位を取得。
課題（中西）：Significance of gene mutations in Wnt signaling pathway in
traditional serrated adenomas of the colon and rectum. PLoS
One 15 (2): e0229262, 2020.
課題（上原）：Glycogen synthase kinase 3β3 participates in acquired
resistance to gemcitabine in pancreatic cancer. Cancer Sci 111(12): 4405-16, 2020.

源と中西宏佳君

上原将大君と源

202１年 4 月 1 日

・上原将大君と Dilireba Bolidong さんが博士研究員に採用された。

2021 年 7 月 3 日

・腫瘍制御研究分野共同研究セミナーと 20 周年記念会の開催予定を延期

【附記１】

した。

2021年109月
月 1日

・小竹優範君（厚生連高岡病院 外科）が研究生に在籍。

2021年11月28日

・腫瘍制御研究分野共同研究セミナーと 20 周年記念会の開催を再延期し【附記１】
2021年11月28日

た。
・源 利成が、日本消化器内視鏡学会北陸支部評議員会で第 31 回日本消化器内視鏡学会北陸セミナーの会長に指名された。

—

5- 5—-

金沢大学がん進展制御研究所 腫瘍制御 (旧：遺伝子診断) 研究分野

【研究分野と活動の概要】
当研究分野は 1998 年に遺伝子診断の旧称で開設され、その後 23 年間にわたって、
消化器がんを中心にがんの多様な生物病態と腫瘍外科学的特性について、基礎と臨床を関係づけるかたちの研究を指向している。そして、その成果を難治がんや希少がんの病態解明と制御に応用するために、学内外のグループと共同研究を進める。

Division Summary
The mission of our division centers on laboratory and clinical research to develop thenovel strategies and modalities for diagnosis and treatment of the gastrointestinal,
refractory and rare cancer types including glioblastoma, bone and soft tissue sarcomas.
Research projects are based on biological characteristics of individual tumor types that are
relevant to their invasive and metastatic potential, resistance to therapy, recurrence andoutcome of patients. Our current efforts are focused on (1) research and development of
the cancer therapy by targeting aberrant glycogen synthase kinase (GSK)3; (2)understanding of malignant phenotypes of cancer by investigating pathological metabolic
properties (eg., aerobic glycolysis, tumor-promoting autophagy); and (3) biological basisof gastrointestinal and refractory cancer, all for clinical translation. We have been alsoestablishing the tissue material resources of human stomach and colorectal cancer for ourown projects described above as well as for studies collaborating with our institutional andmany other research groups. In an immediate couple of years, we have obtained thepreliminary results indicating the putative roles of tumor GSK3 as a molecular hub thatconnects the pathways responsible for tumor invasion and resistance to therapy, thus
enforcing its potential as a major cancer therapeutic target. We are extending this project
toward investigation of the putative roles for GSK3 in promoting esophageal squamouscell carcinoma (ESCC; the major type of esophageal cancer in Asia and Japan) andpancreatic neuroendocrine neoplasms as well as in acquiring chemoresistance in pancreaticcancer. We also have been trying to develop cellular and mouse models predisposing to
ESCC by CRISPR-Cas9-based genome editing of the metabolic enzymes including
glycogen synthase and GSK3. In addition to these projects, we have clarified the genetic
and epigenetic characteristics of traditional serrated adenoma and duodenal non-ampullaryadenoma and adenocarcinoma.
＜2021 年の研究成果，進捗状況及び今後の計画＞
１．glycogen synthase kinase（GSK）3β 阻害によるがん治療法の研究、開発
Wnt 経路抑制因子と認識されている GSK3 が固有の分子経路を介して、がんの悪性形質を推進することを系統的に示してきた。そして、GSK3 阻害のがん治療効果を細胞—

6 —研究のあゆみと業績 2021

レベルと担がん動物で実証した。また，学内外の外科系グループと連携し、膵がん、膠芽腫や骨軟部肉腫などの難治、希少がんで高活性を示す GSK3 が、腫瘍浸潤性と治療（抗がん剤，放射線）不応性の悪性形質を連結することを見出した。一連の研究をもとに、
GSK3 阻害薬品の転用と抗がん剤を併用するがん治療法を開発し、再発膠芽腫（附属病院脳神経外科）と進行膵がん（金沢医科大学病院）を対象とする医師主導臨床研究によりその安全性と抗腫瘍効果を検証した。2021 年には食道扁平上皮がんと抗がん剤耐性獲得膵がんに対する GSK3 阻害の治療効果とメカニズムを明らかにした。また、
GSK3 阻害によるがん治療の概念実証のため、膵内分泌腫瘍（PNET）の共同研究に加えて、米国で臨床試験中の GSK3 阻害剤 9-ING-41 を開発した Actuate 社と共同で治療耐性膵がんの前臨床試験を計画した。
２．がんの代謝特性にもとづく悪性形質の解析研究
GSK3 はグリコーゲン代謝を制御する。また、特定の中間代謝産物が解糖経路と自食作用の接点になるとが知られている。そこで大腸がんを中心に、がん固有の糖代謝（Warburg 効果）に関わる触媒酵素やがん促進性自食作用に対する GSK3 の統合的解析を進めている。これとは別に、内視鏡的にヨード不染を特徴とする食道の扁平上皮
発がん初期の生物学的特性は細胞内グリコーゲンの減少，消失である。そこで、患者由来の正常食道扁平上皮細胞とマウスを対象に、グリコーゲン合成酵素と GSK3 のゲノム編集による食道扁平上皮易発がん状態の誘発を試みる研究を 2018 年に開始し、目的の改変マウスを作出して、経過観察を継続している。また 2021 年には、GSK3 がエネル
ギーセンサー酵素 AMPK をリン酸化により不活性化するという知見をもとに、食道扁平上皮がんにおける両酵素の関連解析を始めた。
３．ヒト消化管がん組織バンクを中心とする大腸がんの分子病理学的研究
消化管がん研究や臨床研究の基礎資源として 2008 年から本事業を開始し、2010 年にこの事業を当研究所ヒトがん組織バンクに継承し現在に至っている。この組織資源の共同利用促進のために、日本医療研究開発機構ゲノム医療支援サイト（http://www.bio
bank.amed.go.jp/biobank/index.html）に情報公開している。山梨大学：竹田 扇らが開発した大気圧イオン化法-質量分析を用いて、大腸がん質量分析診断法開発の共同研究を継続している。大腸組織の質量分析パターンをもとに特有の統計解析と機械学習を組み合わせて非がん/がんの判別（診断）アルゴリズムを構築し、90%以上の感度と特異度による判別を可能にした（論文作成予定）。現在、島津製作所基盤技術研究所と共同で、大腸がんの質量分析—内視鏡診断法の内視鏡デバイス開発に着手した。組織バンク検体を利用して、名古屋市立大学（十二指腸腺腫，早期がん）、香川大学（大腸がんテロメア
解析）と共同研究を開始した。

—

7- 7—-

金沢大学がん進展制御研究所 腫瘍制御 (旧：遺伝子診断) 研究分野

【研究費】 （2021 年 1 月以降の新規，継続，分担と連携を含む外部資金の獲得状況）
研究種目・期間（課題番号）
2020年－2022年度
科学研究費補助金（基盤研究Ｃ）（20K09100）
2019年－2021年度
科学研究費補助金（ 基盤研究Ｂ）（19H03727）
2019年－2021年度
科学研究費補助金（基盤研究Ｃ）（19K07710）
2019年－2021年度
科学研究費補助金（基盤研究Ｃ）（19K08367）
2019年－2021年度
科学研究費補助金（基盤研究Ｃ）（19K08643）

研究代表者

研究分担者
連携研究者

研究課題

宮下知治

太田哲生，
源 利成，
ほか

直接経費
3,200,000 円
GSK3を基軸とした食道発癌機構の間接経費
解明と新規治療戦略の開発
960,000 円

源 利成

直接経費
大腸がんの糖代謝改変と細胞核分13,400,000 円
Richard Wong
裂機構を繋ぐ分子経路の解明とがん
宮下知治
間接経費
制御法開発への応用
4,020,000 円

堂本貴寛

GSK3によるがん促進的糖代謝特3,400,000 円
間接経費性の解明と制御への応用

研究経費

直接経費

1,020,000 円
直接経費

太田亮介

澤田 武，
源 利成

RNAシーケンスによる大腸鋸歯状腺3,400,000 円腫の発癌機構の解明と分子標的治間接経費
療の基盤確立1,020,000 円

直接経費

澤田 武

太田亮介，
ほか

表在性非乳頭部十二指腸腫瘍の発3,400,000 円
癌機構の解明と，進展を予測する内間接経費
視鏡診断体系の確立
1,020,000 円

2021年度金沢大学
がん進展制御研究 佐々木泰史
所共同研究（一般）

澤田 武，
源 利成，
ほか

非乳頭部十二指腸腫瘍における(5%%受容体ファミリーの解析と治療
標的の探索（一般：新規）

350,000 円

2021年度金沢大学
がん進展制御研究 成澤裕子
所共同研究（若手）

松田陽子，
源 利成，
ほか

光子励起顕微鏡を用いた膵癌細胞の血行性肝転移と加齢との関連の解明（若手：継続）

350,000 円

2021年度金沢大学
がん進展制御研究 北村浩一
所共同研究（若手）

松下一之，
源 利成，
ほか

消化器・難治がんのリボソーム生合成の新規メカニズム解明と診断，治療法への応用（若手：継続）

350,000 円

2021年度金沢大学
澤田 武，
がん進展制御研究 久保田英嗣 源 利成，所共同研究（一般）ほか

大腸癌における循環腫瘍DNAを用いた抗EGFR抗体薬耐性の検出と病状モニタリングの確立（一般：継続）

300,000 円

2021年度金沢大学
がん進展制御研究 宮下知治
所共同研究

上原将大，
堂本貴寛，
源 利成

GSK3/STAT3経路を基軸とする膵
神経内分泌腫瘍の病態解明と治療法開発（一般：継続）

300,000 円

2021年度金沢大学
がん進展制御研究 小泉惠太
所共同研究中島日出夫
GSK3の作用機序解析とハイパーサ
堂本貴寛，ーミア/GSK3阻害剤併用の可能性へ
源 利成の探求（一般：継続、採択後辞退）ハ イ パ ー サ ー ミ ア に お け るFam107B250,000 円

期間の総額（間接経費を含む） 36,490,000 円—

8 —研究のあゆみと業績 2021

【研究業績】
Ⅰ．論文発表 [Impact factor 2020/2021]
・英文総説，著書なし
・英文原著

1. Shimasaki T, Yamamoto S, Omura R, Ito K, Nishide Y, Yamada H4, Ohtomo K,
Ishisaka T, Okano K, Ogawa T, Tsuji H, Matsuo Y, Minamoto T, Tomosugi N, Ferain
E, Ochiya T. Novel platform for regulation of extracellular vesicles and metabolites
secretion from cells using a multi-linkable horizontal co-culture plate. Micromachines
12 (11): 1431, 2021. doi: 10.3390/mi12111431 [2.892]
2. Kotake M, Bando H, Kaneko M, Takemura H, Minamoto T, Kawakami K. LOH of thethymidylate synthase locus in combination with genotype has prognostic andpredictive significance in colorectal cancer. Mol Clin Oncol 15: 235, 2021. doi:
10.3892/mco. 2021.2398 [unavailable]
Ⅱ．学会発表・国際学会

1. Takahiro Domoto, Masahiro Uehara, Satoshi Takenaka, Dilireba Bolidong, Tatsuhiko
Furusawa, Osamu Takeuchi, Tomoharu Miyashita, Toshinari Minamoto. GSK3
GSK3β interconnects the malignant properties in therapy-resistant pancreatic cancer. The 10th
International Conference of the International Society of Gastroenterological Carcinogenesis (ISGC 2021), November 26 (Fri), 27 (Sat), 2021, Gifu, Japan (WEB).
2. Masahiro Uehara, Takahiro Domoto, Satoshi Takenaka, Dilireba Bolidong, Osamu
Takeuchi, Tomoharu Miyashita, Toshinari Minamoto. Glycogen synthase kinase(GSK) 3
3β participates in acquired resistance to gemcitabine in pancreatic cancer. The
10th International Conference of the International Society of Gastroenterological
Carcinogenesis
Carcino-genesis(ISGC(ISGC2021),
2021),November
November26
26(Fri),(Fri),27
27(Sat),(Sat),2021,
2021,Gifu,
Gifu,Japan
Japan(WEB).
(WEB).
3. Dilireba Bolidong, Takahiro Domoto, Masahiro Uehara, Hemragul Sabit, Tomoyuki
Okumura, Yoshio Endo, Mitutoshi Nakada, Tomoharu Miyashita, Richard W. Wong,Toshinari Minamoto. Potential therapeutic effect of targeting glycogen syntase kinase(GSK)3β(GSK)3 in esophageal squamous cell carcinoma (ESCC). The 10th International
Conference of the International Society of Gastroenterological Carcinogenesis (ISGC2021), November 26 (Fri), 27 (Sat), 2021, Gifu, Japan (WEB).
・国内（全国）学会

4. 松田陽子，成澤裕子，葉 娟娟，山川けいこ，向井裕理，谷元ミサ，横平政直，本間尚子，源利成．長いテロメアを有する腫瘍の頻度と予後との関連．Incidence and prognostic relevanceof long telomere cancers. 第 110 回日本病理学会総会，2021 年 4 月 22 日（木）－24 日（土），京王プラザホテル，東京．

5. 岩嶋友紀，羽澤勝治，源 利成，Richard Wong．核膜孔複合体による gene-gating 形成機序．
日本生化学会北陸支部第 39 回大会，2021 年 6 月 5 日（土），WEB．

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9- 9—-

金沢大学がん進展制御研究所 腫瘍制御 (旧：遺伝子診断) 研究分野

6. Takahiro Domoto, Satoshi Takenaka, Masahiro Uehara, Dilireba Bolidong, Tatsuhiko
Furukawa, Tomoharu Miyashita, Toshinari Minamoto. Glycogen synthase kinase3 renders pancreatic cancer acquiring resistance to gemcitabine via the STAT3(GSK) 3βactivation. 堂本貴寛，竹中 哲，上原将大，ボリドン ディレレバ，古川龍彦，宮下知治，源 利
成．GSK3β成．GSK3 は STAT3 の活性化を介して膵がんのゲムシタビン耐性獲得に寄与する．第 80回日本癌学会学術総会，2021 年 9 月 30 日（木）－10 月 2 日（土），パシフィコ横浜．

7. Takeshi Sawada, Eiji Kubota, Keishi Nakamura, Naoki Takahashi, Ryosuke Ota,
Masashi Idogawa, Yasushi Sasaki, Takashi Tokino, Toshinari Minamoto, Hiromi
Kataoka. RAS, BRAF and PIK3CA mutations in circulating tumor DNA andcomparison with mutations in tissue in colorectal cancer. 澤田 武，久保田英嗣，中村慶史，高橋直樹，太田亮介，井戸川雅史，佐々木泰史，時野 隆，源 利成，片岡洋望．RAS 野
生型転移性大腸癌患者における循環腫瘍 DNA 中の RAS, BRAF, PIK3CA 変異の同定と腫瘍組織の変異との比較．第 80 回日本癌学会学術総会，2021 年 9 月 30 日（木）－10 月 2日（土），パシフィコ横浜．

8. Masaharu Hazawa, Toshinari Minamoto, Takeshi Suzuki, Richard Wong. NUP153
drives oncogenic TP63 expression through liquid-liquid phase separation mediatedgene-gating in squamous cancer. 羽澤勝治，源 利成，鈴木 健之，ウォング リチャード．
NUP153 は相分離を介した Gene-gating 機構で TP63 の発現を誘導する．第 80 回日本癌学会学術総会，2021 年 9 月 30 日（木）－10 月 2 日（土），パシフィコ横浜．

9. 堂本貴寛，上原将大，竹中 哲，ディリレバ ボリドン，古川龍彦，竹内 修，宮下知治、源 利成．
GSK3β
GSK3 は膵がんの難治性腫瘍形質を連関する．第 32 回日本消化器癌発生学会総会，
2021 年 11 月 26 日（金），27 日（土），岐阜市（WEB）．

10. 上原将大，堂本貴寛，竹中 哲，ディリレバ ボリドン，竹内 修，宮下知治，源 利成．Glycogensynthase kinase（GSK）3βkinase（GSK）3 は膵がんのゲムシタビン獲得耐性に寄与する．第 32 回日本消化器癌発生学会総会，2021 年 11 月 26 日（金），27 日（土），岐阜市（WEB）．

11. ディリレバ ボリドン，堂本貴寛，上原将大，アムラ サビット，奥村知之，遠藤良夫，中田光俊，
宮下知治，リチャード ウォング，源 利成．GSK3β
利成．GSK3 阻害による食道扁平上皮がんの治療効果とメカニズム．第 32 回日本消化器癌発生学会総会，2021 年 11 月 26 日（金），27 日（土），岐阜市（WEB）．

・司会・座長など

12. Akinobu Taketomi, Toshinari Minamoto. Basic Science 5. The 10th International
Conference of the International
International Society
Society ofof Gastroenterological
Gastroenterological Carcino-genesis
Carcinogenesis (ISGC2021), November 26 (Fri), 27 (Sat), 2021, Gifu, Japan (WEB).
O5-1. Hitoshi Masuo: Mitochondria participate in chemoresistance to gemcitabine inhuman pancreatic cancer cells.
O5-2. Takahiro Domoto, Masahiro Uehara, Satoshi Takenaka, Dilireba Bolidong,
Tatsuhiko Furusawa, Osamu Takeuchi, Tomoharu Miyashita, Toshinari Minamoto.
GSK3β
GSK3 interconnects the malignant properties in therapy-resistant pancreatic cancer.
O5-3. Yoshinaga Aoyama, Aya Naiki, Yoichi Matsuo, Aya Nagano, Masayuki Komura,
Hiroyuki Kato, Goro Ueda, Hiromichi Murase, Tomokatsu Kato, Kan Omi, Yuichi
Hayashi, Hiroyuki Imafuji, Kenta Saito, Mamoru Morimoto, Shingo Inaguma, Ryo
Ogawa, Hiroki Takahashi, Shuji Taniguchi, Satoru Takahashi. Chemopreventive effectof lactoferrin on non-alcoholic steatohepatitis and liver carcinogenesis in rats.

