新規Wntシグナル制御系を標的とした抗癌剤の開発

2015 Fiscal Year Annual Research Report

• Project/Area Number: 15H04806
• Research Institution: The University of Tokyo
• Principal Investigator: 藤城 光弘 東京大学, 医学部附属病院, 准教授 (70396745)
• Co-Investigator(Kenkyū-buntansha): 池田 祐一 東京大学, 医学部附属病院, 准教授 (10744419) ; 熊谷 英敏 東京大学, 医学部附属病院, 助教 (20281008) ; 山下 裕玄 東京大学, 医学部附属病院, 講師 (50599397) ; 森田 啓行 東京大学, 医学部附属病院, 講師 (60323573) ; 浅岡 良成 東京大学, 医学部附属病院, 助教 (90431858)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Keywords: 内科 / 消化器内科 / 下部消化管 / 抗癌剤

Outline of Annual Research Achievements

最近の研究により、細胞膜におけるWntシグナル制御機構であるR-spondin-LGR4/5/6-ZNRF3/RNF43系が癌の治療標的となることが示された。そこで申請者らは、R-spondin-LGR4/5/6-ZNRF3/RNF43系を直接的に制御する化合物をスクリーニングするために酵素断片コンプリメンテーション法を応用したアッセイ系を構築した。本申請研究は、このアッセイ系を用いた低分子化合物ライブラリースクリーニングにより新規抗癌剤開発を目指すものである。ベータガラクトシダーゼは、N末端側の6kDaのオメガ断片と残りのC末端側のアルファ断片の2つに分離するとその酵素活性を失うが、両者が会合すると酵素活性が復活する。そこでR-spondin依存的なZNRF3/RNF43とLGR4/5/6の会合をモニターするため、LGR4にベータガラクトシダーゼのオメガ断片を結合させた分子（LGR4-オメガ）及びZNRF3にアルファ断片を結合させた分子（ZNRF3-アルファ）を作成し、HEK293T細胞に同時に一過性に発現させた。この細胞をR-spondinで刺激したところ容量依存的に酵素活性が上昇し、アッセイ系が計画どおりに動いていることが確認された。実際のスクリーニングでは数多くの化合物を取り扱わなければならない為HEK293T細胞のような接着細胞ではなく浮遊細胞の使用が適している。そこで引き続き株化浮遊細胞を用いて同様のアッセイ系の確立を目指した。様々な方法を用いて二つの融合分子の浮遊細胞への導入を試みたが活性を検出するために必要な遺伝子導入効率が得られなかったため方針を転換し、二つの融合分子を導入した接着細胞をアッセイ時には浮遊で使用する方法を着想した。今後はこの方法を用いて、1万の代表的な化合物から構成されるフォーカスライブラリーのスクリーニングを施行する。

Current Status of Research Progress

4: Progress in research has been delayed.

Reason

多数の化合物を扱う実際のスクリーニングでは、接着細胞ではなく浮遊細胞が適しているので、株化浮遊細胞を用いたアッセイ系の確立を目指した。ウイルスベクター、エレクトロポレーション、リポフェクションなど様々な方法を用いて二つの融合分子の浮遊細胞への導入を試みたが、融合遺伝子がかなり大きいこともあり、活性を検出するのに十分な遺伝子導入効率が得られず、計画が遅れた。次に、二つの融合分子を導入した接着細胞をアッセイ時にのみ浮遊で使用する方法を着想し、この方法に適した株化接着細胞をスクリーニングした。この細胞株スクリーニングにも予想外の時間を要した。

Strategy for Future Research Activity

二つの融合分子を導入した接着細胞をアッセイ時には浮遊で使用する方法を着想し、この方法に適した株化接着細胞を細胞株スクリーニングにより探索した。その結果この方法にはHeLa細胞が適していることを見いだした。今回新たに樹立した“HeLa細胞を浮遊状態で使用するアッセイ系”を用いて、今後は1万の代表的な化合物から構成されるフォーカスライブラリーのスクリーニングを実施する。

