微小RNA-34家族抑制EMT逆转肺癌EGFR-TKI获得性耐药的机制研究

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are clinically effective treatments for non-small cell lung cancer (NSCLC) patients with EGFR mutation. However, in almost NSCLC patients, acquired TKI resistance can develop during treatment with TKI, which can lead to treatment failure. It is reported that epithelial-mesenchymal transition (EMT) is a new mechanism leading to chemotherapy and targeted drugs resistance. In our previous study, TKI resistance cell line was established and EMT phenomenon was observed in the resistance cell line. Next, microarray profile analysis of microRNAs was performed in TKI resistant cells and the parental cells respectively. Our results demonstrated that the miR-34 family expressed much lower in the resistant cells than the parental cells. Treatment of TKI resistance cells with miR-34 mimics significantly increased the sensitivity to TKI and down-regulated the expressions of Notch1 and c-Met, which acts to upstream in EMT pathway. These results suggest that miR-34 might reverse TKI resistance through suppressing EMT in NSCLC. The aim of this study is to clarify how miR-34 family are downregulated in the TKI resistant cells and the mechanisms of miR-34 family members suppressing EMT. Eventually, our study will provide a novel way for reversing the TKI resistance through targeting miR-34, Notch1 and c-Met.

EGFR-TKI是治疗EFGR突变型NSCLC患者的首选药物，但几乎所有患者终将发生EGFR-TKI获得性耐药，导致治疗失败。研究表明，肺癌细胞上皮间质转化(EMT)是导致化疗和靶向治疗耐药的新机制。我们在前期研究中，建立了TKI 诱导的耐药细胞,并观察到EMT现象。同时miRNA芯片分析结果显示，与其亲本敏感细胞相比，耐药细胞的miR-34家族表达显著降低。过表达miR-34拟似物，显著提高了TKI的敏感性，同时下调了Notch1、c-Met等EMT的上游调节分子，提示miR-34家族可能通过抑制EMT逆转TKI耐药。本研究旨在进一步明确在TKI获得性耐药细胞中，miR-34家族降低的分子机制，以及miR-34如何抑制耐药细胞发生EMT，并探索以miR-34和Notch1、c-Met为靶点的逆转TKI耐药的新方法。

表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKI）已作为EGFR敏感突变患者的一线治疗方案，但获得性耐药极大限制了该药的适用范围及靶向作用。近年来的研究显示，肿瘤细胞的上皮间质转化（epithelial-to-mesenchymal transition, EMT）和细胞间粘附均可导致肿瘤化疗、靶向及免疫治疗耐药。我们的研究发现，与肿瘤细胞EMT相关的膜受体肝细胞生长因子受体（c-Met）表达阳性提示非小细胞肺癌（NSCLC）患者预后不佳，且为独立预后不良因素，与Notch-1联合检测能够更为准确地评估患者预后。在EGFR敏感突变的NSCLC细胞中，埃克替尼（Icotinib）能够诱导激活Src/IL-6/STAT3通路；而耐药细胞来源的Exosomes可使Src活化进而促进肺癌细胞侵袭转移。局部粘着斑激酶（FAK）促进细胞间粘附，阻止肿瘤细胞脱离原发病灶。我们发现胰蛋白酶处理人胃癌、肺癌等细胞系后FAK表达下调，而FAK的下调促进了胃癌MGC803细胞的解离，验证了FAK具有抑制解离促进粘附作用；进一步研究发现E3泛素连接酶（Cbl-b）通过泛素化降解FAK促进细胞解离；而Icotinib上调了Cbl-b的表达水平；与此同时，过表达p65（转录因子NF-κB家族的主要成员）逆转了Icotinib介导的Cbl-b上调。上述结果关于Cbl-b的作用看似矛盾，实际上FAK除了抑制细胞解离转移，还有一个重要作用是阻止肿瘤细胞失巢凋亡（anoikis），因此Cbl-b的双向调节作用亦可以解释。我们通过对人肺腺癌(LUAD)公共数据库和组织芯片(TMA)比较发现转录辅助因子LBH为良好的独立预后因子，而生物信息学分析表明，LBH与细胞粘附通路显著相关，可上调部分整合素家族成员。综上所述，c-Met、Notch-1和整合素三者均是位于细胞膜上的跨膜蛋白，我们既往的研究已证明脂筏可参与调控肿瘤细胞耐药，因此推测三者可能通过脂筏调控肺癌细胞侵袭转移，进而导致EGFR-TKI获得性耐药，为临床治疗提供了新的策略。