EGFR浆/核转位表达对TKIs耐药的分子机制

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.

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。

酪氨酸激酶抑制剂（TKIs）在表皮生长因子受体（EGFR）突变肺癌患者的靶向治疗中产生耐药性，其潜在机制尚未完全阐明。本研究课题结合细胞培养及动物模型， 应用免疫印迹检测、双荧光素酶、免疫共沉淀检测、质谱分析、实时荧光定量PCR和功能实验等方法，探讨了EGFR的膜/质/核转位在TKIs耐药中的分子机制。结果证实一代TKIs诱导耐药过程中，EGFR由细胞膜转入细胞浆，通过与SIK2的结合作用于Hippo通路，引起Hippo通路的活性抑制，EGFR与YAP在胞浆中结合后可被 YAP 带入到细胞核，进入到细胞核内的EGFR 激活下游靶基因的转录，进一步加重耐药。课题组还发现一代TKIs诱导产生耐药的同时出现p120ctn从细胞膜到细胞质的转位，其分子机制未见报道。我们应用免疫组化、免疫印迹和底物陷阱等方法检测，结果证实第一代TKI的耐药抑制了PTP-PEST表达，促进了p120ctn-Y335磷酸化并减少了p120ctn与E-钙粘蛋白的相互作用，导致p120ctn胞质转位。 总之，EGFR的细胞膜/质/核转位是肺癌TKIs耐药的重要机制之一，PTP-PEST对p120ctn的调控在TKI耐药性中发挥作用，这些结果将为肺癌TKIs耐药机制研究提供新的见解，并可能对肺癌治疗的新策略产生影响。

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