Research Products

• [Journal Article] Safety and Effectiveness of Endovenous Laser Ablation Combined With Ligation for Severe Saphenous Varicose Veins in Japanese Patients. (2016)
  • Author(s): Izumi M, Ikeda Y, Yamashita H, Asaoka Y, Fujishiro M, Shin M, Abo Y.
  • Journal Title: Int Heart J Volume: 57 Pages: 87-90
  • DOI: 10.1536/ihj.15-282
  • Peer Reviewed / Open Access
• [Journal Article] Glucose metabolism in gastric cancer: The cutting-edge. (2016)
  • Author(s): Yuan LW, Yamashita H, Seto Y.
  • Journal Title: World J Gastroenterol. Volume: 22 Pages: 2046-2059
  • DOI: 10.3748/wjg.v22.i6.2046
  • Peer Reviewed / Open Access
• [Journal Article] Is the transthoracic approach no longer a viable option for Siewert type II esophagogastric junction carcinoma? (2016)
  • Author(s): Yamashita H, Seto Y.
  • Journal Title: Translational Gastroenterology and Hepatology Volume: 1 Pages: 14
  • Peer Reviewed
• [Journal Article] Current therapies and investigational drugs for peripheral arterial disease. (2016)
  • Author(s): Suzuki J, Shimamura M, Suda H, Wakayama K, Kumagai H, Ikeda Y, Akazawa H, Isobe M, Komuro I, Morishita R
  • Journal Title: Hypertens Res. Volume: 39 Pages: 183-191
  • DOI: 10.1038/hr.2015.134
  • Peer Reviewed
• [Journal Article] Angiotensin II receptor blocker irbesartan attenuates cardiac dysfunction induced by myocardial infarction in the presence of renal failure. (2016)
  • Author(s): Watanabe R, Suzuki J, Wakayama K, Kumagai H, Ikeda Y, Akazawa H, Komuro I, Isobe M
  • Journal Title: Hypertens Res. Volume: 39 Pages: 237-244
  • DOI: 10.1038/hr.2015.141
  • Peer Reviewed
• [Journal Article] Comparative analysis of upper gastrointestinal endoscopy, double-contrast upper gastrointestinal barium X-ray radiography, and the titer of serum anti-Helicobacter pylori IgG focusing on the diagnosis of atrophic gastritis. (2016)
  • Author(s): Yamamichi N, Hirano C, Takahashi Y, Minatsuki C, Nakayama C, Matsuda R, Shimamoto T, Takeuchi C, Kodashima S, Ono S, Tsuji Y, Fujishiro M, Wada R, Mitsushima T, Koike K.
  • Journal Title: Gastric Cancer. Volume: 19 Pages: 670-675
  • DOI: 10.1007/s10120-015-0515-y
  • Peer Reviewed
• [Journal Article] Guidelines for endoscopic submucosal dissection and endoscopic mucosal resection for early gastric cancer. (2016)
  • Author(s): Ono H, Yao K, Fujishiro M, Oda I, Nimura S, Yahagi N, Iishi H, Oka M, Ajioka Y, Ichinose M, Matsui T.
  • Journal Title: Dig Endosc. Volume: 28 Pages: 3-15
  • DOI: 10.1111/den.12518
  • Peer Reviewed
• [Journal Article] Outcome of salvage surgery for colorectal cancer initially treated by upfront endoscopic therapy. (2016)
  • Author(s): Nozawa H, Ishihara S, Fujishiro M, Kodashima S, Ohtani K, Yasuda K, Nishikawa T, Tanaka T, Tanaka J, Kiyomatsu T, Kawai K, Hata K, Kazama S, Sunami E, Kitayama J, Watanabe T.
  • Journal Title: Surgery. Volume: 159 Pages: 713-720
  • DOI: 10.1016/j.surg.2015.09.008
  • Peer Reviewed
• [Journal Article] Efficacy of lymph node dissection for each station based on esophageal tumor location. (2016)
