核纤层蛋白A/C抑制EMT逆转肺腺癌EGFR-TKI耐药机制

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) have been employed as the first-line treatment for lung adenocarcinoma patients with EGFR-positive mutations, but the EGFR-TKI acquired resistance becomes the bottleneck of continuation of EGFR-TKI therapy in clinic. Epithelial-mesenchymal transition (EMT) is one of an important cause of EGFR-TKI resistance in lung adenocarcinoma patients. The resistant cells restore the sensitivity to EGFR-TKI by reversing EMT. Our previous works showed that laminA/C expression in HCC827/ER cell was significantly lower than that of the parental cell and EMT occurred in the resistant cells. In addition, overexpression of laminA/C in HCC827/ER cells reversed the EMT and restored the sensitivity to Erlotinib. However, the mechanism of laminA/C induced EGFR-TKI resistance in lung adenocarcinoma is still not clear. This project is designed to explore the roles of laminA/C and nuclear lamina binding protein in regulation of histone modification, EMT and signaling pathway. Several techniques, such as immunoprecipitation, protein mass spectrometry, RT-PCR, Western blot, over-expression or interference with laminA/C will be applied. The present study will contribute to clarify the mechanism of laminA/C on inhibition of EMT and reversal of EGFR-TKI resistance, and provide a theoretical basis for the development of a new generation of targeted therapeutic drugs for lung adenocarcinoma.

表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKI）是治疗肺腺癌EGFR阳性突变的一线用药，但获得性耐药成为制约EGFR-TKI继续使用的瓶颈。上皮间质转化（EMT）是引起EGFR-TKI耐药的主要原因之一，逆转耐药细胞EMT可恢复对EGFR-TKI的敏感性。我们前期研究发现在肺腺癌厄洛替尼耐药细胞中核纤层蛋白A/C表达下调且发生EMT，过表达核纤层蛋白A/C可逆转耐药细胞EMT恢复对厄洛替尼的敏感性。本项目拟采用免疫共沉淀、蛋白质谱、RT-PCR、Western blot、过表达或干涉核纤层蛋白A/C等技术，结合药理学方法探讨核纤层蛋白A/C逆转肺腺癌细胞EGFR-TKI耐药作用，进行核纤层蛋白A/C与核纤层结合蛋白相互作用调控组蛋白修饰、EMT及其信号通路研究，阐明核纤层蛋白A/C抑制EMT逆转肺腺癌EGFR-TKI耐药机制，为研发靶向逆转TKI药物获得性耐药的治疗新策略提供理论依据。

表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKI）已成为治疗非小细胞肺癌患者EGFR阳性突变的一线靶向药物，然而大部分患者在接受治疗后10-12个月出现获得性耐药现象，其耐药机制尚未完全阐释清楚。本项目通过厄洛替尼低浓度梯度递增法诱导亲本肺腺癌HCC827细胞6个月建立了HCC827/ER耐药细胞，采用MTT法检测其耐药倍数约350倍。利用RNA-seq分析HCC827与HCC827/ER细胞表达差异基因、GO及KEGG信号通路，采用P≤0.05、q≤0.05、|fold change|≥2的条件，共鉴定出2118个差异基因，其中1242个为上调基因，876个为下调基因。从KEGG信号通路分析结果发现，耐药细胞富集的信号通路主要集中在MAPK、肿瘤通路、细胞粘附、EGFR酪氨酸激酶抑制剂信号通路、细胞迁移和增殖等。通过qRT-PCR、western blot等方法检测了细胞中laminA/C、EMT标志物的表达情况，结果显示LMNA和CDH1（上皮标志物）在HCC827/ER细胞中比HCC827细胞显著下调，然而间质标志物如CDH2、SNAI2、VIM、ZEB1和TWIST1表达显著上调。在此基础上，我们进一步构建了过表达、干涉laminA/C表达慢病毒载体并分别感染HCC827/ER和HCC827细胞，证明laminA/C在厄洛替尼耐药、细胞形态、细胞核形态、EMT转化方面的作用。LMNA在HCC827细胞中低表达能促进E-Cadherin低表达、Vimentin高表达，促进细胞发生EMT转化；LMNA在HCC827/ER细胞中过表达，促进E-Cadherin表达升高、Vimentin表达降低，能逆转EMT转化。随后，我们还利用蛋白质谱技术分析了laminA/C相互作用蛋白，获得亲本细胞与耐药细胞中与laminA/C相互作用的差异蛋白。最后，探讨laminA/C低表达诱导HCC827细胞产生厄洛替尼耐药的分子机制，确定了laminA/C介导EMT相关的信号通路FGFR/MAPK/C-fos。总之，本研究发现核纤层蛋白A/C通过FGFR/MAPK/C-fos信号通路调控肺腺癌细胞EMT导致细胞发生厄洛替尼耐药。该项研究可为研发靶向逆转TKI药物获得性耐药的治疗新策略提供理论依据。