NAFLD/NASH肝組織リン酸化活性プロファイリングと新規治療標的分子の同定

• Project/Area Number: 18H02793
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 53010:Gastroenterology-related
• Research Institution: Kanazawa University
• Principal Investigator: 本多 政夫 金沢大学, 保健学系, 教授 (00272980)
• Co-Investigator(Kenkyū-buntansha): 堀本 勝久 国立研究開発法人産業技術総合研究所, 生命工学領域, 招聘研究員 (40238803) ; 鍵和田 晴美 国立研究開発法人産業技術総合研究所, 生命工学領域, 主任研究員 (40443204)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Project Status: Granted (Fiscal Year 2020)
• Budget Amount: ¥17,290,000 (Direct Cost: ¥13,300,000、Indirect Cost: ¥3,990,000); Fiscal Year 2020: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2019: ¥5,720,000 (Direct Cost: ¥4,400,000、Indirect Cost: ¥1,320,000); Fiscal Year 2018: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000)
• Keywords: 非アルコール性脂肪性肝炎 / リン酸化 / プロテインアレイ

Outline of Annual Research Achievements

申請者らは世界最大のヒトタンパク質発現リソースHuPEX (Human Proteome Expression Resource)（約2万種）を基に高操作性・ハイスループットなプロテインアレイを作製し、正味のチロシンキナーゼ活性を高感度に測定する系を初めて確立した。本研究目的は新技術プロテインアレイを用いて、NAFLD/NASH肝組織のリン酸化プロファイルを明らかにして新規治療標的を同定する。
本年度はphosphoproteome(Dev Cell. 2018 Oct 22;47(2):205-221)のデータベースを基にマウス肝組織における標的リン酸化蛋白の発現解析を行った。マウスのphosphoproteomeデータベースから、Ath+HFD（動脈硬化高脂肪食）マウスで遺伝子発現を認めた32974遺伝子中、3627遺伝子がリン酸化蛋白であることが解った。そのうち、病態の進行と共に発現変動を示す遺伝子は2054個認められた（p<0.001）。30週から60週齢（肝発癌）にかけ、有意に発現上昇するリン酸化蛋白（884個）は線維化・炎症と関連し、発現低下するリン酸化蛋白（1170個）はユビキチンプロテアソーム・HSPなど異常蛋白分解に関わる遺伝子であった。非環式レチノイドの投与により線維化・炎症に関連する約65％の遺伝子で発現の改善が見られた。しかし、MEK1/2、SREBP1、p38MAPK、IRS-1、S100A8など含む約35％の遺伝子では発現の改善が見られなかった。これらの遺伝子のリン酸化の変動を解析中である。Ath+HFDマウス肝組織では12週（脂肪肝10サンプル）、30週（肝線維化10サンプル）、60週（肝発癌10サンプル）のリン酸化アレイを行っている。

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

Ath+HFD(動脈硬化高脂肪食) マウス肝組織における標的リン酸化蛋白の候補を同定した。同肝組織におけるリン酸化プロファイルを施行中である。

Strategy for Future Research Activity

Ath+HFDマウス肝組織の遺伝子発現データとリン酸化アレイのデータを統合し、新規治療標的遺伝子を同定する。
ヒトゲノム倫理委員会の承認が金沢大学及び産総研双方で承認されたため、2019年度からヒトNAFLD/NASH肝組織を用いたリン酸化活性プロファイリングの同定を行う。研究代表者の本多はこれまでに100症例のNAFLD/NASH肝組織のmRNA遺伝子発現プロファイルを明らかにした（Cell Metab 2010, Diabetologia 2014, Liver Int 2015）。得られたデータを基にmRNA遺伝子発現プロファイルとリン酸化活性プロファイリングの数理解析を行う（堀本）。NAS（NAFLD Activity Score）、（Steatosis: 0～3，Lobular inflammation: 0～3，Hepatocyte ballooning: 0～2）及びFibrosis (Stage:0～4)を層別化し、脂肪化・炎症・肝細胞風船様変化及び線維化の進行に伴う遺伝子発現変化及びリン酸化シグナル変化をそれぞれ同定する。また、Matteoni分類やBrunt分類に即した層別化を行い、脂肪化・炎症・線維化進行に関わるシグナルパスウェイを同定する。