  • Author(s): Tachimori Y, Ozawa S, Numasaki H, Fujishiro M, MatsubaraH, Oyama T, Shinoda M, Toh Y, Udagawa H, Uno T, The Registration Committee for Esophageal Cancer of the Japan Esophageal Society.
  • Journal Title: Esophagus. Volume: 13 Pages: 1-7
  • Peer Reviewed / Open Access
• [Journal Article] Flow Cytometric Quantification of Intraperitoneal Free Tumor Cells is a Useful Biomarker in Gastric Cancer Patients with Peritoneal Metastasis. (2015)
  • Author(s): Kitayama J, Emoto S, Yamaguchi H, Ishigami H, Onoyama H, Yamashita H, Seto Y, Matsuzaki K, Watanabe T.
  • Journal Title: Ann Surg Oncol. Volume: 22 Pages: 2336-2342
  • DOI: 10.1245/s10434-014-4238-9.
  • Peer Reviewed
• [Journal Article] Phosphatidylserine-specific phospholipase A1 (PS-PLA1) expression in colorectal cancer correlates with tumor invasion and hematogenous metastasis. (2015)
  • Author(s): Iida Y, Sunami E, Yamashita H, Hiyoshi M, Ishihara S, Yamaguchi H, Inoue A, Makide K, Tsuno NH, Aoki J, Kitayama J, Watanabe T.
  • Journal Title: Anticancer Res. Volume: 35 Pages: 1459-1464
  • Peer Reviewed
• [Journal Article] Solitary fibrous tumor of the greater omentum, mimicking gastrointestinal stromal tumor of the small intestine: a case report. (2015)
  • Author(s): Urabe M, Yamagata Y, Aikou S, Mori K, Yamashita H, Nomura S, Shibahara J, Fukayama M, Seto Y.
  • Journal Title: Int Surg. Volume: 100 Pages: 836-840
  • DOI: 10.9738/INTSURG-D-14-00141.1
  • Peer Reviewed
• [Journal Article] Evaluation of 18F-FDG uptake for detecting lymph node metastasis of gastric cancer: a prospective pilot study for one-to-one comparison of radiation dose and pathological findings. (2015)
  • Author(s): Okumura Y, Aikou S, Onoyama H, Jinbo K, Yamagata Y, Mori K, Yamashita H, Nomura S, Takahashi M, Koyama K, Momose T, Abe H, Matsusaka K, Ushiku T, Fukayama M, Seto Y
  • Journal Title: World J Surg Oncol. Volume: 13 Pages: 327
  • DOI: 10.1186/s12957-015-0743-y
  • Peer Reviewed / Open Access
• [Journal Article] PD-1 Blockade in Tumors with Mismatch-Repair Deficiency. (2015)
  • Author(s): Asaoka Y, Ijichi H, Koike K.
  • Journal Title: N Engl J Med. Volume: 373 Pages: 1979
  • DOI: 10.1056/NEJMc1510353#SA1
  • Peer Reviewed
• [Journal Article] Serum Alpha-Fetoprotein Has High Specificity for the Early Detection of Hepatocellular Carcinoma After Hepatitis C Virus Eradication in Patients. (2015)
  • Author(s): Minami T, Tateishi R, Kondo M, Nakagomi R, Fujiwara N, Sato M, Uchino K, Enooku K, Nakagawa H, Asaoka Y, Kondo Y, Moriya K, Shiina S, Koike K.
  • Journal Title: Medicine (Baltimore). Volume: 94 Pages: e901
  • DOI: 10.1097/MD.0000000000000901
  • Peer Reviewed / Open Access
• [Journal Article] Sarcopenia, intramuscular fat deposition, and visceral adiposity independently predict the outcomes of hepatocellular carcinoma. (2015)
  • Author(s): Fujiwara N, Nakagawa H, Kudo Y, Tateishi R, Taguri M, Watadani T, Nakagomi R, Kondo M, Nakatsuka T, Minami T, Sato M, Uchino K, Enooku K, Kondo Y, Asaoka Y, Tanaka Y, Ohtomo K, Shiina S, Koike K.
  • Journal Title: J Hepatol. Volume: 63 Pages: 131-140
  • DOI: 10.1016/j.jhep.2015.02.031
