T790M及EMT在预测EGFR-TKI继发耐药后化疗疗效中的重要作用及机制

Epidermal growth factor receptor-tyrosine kinase inhibitor(EGFR-TKI) is one of the most important achievement for treatment of non-small cell lung cancer (NSCLC) in the 21st century. However the acquired resistance to EGFR-TKIs has greatly interfered the therapeutic effect. Chemotherapy is the most important strategy for EGFR-TKIs resistant patients, but the prognosis of chemotherapy is still an open question. It is of great importance to select the most optimal cytotoxic drugs for individual patient. Previous studies indicated that the sensitivity of patients to chemotherapy after acquired resistance to EGFR-TKIs was different, and that the resistance mechanisms including T790M mutation and epithelial-mesenchymal transition (EMT) were closely related to the activity of chemotherapy. Accordingly, we presumed for the first time that the different resistance mechanisms would impact the effects of subsequently used chemotherapy. In the present project, we planned to investigate the sensitivity of chemotherapy in different cell lines with T790M and EMT respectively, and to use RT-PCR, Western-blot, siRNA, gene transfection and knockout to assess the effect of T790M and EMT on chemotherapy, so as to improve the prognosis of chemotherapy for NSCLC patients with acquired resistance to EGFR-TKIs.

表皮生长因子酪氨酸激酶抑制剂（EGFR-TKIs）治疗晚期非小细胞肺癌（NSCLC）是21世纪重要的医学成果之一。然而EGFR-TKIs的获得性耐药已成为影响其疗效的重要因素。目前化疗是EGFR-TKIs耐药后的最重要治疗手段，然而化疗疗效的预测至今难以实现，如何选择更有效的化疗方案意义重大。鉴于研究显示：EGFR-TKIs获得性耐药后对不同化疗药物敏感性不同，经典的T790M突变和非经典的上皮－间质转换（EMT）等机制与二线化疗疗效相关，我们首次提出科学假设：EGFR-TKIs的不同耐药机制影响后续化疗疗效。本项目拟采用经典的T790M突变与非经典的EMT阳性的耐药细胞株，研究其对不同化疗药物的敏感性，通过RT-PCR、Western-blot、基因转染及敲除、siRNA干扰等方法阐明T790M和EMT对化疗敏感性的影响，为合理选择化疗方案提高EGFR-TKI耐药后疗效提供科学依据。

化疗仍是EGFR-TKI耐药后的最重要治疗手段，然而化疗疗效的预测和个体化至今难以实现，如何选择更有效的化疗方案意义重大。鉴于既往研究显示：EGFR-TKI获得性耐药后对不同化疗药物敏感性不同，经典的T790M突变和非经典的上皮间质转换（EMT）等机制与二线化疗疗效相关，我们首次提出科学假设：EGFR-TKI的不同耐药机制可能影响后续化疗疗效。本研究通过通过EGFR-TKI诱导肺癌细胞PC-9（EGFR突变型）和H460（EGFR野生型）获得经典的T790M突变的细胞株PC-9/ZD，EMT阳性的耐药细胞株PC-9/GR和H460/ER，CCK8法检测不同肺癌细胞株对化疗药物的敏感性；siRNA干扰下调T790M突变细胞的T790M基因、转染CDH1后逆转耐药细胞EMT，检测各细胞对化疗药物敏感性变化阐明T790M和EMT对化疗敏感性的影响；裸鼠成瘤实验进一步验证化疗疗效；裸鼠肺转移模型检测T790M突变和EMT阳性对裸鼠肺转移、生存的影响。得出如下主要结果：1、对目前治疗NSCLC的第三代化疗药物，EGFR野生型肺癌细胞株H460的敏感性差于EGFR基因突变的细胞株PC-9。2、EGFR-TKI获得性耐药后的PC-9/ZD细胞（T790M突变，EMT阴性）与亲代PC-9细胞相比，对化疗药物（紫杉醇、多西他塞）的敏感增高。PC-9/ZD（T790M突变，EMT阴性）对化疗药物敏感性（紫杉醇、多西他塞）优于PC-9/GR（无T790M突变，EMT阳性）。3、EMT阳性NSCLC细胞株（PC-9/GR和H460/ER）对化疗药物（顺铂、吉西他滨、培美曲塞、紫杉醇、多西他塞）敏感性差于亲代PC-9和H460细胞株；TGF-β1作用肺癌细胞PC-9和H460后对上述化疗药物敏感性下降；PC-9/GR和H460/ER细胞转染CDH1后逆转EMT，可以恢复细胞对上述化疗药物的敏感性。4、EGFR-TKI获得性耐药细胞株，EMT阳性肺癌细胞（PC-9/GR）裸鼠肺转移数目多于T790M突变细胞PC-9/ZD，进一步影响裸鼠的生存。EGFR-TKI获得性耐药后细胞不同耐药机制（T790M突变和EMT阳性）影响后续的化疗疗效，T790M是紫杉类化疗敏感性的指标，EMT与化疗耐药有关；有T790M突变的肺癌细胞株肺转移数目少，预后好；而EMT阳性肺癌细胞株肺转移数目多，生存期短。