—

10 —

研究のあゆみと業績 2021

O5-4. Shohei Okikawa, Yuji Morine, Tetsuya Ikemoto, Yu saito, Shinichiro Yamada,katsuki Miyazaki, Chiharu Nakasu, Mitsuo Shimada. VEGF inhibition reduce TAM'smalignant potential.
O5-5. Kohei Tamura, Yuki Kaneko, Yurie Futoh, Kazuya Takahashi, Yuki Kimura,
Akira Saito, Mineyuki Tojyo, Hideyo Miyato, Hideyuki Ohzawa, Yasushi Saga, Yuji
Takei, Hiroyuki Fujiwara, Joji Kitayama. Activated neutrophiles can suppress
chemotactic migration of activated T lymphocytes.
13. 源 利成．共催セミナー．第 50 回日本消化器がん検診学会東海北陸地方会，2021 年 9 月
25 日（土），アクトシティ浜松 コングレスセンター，浜松市．
コングレスセンター，浜松市市．
松田尚久：内視鏡を大腸がん検診の modality として用いるには

14. 武冨紹信，源 利成．基礎研究 5．第 32 回日本消化器癌発生学会総会，2021 年 11 月 26 日
（金），27 日（土），岐阜市（WEB）．
O5-1. 増尾仁志：ヒト膵癌細胞株におけるゲムシタビン耐性獲得とミトコンドリアの関与．
O5-2. 堂本貴寛，上原将大，竹中 哲，ディリレバ ボリドン，古川龍彦，竹内 修，宮下知治、源利成．GSK3β
利成．GSK3 は膵がんの難治性腫瘍形質を連関する．
O5-3. 青山佳永，内木 綾，松尾洋一，永野愛矢，古村理行，加藤寛之，上田悟郎，村瀬寛之，
齊藤健太，森本 守，稲熊真悟，小川 了，高橋広城，瀧口修司，高橋 智．ラット非アル
コール性脂肪肝炎および肝発癌に対する Lactoferrin の化学予防効果．
O5-4. 沖川昌平，森根裕二，池本哲也，齋藤 裕，山田眞一郎，宮崎克己，仲須千春，島田光生．VEGF 阻害は TAM の悪性度を減じる．
O5-5. 田村昂平，金子勇貴，風當ゆりえ，高橋和也，木村有希，齋藤 晶，東條峰之，宮戸秀世，大澤英之，嵯峨 泰，竹井裕二，藤原寛行，北山丈二：活性化好中球は活性化 T
リンパ球の走化性を抑制する．
・その他（講演，社会・地域貢献を含む）
源 利成．基調講演：GSK3β
利成．基調講演：GSK3 のがん生物学とがん治療抵抗性の一隅．第 29 回日本癌病態治療研究会 パネルディスカッション：癌治療抵抗性の機序解明に基づく新たな治療法の開発，
2021 年 1 月 14 日（木），15 日（金），G メッセ群馬，高崎市．

Ⅲ．学会開催該当なしⅣ．究成果による知的財産権の出願状況
該当なし
Ⅴ．新聞，報道など
該当なし—- 11—11金沢大学がん進展制御研究所 腫瘍制御 (旧：遺伝子診断) 研究分野

【附記１】 腫瘍制御共同研究セミナーと  周年記念会：誌上開催
Division of Translational & Clinical Oncology
Cancer Research InstituteKanazawa University

2021 年 5 月 10 日(月)
ご関係の皆さま：
皆さんそれぞれにお変わりなく、元気に楽しい毎日を過ごされていることでしょう。
いつもお世話になりありがとうございます。
いまからおよそ 2 か月後の 7 月 3 日(土)に開催を予定している共同研究セミナー
2021 と、同日夕にミキサー形式で併催する「七夕の会 2021」を案内したくお便りしました。当研究分野の 20 周年と、私の研究室主任として 20 歳(はたち)の誕生日に相当します。詳細は別紙の開催案をご覧ください。それぞれ異なる領域で活躍されている４名の先生方の講演に続いて、金沢医科大学：前学長の勝田省吾先生にご挨拶をいただくプログラムを計画しています。また、時間が許せば、この 20 年間の研究室の記録や雑感などを講演の合い間に紹介いたします。参加費の暫定額をお知らせします。
常勤の方
15,000 円
それ以外の方 8,000 円＊当日はお釣りを用意しませんので、ご協力ください。

参加費

医療従事者とご高齢の方々を中心にワクチン接種が始まったとはいえ、世のなかが平穏になる兆しは見通せません。このため、皆さんへの案内どころかセミナーやミキサーの開催自体を逡巡しています。このような状況ながら、当日のご参加について皆さんのご意向、ご都合や忌憚のないご意見を、５月 31 日(月)を目途にファクスあるいはメールでご返信ください。ご返信のときに、ファクス番号と、メールを日常的にお使いであればアドレスをお知らせくださると助かります。皆さんのお返事を参考にして、6 月中旬までに当日の開催を再考し、開催あるいは中止の方針が決まりしだいお知らせいたします。あやふやな案内になってすみません。状況をご理解のうえ、ご協
力いただければありがたく思います。
それでは、皆さんがこれからもお健やかに過ごされるよう願っています。そして、
願わくは当日、多くの皆さんがご参加くださることを心待ちにしています。どうぞよ
ろしくお願いします。

金沢大学がん進展制御 研究所 腫瘍制御
金沢大学附属病院がんセンター（併任）Toshinari Minamoto, M.D., Ph.D.
Division of Translational & Clinical Oncology
Cancer Research Institute and Cancer Center
Kanazawa University and Hospital
13-1 Takara-machi, Kanazawa 920-0934, Japan☎ 076-265-2798 📠📠 076-234-4529
消滅が危惧されている千里浜海岸 📨📨 minamoto@staff.kanazawa-u.ac.jp

—

12 —

金沢大学がん進展制御研究所 腫瘍制御
金沢大学附属病院 がんセンター〒920-0934 金沢市宝町 13-1
☎ 076-265-2798 📠📠 076-234-4529
📨📨 minamoto@staff.kanazawa-u.ac.jp

研究のあゆみと業績 2021

開催延期の案内
Division of Translational & Clinical Oncology
Cancer Research InstituteKanazawa University

2021 年 5 月 31 日(月)ご関係のみなさん：
春の連休前に共同研究セミナーの開催をお知らせして、ひと月以上になります。この間（かん）にパンデミックは変容（変異）し、ワクチン接種が混乱しています。それでも皆さんそれぞれにお変わりなく、お元気に毎日を楽しまれていることでしょう。
先の案内に対して、皆さんから参加の可否とともに温かいお便りとご意見をいただ
きました。ありがとうございます。皆さんのお返事とパンデミックの現状を勘案し、
7 月 3 日（土）に開催を予定している共同研究セミナー2021 と、同日夕に併催する「七夕の会 2021」を 2021 年 11 月 27 日（土）に延期いたします。研究会のプログラムや参加費などは現状と変更はありません。なお、今回のセミナーの適時性は大切でありながら、オンライン開催がセミナーや交流会の趣旨にそぐわないことから、これ以上の延期はありません。9 月下旬をめどに当日の開催を再考し、開催あるいは中止の方針が決まりしだい、あらためてお知らせします。また、あいまいな案内になってしましました。とりあえず、11 月 27 日 (土) の午後の予定を確保していただけるとありがたく思います。
このあとも、皆さんがお健やかに過ごされるよう願っています。

金沢大学がん進展制御 研究所 腫瘍制御
金沢大学附属病院がんセンター（併任）Toshinari Minamoto, M.D., Ph.D.
Division of Translational & Clinical Oncology
Cancer Research Institute and Cancer Center
Kanazawa University and Hospital
13-1 Takara-machi, Kanazawa 920-0934, Japan☎ 076-265-2798 📠📠 076-234-4529
消滅が危惧されている千里浜海岸 📨📨 minamoto@staff.kanazawa-u.ac.jp—- 13—13

金沢大学がん進展制御研究所 腫瘍制御
金沢大学附属病院 がんセンター〒920-0934 金沢市宝町 13-1
☎ 076-265-2798 📠📠 076-234-4529
📨📨 minamoto@staff.kanazawa-u.ac.jp

金沢大学がん進展制御研究所 腫瘍制御 (旧：遺伝子診断) 研究分野

開催中止の案内
Division of Translational & Clinical Oncology
Cancer Research InstituteKanazawa University

2021 年 10 月 18 日(月)Re: 共同研究セミナー開催中止と誌上開催の案内
ご関係のみなさん：
５月末に共同研究セミナー開催の延期をお知らせして、４か月以上になり、当地では紅葉を迎えています。奇しくも東京 2020 と歩調を合わせたかのようなパンデミッ
クは休止し、世のなかは平穏になりつつあります。かりにこれが錯覚であるにしても、皆さんそれぞれにお変わりなく、お元気に毎日を楽しまれていることでしょう。
先般、ホテル日航金沢の担当の方と話しました。
そしてその内容は、11 月 27 日（土）に先延ばしした
共同研究セミナー2021 は開催できるものの、同日夕の「七夕の会 2021」はマスクをして、手酌、黙飲食
なら併催可というものでした。これでは楽しくない、
面白くない、自分が描いていたセミナーや交流会の趣旨にそぐわないという身勝手な思いから、中止という決断に至りました。当日の予定を確保していただいている皆さんにお詫びします。それでも、セミナーの発表や司会をお引き受けいただいた方々のご協力を無にしないよう、誌上開催として「研究のあゆみと業績 2021」に記録を掲載します。
このあとパンデミックがどうなろうとも、皆さんがお健やかに過ごされるよう願っています。

金沢大学がん進展制御 研究所 腫瘍制御
金沢大学附属病院がんセンター（併任）Toshinari Minamoto, M.D., Ph.D.
Division of Translational & Clinical Oncology
Cancer Research Institute and Cancer Center
Kanazawa University and Hospital
13-1 Takara-machi, Kanazawa 920-0934, Japan☎ 076-265-2798 📠📠 076-234-4529
消滅が危惧されている千里浜海岸 📨📨 minamoto@staff.kanazawa-u.ac.jp

—

14 —

金沢大学がん進展制御研究所 腫瘍制御
金沢大学附属病院 がんセンター〒920-0934 金沢市宝町 13-1
☎ 076-265-2798 📠📠 076-234-4529
📨📨 minamoto@staff.kanazawa-u.ac.jp

研究のあゆみと業績 2021

－ 誌上（仮）開催 －
進展
金沢大学がん制御研究所腫瘍制御学
共同研究セミナー2021







―七夕の会 2021―

日時：2021 年 11 月 27 日（土）13:30～19:00
場所：ホテル日航金沢〒920-0853 金沢市本町 2-15-1 電話：076-234-1111
主催：金沢大学がん進展制御研究所腫瘍制御学
共催：七夕の会

事務局： 堂本貴寛
金沢大学がん進展制御研究所腫瘍制御学
 〒920-0934 金沢市宝町 13-1
電話：076-265-2798 (9 時～16 時)
 電子メール：tdomoto@staff.kanazawa-u.ac.jp—- 15—15金沢大学がん進展制御研究所 腫瘍制御 (旧：遺伝子診断) 研究分野
進展
金沢大学がん
制 御 研究所腫瘍制御学共同研究セミナー2021

日時：2021 年 11 月 27 日（土） 13:30～19:00
場所：ホテル日航金沢 〒920-0853 金沢市本町 2-15-1 電話：076-234-1111
主催：金沢大学がん進展制御研究所腫瘍制御学
時間プログラム（発表と討論）                敬称略
受付け
13:3014:25-

開会 源 利成 金沢大学がん進展制御研究所 腫瘍制御、七夕の会

14:30-14:55 骨軟部腫瘍の臨床と研究  司 会  保下崎真吾 医療法人社団 下崎整形外科医院／腫瘍制御学  発表者  阿部健作 金沢大学整形外科学／社会医療法人財団 董仙会 恵寿総合病院 整形外科
14:55-15:20 IgG4 関連疾患の臨床と病理  司 会  松田陽子 香川大学 病理病態・生体防御医学講座 腫瘍病理学
  発表者  笠島里美 金沢大学 医薬保健研究域保健学系 病態検査学
15:20-15:45 Non-mass-forming clonal expansion に着目した病理学研究  司 会  中田光俊 金沢大学 医薬保健研究域医学系 脳・脊髄機能制御学  発表者  前田大地 金沢大学 医薬保健系研究域医学系 分子細胞病理学
15:45-15:55

リフレッシュ

15:55-16:10 この 20 年間の雑感とこれから  司 会   発表者  源 利成  金沢大学がん進展制御研究所 腫瘍制御
16:10-17:00 がん研究から志摩半島での地域医療へ  司 会  岡田保典 順天堂大学 整形外科 運動器・腫瘍性疾患病態学講座
  発表者  江角浩安 国民健康保険 志摩市民病院
17:00-17:15 Closing remarks / 閉会のことば  司 会  源 利成 金沢大学がん進展制御研究所腫瘍制御  発表者  勝田省吾 金沢医科大学 前学長
17:30- TF

ミキサー：七夕の会 2021 併催

担当：堂本貴寛 
金沢大学がん進展制御研究所腫瘍制御学   〒920-0934 金沢市宝町 13-1 電話：076-265-2786, 2798 (事務：9-16 時)
   電子メール：tdomoto@staff.kanazawa-u.ac.jp—

16 —研究のあゆみと業績 2021

【目 次】プログラム



骨軟部肉腫の臨床と研究







,J* 関連疾患の臨床と病理







Non-mass-forming clonal expansion に着目した病理学研究







この  年間の雑感とこれから







がん研究から志摩半島での地域医療へ







Closing remarks／閉会のご挨拶







誌上開催七夕の会 酒呑もおよ！

—- 17—17

18
20
22
24

26
28

金沢大学がん進展制御研究所 腫瘍制御 (旧：遺伝子診断) 研究分野

骨軟部腫瘍の臨床と研究
金沢大学整形外科学／社会医療法人財団 董仙会 恵寿総合病院 整形外科
整形外科
健作
阿部健作

阿部

「骨・軟部腫瘍」分野は，整形外科疾患の中でも、その対応、治療が生命に直接関わる重要分野であり、四肢・脊椎の解剖、機能の理解は当然であるが、画像診断、病理診
断、手術手技、化学療法・放射線療法の知識など、とりわけ集学的知識が要求される。
骨・軟部腫瘍に対しては、早期に正しい診断を行い、治療計画を経て、適切な治療（あるいは、腫瘍専門病院への紹介）を開始することが治療成績の向上に必須である 1)。
骨軟部腫瘍の中でも特に悪性腫瘍については、希少性が非常に高く、それにも関わらず組織形態は多彩であるため、新たな治療方法の導入や研究は、他のがん種と比して遅れをとっていると言わざるを得ない。特に化学療法については、近年、悪性骨軟部腫瘍分
野においてもいくつかの新たな化学療法薬や分子標的薬が導入されはしたものの、どれも既存の治療効果を上回るものではなく、この数十年，治療内容に大きな変化を認めていない。それゆえ、臨床・研究双方における大学の役割は非常に大きなものとなっている。
悪性骨軟部腫瘍に対する治療の柱は、多くの場合において、手術と化学療法である。
当教室においても、これらの研究に力を入れてきている。前者は液体窒素処理骨を用いた再建術や腫瘍脊椎骨全摘術などがあり、これらは保険収載されている。他にも臨床応用に向けて、様々な研究が取り組まれている。後者については、骨肉腫に対する新規シ
スプラチン製剤による新規治療の開発 2) や軟骨肉腫に対するザルトプロフェンによる抗腫瘍効果に関する解析 3)などがあり、臨床応用に向けて研究は続いている。その中で、
源先生ご指導のもと、我々は骨軟部肉腫に対する GSK3β を標的とした分子標的治療に関する研究を行ってきた 4, 5)。
GSK3β に関する研究では、骨肉腫においては、GSK3β 阻害薬の使用により Wnt/βcatanin 経路や NF-κB 経路を介して骨肉腫細胞の増殖を抑制していることが示唆され 4)、
滑膜肉腫と線維肉腫においては、CDK-cyclin 複合体を介した細胞周期の進行による生存・増殖と、MMP-2 を介した浸潤が、GSK3β に依存することが示唆された 5)。GSK3β 阻
害効果を持つ薬剤は実際に臨床の場に出ており、drug repositioning の形で臨床応用されることが今後、期待される。
1) 越智隆弘・吉川秀樹（2007） 『最新整形外科学大系 骨・軟部腫瘍および関連疾患』，
中山書店．
2) Igarashi K, Yamamoto N, Odani A, Tsuchiya H, et al. Effectiveness of two novel anionicand cationic platinum complexes in the treatment of osteosarcoma. Anticancer Agents
Med Chem 15(3):390-9, 2015.
3) Higuchi T, Yamamoto N, Yamamoto Y, Tsuchiya H, et al. Anti-tumor effects of anonsteroidal anti-inflammatory drug zaltoprofen on chondrosarcoma via activating
peroxisome proliferator-activated receptor gamma and suppressing matrix
metalloproteinase-2 expression. Cancer Med 7(5):1944-54, 2018.
4) Shimozaki S, Yamamoto N, Domoto T, Minamoto T, Tsuchiya H, et al. Efficacy ofglycogen synthase kinase-3β targeting against osteosarcoma via activation of β-catenin.
Oncotarget 7 (47): 77038-51, 2016.
5) Abe K, Yamamoto N, Domoto T, Minamoto T, Tsuchiya H, et al. Glycogen synthase 3β
as a potential therapeutic target in synovial sarcoma and fibrosarcoma. Cancer Sci 111(2): 429-40, 2020.
—18 —研究のあゆみと業績 2021

阿部健作（あべ けんさく）
恵寿総合病院 整形外科 医長
金沢大学 整形外科 協力研究員
学 歴：2010 年 3 月 金沢大学医学部 卒業
2020 年 6 月 金沢大学大学院医薬保健学総合研究科がん医科学専攻 機能再建学（整形外科学） 修了
2020 年度金沢大学学長賞受賞：同年 9 月 28 日

職 歴：2010 年 4 月 初期臨床研修医（金沢大学附属病院，恵寿総合病院，南砺市民病院，
木島病院）
2012 年 4 月 石川県立中央病院 整形外科 医員
2013 年 4 月 かみいち総合病院 整形外科 医員
2014 年 10 月 金沢大学附属病院 整形外科 医員／大学院生
2015 年 4 月 金沢大学 がん進展制御研究所 協力研究員 併任（〜2020 年 3 月）
2018 年 4 月 金沢赤十字病院 整形外科 医員
2019 年 4 月 浅ノ川総合病院 整形外科 医長
2020 年 4 月 恵寿総合病院 整形外科 医長
2021 年 4 月 金沢大学 整形外科 協力研究員 併任
所属学会と資格
日本整形外科学会 専門医
中部日本整形外科災害外科学会
日本癌治療学会
日本整形外科学会スポーツ医学会
日本関節鏡・膝・スポーツ整形外科学会
日本人工関節学会
その他 日本スポーツ協会認定スポーツドクター
石川県サッカー協会医事委員
ツエーゲン金沢チームドクター—- 19—19

