Establishment of quantitation method for proteome-based phospho-signaling pathway and its application for prediction of drug effect monitoring

• Project/Area Number: 25290054
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Tumor diagnostics
• Research Institution: National Institutes of Biomedical Innovation, Health and Nutrition
• Principal Investigator: Tomonaga Takeshi 国立研究開発法人医薬基盤・健康・栄養研究所, プロテオームリサーチプロジェクト, プロジェクトリーダー (80227644)
• Co-Investigator(Kenkyū-buntansha): ISHIHAMA Yasushi 京都大学, 薬学部, 教授 (30439244) ; MATSUBARA Hisahiro 千葉大学, 医学研究院, 教授 (20282486)
• Co-Investigator(Renkei-kenkyūsha): NAGAYAMA Satoshi 公益財団法人がん研究会, 有明病院消化器外科, 医長 (70362499)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥16,770,000 (Direct Cost: ¥12,900,000、Indirect Cost: ¥3,870,000); Fiscal Year 2015: ¥6,110,000 (Direct Cost: ¥4,700,000、Indirect Cost: ¥1,410,000); Fiscal Year 2014: ¥6,110,000 (Direct Cost: ¥4,700,000、Indirect Cost: ¥1,410,000); Fiscal Year 2013: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
• Keywords: リン酸化プロテオミクス / 薬効予測 / EGFR阻害剤 / 肺がん / プロテオミクス / バイオマーカー / リン酸化タンパク質 / がん / リン酸化タンパク

Outline of Final Research Achievements

Molecular target drugs such as kinase inhibitors for cancer treatment are not uniformly clinically effective, thus sorting the patients that will benefit from the treatments is critical. Moreover, acquired resistance for the drugs is also a critical problem. Therefore, the identification of mechanisms underlying the resistance is urgent need. Systematic quantification of kinase activation is potentially predictive of the response of kinase inhibitors and it can be quantitated by phosphorylation level of their substrates. In this study, we developed a large-scale phosphoproteome quantification method for the kinase substrates and applied for prediction of the kinase activities and drug response. We also examined the mechanism of drug resistance. By the phosphoproteome analysis of several lung cancer cell lines which are sensitive or resistant for the drug currently used in clinic, erlotinib, we identified several phosphoproteins and kinases which are able to predict drug sensitivity.

Research Products

• [Journal Article] Cell surface localization of importin α1/KPNA2 affects cancer cell proliferation by regulating FGF1 signalling (2016)
  • Author(s): Yamada K., Miyamoto Y., Tsujii A., Moriyama T., Ikuno Y., Shiromizu T., Serada S., Fujimoto M., Tomonaga T., Naka T., Yoneda Y., Oka M.
  • Journal Title: Scientific Reports Volume: 6 Issue: 1 Pages: 231410-231410
  • DOI: 10.1038/srep21410
  • Peer Reviewed / Open Access
• [Journal Article] Cell surface localization of importin α1/KPNA2 affects cancer cell proliferation by regulating FGF1 signalling. (2016)
  • Author(s): Yamada K., Miyamoto Y., Tsujii A., Moriyama T., Ikuno Y., Shiromizu T., Serada S., Fujimoto M., Tomonaga T., Naka T., Oka M.
  • Journal Title: Sci Rep Volume: 6 Issue: 1 Pages: 24823-24823
  • DOI: 10.1038/srep24823
  • Peer Reviewed / Open Access
• [Journal Article] Targeted Phosphoproteome Analysis Using Selected/Multiple Reaction Monitoring (SRM/MRM). (2016)
  • Author(s): Adachi, J., Narumi, R., Tomonaga, T
  • Journal Title: Methods Mol Biol Volume: 1394 Pages: 87-100
  • DOI: 10.1007/978-1-4939-3341-9_7
  • ISBN: 9781493933396, 9781493933419
  • Peer Reviewed
• [Journal Article] Quantitative Analysis of Tissue Samples by Combining iTRAQ Isobaric Labeling with Selected/Multiple Reaction Monitoring (SRM/MRM). (2016)
  • Author(s): Narumi, R., Tomonaga, T
  • Journal Title: Methods Mol Biol Volume: 1355 Pages: 85-101
  • DOI: 10.1007/978-1-4939-3049-4_6
  • ISBN: 9781493930487, 9781493930494
  • Peer Reviewed
• [Journal Article] Data for Proteomic Analysis of ATP-Binding Proteins and Kinase Inhibitor Target Proteins Using an ATP Probe. (2015)
  • Author(s): Adachi, J., Kishida, M., Watanabe, S., Hashimoto, Y., Fukamizu, K., and Tomonaga, T.