  • Peer Reviewed / Open Access
• [Journal Article] Validation of the Japanese version of HFS-14, a disease-specific quality of life scale for patients suffering from hand-foot syndrome. (2015)
  • Author(s): Mikoshiba N, Yamamoto-Mitani N, Sato K, Asaoka Y, Ohki T, Ohata M, Miyashita M.
  • Journal Title: Support Care Cancer. Volume: 23 Pages: 2739-2745
  • DOI: 10.1007/s00520-015-2638-4
  • Peer Reviewed
• [Journal Article] Comparison of improved prognosis between hepatitis B- and hepatitis C-related hepatocellular carcinoma. (2015)
  • Author(s): Minami T, Tateishi R, Shiina S, Nakagomi R, Kondo M, Fujiwara N, Mikami S, Sato M, Uchino K, Enooku K, Nakagawa H, Asaoka Y, Kondo Y, Yoshida H, Koike K.
  • Journal Title: Hepatol Res. Volume: 45 Pages: E99-E107
  • DOI: 10.1111/hepr.12468
  • Peer Reviewed
• [Journal Article] Neuromedin S-producing neurons act as essential pacemakers in the suprachiasmatic nucleus to couple clock neurons and dictate circadian rhythms. (2015)
  • Author(s): Lee IT, Chang AS, Manandhar M, Shan Y, Fan J, Izumo M, Ikeda Y, Motoike T, Dixon S, Seinfeld JE, Takahashi JS, Yanagisawa M
  • Journal Title: Neuron Volume: 85 Pages: 1086-1102
  • DOI: 10.1016/j.neuron.2015.02.006
  • Peer Reviewed / Open Access
• [Journal Article] Toward further prevention of bleeding after gastric endoscopic submucosal dissection. (2015)
  • Author(s): Mochizuki S, Ikeda Y, Arai T, Matsuo K
  • Journal Title: Dig Endosc. Volume: 27 Pages: 295-297
  • DOI: 10.1111/den.12441
  • Peer Reviewed
• [Journal Article] Recent clinical management of antithrombotic agents for gastrointestinal endoscopy after revision of guidelines in Japan. (2015)
  • Author(s): Ono S, Fujishiro M, Ikeda Y, Komuro I, Koike K
  • Journal Title: Dig Endosc. Volume: 27 Pages: 649-656
  • DOI: 10.1111/den.12498
  • Peer Reviewed
• [Journal Article] Quantitative measurement of GPCR endocytosis via pulse-chase covalent labeling. (2015)
  • Author(s): Kumagai H, Ikeda Y, Motozawa Y, Fujishiro M, Okamura T, Fujio K, Okazaki H, Nomura S, Takeda N, Harada M, Toko H, Takimoto E, Akazawa H, Morita H, Suzuki J, Yamazaki T, Yamamoto K, Komuro I, Yanagisawa M
  • Journal Title: PLoS One Volume: 10 Pages: e0129394
  • DOI: 10.1371/journal.pone.0129394
  • Peer Reviewed / Open Access
• [Journal Article] Monitoring beta-arrestin recruitment via beta-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors. (2015)
  • Author(s): Ikeda Y, Kumagai H, Okazaki H, Fujishiro M, Motozawa Y, Nomura S, Takeda N, Toko H, Takimoto E, Akazawa H, Morita H, Suzuki J, Yamazaki T, Komuro I, Yanagisawa M
  • Journal Title: PLoS One Volume: 10 Pages: e0127445
  • DOI: 10.1371/journal.pone.0127445
  • Peer Reviewed / Open Access
• [Journal Article] SH2B1: a new player in the regulation of cardiac hypertrophic response in failing hearts. (2015)
  • Author(s): Ikeda Y, Takimoto E, Komuro I
  • Journal Title: Cardiovasc Res. Volume: 107 Pages: 197-199
  • DOI: 10.1093/cvr/cvv172
  • Peer Reviewed
• [Journal Article] Acute myocardial infarction that resulted from poor adherence to medical treatment for giant coronary aneurysm: the importance of patient education in the chronic phase of Kawasaki disease. (2015)
