EGFR浆/核转位表达对TKIs耐药的分子机制

TKIs治疗引起Hippo通路活性抑制而耐药，但机制不清。我们发现EGFR膜表达患者TKIs治疗后PFS明显长于胞浆表达者，用TKIs诱导肺癌细胞耐药后，其EGFR发生细胞膜→浆→核转位的同时Hippo通路活性下降，质谱分析显示EGFR与抑制Hippo活性的SIK2结合，推测浆中的EGFR通过自身的SH3结构与SIK2的PxxP基序结合而抑制Hippo通路活性使YAP入核引起耐药；YAP的WW结构域与EGFR的PPxY基序结合将后者携带入核，后者启动下游靶基因转录而加重耐药。我们将构建和转染SIK2-ΔPxxP和YAP-ΔWW等突变质粒和其它干扰序列，用GST-Pulldown、荧光素酶报告基因等方法揭示EGFR的转位引起Hippo活性抑制、产生耐药的机制，并在裸鼠体内对这一机制进行验证。本项目从全新的角度揭示EGFR突变的非小细胞肺癌患者对TKIs的耐药机制，具有重要的理论和现实意义。

The mechanisms of TKIs' resistance caused by Hippo pathway are still unclear.Patients with membranous EGFR have a long PFS than those only have cytoplasmic EGFR. EGFR will translocate from membrane to nucleus after treating with TKIs and Hippo pathway's activity will be inhibited. Mass spectrometry analysis shows that EGFR can combine with SIK2(a Hippo pathway inhibited protein).We confer that cytoplasmic EGFR can combine PxxP motif of SIK2 by its SH3 domain and inhibit Hippo pathway activity. YAP take EGFR from cytoplasm to nucleus after combined with PPxY motif of EGFR by its WW domain,which activates target genes and leads to TKIs resistance. We'll construct and tranfect SIK2-ΔPxxP and YAP-ΔWW mutated plasmids and its interference sequences,then use GST-Pulldown、Luciferase reporter gene to prove the inhibition of Hippo pathway caused by EGFR translocation and test it in nude mice.This project prove TKIs resistance in NSCLC patients with EGFR mutation from a whole new aspect, which has important theoretical and practical significance.