Department of Surgery, Osaka University Graduate School of Medicine, Suita, Japan; Laboratory for Immune Signal, National Institute of Biomedical Innovation, Ibaraki, Japan.
Int J Cancer. 2013 Dec 1;133(11):2737-43. doi: 10.1002/ijc.28282. Epub 2013 Jun 14.
Despite the revolutionary effects of imatinib on advanced gastrointestinal stromal tumors (GISTs), most patients eventually develop disease progression following primary resistance or acquired resistance driven by secondary-resistant mutations. Even in radiographically vanishing lesions, pathology has revealed persistent viable cells during imatinib therapy, which could lead to the emergence of drug-resistant clones. To uncover the mechanisms underlying these clinical issues, here we examined imatinib-induced phosphoproteomic alterations in GIST-T1 cells, using our quantitative tyrosine phosphoproteomic analysis method, which combined immunoaffinity enrichment of phosphotyrosine-containing peptides with isobaric tags for relative and absolute quantitation (iTRAQ) technology. Using this approach, we identified 171 tyrosine phosphorylation sites spanning 134 proteins, with 11 proteins exhibiting greater than 1.5-fold increases in tyrosine phosphorylation. Among them, we evaluated FYN and focal adhesion kinase (FAK), both of which are reportedly involved in proliferation and malignant alteration of tumors. We confirmed increased tyrosine phosphorylation of both kinases by western blotting. Inhibition of FYN and FAK phosphorylation each increased tumor cell sensitivity to imatinib. Furthermore, a FAK-selective inhibitor (TAG372) induced apoptosis of imatinib-resistant GIST-T1 cells and decreased the imatinib IC50 . These results indicate that FYN or FAK might be potential therapeutic targets to overcome resistance to imatinib in GISTs. Additionally, we showed that the iTRAQ-based quantitative phosphotyrosine-focused phosphoproteomic approach is a powerful method for screening phosphoproteins associated with drug resistance.
尽管伊马替尼对晚期胃肠道间质瘤（GIST）具有革命性的影响，但大多数患者最终会因原发性耐药或继发耐药突变导致的获得性耐药而发生疾病进展。即使在影像学上消失的病变中，在伊马替尼治疗期间，病理学已经揭示了持续存在的有活力的细胞，这可能导致耐药克隆的出现。为了揭示这些临床问题的机制，我们在这里使用我们的定量酪氨酸磷酸化蛋白质组学分析方法，即免疫亲和富集含磷酸酪氨酸肽与同重同位素标记相对和绝对定量（iTRAQ）技术，研究了伊马替尼诱导的 GIST-T1 细胞磷酸蛋白质组变化。使用这种方法，我们鉴定了 171 个跨越 134 种蛋白质的酪氨酸磷酸化位点，其中 11 种蛋白质的酪氨酸磷酸化增加了 1.5 倍以上。在这些蛋白质中，我们评估了 FYN 和粘着斑激酶（FAK），据报道它们都参与了肿瘤的增殖和恶性改变。我们通过 Western blot 证实了这两种激酶的酪氨酸磷酸化增加。抑制 FYN 和 FAK 磷酸化均可增加肿瘤细胞对伊马替尼的敏感性。此外，FAK 选择性抑制剂（TAG372）诱导伊马替尼耐药 GIST-T1 细胞凋亡，并降低伊马替尼的 IC50。这些结果表明，FYN 或 FAK 可能是克服 GIST 对伊马替尼耐药的潜在治疗靶点。此外，我们表明，基于 iTRAQ 的定量磷酸酪氨酸聚焦磷酸蛋白质组学方法是筛选与耐药相关的磷酸蛋白质的有力方法。