金沢大学がん進展制御研究所 腫瘍制御 (旧：遺伝子診断) 研究分野

IgG4 関連疾患の臨床と病理金沢大学 医薬保健研究域保健学系 病態検査学

笠島里美

IgG4 関連疾患（IgG4-related disease; IgG4-RD）は、血清の IgG4 高値、組織での多数の IgG4 陽性の形質細胞浸潤と線維増生を特徴とする疾患群であり、2004 年に初めて膵
臓で報告され、以後 2000 年代には、全身多数の臓器で IgG4-RD が同時性、異時性に発生することが報告された。我々は、血管外膜肥厚の高度な炎症性腹部大動脈瘤（inflammatory abdominal aortic aneurysm; IAAA）の約半数が IgG4-RD であることを世界で初めて報告し 1)、その後も、冠動脈瘤, 胸部大動脈疾患の一部、大腿動脈瘤も
IgG4-RD が原因であることを明らかにし、血管領域での IgG4-RD の臨床病理学的特徴の解明に寄与してきた．2010 年代からは，IgG4-RD の病因として、helper T（Th）2 細胞優位の免疫状態やその反応性の制御性 TT細胞の亢進が注目され、Th2細胞の亢進が注目され、Th2関連サイトカイン(
関連サイトカイン（interleukin [IL]-4, IL-5, IL-13）、炎症制御性サイトカイン（IL-10, TGF-β）の検討が各臓
器の IgG4-RD で進められた。一方で、一般に動脈疾患では粥腫性動脈硬化症に伴うとされる炎症性サイトカインの上昇、特に IL-6 の上昇が知られる。IgG4 関連血管病変では、動脈硬化性大動脈瘤及び解剖例の正常例と比較して、血清 IL-6 が有意に上昇し、血管外膜では組織球の IL-6 産生が高度であり、動脈硬化性に関わるサイトカイン産生に加えて、“炎症性”大動脈瘤としての特徴を示した 2)。これは血管病変のない他臓器の IgG4RD と比較して特徴的な像であった。
IgG4 関連血管病変の外科的治療症例が蓄積されるに従い、血管内治療後には、動脈周囲の線維性肥厚の増悪、発熱や腹痛が術後の持続、瘤径拡大などが多い傾向があり、治療方針選択の際の問題となっている。IgG4 関連血管病変の開腹手術による人工血管置換術ではほぼ全例が完治する一方で、血管内治療例の約半数が術後 2 年以内に何らかの増悪を示した。血管内治療後に血清 IgG4 値が上昇する群は、血管径拡大、血清 IL-6、血清 matrix metalloproteinase（MMP）-9 が上昇しており、血管内治療では残存する血管壁に IgG4-RD の活動性炎症が残ることが，術後の増悪因子であると想定された 3)。IgG4 関連動脈瘤では、動脈硬化性大動脈瘤及び解剖例の正常例と比較して、組織でも外膜の MMP-2，MMP-9 産生が亢進しており、リンパ濾胞形成に関わる免疫細胞（濾胞樹状細胞）、抑制型組織球、形質細胞型樹状細胞など多彩な免疫細胞がMMPs 産生に関与していることを報告した 4)。
現在は、血管外膜のリンパ濾胞の発達が IgG4 関連血管病変の活動性の指標になるとして検討中である。また、IgG4 関連血管病変は，腹部大動脈やその分枝が好発部位であるが、高安病などの‘いわゆる大血管炎’のよく起こる胸部大動脈，頸部動脈では頻度が低い理由も長年不明であった。我々は、正常血管の自然免疫細胞の分布に注目し、IgG4 関連血管病変の局在についての解明を進めている。
1) Kasashima S, Zen Y, Kawashima A, et al. Inflammatory abdominal aortic aneurysms: close
relationship to IgG4-relared periaortitis. Am J Surg Pathol. 2008;32(2):197-204.
2) Kasashima S, Kawashima A, Zen Y, et al. Upregulated interleukins (IL-6, IL-10, and IL-13) inimmunoglobulin G4-related aortic aneurysm patients. J Vasc Surg 2018;67(4):1248-1262.
3) Kasashima S, Kasashima F, Kawashima A, et al. Clinical outcomes after endovascular and open
surgery repair of immunoglobulin G4-related or non-related inflammatory aortic aneurysms. J
Endovasc Ther 2017;24(6):833-845.
4) Kasashima S, Kawashima A, Kasashima F, et al. Adventitial matrix metalloproteinase
production and distribution of immunoglobulin G4-related abdominal aortic aneurysms. J Vasc
Surgery Vasc Sci 2020;1:151-165.
—

20 —研究のあゆみと業績 2021

笠島里美（かさしま さとみ）
金沢大学医薬保健研究域保健学系病態検査学 教授

学歴：1994 年 金沢大学医学部医学科 卒業
1998 年 金沢大学大学院医学研究科病理学課程（病理学第一）修了
職歴：
1998 年 金沢大学医学部病理学第一 協力研究員
2004 年 金沢大学附属病院病理部 医員
2007 年 国立病院機構金沢医療センター 臨床検査科科長
金沢大学がん研究所研究協力員
2017 年 金沢大学医薬保健研究域保健学系病態検査学 准教授
2021 年 金沢大学医薬保健研究域保健学系病態検査学 教授
所属学会と資格：
日本病理学会（死体解剖認定医，認定病理医，専門医研修指導医，評議員）
日本臨床細胞学会（細胞診専門医，教育研修指導医，石川県支部総会幹事）
日本臨床検査医学会（臨床検査専門医，臨床検査管理医）
IgG4 関連疾患学会（IgG4 関連心血管診断基準作成委員）—- 21—21

金沢大学がん進展制御研究所 腫瘍制御 (旧：遺伝子診断) 研究分野

Non-mass-forming clonal expansion に着目した病理学研究
金沢大学 医薬保健系研究域 分子細胞病理学

前田大地

私は、2021 年 4 月に金沢大学に着任し、分子細胞病理学分野を担当しております。本セミナーでは、私のこれまでのキャリアと研究内容を振り返りつつ、現在、特に注力しているテーマである”non-mass-forming clonal expansion”について言及したいと考えています。
私は外科病理医で、大学院時代から一貫して婦人科領域の腫瘍性疾患をテーマとし、
主に背景遺伝子異常を探索する研究を行ってきました。その間、卵巣癌の領域では、純形態学的アプローチによってその多くが卵管上皮由来であることが実証され、発生母地に関するパラダイムシフトが起こりました。具体的には卵巣 high-grade serous carcinoma
症例の多くにおいて卵管上皮内癌（先行病変）の併存が確認され、両者に共通の TP53
変異が存在することが示された訳ですが、同時に正常卵管上皮にも一定頻度で“p53signature”と呼ばれる微小変異細胞集塊が存在することが知られるようになりました。p53
signature は今まで単なる正常卵管上皮の一部として見過ごされてきた（病変と呼ぶのも憚られる）存在です。ただし、「卵管上皮細胞に TP53 変異が生じるだけでは腫瘤を形成
せず、悪性化もきたさない」ことを示唆する点が特異です。p53 signature との遭遇をきっかけに、私の興味は、正常組織中に存在する変異細胞集塊へとシフトしました。「正常卵管上皮には TP53 に限らず、様々な遺伝子変異が生じているのではないか」という仮説を立て、検証した結果、約 2 割に CTNNB1 変異細胞集塊が存在することが判明し、β-cateninsignature と名付けて報告しました。次世代シーケンスを用いた研究においても、全身諸臓器の非腫瘍部に低 allele frequency で癌関連遺伝子の変異が検出されることが明らかになってきた今、病理組織検体において変異細胞の局在を示す研究系はまずます重要になってきています。
さて後半では、non-mass-forming clonal expansion の別の例としてハンナ型間質性膀胱炎（HIC）を取り上げます。私はある一例において軽鎖制限を確認したのをきっかけに、
HIC 患者の膀胱で B-cell の clonal expansion が高頻度で起きていることを見出し、以降、継続して研究を行ってきています。HIC の組織像は単なる慢性炎症を示すもので、長期経過を見ても悪性リンパ腫は発生しません。Clonality が証明されたら「腫瘍」と言えるのか。あるいは腫瘍原性遺伝子変異が確認されたら「腫瘍性病変」なのか。従来は、臨床的に腫瘤形成性病変として捉えられ、また組織学的に均一な細胞が集塊を作ってくるよ
うな病態をもって「腫瘍」が定義されてきましたが、ポストゲノム時代に入ってその定義は揺らいできています。ただし、「腫瘍なのか非腫瘍なのか」というポイントに拘泥せず、全身諸臓器で生じている特定の細胞の clonal expansion に着目することで、HIC を含め、今まで成因不明とされていた疾患の病態解明の糸口がつかめるのではないか、と私は考えております。今後の研究の展望も含めてお話しさせていただければ深甚に存じます。
1. Maeda D, Akiyama Y, Morikawa T, Kunita A, Ota Y, Katoh H, Niimi A, Nomiya A,
Ishikawa S, Goto A, Igawa Y, Fukayama M, Homma Y. Hunner-type (classic)
interstitial cystitis: a distinct inflammatory disorder characterized by pancystitis, withfrequent expansion of clonal B-cells and epithelial denudation. PLoS One. 10 (11):e0143316, 2015.
2. Koyama K, Maeda D, Kito M, Tamura D, Kudo-Asabe Y, Katoh H, Ishikawa S, Nanjo
H, Terada Y, Goto A. Clinicopathological and molecular analyses of linearly expanded
epithelial cells with β-catenin alterations, "β-catenin signature", in the normal fallopian
tube. Histopathology. 77 (6): 880-9, 2020.
—22 —研究のあゆみと業績 2021

前田大地（まえだ だいち）
金沢大学医薬保健研究域医学系分子細胞病理学分野 教授
学   歴2004 年 3 月 東京大学医学部医学科卒業
2010 年 3 月 東京大学大学院医学系研究科博士課程修了
職   歴
2004 年 4 月 東京大学医学部附属病院初期臨床研修医
2005 年 4 月 三井記念病院初期臨床研修医
2006 年 4 月 三井記念病院病理部医員
2010 年 4 月 東京大学大学院医学系研究科人体病理学・病理診断学分野 助教
2012 年 4 月 東京大学医学部附属病院病理部 助教
2014 年 4 月 秋田大学大学院医学系研究科器官病態学講座 准教授
2018 年 4 月 大阪大学大学院医学系研究科先端ゲノム医療学共同研究講座 特任教授
2021 年 4 月 金沢大学医薬保健研究域医学系分子細胞病理学分野 教授
所属学会と資格
・日本病理学会 （専門医、学術評議員、Pathology International 刊行委員、分子病理専門医制度運営委員会委員、分子病理専門医資格審査委員会委員）
・日本婦人科病理学会 （理事）・日本臨床細胞学会
・日本間質性膀胱炎研究会
・日本婦人科腫瘍学会（代議員、卵巣がん治療ガイドライン 2020 年版評価委員）
・JSAWI (Japanese Society for the Advancement of Women’s Imaging) （世話人）
・日本癌学会—- 23—23金沢大学がん進展制御研究所 腫瘍制御 (旧：遺伝子診断) 研究分野

この 20 年間の雑感とこれから（仮題）金沢大学がん進展制御研究所

源 利成

私が 2001 年 7 月の金沢大学がん研究所教授就任を茶崖に報告してから 1)、やがて20 年が経過します。今回、本誌への寄稿依頼が届いたのは奇遇です。せっかくの機会
なので、これまでといま、そしてこれからのことについて認（したた）めます。
私は卒後すぐに、金沢の大学院で病理学を修め、在籍中の２年間は東京医科歯科大学難治疾患研究所に出向しました。この時期にこれまでで唯一、順天堂と交流しました。
その後は 2008 年まで金沢大学がん研究所と附属病院（2001 年に金沢大学病院に統合）
で消化器・腫瘍外科の修練と研究に従事しました。途中、国立がんセンター研究所と米国
マントサイナイ医科大学に出向した期間のほかは、およそ 15 年間に消化器や乳腺、甲状腺のがんを中心に 400 数十人の患者さんを執刀する機会に恵まれました。1998 年春から現在の研究室を併任し始めましたので、2008 年 7 月のがん研外科診療科の廃止までの10 年間は外科とがん研究の掛けもちでした。それ以降は現在まで、消化器がんや難治・
希少がんの橋渡し研究に軸足を移し、大学病院がんセンターで診療を続けています。
外科の恩師からはつぎのような教えを受けました。それは「手術は知識、技量、経験を遺憾なく発揮すべき場であり、外科医を志す医師がその習熟と向上に努力すべきことは言うまでもない。日常診療で外科医が患者さんを診察し、手術し、社会復帰させるまでの一連の過程には、患者さんと医師との信頼関係が最も重要であり、単に手術を行うという技術的な面だけでなく、外科医としての情熱と人間性が要求される。」というものです。また、研究が少し軌道に乗りかけたころ、米国研究者や米国癌学会とトラブルがあったとき、国立がんセンターの恩師から、つぎのような手紙が届きました。その内容は「国際的－international－ということは、各国の歴史、文化が確認して成り立っている美名です。中性の水のようなものではなく、国際化という言葉は商売の駆け引きや民族の勢力争いのとき、最もしばしば使われます。パスツールが云った科学には国境がなくとも、科学者には国境がある、ということです。日本人は人が好いので、結局、西洋人に手玉にとられるのです。
西洋にはない仕事を早くして、早く発表することが唯一無二の方策です。元気を出して、日本の科学のためにご健闘ください。」という、叱咤です。これまでの仕事とこれらの助言を踏まえ、臨床と研究に関する自身の考えを駄文にまとめました 2)。ご関心があれば、私か茶崖編集部に電子版別刷りをご請求ください。
人生のおよそ３分の２におよぶ期間、外科、研究ともに大講座ではなく、一貫して小さなグループで活動してきました。それでも多くの仲間に出会い（次頁の写真はそのお一人の人事のお祝いです）、いただいた公的・民間の研究資金は 8 億円を超えています 3)。
ありがたいことと国民や仲間のみなさんに感謝しながら、大学で残された３年間をこれからどうしようかと思案しています。
1) 源 利成．腫瘍外科医の遺伝子診断事始め．茶崖 105: 6-9, 2002.
2) 源 利成．巻頭言：研究力と臨床力 Integrative biomedical research and clinical activity．
金沢大学十全医会誌 124 (2): 19, 2015.
3) 金沢大がん研腫瘍制御 HP：http://ganken.cri.kanazawau.ac.jp/shuyoseigyo/index.html※本文は、順天堂大学同窓会誌：茶崖（さがい）第 181 号（2021 年 4 月）の寄稿文から転載

—

24 —

研究のあゆみと業績 2021

源 利成（みなもと としなり）金沢大学がん進展制御研究所 腫瘍制御研究分野 教授
金沢大学附属病院がんセンター 併任
学 歴：1983 年 3 月 順天堂大学医学部 卒業
1987 年 3 月 金沢大学大学院医学研究科（病理学第一）修了
職 歴：1987 年 4 月 金沢大学がん研究所附属病院外科 医員
1990 年 1 月 富山逓信病院外科 勤務
1991 年 1 月 金沢大学がん研究所外科部 / 同附属病院外科 助手
1992 年 4 月 国立がんセンター研究所生化学部 研究員
1994 年 4 月 金沢大学がん研究所外科部 / 同附属病院外科 助手（復職）1995 年 11 月 American Health Foundation, Naylor Dana 疾病予防研究所 博士研究員
1997 年 1 月 ニューヨーク市立大学マントサイナイ医学校がんセンター 客員講師
1997 年 11 月 金沢大学がん研究所腫瘍外科 / 同附属病院腫瘍外科 助手（復職）1998 年 4 月 金沢大学がん研究所腫瘍制御（旧：遺伝子診断）助教授
金沢大学がん研究所附属病院腫瘍外科 助教授（併任）2001 年 7 月 金沢大学がん研究所腫瘍制御（旧：遺伝子診断）教授
金沢大学附属病院腫瘍外科（現：がんセンター）教授（併任）
所属学会と資格
基礎系 日本癌学会（評議員）
日本消化器癌発生学会（理事，評議員）
米国癌学会（AACR）：Corresponding Member
国際消化器発がん学会（理事）日本病理学会（死体解剖認定医：病理解剖），ほか
橋渡系 日本癌病態治療研究会（世話人）
日本分子腫瘍マーカー研究会（世話人）
臨床系 日本外科学会（指導医 2017 年まで，専門医 [認定登録医]）
日本消化器外科学会（指導医，専門医（~2015 年）[認定登録医]，認定医）
日本消化器内視鏡学会（本部評議員，指導医，専門医）
日本消化器病学会（本部学術評議員，指導医，専門医）
日本消化器がん検診学会（代議員，東海北陸支部幹事，指導医，認定医）
日本癌治療学会
日本胃癌学会
大腸癌研究会，ほか
その他 日本がん治療認定医機構 暫定教育医
日本消化器外科学会 消化器がん外科治療認定医
日本がん治療認定医機構 がん治療認定医
石川県予防医学協会：集検事業管理指導委員会 胃がん部会委員，大腸がん部会委員長
石川県成人病予防センター：大腸集団検診指導委員会委員
石川県教育委員会ｽｰﾊﾟｰｻｲｴﾝｽﾊｲｽｸｰﾙ（SSH）事業 運営指導委員，ほか—- 25—25

金沢大学がん進展制御研究所 腫瘍制御 (旧：遺伝子診断) 研究分野

源 利成 教授 就任 20 周年に寄せて
金沢医科大学 元学長

勝田省吾

この度、源 利成先生が教授就任 20 周年を迎えられたことを心からお祝い申し上げます。誠におめでとうございます。
金沢大学医学部第一病理学教室（現分子細胞病理学）の同門の一人として、第一病理と源先生との思い出などを述べたいと思います。
１．第一病理学教室で学んだ「厳しさ」と「競合と調和」
私は臨床の教室に入る前に病理学を学びたいと考え、医学部を卒業後、大学院医学研究科病理学第一に進学しました。病理学第一講座を主宰されていたのは、当時では先端的技術であった電子顕微鏡を用いたコラーゲン線維や結合組織の研究で有名な梶川欽一郎教授でした。私は実験的動脈硬化症の超微構造的研究で学位を取得しました。
学位論文の最終稿が出来た時、教授室で梶川先生から教室に残るよう声をかけられ、その場で「はい」と答えたことが懐かしく思い出されます。梶川先生の学問に対して妥協を許さない厳しいご指導は、私の心に一本のしっかりした「芯」を作って下さいました。
1983 年 4 月、梶川教授の後を中西功夫教授が引き継がれ、私は助教授として 11 年
間、仕事をさせて頂きました。中西教授は従来の超微構造形態学に加えて、生化学、細胞生物学、分子生物学や遺伝子解析など新しい研究手法を取り入れたダイナミックな先
端的医学研究を指向されました。そして、スタッフ個人の研究意欲と素質を大切にしたオーケストラ型の教室を目指され、「競合と調和」を合い言葉に教室運営を心がけられました。これまで全国でご活躍の旧第一病理の皆さんの心には、梶川先生と中西先生のDNA がしっかりと受け継がれていると思っております。
２．源 利成先生のご活躍は病理学第一の誇り
利成先生の活躍は病理学第一の誇り
源先生は 1983 年 4 月、中西功夫教授にとって最初の大学院生として病理学第一に入学されました。中西教授の期待を背負って、翌年 4 月より結合組織の生化学的研究の第一人者である東京医科歯科大学難治疾患研究所の永井 裕教授の研究室に 2 年間出向し、結合組織、特にコラーゲンの生化学的研究に従事し、抗コラーゲン抗体の精製など多くの成果を挙げられました。源先生の修得された生化学的研究手法は、その後の第一病理の発展に大きく貢献しました。私自身も源先生が第一病理に導入された生化学
第一病理の発展に大きく貢献されました。私自身も源先生が第一病理に導入された生化
的
研研
究究
のの
恩恩
恵恵
をを
受受
けけ
たた
一一
人人
でで
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ルで あ る 、Arteriosclerosis, Thrombosis and Vascular Biology に動脈硬化病変における型別コラー
ゲンの局在を発表することが出来ました。また、培養平滑筋細胞のコラーゲン合成に関する研究も進めることが出来、私達の研究に新しい分野を開いていただきました。
源先生は 1987 年 4 月、金沢大学がん研究所外科の磨伊正義教授の下に移籍されました。研究と臨床の分野で多くの実績を残され、2001
年 7 月金沢大学がん研究所の教授にた。研究と臨床の分野で多くの素晴らしい実績を残され、2001
年 7 月金沢大学がん研究就任されました。先生は教授就任後、毎年、年報「研究のあゆみと業績」を発行され、教室所の教授に就任されました。先生は教授就任後、毎年、年報「研究のあゆみと業績」を発行の活動状況を届けて下さいました。学内外のグループとの共同研究、研究業績、外部資金され、教室の活動状況を届けて下さいました。学内外のグループとの共同研究、研究業績、の獲得、特許出願など着実に成果を挙げておられ、教室の発展ぶりが分かり、第一病理の外部資金の獲得状況など着実に成果を挙げておられ、教室の発展ぶりが分かり、第一病同門の一人して大変嬉しく思っておりました。また、毎年「七夕の会」の開催など、いろいろ
理の同門の一人して大変嬉しく思っておりました。また、毎年「七夕の会」の開催など、いろな機会を通して、仲間を大切に絆を深めておられる源先生のご努力に敬意を表します。
いろな機会を通して、仲間を大切に絆を深めておられる源先生のご努力に敬意を表します。
今後とも源先生の主な研究テーマ「Wnt 経路抑制因子である glycogen synthase kinase（GSK）3β 阻害によるがん治療法の研究、開発」を発展させ、がん治療に繋がる成果を挙阻害によるがん治療法の研究、開発」を発展させ、がん治療に繋がる素晴らしい成果を挙げられることを心より祈念しております。
げられることを心より祈念しております。
—

