Exon4-deletion Variant Of Epidermal Growth Factor Receptor Enhances Invasiveness And Cisplatin Resistance In Epithelial Ovarian Cancer

Ovarian cancer is the leading cause of death among gynecologic cancers and the fifth leading cause of all cancer-related deaths among women. Although survival has increased slightly over the past25years,5-year survival of ovarian cancer patients remains below50%. The poor prognosis of ovarian cancer is attributed to its vicious symptoms induced by widespread metastasis and resistance to chemotherapy in patients with advanced disease. There are three major types of human ovarian cancers: epithelial, stromal and germ cell. The majority of ovarian cancers are epithelial carcinomas.Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein that belongs to the receptor tyrosine kinase family of growth factor receptors and participates in important physiological process such as cell survival, proliferation, and motility. Overexpression of the EGFR has been associated with advanced disease and poor survival outcome of patients in a large of human tumor types, such as breast cancer, lung cancer, liver cancer, prostate cancer and ovarian cancer. EGFR coding sequence alterations also are frequently found in human cancers. Most of the variants with deletions in the extracellular domain correlate with poor survival. Generally, these variants are constitutively active and confer the tumor cells a growth advantage and increased malignant potential.Recently, de4EGFR, a variant of epidermal growth factor receptor (EGFR) with exon4deletion was identified in glioblastoma and ovarian cancer. However, its biological function on ovarian cancer is still not clear. In this study, the expression profile of de4EGFR and its contribution to epithelial ovarian cancer cells proliferation, invasiveness and drug resistance were studied. Part One The expression and biological behavior of de4EGFR in epithelial ovarian cancerObjective To study the expression profile of de4EGFR in the tissue of epithelial ovarian cancer and its the contribution to the biological behavior as proliferation and invasiveness in epithelial ovarian cancer.Methods Human normal ovarian tissue (n=12) and epithelial ovarian cancer tissues (n=72) were obtained and the expression of de4EGFR of the samples were detected by RT-PCR. The EGFR and de4EGFR expression lentivirus were produced and used to transfect three different epithelial ovarian cancer target cell lines(SKOV3, CAOV3, ES2). The biological behavior of the epithelial ovarian cancer cell trasfected with lentivirus was thus observed. The proliferation assay was tested by CCK-8kit; the migration and invation assay was tested by Transwell assay. In vivo tumor growth assay was used to evalutate the influence of de4EGFR to the volume and weight of the tumor, which was performed by subcutaneous inoculation of ovarian cancer cells into nude mice.Results Our results showed that48.6%(35/72) of epithelial ovarian cancer tissues had de4EGFR expression. The de4EGFR expression was not observed in normal ovarian tissues. In clinical stage, de4EGFR expression in cancer tissues of stage â…¢-â…¤ was significantly higher than cancer tissues of stage â… -â…¡ (P<0.05). Growth curves demonstrated that, similar to EGFR, compared to the GFP control, de4EGFR significantly increased cell proliferation (P<0.05or P<0.01). In tumor formation assay in vivo, both EGFR and de4EGFR promoted tumor growth when compared to GFP controls (P<0.01and P<0.05) while no significant difference was observed between EGFR group and de4EGFR group. A transwell migration and invasion assay using SKOV3transfectants showed that EGFR induced a two-fold increase in cell invasion compared to the GFP control. Notably, de4EGFR induced higher cell invasion ratios than EGFR did (P<0.001). Similar results were obtained in invasion experiments using CAOV3and ES2 transfectants (P<0.001).Conclusions These results demonstrate that de4EGFR expression was observed in epithelial ovarian cancer tissues and the expression ratio positively correlated with clinical stages. de4EGFR promoted epithelial ovarian cancer cells proliferation and had a higher invasiveness-promoting capacity than EGFR in epithelial ovarian cancer cells.Part TwoThe molecular mechanism of the de4EGFR that enhances invasiveness in epithelial ovarian cancer cellObjective To study the molecular mechanism of the de4EGFR that enhances invasiveness in epithelial ovarian cancer cell.Methods The expression of de4EGFR was transfected with lentivirus in three ovarian cancer cell lines(SKOV3, CAOV3, ES2). We observed the autophosphorylation of de4EGFR with or without EGF stimulation and the downstream signaling pathways by Western blot. We also observed the other invasiveness-promoting molecules as Src, FAK, MMP-9and E-cadherin. FAK was downregulated by siRNA in epithelial ovarian cancer cell line SKOV3and Western Blot test was used to detect the effect of FAK on the activation and inhibiton of multiple signaling pathways and transwell assay was used to observe the effect on the invasion of the epithelial ovarian cancer cells.Results We observed that de4EGFR could undergo autophosphorylation and was not sensitive to EGF stimulation. In comparison with GFP and EGFR transfectants, de4EGFR transfectants significantly upregulated the ERK, AKT, FAK and Src phosphorylation and MMP-9expression while downregulated the expression of E-cadherin. Moreover, our results demonstrated that the siRNA-mediated downregulation of FAK significantly suppressed the invasion in SKOV3transfected with de4EGFR cells (P<0.05). Additionally, the FAK siRNA significantly attenuated Src, ERK, AKT, MMP-9 activation, and upregulated E-cadherin expression in SKOV3and ES2transfected with de4EGFR.Conclusions These results showed that de4EGFR upregulated FAK phosphorylation and expression which lead to activation of ERK/AKT. Subsequently, constitutive activation of ERK/AKT increases MMP-9expression and inhibits E-cadherin expression to promote epithelial ovarian cancer cells invasiveness.Part Threede4EGFR enhances cisplatin resistance in epithelial ovarian cancer cellObjective To study the contribution of de4EGFR to cisplatin resistence in epithelial ovarian cancer cell.Methods Drug sensitivity of the cells to cisplatin was assayed by CCK-8Kit in SKOV3and CAOV3transfected with GFP, EGFR and de4EGFR. Apoptosis was assessed by using of an Annexin V-coupled fluorescein isothiocyanate (FITC) apoptosis detection kit-1in these same cells. The involved signaling pathways as AKT, ERK and proteins associated with apoptosis as Bcl-2, Bad were assyed by Western Blot with or without cisplatin.Results de4EGFR transfectants of SKOV3were significantly less cisplatin inhibited growth and induced apotosis than both GFP (P<0.001) and EGFR (P<0.01) transfectants, although EGFR transfectants have an increased resistance to cisplatin when compared to GFP (P<0.05). Similar results were observed in CAOV3transfectants. Moreover, compared with EGFR and GFP transfectants, de4EGFR transfectants had higher phosphorylated ERK and AKT levels treated with cisplatin. Additionally, compared with EGFR and GFP, de4EGFR induced higher level of Bcl-2expression and lower level of Bad expression treated with cisplatin. Similar results were obtained in the experiments using CAOV3transfectants.Conclusions Our results showed that de4EGFR could significantly activate ERK and AKT phosphorylation which can enhance cisplatin resistance in epithelial ovarian cancer cell through upregulation of Bcl-2and downregulation of Bad.