Augmentation of multiple protein kinase activities associated with secondary imatinib resistance in gastrointestinal stromal tumors as revealed by quantitative phosphoproteome analysis
Augmentation of multiple protein kinase activities associated with secondary imatinib resistance in gastrointestinal stromal tumors as revealed by quantitative phosphoproteome analysis
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DOI：
10.1016/j.jprot.2014.12.012
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发表时间：
2015-02-06
影响因子：
3.3
通讯作者：
Hirano, Hisashi
中科院分区：
文献类型：
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作者：
Nagata, Kayoko;Kawakami, Takao;Hirano, Hisashi
Kit受体酪氨酸激酶基因（KIT）的突变导致蛋白（KIT）的组成性激活，与胃肠道间质瘤（GIST）的发生有因果关系。伊马替尼是一种靶向抗癌药物，通过抑制KIT的激酶活性对GIST发挥治疗作用。然而，这种药物的长期给药会导致伊马替尼耐药GIST的出现。我们使用基于细胞的GIST模型系统进行了定量磷酸化蛋白质组分析，该模型系统包括伊马替尼敏感的GIST细胞系（GIST 882）、伊马替尼治疗下的GIST 882（GIST 882-IM）和继发性伊马替尼耐药的GIST 882（GIST 882-R）。使用基于二氧化钛的亲和层析或抗磷酸酪氨酸免疫沉淀从每个细胞系中纯化磷酸化肽，然后进行基于LC-MS/MS的定量磷酸蛋白质组分析。使用这种方法，我们确定了信号转导途径的多个元件，特别是KIT和EGFR的激酶活性的增强。尽管这些元件在GIST 882-R中上调，但在继发性伊马替尼耐药GIST细胞中未发现额外突变的KIT mRNA。伊马替尼联合EGFR抑制剂吉非替尼治疗GIST 882-R，部分抑制了细胞生长，提示EGFR可能参与了GIST继发性伊马替尼耐药的形成。该研究的目的是研究GIST对伊马替尼的获得性耐药机制，伊马替尼是一种抑制KIT蛋白激酶活性的分子靶向药物，已被批准用于治疗GIST。在伊马替尼耐药的GIST细胞中，我们观察到KIT表达升高，其激酶活性恢复，以及多种增殖信号通路的激活。我们的研究结果表明，即使是所谓的“分子靶向”药物的影响，是广泛的，而不是收敛的，并且这种药物在连续给药过程中的作用机制是非常复杂的。(C)2014爱思唯尔有限公司版权所有。
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). Phosphoxylated 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.Biological significanceIn 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. (C) 2014 Elsevier B.V. All rights reserved.