26 —研究のあゆみと業績 2021

勝田省吾（かつだ しょうご）
金沢医科大学名誉教授、元学長
学 歴：1971 年 9 月 金沢大学医学部卒業
1976 年 3 月 金沢大学大学院医学研究科博士課程修了
略 歴：1976 年 4 月 金沢大学医学部助手（病理学第一）1977 年 11 月 金沢大学医学部講師（病理学第一）1979 年 4 月 金沢大学医療技術短期大学部助教授
1983 年 7 月 金沢大学医学部助教授（病理学第一）1989 年
年 55 月
月 文部省在外研究員（米国ワシントン大学病理学教室）
文部省在外研究員（米国ワシントン大学病理学教室）
1989 年 1 月 米国ワシントン大学 Research Fellow（～1990 年 6 月）
1994 年 7 月 金沢医科大学教授（病理学Ⅱ）2002 年 4 月 金沢医科大学副学長（～2004 年 8 月）
2007 年 9 月 金沢医科大学学長補佐（～2010 年 8 月）
2008 年 4 月 金沢医科大学国際交流センター長（～2010 年 8 月）
2010 年 9 月 金沢医科大学学長（～2016 年 8 月）
2013 年 9 月 金沢医科大学一般教育機構長（～2015 年 8 月）
称号・叙勲：
2014 年 10 月 ロシア・ヤロスラブリ医科大学名誉博士
2020 年 11 月 瑞宝中授章受章
所属学会：
2011 年 4 月 日本動脈硬化学会功労会員
2012 年 4 月 日本病理学会功労会員
主な原著論文（５編）：
1. Ross R, Masuda J, Raines EW, Gown AM, Katsuda S, Sasahara M, Malden T, Masuko
MasakoH, Sato H: Localization of PDGF-B
PDGF-β protein in macrophages in all phases ofatherosclerosis. Science 248: 1009-1012, 1990.
2. Katsuda S, Okada Y, Minamoto T, Oda Y, Matsui Y, Nakanishi I: Collagens in humanatherosclerosis: Immunohistochemical
immunohistochemical analysis using collagen type specific antibodies.
Arterioscler
Anterioscler Thromb
Thromb Vasc
Vasc Biol
Biol 12:
12: 494-502,
494-502, 1992.
1992.
3. Katsuda S, Boyd HC, Flinger BC, Ross R, Gown AM: Human atherosclerosis Ⅲ.
Immunocytochemical
Immuno-cytochemicalanalysis
analysisofofthe
thecellcellcomposition
compositionofoflesions
lesionsofofyoung
youngadults.
adults.Am
AmJ
PatholJ Pathol140:
140:907-914,1992.
907-914,1992.
4. Katsuda S, Coltrera MC, Ross R, Gown AM: Human atherosclerosis Ⅳ. Immunocytochemical analysis of cell activation and proliferation in lesions of young adults.
Am J Pathol 142: 1787-1793, 1993.
5. Katsuda S, Okada Y, Okada Y, Imai K, Nakanishi I : Matrix metalloproteinase-9 (92gelatinate/type Ⅳ collagenase equals gelatinase B)β) can degrade arterial elastin. Am
kd gelatinase/type
J Pathol 145: 1208-1218, 1994.
著書
勝田省吾 「金沢医科大学 学生の自信と誇りを育む」 金沢医科大学出版局 2019 年
3 月 15 日発行—- 27—27

金沢大学がん進展制御研究所 腫瘍制御 (旧：遺伝子診断) 研究分野

【誌上開催：七夕の会】

出典： http://kataoka-tsurutaro.com/products/detail.php?product_id=5—28 —研究のあゆみと業績 2021

【附記２】 金沢大がん研 News Letter（抜粋）：共同研究者紹介

金沢大学がん進展制御研究所
Cancer Research Institute Kanazawa University

Vol.15 October 2021

Contents
所長挨拶

クラウドファンディングに挑戦しました！
シンポジウム・研究会の開催
共同研究者の紹介
香川大学医学部

松田

陽子

教授

金沢大学がん進展制御研究所

源

利成

教授

善岡

克次

教授

神村

栄吉

先生

高校生へ向けて研究紹介
シグナル伝達研究分野
特別寄稿 宝町から角間へ移転10年

令和３年度

共同研究採択課題一覧

これまでの受賞・表彰／論文業績
少し足を延ばして石川歴史探訪

01
02
05
07
09
11
12
13
14

写

真：金沢城玉泉院丸庭園（左）［ 写真提供：石川県観光連盟］、金沢駅鼓門1（ 右）「写真提供：金沢市」
研究画像：Lgr5遺伝子陽性のマウス胃正常組織幹細胞（左）と胃がん幹細胞（右）に発現するSox9遺伝子（青；細胞核、緑；Lgr5遺伝子、赤；Sox9遺伝子）［提供：村上和弘助教］※金沢大学がん進展制御研究所共同利用・共同研究拠点の承諾を得て転載。

—- 29—29金沢大学がん進展制御研究所 腫瘍制御 (旧：遺伝子診断) 研究分野

共同研究者の紹介
教授

松田

松田教授と源教授は2021年度

陽子

MATSUDAYOKO

香川大学医学部

共同研 究からはじまった
源先生との出会い

腫瘍病理学

金沢大学がん進展制御研究所腫瘍制御研究分野の源 利成教授との共同研究のご縁で、このような執筆の機会
いしわたをいただき厚く御礼申しあげます。私は日本医科大学病理学教室にて、石渡俊行准教授とともに膵癌の新規治療薬
開 発に関する研 究を行っておりました時に、金 沢 大 学がん進 展 制 御 研 究 所の共同研 究の公 募のお話を耳に挟み、ホームページを拝見した際に、膵臓癌の研究に関する源 利成教授の研究成果に大きな感銘を受けました。当たって砕ける覚悟で源先生にご連絡差し上げたところ、直ぐに温かいお返事をいただき、また私の研究を直接ご相談させていただく機会をいただきました。その後、毎年の共同研究の申請や金沢での成果発表会、及び日本癌学会で多大なご指導をいただきましたこと、この場をお借りして深謝申し上げます。
2 0 1 5 年に私が東 京 都 健 康 長 寿 医 療センターに移りましてからも、源 先 生との共同研 究を継 続させていただきました。2018年には、東京都健康長寿医療センター第4回 老年病理学研究セミナーにて、「GSK3βとがん生物学」について源先生のご講演を賜りました。講演会では、小さな研究から積み重ねて大きな成果を獲得した経緯や、基礎研究か
ら臨床に還元する方法を具体的にご教示いただきました。
また、源先生の様々な研究が一見、別々のように見えていても、最終的には一つの方向性を持つ大きな研究を実践されていることに、感銘を受けると同時に、私自身の研究の道筋が開けたことを感じました。2019年に香川大学腫瘍病理学の教授に就任することになった際には、源先生からお祝いと激 励のお言 葉をいただきました（ 写 真 ）。香川大 学でも、源 先 生から多 大なご指 導、
ご支 援をいただき、膵 癌、大 腸癌、胃癌の病理研究を継続させていただいております。
源先生は毎年、「七夕の会」という研究会を主宰され、さらに年次研究成果報告集を刊行されております。そのたびに、私は叱咤激励を受け、いつか源先生のような研究者になりたいと改めて思います。そのため、私も年に一度の研究会を香川で立ち上げました。今後も自分にできることを少しずつ着実に進め、癌の予後改善に少しでも貢献できるよう、
病理医の立場からの研究に邁進する所存です。
源 先 生との共 同 研 究 から始まった様々な成 果は、源 先 生をはじめ、教 室の皆 様、金 沢 大 学がん
進 展 制 御 研 究 所の皆 様 、そして共 同 研 究として
御 採 択いただいたお蔭です。
この場を借りて心より感 謝申し上げるとともに、
これまでの御 恩を論 文
等でお返ししていきたいと思います。最 後になりましたが、貴 研 究 所 並びに皆 様の益々のご 発 展を
心より祈 念しております 。今 後とも御 指 導・御 鞭 撻の程何卒お願い申し上げます。
2019年2月金沢にて

07

Vol.15 October 2021

—

30 —

研究のあゆみと業績 2021採択課題で共同研究をすすめています。
教

授

源

利成

MINAMOTO TOSHINARI

金沢大学がん進展制御研究所

松 田 陽 子さんとの８年 間腫瘍制御研究分野

第24回日本消化器癌発生学会総会（2013年9月5日、6日：石川県立音楽堂）の開催準備にかまけていた2013年3月
11日、当時は日本医科大学病理学教室の講師を務められていた松田陽子さんから丁寧なメールが届きました。それは、膵がんの悪性形質に関する共同研究の提案でした。当研究所の共同研究の募集が始まって間もないころで、はじめてみずしらずの研究者から共同研究の提案が来たわけです。それまでは付きあいの狭かった（いまも狭い）、
しかも不愛想な私には新鮮なできごとでした。そして、共同研究課題の採択後の同年6月13日、
日本医科大学病理学教室で初めて松田さんに会いました。私自身、大学院で病理学を専攻したこともあって、とても初対面とは思えないほど打ち解
けて共同研究の相談や雑談をして、つぎの長津田の東工大へ向かうまでの小一時間があっという間に過ぎました。9月に金沢で担当した学会（上記）に松田さんが参加してくださり、とても楽しい思い出になりました。
いしわた

翌2014年に松田さんは教室の上司であった石渡俊行氏とともに、東京都健康長寿医療センター病理診断科に異動され、私どもとの共同研究を継続してくださいました。
これがご縁になって、当研究分野の開設から満15年にあたる
2016年7月9日に開催した共同研究セミナー2016で松田さんに講演していただき（写真）、併催した七夕の会2016で親睦を深めました。
ここからさらに交流が続き、私は2018年4月に同センター研究所の協力（特任）研究員を委嘱され、10月12日に老年病理学研究セミナーで講演の機会をいただきました。その半年後、2019年2月27日の共同研究成果
報告会に演者として参加されたとき、松田さんが香川大学病理学教室の教授に内定したとの朗報がありました。仲間の活躍は手放しで嬉しいものです。夕刻、居酒屋で少人数の宴席ではあったものの、ささやかなお祝いをしました。いまのパンデミックが始まるちょうど１年前でした。
松田さんとの出会いをきっかけに、山梨大学、千葉大学、久留米大学、埼玉医科大学、札幌医科大学、名古屋市立大学、鶴見大学から続々と共同研究の提案が届いています。松田さんと出会ってからこの８年間、私には思いもかけな
かったことばかりです。不愛想で人づきあいの悪い私の狭い料簡と視野を見開かせてくれた松田さんは、いまでは私にはかけがえのない仲間のひとりです。

腫瘍制御研究分野開設15周年共同研究セミナー 2016年7月9日（土） 於：ホテル日航金沢

Vol.15 October 2021—

- 31—3108金沢大学がん進展制御研究所 腫瘍制御 (旧：遺伝子診断) 研究分野

【附記３】 学位論文： 2021 年３月修了
Nakanishi H, Sawada T, Kaizaki Y, Ota R, Suzuki H, Yamamoto E,
Aoki H, Eizuka M, Hasatani K, Takahashi N, Inagaki S, Ebi M, Kato H,
Kubota E, Kataoka H, Takahashi S, Tokino T, Minamoto T, Sugai T,
Sasaki Y. Significance of gene mutations in Wnt signaling pathway in
traditional serrated adenomas of the colon and rectum. PLoS One 15(2): e0229262, 2020. doi: 10.1371/journal.pone.0229262.
Recent studies have shown that colorectal serrated lesions, which include sessile serrated
adenomas (SSAs) and traditional serrated adenomas (TSAs), are precursors of colorectal
cancer. However, the molecular mechanisms underlying the carcinogenesis, particularly in
TSAs, remain largely uncharacterized. To clarify their molecular and clinicopathologicalcharacteristics, we performed mutation and methylation analyses of cancer-associated genes in
78 serrated lesions, including TSAs, SSAs and microvesicular hyperplastic polyps. Target exon
sequence analysis was performed with 39 genes, including genes known to be frequently
mutated in colorectal cancers and/or serrated lesions. We also used bisulfite pyrosequencing toassess the methylation status of various cancer-associated genes and marker genes of the CpG
island methylator phenotype (CIMP). The prevalence of mutations in genes associated with
Wnt signaling was significantly higher in TSAs than SSAs (65% vs. 28%, p<0.01). Among
those, RNF43 mutations were observed in 38% of TSAs and 17% of SSAs. In
immunohistochemical studies of 39 serrated lesions, the prevalence of abnormal nuclear βcatenin accumulation was significantly higher in TSAs (57%) than SSAs (8%) (P=0.01).
SMOC1 methylation was detected in 54% of TSAs but in no SSAs (p<0.01). Additionally,
SMOC1 methylation was more prevalent among TSAs with KRAS mutation (82%) than with
BRAF mutation (38%, p=0.03). Lesions with CIMP-high or RNF43 mutations were detected
only in TSAs with BRAF mutation, suggesting two distinct carcinogenic pathways in TSAs.
Mutations in genes associated with Wnt signaling play a greater role in the carcinogenesis of
TSAs than SSAs.

Uehara M, Domoto T, Takenaka S, Bolidong D, Takeuchi O, Miyashita
Glycogen synthase
synthase kinasekinase 33β partici-pates
participates in acquired
T, Minamoto T. Glycogenresistance to gemcitabine in pancreatic cancer. Cancer Sci 111 (12):4405-16, 2020. doi: 10.1111/cas.14668
Acquisition of resistance to gemcitabine is a challenging clinical and biologicalhallmark property of refractory pancreatic cancer. Here, we investigated whether
glycogen synthase kinase (GSK)-3β, an emerging therapeutic target in various cancer types, ismechanistically involved in acquired resistance to gemcitabine in human pancreatic cancer.
This study included 3 gemcitabine-sensitive BxPC-3 cell-derived clones (BxG30, BxG140,
BxG400) that acquired stepwise resistance to gemcitabine and overexpressed ribonucleotide
reductase (RR)M1. Treatment with GSK3β-specific inhibitor alone attenuated the viability andproliferation of the gemcitabine-resistant clones, while synergistically enhancing the efficacy of
gemcitabine against these clones and their xenograft tumors in rodents. The gemcitabineresensitizing effect of GSK3β inhibition was associated with decreased expression of RRM1,
reduced phosphorylation of Rb protein, and restored binding of Rb to the E2 transcription
factor (E2F)1. This was followed by decreased E2F1 transcriptional activity, which ultimately
suppressed the expression of E2F1 transcriptional targets including RRM1, CCND1 encoding
cyclin D1, thymidylate synthase, and thymidine kinase 1. These results suggested that GSK3β
participates in the acquisition of gemcitabine resistance by pancreatic cancer cells via
impairment of the functional interaction between Rb tumor suppressor protein and E2F1 prooncogenic transcription factor, thereby highlighting GSK3β as a promising target in refractorypancreatic cancer. By providing insight into the molecular mechanism of gemcitabine
resistance, this study identified a potentially novel strategy for pancreatic cancer chemotherapy.
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32 —研究のあゆみと業績 2021

RESEARCH ARTICLE

Significance of gene mutations in the Wnt
signaling pathway in traditional serrated
adenomas of the colon and rectum
Hiroyoshi Nakanishi1☯, Takeshi Sawada ID1,2☯*, Yasuharu Kaizaki3, Ryosuke Ota1,
Hiromu Suzuki4, Eiichiro Yamamoto4, Hironori Aoki4, Makoto Eizuka5, Kenkei Hasatani6,
Naoki Takahashi7, Satoko Inagaki8, Masahide Ebi9, Hiroyuki Kato10, Eiji Kubota2,
Hiromi Kataoka2, Satoru Takahashi10, Takashi Tokino11, Toshinari Minamoto1,
Tamotsu Sugai5, Yasushi Sasaki ID12*

a1111111111
a1111111111
a1111111111
a1111111111
a1111111111

OPEN ACCESS
Citation: Nakanishi H, Sawada T, Kaizaki Y, Ota R,
Suzuki H, Yamamoto E, et al. (2020) Significanceof gene mutations in the Wnt signaling pathway in
traditional serrated adenomas of the colon andrectum. PLoS ONE 15(2): e0229262. https://doi.
org/10.1371/journal.pone.0229262

1 Division of Translational and Clinical Oncology, Cancer Research Institute, Kanazawa University,
Kanazawa, Japan, 2 Department of Gastroenterology and Metabolism, Nagoya City University Graduate
School of Medical Sciences, Nagoya, Japan, 3 Department of Pathology, Fukui Prefectural Hospital, Fukui,Japan, 4 Department of Molecular Biology, Sapporo Medical University School of Medicine, Sapporo, Japan,
5 Department of Molecular Diagnostic Pathology, Iwate Medical University School of Medicine, Morioka,
Japan, 6 Department of Gastroenterology, Fukui Prefectural Hospital, Fukui, Japan, 7 Department of
Gastroenterology, Saitama Cancer Center, Saitama, Japan, 8 Department of Advanced Research inCommunity Medicine, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Japan,
9 Department of Gastroenterology, Aichi Medical University, Nagakute, Japan, 10 Department ofExperimental Pathology and Tumor Biology, Nagoya City University Graduate School of Medical Sciences,
Nagoya, Japan, 11 Department of Medical Genome Sciences, Research Institute for Frontier Medicine,
Sapporo Medical University, Sapporo, Japan, 12 Division of Biology, Department of Liberal Arts andSciences, Center for Medical Education, Sapporo Medical University, Sapporo, Japan☯ These authors contributed equally to this work.
* takeshisawada@icloud.com (TS); yasushi@sapmed.ac.jp (YS)

