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New findings of kinase switch in gastrointestinal stromal tumor under imatinib using phosphoproteomic analysis

Takahashi, Tsuyoshi and Serada, Satoshi and Urase, Maiko and Fujimoto, Minoru and Miyazaki, Yasuaki and Ikezoe, Takayuki and Taguchi, Takahiro and Kurokawa, Yukinori and Yamasaki, Makoto and Miyata, Hiroshi and Takiguchi, Shuji and Mori, Masaki and Doki, Yuichirou and Naka, Tetsuji and Nishida, Toshirou (2013) New findings of kinase switch in gastrointestinal stromal tumor under imatinib using phosphoproteomic analysis. International Journal of Cancer.

Abstract

Despite the initial effectiveness of the imatinib for gastrointestinal stromal tumors (GISTs), patients can’t stop it and eventually relapse by acquired drug resistance. Pathologically, we find some viable cells under the imatinib therapy even in the disappearance region in radiological examination, and these cells are thought to cause these clinical problems. To discover these mechanisms, we investigated the phospho-proteome alternations induced by imatinib treatment using GIST-T1 cell. Because some of tyrosine kinases might be alternatively activated after imatinib exposure, it is quite important to discover increased phosphorylation levels of proteins at proteome scale, and elucidate these proteins with drug resistance. With or without imatinib treatment, extracted proteins from GIST-T1 cells were digested with trypsin and tyrosine-phosphorylated peptides were purified using an immunoaffinity-enriched and labeled with iTRAQ reagent followed by mass spectral analysis. By phosphoproteomic approach, 171 tyrosine phosphorylation sites spanning 134 proteins were identified. Among them, increased tyrosine phosphorylation levels of Fyn(Y420) and focal adhesion kinase (FAK) (Y576) were detected. Increased phosphorylation levels were also confirmed by western blot analysis and knockdown of Fyn or FAK increased the sensitivity toward imatinib. Furthermore, we found that the anti-proliferative effect of imatinib significantly enhanced imatinib resistant GIST-T1 cells which showed constitutive phosphorylation of FAK, treating with FAK selective kinase inhibitor. These results indicate that iTRAQ based quantitative phospho-tyrosine focused phosphoproteomic approaches are powerful method to screen phosphpproteins associated with drug resistance and Fyn or FAK might be potential therapeutic target for the overcoming acquired resistance to imatinib in GIST.

Item Type: Article
Date Deposited: 13 Oct 2015 13:14
Last Modified: 13 Oct 2015 13:14
URI: https://oak.novartis.com/id/eprint/8952

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