ユビキチン化蛋白の網羅的解析による炎症性発癌メカニズムの解明

• Project/Area Number: 17K19675
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Research Field: Organ-based internal medicine and related fields
• Research Institution: Tokyo Medical and Dental University
• Principal Investigator: 鬼澤 道夫 東京医科歯科大学, 大学院医歯学総合研究科, 寄附講座助教 (30783352)
• Co-Investigator(Kenkyū-buntansha): 渡辺 守 東京医科歯科大学, 大学院医歯学総合研究科, 教授 (10175127)
• Project Period (FY): 2017-06-30 – 2020-03-31
• Project Status: Granted (Fiscal Year 2018)
• Budget Amount: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000); Fiscal Year 2019: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000); Fiscal Year 2018: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000); Fiscal Year 2017: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
• Keywords: 大腸腫瘍 / タンパク / 大腸癌

Outline of Annual Research Achievements

炎症性腸疾患合併大腸癌(炎症性大腸癌)は、炎症性腸疾患(IBD)の長期罹患に伴って高率に合併することが知られており、本邦でのIBD患者数の増加に伴い、その予防法及び治療法の開発が求められている。炎症性大腸癌の発生機序は、通常の大腸癌（孤発性大腸癌）とは大きく異なると考えられているものの、その詳細は明らかではない。本研究では翻訳後修飾の視点から、IBDにおける大腸上皮の腫瘍化の機序を、孤発性大腸腫瘍との比較も交えて明らかにすることを目的とする。炎症性大腸腫瘍誘導にあたっては、マウスにアゾキシメタンを投与したのち、デキストラン硫酸による腸炎を3回繰り返した。発症した大腸腫瘍を摘出し、EDTA溶液で腫瘍を遊離させマトリゲルに懸濁・播種し、Wnt3a及びRspondin-1非存在下で培養を行った結果、複数の初代培養株を樹立し得た。培養細胞でβ-カテニンの遺伝子変異を検索した結果、32番目のアスパラギン酸や37番目のセリンの変異を認め、疾患マウス作成のため37番目のセリンの変異（S37F）についてCRISPR/Cas9による変異マウスの作成を試みたが、変異体を得ることが出来なかった。また、質量分析を用いたユビキチン化タンパクの網羅的同定において、非腫瘍部大腸上皮に比べて炎症性大腸腫瘍組織で増加が示唆されたユビキチン化タンパクのうちVimentinやNEDD4等について、減少が示唆されたうちSodium/potassium-transporting ATPase subunit alpha-1等について、発現・機能解析を行っている。

Current Status of Research Progress

3: Progress in research has been slightly delayed.

Reason

炎症性大腸腫瘍及び孤発性大腸腫瘍組織における、網羅的ユビキチン化タンパクの解析を行い、腫瘍化に伴って変化するユビキチン化タンパクを多数同定した。そのうち複数のタンパクについて解析が進行しているが、当初予定していた腫瘍発症に関わる因子を見出せていないため、解析の際の効率的な培養法を検討中である。

Strategy for Future Research Activity

大腸腫瘍の網羅的ユビキチン化タンパクの解析から得られた結果より、解析範囲を広げ、腫瘍化に伴って変化するユビキチン化タンパクの発現や生化学的特徴、大腸腫瘍発症のメカニズムを明らかにする。また大腸腫瘍解析を容易とする培養システムの構築を試み、解析を加速させる。

