癌細胞の脆弱性と頑強性の構成的理解

• Project/Area Number: 16KT0069
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Multi-year Fund
• Section: 特設分野
• Research Field: Constructive Systems Biology
• Research Institution: Center for Novel Science Initatives, National Institutes of Natural Sciences (2018-2019); Okazaki Research Facilities, National Institutes of Natural Sciences (2016-2017)
• Principal Investigator: 青木 一洋 大学共同利用機関法人自然科学研究機構(新分野創成センター、アストロバイオロジーセンター、生命創成探究, 生命創成探究センター, 教授 (80511427)
• Project Period (FY): 2016-07-19 – 2020-03-31
• Project Status: Discontinued (Fiscal Year 2019)
• Budget Amount: ¥18,330,000 (Direct Cost: ¥14,100,000、Indirect Cost: ¥4,230,000); Fiscal Year 2019: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000); Fiscal Year 2018: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000); Fiscal Year 2017: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000); Fiscal Year 2016: ¥7,410,000 (Direct Cost: ¥5,700,000、Indirect Cost: ¥1,710,000)
• Keywords: oncogene addiction / KRas / BRaf / ERK / intrinsic resistance / Oncogene addiction / Intrinsic resistance / Ras / 遺伝子編集

Outline of Annual Research Achievements

癌遺伝子中毒（oncogene addiction）の構成的な理解を目的として研究を進めた。遺伝子編集技術によりヒト正常乳腺上皮細胞MCF-10A細胞にKRas遺伝子、またはBRaf遺伝子に癌で見つかる活性化変異を片アレルに導入した細胞株を入手した。このKRas遺伝子、BRaf遺伝子をsiRNAやCRISPR/Cas9によりKD、またはKOしたが、細胞死は起きなかったことから、これらの細胞株においてはまだoncogene addictionが起きていないことが分かった。これらの細胞株はERKのリン酸化がやや上昇していたが、soft agarアッセイや増殖曲線、MEK阻害剤に対する感受性などを調べた結果、野生株と大差がないことが分かった。これらの結果から、oncogene addictionが起きるためには、癌遺伝子に変異が入るだけでなく、その後のなんらかの変化（エピジェネティックな変化など）が必要であることが示唆された。
一方、MCF-10A細胞に癌遺伝子変異を導入したKRas遺伝子を過剰発現させた細胞株を樹立したところ、これらの細胞株についてはsoft agarアッセイや増殖曲線などに対する応答が野生株よりも向上しており、一般的な癌に見られる特性が観察された。しかしながら、過剰発現させたKRas遺伝子をsiRNAによりKDしたとしても細胞死は観察されず、oncogene addictionは起きていないことが分かった。これらの結果から、癌遺伝子を過剰発現させると癌に特有の特性である細胞増殖や足場非依存性は獲得できるがoncogene addictionは起きないことが分かり、oncogene addictionが起きるためには、なんらかの変化が重要であることが示唆された。

