The Role Of Cancer Stem Cells In EMT-induced Acquired Resistance To EGFR-TKIs In NSCLC Cells

Background and ObjectiveWith increasing incidence and mortality, cancer is the leading cause of death in China. Among all types of cancers, lung cancer is the most common incident cancer and the leading cause of cancer death. In recent years, molecular targeted therapies have become a hot topic and have been widely used in clinic. Among them, The epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), in which gefitinib(Iresa) is one of the representatives, are widely used in the first-or even second-line therapy of advanced NSCLC patients with EGFR mutations. With these drugs, the median progress free survivual (PFS) and overall survivual of the patients are significantly prolonged. But the EGFR-TKIs NSCLC recipients lead a progress free life for about merely 8 to 10 months,which means that the tumours are resistant to these agents. Therefore, to find out the definite mechanisms of acquired resistance is of great imoportance for clinical therapy.These days, it is commonly recepted that the major mechanisms of acquired resistance to EGFR-TKIs of NSCLC with EGFR mutation include the amplification of MET gene and the secondary mutation of EGFR gene, which account for about 60% of acquired resistance. However, about 40% of acquired resistant mechanism remain unknown. Studies show that the unclear mechanisms may include the overexpression of hepatocyte growth factor(HGF), the downregulation or absence of protein tyrosine phosphatase gene (PTEN), the abnormal activation of moleculars within EGFR downstream signaling pathway, the activation of bypass EGFR tyrosine kinase receptor signal pathway, EML4-ALK fusion gene and EMT, etc.We have previously demonstrated that epithelial-mesenchymal transitions (EMT) were seen in both wild type of NSCLC cell line H460 and mutated type of NSCLC cell line PC-9, and we then unprecedentedly transfected CDH1 gene into targeting cells H460/ER and PC-9/AB to reverse EMT and just fond that the transfected cells were re-sensive to EGFR-TKIs compared to their parental cells, which consolidated the conception that EMT play an important role in acquired resistance of NSCLC to EGFR-TKIs. Epithelial-mesenchymal transitions (EMT) is a biological process characterised by down-regulation of epithelial markers (such as e-cadherin) and up-regulation of mesenchymal markers (such as vimentin), in which a number of factors involved in this complex pathological reaction, not only plays crucial roles in the process of tumor development, tumor cells in situ invasion and metastasis,but is closely related to the prognosis of NSCLC and EGFR-TKIs sensitivity.It has been corroborated that the cells with properties of stem-cell (cancer stem cell, CSC) increase both in normal and cancer cells when the EMT process is triggered. Mani SA, et al reported that the induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the acquisition of mesenchymal traits and in the expression of stem-cell markers. Contrarily, stem cell-like cells isolated from HMLE cultures and human mammary glands or mammary carcinomas have undergone an EMT. Polyak K, et al 211also demonstrated that transitions between epithelial and mesenchymal states would generate multiple, distinct cellular subpopulations with sternness. Pirozzi G, etc discover that the induction of EMT by TGF-β1 exposure, in primary lung cancer cell line results in the acquisition of mesenchymal profile and in the expression of stem cell markers. All of these indicate that EMT associates with CSCs. Lately, studies have shown that CSCs not only contribute to the process of tumor in situ invasion and metastasis, they also takes part in tumour recurrence and the resistance to threpies including EGFR-TKIs. Hashida S, etc established various afatinib-resistant cell lines and investigated the resistance mechanisms. The study found that several cell lines displayed EMT features and overexpress putative stem cell marker, such as ALDH1A1 and ABCB1. Ghosh G, ect also found that the erlotinib resistant cell line contained a population of cells with properties similar to CSCs.These cells were found to be less sensivive towards erlotinib treatment. Therefore, we unprecedentedly suppose that EMT mediated acquired resistance to EGFR-TKIs in NSCLC possibly through the up-regulation of CSCs.In recent years CSCs have emerged as the focus of intense investigations in cancer research. CSCs are a small subpopulation of cells within tumors with capabilities of self-renewal, differentiation, and tumorigenicity when transplanted into an animal host. Three key observations classically define the existence of a CSC population:1.Only a minority of cancer cells within each tumor are usually endowed with tumorigenic potential when transplanted into