Research Products

• [Journal Article] Genetic and environmental factors drive personalized medicine for Crohn’s disease. (2019)
  • Author(s): Oshima Shigeru、Watanabe Mamoru
  • Journal Title: J Clin Invest. Volume: 128 Pages: 4758-4760
  • DOI: 10.1172/jci124303
  • Peer Reviewed
• [Journal Article] Ubiquitin D is up-regulated by synergy of Notch signaling and TNF-α in the inflamed intestinal epithelia of IBD patients. (2019)
  • Author(s): Kawamoto Ami、Nagata Sayaka、Anzai Sho、Takahashi Junichi、Kawai Mao、Hama Minami、Nogawa Daichi、Yamamoto Kouhei、Kuno Reiko、Suzuki Kohei、Shimizu Hiromichi、Hiraguri Yui、Yui Shiro、Oshima Shigeru、Tsuchiya Kiichiro、Nakamura Tetsuya、Ohtsuka Kazuo、Kitagawa Masanobu、Okamoto Ryuichi、Watanabe Mamoru
  • Journal Title: J Crohns Colitis Volume: 13 Pages: 495-509
  • DOI: 10.1093/ecco-jcc/jjy180
  • Peer Reviewed
• [Journal Article] Novel polyubiquitin imaging system, PolyUb-FC, reveals that K33-linked polyubiquitin is recruited by SQSTM1/p62. (2018)
  • Author(s): Yoichi Nibe, Shigeru Oshima, Masanori Kobayashi, Chiaki Maeyashiki, Yu Matsuzawa, Kana Otsubo, Hiroki Matsuda, Emi Aonuma, Yasuhiro Nemoto, Takashi Nagaishi, Ryuichi Okamoto, Kiichiro Tsuchiya, Tetsuya Nakamura, Shinichiro Nakada, Mamoru Watanabe
  • Journal Title: Autophagy Volume: 22 Pages: 347-358
  • DOI: 10.1080/15548627.2017.1407889
  • Peer Reviewed / Open Access
• [Journal Article] Contribution of ATOH1+ cells to the homeostasis, repair and tumorigenesis of the colonic epithelium. (2018)
  • Author(s): Ishibashi Fumiaki、Shimizu Hiromichi、Nakata Toru、Fujii Satoru、Suzuki Kohei、Kawamoto Ami、Anzai Sho、Kuno Reiko、Nagata Sayaka、Ito Go、Murano Tatsuro、Mizutani Tomohiro、Oshima Shigeru、Tsuchiya Kiichiro、Nakamura Tetsuya、Watanabe Mamoru、Okamoto Ryuichi
  • Journal Title: Stem Cell Rep. Volume: 10 Pages: 27-42
  • DOI: 10.1016/j.stemcr.2017.11.006
  • Peer Reviewed / Open Access
• [Journal Article] YAP/TAZ-dependent reprogramming of colonic epithelium links ECM remodeling to tissue regeneration. (2018)
  • Author(s): Yui S、Azzolin L、Maimets M、Pedersen MT、Fordham RP、Hansen SL、Larsen HL、Guiu Jordi、Alves MRP、Rundsten CF、Johansen JV、Li Y、Madsen CD、Nakamura T、Watanabe M、Nielsen OH、Schweiger PJ、Piccolo S、Jensen KB
  • Journal Title: Cell Stem Cell Volume: 22 Pages: 35-49
  • DOI: 10.1016/j.stem.2017.11.001
  • Peer Reviewed / Open Access
• [Journal Article] Single cell analysis of Crohn’s disease patient-derived small intestinal organoids reveals disease activity-dependent modification of stem cell properties. (2018)
  • Author(s): Suzuki Kohei、Murano Tatsuro、Shimizu Hiromichi、Ito Go、Nakata Toru、Fujii Satoru、Ishibashi Fumiaki、Kawamoto Ami、Anzai Sho、Kuno Reiko、Kuwabara Konomi、Takahashi Junichi、Tsuchiya Kiichiro、Nakamura Tetsuya、Ohtsuka Kazuo、Watanabe Mamoru、Okamoto Ryuichi
  • Journal Title: J Gastroenterol. Volume: 印刷中 Pages: 1035-1047
  • DOI: 10.1007/s00535-018-1437-3
  • Peer Reviewed / Open Access
• [Journal Article] B cell activation in the cecal patches during the development of an experimental colitis model (2018)
  • Author(s): Taro Watabe, Takashi Nagaishi, Naoya Tsugawa, Yudai Kojima, Nisha Jose, Akinori Hosoya, Michio Onizawa, Yasuhiro Nemoto, Shigeru Oshima, Tetsuya Nakamura, Hajime Karasuyama, Takahiro Adachi, Mamoru Watanabe
  • Journal Title: Biochem Biophys res Commun. Volume: 496 Pages: 367-373
  • DOI: 10.1016/j.bbrc.2018.01.053
  • Peer Reviewed / Open Access
• [Presentation] UC-related and segment-specific properties of patientderived colonic organoids. (2019)
  • Author(s): Suzuki K, Shimizu H, Kawai M, Takahashi J, Anzai S, Kawamoto A, Nagata S, Hiraguri Y, Yui S, Tsuchiya K, Nakamura T, Ohtsuka K, Okamoto R, Watanabe M
  • Organizer: ECCO 2019
