title: Augmentation of multiple protein kinase activities associated with secondary imatinib resistance in gastrointestinal stromal tumors as revealed by quantitative phosphoproteome analysis.
abstract: Mutations in the Kit receptor tyrosine kinase gene (KIT), which result in constitutive activation of the protein (KIT), are causally related to the development of gastrointestinal stromal tumors (GISTs). Imatinib, a targeted anticancer drug, exerts a therapeutic effect against GISTs by repressing the kinase activity of KIT. Long-term administration of this drug, however, causes the emergence of imatinib-resistant GISTs. We performed quantitative phosphoproteome analysis using a cell-based GIST model system comprising an imatinib-sensitive GIST cell line (GIST882), GIST882 under treatment with imatinib (GIST882-IM), and secondary imatinib-resistant GIST882 (GIST882-R). Phosphorylated peptides were purified from each cell line using titania-based affinity chromatography or anti-phosphotyrosine immunoprecipitation, and then subjected to LC-MS/MS based quantitative phosphoproteome analysis. Using this method we identified augmentation of the kinase activities of multiple elements of the signal transduction pathway, especially KIT and EGFR. Although, these elements were up-regulated in GIST882-R, no additionally mutated KIT mRNA was found in secondary imatinib-resistant GIST cells. Treatment of GIST882-R with imatinib in combination with gefitinib, an EGFR inhibitor, partially prevented cell growth, implying that EGFR may be involved in acquisition of secondary imatinib resistance in GIST.
  
  
  CONCLUSIONS
  In this study, we performed a quantitative phosphoproteome analysis using a cell culture-based GIST model system. The goal of the study was to investigate the mechanism of acquired resistance in GISTs against imatinib, a molecularly targeted drug that inhibits kinase activity of the KIT protein and that has been approved for the treatment of GISTs. In imatinib-resistant GIST cells, we observed elevated expression of KIT and restoration of its kinase activity, as well as activation of multiple proliferative signaling pathways. Our results indicate that the effects of even so-called 'molecularly targeted' drugs, are broad rather than convergent, and that the mechanisms of action of such drugs during continuous administration are extremely complex.
authors.affiliation: Advanced Medical Research Center/Graduate School of Medical Life Science, Yokohama City University, 3-9 Fukuura, Kanazawa, Yokohama 236-0004, Japan.
authors.forename: Kayoko
authors.initials: K
authors.lastname: Nagata
authors.affiliation: Advanced Medical Research Center/Graduate School of Medical Life Science, Yokohama City University, 3-9 Fukuura, Kanazawa, Yokohama 236-0004, Japan; Biomolecule Analysis Group, Medical ProteoScope Company, 3-9 Fukuura, Kanazawa, Yokohama 236-0004, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Kawakami
authors.affiliation: Advanced Medical Research Center/Graduate School of Medical Life Science, Yokohama City University, 3-9 Fukuura, Kanazawa, Yokohama 236-0004, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Kurata
authors.affiliation: Advanced Medical Research Center/Graduate School of Medical Life Science, Yokohama City University, 3-9 Fukuura, Kanazawa, Yokohama 236-0004, Japan.
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Kimura
authors.affiliation: Advanced Medical Research Center/Graduate School of Medical Life Science, Yokohama City University, 3-9 Fukuura, Kanazawa, Yokohama 236-0004, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Suzuki
authors.affiliation: Institute of Advanced Energy/Graduate School of Energy Science, Kyoto University, Gokasho, Uji Kyoto 611-0011, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nagata
authors.affiliation: Molecular Pathology and Genetics Division, Kanagawa Cancer Center Research Institute, 1-1-2 Nakao, Asahi, Yokohama 252-0373, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Sakuma
authors.affiliation: Molecular Pathology and Genetics Division, Kanagawa Cancer Center Research Institute, 1-1-2 Nakao, Asahi, Yokohama 252-0373, Japan.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Miyagi
authors.affiliation: Advanced Medical Research Center/Graduate School of Medical Life Science, Yokohama City University, 3-9 Fukuura, Kanazawa, Yokohama 236-0004, Japan. Electronic address: hirano@yokohama-cu.ac.jp.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hirano
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D001549:Benzamides / Q000494:pharmacology*; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D005770:Gastrointestinal Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D000068877:Imatinib Mesylate; D009363:Neoplasm Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics; D011743:Pyrimidines / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D001549:Benzamides; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D010879:Piperazines; D020543:Proteome; D011743:Pyrimidines; D000068877:Imatinib Mesylate; D011494:Protein Kinases
keywords: Acquired resistance; GIST; Imatinib; Phosphoproteomics; Targeted therapy