The Role Of EMT In The Acquired Resistance Of Non-small Cell Lung Cancer To EGFR-TKIs And Associated Mechanisms

Background and ObjectiveLung cancer is a disease with the highest morbidity and mortality in malignant tumors worldwide, which has become the leading cause of death in cancer. In recent years, molecular target therapy has become a hot topic and widely used in clinic. The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), in which gefitinib is one of the representatives, are used in the first- and second- therapy of advanced NSCLC patients with EGFR mutations. Efficiency ratio of EGFR-TKIs for NSCLC with EGFR wild-type also reaches 5%-6%. However, the emergence of acquired resistance has blocked further application of EGFR-TKIs, which leads to a median program of free survivual (PFS) of 8-10 months. Therefore, to find out the mechanism of acquired resistance is important for clinical therapy.Now, it is well known that the major mechanisms of acquired resistance of NSCLC with EGFR mutation to EGFR-TKIs include the secondary mutation of EGFR gene and the amplification of MET gene. This two mechanisms account for about 60% of acquired resistance. However,40% of acquired resistant mechanism of EGFR mutant NSCLC and the resistant mechanism of EGFR wild-type NSCLC remain unknown. The unclear mechanisms may include the activation of unexpected EGFR tyrosine kinase receptor signal pathway, the abnormal activation of moleculars in EGFR downstream signaling pathway, hepatocyte growth factor over-expression, EML4-ALK fusion gene, the absence of protein tyrosine phosphatase gene (PTEN) and EMT, etc.It has been reported that epithelial-mesenchymal transitions (EMT) were seen in wild type of NSCLC H460 and played an important role in acquired resistance of NSCLC to EGFR-TKIs. Epithelial-mesenchymal transitions (EMT) characteristic with down-regulation of epithelial markers (such as e-cadherin) and up-regulation of mesenchymal markers (such as vimentin) is a biological process, which is usually associated with the activation of some signaling pathways. EMT, in which a number of factors involved in this complex pathological reaction, not only plays crucial roles in embryonic development, chronic inflammation, tissue reconstruction and the development of many chronic diseases but also takes part in the process of tumor development, tumor cells in situ invasion and metastasis. What is more, EMT is closely related to the prognosis of NSCLCand EGFR-TKIs sensitivity.Thomson, etc reported that in EGFR wild-type NSCLC cells, cells expressing E cadherin/beta -catenin was more sensitive to EGFR-TKIs, and those expressing vimentin/fibronectin NSCLC cell was insensitive to EGFR-TKIs. Rho, etc[13] established the EGFR-TKIs resistant NSCLC A549/GR cell lines, the resistance is 7.7 times in A549/GR cell lines, compared to A549 cell lines; Further more, A549/ GR cells showed the EMT phenotypic characteristics.In the elotinib resistant non-small cell lung cancer cell lines H460 and Calu6, epithelial marker protein E cadherin expression are reduced, and in the elotinib sensitive cell lines H292,E-cadherin is high expression. Among gefitinib resistant lung cancer cell lines H157, H1730, A549, H520, epithelial marker protein E-cadherin, EpCAM, claudin4 and caludin7 are generally low expressing, but in the sensitive cell lines H322, H358, H1648, these protein expression is higher.Yauch etc found that, no matter in EGRF wild type or EGFR mutation type of NSCLC cells, the level of e-cadherin expression were closely associated with cellular sensitivity to EGFR-TKIs. By gene transfection to increase the level of e-cadherin could improve the sensitivity of tumor cellsto EGFR TKIs.Consistent with the basic research results, level of E-cadherin expression can be used as one of predict biomarkers to EGFR TKIs in the treatment of NSCLC patients. The results of an international multicenter phase Ⅲ clinical study shows that:EGFR-TKIs combined with chemotherapy in the treatment of NSCLC, patients expressing e-cadherin presents longer disease progression time. When gefetinib was in the first-line treatment in NSCLC, patients with e-cadherin expression positive achieved longer overall survival time with significantly than those with 3-cadherin expression negative.Many transcription factors are closely involved in the occurrence of EMT, for example, Slug, Twist, ZEB1, Smad and NF-B. It is found that EMT-related transcription factors (ZEB1, slug) also play a key role in process of the acquired drug resistance to EGFR-TKIs, targeting to eliminate the expression of slug could reverse the mesenchymal characteristics, restore the sensitivity to EGFR-TKIs in resistant cell lines.At present, the molecular biology mechanism of reduction of sensitivity to EGFR TKIs in NSCLC cells after the occur of EMT has not yet been elucidated. EMT might reduce the tumor cell growth or the dependency of EGFR signaling pathway, the mesenchymal phenotype cells can continuously induce Akt activation, trigger the PI3K/Akt signaling pathway activation, which leads to the resistance to EGFR-TKIst. At the moment, the concrete mechanism of EMT takes part in the acquired resistance of NSCLC to EGFR TKIs remains to be further studied.Therefore, the purpose of this study is to observe the effects of EMT on the acquired resistance to EGFR-TKIs by using lentiviral vectors of targeting CDH1 gene to over expressing of e-cadherin and reversing EMT in EGFR wild-type and mutant-type NSCLC cells, and then further investigating the molecular mechanism and important role of EMT on the EGFR-TKIs-resistance.Methods1. EGFR mutant-type human lung adenocarcinoma PC-9 and its EGFR-TKIs resistant PC-9/AB cells were used. The EGFR and K-RAS gene mutation were examined by qPCR-HRM. MTT assay was used to measure the