  • Journal Title: Data in Brief Volume: 5 Pages: 726-729
  • DOI: 10.1016/j.dib.2015.10.018
  • Peer Reviewed / Open Access
• [Journal Article] Nuclear accumulation of annexin A2 contributes to chromosomal instability by coilin-mediated centromere damage (2015)
  • Author(s): Kazami, T., Nie, H., Satoh, M., Kuga, T., Matsushita, K., Kawasaki, N., Tomonaga, T*., and Nomura, F
  • Journal Title: Oncogene Volume: in press Issue: 32 Pages: 4177-4189
  • DOI: 10.1038/onc.2014.345
  • Peer Reviewed
• [Journal Article] Differential Proteome Analysis Identifies TGF-beta-Related Pro-Metastatic Proteins in a 4T1 Murine Breast Cancer Model. (2015)
  • Author(s): Sato, M., Matsubara, T., Adachi, J., Hashimoto, Y., Fukamizu, K., Kishida, M., Yang, Y.A., Wakefield, L.M., and Tomonaga, T
  • Journal Title: PloS One Volume: 10 Issue: 5 Pages: e0126483-e0126483
  • DOI: 10.1371/journal.pone.0126483
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] c-Abl induces stabilization of histone deacetylase 1 (HDAC1) in a kinase activity-dependent manner. (2015)
  • Author(s): Kazumasa Aoyama, Noritaka Yamaguchi, Ryuzaboro Yuki, Mariko Morii, Sho Kubota, Kensuke Hirata, Kohei Abe, Takuya Honda, Takahisa Kuga, Yuuki Hashimoto, Takeshi Tomonaga, and Naoto Yamaguchi
  • Journal Title: Cell Biology International Volume: 39 Issue: 4 Pages: 446-456
  • DOI: 10.1002/cbin.10413
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Role for Tyrosine Phosphorylation of A-kinase Anchoring Protein 8 (AKAP8) in its Dissociation from Chromatin and the Nuclear Matrix (2015)
  • Author(s): Kubota S, Morii M, Yuki R, Yamaguchi N, Yamaguchi H, Aoyama K, Kuga T, Tomonaga T, Yamaguchi N.
  • Journal Title: J Biol Chem Volume: 290 Issue: 17 Pages: 10891-904
  • DOI: 10.1074/jbc.m115.643882
  • Peer Reviewed / Open Access
• [Journal Article] c-Abl-mediated tyrosine phosphorylation of JunB is required for Adriamycin-induced expression of p21. (2015)
  • Author(s): Noritaka Yamaguchi, Ryuzaburo Yuki, Sho Kubota, Kazumasa Aoyama, Takahisa Kuga, Yuuki Hashimoto, Takeshi Tomonaga, and Naoto Yamaguchi.
  • Journal Title: Biochemical Journal Volume: 471 Issue: 1 Pages: 67-77
  • DOI: 10.1042/bj20150372
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Transcriptional Dynamics of Immortalized Human Mesenchymal Stem Cells during Transformation. (2015)
  • Author(s): Takeuchi, M., Higashino, A., Takeuchi, K., Hori, Y., Koshiba-Takeuchi, K., Makino, H., Monobe, Y., Kishida, M., Adachi, J., Takeuchi, J., Tomonaga, T., Umezawa, A., Kameoka, Y., and Akagi, K.
  • Journal Title: PloS one Volume: 10 Issue: 5 Pages: 0126562-0126562
  • DOI: 10.1371/journal.pone.0126562
  • NAID: 120005947126
  • Peer Reviewed / Open Access
• [Journal Article] Differential proteome analysis identifies TGF-β-related pro-metastatic proteins in a 4T1 murine breast cancer model. (2015)
  • Author(s): Sato M, Matsubara T, Adachi J, Hashimoto Y, Kishida M, Yang Y, Wakefield LM, Tomonaga T*
  • Journal Title: PLoS One Volume: in press
  • Peer Reviewed / Open Access
• [Journal Article] Proteome-wide discovery of unknown ATP-binding proteins and kinase inhibitor target proteins using an ATP probe. (2014)
  • Author(s): Jun Adachi, Marina Kishida, Shio Watanabe, Yuuki Hashimoto, Kazuna Fukamizu, Takeshi Tomonaga.