immunodeficient mice.2. Tumorigenic cancer cells are characterized by a distinctive profile of surface markers and can be differentially and reproducibly isolated from nontumorigenic ones by means of flow cytometry or other immunoselection procedures.3. Tumors grown from tumorigenic cells contain mixed populations of tumorigenic and nontumorigenic cancer cells, thus recreating the full phenotypic heterogeneity of the parent tumor. Studies have identified that CSCs exist in several tumour types, such as breast, brain, haematological malignancies as well as in lung cancer.Within NSCLC, CSCs are usually characterized by expression of cell surface proteins, such as clusters of differentiation 44,133 (CD44, CD 133), and they are always rich in side population (SP).Therefore, the purpose of this study is to quantify CSCs in PC-9 (exon 19del E746-A750) and the corresponding gefitinib-resistant cell line PC-9/AB undergoing EMT to verify our assumption that CSCs along with EMT play an important role in the EGFR-TKIs resistance of NSCLC.We also test the proportion of CSCs in both PC-9/AB-CDH and PC-9/TGF-β1 to consolidate the assumption.Methords1、To verify that the acquired resistance of PC-9/AB to gefitinib was mediated by EMT, we firstly prepared cell lines as below:The EGFR del E746-A750 mutated human lung adenocarcinoma PC-9 cell line and gefitinib acquired resistance cell line PC-9/AB were used in this study, PC-9 that had been treated with TGF-J31 for up to 2 weeks was named PC-9/TGF-β1, The stable E-cadherin over-expression cell line PC-9/AB-CDH 1 was established by transforming gene-CDH1 with lentivirus in PC-9/AB. We secondly test their sensitivities to gefitinib with CCK8. Thirdly, EMT was tested among the cell lines via morphology and western blotting. To rule out the possibility that T790M mutation and MET amplification would contribute to gefitinib resistance, we finally tested T790M mutation and MET amplification by respective qPCR-HRM and FISH.2. To test which of the CD44 and CD 133 can be a CSC marker, we firstly ascertain that all the cell lines express CD44 and CD 133. Secondly we ues MACS (magnetic-activated cell sorting) to sorted the cells into four subgroubs:CD44+^ CD44-、CD 133+and CD133-. Thirdly, sphere formation assay, tumorigenicity assay using NOD/SCID mice and flow cytometry for antibody analysis were used to explore whether CD44 or CD 133 can be the marker of CSC.3. With the marker confirmed, we then quantified CSCs within PC-9 and PC-9/AB using flow cytometry for antibody analysis. Meanwhile, sphere formation assay and side-population analysis were also use for supplementary measurement.4. To corroborate the role of CSCs in EMT-induced acquired resistance to EGFR-TKIs in NSCLC logically, firstly we transfect gene-CDH1 in PC-9/AB. Then their sensitivities to gefitinib were tested with CCK8. Thirdly, EMT was tested among the cell lines via morphology and western blotting. To rule out the possibility that T790M mutation and MET amplification would contribute to gefitinib resistance, we also tested T790M mutation and MET amplification by respective qPCR-HRM and FISH. Finally, we then quantify CSCs within PC-9/AB and PC-9/AB-CDH1 using flow cytometry for antibody analysis. Meanwhile, sphere formation assay and side-population analysis were also use for supplementary measurement.5. To corroborate the role of CSCs in EMT-induced acquired resistance to EGFR-TKIs in NSCLC logically, We also established the cell line PC-9/TGF-β1, which had been treated with TGF-β1 for up to 2 weeks. We then quantify CSCs within PC-9 and PC-9/TGF-β1 using flow cytometry for antibody analysis. Meanwhile, sphere formation assay and side-population analysis were also use for supplementary measurement.6. Statistical analysis was done with Graphpad Prism 5 software. All data are presented as mean±SEM except other stated. When two groups were compared, the Student t-test was used (P< 0.05 was considered significant). Each experiment was repeated three or four times, depending upon the particular experiment.Results1. Acquired resistance of PC-9/AB to gefitinib is mediated by EMT Compared to PC-9, PC-9/AB shows a notable resistance to gefitinib (IC50 was 0.07±0.04μM and 10.85±2.64μM respectively, P<0.01). In accordance with our previous study, PC-9/AB cells lost its epithelial morphology assuming a fibroblast-like appearance, which is accompanied by a downregulation of E-cadherin and an upregulation of vimentin as shown by western blotting. There is no T790M mutation and MET amplification in PC-9/AB by qPCR-HRM and FISH. Compared to PC-9/AB, the sensitivity to gefitinib was recovered when EMT had been reversed by transforming gene-CDH1 (PC-9/AB-CDH1) (IC50 was 10.85±2.64μM and 1.31±0.47μM respectively, P<0.05). The sensitivity to gefitinib decreased when PC-9 undergo an EMT driven