Research Products

• [Journal Article] Single-cell quantification of the concentrations and dissociation constants of endogenous proteins (2019)
  • Author(s): Akira T Komatsubara, Yuhei Goto, Yohei Kondo, Michiyuki Matsuda, Kazuhiro Aoki
  • Journal Title: Journal of Biological Chemistry Volume: 294 Issue: 15 Pages: 6062-6072
  • DOI: 10.1074/jbc.ra119.007685
  • Peer Reviewed
• [Journal Article] Cell-to-Cell Heterogeneity in p38-Mediated Cross-Inhibition of JNK Causes Stochastic Cell Death (2018)
  • Author(s): Miura Haruko、Kondo Yohei、Matsuda Michiyuki、Aoki Kazuhiro
  • Journal Title: Cell Reports Volume: 24 Issue: 10 Pages: 2658-2668
  • DOI: 10.1016/j.celrep.2018.08.020
  • Peer Reviewed / Open Access
• [Journal Article] Composite regulation of ERK activity dynamics underlying tumour-specific traits in the intestine (2018)
  • Author(s): Muta Yu、Fujita Yoshihisa、Sumiyama Kenta、Sakurai Atsuro、Taketo M. Mark、Chiba Tsutomu、Seno Hiroshi、Aoki Kazuhiro、Matsuda Michiyuki、Imajo Masamichi
  • Journal Title: Nature Communications Volume: 9 Issue: 1 Pages: 2174-2174
  • DOI: 10.1038/s41467-018-04527-8
  • NAID: 120006488609
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Live-cell Imaging with Genetically Encoded Protein Kinase Activity Reporters (2018)
  • Author(s): Maryu G, Miura H, Uda Y, Komatsubara AT, Matsuda M, Aoki K
  • Journal Title: Cell Structure and Function Volume: 43 Issue: 1 Pages: 61-74
  • DOI: 10.1247/csf.18003
  • NAID: 130006725957
  • ISSN: 0386-7196, 1347-3700
  • Peer Reviewed / Open Access
• [Journal Article] Propagating Wave of ERK Activation Orients Collective Cell Migration (2017)
  • Author(s): Aoki Kazuhiro、Kondo Yohei、Naoki Honda、Hiratsuka Toru、Itoh Reina E.、Matsuda Michiyuki
  • Journal Title: Developmental Cell Volume: 43 Issue: 3 Pages: 305-317.e5
  • DOI: 10.1016/j.devcel.2017.10.016
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Efficient synthesis of phycocyanobilin in mammalian cells for optogenetic control of cell signaling (2017)
  • Author(s): Uda Youichi、Goto Yuhei、Oda Shigekazu、Kohchi Takayuki、Matsuda Michiyuki、Aoki Kazuhiro
  • Journal Title: Proc Natl. Acad. Sci. U.S.A. Volume: 114 Issue: 45 Pages: 11962-11967
  • DOI: 10.1073/pnas.1707190114
  • Peer Reviewed
• [Journal Article] Two New FRET Imaging Measures: Linearly Proportional to and Highly Contrasting the Fraction of Active Molecules. (2016)
  • Author(s): Yamao M, Aoki K, Yukinawa N, Ishii S, Matsuda M, Naoki H.
  • Journal Title: PLoS One Volume: 11 Issue: 10 Pages: e0164254-e0164254
  • DOI: 10.1371/journal.pone.0164254
  • NAID: 120005853281
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Visualization of Neuregulin 1 ectodomain shedding reveals its local processing in vitro and in vivo. (2016)
  • Author(s): Kamezaki A, Sato F, Aoki K, Asakawa K, Kawakami K, Matsuzaki F, Sehara-Fujiwara A.
  • Journal Title: Scientific Reports Volume: 6 Issue: 1 Pages: 28873-28873
  • DOI: 10.1038/srep28873
  • NAID: 120005819488
  • Peer Reviewed / Open Access
• [Journal Article] Multiplexed Fluorescence Imaging of ERK and Akt Activities and Cell-cycle Progression (2016)
  • Author(s): Maryu G, Matsuda M, Aoki K
  • Journal Title: Cell Structure and Function Volume: 41 Issue: 2 Pages: 81-92
  • DOI: 10.1247/csf.16007
  • NAID: 130005165901
  • ISSN: 0386-7196, 1347-3700
  • Peer Reviewed / Open Access
• [Journal Article] Synergistic antitumor effects of combination of PI3K/mTOR and MEK inhibition (SAR245409 and pimasertib) in mucinous ovarian carcinoma cells by fluorescence resonance energy transfer imaging. (2016)
  • Author(s): Inaba K, Oda K, Aoki K, Sone K, Ikeda Y, Miyasaka A, Kashiyama T, Fukuda T, Makii C, Arimoto T, Wada-Hiraike O, Kawana K, Yano T, Osuga Y, Fujii T
  • Journal Title: Oncotarget Volume: 7 Issue: 20 Pages: 29577-29591
  • DOI: 10.18632/oncotarget.8807
  • NAID: 120005820854
  • Peer Reviewed / Open Access
• [Presentation] Visualization, quantification, and manipulation of cell signaling by imaging. (2017)
  • Author(s): Aoki K
  • Organizer: ConBio 2017
  • Invited
• [Presentation] Propagation wave of ERK activation orients collective cell migration. (2017)
  • Author(s): Aoki K
  • Organizer: 50th Annual Meeting of the Japanese Society of Developmental Biologists
  • Invited
• [Remarks] プレスリリース概要
  • URL: http://www.nibb.ac.jp/pressroom/news/2019/04/26.html
• [Remarks] 細胞の集団移動の方向性を決める仕組みの発見 ～ERK分子活性の細胞間伝搬とは逆方向に細胞が動く～
  • URL: http://www.nibb.ac.jp/press/2017/11/07.html
• [Remarks] 赤色光/近赤外光による細胞機能の光操作の簡便化に成功
  • URL: http://www.nibb.ac.jp/press/2017/10/24.html