  • Journal Title: Journal of proteome research Volume: 13 Issue: 12 Pages: 5461-5470
  • DOI: 10.1021/pr500845u
  • Peer Reviewed
• [Journal Article] Discovery of colorectal cancer biomarker candidates by membrane proteomic analysisand subsequent verification using selected reaction monitoring and tissue microarrayanalysis (2014)
  • Author(s): Kume H, Muraoka S, Kuga T, Adachi J, Narumi R, Watanabe S, Kuwano M, Kodera Y, Matsushita K, Fukuoka J, Masuda T, Ishihama Y, Matsubara H, Nomura F, Tomonaga T.
  • Journal Title: Mol Cell Proteomics. Volume: M113 Issue: 6 Pages: 37093-37093
  • DOI: 10.1074/mcp.m113.037093
  • Peer Reviewed
• [Journal Article] Lamin B2 prevents chromosome instability by ensuring proper mitotic chromosome segregation (2013)
  • Author(s): Kuga, T., Nie, H., Kazami, T., Satoh, M., Matsushita, K., Nomura, F., Maeshima, K., Nakayama, Y., Tomonaga, T.
  • Journal Title: Oncogenesis Volume: 3 Issue: 3 Pages: e94-e94
  • DOI: 10.1038/oncsis.2014.6
  • Peer Reviewed
• [Journal Article] Absolute Quantitation of Low Abundance Plasma APL1β peptides at Sub-fmol/mL Level by SRM/MRM without Immunoaffinity Enrichment. (2013)
  • Author(s): Sano S, Tagami S, Hashimoto Y, Yoshizawa-Kumagaye K, Tsunemi M, Okochi M, Tomonaga T.
  • Journal Title: J Proteome Res. Volume: 13 Issue: 2 Pages: 10121020-10121020
  • DOI: 10.1021/pr4010103
  • Peer Reviewed
• [Journal Article] A novel mechanism of keratin cytoskeleton organization through casein kinase I alpha and FAM83H in colorectal cancer. (2013)
  • Author(s): Kuga, T., Kume, H., Kawasaki, N., Sato, M., Adachi, J., Shiromizu, T., Hoshino, I., Nishimori, T., Matsubara, H., and Tomonaga T.
  • Journal Title: J Cell Sci Volume: 126 Pages: 4721-31
  • DOI: 10.1242/jcs.129684
  • Peer Reviewed
• [Journal Article] Formation of long and winding nuclear F-actin bundles by nuclear c-Abl tyrosine kinase. (2013)
  • Author(s): Aoyama K., Yuki R., Horiike Y., Kubota S., Yamaguchi N., Morii M., Ishibashi K., Nakayama Y., Kuga T., Hashimoto Y., Tomonaga T, and Yamaguchi N.
  • Journal Title: Exp Cell Res Volume: 319 Issue: 20 Pages: 3251-68
  • DOI: 10.1016/j.yexcr.2013.09.003
  • Peer Reviewed
• [Journal Article] Phosphorylation of KRAB-associated Protein 1 (KAP1) at Tyr-449, Tyr-458, and Tyr-517 by Nuclear Tyrosine Kinases Inhibits the Association of KAP1 and Heterochromatin Protein 1alpha (HP1alpha) with Heterochromatin. (2013)
  • Author(s): Kubota, S., Fukumoto, Y., Aoyama, K., Ishibashi, K., Yuki, R., Morinaga, T., Honda, T., Yamaguchi, N., Hashimoto, Y., Kuga, T., Tomonaga T, and Yamaguchi, N.