Research Products

• [Journal Article] Three renal failure cases successfully treated with ombitasvir/paritaprevir/ritonavir for genotype 1b hepatitis C virus reinfection after liver transplantation (2019)
  • Author(s): Orita N, Shimakami T, Sunagozaka H, Horii R, Nio K, Terashima T, Iida N, Kitahara M, Takatori H, Kawaguchi K, Kitamura K, Arai K, Yamashita T, Sakai Y, Yamashita T, Mizukoshi E, Honda M, Kaneko S.
  • Journal Title: Clinical Journal of Gastroenterology Volume: 12 Pages: 63-70
  • DOI: 10.1007/s12328-018-0884-y
  • Peer Reviewed / Open Access
• [Journal Article] Characteristics of Immune Response to Tumor-Associated Antigens and Immune Cell Profile in Patients With Hepatocellular Carcinoma (2019)
  • Author(s): Y Inada, E Mizukoshi, T Seike, T Tamai, N Iida, M Kitahara, T Yamashita, K Arai, T Terashima, K Fushimi, T Yamashita, M Honda, S Kaneko.
  • Journal Title: Hepatology Volume: 69 Pages: 653-665
  • DOI: 10.1002/hep.30212
  • Peer Reviewed / Open Access
• [Journal Article] POGLUT1, the putative effector gene driven by rs2293370 in primary biliary cholangitis susceptibility locus chromosome 3q13.33. (2019)
  • Author(s): Hitomi, Y. Ueno, K. Kawai, Y. Nishida, N. , Umemura, T. et al
  • Journal Title: Sci Rep. Volume: 9 Pages: 102-102
  • DOI: 10.1038/s41598-018-36490-1
  • NAID: 120006715480
  • Peer Reviewed / Open Access
• [Journal Article] Serum aldo?keto reductase family 1 member B10 predicts advanced liver fibrosis and fatal complications of nonalcoholic steatohepatitis (2019)
  • Author(s): M Kanno, K Kawaguchi, M Honda, R Horii, H Takatori, T Shimakami, K Kitamura, K Arai, T Yamashita, Y Sakai, T Yamashita, E Mizukoshi, S Kaneko.
  • Journal Title: Journal of Gastroenterology Volume: -
  • DOI: 10.1007/s00535-019-01551-3
  • Peer Reviewed / Open Access
• [Journal Article] Notch signaling facilitates hepatitis B virus covalently closed circular DNA transcription via cAMP response element-binding protein with E3 ubiquitin ligase-modulation (2019)
  • Author(s): Wang Z, Kawaguchi K, Honda M, Hashimoto S, Shirasaki T, Okada H, Orita N, Shimakami T, Yamashita T, Sakai Y, Mizukoshi E, Murakami S, Kaneko S.
  • Journal Title: Scientific reports Volume: 9 Pages: 1621-1621
  • DOI: 10.1038/s41598-018-38139-5
  • Peer Reviewed / Open Access
• [Journal Article] Distinct chemotherapy-associated anti-cancer immunity by myeloid cells inhibition in murine pancreatic cancer models (2019)
  • Author(s): Sakai Y, Miyazawa M, Komura T, Yamada T, Nasti A, Yoshida K, Takabatake H, Yamato M, Yamashita T, Yamashita T, Mizukoshi E, Okuzono M, Ho TTB, Kawaguchi K, Wada T, Honda M, Kaneko S.
  • Journal Title: Cancer Science Volume: 110 Pages: 903-912
  • DOI: 10.1111/cas.13944
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Adipose tissue‐derived stem cells prevent fibrosis in murine steatohepatitis by suppressing IL‐17‐mediated inflammation (2019)
  • Author(s): Yamato Masatoshi、Sakai Yoshio、Mochida Hatsune、Kawaguchi Kazunori、Takamura Masayuki、Usui Soichiro、Seki Akihiro、Mizukoshi Eishiro、Yamashita Taro、Yamashita Tatsuya、Ishida Kousuke、Nasti Alessandro、Tuyen Ho Thuy Bich、Komura Takuya、Yoshida Keiko、Wada Takashi、Honda Masao、Kaneko Shuichi
  • Journal Title: Journal of Gastroenterology and Hepatology Volume: -
  • DOI: 10.1111/jgh.14647
  • Peer Reviewed / Open Access
• [Journal Article] Serum C16:1n7/C16:0 ratio as a diagnostic marker for non‐alcoholic steatohepatitis (2019)