cell proliferation. Wound-healing assay and transwell assay were used to determine the invasive and migratory capabilities of cells. The protein expressions of EGFR (epidermal growth factor receptor), p-EGFR, e-cadherin, vimentin and snail were determined by western blotting.2. GV218 plasmid was used to construct both over-expression vector targeting to CDH1 gene and its negtive control vector. The vectors were identified by PCR and gene sequencing. The identified vectors was given to transfect 293T cells and then been screened for effective targets by western blot. The effective vector was chosen to be packaged with 293T cells and the virus titer was determinated. The virus acquired was transfected into targeting cells H460/ER and PC-9/AB. Real-time quantitative RT-PCR and Western Blot detected the inhibited effeciency of e-cadherin in H460/ER and PC-9/AB cells.3. After CDH1 gene was transfected into targeting cells H460/ER and PC-9/AB, MTT assay was used to measure the cell proliferation. Wound-healing assay and transwell assay were used to determine the invasive and migratory capabilities of cells. The protein expressions of E-Cadherin, Vimentin and Snail were determined by Western blotting.4. After CDH1 gene was transfected into targeting cells H460/ER and PC-9/AB, MTT assay was used to measure the cell proliferation.The protein expressions of EGFR, p-EGFRwere determined by Western blotting.5. The results analyzed by SPSS13.0 were expressed as means ± S.D.s.Two groups were analyzed by t-test and Multiple groups were analyzed by one-way ANOVA followed by LSD multiple comparison test.Results1. EGFR and K-ras gene states were determinated in the cells.EGFR gene exon 18, exon 20, exon 21 and K-RAS gene were all wild-type, while EGFR gene exon 19 were mutant-type and in PC-9/AB cells, nether T790M mutation nor cMET gene amplification were detected. Our former research shows that no gene mutation was detected in H460/ER cells.2. The sensitivities of PC-9/AB cells to EGFR-TKIs were both decreased.The two cell lines grew multiply indefinitely with cultured by ordinary medium. The proliferative effects of PC-9 and PC-9/AB cells on Gefitinib are also concentration dependent. Compared with PC-9 cells, the sensitivity to EGFR-TKIs of PC-9/AB was significantly decreased. The IC50 values of PC-9/AB and PC-9 cells were 7.23±6.89μmol/L and 0.04±0.00μmol/L (P<0.05)3. Morphological changes happened in the cells.Compared with PC-9 cells, PC-9/AB cells displayed mesenchymal status with fusiform shaped and more pseudopods among the cells.4. The capability of invasion and migration was inhanced in the PC-9/AB cells.As shown in the wound-healing assay, the distance of scratches edge of PC-9 cells was significantly extended than that of PC-9/AB cells (P<0.05).It was observed in the transwell assay that there were less cells invaded through the membrane-coated commercial Matrigel in the PC-9cells.5. The expressions of EMT proteins and EGFR were determined.The result determined by Western Blot shows that vimentin and Snail were in creased in the PC-9/AB cells (P<0.05) and the expression of EGFR, E-cadherin andβ-catenin were decreased significantly (P<0.05).7. The lentiviral vector of over-expression targeting CDH1 gene was constructed.Two lentiviral vectors of over-expression targeting CDh1 gene (named LV-CDH1(5386-1)-P1、LV-CDH1(5386-1)-P2) and their negtive control vector were successfully constructed. The effective vector LV-CDH1(5386-1) was identified and packaged. LV-CDH1(5386-1) had significant inhibitory effect on H460/ER and PC-9/AB cells, and the increasing rate of mRNA was 16 times in H460/ER cells and 4 times in PC-9/AB cells respectively (P< 0.05),with the level of E-cadherin significantly increased.8. Morphological change in cells After CDH1 gene was transfected into targeting cells H460/ER and PC-9/AB.Compared with negative control H460/ER and PC-9/AB cells, H460/ER-CDH1 cells and PC-9/AB-CDH1 cells displayed epithelial status with pebble shaped and less pseudopods among the cells.9. The expression change of EMT proteins in cells After CDH1 gene was transfected into targeting cells H460/ER and PC-9/AB.The result determined by Western Blot shows that vimentn and Snail were decreased in the H460/ER-CDH1 cells and PC-9/AB-CDH1 cells (P< 0.05) and the expression ofβ-catenin was increased significantly (P< 0.05).10. The sensitivities to EGFR-TKIs in cells After CDH1 gene was transfected into targeting cells H460/ER and PC-9/AB.The four cell lines grew multiply indefinitely with cultured by ordinary medium. The proliferative effects of PC-9/AB-NC and PC-9/AB-CDH1 cells on Gefitinib are concentration dependent. Compared with PC-9/AB-NC cells, the sensitivity to EGFR-TKIs of PC-9/AB-CDH1 was significantly increased. The IC50 values of PC-9/AB-NC and PC-9/AB-CDH1 cells were 8.68±0.44μmol/L and 0.70±0.22mol/L (P <0.05). The proliferative effects of H460/ER-NC and H460/ER-CDH1 cells on Gefitinib are also concentration dependent. Compared with H460/ER-NC cells, the sensitivity to EGFR-TKIs of H460/ER -CDH1 was significantly increased. The IC50 values of H460/ER -NC and H460/ER -CDH1 cells were 53.72±12.95μmol/L and 7.51±1.12mol/L (P<0.05)11. The expression change of EGFR,p-EGFR in cells After CDH1 gene was transfected into targeting cells H460/ER and PC-9/AB.The result determined by Western Blot shows that EGFR and p-EGFR were increased in the H460/ER-CDH1 cells and PC-9/AB-CDH1 cells (P<0.05).ConclusionThe human lung adenocarcinoma cells PC-9/AB acquire the resistance to EGFR-TKIs accompanied by EMT. Reversing the EMT of the resistant cells could induce recovering sensitivity to EGFR-TKIs. EMT play an important role in the NSCLC cells acquired the resistance to EGFR-TKIs, and the reduction of EGFR and p-EGFR maybe one of its possible mechanism.