Editor: Chunming Liu, University of Kentucky,
UNITED STATESAbstract

Received: October 24, 2019

Recent studies have shown that colorectal serrated lesions, which include sessile serrated
adenomas (SSAs) and traditional serrated adenomas (TSAs), are precursors of colorectal
cancer. However, the molecular mechanisms underlying the carcinogenesis, particularly in
TSAs, remain largely uncharacterized. To clarify their molecular and clinicopathologicalcharacteristics, we performed mutation and methylation analyses of cancer-associatedgenes in 78 serrated lesions, including TSAs, SSAs and microvesicular hyperplastic polyps.
Target exon sequence analysis was performed with 39 genes, including genes known to be
frequently mutated in colorectal cancers and/or serrated lesions. We also used bisulfite pyrosequencing to assess the methylation status of various cancer-associated genes andmarker genes of the CpG island methylator phenotype (CIMP). The prevalence of mutations
in genes associated with Wnt signaling was significantly higher in TSAs than SSAs (65% vs.
28%, p < 0.01). Among those, RNF43 mutations were observed in 38% of TSAs and 17% of
SSAs. In immunohistochemical studies of 39 serrated lesions, the prevalence of abnormal
nuclear β-catenin accumulation was significantly higher in TSAs (57%) than SSAs (8%)(P = 0.01). SMOC1 methylation was detected in 54% of TSAs but in no SSAs (p < 0.01).
Additionally, SMOC1 methylation was more prevalent among TSAs with KRAS mutation(82%) than with BRAF mutation (38%, p = 0.03). Lesions with CIMP-high or RNF43 mutations were detected only in TSAs with BRAF mutation, suggesting two distinct carcinogenic

Accepted: February 2, 2020
Published: February 24, 2020
Copyright: © 2020 Nakanishi et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which
permits unrestricted use, distribution, andreproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: Sequence data has
been deposited at the Japanese Genotypephenotype Archive (JGA, http://trace.ddbj.nig.ac.jp/jga), which is hosted by the DNA DataBank ofJapan (DDBJ), under accession number
JGAS00000000217.
Funding: This study was supported in part by
JSPS KAKENHI Grant Numbers JP16K09304 (TS),JP17K09374 (SI), and JP19K08367 (RO), Grantin-Aid from the Japanese Foundation for Researchand Promotion of Endoscopy (TS), Extramural

PLOS ONE | https://doi.org/10.1371/journal.pone.0229262 February 24, 2020

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金沢大学がん進展制御研究所 腫瘍制御 (旧：遺伝子診断) 研究分野

Wnt signaling in colorectal serrated lesions

Collaborative Research Grant of Cancer ResearchInstitute, Kanazawa University (TS), and a Grant-inAid from the Takeda Science Foundation (TS).
There was no additional external funding received
for this study. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing interests: The authors have declaredthat no competing interests exist.
Abbreviations: A, ascending colon; C, cecum;
CGH, comparative genomic hybridization; CIMP,
CpG island methylator phenotype; CIMP-H, CpG
island methylator phenotype-high; CIN,
chromosomal instability; CNV, copy number
variation; CRC, colorectal cancer; D, descending
colon; FFPE, formalin-fixed, paraffin-embedded;
IGV, Integrative Genomics Viewer; InDel, insertionand deletion; LINE, long interspersed nucleotide
elements; MSI, microsatellite instability; MSS,
microsatellite stable; MVHP, microvesicular
hyperplastic polyp; R, rectum; S, sigmoid colon;
SNV, single nucleotide variant; SSA, sessile
serrated adenoma; T, transverse colon; TSA,
traditional serrated adenoma.

pathways in TSAs. Mutations in genes associated with Wnt signaling play a greater role inthe carcinogenesis of TSAs than SSAs.

Introduction
Colorectal cancer (CRC) is a major cause of cancer-related death worldwide. Recent molecularpathological studies, including The Cancer Genome Atlas project, have shown that CRCs areheterogeneous diseases that arise via different molecular pathways [1,2]. Most (80–85%) sporadic CRCs are classified as non-hypermutated or microsatellite stable (MSS) tumors anddevelop through accumulation of multiple genetic and epigenetic alterations [1,2], including
mutation of oncogenes and tumor suppressor genes, as well as chromosomal instability [3,4].
The remaining 15–20% of sporadic CRCs are classified as hypermutated tumors and mainly
exhibit microsatellite instability (MSI) and concurrent hypermethylation in multiple loci [2],which is referred to as the CpG island methylator phenotype (CIMP) and is closely associatedwith BRAF mutation [5–7].
Recent studies of the molecular and clinicopathological characteristics of colorectal premalignant lesions have provided insight into the pathogenesis of CRCs as well as clues to prevention and treatment [8–10]. Since establishing the pathological classification of serrated
colorectal lesions as hyperplastic polyps, traditional serrated adenomas (TSAs) or sessile serrated adenomas (SSAs) [11–13], a number of studies have demonstrated that SSAs are associated with BRAF mutation and CIMP, and that they are precursors of MSI-positive CRCs,
which are frequently located in the proximal colon [6,9,10,14–17]. However, less is known
about the biological and clinical characteristics of TSAs, although they are also considered to
be premalignant lesions and reportedly exhibit BRAF or KRAS mutations and aberrant DNA
methylation [16–21]. In addition, several recent studies have reported PTPRK-RSPO3 fusionand somatic mutations of RNF43 in TSAs [22–24]. Because only a small number of analyses
have investigated gene mutations in serrated lesions [23,25,26], the molecular mechanisms
underlying carcinogenesis, especially in TSAs, are still not well characterized. Although it has
been suggested based on immunohistochemical studies that dysregulation of the Wnt signaling pathway contributes to carcinogenesis in serrated lesions [20,21,23,27–29], mutation ofindividual gene within this pathway have not been investigated.
To clarify the molecular and clinicopathological characteristics of colorectal serrated
lesions, we assessed the mutation of genes associated with Wnt signaling as well as other genes
reportedly mutated in serrated lesions [26] and advanced CRCs [2]. We also investigatedmutations in genes associated with oncogene-induced senescence, which have been reported
as germline mutations in patients with multiple SSAs [30], and we performed immunohistochemical studies of β-catenin expression to assess activation of the Wnt signaling pathway.
Finally, we investigated DNA methylation of cancer-associated genes including SMOC1 as
well as CIMP marker genes in TSAs because it was reported that SMOC1 is specifically methylated in TSAs [31].

Materials and methods
Patients and tissue samples
Specimens of colorectal serrated lesions (n = 78) were obtained from 78 Japanese patients
who underwent endoscopic mucosal resection at Nagoya City University Hospital, Fukui
Prefectural Hospital, or Komatsu Municipal Hospital. This study was approved by the

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Institutional Review Board at each hospital as well as Kanazawa University and Sapporo Medical University.

Endoscopic analysis
High-resolution magnifying endoscopes (CF260AZI; Olympus, Tokyo, Japan) were used for
all colonoscopic examinations. The morphology of colorectal lesions was determined according to the Paris classification [32]. All lesions detected during colonoscopy were observed athigh magnification using indigo carmine dye, after which samples were collected throughendoscopic mucosal resection for histological analysis. Tumor locations were defined as proximal colon (cecum, ascending colon, transverse colon) or distal colon (descending colon, sigmoid colon, and rectum).

Histological analysis
Histological diagnosis of tumors was done at each facility, after which the histological findings
for all specimens were reviewed by a board certified pathologist (Sugai T) who was blinded tothe clinical and molecular information. Serrated lesions, including microvesicular hyperplastic
polyps (MVHPs), SSAs and TSAs, were classified according to WHO classification criteria[33]. Mixed serrated lesion composed of TSA and tubulovillous adenoma was classified as TSA
in our analyses. The clinicopathological features of the lesions are summarized in Table 1.
Table 1. Clinicopathological features of the serrated lesions in this study.
Patients (n = 78)
Age (y, mean ± SD)

65.3 ± 10.9

Sex, n (%)
Male

51 (65)

Female27 (35)

Lesions (n = 78)
Location, n (%)
Proximal42 (54)

Distal36 (46)

Bowel subsites, n (%)
Cecum

13 (17)

Ascending colon

22 (28)Transverse colon

7 (9)Descending colon

5 (6)Sigmoid colon

20 (26)

Rectum11 (14)Morphology, n (%)
0-Ip

15 (19)0-Is

47 (60)0-IIa

16 (21)

Histology, n (%)
MVHP

23 (30)TSA

36 (46)

TSA + TVA

1 (1)

SSA

18 (23)

MVHP, microvesicular hyperplastic polyp; TSA, traditional serrated adenoma; TVA, tubulovillous adenoma; SSA,
sessile serrated adenoma.
https://doi.org/10.1371/journal.pone.0229262.t001

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DNA preparation
DNA was isolated from formalin-fixed, paraffin-embedded (FFPE) tissue sections using a
QIAamp DNA FFPE Tissue kit (Qiagen, Hilden, Germany) following the manufacturer’s
instructions. A TaqMan RNase P Detection Reagents kit (Thermo Fisher Scientific, Waltham,MA) was used to quantify the purified DNA.

Semiconductor-based next-generation sequencing
A customized panel, encompassing all the exons of 39 cancer-related genes, including genes
frequently reported to be involved in advanced CRCs and serrated lesions [2,25,26], was created using the Ion Torrent System with an Ion AmpliSeq Designer (Thermo Fisher Scientific)(S1 Fig). Genes within oncogene-induced senescence pathways detected in patients with multiple serrated polyps were also investigated [30]. The assay design consisted of 1,455 amplicons
ranging from 125 to 175 bp in length, covering 94% of the 112.8 kb target sequence.
Library preparation and sequencing with the Ion Torrent sequencer were performed as previously described [34–36]. The templates were sequenced after emulsion PCR was performed
with 24 samples per Ion PI chip using the Ion PI HI-Q Chef kit (Thermo Fisher Scientific).

Identification of somatic mutations and copy number variations (CNVs)
Human genome build 19 (hg19) was used as a reference. Signal processing, mapping to thehg19 reference, and quality control were performed using Torrent Suite version 5.0 (Thermo
Fisher Scientific). Somatic mutations (point mutations, insertions, and deletions) weredetected using Ion Reporter Software 5.0 (Thermo Fisher Scientific). Because matched normal
controls were not available, the control sequence data provided by Thermo Fisher Scientificwere used as a control. Pathogenic status of the variant was stated if it was a missense variant
with < 0.1% global minor allele frequency in dbSNP or the 1000 Genomes Project database
and/or the variant was registered as pathogenic in ClinVar or COSMIC databases. Variants
with allele frequencies between 0.4 and 0.6 or > 0.9 were considered germline variants unlesslisted as a pathogenic variant. Additionally, if the same variants were detected in multiple
samples, these variants were considered germline variants unless occurring at a known
hotspot variant in databases. Integrative Genomics Viewer (IGV) software (http://software.
broadinstitute.org/software/igv/) was used to filter out possible strand-specific errors, such as
a mutation that was detected in the forward or reverse DNA strand but not in both strands.
CNV detection was also performed with the Ion Reporter Software using an algorithm basedon the Hidden Markov Model. Recurrent genomic regions with CNVs were identified using
copy numbers greater than 3 and less than 1 for gains and losses, respectively.

DNA methylation analysis
DNA methylation was analyzed using bisulfite pyrosequencing as described previously[37,38]. Briefly, genomic DNA (1 μg) was modified with sodium bisulfite using an EpiTect
Bisulfite kit (Qiagen). Pyrosequencing was then carried out using a PSQ 96MA system (Qiagen) with a Pyro Gold Reagent kit (Qiagen), and the results were analyzed using Pyro Q-CpG
software (Qiagen). A cutoff value of 15% was used to define genes as methylation-positive.
Using five classic CIMP markers (MINT1, MINT2, MINT12, MINT31 and MLH1) and
CDKN2A (p16), tumors were defined as CIMP-positive (three or more loci showed methylation) or CIMP-high (CIMP-H, four or more loci showed methylation). Methylation of
SMOC1, GALNT14, SFRP1, SFRP2, IGFBP7, SOX5 and long interspersed nucleotide element 1

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Wnt signaling in colorectal serrated lesions(LINE-1) was also analyzed using bisulfite pyrosequencing. The primer sequences used wereas previously reported [9,31,39].

Immunohistochemistry
Immunohistochemical studies of β-catenin expression were performed as previously describedwith 39 serrated lesions, including 14 TSAs, 13 SSAs and 12 MVHPs [40]. A mouse anti-βcatenin monoclonal antibody (1:1000 dilution, Clone 14; BD Biosciences, San Jose, CA) wasused. β-catenin expression was semi-quantitatively evaluated in tumor cells with β-cateninpositive nuclei, and positive nuclear accumulation was defined as staining of more than 10% of
tumor cell nuclei throughout the lesions, as reported previously [40]. All slides were evaluatedby two independent pathologists (YK and TM) who were blinded to the clinical and moleculardata.

Statistical analysis
Continuous data were analyzed using t-tests (for two groups) or ANOVA with a post hoc
Tukey’s HSD test (for more than two groups). Fisher’s exact test and logistic regression wereused to assess the association between categorical variables. Values of P < 0.05 were considered statistically significant. All statistical analyses were performed using SPSS 20 (IBM Corporation, Somers, NY) and GraphPad Prism 6 (GraphPad Software, La Jolla, CA).

Results
Clinicopathological characteristics of serrated lesions
The clinicopathological and molecular characteristics of the colorectal serrated lesions analyzed in this study are summarized in Tables 1 and 2. The majority of MVHPs (17/23, 74%)
and SSAs (15/18, 83%) were located in the proximal colon, from the cecum to the transverse
colon, while TSAs were more prevalent in the distal colon (27/37, 73%), especially the sigmoid
colon and rectum (14 cases and 11 cases, respectively). On endoscopic observation, more than
one-third (14/37, 38%) of TSAs were protruding, pedunculated (0-Ip) lesions and differed significantly from SSAs, most of which were protruding, sessile (0-Is) lesions (16/18, 88%)
(Table 2).

Targeted amplicon sequencing of colorectal serrated lesions
We performed semiconductor sequencing of all exons in 39 cancer-related genes that werepreviously detected in 78 colorectal serrated lesions and advanced CRCs. The sequencing
overview, including reads, coverage, and uniformity of the read coverage distribution, isshown in S1 Table. Each FFPE sample underwent an average of 2.7 million sequencing reads
after quality filtering. A mean coverage depth of 1722.5 reads (100.2–5126.0) per base wasobserved.
All single nucleotide variants (SNVs) and insertions and deletions (InDels) detected
through bioinformatics analysis underwent visual inspection using the IGV for confirmation.
We identified a mean of 2.4 somatic nonsynonymous mutations (range 0–13) per sample(S2 Table). The 11 most commonly mutated genes in serrated lesions are depicted in Fig 1.
BRAF was the most frequently mutated gene in serrated lesions (68%, 53 of 78 cases), followed
by RNF43 (26%), KRAS (21%), and APC (10%). BRAF V600E mutations were detected in 52
samples, and an N581S mutation was detected in 1 sample accompanied by a KRAS Q61H
mutation (Patient 60 in Fig 1). The BRAF V600E mutation and KRAS mutation appeared to be
mutually exclusive, as they were never detected in the same sample (Fig 1). RNF43 mutations

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Table 2. Clinicopathological and molecular characteristics of the respective serrated lesion.
No. of cases

MVHP

SSA

TSA

23

18

37

p value (SSA vs TSA)

Sex (male/female)

16/7

9/9

26/11

0.24

Age (y, mean ± SD)

66 ± 9.1

63.3 ± 10.6

65.8 ± 11.8

0.44

Proximal (C/A/T)

17 (6/10/1)

15 (6/5/4)

10 (1/7/2)<0.01

Distal (D/S/R)

6 (1/5/0)

3 (2/1/0)

27 (2/14/11)

11.3 ± 7.0

11.2 ± 4.7

12.2 ± 5.4

0.5

0-Ip

0 (0)

1 (6)

14 (38)

0.01

0-Is

13 (57)16 (88)18 (49)0-IIa

10 (43)1 (6)5 (13)

BRAF V600E mutation

14 (61)14 (78)24 (65)

0.37

KRAS mutation

4 (17)

1 (6)

11 (30)0.08

RNF43 mutation

3 (13)

3 (17)

14 (38)0.13

APC mutation

1 (4)

0 (0)

7 (19)

0.08

WNT signaling associated genes

9 (39)

5 (28)

24 (65)

<0.01

CIMP

9 (39)

8 (44)

16 (43)

1

CIMP-high

5 (22)

4 (22)

10 (27)1

SMOC1 methylation

0 (0)

0 (0)

20 (54)<0.01

Tumor location, n (bowel subsite)

Tumor size (mm, mean ± SD)
Morphology, n (%)

Gene mutation/epigenetic alteration, n (%)

MVHP, microvesicular hyperplastic polyp; SSA, sessile serrated adenoma; TSA, traditional serrated adenoma; C, cecum; A, ascending colon; T, transverse colon; D,descending colon; S, sigmoid colon; R, rectum; CIMP, CpG island methylator phenotype
https://doi.org/10.1371/journal.pone.0229262.t002

were detected in 38% of TSAs and 17% of SSAs. Nonsense or frameshift mutations in RNF43
were seen in 30% of TSAs and 11% of SSAs. Most of the mutations were located upstream of
the ring finger domain of RNF43 (Fig 2). We also found APC mutations in 7 of 37 TSAs (19%),
but none in SSAs. Protein-truncating (nonsense and frameshift) mutations of APC weredetected in 14% of TSAs (Fig 1). Overall, the frequency of mutations in Wnt pathway components was significantly higher in TSAs than in SSAs (65% vs. 28%, P < 0.01) (Fig 3, Table 2).
In addition, a GNAS R201H mutation was found in 1 TSA, while 1 SSA and 3 MVHPs harbored a GNAS V334G mutation, though the biological significance of the latter is unknown(Fig 3A, S2 Table).

Target amplicon sequencing detects CNVs
We also detected CNVs in segments of the genome that could be duplicated or deleted fromthe sequencing data (Fig 1). In all samples, the genes most frequently affected by copy number
gains were KRAS (42%), CTNNB1 (28%), and HRAS (24%), while the genes most frequently
affected by copy number losses were DKK1 (53%), CDKN2A (41%), TP53 (31%), and XAF1(31%). In addition, copy number loss at the APC gene locus was seen in one TSA (S2 Fig). In
total, at least one CNV (loss or gain) affecting a Wnt pathway component was found in 49% of
TSAs and 67% of SSAs. When considered together with the gene mutations, at least one genomic abnormality affecting genes associated with Wnt signaling was seen in 84% of TSAs and
78% of SSAs. Among these, the most frequently affected genes associated with Wnt signaling
were CTNNB1 (gain at 3p22.1), which encodes β-catenin, and DKK1 (loss at 10q21.1), which
encodes Dkk-1, a negative regulator of Wnt signaling (S2 Fig).