by TGF-β1 (PC-9/TGFβ1) (IC50 was 0.07±0.04μM and 6.73±2.19μM respectively,P<0.01)2. CD 133 can be a CSC marker of PC-9 and its derived cell lines.Studies have shown that CD44 and CD 133 can be the CSC marker of NSCLC cell lines, so we firstly try to confirm that all the cell lines express CD44 and CD 133. It turned out as expected that all the cell lines above expressed CD44 and CD 133, among which CD44+cells in PC-9、PC-9/AB、PC-9/AB-CDH andPC-9/TGF-β1 accounted for 94.1%、97.6%、95.5% and 98.4% respectively while CD133+cells 0.11%、1.2%、0.26% and 0.75% respectively. We then sorted PC-9/AB into four groups (CD44+、CD44-、CD 133+ and CD133-) by MACS and got on with sphere formation assay, tumorigenicity assay using NOD/SCID mice and flow cytometry for antibody analysis to explore whether CD44 or CD133 can be the CSC marker. Flow cytometry for antibody analysis showed that MACS was of high efficiency in cell sorting, by which the purity of sorted CD44+ and CD133+ cells were up to 94.2% and 96.6%. The sorted CD44+ and CD133+cells were again got on with flow cytometry for antibody analysis after cultured regularly for 2 weeks. Results showed that:1. CD133+cells can generate both CD133+ and CD133-cells while CD133-cells can only generate CD 133- cells.2. Both CD44+ and CD44-cells can generate CD44- and CD44+ cells. Sphere formation assay showed that:1. spheres generated by CD133+ cells were of great significance as compared to CD133-cells ((452±10.60) and (4.67±0.88), P<0.000).2. spheres generated by CD44+ cells were comparable to CD44-cells ((76±12) and (65±15), P=0.446). Tumorigenicity assay using NOD/SCID mice showed that:1. Tumors formed only with 103 CD 133+ cells while it is cannot with 106 CD 133-cells.2. Tumors hardly formed with the minimum 105 CD44+ or CD44-cells. All above draw a conclusion that CD 133 instead of CD44 can be the CSC marker of PC-9 and its derived cell line.3. An increase of CSCs display in PC-9/AB, as compared to PC-9.With the marker confirmed, we then quantified CSCs within PC-9 and PC-9/AB. Flow cytometry for antibody analysis showed that CD 133+ cells raise markedly (evidently,observably,remarkebly) in PC-9/AB in contrast to PC-9, (1.20%±0.22%) and (0.11%±0.02%) respectively, P=0.008. Side-population analysis revealed that the percentage of side population in PC-9/AB increased observably as compared to PC-9, (1.32%±0.24%) and (0.14%±0.03%) respectively,P=0.008. In contrast to PC-9, spheres in PC-9/AB also increased significantly, (71±9.5) and (18±4.5) tespectively,P=0.007.All above conformably announce that CSCs in the gefitinib resistant PC-9/AB undergoing EMT are much more than gefitinib sensitive PC-9.4. CSCs decrease when EMT sustained in PC-AB was reversed.To corroborate the role of CSCs in EMT-induced acquired resistance to EGFR-TKIs in NSCLC logically, we also quantified CSCs in PC-9/AB-CDH1. Flow cytometry for antibody analysis showed that CD133+ cells decreased sharply in PC-9/AB-CDH1 in contrast to PC-9/AB, (0.26%±0.05%) and (1.20%±0.22%) respectively, P=0.014. Side-population analysis revealed that the proportion of side population in PC-9/AB-CDH1 decreased evidently as compared to PC-9/AB, (0.34%±0.10%) and (1.32%±0.24%) respectively, P=0.019. In contrast to PC-9/AB, spheres in PC-9/AB-CDH1 also decreased significantly, (23±6.1) and (71±9.5) respectively,P=0.013. All above conformably announce that CSCs in EMT free PC-9/AB-CDH1 drop observably as compared to parental cell line PC-9/AB.5. CSCs increase when EMT was triggered in PC-9/TGF-β1.To corroborate the role of CSCs in EMT-induced acquired resistance to EGFR-TKIs in NSCLC logically, we also quantified CSCs in PC-9/TGF-β1. Flow cytometry for antibody analysis showed that CD 133+ cells increased in PC-9/TGF-β1 in contrast to PC-9, (0.75%±0.14%) and (0.11%±0.02%) respectively, P=0.01. Side-population analysis revealed that the proportion of side population in PC-9/TGF-β1 increased evidently as compared to PC-9, (0.96%±0.22%) and (0.14%±0.03%) respectively, P=0.022. In contrast to PC-9, spheres in PC-9/TGF-β1 also increased significantly, (60±11.27) and(18±4.5) respectively,P=0.026. All above conformably announce that CSCs increase when EMT was triggered by TGF-β1 as compared to parental cell line PC-9.ConclusionIn this study we reveal that:EMT appears in NSCLC cell line PC-9 during the inducement of gefitinib resistant cell line PC-9/AB, which is free of T790M mutation and MET amplification and accompanied with an increase of CSCs. Reversing EMT of PC-9/AB by transfected CDH1 gene results in the sensitiveness to gefitinib and a decrease of CSCs. Inducing EMT of PC-9 by treating with TGF-β1 leads to the insensitiveness to gefitinib and an increase of CSCs. EMT plays an important role in the acquired resistance to EGFR-TKIs in NSCLC, possibly through the up-regulation of CSCs.