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

发布时间：2018-06-15 07:04

本文选题：表皮生长因子酪氨酸激酶抑制剂 + 非小细胞肺癌　；参考：《第二军医大学》2016年博士论文

【摘要】：研究背景:表皮生长因子酪氨酸激酶抑制剂(EGFR-TKI)已经成为EGFR基因突变的晚期非小细胞肺癌(NSCLC)的标准治疗之一,然而EGFR-TKI的获得性耐药已成为影响其疗效的瓶颈。目前化疗仍是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。研究方法: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;通过RT-PCR、Western-blot检测EGFR-TKI获得性耐药机制;si RNA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。研究结果:1、对目前治疗NSCLC的第三代化疗药物(吉西他滨、培美曲塞、紫杉醇、多西他塞、伊力替康),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;而对顺铂的敏感性两者无差异。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,但对化疗药物(顺铂、吉西他滨、培美曲塞)的药物敏感性无差异。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、特异性下调EGFR和T790M突变基因后,NSCLC细胞株PC-9/ZD对化疗药物(紫杉醇、多西他塞)敏感性均下降。4、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。5、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(EMT阳性PC-9/GR细胞肺转移裸鼠生存期短于T790M突变肺转移裸鼠)。结论: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;T790M突变、EMT阳性还是远处转移和预后的指标,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。
[Abstract]:Background: EGFR-TKI, an inhibitor of epidermal growth factor tyrosine kinase, has become one of the standard treatments for advanced non-small cell lung cancer (NSCLC) with EGFR gene mutation. However, the acquired resistance of EGFR-TKI has become a bottleneck affecting its efficacy. At present, chemotherapy is still the most important treatment method after EGFR-TKI resistance. However, it is difficult to predict and individualize the effect of chemotherapy, so how to choose a more effective chemotherapy regimen is of great significance. Since previous studies have shown that the sensitivity to different chemotherapeutic agents is different after acquired drug resistance, the mechanisms of classical T790M mutation and non-classical epithelial interstitial transition (EMTT) are related to the efficacy of second-line chemotherapy. For the first time, we suggest that different drug resistance mechanisms of 1: EGFR-TKI may affect the efficacy of subsequent chemotherapy. Methods: the chemosensitivity of different lung cancer cell lines to chemotherapeutic drugs was detected by EGFR-TKI induced PC-9 EGFR mutation) and H460 EGFR wild-type). The classical T790M mutant cell lines PC-9 / ZDN-EMT positive drug resistant cell lines PC-9% gr and H460% ERCCK8 were used to detect the chemosensitivity of different lung cancer cell lines to chemotherapeutic drugs. EGFR-TKI acquired drug resistance mechanism (EGFR-TKI) was detected by RT-PCR Western-blot. The T790M gene was down-regulated by siRNA interference in EGFR-TKI mutant cells. After transfection of CDH1, the EMTs were reversed. The effects of T790M and EMT on the chemosensitivity of EGFR-TKI cells were investigated. T790M mutation and EMT positive effect on lung metastasis and survival of nude mice were detected in nude mice lung metastasis model. The third generation of chemotherapy drugs (gemcitabine, pemetrexed, paclitaxel, docetaxel, docetaxel) for current treatment of NSCLC, The sensitivity of wild-type lung cancer cell line H460 with EGFR gene mutation was lower than that of PC-9 cell line with EGFR gene mutation, but the sensitivity to cisplatin was not different between H460 cell line and PC-9 cell line, but the sensitivity to cisplatin was not different between H460 cell line and PC-9 cell line, but the sensitivity to cisplatin was not different between H460 cell line and PC-9 cell line. Sensitivity to chemotherapeutic drugs (paclitaxel, docetaxel) was increased, but there was no difference in sensitivity to chemotherapeutic drugs (cisplatin, gemcitabine, pemetrexide). PC-9 / ZDT790M mutation EMT was negative for chemotherapeutic drugs (paclitaxel, paclitaxel). Docetaxel was superior to PC-9 / GR( no T790M mutation EMT positive, down-regulated the sensitivity of EGFR and T790M mutant PC-9 / ZD to chemotherapeutic drugs (paclitaxel, docetaxel). 4% EMT positive NSCLC cell lines PC-9% GR and H460% ERR) were more sensitive to chemotherapeutic drugs (cisplatin, docetaxel). Gemcitabine, pemetrexed, paclitaxel, The sensitivity of PC-9 and H460 cells to these chemotherapeutic drugs decreased after transfection of PC-9 / gr and H460 / ER cells, and then reversed EMTs, which could restore the sensitivity of the cells to the above chemotherapeutic drugs after transfection of PC-9 and H460 cell lines with TGF- 尾 1. The number of lung metastasis in nude mice was more than that in T790M mutant PC-9 / ZD, which further affected the survival of Nude mice with EMT positive PC-9 / gr cells and the survival time of Nude mice with EMT positive PC-9 / gr cells was shorter than that of T790M mutant lung metastasis nude mice. Conclusion the T790M mutation and EMT positive mechanism of different resistance mechanisms after acquired drug resistance in the cells of 10% EGFR-TKI affect the therapeutic effect of subsequent chemotherapy. T790M is an indicator of the chemosensitivity of Taxus chinensis. The relationship between EMT and chemotherapeutic resistance is related to the EMT of T790M mutation or distant metastasis and prognosis. Lung cancer cell lines with T790M mutation had fewer metastatic numbers and better prognosis, while EMT positive lung cancer cell lines had more metastatic numbers and shorter survival time.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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