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Fig 1. Summary of somatic mutations and CNVs across 78 serrated lesions. Profiles of gene mutations and CNVs within individual samples aregrouped with respect to histological types. In the upper panel, the top row indicates the histology, the second row indicates the tumor location, and thethird row indicates the CIMP status. Columns correspond to the individual cases. In the middle panels, frequently mutated genes, colored to indicate
the type of mutation, and their mutational frequency are shown. In the lower panels, CNVs frequently detected in colorectal serrated lesions are shown.
https://doi.org/10.1371/journal.pone.0229262.g001

Methylation analysis of CIMP markers, cancer-associated genes, and LINE1
We next assessed methylation of CIMP markers and genes known to be frequently methylated
in CRCs (S3 Table) [9]. When comparing TSA and SSA, the prevalence of CIMP statuses(CIMP-positive or CIMP-H) did not significantly differ (Table 2, Fig 3A). Notably, however,SMOC1 methylation was detected in 20 of 37 TSAs (54%) but in none of the 18 SSAs tested(P < 0.01) (Table 2, Fig 3A). By contrast, the frequencies of lesions showing methylation ofother cancer-associated genes did not significantly differ between TSAs and SSAs. There wereno lesions in which MLH1 methylation was positive in the present study.
We found that the levels of SMOC1, SFRP1 and SFRP2 methylation were significantly
higher in TSAs than in SSAs or MVHPs (S3 Fig). In addition, the level of SOX5 methylation
was significantly higher in TSAs than in MVHPs. Levels of LINE-1 methylation, which wasmeasured to evaluate global DNA hypomethylation in the lesions, did not correlate with thehistological types of serrated lesions (S3 Fig).

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Fig 2. Distributions of RNF43 mutations in detected in TSAs in the present study. Previously reported TSA mutations with or without
dysplasia as well as MSS and MSI-high CRCs are shown for reference [23,24,41].
https://doi.org/10.1371/journal.pone.0229262.g002

Clinicopathological and molecular characteristics of TSAs with KRAS or
BRAF mutation
We also compared clinicopathological and molecular characteristics of TSAs with KRAS or
BRAF (V600E) mutation. It appears that TSAs with BRAF mutation were more likely to belocated in the proximal colon than those with KRAS mutation, though the difference was notstatistically significant (P = 0.12) (Table 3). Regarding gene mutations, RNF43 mutation was
frequently detected in TSAs with BRAF mutation, but not in TSAs with KRAS mutation(P < 0.01). Lesions with CIMP-H were also found only in TSAs with BRAF mutations (38%vs. 0%, P = 0.03). Although lesions positive for SMOC1 methylation were seen in both groups,
SMOC1 methylation was significantly more prevalent among TSAs with KRAS mutations thanthose with BRAF mutations (82% vs. 38%, P = 0.03) (Table 3). In addition, levels of SFRP1

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Fig 3. Gene mutations leading to dysregulation of Wnt signaling pathway in serrated lesions. (A) Representative mutation andmethylation profile across 78 serrated lesions. Blue coloration indicates gene mutations affecting Wnt pathway components, while redcoloration indicates positivity for gene mutations or methylation. (B) Frequencies of gene mutations leading to dysregulation of Wnt
signaling in TSAs and SSAs. Alteration frequencies are expressed as percentages of all cases. Frequencies of gene mutations in TSAs areshown on the left, while those in SSAs are shown on the right. Red denotes activated genes and blue denotes inactivated genes.
https://doi.org/10.1371/journal.pone.0229262.g003

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Table 3. Clinicopathological and molecular characteristics of the KRAS- and BRAF- mutant TSAs.
BRAF mutant TSA

KRAS mutant TSA

No. of cases

24

11

p valueSex (male/female)

16/8

9/2

0.45

Age (y, mean ± SD)

65.9 ± 8.7

63.1 ± 15.9

0.61

Proximal (C/A/T)

9 (1/7/1)

1 (0/0/1)

0.12

Distal (D/S/R)

15 (2/9/4)

10 (0/4/6)

11.9 ± 4.5

12.1 ± 5.9

0.92

0-Ip

10 (42)3 (27)

0.26

0-Is

12 (50)5 (46)

0-IIa

2 (8)

3 (27)

RNF43 mutation

13 (54)0 (0)< 0.01

APC mutation

4 (17)

3 (27)

0.65

WNT signaling associated genes

17 (71)

6 (55)

0.35

CIMP

12 (50)3 (27)

0.28

CIMP-high

9 (38)

0 (0)

0.03

SMOC1 methylation

9 (38)

9 (82)

0.03

Tumor location, n (bowel subsite)

Tumor size (mm, mean ± SD)
Morphology, n (%)

Gene mutation/epigenetic alteration, n (%)

TSA, traditional serrated adenoma; C, cecum; A, ascending colon; T, transverse colon; D, descending colon; S, sigmoid colon; R, rectum; CIMP, CpG island methylator
phenotype
https://doi.org/10.1371/journal.pone.0229262.t003

methylation were significantly higher in TSAs with KRAS mutation than in those with BRAF
mutation. On the other hand, levels of IGFBP7 methylation was significantly higher in TSAs
with BRAF mutation than those with KRAS mutation (S4 Fig).

Immunohistochemistry
Of 39 serrated lesions analyzed, 12 (31%) showed positive nuclear accumulation, while 27(69%) showed membranous expression of β-catenin. The abnormal nuclear accumulation wasobserved in 8/14 TSAs (57%), 1/13 (8%) of SSAs, and 3/12 (25%) MVHPs (S4 Table, S5 Fig).
When compared with SSAs, the nuclear accumulation of β-catenin was more prevalent in
TSAs (57% vs. 8%, P = 0.01). Across all lesions, the prevalence of nuclear β-catenin accumulation was significantly higher in lesions with RNF43 mutations than in those without mutations(83% vs. 21%, P < 0.01). When we focused only on TSAs, the prevalence of nuclear β-catenin
accumulation was significantly higher in TSAs with a BRAF V600E mutation than those with
KRAS mutation (7/8 vs. 1/5, P = 0.01).

Discussion
In the present study, we used targeted next-generation sequencing to assess the mutation ofgenes associated with Wnt signaling. We found that these mutations occurred with higher frequency in TSAs than SSAs, which is suggestive of the importance of Wnt signaling in the pathogenesis of TSAs. Sekine et al. reported genetic alterations that included PTPRK-RSPO3
fusions among Wnt pathway components in 71% of TSAs [23]. If PTPRK-RSPO3 fusions had
also been investigated in the present study, the frequency of genetic alterations would havebeen even higher. RNF43 encodes E3 ubiquitin ligase, which negatively regulates Wnt

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signaling. We detected RNF43 mutations in 38% of TSAs and 17% of SSAs, which is consistent
with earlier reports [23,24,26]. Most mutations, especially protein-truncating mutations, weresituated upstream of the ring finger domain without clustering, as was shown in previous studies of TSAs with or without dysplasia [23,24]. Two major frameshift mutations (Arg117fs andGly659fs) have been reported in CRCs with MSI [41], while CRCs with BRAF mutation/MSS
had mutation profiles similar to TSAs [24,41]. This finding may indicate that TSAs belong to a
carcinogenic pathway that is distinct from the SSA pathway (serrated-neoplasia pathway),
making them possible precursors of BRAF mutated/MSS CRCs. We also sequenced the entire
region of the APC gene and found protein-truncating mutations in 14% of TSAs, which is consistent with a report from Sekine et al. (13%) [23], though they only investigated frequently
mutated regions of the gene. Protein truncating mutations of RNF43 and APC were found to
be mutually exclusive, suggesting the importance of both genes to Wnt signaling during carcinogenesis in TSAs. By contrast, the other genes encoding Wnt pathway components, including
CTNNB1, FBXW7, LRP5, AMER1, and AXIN2, harbored mutations in only small fractions oftumors.
Previous studies have shown that there is wide variation in the rate of β-catenin positivity
in both TSAs [21,23,27–29] and SSAs [29]. We showed in the present study that nuclear βcatenin accumulation is significantly more prevalent in TSAs than SSAs. This is likely associated with a high prevalence of mutations in the Wnt signaling pathway, especially in RNF43.
These results may thus support the greater significance of RNF43 mutation and Wnt signaling
pathway activation in the carcinogenesis of TSAs than SSAs. Alternatively, it is possible thatRNF43 mutation represents subgroups in which Wnt signaling is activated by other genetic or
epigenetic mechanisms.
Although an earlier study using a comparative genomic hybridization (CGH) microarray
reported that CNVs were found only infrequently in colorectal precursor lesions including serrated lesions [9], CNVs in colorectal serrated lesions have not been thoroughly investigated.
The present study demonstrated the frequent occurrence of CNVs at the CTNNB1 and DKK1loci among Wnt signaling pathway associated genes. In addition, copy number losses at 17p(the TP53 locus) are reportedly associated with progression of tumors from conventional-typeadenoma to carcinoma and are frequently found in advanced CRCs [2,3]. This result may indicate the importance of TP53 to carcinogenesis of serrated lesions. Because recent studies suggest that somatic CNVs at oncogenic loci are not always associated with gene expression[2,42,43], validation of the effect of CNVs through comparison with expression data is needed.
The prevalence of CIMP-positive or CIMP-H lesions did not significantly differ between
SSAs and TSAs in this study. CIMP was detected in up to 79% of TSAs in previous studies[16,17]. One recent study reported that the prevalence of CIMP-H was significantly higher in
SSAs than TSAs [44], which is inconsistent with our results. This difference likely reflects differences in the sample cohort, or may be due to a difference in the CIMP markers analyzed or
the method used for methylation analysis (pyrosequencing vs. MethyLight). Interestingly, levels of DNA methylation in SFRP1 and SFRP2 were higher in TSAs than in SSAs or MVHPs.
Epigenetic inactivation of SFRP family genes, including SFRP1 and SFRP2, occurs early during
CRC progression and enables constitutive Wnt signaling in CRCs [45]. It was recently
reported that levels of SFRP1 and SFRP2 transcription correlate inversely with the methylationlevels in samples of gastric mucosa, with or without H. pylori infection, as well as backgroundmucosa in gastric cancers [46]. It is possible that SFRP1 and SFRP2 methylation contributes
more significantly to carcinogenesis in TSAs than SSAs, although these phenomena may be
influenced by the sample cohort, the degree of contamination by non-neoplastic cells, andmethodology. We also analyzed the methylation status of SMOC1 in a patient cohort different
from the one examined in an earlier study [31]. We found that SMOC1 methylation was highly

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specific for TSAs, and the methylation level was significantly higher in TSAs than SSAs. Thus,
SMOC1 methylation may be a potential marker to distinguish TSAs from other serrated
polyps.
Recent reports suggest that TSAs belong to a heterogenous category and develop throughat least two different neoplastic progression pathways. It is also suggested that lesions with
BRAF mutation and those with KRAS mutation exhibit different clinicopathological andmolecular characteristics [18–21]. In the present study, TSAs with BRAF mutation were preferentially located in the proximal colon, while those with KRAS mutation were preferentiallylocated in the distal colon and rectum. In addition, TSAs with BRAF mutation were more
likely to be CIMP-positive than those with KRAS mutation. CIMP-H positivity was only
detected in TSAs with BRAF mutation, which is consistent with an earlier study [21]. Lesions
with RNF43 mutation were also found only in TSAs with BRAF mutation, while the prevalence of SMOC1 methylation was significantly higher in TSAs with KRAS mutation than with
BRAF mutation. The level of IGFBP7 methylation is significantly higher in TSAs with BRAF
mutation than with KRAS mutation, though the frequency of methylation-positive IGFBP7
cases did not significantly differ between the two groups. IGFBP7 has been shown to play acentral role in BRAF-induced senescence and to be a direct target of TP53 [47]. Although
methylation of IGFBP7 has been investigated in specific histological types of serrated lesions[10,48], IGFBP7 methylation status has not been compared between these two TSA subtypes.
In addition to these differences in genetic and epigenetic alterations, the difference in theprevalence of β-catenin expression further supports there being two different neoplastic pathways for TSAs. Determining whether TSAs with KRAS or BRAF mutation belong to different
neoplastic pathways with different malignant potentials will require further clinicopathological and molecular analyses.
The present study has several limitations, including a relatively small sample size, lack ofdata on fusion genes, and a lack of normal background samples for mutational analysis to ruleout single nucleotide polymorphisms. Nonetheless, we were able to make several important
observations. First, the mutational status of genes involved in Wnt signaling differs amongcolorectal serrated polyps, depending on TSA histology, which likely results in the differencesin nuclear β-catenin expression. Second, we confirmed that SMOC1 methylation is very specific to TSAs. Third, we detected significant differences in clinicopathological and molecularvariables between TSAs with KRAS or BRAF mutation, which may indicate the presence ofseparate carcinogenic pathways among TSAs. By comparing gene expression data from CRCs
and SSAs, an earlier study found that a particular subtype of CRCs with a poor-prognosis
developed from serrated lesions [49]. Additionally, previous studies have also shown that there
are similarities between the gene expression profiles of SSAs and those of MVHPs and MSI
CRCs, which supports the concept of a serrated-neoplasia pathway [50,51]. Comprehensive
gene expression studies together with analyses of the genetic and epigenetic alterations in
TSAs and comparison of those data with other serrated polyps or CRCs could potentiallyestablish the molecular carcinogenesis pathway in TSAs.

Supporting information
S1 Fig. List of genes in the custom AmpliSeq gene panel used in this study.
(PDF)
S2 Fig. Gene mutations and CNVs in association with Wnt signaling across 78 colorectal
serrated lesions.
(PDF)

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Wnt signaling in colorectal serrated lesions

S3 Fig. Levels of methylation of the indicated genes and LINE-1 in the indicated histological types of colorectal serrated lesions.
(PDF)
S4 Fig. Levels of methylation of the indicated genes and LINE-1 in TSAs with BRAF or
KRAS mutation.
(PDF)
S5 Fig. Representative views of hematoxylin and eosin (left) and β-catenin (right) expression in SSA and TSA. (A, B) SSA showing membranous localization of β-catenin. (C, D) TSA
showing nuclear accumulation of β-catenin.
(TIF)
S1 Table. Summary of targeted amplicon sequencing data.
(XLSX)S2 Table. Somatic nonsynonymous mutations found in 78 colorectal serrated lesions.
(XLSX)S3 Table. Methylation profiles and CIMP status in colorectal serrated lesions.
(XLSX)S4 Table. Nuclear β-catenin expression in colorectal serrated lesions.
(XLSX)

Acknowledgments
The authors thank Mutsumi Toyota, Akina Omori, Toshiki Kenuka, Aki Iwata and Shigenori
Wakita for their excellent technical assistance; Takahito Katano, Mamoru Tanaka, Hirotaka
Nishiwaki, Keiji Ozeki, Hironobu Tsukamoto, Tsutomu Mizoshita, Yoshinori Mori, Hiroyuki
Aoyagi, Shigetsugu Tsuji, Masashi Yoshimitsu, Kazuhiro Miwa, Yoshinori Goto and Yutaka
Matano for providing samples; and Dr. William F. Goldman for editing the manuscript.

Author Contributions
Conceptualization: Takeshi Sawada.
Data curation: Takeshi Sawada.
Formal analysis: Hiroyoshi Nakanishi.
Investigation: Hiroyoshi Nakanishi, Takeshi Sawada, Yasuharu Kaizaki, Ryosuke Ota, Hiromu Suzuki, Eiichiro Yamamoto, Hironori Aoki, Makoto Eizuka, Satoko Inagaki, Toshinari Minamoto, Tamotsu Sugai, Yasushi Sasaki.
Methodology: Takeshi Sawada, Hiromu Suzuki, Takashi Tokino, Toshinari Minamoto,Yasushi Sasaki.
Resources: Yasuharu Kaizaki, Kenkei Hasatani, Naoki Takahashi, Masahide Ebi, Hiroyuki
Kato, Eiji Kubota, Hiromi Kataoka, Satoru Takahashi.
Supervision: Takeshi Sawada, Toshinari Minamoto.
Writing – original draft: Takeshi Sawada.
Writing – review & editing: Hiromu Suzuki, Toshinari Minamoto, Yasushi Sasaki.

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Wnt signaling in colorectal serrated lesions

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Received: 21 July 2020

| Revised: 11 September 2020 | Accepted: 19 September 2020

DOI: 10.1111/cas.14668

ORIGINAL ARTICLE

Glycogen synthase kinase-3β participates in acquired
resistance to gemcitabine in pancreatic cancer
Masahiro Uehara1
Osamu Takeuchi3| Takahiro Domoto1| Satoshi Takenaka1,2 | Dilireba Bolidong1 || Tomoharu Miyashita2,4 | Toshinari Minamoto1

1
Division of Translational and ClinicalOncology, Cancer Research Institute,
Kanazawa University, Kanazawa, Japan2

Department of Gastroenterological
Surgery, Graduate School of Medical
Sciences, Kanazawa University, Kanazawa,Japan
3
Biomedical Laboratory, Department ofResearch, Kitasato University Kitasato
Institute Hospital, Tokyo, Japan
4

Department of Surgical Oncology,
Kanazawa Medical University, Ishikawa,Japan
Correspondence
Toshinari Minamoto, Division ofTranslational and Clinical Oncology, Cancer
Research Institute, Kanazawa University,
13-1 Takara-machi, Kanazawa 920-0934,
Japan.
Email: minamoto@staff.kanazawa-u.ac.jp
Funding information
Ministry of Education, Culture, Sports,
Science and Technology-Japan; Japan
Society for the Promotion of Science

Abstract
Acquisition of resistance to gemcitabine is a challenging clinical and biological hallmark property of refractory pancreatic cancer. Here, we investigated whether glycogen synthase kinase (GSK)-3β, an emerging therapeutic target in various cancer
types, is mechanistically involved in acquired resistance to gemcitabine in humanpancreatic cancer. This study included 3 gemcitabine-sensitive BxPC-3 cell-derived
clones (BxG30, BxG140, BxG400) that acquired stepwise resistance to gemcitabineand overexpressed ribonucleotide reductase (RR)M1. Treatment with GSK3β-specificinhibitor alone attenuated the viability and proliferation of the gemcitabine-resistantclones, while synergistically enhancing the efficacy of gemcitabine against theseclones and their xenograft tumors in rodents. The gemcitabine-resensitizing effectof GSK3β inhibition was associated with decreased expression of RRM1, reduced
phosphorylation of Rb protein, and restored binding of Rb to the E2 transcription
factor (E2F)1. This was followed by decreased E2F1 transcriptional activity, which
ultimately suppressed the expression of E2F1 transcriptional targets including RRM1,
CCND1 encoding cyclin D1, thymidylate synthase, and thymidine kinase 1. These results suggested that GSK3β participates in the acquisition of gemcitabine resistanceby pancreatic cancer cells via impairment of the functional interaction between Rb
tumor suppressor protein and E2F1 pro-oncogenic transcription factor, thereby highlighting GSK3β as a promising target in refractory pancreatic cancer. By providing
insight into the molecular mechanism of gemcitabine resistance, this study identified
a potentially novel strategy for pancreatic cancer chemotherapy.
KEYWORDS

acquired resistance, gemcitabine, glycogen synthase kinase-3β, pancreatic cancer,
ribonucleotide reductase M1

Abbreviations: CDK, cyclin-dependent kinase; dCK, deoxycytidine kinase; dFdC, 2′-2′-difluoro-2′-deoxycytidine; dFdCDP, dFdC diphosphate; E2F1, E2 transcription factor 1; EMT,
epithelial-mesenchymal transition; FOLFIRINOX, combination of folate, 5-FU, irinotecan, and oxaliplatin; FU, fluorouracil; GSK3β, glycogen synthase kinase-3β; hENT1, humanequilibrative NT1; IHC, immunohistochemistry; IP, immunoprecipitation; nab, nanoparticle albumin-bound; NT, nucleoside transporter; PS, performance status; qRT-PCR, quantitative
reverse transcription-polymerase chain reaction; Rb, retinoblastoma gene/tumor suppressor protein; RRM1, ribonucleotide reductase M1; S, serine; TK1, thymidine kinase 1; TP53INP1,
tumor protein p53 inducible nuclear protein 1; TS, thymidylate synthase; WB, Western blotting; Y, tyrosine.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution inany medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.
Cancer Science. 2020;111:4405–4416.

wileyonlinelibrary.com/journal/cas

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4406|

UEHARA Et Al.

