ヒト大腸上皮培養による大腸癌幹細胞の分化破綻機構解析

• Project Area: Development of Novel Treatment Strategies Targeting Cancer Stem Cells
• Project/Area Number: 23130506
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Tokyo Medical and Dental University
• Principal Investigator: 土屋 輝一郎 東京医科歯科大学, 医歯(薬)学総合研究科, 寄附講座講師 (40376786)
• Project Period (FY): 2011-04-01 – 2013-03-31
• Project Status: Completed (Fiscal Year 2012)
• Budget Amount: ¥14,820,000 (Direct Cost: ¥11,400,000、Indirect Cost: ¥3,420,000); Fiscal Year 2012: ¥7,410,000 (Direct Cost: ¥5,700,000、Indirect Cost: ¥1,710,000); Fiscal Year 2011: ¥7,410,000 (Direct Cost: ¥5,700,000、Indirect Cost: ¥1,710,000)
• Keywords: Atoh1 / 癌幹細胞 / 炎症発がん / 大腸上皮培養 / がん幹細胞可視化 / 粘液産生がん

Outline of Annual Research Achievements

本計画は同時に開発した大腸上皮細胞の初代培養系を応用し癌化モデルによる正常幹細胞由来癌幹細胞の形成を空間的・経時的に解析することで癌幹細胞の特異性、機能維持機構を解明することを目的とする。今年度は計画に従いマウス由来大腸上皮の初代培養を行い、in vitroにおける炎症発がんモデルを確立するため、初代培養細胞にAOM処理を行い、さらに種々のサイトカインにて刺激するなど条件検討を行った。小腸初代培養細胞においてTLR5は基底側に限局して発現しており、フラジェリン添加によりサイトカインを産生することが明らかとなった。一方でTLR4は管腔側に限局して局在しLPS添加による応答は認められなかった。しかし大腸上皮初代培養細胞ではフラジェリン、LPSの両方に応答することが判明し、NFkBシグナルが作動することを確認した。また炎症発がんの特徴である粘液産生がんに着目し、粘液産生に寄与するAtoh1蛋白の機能解析を行い、粘液産生のみならずがん幹細胞性質、抗がん剤耐性機構まで関与することを明らかとした。
以上の成果は当初の研究計画をおおむね順調に遂行しており、ヒト大腸初代培養の確立および幹細胞可視化への条件検討は炎症発がんのモデルだけでなく、種々の病態、疾患予測に有用であると期待される。

