マイクロサテライト不安定性を呈する発癌機構の統合的理解と新たな治療体系の確立

2013 Fiscal Year Annual Research Report

• Project/Area Number: 25253080
• Research Category: Grant-in-Aid for Scientific Research (A)
• Research Institution: The University of Tokyo
• Principal Investigator: 渡邉 聡明 東京大学, 医学部附属病院, 教授 (80210920)
• Co-Investigator(Kenkyū-buntansha): 佐藤 俊朗 慶應義塾大学, 医学部, 准教授 (70365245)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Keywords: マイクロサテライト不安定性 / 大腸鋸歯状腺腫 / 大腸幹細胞 / エピジェネティクス

Research Abstract

本年度の予定通り，大腸鋸歯状病変の内視鏡生検サンプルより，過形成ポリープおよび鋸歯状腺腫オのルガノイド株を樹立した．また，同様に手術検体サンプルよりMSI大腸癌のオルガノイド株を樹立し，大腸癌発癌において提唱されているserrated pathwayについて，各ステップのオルガノイド株を樹立することに成功した．樹立した全てのオルガノイド株について，BRAF遺伝子解析，マイクロサテライト解析，CpGアイランドのメチル化解析を行った．BRAF遺伝子変異解析はSangerシーケンスによりV600E変異の有無を確認した．マイクロサテライト解析については，5つのマイクロサテライト領域を蛍光標識プライマーを使用したPCRにて増幅し，それぞれのフラグメント解析を行った．CpGアイランドメチル化解析については，スループットを重視しリアルタイムPCRによるMethylight法を使用し，WeisenbergerらのCIMPパネルを使用してCIMPの有無を判定した．その結果，樹立した過形成ポリープオルガノイド，鋸歯状腺腫オルガノイドはいずれもBRAF遺伝子変異，CpGアイランドのメチル化（CIMP）を認めたが，MSIは認めなかった．MSI大腸癌については66%にBRAF遺伝子変異およびCIMP highを認めた．さらに，それぞれのオルガノイド株よりgenomic DNAを抽出し，次世代シーケンサーによるターゲットリシーケンスないしエキソームシーケンスを行った．

Current Status of Research Progress

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

Reason

本年度の研究については，大腸鋸歯状腺腫よりオルガノイド株を樹立し，BRAF遺伝子解析，CIMP解析，MSI解析を定型的に行い，最終的に次世代シーケンスを行うところまで成功しており，当初の予定通り進行している．しかしながら，過形成ポリープ，鋸歯状腺腫については疾患の性質上，2例のサンプル数にとどまっている．翌年度以降の研究には支障ないため，並行しつつさらにサンプル数を蓄積したい．

Strategy for Future Research Activity

In vitroにおけるserrated pathwayの再現を目指す．BRAF遺伝子変異はserrated pathwayにおけるinitiation stepであると考えられており，最新の遺伝子編集技術であるCRISPR/Cas9システムを使用し，正常大腸上皮オルガノイドにBRAF遺伝子変異のノックインを行う．当研究室では既に複数の癌抑制遺伝子についてCRISPR/Cas9システムを使用した遺伝子ノックアウトに成功しており，同様の方法論にてノックインも可能であると考えている．得られたBRAF変異オルガノイドを使用し，CpGアイランドのメチル化を惹起する条件を検討する．具体的には低酸素，酸性，低グルコース等を想定している．これら条件にてメチル化が起こらない時の対応として，メチル化シトシンの脱メチル化を阻害するIDH遺伝子変異，TET遺伝子変異を導入することでメチル化シトシンの蓄積をもたらし，擬似的にCIMPを誘導することを考慮している．最終的にはミスマッチ修復遺伝子のサイレンシングによりMSIを来すに至ることを確認し，今後のMSI大腸癌予防および治療に役立てたい．