1 | I NTRO D U C TI O Nepithelial-mesenchymal transition (EMT) in untransformed cells,
GSK3β has long been hypothesized to suppress tumor development

Pancreatic cancer is one of the most intractable cancer types, with lit1,2

tle improvement in the survival rate over the past decades.

and progression.13 Previous studies on the putative tumor-suppres-Patients

sive roles of GSK3β have shown that it is inactivated by S9 phos-with locally advanced tumors and those with metastatic tumors ac-

phorylation in various oncogenic pathways. However, there has

count for approximately one-third and one-half, respectively, of all

been no evidence showing that active GSK3β suppresses tumori-pancreatic cancer cases.1 Due to the extreme difficulty in obtaining

genesis, or that GSK3β inhibition promotes tumor development andan early diagnosis,3 only 10%-15% of newly identified pancreatic can-progression (reviewed in Ref.15). In contrast, many studies by ourcer patients present with resectable or borderline resectable tumorgroup and others have demonstrated direct tumor-promoting roles(stage I or II). Most patients have local disease recurrence and/or dis-for GSK3β as well as therapeutic effects following its inhibition intant metastasis following surgery. Therefore, most patients undergo

at least 25 different cancer types15-17 including pancreatic cancer.18

nonsurgical therapies including conventional chemotherapy, radiation,

In addition to its therapeutic effect, we and others have shown thatand molecular-targeted therapies.1 More recently, clinical trials with

GSK3β inhibition sensitizes pancreatic cancer cells to gemcitabine

immune checkpoint blockades and precision therapies have also been

through the impairment of DNA damage repair and cell cycle regula-conducted.4,5 Despite multimodal combinations of these therapies, thetion.19-21 However, none of the earlier studies modeled the acquired

5-year overall survival rate for pancreatic cancer is at most 3%-15%.2,6

resistance to gemcitabine in pancreatic cancer that is frequently

Chemotherapeutic agents used for pancreatic cancer patientsencountered in the clinical setting. To more directly investigate theinclude gemcitabine alone or in combination with nanoparticle al-role of GSK3β in the acquisition of gemcitabine resistance, we estab-bumin-bound (nab)-paclitaxel, TS-1 (a fluorouracil [FU] prodrug),

lished cell clones derived from a gemcitabine-sensitive human pan-

FOLFIRINOX (sequential combination of folate, 5-FU, irinotecan,creatic cancer BxPC-3 cell line that had acquired stepwise resistanceand oxaliplatin) and nanoliposomal irinotecan with 5-FU and folate.7

to gemcitabine. This system models the development of gemcitabine

Currently, gemcitabine-based regimens are the most frequently

resistance observed in clinical pancreatic cancer. 22

prescribed in the first-line setting and remain one of the standard
therapies for pancreatic cancer.7,8 However, most patients are initially resistant or soon acquire resistance to gemcitabine, therefore
obtaining little survival benefit from this treatment.9 Therefore, theidentification of mechanism-based strategies and therapeutic tar-

2 | M ATE R I A L S A N D M E TH O DS
2.1 | Cell lines

gets to overcome gemcitabine resistance has become an active areafor biological and clinical research.10

This study investigated the human pancreatic cancer cell line

In the present study we hypothesized that glycogen synthase

BxPC-3 and its derivative clones that acquired stepwise resistancekinase (GSK)-3β was a candidate molecular target that may affectto gemcitabine (BxG30, BxG140, and BxG400 in increasing order

the efficacy of chemotherapeutic agents. GSK3β is an isoform of theof resistance). 22 The cloned cells were confirmed to sustain theirmultifunctional GSK3 family serine/threonine kinases and regulates

resistance without supplementation of gemcitabine in the medium

a diverse array of fundamental cellular pathways by phosphorylation

for at least 2 months. The cells were maintained in RPMI 1640

and interaction with dozens of structural and functional molecules.11

medium supplemented with 10% fetal bovine serum and antibiot-

GSK3β activity is finely controlled by differential phosphorylation of

ics (100 unit/mL penicillin G and 0.1 mg/mL streptomycin) (Gibco)

serine (S)9 (inactive) and tyrosine (Y)216 (active) residues. It is con-at 37°C with 5% CO2 . All subsequent experiments, except for thestitutively active in normal cells, but in many circumstances negative

mouse xenograft study, were completed within 2 months after ini-

regulation of its activity allows cells to maintain vital activity andtiation from frozen cell cultures.

homeostasis in response to various stimuli.12 Deregulation of GSK3β
expression and activity has been implicated in the pathogenesis andprogression of common diseases including diabetes mellitus, neurodegenerative disorders, and various inflammatory and immunologi-

2.2 | Quantitative reverse transcription-polymerase
chain reaction (qRT-PCR)

cal conditions.12,13 Such diverse roles in normal cells and in diseases
have highlighted GSK3β as a potentially attractive drug target andTotal RNA was extracted from the cells using ISOGEN (Wako).

has led to the development of inhibitors.14 At present, several ear-Complementary DNA was synthesized from total RNA using aly-phase clinical trials have been evaluating some of these inhibitors

Reverse Transcription Kit (Promega). qRT-PCR was performed using

and lithium (an ATP non-competitive and non-specific GSK3 inhib-

the Stratagene Mx3000P system (Agilent Technologies) and SYBRitor) for neurodegenerative disorders and various cancer types,

Premix Ex Taq (TaKaRa Bio) with sets of sense and anti-sense prim-

but none of them has been approved for clinical use (reviewed iners (FASMAC) for the respective genes, as shown in Supporting

Ref.15,16).

Information Table S1. Relative mRNA expression of each gene was

Based on its known functions against pro-oncogenic pathways(eg,

Wnt/β-catenin,

hedgehog,

Notchsignaling)and—calculated using the ΔCt method and calibrated against the expression of GAPDH as an internal control.

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2.3 | Western blotting (WB)

2.6 | Effects of GSK3β inhibitor on thesusceptibility of cancer cells to gemcitabine

Cellular protein was extracted from cultured cells using a lysis buffer(CelLytic-MT; Sigma-Aldrich) with a mixture of protease and phos-

IC50 values at 72 h following treatment with gemcitabine and AR-

phatase inhibitors (Sigma-Aldrich). A 20 μg aliquot of protein extract

A014418 were measured by WST-8 assay for BxPC-3, BxG30,

was subjected to WB for the proteins of interest, as reported previ-

BxG140, and BxG400 cells. These cells were then treated with gem-

ously. 20 The expression of β-actin was monitored as a control for thecitabine at the doses close to the respective IC50 in combination withloading amount of protein sample. Primary antibodies used at theirDMSO or AR-A014418 at the concentrations shown in Table S3. The

respective dilutions are shown in Table S2.

combined effects of gemcitabine and AR-A014418 on the viability ofcells were determined as being additive, synergistic, or antagonistic
using the isobologram method. 25

2.4 | Analyses for cell survival,
proliferation, and apoptosis

2.7 | Immunoprecipitation (IP) analysis

BxPC-3 cells and cells from the gemcitabine-resistant clones (BxG30,
BxG140, BxG400) were seeded into 96-well plates and treated with

BxG400 cells were treated with DMSO or 25 μmol/L AR-A014418

gemcitabine (Sigma-Aldrich) at different concentrations. Two otherfor 24 h. The nuclear fraction of these cells was isolated usingbatches of the same cells were passaged in the absence of gemcit-

Nuclear Complex Co-IP Kit (Active Motif) and pre-cleaned with pro-abine for 1 or 2 months, respectively, and then treated with gemcit-

tein G magnetic beads slurry (Cell Signaling Technologies). Nuclear

abine. In these 3 sets of experiments, the relative numbers of viable

extracts from the respective cells were divided into aliquots and im-

cells at designated time points following treatment with gemcitabinemunoprecipitated with a mixture of non-immune mouse and rabbit

were determined using the WST-8 assay kit (Cell Counting Kit-8;

IgG, mouse anti-Rb, and rabbit anti-E2F1 antibodies, respectively,

Dojindo). These cells were also treated with DMSO (Sigma-Aldrich)according to our previous study. 26 An aliquot of nuclear extract

23

or(input) and the immunoprecipitated material was analyzed by WB

SB-216763 (Sigma-Aldrich)24 at the indicated final concentration inusing anti-Rb and anti-E2F1 antibodies. The primary antibodies used

the medium. The relative number of viable cells at the designatedat the respective dilutions are shown in Table S2.

or with one of the GSK3β inhibitors: AR-A014418 (Calbiochem)

time points was measured as described above. The concentrations of
GSK3β inhibitors used in this study are within the reported range ofpharmacologically relevant doses. 23,24 After treatment with DMSO

2.8 | Luciferase reporter assay

or GSK3β inhibitor, the relative numbers of proliferating and apoptotic cells were determined using the Click-iT EdU Proliferation

Transcriptional activity of E2F1 was determined by luciferase re-Assay for Microplates kit (Thermo Fisher Scientific) and the Cellular

porter assay. The respective cancer cells were co-transfected withDNA Fragmentation ELISA kit (Roche Diagnostics), respectively. The

E2F1 firefly luciferase vector (E2F1(3) Luciferase Reporter Vector,

mean numbers of viable cells, EdU-labeled proliferating cells, andPanomics) and internal control Renilla luciferase vector (pRL-SV40

apoptotic cells from triplicate experiments were calculated together

Vector, Promega) in accordance with the manufacturers’ proto-

with their standard deviations (SDs). They were compared statisti-

cols. At 72 h after transfection, the cells were treated with DMSO

cally to the same cells treated with gemcitabine at different concen-

or 25 μmol/L AR-A014418 for 24 h. The cells were then examinedtrations, as well as to cells treated with DMSO or GSK3β inhibitors.

for activities of both luciferases using the Dual-Luciferase Reporter
Assay System (Promega) and Fluoroscan ascent FL instrument(Dainippon Sumitomo Pharmaceutical). The relative transcriptional

2.5 | RNA interference (RNAi)

activity of E2F1 was determined by normalizing firefly luciferase activity with Renilla luciferase activity in the same cells.

siRNA specific for human GSK3β (GSK3β Validated Stealth RNAi)
and negative control siRNA (Stealth RNAi Negative Control Low GC
duplex) were obtained from Invitrogen. The specificity of GSK3β-

2.9 | Animal study

specific siRNA was validated in our previous study. 20 Cells weretransfected with 20 nmol/L of either siRNA using Lipofectamine

The therapeutic effects of gemcitabine and GSK3β inhibitor, either

RNAiMAX reagent (Invitrogen). The efficiency of RNA interference

alone or in combination, were examined in gemcitabine-sensitivewas evaluated by WB with an antibody that recognized both GSK3α

BxPC-3 cells and in the most gemcitabine-resistant BxG400 cells

and GSK3β (Table S2). The effects of GSK3β knockdown on the rela-grown as xenografts in athymic mice. In total, 1 × 106 BxPC-3 ortive numbers of surviving, proliferating, and apoptotic cells were ex-

BxG400 cells suspended in 50 μL of phosphate buffered saline wereamined and compared with the same cancer cells transfected with

subcutaneously inoculated into each of 53 athymic mice (Charles

control or GSK3β-specific siRNA for 72 h as described above.

River Laboratories, Japan). The mice were randomly assigned to 4

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groups and given intraperitoneal injections of 100 μL of 75% DMSO,
gemcitabine (20 mg/kg body weight), or AR-A014418 (2 mg/kg
body weight) alone or in combination, respectively, twice a week for
7-10 wk. Assuming that total body fluid in mice accounted for c. 60%of their body weight, the AR-A014418 dose of 2 mg/kg body weightcorresponded to a concentration of c. 10 μmol/L in culture medium,
which was within the known pharmacological dose range for thisagent. 23 The dose of gemcitabine corresponded closely to the standard clinical dose (1000 mg/m2 body surface area). Throughout theexperiment, all mice were carefully observed every day for adverse
events and their body weight was monitored. Tumors were measured in 2 dimensions, twice a week. Tumor volume (cm3) was calculated using the formula: 0.5 × S2 × L, where S is the smallest tumor
diameter (cm) and L is the largest (cm). The design and protocol of theanimal experiment and changes in body weight of mice during treatment are shown in Figure S1. All animal experiments were undertaken in accordance with the Japanese animal ethics guidelines. 27
The protocol was approved by the Institute for Experimental Animal
Work, Kanazawa University Advanced Science Research Center.
At necropsy, tumors were removed, fixed in 10% paraformaldehyde and embedded in paraffin for histopathological and immunohistochemical staining. Representative paraffin sections of
the tumors were stained with H&E and immunostained using theavidin-biotin-peroxidase complex (ABC) method as described previously. 28 The primary antibodies used at the dilutions for immunohistochemistry (IHC) are shown in Table S2.

3 | R E S U LT S

F I G U R E 1 Efficacy of gemcitabine and expression of RRM1 in
BxPC-3 cells and cells from gemcitabine-resistant clones (BxG30,
BxG140, BxG400). A, Respective cells were treated with H2O orthe indicated concentrations of gemcitabine for the designatedtimes in triplicate. The relative mean number of viable cells at eachtime point is shown with SDs. *P < .05. B, Relative expression of
RRM1 mRNA in BxPC-3 and the gemcitabine-resistant clones.
Values shown are the means ± SDs of triplicate measurements.
*P < .05. C, Comparison of RRM1 expression between BxPC-3 cells
and the gemcitabine-resistant clones by Western blotting. β-actin
expression was monitored as a loading control in each sample

3.1 | Efficacy of gemcitabine on BxPC-3 cellderived clones with stepwise acquired resistance togemcitabine

and BxG400 cells as the level of resistance to gemcitabine increased

2.10 | Statistical analysis
The results are presented as mean ± SD. Data were analyzed using
Student t test in a two-tailed analysis. Statistical significance wasdefined as values of P < .05.

for gemcitabine resistance,9,29 increased significantly in the BxG140(Figure 1B,C).

The IC50 values for gemcitabine in BxPC-3 cells and for the gemcitabine-resistant BxG30, BxG140, and BxG400 cells were 1.28 ng/mL,
39 ng/mL, 370 ng/mL, and 2000 ng/mL, respectively (Figure 1A).
Consistent with our previous study, 22 the results confirmed that

3.2 | Effects of GSK3β inhibition on thegemcitabine-resistant clones

the persistent and stepwise gemcitabine resistance was retained
during long-term frozen storage of these resistant clones. We also

We previously reported the constitutive activation of GSK3β indetermined the IC50 values of gemcitabine in the same cells fol-

human pancreatic cancer cells, including BxPC-3, and the therapeutic

lowing continuous culture in the absence of gemcitabine for 1 or 2

effect of GSK3β inhibition against these cancer cells.19,20 We there-

months, respectively (Figure S2). This indicated that gemcitabine re-fore evaluated the effects of GSK3β inhibition on 3 gemcitabine-sistance was present for at least 2 months after frozen cell culture,

resistant clones. Gemcitabine-resistant cells showed a higher level

thus allowing subsequent experiments. mRNA and protein expres-of cell survival and proliferation and a lower frequency of apopto-

sion of ribonucleotide reductase M1 (RRM1), a known biomarker

sis compared with their parent BxPC-3 cells (Figures 2 and S3). The

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(Figures 2B,C, S3B,C, and S4B,C). Such difference suggested thatthese resistant clones might also acquire the phenotype invulnerable to apoptosis-inducing stimuli. Alternatively, they might havemore dependence on the cell proliferation signal mediated by cyclin D1 and cyclin-dependent kinase (CDK)4/6 as we previously
observed in pancreatic cancer cells that were primarily unresponsive to gemcitabine. 20 The effects of GSK3β RNAi on cell survival,
proliferation, and apoptosis (Figure S4) were less marked than the
GSK3β inhibitors (Figures 2 and S3). In many cases, the effect ofan enzyme largely depends on its biochemical or catalytic activity
rather than its level of expression. The biological effect of RNAi
depends on the efficiency of siRNA transfection as well as on thesubsequent knockdown of the target molecule. In line with thesenotions, our previous study using an in vitro kinase assay showedthat the GSK3β inhibitor AR-A014418 inactivated GSK3β in humanpancreatic cancer cells (including BxPC-3 cells) within 1 h after
treatment, while the GSK3β-specific siRNA took longer than 48 h
to efficiently, but not completely, deplete GSK3β expression in
the same cells. 20 Therefore, it is conceivable that pharmacological
GSK3β inhibitors more promptly and efficiently inhibit GSK3β incells, leading to a more prominent biological effect on cells compared with GSK3β RNAi.