  • Author(s): Motozawa Y, Uozumi H, Maemura S, Nakata R, Yamamoto K, Takizawa M, Kumagai H, Ikeda Y, Komuro I, Ikenouchi H
  • Journal Title: Int Heart J. Volume: 56 Pages: 551-554
  • DOI: 10.1536/ihj.15-155
  • Peer Reviewed / Open Access
• [Journal Article] Biased agonism of the angiotensin II type I receptor: a potential strategy for the treatment of acute heart failure. (2015)
  • Author(s): Ikeda Y, Kumagai H, Motozawa Y, Suzuki J, Komuro I
  • Journal Title: Int Heart J. Volume: 56 Pages: 485-488
  • DOI: 10.1536/ihj.15-256
  • Peer Reviewed / Open Access
• [Journal Article] A food-derived flavonoid luteolin protects against angiotensin II-induced cardiac remodeling. (2015)
  • Author(s): Nakayama A, Morita H, Nakao T, Yamaguchi T, Sumida T, Ikeda Y, Kumagai H, Motozawa Y, Takahashi T, Imaizumi A, Hashimoto T, Nagai R, Komuro I
  • Journal Title: PLoS One Volume: 10 Pages: e0137106
  • DOI: 10.1371/journal.pone.0137106
  • Peer Reviewed / Open Access
• [Journal Article] Next generation ARBs: going beyond modulation of the renin-angiotensin system. (2015)
  • Author(s): Ikeda Y, Kumagai H, Motozawa Y, Suzuki J, Komuro I
  • Journal Title: Int Heart J. Volume: 56 Pages: 585-586
  • DOI: 10.1536/ihj.15-295
  • Peer Reviewed / Open Access
• [Journal Article] Polyglycolic acid sheets and fibrin glue decrease the risk of bleeding after endoscopic submucosal dissection of gastric neoplasms (with video). (2015)
  • Author(s): Tsuji Y, Fujishiro M, Kodashima S, Ono S, Niimi K, Mochizuki S, Asada-Hirayama I, Matsuda R, Minatsuki C, Nakayama C, Takahashi Y, Sakaguchi Y, Yamamichi N, Koike K.
  • Journal Title: Gastrointest Endosc. Volume: 81 Pages: 906-912
  • DOI: 10.1016/j.gie.2014.08.028
  • Peer Reviewed
• [Journal Article] Polyglycolic acid sheets with fibrin glue can prevent esophageal stricture after endoscopic submucosal dissection. (2015)
  • Author(s): Sakaguchi Y, Tsuji Y, Ono S, Saito I, Kataoka Y, Takahashi Y, Nakayama C, Shichijo S, Matsuda R, Minatsuki C, Asada-Hirayama I, Niimi K, Kodashima S, Yamamichi N, Fujishiro M, Koike K.
  • Journal Title: Endoscopy. Volume: 47 Pages: 336-340
  • DOI: 10.1055/s-0034-1390787
  • Peer Reviewed
• [Journal Article] Thienopyridine derivatives as risk factors for bleeding following high risk endoscopic treatments: Safe Treatment on Antiplatelets (STRAP) study. (2015)
  • Author(s): Ono S, Fujishiro M, Yoshida N, Doyama H, Kamoshida T, Hirai S, Kishihara T, Yamamoto Y, Sakae H, Imagawa A, Hirano M, Koike K.
  • Journal Title: Endoscopy. Volume: 47 Pages: 632-637
  • DOI: 10.1055/s-0034-1391354
  • Peer Reviewed
• [Journal Article] Helicobacter pylori infection is not associated with fatty liver disease including non-alcoholic fatty liver disease: a large-scale cross-sectional study in Japan. (2015)
  • Author(s): Okushin K, Takahashi Y, Yamamichi N, Shimamoto T, Enooku K, Fujinaga H, Tsutsumi T, Shintani Y, Sakaguchi Y, Ono S, Kodashima S, Fujishiro M, Moriya K, Yotsuyanagi H, Mitsushima T, Koike K.