  • Author(s): Yamada Kazutoshi、Mizukoshi Eishiro、Seike Takuya、Horii Rika、Terashima Takeshi、Iida Noriho、Kitahara Masaaki、Sunagozaka Hajime、Arai Kuniaki、Yamashita Tatsuya、Honda Masao、Takamura Toshinari、Harada Kenichi、Kaneko Shuichi
  • Journal Title: Journal of Gastroenterology and Hepatology Volume: Epub ahead of print
  • DOI: 10.1111/jgh.14654
  • Peer Reviewed / Open Access
• [Journal Article] Development of novel diagnostic system for pancreas cancer including early stages measuring mRNA of whole blood cells (2019)
  • Author(s): Sakai Yoshio、Honda Masao、Matsui Shigeyuki、Komori Osamu、Murayama Toshinori、Fujiwara Tadami、Mizuno Masaaki、Imai Yasuhito、Yoshimura Kenichi、Nasti Alessandro、Wada Takashi、Iida Noriho、Kitahara Masaaki、Horii Rika、Toshikatsu Tamai、Nishikawa Masashi、Okafuji Hirofumi、Mizukoshi Eishiro、et al
  • Journal Title: Cancer Science Volume: 110 Pages: 1364-1388
  • DOI: 10.1111/cas.13971
  • Peer Reviewed / Open Access
• [Journal Article] Analysis of the liver functional reserve of patients with advanced hepatocellular carcinoma undergoing sorafenib treatment: prospects for regorafenib therapy. (2018)
  • Author(s): Terashima T, Yamashita T, Sunagozaka H, Arai K, Kawaguchi K, Kitamura K, Yamashita T, Sakai Y, Mizukoshi E, Honda M, Kaneko S.
  • Journal Title: Hepatology Research Volume: 48 Pages: 956-966
  • DOI: 10.1111/hepr.13196
  • Peer Reviewed / Open Access
• [Journal Article] Immune responses against tumour-associated antigen-derived cytotoxic T lymphocyte epitopes in cholangiocarcinoma patients (2018)
  • Author(s): Kida A, Mizukoshi E, Tamai T, Kitahara M, Yamashita T, Arai K, Terashima T, Iida N, Fushimi K, Kaneko S.
  • Journal Title: Liver International Volume: 38 Pages: 2040-2050
  • DOI: 10.1111/liv.13885
  • Peer Reviewed / Open Access
• [Journal Article] Prevention of hepatocellular carcinoma by targeting MYCN-positive liver cancer stem cells with acyclic retinoid. (2018)
  • Author(s): Qin XY, Suzuki H, Honda M, Okada H, Kaneko S, Inoue I, Ebisui E, Hashimoto K, Carninci P, Kanki K, Tatsukawa H, Ishibashi N, Masaki T, Matsuura T, Kagechika H, Toriguchi K, Hatano E, Shirakami Y, Shiota G, Shimizu M, Moriwaki H, Kojima S
  • Journal Title: Proc Natl Acad Sci U S A. Volume: 115 Pages: 4969-4974
  • DOI: 10.1073/pnas.1802279115
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Genome-wide association study identified new susceptible genetic variants in HLA class I region for hepatitis B virus-related hepatocellular carcinoma. (2018)
  • Author(s): awai H, Nishida N, Sugiyama M, Murata K, Hino K, Nishina S, Matsumoto A, Tanaka E, Sakamoto N, Ogawa K, Yamamoto K, Tamori A, Yokosuka O, Kanda T, Sakaida I, Itoh Y, Eguchi Y, Oeda S, Mochida S, Yuen MF, Seto WK, Poovorawan Y, Posuwan N, Mizokami M, Tokunaga K
  • Journal Title: Scientific Reports Volume: 8 Pages: 7958-7958
  • DOI: 10.1038/s41598-018-26217-7
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Unexpected Replication Boost by Simeprevir for Simeprevir-Resistant Variants in Genotype 1a Hepatitis C Virus (2018)
  • Author(s): Murai K, Shimakami T, Welsch C, Shirasaki T, Liu F, Kitabayashi J, Tanaka S, Funaki M, Omura H, Nishikawa T, Suminyadorj A, Honda M, Kaneko S.
  • Journal Title: Antimicrobial Agents and Chemotherapy Volume: 印刷中
  • DOI: 10.1128/aac.02601-17
