研究室リレー紹介

病態制御医学講座 内科学(２)分野

消化器内科

脳神経内科

中本 安成、大谷 昌弘、髙橋 和人、内藤 達志、野阪 拓人、田中 知子、赤澤 悠、

濵野 忠則、榎本 崇一、遠藤 芳徳、菅田 亮太郎、山本 有紗、木村 剛徳、山下 順貴、内藤 豊智、野宮 廣貴

北﨑 佑樹、佐々木 宏仁、堀内 裕介

Hepatocellular carcinoma progression promoted by 5-lipoxygenase
activity in CD163(+) tumor-associated macrophages

Longer Survival and Preserved Liver Function after Proton Beam
Therapy for Patients with Unresectable Hepatocellular Carcinoma

Biomed Pharmacother. 2023 Jun;162:114592.

Curr Oncol. 2023 Mar 30;30(4):3915-3926.

Takuto Nosaka, Yasunari Nakamoto et al.

Takuto Nosaka, Yasunari Nakamoto et al.

Arachidonic acid 5-lipoxygenase (5-LOX), an enzyme that synthesizes leukotrienes (LTs), is involvedin cancer development including proliferation, invasion, metastasis and drug resistance. However, thefunctional role of 5-LOX in hepatocellular carcinoma (HCC) remains to be elucidated. In this study, weanalyzed the contribution of 5-LOX in HCC progression and investigated the potential of targeted
therapy. Analysis of 86 resected HCC specimens and the clinical data of 362 cases of liver cancerfrom The Cancer Genome Atlas Liver Hepatocellular Carcinoma dataset, showed that 5-LOX
expression was associated with postoperative survival. The cancer proliferative and stem cell potential
were correlated with the levels of 5-LOX in CD163(+) tumor-associated macrophages (TAMs). In an
HCC mouse model, CD163(+) TAMs expressed 5-LOX and produced LTB4 and LTC/D/E4; the 5-LOX
inhibitor, zileuton, suppressed HCC progression. LTB4 and LTC/D/E4 promoted cancer proliferationand stem cell capacity via phosphorylation of extracellular signal-regulated kinase 1/2 and stem cellassociated genes. Taken together, we identified a novel mechanism of HCC progression in which
CD163(+) TAMs express 5-LOX and produce LTB4 and LTC/D/E4, thereby enhancing the proliferativeand stem cell potential of HCC cells. Furthermore, inhibition of 5-LOX activity regulates HCC
progression, suggesting it has potential as a new therapeutic target.

Background: Proton beam therapy (PBT) has been recently reported to achieve excellent tumor
control with minimal toxicity in patients with unresectable hepatocellular carcinoma (HCC).
Radiofrequency ablation (RFA) combined with transcatheter arterial chemoembolization (TACE) wasinvestigated for larger HCC. This study was designed to evaluate the therapeutic effect of PBT onunresectable HCC in comparison with TACE combined with RFA.
Methods: We retrospectively analyzed 70 patients with HCC which was difficult to control by surgicalresection or RFA monotherapy, 24 patients treated with PBT and 46 patients with TACE plus RFA. The
therapeutic effects were assessed as local progression-free survival (PFS) and overall survival (OS).
Results: The local PFS was more than 65% in 60 months for PBT and TACE plus RFA. The patientstreated with PBT showed 82% OS at 60 months post-treatment. In contrast, those treated with TACE
plus RFA showed 28% OS. When comparing the changes of ALBI scores in patients with different
severities of chronic liver disease, the scores of PBT-treated patients were maintained at the baseline;
however, those of TACE plus RFA-treated patients worsened after the treatments.
Conclusions: The results indicated that PBT may show better benefits than TACE plus RFA therapy
in terms of OS in patients with unresectable HCC by sparing the non-tumor liver tissues.

ab76 y.o., M, 3.3 cm, 0.8 cm, Vp0, Vv0, BCLC-C,NBNC-LC, Child-Pugh Class A (5), mALBI grade 1

Overall survival

Proton beam therapy
Probability of survival rate (%)

PBT

#1

TACE+RFA

100p = 0.0267
Hazard ratio, 3.47
(95% CI, 1.55–7.77)

50

#2

00

10

2030Months

40

5060

PBT

24

21

16

13

75

4

TACE+RFA

46

4240

29

21136c2

PBT #2

ALBI grade 3

PBT #10ALBI score

grade 2-2grade 1-3Months

5048

46

44

4240

3836343230

28

2624

22

20

18161412108642-40ALBI score-1Changes in ALBI score

Number at risk

N.S.