Research Products

• [Journal Article] Prediction of response to preoperative chemoradiotherapy in rectal cancer by using reverse transcriptase polymerase chain reaction analysis of four genes. (2014)
  • Author(s): Watanabe T, Kobunai T, Akiyoshi T, Matsuda K, Ishihara S, Nozawa K.
  • Journal Title: Dis Colon Rectum Volume: 57 Pages: 23-31
  • DOI: 10.1097/01.dcr.0000437688.33795.9d
  • Peer Reviewed
• [Journal Article] Changes in the plasma levels of cytokines/chemokines for predicting the response to chemoradiation therapy in rectal cancer patients. (2014)
  • Author(s): Tada N, Tsuno NH, Kawai K, Murono K, Nirei T, Ishihara S, Sunami E, Kitayama J, Watanabe T.
  • Journal Title: Oncol Rep Volume: 31 Pages: 463-71
  • DOI: 10.3892/or.2013.2857
  • Peer Reviewed
• [Journal Article] KLF5 Regulates the Integrity and Oncogenicity of Intestinal Stem Cells. (2014)
  • Author(s): Nakaya T, Ogawa S, Manabe I, Tanaka M, Sanada M, Sato T, Taketo MM, Nakao K, Clevers H, Fukayama M, Kuroda M, Nagai R.
  • Journal Title: Cancer Res Volume: Epub ahead of print Pages: in press
  • Peer Reviewed
• [Journal Article] Macrophages and dendritic cells emerge in the liver during intestinal inflammation and predispose the liver to inflammation. (2014)
  • Author(s): Mikami Y, Mizuno S, Nakamoto N, Hayashi A, Sujino T, Sato T, Kamada N, Matsuoka K, Hisamatsu T, Ebinuma H, Hibi T, Yoshimura A, Kanai T.
  • Journal Title: PLoS One Volume: 9(1) Pages: e84619
  • DOI: 10.1371/journal.pone.0084619.
  • Peer Reviewed
• [Journal Article] Clinical benefit of surgery for stage IV colorectal cancer with synchronous peritoneal metastasis. (2014)
  • Author(s): Kobayashi H, Kotake K, Funahashi K, Hase K, Hirata K, Iiai T, Kameoka S, Kanemitsu Y, Maeda K, Murata A, Ohue M, Shirouzu K, Takahashi K, Watanabe T, Yano H, Yatsuoka T, Hashiguchi Y, Sugihara K; Study Group for Peritoneal Metastasis from Colorectal Cancer by the Japanese Society for Cancer of the Colon and Rectum.
  • Journal Title: J Gastroenterol Volume: 49(4) Pages: 646-54
  • DOI: 10.1007/s00535-013-0820-3.
  • Peer Reviewed
• [Journal Article] Salvage Gastrectomy After Intravenous and Intraperitoneal Paclitaxel (PTX) Administration with Oral S-1 for Peritoneal Dissemination of Advanced Gastric Cancer with Malignant Ascites. (2014)
  • Author(s): Kitayama J, Ishigami H, Yamaguchi H, Yamashita H, Emoto S, Kaisaki S, Watanabe T.
  • Journal Title: Ann Surg Oncol Volume: 21(2) Pages: 539-46
  • DOI: 10.1245/s10434-013-3208-y.
  • Peer Reviewed
• [Journal Article] CD90(+) mesothelial-like cells in peritoneal fluid promote peritoneal metastasis by forming a tumor permissive microenvironment. (2014)
  • Author(s): Kitayama J, Emoto S, Yamaguchi H, Isigami H, Watanabe T.
  • Journal Title: PloS One Volume: 9(1) Pages: e86516
  • DOI: 10.1371/journal.pone.0086516.
  • Peer Reviewed
• [Journal Article] Flow cytometric quantification of intraperitoneal free tumor cells in patients with peritoneal metastasis. (2014)
  • Author(s): Kitayama J, Emoto S, Yamaguchi H, Ishigami H, Kamei T, Yamashita H, Seto Y, Matsuzaki K, Watanabe T.
  • Journal Title: Cytometry B Clin Cytom Volume: 86(1) Pages: 56-62
  • DOI: 10.1002/cyto.b.21126.
  • Peer Reviewed
• [Journal Article] Colorectal cancer and hypercoagulability. (2014)
  • Author(s): Kawai K, Watanabe T.
  • Journal Title: Surg Today Volume: 44(5) Pages: 797-803
  • DOI: 10.1007/s00595-013-0606-5.
  • Peer Reviewed
• [Journal Article] Thrombocytosis before pre-operative chemoradiotherapy predicts poor response and shorter local recurrence-free survival in rectal cancer. (2014)
  • Author(s): Kawai K, Kitayama J, Tsuno NH, Sunami E, Watanabe T.
  • Journal Title: Int J Colorectal Dis Volume: 28(4) Pages: 527-535
  • DOI: 10.1007/s00384-012-1594-4.
  • Peer Reviewed
• [Journal Article] Intraperitoneal administration of cisplatin via an in situ cross-linkable hyaluronic acid-based hydrogel for peritoneal dissemination of gastric cancer. (2014)
  • Author(s): Emoto S, Yamaguchi H, Kamei T, Ishigami H, Suhara T, Suzuki Y, Ito T, Kitayama J, Watanabe T.
  • Journal Title: Surg Today Volume: 44(5) Pages: 919-26
  • DOI: 10.1007/s00595-013-0674-6.