3.3 | Combined effect of gemcitabine and
GSK3β inhibitor
Molecular-targeted therapeutics are preferably prescribed in combination with conventional chemotherapeutic agents and/or radiaF I G U R E 2 Effects of GSK3β inhibition on viability, proliferation,
and apoptosis in BxPC-3 cells and the gemcitabine-resistant clones.
A, Respective cells were treated with DMSO or the indicatedconcentration of AR-A014418 for the designated times. The
relative number of viable cells at each time point was measured
by WST-8 assay. B,C, EdU-positive proliferating cells (B) and therelative number of apoptotic cells (C) were compared between
the respective cells treated with DMSO or AR-A014418. A-C,
The mean value from triplicate experiments was scored with SDs.
*P < .05

tion and with other targeted agents. This allows the optimization oftherapeutic efficacy and the minimization of undesired effects, aswell as preventing the acquisition of therapy resistance.30 We previously showed that deregulated GSK3β renders pancreatic cancer
cells intrinsically unresponsive to gemcitabine via the tumor protein p53 inducible nuclear protein (TP53INP)1-mediated DNA damage repair machinery and the impairment of Rb-mediated cell cycle
regulation.19,20 Therefore, we investigated whether GSK3β inhibition
affected cancer cells that had acquired resistance to gemcitabine.
AR-A014418 at 25 μmol/L alone showed adequate and similar therapeutic effects against BxG30, BxG140, and BxG400 cells

GSK3β inhibitors AR-A014418 and SB-216763 reduced the viability of(Figure 2). We therefore examined the therapeutic effects of all

BxPC-3 cells and the gemcitabine-resistant clones in a dose-depend-

combinations of AR-A014418 at a dose range of 1.56-25 μmol/L

ent and time-dependent manner, with similar IC50 values irrespective

and of gemcitabine at different doses in accordance with the IC50

of gemcitabine resistance (Figures 2A and S3A). The GSK3β inhibitorsof the respective gemcitabine-resistant clones (Table S3). Whenalso suppressed the proliferation of all cancer cells and induced their

BxG30, BxG140, and BxG400 cells were treated with gemcitabine

apoptosis (Figures 2B,C and S3B,C). Similar effects were observed in

at increasing doses, the combination with AR-A014418 reduced

these cancer cells following depletion of GSK3β (Figure S4). These re-the IC50 of gemcitabine in a dose-dependent fashion (Figure S5

sults indicated that GSK3β sustained the survival and proliferation of

and Table S4). Analysis of the data using the isobologram method25

BxPC-3 cells and of derived gemcitabine-resistant clones.

showed that AR-A014418 in combination with gemcitabine was syn-Compared with the parental BxPC-3 cells that are prone

ergistic against cancer cells from all gemcitabine-resistant clones

to going apoptosis following inhibition of GSK3β, the effect of(Figure 3A). Knockdown of GSK3β also significantly enhanced the

GSK3β inhibition on cell proliferation was more prominent thaneffects of gemcitabine against these cancer cells (Figure 3B), al-on apoptosis in the BxPC-3-derived gemcitabine-resistant clones

though the combined effects were not amenable to isobologram

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expression of RRM1 at both mRNA and protein levels in gemcitabineresistant cancer cells (Figure 4A,B), although the levels of RRM1 expression in BxG140 and BxG400 cells were still higher than in BxPC-3
and BxG30 cells at 72 h after treatment with AR-A014418 (Figure S6).
RRM1 is a transcriptional target for E2F1. The Rb tumor suppressor
protein normally traps E2F1, thereby repressing its transcriptional activity.31,32 We therefore hypothesized that GSK3β may interfere with
the Rb-mediated negative regulation of E2F1 during acquisition of resistance to gemcitabine in pancreatic cancer cells.
We have previously shown that expression of cyclin D1 and
CDK4 decreased in pancreatic cancer cells (including BxPC-3) concurrently with their sensitization to gemcitabine via GSK3β inhibition.20
Treatment with AR-A014418 consistently decreased the expression of
cyclin D1 and CDK4 in cells from BxPC-3-derived gemcitabine-resistant clones (Figure 4B). As the cyclin D1-CDK4/6 complex negatively
regulates the tumor suppressor function of Rb via its phosphorylation, we next investigated the expression and phosphorylation of Rbin these cells. Phosphorylation of Rb (pRbS807/811) is known to affectits ability to bind to E2F132 and was progressively higher in the gemcitabine-resistant clones compared with BxPC-3 cells. Treatment with
AR-A014418 decreased the level of pRbS807/811 but did not affect theexpression of Rb and E2F1 in the same cells (Figure 5A). This result suggested that GSK3β-mediated loss of Rb function may be involved in theacquisition of resistance to gemcitabine in pancreatic cancer.

3.5 | Changes in E2F1 transcriptional activity
following GSK3β inhibition in gemcitabine-resistantcancer cells
F I G U R E 3 Effect of gemcitabine in combination with GSK3βinhibitor or GSK3β-RNAi on BxPC-3 cells and gemcitabineresistant clones. A, The respective cells were treated with various
combinations of gemcitabine and/or AR-A014418 as shownin Table S3. The combined effect at 72 h after treatment wasmeasured by WST-8 assay and analyzed by the isobologram
method at respective doses (closed circle). B, The respective cells
were transfected with non-specific (N) or GSK3β-specific siRNA(S) and treated with the indicated concentrations of gemcitabine(GEM) for 72 h. Mean relative number of viable cells in triplicate
were scored with SDs and compared between the same cells withdifferent treatments. *P < .05

Further to the results shown above, we next examined whether GSK3βaffected the ability of Rb to bind to E2F1, as well as evaluating thetranscriptional activity of E2F1 in gemcitabine-resistant cancer cells.
Following treatment with DMSO or 25 μmol/L AR-A014418, nuclear
extracts from the most resistant BxG400 cells were immunoprecipitated with antibodies to Rb and E2F1. WB of these immunoprecipitates showed that GSK3β inhibition increased binding between Rb andE2F1 in the cell nuclei (Figure 5B). Luciferase reporter assay showedthat transcriptional activity of E2F1 in BxG140 and BxG400 cells
was significantly higher than in BxPC-3 cells. Treatment with GSK3βinhibitor reduced the transcriptional activity of E2F1 (Figure 5C) andanalysis. These results indicated that GSK3β inhibition could circum-

the expression of its target genes including RRM1 (Figure 4A), CCND1

vent acquired resistance to gemcitabine in pancreatic cancer cells.

(encoding cyclin D1), TS and thymidine kinase 1 (TK1) in both BxG140
and BxG400 cells (Figure 5D). These results suggested that GSK3β
may alter the functional interaction between Rb and E2F1 during the

3.4 | Changes in molecular characteristics ofgemcitabine-resistant cancer cells following
GSK3β inhibitionacquisition of gemcitabine resistance in pancreatic cancer.

To improve the treatment of refractory pancreatic cancer, it is impor-

3.6 | Effects of GSK3β inhibitors on BxPC-3 andBxG400 xenografts in micetant to clarify the molecular mechanism by which GSK3β inhibition alters the acquired resistance of pancreatic cancer cells to gemcitabine.

As a prerequisite for the clinical translation of GSK3β-targeted ther-

Strikingly, inhibition of GSK3β expression and activity decreased theapy for pancreatic cancer patients with resistance to gemcitabine,

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F I G U R E 4 Changes in expressionof RRM1, GSK3β and cell cycle
regulatory molecules in BxPC-3 cells
and gemcitabine-resistant clones
following GSK3β inhibition. A, The
relative expression of RRM1 mRNA wascompared between the respective cells
treated with DMSO or AR-A014418
for 24 h (left panel), and between cells
transfected with non-specific or GSK3βspecific siRNA (right panel). The meanvalue from triplicate experiments wasscored with SDs. *P < .05. B, Western
blotting analysis for expression of GSK3β,
RRM1 and cell cycle regulatory molecules(cyclin D1, CDK4, CDK6) and for GSK3βphosphorylation (pGSK3βY216) in therespective cells treated with DMSO or
AR-A014418 for 24 h. β-actin expression
was monitored as a loading control in eachsamplewe tested the efficacy of gemcitabine and AR-A014418, either

4 | D I S CU S S I O N

alone or in combination, against BxPC-3 and BxG400 cell xenograft
tumors in athymic mice (Figure S1A). Xenograft tumors of BxG400

Current first-line chemotherapy for locally advanced and meta-

cells grew faster than BxPC-3 tumors and were unresponsive

static pancreatic cancers consisted of 2 combination protocols,

to gemcitabine at 20 mg/kg, which was almost equivalent to the

FOLFILINOX and nab-paclitaxel with gemcitabine. Both have beenstandard clinical dose. Due to animal ethics issues, we euthanizedshown to improve the efficacy of gemcitabine monotherapy.33,34

the sham (DMSO)-treated mice with BxG400 xenografts and those

The only approved second-line regimen for patients who failed gem-

treated with gemcitabine at 6 and 8 wk, respectively, after treat-citabine-based therapy is nanoliposomal irinotecan with 5-FU andment. Compared with treatment with either AR-A014418 or gem-

folate.35 Importantly, however, no study has yet shown a significant

citabine alone, treatment of mice with the 2 agents in combination

improvement in outcome from any of the combination regimens oversignificantly reduced tumor growth in a time-dependent manner

gemcitabine alone in patients with poor (≥2) PS as defined by the(Figure 6). We observed no serious adverse events in the 4 groups

Eastern Cooperative Oncology Group. As the vast majority of pan-of mice during treatment, and there were no statistically significant

creatic cancer patients present with a PS ≥ 2, gemcitabine therefore

differences in mean body weight between the groups (Figure S1B).

remains the standard of treatment for pancreatic cancer.7,8 This in

At necropsy, gross observation and histological examination showedturn has attracted growing attention to the problem of gemcitabine

no lesions, primary tumors, or metastatic tumors in the major vital

resistance.9,10

organs of all mice.

Putative biochemical mechanisms of gemcitabine resistance in-

IHC examination of the tumors removed from sham (DMSO)

clude the decreased expression of human equilibrative nucleosideand gemcitabine-treated mice showed higher levels of active

transporter-1 (hENT1) that is indispensable for cellular uptake of

GSK3β (pGSK3β Y216) and RRM1 expression in BxG400 tumors

gemcitabine,36 the inactivation of deoxycytidine kinase (dCK) thatthan in BxPC-3 tumors. Treatment with AR-A014418 alone or in

is a late-limiting enzyme for metabolic activation of gemcitabine,37

combination with gemcitabine decreased the pGSK3β

Y216

level

and the overexpression of RRM1 that sustains DNA synthesis, thus

and RRM1 expression (Figure 7). Similar to the results from cellcounteracting the pharmacological action of gemcitabine. 29 These

culture studies (Figures 4B and 5A), cyclin D1 expression and Rb

molecular alterations found in the tumors were associated with poor

phosphorylation (pRbS807/811) in BxG400 tumors treated withsurvival of pancreatic cancer patients undergoing treatment withDMSO or gemcitabine alone were higher than in BxPC-3 tumors

gemcitabine in various clinical settings.38,39 Recent experimental

treated with the same agents, but were reduced following treat-approaches aimed at overcoming the acquired resistance to gem-

ment with AR-A014418 alone or in combination with gemcitabine

citabine include targeting of hENT1 expression by TS inhibitor and(Figure S7).

bypassing nucleoside transporters by prodrugs. Other approaches

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F I G U R E 5 Effects of GSK3β inhibitor on the expression and phosphorylation of Rb, its binding to E2F1 and the transcriptional activityof E2F1. A, Western blotting (WB) analysis for expression of Rb, E2F1 and phosphorylated of Rb (pRBS807/811) in cells treated with DMSO
or AR-A014418 for 24 h. β-actin expression was monitored as a loading control in each sample. B, Immunoprecipitates (IP) from nuclear
extracts of DMSO-treated or AR-A014418-treated BxG400 cells with non-immune mouse/rabbit IgG and the antibody to Rb or E2F1 wereanalyzed by WB with the indicated antibodies. C, Relative transcriptional activity of E2F1 in the respective cells treated with DMSO or ARA014418 was measured by luciferase reporter assay. D, Relative expression of CCND1 (cyclin D1), TK1, and TS mRNA in the respective cells
treated with DMSO or AR-A014418. C, D, Data are the mean values with SDs of triplicate experiments. *P < .05

included improving the intracellular delivery of gemcitabine by con-

GSK3β inhibitor synergistically enhanced the efficacy of gemcit-

jugating it with nanocarriers, as well as molecular targeting of theabine against all of the resistant clones in culture and against the

pro-oncogenic Wnt, hedgehog and Notch signaling that are reacti-

most resistant BxG400 xenografts in mice. These effects of GSK3βvated during acquired resistance to gemcitabine (reviewed in Ref.

inhibition were associated with decreased expression of RRM1, sug-9,10). However, there have been few attempts at targeting RRM1 togesting that impairment of the transcriptional activity of E2F1 mayenhance the efficacy of gemcitabine, even though it is recognized as

be responsible for the “resensitization” of resistant clones to gem-a potentially strong target for overcoming gemcitabine resistance.40

citabine. GSK3β inhibition consistently attenuates E2F1 transcrip-

The gemcitabine-resistant clones (BxG30, BxG140, BxG400)

tional activity, resulting in decreased expression of its transcriptional

used in this study were established from gemcitabine-sensitive

targets including RRM1, CCND1, TS, and TK1. As we previously re-pancreatic cancer BxPC-3 cells and were characterized by overex-

ported, 20 GSK3β inhibition decreases the expression of cyclin D1

pression of RRM1, but only minor alterations in the expression of

and CDK4 and phosphorylation of Rb, thereby restoring the binding

hENT1, dCK, RRM2, and Bcl2. 22 This suggested that there may beof Rb to E2F1 in tumor cell nuclei. Consequently, our results suggest

dependency of these resistant clones on RRM1 for the acquisition

that the mechanism whereby GSK3β confers acquired resistance toof resistance to gemcitabine. We showed that GSK3β inhibitors

gemcitabine in pancreatic cancer is via disturbance of the physiolog-

alone exerted therapeutic effects against BxPC-3 cells as well as

ical (tumor-suppressive) interaction between Rb and E2F1.

against gemcitabine-resistant clones with a similar IC50. Notably,

The levels of RRM1 expression in the gemcitabine-resistantwhen combined with various concentrations of gemcitabine, theBxG140 and BxG400 cells decreased during longer treatment

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F I G U R E 6 Efficacy of gemcitabineand GSK3β inhibitor on BxPC-3 andBxG400 xenograft tumors in athymic
mice. A, Time course of mean xenograft
tumor volume with SDs in mice treatedwith DMSO, gemcitabine alone, ARA014418 alone, or the gemcitabine/
AR-A014418 combination. Mice with
the BxG400 xenograft and treated withDMSO or gemcitabine were euthanized at
6 and 8 wk, respectively, after treatment
because of animal experiment ethicalissue. *P < .05. B, Gross appearance of
xenograft tumors removed at autopsy
from the micewith AR-A014418, but they were still higher than in BxPC-3 andFurthermore, combination with GSK3β inhibitor may potentially

BxG30 cells (Figure S6). Isobologram analysis showed synergis-

enhance the efficacy of FORFIRINOX, nab-paclitaxel with gemcit-

tic therapeutic effects of gemcitabine and AR-A014418 in com-abine, nanoliposomal irinotecan with 5-FU, and folate, as well as

bination against both resistant clones (Figure 3A). Nevertheless,ionizing radiation. In addition to pancreatic ductal adenocarcinoma,

AR-A014418 could substantially, but not completely, reverse thea previous study reported that GSK3β participates in tumor progres-resistance to gemcitabine in BxG140 and BxG400 cells (Figure S5)sion and resistance to everolimus, an inhibitor of mechanistic target

and BxG400 xenograft tumors in rodents (Figure 6). An interme-of rapamycin complex 1, in pancreatic neuroendocrine neoplasm.41

diate metabolite of gemcitabine, dFdCDP (2′-2′-difluoro-2′-de-Collectively, GSK3β may potentially play broader pathologic roles inoxycytidine diphosphate), potently binds to and inhibits RRM1,

pancreatic malignancy.

thereby exerting its therapeutic effect via decrease of compet-

The proposed biological mechanisms for gemcitabine resis-

ing deoxyribonucleotide pools necessary for DNA synthesis (re-tance in cancer cells include pro-invasive capacity and cancer

viewed in Ref.9). Our results may therefore imply that the amountstemness phenotypes (reviewed in Ref. 9,10). Previous studies

of RRM1 in the resistant clones treated with both gemcitabine andhave shown that gemcitabine-resistant pancreatic cancer cells ac-

AR-A014418 exceeded the dose of gemcitabine. Collectively, it isquire a pro-invasive phenotype such as EMT,42 thus contributing

suggested that the remaining RRM1 in the resistant clones after

to acquired resistance.43,44 Based on the notion of an interconnec-

treatment with GSK3β inhibitor may still contribute to gemcitabinetion between cancer invasion and therapy resistance,45 we pre-resistance. As we previously reported,

22

resistance to gemcitabine

viously showed that GSK3β facilitates both pro-invasive capacity

in these clones was not only dependent largely on RRM1, but also

and resistance to chemotherapy in pancreatic cancer 20 and glio-on the other known factors (such as hENT1, dCK, RRM2, and Bcl2)

blastoma.46,47 It has also been reported that gemcitabine treat-and probably on unknown factors. Accordingly, future systematic

ment promotes pancreatic cancer stemness through a distinct

analysis of RRM1 and these factors is necessary to clarify whether

molecular pathway.48 In light of the mounting evidence for tu-

any remaining RRM1 in the resistant clones (BxG140, BxG400)

mor-promoting roles of GSK3β, we propose that GSK3β functions

following the treatment with GSK3β inhibitor contributes to theas a molecular hub that integrates therapy resistance, pro-invasive

gemcitabine resistance in these cells.

capacity, and the cancer stemness phenotype in refractory cancer,

As previously reported, the gemcitabine-resistant clones exam-as represented by pancreatic cancer.15,16 This cancer type is also

ined in this study were also resistant to 5-FU, cisplatin, irinotecancharacterized biologically by a desmoplastic and immunosuppres-(CPT-11), and docetaxel. 22 As TS and TK1 are known biomarkers forsive tumor microenvironment that has emerged as a robust barrier

the efficacy of 5-FU and ionizing radiation, respectively, GSK3β may

to various therapeutic agents and radiation.49,50 Recent evidence

participate in cross-resistance to multiple chemotherapeutic agents.

has suggested that GSK3β plays an active role in establishing the

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金沢大学がん進展制御研究所 腫瘍制御 (旧：遺伝子診断) 研究分野
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UEHARA Et Al.

F I G U R E 7 Histological andimmunohistochemical findings ofxenograft tumors of BxPC-3 (A) andBxG400 (B) cells in mice treated with
DMSO, gemcitabine alone, AR-A014418
alone or the gemcitabine/AR-A014418
combination. Serial sections of thetumors were stained with H&E andimmunostained for GSK3β, pGSK3βY216,
and RRM1. Scale bars, 100 μm

immunosuppressive tumor environment (reviewed in Ref. 16).

Australia) for critical review and editing of the manuscript. This

Therefore, further research in this area is important for under-

study was supported by Grants-in-Aid for Scientific Research fromstanding the broader biological mechanisms of GSK3β-mediatedthe Ministry of Education, Culture, Sports, Science and TechnologyJapan and from the Japan Society for the Promotion of Science

therapy resistance in pancreatic cancer.

(T. Minamoto).
AC K N OW L E D G M E N T S
We acknowledge Dr. Ilya V. Pyko in our laboratory for assistance with

D I S C LO S U R E

animal experiments and Dr. Barry Iacopetta (University of Western

The authors have no conflict of interest.

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研究のあゆみと業績 2021

| 4415

UEHARA Et Al.

ORCID
Masahiro Uehara

https://orcid.org/0000-0002-9874-2672

Takahiro Domoto

https://orcid.org/0000-0002-8040-7630

Osamu Takeuchi

https://orcid.org/0000-0003-3826-8654

Toshinari Minamoto

22.

23.

https://orcid.org/0000-0003-4168-996X

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S U P P O R T I N G I N FO R M AT I O N
Additional supporting information may be found online in the
Supporting Information section.

How to cite this article: Uehara M, Domoto T, Takenaka S,
et al. Glycogen synthase kinase-3β participates in acquired
resistance to gemcitabine in pancreatic cancer. Cancer Sci.
2020;111:4405–4416. https://doi.org/10.1111/cas.14668

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