Hippo通路基因遗传变异介导EGFR-TKI耐药及其作为临床疗效和预后判断标志物研究

EGFR-TKI resistance is an important cause of poor clinical efficacy and prognosis in NSCLC patients, however its mechanism has not been completely elucidated. Till now, there is no study to report the relationship between Hippo pathway gene variation and EGFR-TKI efficacy, prognosis and its molecular mechanism. Results of our previous study showed that six genetic loci in Hippo pathway genes were significantly associated with poor clinical objective response rate, disease control rate and progression-free survival in dominant genetic model in NSCLC patients undergoing EGFR-TKI therapy, mRNA and protein levels of YAP1 were significantly higher in mutated genotypes of rs1820453 than the wild genotype in NSCLC tissue and cell lines. Thus, we hypothesized that genetic variation of hippo pathway gene activated downstream targets(Bcl-2 family and EMT related genes), inhibiting apoptosis and promoting the progression of epithelial-mesenchymal transition in NSCLC cell, contributed to EGFR-TKI resistance, and the significant genetic variations might to be biomarkers for clinical efficacy and prognosis in NSCLC patients treated by EGFR-TKI. We tried to investigate relationship between trait genetic loci of Hippo pathway genes and efficacy of EGFR-TKI as well as prognosis of patients and to explore molecular mechanism of the significant locus involved in EGFR-TKI resistance in clinic, tissue, cell and animal levels. This study is expected to expand the understanding of EGFR-TKI resistance in aspect of Hippo pathway gene variation, and to explore molecular biomarker for precise section and predicting clinical efficacy and survival in NSCLC patients.

EGFR-TKI耐药是导致NSCLC患者不良疗效和预后的重要原因，其耐药机制尚不清楚，有关Hippo通路基因遗传变异与EGFR-TKI疗效和预后研究亦未见报道。我们前期研究发现，此通路6个基因遗传位点在显性模型下与EGFR-TKI应答和疾病控制率以及患者PFS显著负相关;携带rs1820453不同基因型的组织和细胞系中YAP1mRNA和蛋白水平差异明显。为此，我们提出假说：Hippo通路基因显著相关遗传位点激活下游通路蛋白，抑制NSCLC细胞凋亡并促进其上皮间质转化，介导EGFR-TKI耐药，其可作为疗效和预后判断标志物。为验证此假说，本研究拟从临床、组织、细胞和动物整体水平探讨此通路基因遗传位点与药物疗效和患者预后关系；明确显著相关位点介导其耐药的分子机制。本研究将从Hippo通路基因变异这个新视点探讨EGFR-TKI耐药机制，为其精准筛选适用患者、临床疗效和预后判断提供新的分子标志物。

EGFR-TKI耐药是导致NSCLC患者不良疗效和预后的重要原因，其耐药机制尚不清楚，有关Hippo通路基因遗传变异与EGFR-TKI疗效和预后研究亦未见报道。本研究拟从临床、组织、细胞和动物整体水平探讨此通路基因遗传位点与药物疗效和患者预后关系；从体内和体外两个层面明确显著相关位点介导其耐药的分子机制。临床研究中，我们发现并验证了rs1820453 TT基因型的NSCLC患者的临床反应率和疾病控制率显着高于TG / GG基因型患者。 Kaplan-Meier曲线，单因素和多因素Cox回归分析表明rs1820453的TT基因型和G等位基因与患者生存率显着相关，TT基因型患者的预后优于G等位基因病例。此外，rs1820453的TT基因型分别与癌症浸润和淋巴结转移负相关。在组织和细胞系中发现rs1820453位点TT基因型携带者YAP1基因mRNA表达量和YAP1蛋白水平明显低于TG/GG基因型携带者。进一步研究发现FOXN1转录因子与YAP1基因启动子区rs1820453位点结合，预测发现G和T等位基因与FOXN1的预测结合能力分别为10.687和2.322。通过荧光素酶报告基因试验发现此位点位点 G 等位基因载体的荧光素酶活性显著高于T等位基因载体。上述研究结果揭示YAP1 rs1820453位点可作为EGFR-TKI临床疗效预测和NSCLC患者生存预后判断的独立生物标志物；此位点G和T等位基因通过影响FOXN1转录因子结合，影响YAP1基因启动子区活性，诱导YAP蛋白紊乱表达，抑制Hippo通路，促进EGFR-TKI耐药和患者不良预后水平。