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Distinct notch signaling expression patterns between nucleoside and nucleotide analogues treatment for hepatitis B virus infection (2018)
  • Author(s): Wang Z, Kawaguchi K, Honda M, Sakai Y, Yamashita T, Mizukoshi E, Kaneko S.
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 501 Pages: 682-687
  • DOI: 10.1016/j.bbrc.2018.04.236
  • Peer Reviewed / Open Access
• [Journal Article] Serum Wisteria floribunda agglutinin-positive Mac-2 binding protein predicts hepatocellular carcinoma incidence and recurrence in nucleos(t)ide analogue therapy for chronic hepatitis B (2018)
  • Author(s): Kawaguchi K, Honda M, Ohta H, Terashima T, Shimakami T, Arai K, Yamashita T, Sakai Y, Yamashita T, Mizukoshi E, Komura T, Unoura M, Kaneko S
  • Journal Title: Journal of Gastroenterology Volume: ePub ahead Pages: 740-751
  • DOI: 10.1007/s00535-017-1386-2
  • Peer Reviewed / Open Access
• [Journal Article] Immune responses of human T lymphocytes to novel hepatitis B virus-derived peptides (2018)
  • Author(s): Yamamiya D, Mizukoshi E, Kaji K, Terashima T, Kitahara M, Yamashita T, Arai K, Fushimi K, Honda M, Kaneko S.
  • Journal Title: PLOS ONE Volume: 13
  • DOI: 10.1371/journal.pone.0198264
  • Peer Reviewed / Open Access
• [Journal Article] Immune Condition of Colorectal Cancer Patients Featured by Serum Chemokines and Gene Expressions of CD4+ Cells in Blood (2018)
  • Author(s): Komura T, Yano M, Miyake A, Takabatake H, Miyazawa M, Ogawa N, Seki A, Honda M, Wada T, Matsui S, Kaneko S, Sakai Y.
  • Journal Title: Canadian Journal of Gastroenterology and Hepatology Volume: 7436205 Pages: 7436205-7436205
  • DOI: 10.1155/2018/7436205
  • Peer Reviewed / Open Access
• [Journal Article] The osteopontin-CD44 axis in hepatic cancer stem cells regulates IFN signaling and HCV replication (2018)
  • Author(s): Shirasaki T, Honda M, Yamashita T, Nio K, Shimakami T, Shimizu R, Nakasyo S, Murai K, Shirasaki N, Okada H, Sakai Y, Sato T, Suzuki T, Yoshioka K, Kaneko S.
  • Journal Title: Scientific Reports Volume: 8 Pages: 13143-13143
  • DOI: 10.1038/s41598-018-31421-6
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Biological characteristics of gene expression features in pancreatic cancer cells induced by proton and X-ray irradiation (2018)
  • Author(s): Fujinaga H, Sakai Y, Yamashita T, Arai K, Terashima T, Komura T, Seki A, Kawaguchi K, Nasti A, Yoshida K, Wada T, Yamamoto K, Kume K, Hasegawa T, Takata T, Honda M, Kaneko S.
  • Journal Title: International Journal of Radiation Biology Volume: 17 Pages: 1-9
  • DOI: 10.1080/09553002.2019.1558297
  • Peer Reviewed / Open Access
• [Presentation] NASH患者における肝線維化と血清AKR1B10との関連 (2018)
  • Author(s): 川口和紀，神野正隆，本多政夫，金子周一
  • Organizer: 第104回日本消化器病学会総会
• [Presentation] ペレチノインの作用機序からみた肝癌化学予防療法の個別化医療の可能性 (2018)
  • Author(s): 山下太郎，本多政夫，金子周一
  • Organizer: 第104回日本消化器病学会総会
• [Presentation] 肝癌治療後C型慢性肝疾患に対する抗ウイルス薬治療後の肝癌再発に関する検討 (2018)
  • Author(s): 舩木雅也，島上哲朗，堀井里和，砂子阪肇，荒井邦明，山下太郎，酒井佳夫，山下竜也，水腰英四郎，本多政夫，金子周一
  • Organizer: 第104回日本消化器病学会総会
• [Presentation] HBV cccDNAに作用する転写因子を標的とした抗HBV効果 (2018)
  • Author(s): 川口和紀，本多政夫，金子周一
  • Organizer: 第104回日本消化器病学会総会
• [Presentation] 難治性腹水を伴う非代償性肝硬変におけるトルバプタン導入後の肝癌治療と予後について (2018)
  • Author(s): 朝日向良朗，堀井里和，丹尾幸樹，飯田宗穂，北原征明，寺島健志，鷹取元，荒井邦明，川口和紀，北村和哉，山下太郎，酒井佳夫，山下竜也、水腰英四郎，本多政夫，金子周一
  • Organizer: 第54回日本肝臓学科総会
• [Presentation] HBV生活環におけるNotchシグナルを介した核酸アナログの作用点に関する検討 (2018)
  • Author(s): 川口和紀，本多政夫，金子周一
  • Organizer: 第54回日本肝臓学科総会
