New findings of kinase switching in gastrointestinal stromal tumor under imatinib using phosphoproteomic analysis.
New findings of kinase switching in gastrointestinal stromal tumor under imatinib using phosphoproteomic analysis.
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使用磷酸蛋白质组学分析在伊马替尼治疗下胃肠道间质瘤中激酶转换的新发现。
DOI：
10.1002/ijc.28282
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发表时间：
2013
期刊：
影响因子：
6.4
通讯作者：
Nishida T.
中科院分区：
文献类型：
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作者：
Takahashi T;Serada S;Ako M;Fujimoto M;Miyazaki Y;Nakatsuka R;Ikezoe T;Yokoyama A;Taguchi T;Shimada K;Kurokawa Y;Yamasaki M;Miyata H;Nakajima K;Takiguchi S;Mori M;Doki Y;Naka T;Nishida T.
尽管伊马替尼对晚期胃肠道间质瘤（GIST）具有革命性的作用，但大多数患者最终在原发性耐药或由继发性耐药突变驱动的获得性耐药后出现疾病进展。即使在放射学上消失的病变中，病理学也显示在伊马替尼治疗期间存在持续的活细胞，这可能导致耐药克隆的出现。为了揭示这些临床问题背后的机制，我们在这里使用我们的定量酪氨酸磷酸蛋白质组分析方法检查了伊马替尼诱导的 GIST-T1 细胞中的磷酸蛋白质组改变，该方法将含磷酸酪氨酸的肽的免疫亲和富集与用于相对和绝对定量 (iTRAQ) 技术的同量异位标签相结合。使用这种方法，我们鉴定了 134 个蛋白质的 171 个酪氨酸磷酸化位点，其中 11 个蛋白质的酪氨酸磷酸化增加了 1.5 倍以上。其中，我们评估了 FYN 和粘着斑激酶 (FAK)，据报道这两者都与肿瘤的增殖和恶性改变有关。我们通过蛋白质印迹证实了两种激酶的酪氨酸磷酸化增加。抑制 FYN 和 FAK 磷酸化均会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）诱导伊马替尼耐药的GIST-T1细胞凋亡并降低伊马替尼IC50。这些结果表明，FYN 或 FAK 可能是克服 GIST 伊马替尼耐药性的潜在治疗靶点。此外，我们还表明，基于 iTRAQ 的定量磷酸酪氨酸磷酸蛋白质组学方法是筛选与耐药性相关的磷蛋白的有效方法。
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.