  • Journal Title: J Biol Chem Volume: 288, Issue: 24 Pages: 17871-17883
  • DOI: 10.1074/jbc.m112.437756
  • Peer Reviewed
• [Presentation] プロテオミクスを用いたバイオマーカー探索とPrecision Medicineへの応用 (2016)
  • Author(s): 朝長 毅
  • Organizer: 第44回 京阪泌尿器腫瘍セミナー
  • Place of Presentation: 大阪
  • Year and Date: 2016-02-08
  • Invited
• [Presentation] 定量プロテオミクスの創薬及び個別化医療への応用 (2016)
  • Author(s): 朝長 毅
  • Organizer: 第13回 神戸ポートアイランド創薬フォーラム
  • Place of Presentation: 神戸臨床研究情報センター
  • Year and Date: 2016-01-26
  • Invited
• [Presentation] 定量プロテオミクスを用いた疾患バイオマーカー探索とその医療への応用 (2015)
  • Author(s): 朝長 毅
  • Organizer: 日本プロテオーム学会2015年会
  • Place of Presentation: くまもと森都心プラザ
  • Year and Date: 2015-07-23
  • Invited
• [Presentation] 定量プロテオミクスの医療への応用 (2015)
  • Author(s): 朝長 毅
  • Organizer: 第42回BMSカンファレンス
  • Place of Presentation: 岐阜グランドホテル
  • Year and Date: 2015-07-06
  • Invited
• [Presentation] Verification of predictive biomarker candidates of colorectal cancer metastasis in serum extracellular vesicles by selected reaction monitoring based targeted proteomics. (2015)
  • Author(s): Tomonaga T, Kume H
  • Organizer: International Extracellular Vesicles Conference
  • Place of Presentation: Washington DC, USA
  • Year and Date: 2015-04-23
  • Int'l Joint Research
• [Presentation] 定量プロテオミクスの医療への応用 (2015)
  • Author(s): 朝長 毅
  • Organizer: 第150回 質量分析関西談話会
  • Place of Presentation: 大阪
  • Year and Date: 2015-03-14
  • Invited
• [Presentation] Identification of predictive biomarker candidates of colorectal cancer metastasis in serum extracellular vesicles based on membrane quantitative proteomics of cancer tissues. (2014)
  • Author(s): Tomonaga T, Kume H
  • Organizer: Australasia Extracellular Vesicles Conference
  • Place of Presentation: Kerns, Australia
  • Year and Date: 2014-11-20
• [Presentation] Discovery of colorectal cancer biomarker candidates by membrane proteomic analysis and subsequent verification using selected reaction monitoring (2014)
  • Author(s): Tomonaga T, Kume H
  • Organizer: Human Proteome Organization (HUPO) 13th Annual World Congress
  • Place of Presentation: Madrid, Spain
  • Year and Date: 2014-10-05 – 2014-10-08
• [Presentation] Discovery of colorectal cancer biomarker candidates by membrane proteomic analysis of human tissue samples and subsequent verification using SRM/MRM (2014)
  • Author(s): Tomonaga T
  • Organizer: The 13th HUPO World Congress Post Congress Workshop
  • Place of Presentation: Segovia, Spain
  • Year and Date: 2014-09-09
  • Invited
• [Presentation] 質量分析計を用いたバイオマーカー定量法の実用化（医療への応用） (2014)
  • Author(s): 朝長 毅
  • Organizer: レドックス・ライフイノベーション第170委員会
  • Place of Presentation: 宮崎
  • Year and Date: 2014-08-22
  • Invited
• [Presentation] 質量分析計を用いたバイオマーカー定量法の創薬への応用を目指して (2014)
  • Author(s): 朝長 毅
  • Organizer: 第12回日本プロテオーム学会2014年会
  • Place of Presentation: つくば
  • Year and Date: 2014-07-17
  • Invited
• [Presentation] 質量分析計を用いたバイオマーカー定量法の実用化（医療への応用） (2014)
  • Author(s): 朝長 毅
  • Organizer: 第14回日本蛋白質科学会
  • Place of Presentation: 神奈川
  • Year and Date: 2014-06-25
  • Invited
• [Presentation] 質量分析計を用いたバイオマーカー定量法の医療への応用 (2014)
  • Author(s): 朝長 毅
  • Organizer: 第10回日本臨床プロテオーム研究会
  • Place of Presentation: 東京
  • Year and Date: 2014-05-10
  • Invited
• [Presentation] 質量分析計を用いたバイオマーカー定量法の実用化 (2014)
  • Author(s): 朝長 毅
  • Organizer: 第11 回 北里疾患プロテオミクス研究会
  • Place of Presentation: 神奈川
• [Presentation] ターゲットプロテオミクス～Beyond SRM～ (2013)
  • Author(s): 足立 淳、朝長 毅
  • Organizer: 第86回日本生化学会大会
  • Place of Presentation: 横浜
• [Presentation] 質量分析計を用いたバイオマーカー定量法の実用化への挑戦 (2013)
  • Author(s): 朝長 毅
  • Organizer: 第10 回 千葉疾患プロテオミクス研究会
  • Place of Presentation: 東京
• [Presentation] 自己抗体ファージライブラリを用いたスキルス胃癌の診断バイオマーカーの探索 (2013)
  • Author(s): 村岡 賢，久米秀明，西塚 哲，若林 剛，星野 敢，松原久裕，朝長 毅
  • Organizer: 第72回日本癌学会学術総会
  • Place of Presentation: 横浜
• [Presentation] マウス同所移植モデルによる大腸癌高浸潤性細胞のプロテオーム解析 (2013)
  • Author(s): 白水 崇、足立 淳、八木 滋雄、朝長 毅
  • Organizer: 第72回日本癌学会学術総会
  • Place of Presentation: 横浜
• [Presentation] ATP Accessibility Screening (AAS), A High-Throughput and High-Resolution Kinase Analysis Platform for Signaling Research. (2013)
  • Author(s): Adachi J, Higo D, Watanabe S, Kuwano M, Hashimoto Y, Tomonaga T.
