上皮间质转化在非小细胞肺癌EGFR-TKIs获得性耐药中的作用研究∗

摘要

Objective To explore the role of epithelial⁃mesenchymal transition( EMT) in the acquired resistance to epidermal growth factor receptor⁃tyrosine kinase inhibitors ( EGFR⁃TKIs ) in non⁃small cell lung cancer ( NSCLC ) . Methods The EGFR del E746⁃A750⁃mutated human lung adenocarcinoma PC9/AB cell line and the EGFR wild⁃type H460/ER cell line were used in this study. The role of EMT in the acquired resistance to EGFR⁃TKIs was investigated by establishing stable E⁃cadherin over⁃expression cell lines( PC9/AB⁃CDH1 and H460/ER⁃CDH1) by transforming gene⁃CDH1 with lentivirus. MTT assay was used to measure the cell pro⁃liferation. Wound⁃healing assay and Transwell assay were adopted to determine the migration and invasion ability of the cells. The mR⁃NA and protein expressions of E⁃cadherin, Vimentin, Snail,β⁃catenin and EGFR were determined by the real⁃time fluorescence quan⁃titative PCR( qRT⁃PCR) and Western blotting, respectively. Results EMT( low E⁃cadherin and high Vimentin) was found in H460/ER and PC9/AB cells, neither T790M mutation nor c⁃Met amplification were detected. Over⁃expression of E⁃cadherin in both PC9/AB⁃CDH1 and H460/ER⁃CDH1 cells reversed morphological signature of EMT. Reversing of EMT remarkably increased the sensitivity to EGFR⁃TKIs in PC9/AB⁃CDH1and H460/ER⁃CDH1 cells. Compared with PC9/AB cells, the sensitivity to gefitinib in PC9/AB⁃CDH1 cells increased 11�4 folds. The half⁃inhibition concentration(IC50)of PC9/AB⁃CDH1 and PC9/AB cells was (0�70±0�22)μmol/L and (8�68±0�44)μmol/L with statistical significance(P<0�05). Compared with H460/ER cells, the sensitivity to erlotinib in H460/ER⁃CDH1 cells increased 6�1 folds. The IC50 of H460/ER⁃CDH1 and H460/ER cells was ( 7�51 ± 1�12) μmol/L and ( 53�72 ± 12�95) μmol/L with statistical significance(P<0�05). The expressions of EGFR and its phosphorylation form in both PC9/AB⁃CDH1and H460/ER⁃CDH1 cells were significantly increased( P<0�05) . Conclusion It demonstrates that reversing of EMT can re⁃verse the acquired gefitinib/erlotinib⁃resistance in NSCLC, which suggests that EMT plays an important role in the acquired resistance to EGFR⁃TKIs in NSCLC, possibly through down⁃regulating the phosphorylation of EGFR.%目的：探讨上皮间质转化（ EMT）在非小细胞肺癌（ NSCLC）对表皮生长因子受体⁃酪氨酸激酶抑制剂（ EGFR⁃TKIs）获得性耐药中的作用及可能机制。方法选用EGFR基因19号外显子突变型吉非替尼耐药细胞PC9／AB和EGFR野生型厄洛替尼耐药细胞H460／ER，通过基因转染获得E⁃cadherin稳定过表达细胞PC9／AB⁃CDH1和H460ER⁃CDH1。四甲基偶氮唑盐（MTT）法检测细胞的增殖情况，划痕实验及Transwell侵袭实验检测细胞迁移和侵袭能力，实时荧光定量PCR（qRT⁃PCR）和蛋白印迹法检测EMT相关分子、EGFR信号通路分子的mRNA和蛋白表达水平。结果 PC9／AB和H460／ER细胞未发生T790M突变和c⁃Met基因扩增，但发生了EMT，表现为E⁃cadherin表达降低和Vimentin表达增加。通过基因转染提高E⁃cadherin表达水平能逆转PC9／AB和H460／ER耐药细胞的EMT，使其对EGFR⁃TKIs的敏感性增加， PC9／AB⁃CDH1细胞较PC9／AB对吉非替尼的敏感性增加约11�4倍，其半数抑制浓度（IC50）分别为（0�70±0�22）μmol／L和（8�68±0�44）μmol／L，差异有统计学意义（P＜0�05）； H460／ER⁃CDH1较H460／ER对厄洛替尼的敏感性增加约6�1倍，其 IC50分别为（7�51±1�12）μmol／L和（53�72±12�95）μmol／L，差异有统计学意义（P＜0�05）。同时，逆转EMT后，细胞的EGFR、p⁃EGFR的mRNA和蛋白表达量均增加，差异有统计学意义（ P＜0�05）。结论阻断耐药细胞的EMT可逆转NSCLC对EGFR⁃TKIs的获得性耐药，EMT在NSCLC对EGFR⁃TKIs的获得性耐药中起重要作用，其机制可能与EGFR磷酸化水平降低有关。