• [Presentation] C型慢性肝疾患に対する第2世代DAAの治療成績とDAA非著効例に対する再治療成績に関する解析 (2018)
  • Author(s): 島上哲朗，本多政夫，金子周一
  • Organizer: 第54回日本肝臓学科総会
• [Presentation] 当科における抗ミトコンドリア抗体陰性原発性胆汁性胆管炎の検討 (2018)
  • Author(s): 岡藤啓史，北村和哉，朝日向良朗，高田昇，堀井里和，北原征明，飯田宗穂，鷹取 元，島上哲朗，荒井邦明，川口和紀，山下太郎，酒井佳夫，山下竜也，水腰英四郎，本多政夫，金子周一
  • Organizer: 第54回日本肝臓学科総会
• [Presentation] B型慢性肝炎患者の核酸アナログ投与下における肝線維化と肝発癌および再発予測 (2018)
  • Author(s): 川口和紀，本多政夫，金子周一
  • Organizer: JDDW2018
• [Presentation] NASHにおける血清バイオマーカーを組み合わせた肝発癌および重症度予測 (2018)
  • Author(s): 神野正隆，川口和紀，本多政夫，金子周一
  • Organizer: JDDW2018
• [Presentation] Characteristics and Functional Regulation of Dendritic Cells in Hepatitis B Patients (2018)
  • Author(s): Atsushi Yonejima, Eishiro Mizukoshi, Kiichiro Kaji, Toshikatsu Tamai, Hidetoshi Nakagawa, Takeshi Terashima, Noriho Iida, Masaaki Kitahara, Kuniaki Arai, Tatsuya Yamashita, Masao Honda, Shuichi Kaneko
  • Organizer: AASLD2018
  • Int'l Joint Research
• [Presentation] Mint3 is a novel molecular target expressed in well-differentiated AFP-negative HCC for prevention of cancer progression (2018)
  • Author(s): Nishitani Masaki, Yamashita Taro, Okada Hikari, Nio Kouki, Suda Tsuyoshi, Takatori Hajime, Honda Masao, Sakamoto Takeharu, Kaneko Shuichi
  • Organizer: AASLD2018
  • Int'l Joint Research
• [Presentation] A Liver-Derived Secretory Protein, LECT2, Enhances the Innate Immune Response and Suppresses RIG-I Mediated Virus Infection (2018)
  • Author(s): Takayoshi Shirasaki, Masao Honda, Kazuhisa Murai, Tetsuro Shimakami, Hirofumi Misu, Toshinari Takamura, Shuichi Kaneko
  • Organizer: AASLD2018
  • Int'l Joint Research
• [Presentation] Levocarnitine improves Liver Histology and the Development of Hepatocellular Carcinoma in the Atherogenic High-Fat Diet Nash-HCC Mouse Model (2018)
  • Author(s): Hikari Okada, Masao Honda, Hajime Sunakozaka, Shuichi Kaneko
  • Organizer: AASLD2018
  • Int'l Joint Research
• [Presentation] Adipose tissue derived stromal/stem cells alleviated hepatic fibrosis development induced by IL17-mediated stellate cell activation in NASH mice (2018)
  • Author(s): Masatoshi Yamato, Yoshio Sakai, Hatsune Mochida, Takuya Komura, Alessandro Nasti, Akihiro Seki, Kazunori Kawaguchi, Takashi Wada, Masao Honda, Shuichi Kaneko
  • Organizer: AASLD2018
  • Int'l Joint Research
• [Presentation] Immune response to tumor-associated antigens and immune vell profiles in NASH-related hepatocellular carcinoma (2018)
  • Author(s): Takuya Seike, Eishiro Mizukoshi, Yuki Inada, Toshikatsu Tamai, Noriho Iida, Masaaki Kitahara, Tatsuya Yamashita, Kuniaki Arai, Takeshi Terashima, Taro Yamashita, Masao Honda, Shuichi Kaneko
  • Organizer: AASLD2018
  • Int'l Joint Research
• [Presentation] Inactivation of transcriptional repressor capicua confers chemoresistance to sorafenib in hepatocellular carcinoma (2018)
  • Author(s): Tomomi Hashiba, Taro Yamashita, Hikari Okada, Kouki Nio, Takehiro Hayashi, Masao Honda, Shuichi Kaneko
  • Organizer: AASLD2018
  • Int'l Joint Research
• [Presentation] Elevation of serum aldo-keto reductase family 1 member B10 is associated with advanced liver fibrosis in non-alcoholic steatohepatitis (2018)
  • Author(s): Kanno Masataka, Kazunori Kawaguchi, Masao Honda, Rika Horii, Hajime Takatori, Tetsuro Shimakami, Kazuya Kitamura, Kuniaki Arai, Taro Yamashita, Yoshio Sakai, Tatsuya Yamashita, Eishiro Mizukoshi, Shuichi Kaneko
  • Organizer: AASLD2018
  • Int'l Joint Research