10-10 1 2 3 4 5 6 7 8 9 10 11 12Months

Number at risk
24 24 24 24 23 22 21 20 19 18 18 18 18

Regulatory function of interferon-inducible 44-like for hepatitis Bvirus covalently closed circular DNA in primary human hepatocytes

Molecular signature of hepatitis B virus regulation by interferon-γ inprimary human hepatocytes

Hepatol Res. 2022 Feb;52(2):141-152.

Hepatol Res. 2020 Mar;50(3):292-302.

Takuto Nosaka, Yasunari Nakamoto et al.

Takuto Nosaka, Yasunari Nakamoto et al.

Aim: Curing hepatitis B virus (HBV) infection requires elimination of covalently closed circular DNA(cccDNA). Interferon (IFN)-γ has noncytolytic antiviral potential; however, elimination of cccDNA could
not be achieved. To enhance the regulatory effect, we comprehensively analyzed the host factors
associated with cccDNA amplification and IFN-γ and IFN-α effects using an in vitro HBV infection
system showing various transcription levels.
Methods: Primary human hepatocytes were infected with HBV using genomic plasmids carrying thebasic core promoter mutation A1762T/G1764A and/or the precore mutation G1896A and treated withIFN-γ and IFN-α. Comprehensive and functional studies involving microarray and small interfering
RNA analysis revealed the host factors related to cccDNA regulation.
Results: The HBV infection system reproduced the HBV life cycle and showed various propagationlevels. Microarray analysis revealed 53 genes correlated with the cccDNA levels. Of the 53 genes,expression of IFN-induced protein 44-like (IFI44L) was significantly upregulated by IFN-γ and IFN-α.
The anti-HBV effect of IFI44L is exerted regardless of IFN-γ or IFN-α by inhibiting the activation ofnuclear factor-κB and signal transducer and activator of transcription 1 pathways.
Conclusions: Using the in vitro HBV infection system, an IFN-inducible molecule, IFI44L, associatedwith cccDNA amplification, was identified. These results suggest an innovative molecular strategy forthe regulation of HBV cccDNA by controlling a novel host factor, IFI44L.

Aim: A complete cure for chronic hepatitis B virus (HBV) infection requires elimination of covalently
closed circular DNA; however, this remains to be clinically achieved. Interferon (IFN)-γ, a type II IFN, is
produced by intrahepatic cytotoxic T lymphocytes and has non-cytolytic antiviral potential. However,
the mechanism by which IFN-γ regulates HBV infection has not been fully elucidated. Thus, wedeveloped an in vitro HBV infection assay system and analyzed the molecular signature of HBVregulation by IFN-γ.
Methods: The in vitro HBV infection assay system was established in primary human hepatocytes
infected with HBV derived from the plasmid containing 1.3-mer HBV genome, and treated with IFN-γ.
The antiviral effects and signaling pathways of IFN-γ were examined using microarray, and assessedby siRNA knockdown experiments of the related genes.
Results: IFN-γ treatment suppressed both HBV propagation and transcription as efficiently as IFN-α.
Microarray analysis showed that IFN-γ stimulation induced the activation of both IFN-γ and IFN-α
signaling, regulating HBV covalently closed circular DNA. HBV production was decreased by IFN-γ
through Janus kinase/signal transducer and activator of transcription signaling and interferonstimulated genes, such as 2'-5'-oligoadenylate synthase 2 and apolipoprotein B mRNA editing enzyme
catalytic subunit 3G.
Conclusions: IFN-γ can suppress HBV propagation and transcription in hepatocytes by activating
specific intracellular signaling pathways in hepatocytes, and suggests the future application of theseparticular signaling pathways or genes for the complete elimination of HBV.

Human hepatocyteIFN-γ/α

HBV

PXB

Plasmamembrane

PXB cell

HBV infection
Genotype C2 High t.

Cytoplasm

HBV infection
IFN-γ Day23

STAT1
pathway ISG

IKKαIFI44L

IKKβ

STAT1

NF-κB
pathway

Nucleus

STAT1

NF-κB

(Agilent Expression GE 8x60K v3 Microarray)

60,902 RNAs

Interferon gamma signaling
Interferon alpha-beta signaling

Control

IFI44L

IFN-γ

IFN-αβpathway

NF-κB

RNA microarray

IFN-α
ISG
OAS2
APOBEC3G

cccDNA

Alveolar Macrophages Drive Hepatocellular Carcinoma Lung
Metastasis by Generating Leukotriene B4Gene expression profiling of hepatocarcinogenesis in a mousemodel of chronic hepatitis B

J Immunol. 2018 Mar 1;200(5):1839-1852.