Research Progress Status

24年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

24年度が最終年度であるため、記入しない。

Research Products

• [Journal Article] The acquisition of malignant potential in colon cancer is regulated by the stabilization of Atonal homolog 1 protein. (2013)
  • Author(s): Kano Y
  • Journal Title: Biochem. Biophys. Res. Commun. Volume: 432 Issue: 1 Pages: 175-181
  • DOI: 10.1016/j.bbrc.2013.01.034
  • Peer Reviewed / Open Access
• [Journal Article] Bone marrow-mesenchymal stem cells are a major source of interleukin-7 and sustain colitis by forming the niche for colitogenic CD4+ memory T cells. (2013)
  • Author(s): Nemoto Y, Nakamura T, Watanabe M et al
  • Journal Title: Gut. Volume: 62 Issue: 8 Pages: 1142-1152
  • DOI: 10.1136/gutjnl-2012-302029
  • Peer Reviewed
• [Journal Article] Modifiedsingle-operator method for double-balloon endoscopy (2012)
  • Author(s): Araki A, Suzuki S, Tsuchiya K, OshimaS, Okada E, Watanabe M
  • Journal Title: Dig Endosc Volume: 24(6) Issue: 6 Pages: 470-4
  • DOI: 10.1111/j.1443-1661.2012.01321.x
  • Peer Reviewed
• [Journal Article] Endoscopic ultrasound with double-balloon endoscopy for the diagnosis of inverted Meckel'sdiverticulum: a case report (2012)
  • Author(s): Araki A, Tsuchiya K, Oshima S, Okada E,Suzuki S, Akiyama JM, Fujii T, OkamotoR, Watanabe M
  • Journal Title: J Med Case Rep Volume: 6(1) Issue: 1 Pages: 328-328
  • DOI: 10.1186/1752-1947-6-328
  • Peer Reviewed
• [Journal Article] Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+stem cell (2012)
  • Author(s): Yui S, Nakamura T, Sato T, Nemoto Y, Mizutani T, Zheng X, Ichinose S, Nagaishi T, Okamoto R, Tsuchiya K, Clevers H, and Watanabe M
  • Journal Title: Nature Medicine Volume: 18 Issue: 4 Pages: 618-623
  • DOI: 10.1038/nm.2695
  • Peer Reviewed
• [Journal Article] Real-time analysis of P-glycoprotein-mediated drug transport across primary intestinal epithelium three-dimensionally cultured in vitro (2012)
  • Author(s): Mizutani T, Nakamura T, et al
  • Journal Title: Biochem Biophys Res Commun Volume: 419 Issue: 2 Pages: 238-243
  • DOI: 10.1016/j.bbrc.2012.01.155
  • Peer Reviewed
• [Journal Article] The development of colitogenic CD4+ T cells is regulated by IL-7 in collaboration with natural killer cell function in a murine model of colitis (2012)
  • Author(s): Yamaji O, Totsuka T
  • Journal Title: J Immunol Volume: 188 Issue: 6 Pages: 2524-2536
  • DOI: 10.4049/jimmunol.1100371
  • Peer Reviewed
• [Journal Article] Luminal CD4+ T cells penetrate gut epithelial monolayers and egress from lamina propria to blood circulation (2011)
  • Author(s): Nemoto Y, Watanabe M
  • Journal Title: Gastroenterology Volume: 141 Issue: 6 Pages: 2130-2139
  • DOI: 10.1053/j.gastro.2011.08.035
  • Peer Reviewed
• [Journal Article] Upregulated IL-7Rα expression on colitogenic memory CD4+ T cells may participate in the development and persistence of chronic colitis (2011)
  • Author(s): Shinohara, T., et al
  • Journal Title: J.Immunol. Volume: 186 Issue: 4 Pages: 2623-2632
  • DOI: 10.4049/jimmunol.1000057
  • Peer Reviewed
• [Journal Article] Suppression of Hath1 gene expression directly regulated by Heslvia Notch signaling is associated with goblet cell depletion in ulcerative colitis (2011)
  • Author(s): Zheng X, Nakamura T, et al
  • Journal Title: Inflamm Bowel Dis Volume: 17 Issue: 11 Pages: 2251-2260
  • DOI: 10.1002/ibd.21611
  • Peer Reviewed
• [Journal Article] Longitudinal cell formation in the entire human small intestine is correlated with the localization of Hath1 and Klf4 (2011)
  • Author(s): Iwasaki M, Tsuchiya K, Suzuki S, Watanabe M, et al
