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.

Biological significance: 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.

Keywords: Acquired resistance; GIST; Imatinib; Phosphoproteomics; Targeted therapy.