The Important Role Of T790M And EMT In Prognosis Of Chemotherapy In NSCLC With Acquired Resistance Of EGFR-TKI

Background: Epidermal growth factor receptor-tyrosine kinase inhibitor(EGFRTKI) is one of the most important achievement for treatment of non-small cell lung cancer(NSCLC) in the 21 st century. However the acquired resistance to EGFR-TKI has greatly interfered the therapeutic effect. Chemotherapy is the most important strategy for EGFR-TKI 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-TKI was different, and that the resistance mechanisms including T790 M 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.Methods: In the present project, through the EGFR-TKI induced lung cancer cells PC-9(EGFR mutation) and H460(EGFR wild), we had two kinds of acquired resistant lung cancer cells: PC-9/ZD is T790 M mutation and EMT negative; PC-9/GR and H460/ER are non-T790 M mutation and EMT positive. We planned to investigate the sensitivity of chemotherapy in different cell lines with T790 M and EMT respectively by CCK8 assay, and to use RT-PCR, Western-blot to detect EMT, si RNA to downregulate the T790 M, CDH1 transfected to acquired resistant lung cancer cell to reversal EMT to assess the effect of T790 M and EMT on chemotherapy, so as to improve the prognosis of chemotherapy for NSCLC patients with acquired resistance to EGFR-TKI. Nude mice transplantation tumor experiment were performed to verify the curative effect of chemotherapy. Lung metastatic model in nude mice were performed to examine metastatic capability in vivo and the impact on survival.Results: 1. The IC50 of most chemotherapeutics(Gemcitabine, pemetrexed, Taxol,Docetaxel, Irinotecan) in NSCLC PC-9 cells with EGFR mutations was significantly lower than H460 cell lines with wild-type(p < 0.05); There were no significant differences in IC50 of cisplatin between PC-9 and H460 cells(p>0.05). 2. Sensitivity of chemotherapeutics(Taxol, Docetaxel) in PC-9/ZD cell lines(T790M and EMT negative)was higher than parental PC-9 cell lines and PC-9/GR(EMT positive and non-T790M)(p < 0.05), while the sensitivity of chemotherapeutics(Cisplatin, Gemcitabine,Pemetrexed) in PC-9/ZD cell lines was similar to parental PC-9 cell lines. 3. Specificitysilencing EGFR and T790 M by si RNA in PC-9/ZD cell lines resulted in a lower sensitivity to chemotherapeutics(Taxol, Docetaxel)(p < 0.05). 4. The IC50 of chemotherapeutics(cisplatin, pemetrexed, Taxol, Docetaxel, Gemcitabine) in EGFR-TKI acquired resistance lung cell lines PC-9/GR and H460/ER(EMT positive and non-T790M) was higher than their parental PC-9 and H460 cell lines(p < 0.05).Inducing EMT by TGF-β1 in PC-9 and H460 cells decreased sensitivity to previous chemotherapeutics. Reversing EMT by E-cadherin overexpression in PC-9/GR and H460/ER cells restored sensitivity to previous chemotherapeutics. 5. Lung metastatic model in nude mice displayed the number of metastatic nodules in the lung of PC-9/GR was larger than the number of metastatic nodules of PC-9/ZD(p<0.05). The nude mice of PC-9/GR had short survival time than the nude mice of PC-9/ZD(p<0.05).Conclusion: The different resistance mechanisms(T790M mutation and EMT positive) impact the effects of subsequently used chemotherapy, lung cancer cells with T790 M mutation has more sensitivity to taxoid anticancer drugs, while EMT positive lung cancer cells were resistant to chemotherapeutics. T790 M mutation and EMT positive could serve as metastatic and prognostic factor. Lung cancer with T790 M has small number of lung metastasis, and longer survival time; While EMT plays crucial roles in the metastasis and shorter survival.