  • Journal Title: J Gastroenterol Volume: 46 Issue: 2 Pages: 191-202
  • DOI: 10.1007/s00535-010-0346-x
  • NAID: 10029027237
  • Peer Reviewed
• [Journal Article] Analysis of interferon signaling by infectious hepatitis C virus clones with substitutions of core amino acids 70 and 91 (2011)
  • Author(s): Funaoka Y, Sakamoto N, Suda G, Itsui Y, Nakagawa M, Kakinuma S, Watanabe T, Nitta S, Kitazume A, Kiyohashi K, Murakawa M, Azuma S, Tsuchiya K,Watanabe M
  • Journal Title: J Virol Volume: 85 Issue: 12 Pages: 5986-94
  • DOI: 10.1128/jvi.02583-10
  • Peer Reviewed
• [Journal Article] MR enterocolonography is useful for simultaneous evaluation of small and large intestinal lesions in Crohn's disease (2011)
  • Author(s): Hyun SB, Kitazume Y, Suzuki S, Watanabe M, et al
  • Journal Title: Inflam Bowel Dis Volume: 17 Issue: 5 Pages: 1063-1072
  • DOI: 10.1002/ibd.21510
  • Peer Reviewed
• [Journal Article] Inhibitory effect of a triterpenoid compound, with or without alpha interferon, on hepatitis C virus infection (2011)
  • Author(s): Watanabe T, Sakamoto N, Nakagawa M, Azuma S, Watanabe M, et al.
  • Journal Title: Antimicrob Agents Chemother Volume: 55(6) Issue: 6 Pages: 2537-2545
  • DOI: 10.1128/aac.01780-10
  • Peer Reviewed
• [Presentation] The acquisition of cancer stemness in colon cancer by the Atoh1 protein stabilization. (2012)
  • Author(s): Kano Y, Tsuchiya K, Horita N, Zheng X, Okamoto R, Nakamura T, Watanabe M
  • Organizer: DDW2012
  • Place of Presentation: San Diego, USA
  • Year and Date: 2012-05-19
• [Presentation] Flagellin response via TLR5 on basolateral membrane of primary intestinal epithelial cells is regulated by Notch signaling. (2012)
  • Author(s): Tsuchiya K, Zheng X, Kano Y, Okamoto R, Nakamura T, Watanabe M
  • Organizer: DDW2012
  • Place of Presentation: San Diego, USA
  • Year and Date: 2012-05-19
• [Presentation] The stabilization of Atoh1 protein in colon cancer acquires cancer stemness and chemoresistance. (2012)
  • Author(s): Tsuchiya K, Kano Y, Mizutani T, Yui S, Nakamura T, Watanabe M
  • Organizer: 第10回 幹細胞シンポジウム
  • Place of Presentation: 淡路島
• [Presentation] The stabilization of Atoh1 protein in colitic cancer induces cancer stemness and chemoresistance. (2012)
  • Author(s): Tsuchiya K, Kano Y, Watanabe M
  • Organizer: 第71回日本癌学会学術総会
  • Place of Presentation: 札幌
• [Presentation] Intestinal stem cells and cancer stem cells. (2012)
  • Author(s): Tsuchiya K
  • Organizer: Cancer Stem Cell Symposium
  • Place of Presentation: 福岡
  • Invited
• [Presentation] 大腸における幹細胞維持とがん幹細胞発現機構 (2012)
  • Author(s): 土屋輝一郎、加納嘉人、中村哲也、渡辺 守
  • Organizer: 第44回日本臨床分子形態学会総会・学術集会
  • Place of Presentation: 高知
  • Invited
• [Presentation] Flagellin via TLR5 on basolateral membrane of primary intestinal epithelial cells. (IEC) shows the role of IEC in the response to bacteria (2011)
  • Author(s): Tsuchiya K
  • Organizer: UEGW2011
  • Place of Presentation: Stockholm
  • Year and Date: 2011-10-26
• [Presentation] The stabilization of Atoh1 protein in colorectal cancer mimics raucinous adenocarcinoma (2011)
  • Author(s): 土屋輝一郎
  • Organizer: 第70回日本癌学会学術総会
  • Place of Presentation: 名古屋
  • Year and Date: 2011-10-04
• [Presentation] IBDにおける消化管上皮の分化制御と免疫応答 (2011)
  • Author(s): 土屋輝一郎
  • Organizer: 第39回日本臨床免疫学会総会
  • Place of Presentation: 東京(招待講演)
  • Year and Date: 2011-09-17
• [Presentation] Cdx2 and Hes1 regulate Hath1 gene expression independently of each other, associating with goblet cell content in Ulcerative Colitis (2011)
  • Author(s): Zheng X, Tsuchiya K
  • Organizer: DDW 2011
  • Place of Presentation: Chicago
  • Year and Date: 2011-05-09