  • Int'l Joint Research
• [Presentation] Synergy of Notch signalling and TNF-α in the inflamed intestinal epithelia of IBD patients leads to up-regulation of UBD, a ubiquitin-like protein. (2019)
  • Author(s): Kawamoto A, Nagata S, Anzai S, Takahashi J, Kawai M, Hama M, Nogawa D, Yamamoto K, Kuno R, Suzuki K, Shimizu H, Hiraguri Y, Yui S, Oshima S, Tsuchiya K, Nakamura T, Ohtsuka K, Kitagawa M, Okamoto R, Watanabe M
  • Organizer: ECCO 2019
  • Int'l Joint Research
• [Presentation] TP53 mutation in human colonic organoids acquires resistance to in vitro long-term inflammation. (2019)
  • Author(s): Tsuchiya K, Watanabe S, Shirasaki T, Nishimura R, Katsukura N, Hibiya S, Okamoto R, Nakamura T, Watanabe M
  • Organizer: ECCO 2019
  • Int'l Joint Research
• [Presentation] Fetalization of colonic epithelium medicated by YAP/TAZ links ECM remodeling to tissue regeneration. (2018)
  • Author(s): Yui S, Okamoto R, Tsuchiya K, Nakamura T, Watanabe M
  • Organizer: GI Research Academy 2018
  • Int'l Joint Research
• [Presentation] Single-cell level analysis of organoids derived from CD patients reveals disease-status related modifications of small intestinal stem cells. (2018)
  • Author(s): Suzuki K, Kuwabara K, Takahashi J, Anzai S, Kuno R, Kawamoto A, Ishibashi F, Nagata S, Hiraguri Y, Yui S, Tsuchiya K, Nakamura T, Ohtsuka K, Okamoto R, Watanabe M
  • Organizer: DDW 2018
  • Int'l Joint Research
• [Presentation] Analysis of ileocecal immune response in an animal model of colitis using intra-vital imaging. (2018)
  • Author(s): Watabe T, Nagaishi T, Tsugawa N, Kojima Y, Jose N, Hosoya A, Adachi T, Watanabe M
  • Organizer: DDW 2018
  • Int'l Joint Research
• [Presentation] Identification of lesion-specific epithelial function of ulcerative colitis by comparing colonic organoids from lesion and non-lesion parts of same patients. (2018)
  • Author(s): Shirasaki T, Tsuchiya K, Nishimura R, Watanabe S, Hibiya S, Katsukura N, Okamoto R, Nakamura T, Watanabe M
  • Organizer: DDW 2018
  • Int'l Joint Research
• [Presentation] Human colonic organoid treated with inflammatory factors might mimic the pathophysiology of epithelial cells in ulcerative colitis. (2018)
  • Author(s): Hibiya S, Tsuchiya K, Watanabe S, Nishimura R, Shirasaki T, Oshima S, Okamoto R, Nakamura T, Watanabe M
  • Organizer: DDW 2018
  • Int'l Joint Research
• [Presentation] Mice overexpressing MKL1 in macrophages are susceptible to the development of colitis. (2018)
  • Author(s): An J, Nagaishi T, Watabe T, Naruse TK., Watanabe M, Kimura A
  • Organizer: DDW 2018
  • Int'l Joint Research
• [Presentation] Mouse colonic secretory cells de-differentiate into intestinal stem cells and promote mucosal repair through activation of NF-KB signaling. (2018)
  • Author(s): Ishibashi F, Shimizu H, Kawamoto A, Suzuki K, Anzai S, Kuwabara K, Takahashi J, Nagata S, Oshima S, Tsuchiya K, Nakamura T, Watanabe M, Okamoto R
  • Organizer: ISSCR 2018
  • Int'l Joint Research
• [Presentation] Analysis of cecal immune response in a murine model of colitis using intravital imaging. (2018)
  • Author(s): Nagaishi T, Watabe T, Tsugawa N, Kojima Y, Jose N, Yamada D, Adachi T, Watanabe M
  • Organizer: FOCIS 2018
  • Int'l Joint Research
• [Presentation] Mice overexpressing MKL1 in macrophages are susceptible to DSS-induced colitis. (2018)
  • Author(s): An J, Nagaishi T, Watabe T, Naruse TK., Watanabe M, Kimura
  • Organizer: FOCIS 2018
  • Int'l Joint Research
• [Presentation] Overexpression of MKL1 in Macrophages leads to the development of murine colitis. (2018)
  • Author(s): An J, Nagaishi T, Watabe T, Naruse TK., Watanabe M, Kimura A
  • Organizer: MICS 2018
  • Int'l Joint Research