PLoS One. 2017 Oct 2;12(10):e0185442.

Takuto Nosaka, Yasunari Nakamoto et al.

Takuto Nosaka, Yasunari Nakamoto et al.

Macrophages in lungs can be classified into two subpopulations, alveolar macrophages (AMs) andinterstitial macrophages (IMs), which reside in the alveolar and interstitial spaces, respectively.
Accumulating evidence indicates the involvement of IMs in lung metastasis, but the roles of AMs inlung metastasis still remain elusive. An i.v. injection of a mouse hepatocellular carcinoma (HCC) cellline, BNL, caused lung metastasis foci with infiltration of AMs and IMs. Comprehensive determinationof arachidonic acid metabolite levels revealed increases in leukotrienes and PGs in lungs in thismetastasis model. A 5-lipoxygenase (LOX) inhibitor but not a cyclooxygenase inhibitor reduced thenumbers of metastatic foci, particularly those of a larger size. A major 5-LOX metabolite, LTB4,augmented in vitro cell proliferation of human HCC cell lines as well as BNL cells. Moreover, in thislung metastasis course, AMs exhibited higher expression levels of the 5-LOX and LTB4 than IMs.
Consistently, 5-LOX-expressing AMs increased in the lungs of human HCC patients with lungmetastasis, compared with those without lung metastasis. Furthermore, intratracheal clodronate
liposome injection selectively depleted AMs but not IMs, together with reduced LTB4 content andmetastatic foci numbers in this lung metastasis process. Finally, IMs in mouse metastatic fociproduced CCL2, thereby recruiting blood-borne, CCR2-expressing AMs into lungs. Thus, AMs can be
recruited under the guidance of IM-derived CCL2 into metastatic lungs and can eventually contributeto the progression of lung metastasis by providing a potent arachidonic acid-derived tumor growthpromoting mediator, LTB4.

Background: Hepatocellular carcinoma (HCC) is a common complication of chronic viral hepatitis. In
support of this notion, we have reported that hepatitis B surface antigen (HBsAg)-specific CD8+ T
lymphocytes critically contribute to inducing chronic liver cell injury that exerts high carcinogenic
potential in a hepatitis B virus (HBV) transgenic mouse model. The dynamics of the molecularsignatures responsible for hepatocellular carcinogenesis are not fully understood. The current study
was designed to determine the serial changes in gene expression profiles in a model of chronic
immune-mediated hepatitis.
Methods: Three-month-old HBV transgenic mice were immunologically reconstituted with bonemarrow cells and splenocytes from syngeneic nontransgenic donors. Liver tissues were obtained
every three months until 18 months at which time all mice developed multiple liver tumors. Nitrative
DNA lesions and hepatocyte turnover were assessed immunohistochemically. Gene expression
profiles were generated by extracting total RNA from the tissues and analyzing by microarray.
Results: The nitrative DNA lesions and the regenerative proliferation of hepatocytes were increased
during the progression of chronic liver disease. In a gene expression profile analysis of liver samples,the chemokine- and T cell receptor (TCR)-mediated pathways were enhanced during chronic hepatitis,
and the EGF- and VEGF-mediated pathways were induced in HCC. Among these molecules, theprotein levels of STAT3 were greatly enhanced in all hepatocyte nuclei and further elevated in thecytoplasm in HCC tissue samples at 18 months, and the levels of phosphorylated TP53 (p-p53-Ser 6and -Ser 15) were increased in liver tissues.
Conclusions: HBV-specific immune responses caused unique molecular signatures in the livertissues of chronic hepatitis and triggered subsequent carcinogenic gene expression profiles in amouse model. The results suggest a plausible molecular basis responsible for HBV-induced immune
pathogenesis of HCC.

Day 21CCR210 mm

Progenitor ofAM

CCL2

HBV特異的免疫による慢性肝炎発癌マウスモデル

TCD8+

Proliferation

IFNγ

HBsAg

1 wk

9 mos

18 mosInterstitialmacrophage
Cancer cell

LTB4

Alveolarmacrophage (AM)HCC(破裂)肝臓の分子病態Pre

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http://www-n.med.u-fukui.ac.jp/laboratory/second-internal/

https://www.youtube.com/watch?v=yCdzmk8npIw