  • Peer Reviewed
• [Journal Article] Selective Lateral Pelvic Lymph Node Dissection in Patients with Advanced Low Rectal Cancer Treated with Preoperative Chemoradiotherapy Based on Pretreatment Imaging. (2014)
  • Author(s): Akiyoshi T, Ueno M, Matsueda K, Konishi T, Fujimoto Y, Nagayama S, Fukunaga Y, Unno T, Kano A, Kuroyanagi H, Oya M, Yamaguchi T, Watanabe T, Muto T.
  • Journal Title: Ann Surg Oncol Volume: 21(1) Pages: 189-96
  • DOI: 10.1245/s10434-013-3216-y.
  • Peer Reviewed
• [Journal Article] Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis. (2013)
  • Author(s): Huch M, Bonfanti P, Boj SF, Sato T, Loomans CJ, van de Wetering M, Sojoodi M, Li VS, Schuijers J, Gracanin A, Ringnalda F, Begthel H, Hamer K, Mulder J, van Es JH, de Koning E, Vries RG, Heimberg H, Clevers H.
  • Journal Title: EMBO J Volume: 32(20) Pages: 2708-21
  • DOI: 10.1038/emboj.2013.204.
  • Peer Reviewed
• [Journal Article] A single strain of Clostridium butyricum induces intestinal IL-10-producing macrophages to suppress acute experimental colitis in mice. (2013)
  • Author(s): Hayashi A, Sato T, Kamada N, Mikami Y, Matsuoka K, Hisamatsu T, Hibi T, Roers A, Yagita H, Ohteki T, Yoshimura A, Kanai T.
  • Journal Title: Cell Host Microbe Volume: 13(6) Pages: 711-22
  • DOI: 10.1016/j.chom.2013.05.013.
• [Journal Article] Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. (2013)
  • Author(s): Sato T, Clevers H.
  • Journal Title: Science Volume: 340(6137) Pages: 1190-4
  • DOI: 10.1126/science.1234852.
  • Peer Reviewed
• [Journal Article] IL-22-producing RORγt-dependent innate lymphoid cells play a novel protective role in murine acute hepatitis. (2013)
  • Author(s): Matsumoto A, Kanai T, Mikami Y, Chu PS, Nakamoto N, Ebinuma H, Saito H, Sato T, Yagita H, Hibi T.
  • Journal Title: PLoS One Volume: 8(4) Pages: e62853
  • DOI: 10.1371/journal.pone.0062853.
  • Peer Reviewed
• [Journal Article] In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. (2013)
  • Author(s): Huch M, Dorrell C, Boj SF, van Es JH, Li VS, van de Wetering M, Sato T, Hamer K, Sasaki N, Finegold MJ, Haft A, Vries RG, Grompe M, Clevers H.
  • Journal Title: Nature Volume: 494(7436) Pages: 247-50
  • DOI: 10.1038/nature11826.
  • Peer Reviewed
• [Journal Article] A phase 2 trial of intravenous and intraperitoneal paclitaxel combined with S-1 for treatment of gastric cancer with macroscopic peritoneal metastasis. (2013)
  • Author(s): Yamaguchi H, Kitayama J, Ishigami H, Emoto S, Yamashita H, Watanabe T.
  • Journal Title: Cancer Volume: 119(18) Pages: 3354-8
  • DOI: 10.1002/cncr.28204.
  • Peer Reviewed
• [Journal Article] KRAS Mutational Status in Japanese Patients with Colorectal Cancer: Results from a Nationwide, Multicenter, Cross-sectional Study. (2013)
  • Author(s): "Watanabe T, Yoshino T, Uetake H, Yamazaki K, Ishiguro M, Kurokawa T, Saijo N, Ohashi Y, Sugihara K. "
  • Journal Title: Jpn J Clin Oncol Volume: 43(7) Pages: 706-712
  • DOI: 10.1093/jjco/hyt062.
  • Peer Reviewed
• [Journal Article] Hepatic steatosis is associated with lower incidence of liver metastasis from colorectal cancer. (2013)
  • Author(s): Murono K, Kitayama J, Tsuno NH, Nozawa H, Kawai K, Sunami E, Akahane M, Watanabe T.
  • Journal Title: Int J Colorectal Dis Volume: 28(8) Pages: 1065-1072
  • DOI: 10.1007/s00384-013-1656-2.
  • Peer Reviewed
• [Journal Article] Safety and factors contributing to the difficulty of laparoscopic surgery for rectal cancer treated with preoperative chemoradiotherapy. (2013)
  • Author(s): Ishihara S, Watanabe T, Fukushima Y, Akahane T, Horiuchi A, Shimada R, Nakamura K, Hayama T, Yamada H, Nozawa K, Matsuda K, Hashiguchi Y.
  • Journal Title: Tech Coloproctol Volume: 18(3) Pages: 247-255
  • DOI: 10.1007/s10151-013-1048-1.
  • Peer Reviewed
• [Journal Article] Hypoxia enhances colon cancer migration and invasion through promotion of epithelial-mesenchymal transition. (2013)
  • Author(s): Hongo K, Tsuno NH, Kawai K, Sasaki K, Kaneko M, Hiyoshi M, Murono K, Tada N, Nirei T, Sunami E, Takahashi K, Nagawa H, Kitayama J, Watanabe T.
  • Journal Title: J Surg Res Volume: 182(1) Pages: 75-84
  • DOI: 10.1016/j.jss.2012.08.034.
  • Peer Reviewed