• [Presentation] Development of a new mouse model of short bowel syndrome that may allow for the assessment of therapeutic efficacy of heterotopic transplantation of small intestinal organoids. (2018)
  • Author(s): Matsumoto Y, Watanabe M, Nakamura T
  • Organizer: 5th TERMIS World Congress 2018
  • Int'l Joint Research
• [Presentation] Crohn’s disease patient-derived small intestinal organoids reveal disease-status related modification of stem cell properties (2018)
  • Author(s): Suzuki K, Murano T, Hiraguri Y, Takahashi J, Shimizu H,Anzai S, Kuwabara K, Kawamoto A, Ishibashi F, Yui S, Tsuchiya K, Nakamura T, Ohtsuka K, Watanabe M, Okamoto R
  • Organizer: 5th TERMIS World Congress 2018
  • Int'l Joint Research
• [Presentation] Regenerative medicine for the treatment of inflammatory bowel disease. (2018)
  • Author(s): Okamoto R, Watanabe M
  • Organizer: 5th TERMIS World Congress 2018
  • Int'l Joint Research
• [Presentation] Ectopic expression of reg3a in the mice distal Colon is mediated by interactions between notch and Il-22 signaling pathways, and promotes tissue repair By the augmentation of EGFR signaling. (2018)
  • Author(s): Ishibashi F, Kuwabara K, Kawamoto A, Anzai S, Takahashi J, Nagata S, Shimizu H, Yui S, Oshima S, Tsuchiya K, Nakamura T, Watanabe M, Okamoto R
  • Organizer: UEGW 2018
  • Int'l Joint Research
• [Presentation] Long-lived secretory cells residing in the mouse proximal colon serve as reserve stem cells under DNA damage-induced mucosal injury. (2018)
  • Author(s): Kuwabara K, Ishibashi F, Kawamoto A, Anzai S, Takahashi J, Nagata S, Shimizu H, Yui S, Oshima S, Tsuchiya K, Nakamura T, Watanabe M, Okamoto R
  • Organizer: UEGW 2018
  • Int'l Joint Research
• [Presentation] Tp53 mutation enhances cell proliferation and stemness in human colon epithelial organoids and promotes a resistance against long-term inflammation. (2018)
  • Author(s): Watanabe S, Tsuchiya K, Shirasaki T, Hibiya S, Nishimura S, Katsukura N, Oshima S, Okamoto R, Tetsuya N, Watanabe M
  • Organizer: UEGW 2018
  • Int'l Joint Research
• [Presentation] Lesion-specific gene expression in the Epithelial cells of Crohn’s disease by comparing Small intestinal organoids from active and inactive Lesion in the same patient. (2018)
  • Author(s): Hibiya S, Tsuchiya K, Nishimura R, Shirasaki T, Watanabe S, Katsukura N, Okamoto R, Nakamura T, Watanabe M
  • Organizer: UEGW 2018
  • Int'l Joint Research
• [Presentation] B cell-mediated ileocecal immune response is activated in the early phase of colitis development. (2018)
  • Author(s): Kojima Y, Nagaishi T, Watabe T, Tsugawa N, Jose N, Yamada D, Hosoya A, Adachi T, Watanabe M
  • Organizer: UEGW 2018
  • Int'l Joint Research
• [Presentation] Mice overexpressing MKL1 in macrophages experience fulminant colitis. (2018)
  • Author(s): An J, Nagaishi T, Watabe T, Naruse TK., Watanabe M, Kimura A
  • Organizer: UEGW 2018
  • Int'l Joint Research
• [Presentation] Downregulated APL expression in T cells in the setting of chronic colitis. (2018)
  • Author(s): Kojima Y, Nagaishi T, Watabe T, Yamada D, Tsugawa N, Jose N, Hosoya A, Shimizu M, Onizawa M, Watanabe M
  • Organizer: 第47回日本免疫学会学術集会
  • Int'l Joint Research
• [Presentation] Regeneration of intestinal epithelial cell and IBD (2018)
  • Author(s): Watanabe M
  • Organizer: Falk symposium 2018
  • Int'l Joint Research / Invited
• [Presentation] Intestinal Epithelial Stem Cell Organoid and IBD. (2018)
  • Author(s): Watanabe M
  • Organizer: 第55回日本消化器免疫学会
  • Int'l Joint Research / Invited
• [Presentation] Introduction to IBD Centre at Tokyo Medical and Dental University. (2018)
  • Author(s): Watanabe M
  • Organizer: IBD Center of Excellence
  • Int'l Joint Research / Invited