miR-125a-5p regulation increases phosphorylation of FAK that contributes to imatinib resistance in gastrointestinal stromal tumors
miR-125a-5p regulation increases phosphorylation of FAK that contributes to imatinib resistance in gastrointestinal stromal tumors
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DOI：
10.1016/j.yexcr.2018.08.028
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发表时间：
2018-10-01
影响因子：
3.7
通讯作者：
Lui, Weng-Onn
中科院分区：
文献类型：
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作者：
Huang, Wen-Kuan;Akcakaya, Pinar;Lui, Weng-Onn
甲磺酸伊马替尼的使用大大改善了胃肠道间质瘤（GIST）患者的临床结局。然而，伊马替尼耐药仍然是一个主要的临床挑战，其分子机制尚未完全了解。我们先前已经表明，miR-125a-5p及其mRNA靶点PTPN18调节GIST细胞中的伊马替尼反应。在此，我们评估了磷酸化FAK（pFAK）作为PTPN 18的候选下游靶点，以及这种调节与GIST中伊马替尼耐药的可能关联。使用Western印迹分析，在用靶向PTPN18或miR-125a-5p模拟物的短发夹RNA或短干扰RNA转染的GIST 882细胞、伊马替尼抗性GIST 882R亚克隆和临床样品中评估FAK和pFAK表达。使用FAK抑制剂14（FAKi）阻断FAK磷酸化，并使用WST-1测定和裂解的PARP表达评价对细胞活力和凋亡的影响。FAK和pFAK表达与伊马替尼耐药、KIT突变和患者结局的临床相关性通过Fisher精确检验或对数秩检验进行评估。miR-125a-5p的过表达和PTPN 18的沉默增加了GIST细胞中pFAK的表达，但不增加FAK的表达。与伊马替尼敏感的亲本细胞相比，在具有获得性伊马替尼抗性的GIST 882 R亚克隆中观察到更高的pFAK表达。FAKi治疗伊马替尼耐药GIST 882 R细胞降低细胞活力，增加伊马替尼治疗后的细胞凋亡。此外，FAKi可以挽救由miR-125a-5p过表达介导的伊马替尼耐药效应。在临床样本中，FAK和pFAK高表达与KIT突变状态相关，FAK高表达也与GIST转移相关。总生存期较短的病例中pFAK水平较高。我们的研究结果强调了miR-125a-5p调控及其下游靶点pFAK在GIST中对伊马替尼耐药的重要作用。pFAK和FAK可能对GIST的预后有一定价值。
The use of imatinib mesylate has greatly improved the clinical outcome for gastrointestinal stromal tumor (GIST) patients. However, imatinib resistance is still a major clinical challenge, and the molecular mechanisms are not fully understood. We have previously shown that miR-125a-5p and its mRNA target PTPN18 modulate imatinib response in GIST cells. Herein, we evaluated phosphorylated FAK (pFAK) as a candidate downstream target of PTPN18 and the possible association of this regulation with imatinib resistance in GIST. FAK and pFAK expressions were evaluated in GIST882 cells transfected with short hairpin RNA or short interfering RNA targeting PTPN18 or miR-125a-5p mimic, imatinib-resistant GIST882R subclones and clinical samples using Western blot analyses. FAK phosphorylation was blocked using the FAK inhibitor 14 (FAKi) and the effects on cell viability and apoptosis were evaluated using WST-1 assay and cleaved PARP expression. Clinical associations of FAK and pFAK expression with imatinib resistance, KIT mutation and patient outcome were assessed by Fisher's exact test or log-rank test. Over-expression of miR-125a-5p and silencing of PTPN18 increased pFAK, but not FAK, expression in GIST cells. Higher pFAK expression was observed in the GIST882R subclones with acquired imatinib resistance compared to their imatinib-sensitive parental cells. Treatment with FAKi in imatinib-resistant GIST882R cells reduced cell viability and increased apoptosis upon imatinib treatment. Additionally, FAKi could rescue the imatinib resistance effect mediated by miR-125a-5p over-expression. In clinical samples, high FAK and pFAK expressions were associated with KIT mutation status, and high FAK expression was also associated with metastasis in GIST. Higher pFAK was found in cases with shorter overall survival. Our findings highlight an important role for miR-125a-5p regulation and its downstream target pFAK for imatinib resistance in GIST. pFAK and FAK may have prognostic values in GIST.