  • Journal Title: BMC Gastroenterol. Volume: 15 Pages: 247
  • DOI: 10.1186/s12876-015-0247-9
  • Peer Reviewed / Open Access
• [Journal Article] Trend and risk factors of diverticulosis in Japan: age, gender, and lifestyle/metabolic-related factors may cooperatively affect on the colorectal diverticula formation. (2015)
  • Author(s): Yamamichi N, Shimamoto T, Takahashi Y, Sakaguchi Y, Kakimoto H, Matsuda R, Kataoka Y, Saito I, Tsuji Y, Yakabi S, Takeuchi C, Minatsuki C, Niimi K, Asada-Hirayama I, Nakayama C, Ono S, Kodashima S, Yamaguchi D, Fujishiro M, Yamaji Y, Wada R, Mitsushima T, Koike K.
  • Journal Title: PLoS One. Volume: 10 Pages: e0123688.
  • DOI: 10.1371/journal.pone.0123688
  • Peer Reviewed / Open Access
• [Journal Article] PLANETARIUM Study Group. Long-term efficacy and safety of rabeprazole in patients taking low-dose aspirin with a history of peptic ulcers: a phase 2/3, randomized, parallel-group, multicenter, extension clinical trial. (2015)
  • Author(s): Fujishiro M, Higuchi K, Kato M, Kinoshita Y, Iwakiri R, Watanabe T, Takeuchi T, Sugisaki N, Okada Y, Ogawa H, Arakawa T, Fujimoto K
  • Journal Title: J Clin Biochem Nutr. Volume: 56 Pages: 228-239
  • DOI: 10.3164/jcbn.15-1
  • Peer Reviewed / Open Access
• [Journal Article] Foam plombage: a novel technique for optimal fixation of polyglycolic acid sheets positioned using "clip and pull" after esophageal endoscopic submucosal dissection. (2015)
  • Author(s): Ono S, Sakaguchi Y, Tsuji Y, Kodashima S, Yamamichi N, Fujishiro M, Koike K
  • Journal Title: Endoscopy. Volume: 47 Pages: E435-436
  • DOI: 10.1055/s-0034-1392612
  • Peer Reviewed
• [Journal Article] Preventing esophageal stricture after endoscopic submucosal dissection: steroid injection and shielding with polyglycolic acid sheets and fibrin glue. (2015)
  • Author(s): Kataoka Y, Tsuji Y, Sakaguchi Y, Kodashima S, Yamamichi N, Fujishiro M, Koike K.
  • Journal Title: Endoscopy. Volume: 47 Pages: E473-474
  • DOI: 10.1055/s-0034-1392975
  • Peer Reviewed
• [Journal Article] Comprehensive Registry of Esophageal Cancer in Japan, 2007 (2015)
  • Author(s): Tachimori Y, Ozawa S, Numasaki H, Fujishiro M, MatsubaraH, Oyama T, Shinoda M, Toh Y, Udagawa H, Uno T, The Registration Committee for Esophageal Cancer of the Japan Esophageal Society.
  • Journal Title: Esophagus. Volume: 12 Pages: 101-129
  • Peer Reviewed
• [Journal Article] Comprehensive Registry of Esophageal Cancer in Japan, 2008 (2015)
  • Author(s): Tachimori Y, Ozawa S, Numasaki H, Fujishiro M, MatsubaraH, Oyama T, Shinoda M, Toh Y, Udagawa H, Uno T, The Registration Committee for Esophageal Cancer of the Japan Esophageal Society.
  • Journal Title: Esophagus. Volume: 12 Pages: 130-157
  • Peer Reviewed
• [Journal Article] Video of the Month: A Novel Endoscopic Full-Thickness Resection for Early Gastric Cancer. (2015)
  • Author(s): Niimi K, Aikou S, Kodashima S, Yagi K, Oya S, Yamaguchi D, Yamashita H, Yamamichi N, Fujishiro M, Koike K, Seto Y.
  • Journal Title: Am J Gastroenterol. Volume: 110 Pages: 1535
  • DOI: 10.1038/ajg.2015.226
  • Peer Reviewed