  • Organizer: Human Proteome Organization (HUPO) 12th annual world congress
  • Place of Presentation: 横浜
• [Presentation] Discovery and subsequent validation of biomarkers for colorectal cancer by large-scale proteomic analysis and tissue microarray analysis. (2013)
  • Author(s): Kume H, Muraoka S, Hashimoto Y, Watanabe S, Masuda T, Ishihama Y, Fukuoka J, Kodera Y, Matsushita K, Matsubara H, Tomonaga T.
  • Organizer: Human Proteome Organization (HUPO) 12th annual world congress
  • Place of Presentation: 横浜
• [Presentation] Proteomic Analysis of Highly Invasive Colorectal Cancer Cells Established byOrthotopic Xenograft Mouse Model. (2013)
  • Author(s): Shiromizu T, Adachi J, Yagi S, Hoffman RM, Tomonaga T.
  • Organizer: Human Proteome Organization (HUPO) 12th annual world congress
  • Place of Presentation: 横浜
• [Presentation] Development of High-throughput Screening System Using Autoantibody Library for Discovery of Scirrhous Gastric Cancer Biomarker. (2013)
  • Author(s): Muraoka S, Kume H, Saitoh H, Enomoto Y, Ito Y, Nishizuka S, Wakabayashi G, Hoshino I, Matsubara H, and Tomonaga T.
  • Organizer: Human Proteome Organization (HUPO) 12th annual world congress
  • Place of Presentation: 横浜
• [Presentation] Quantitative Phosphoproteome Analysis of Cultured Stomach Cancer Cell Lines Aimed at Development of Biomarkers for Prediction of Drug Efficacy. (2013)
  • Author(s): Hashiguchi K, Muraoka S, Adachi J, Sato M, Kuga T, Watanabe R, Shiromizu T, Hashimoto Y, Nagano M, Kishida M, Tomonaga T.
  • Organizer: Human Proteome Organization (HUPO) 12th annual world congress
  • Place of Presentation: 横浜
• [Presentation] Quantitative Proteome Analysis with Isotope Dimethyl Labeling to Identify TGF-beta-mediated Tumor Proteins Using the Metastatic Mouse Breast Cancer Model. (2013)
  • Author(s): Sato M, Adachi J, Tomonaga T.
  • Organizer: Human Proteome Organization (HUPO) 12th annual world congress
  • Place of Presentation: 横浜
• [Presentation] Phospho-profiling of mTOR inhibitor-treated renal cell carcinoma (RCC) cell lines and its application for drug response-efficacy biomarkers. (2013)
  • Author(s): Watanabe R, Hashimoto Y, Kishida M, Matsubara M, Adachi J, Tomonaga T.
  • Organizer: Human Proteome Organization (HUPO) 12th annual world congress
  • Place of Presentation: 横浜
• [Presentation] A Novel Mechanism of Keratin Cytoskeleton Organization Through Casein Kinase I Alpha and FAM83H in Colorectal Cancer: Interactome Analysis of FAM83H (2013)
  • Author(s): Kuga T, Kume H, Kawasaki N, Sato M, Adachi J, Shiromizu T, Hoshino I, Matsubara H, Tomonaga T. .
  • Organizer: Human Proteome Organization (HUPO) 12th annual world congress
  • Place of Presentation: 横浜
• [Presentation] A Kinase Activity-Estimating Method Using LC-MS/MS. (2013)
  • Author(s): Nagano M, Kuga T, Adachi J, Tomonaga T.
  • Organizer: Human Proteome Organization (HUPO) 12th annual world congress
  • Place of Presentation: 横浜
• [Remarks] 国立研究開発法人医薬基盤・健康・栄養研究所 プロテオームリサーチプロジェクト
  • URL: http://www.nibiohn.go.jp/proteome/index.html
• [Patent(Industrial Property Rights)] ペプチド又はタンパク質の分画方法 (2015)
  • Inventor(s): 朝長 毅、足立 淳、宮崎将太
  • Industrial Property Rights Holder: ジーエルサイエンス 株式会社
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2015-134482
  • Filing Date: 2015-07-03