The Effects And Role Of SOX3 In Epithelial Ovarian Cancer Cell Proliferation And Metastasis

Ovarian cancer(OC)is one of the most common gynecologic malignancies.It ranks the 5th among the leading types of female cancer death in United States in 2011.According to the American Cancer Society,there were estimated 21,290 diagnosed cases accounting for 2.6% of all new women cancer cases,and 14,180 deaths accounting for 5% of all women deaths in 2015.Among all types of ovarian cancer cases,epithelial ovarian cancer(EOC)accounts for 80%-90% of them.With the development of medical technologies,the diagnosis and treatments are constantly improving over the past 50 years,however,the five-year survival rates of ovarian cancer remain below 50%.Due to the lack of understanding of the molecular and genetic basis,large amount of EOC cases were not diagnosed until metastasis had been noticed,leading to poor prognosis in patients.Therefore,therapeutic strategies that focusing on the initiation and progression of EOC are crucial to the treatment of patients.To our best knowledge,the molecular mechanisms responsible for epithelial ovarian carcinogenesis and progression are still unknown.The gene of SOX stands for the mammalian sex-determining region Y factor,SRY/Sry,which contains the HMG-box and encodes transcription factors that interact with DNA through a highly conserved HMG DNA-binding domain.Human SOX3 is a member of the SOXB1 subgroup and maps to chromosome Xq27.1..SOX genes areinvolved in regulations related to diverse developmental processes including sex determination,gender differentiation,neurogenesis,chondrogenesis,thymus cell differentiation and testis development.Recently,several studies showed that SOX genes could contribute to cancer development in several tumor types such as cerebral tumor,breast cancer,pancreatic carcinoma and prostatic carcinoma.Xia.et al first provided the evidence of the involvement of SOX3 in cellular transformation in 2000,demonstrating that the aberrant SOX3 expression led to oncogenic transformation of chicken embryonic fibroblasts,suggesting SOX3 can induce abnormal cell growth and promote oncogenesis.Later on,in the genome-based analysis of retroviral insertion sites,SOX3 was identified as a proto-oncogene,which may result in T-cell lymphomas once retroviral DNA integrate into the genome of mouse.Our study,furthermore,suggested that SOX3 gene might play a role in tumorigenesis.We performed the experiments in three parts:(1)The expression of SOX3 in epithelia ovarian cancer cells and tissues was assessed and correlated with clinicopathological parameters.(2)We investigated the effects of SOX3 in proliferation,apoptosis,adhesion ? migration and invasion by either depleting its expression using shRNA or overexpressing it via plasmid transfection in human epithelial ovarian cancer cell lines.(3)We also investigated the potential role of SOX3 functions as an oncogene in promoting epithelial ovarian cancer by targeting Src Kinase.Part IThe expression of SOX3 in epithelial ovarian cancer and the clinical analysis Objective: To assess the expression of SOX3 in epithelial ovarian cancer cells and tissues and correlated with clinicopathological parameters.Methods: We used RT-PCR,Western Blotting and Cell immunofluorescence to screen epithelial ovarian cancer cell lines in which endogenous SOX3 was expressed.SOX3 expression was examined by immunohistochemistry in clinical samples.We calculated a composite histoscore to account for both stain intensity and uniformity.The expression of SOX3 in epithelial ovarian cancer tissues was assessed and correlated with clinicopathological parameters.Results:(1)The expression of SOX3 was relatively high in the highly metastatic cell line HO8910-pm and SKOV3-ip compared with its parent ovarian cancer cell line HO8910 and SkOV3(p<0.01).ES2 cells expressed SOX3 moderately.The expression of SOX3 was relatively low in the benign ovarian serous cystadenoma eternalized cell line MCV152 and the immortal human ovarian epithelial cell line Moody.(2)SOX3was located mainly in the nuclei.Normal ovarian tissue samples were negative for SOX3 expression.Ovarian cystadenoma samples were partial positive for SOX3 expression.Borderline ovarian cystadenoma sample with less than 40% of SOX3 expression.EOC sample with more than 70% of the tumor cells exhibiting expression of SOX3.109(76.8%)of the 142 EOC samples were positive for expression of SOX3.In addition,SOX3-positive epithelial cells were detected in 25%(7/28)of borderline ovarian tumors and 9.1%(3/33)of ovarian cystadenoma respectively.None of thenormal ovarian tissue samples was positive for SOX3 expression.Significant statistical difference of SOX3 expression was achieved between the normal ovarian epithelia and carcinomas(P<0.001).Significant statistical differences were showed between groups of EOC and cystadenoma(P<0.001).(3)SOX3-positive expression was significantly associated with histological grade(P=0.018)and FIGO stage(P=0.004).Specifically,the SOX3-positive expression was relatively higher in less differentiated tumors(63/74,85.1%)and FIGO stage II-IV tumors(94/115,81.7%).However,there were no significant associations between the SOX3-positive expression and histological types,age,serum CA125 levels or ascites levels.(4)Patients with SOX3-positive expression had shorter OS(P=0.029)and DFS(P=0.002)durations and lower survival rates than those with SOX3-negative expression in EOC respectively,suggesting that high level of SOX3 might predict poor prognosis in human ovarian carcinoma.The SOX3 expression status was an independent predictor of OS duration in patients with EOC when challenged by the covariates age at diagnosis,histological type,tumor grade,FIGO stage,serum CA125 level and ascites level.Patients with SOX3-positive expression had a higher risk of death(OR,4.02;95% CI,1.03-15.71;P=0.045)than patients with SOX3-negative expression.However,the SOX3 expression status was not significantly associated with DFS duration(OR,1.57;95% CI,0.35-6.99;P=0.554).In addition,patients with FIGO stage II-IV tumors had a higher risk of death(OR,3.91;95% CI,1.15-13.24;P=0.029)and recurrence(OR,4.65;95% CI,1.19-18.25;P=0.027)than those with FIGO stage I tumors.Part IIThe effect of SOX3 on biological function of epithelial ovarian cancer cells Objective: To investigate the effect of SOX3 on the biological function of epithelial ovarian cancer cells.Methods: We investigated the role of SOX3 in migration,invasion,proliferation,adhesion and apoptosis using shRNA knockdown or plasmid expression experiments in human EOC cell lines.Cell proliferation was determined by CCK-8.The transwell migration and invasion assays were performed to study the migratory and invasive ability of epithelial ovarian cancer cells.The clone formation assay was performed to test the transformation of tumor cells.The ECM adhesion assay was performed to test the adhesion of tumor cells.Results:(1)The relative expression levels of SOX3 in HO8910 was 4.04 in the SOX3 overexpression cells.The relative expression levels of SOX3 in SKOV3 and SKOV3-ip were 0.23 and 0.15,respectively,in the SOX3 knockdown cells.(2)CCK8assays showed that overexpression of SOX3 increased the cell proliferation rate,while inhibition of it reduced proliferation rate of cells.(3)In the cell colony formation assays,compared with both negative control cells and wild type cells,colonies of HO8910-exSOX3 cells were significantly more(p<0.01),and colonies were fewer in SKOV3-siSOX3 cells(p<0.01)and SKOV3-ip-siSOX3 cells(p<0.01).(4)HO8910-exSOX3 cells had strengthened abilities of migration and invasion,while SKOV3-siSOX3 and SKOV3-ip-siSOX3 cells gained weakened migratory and metastatic potentials.(5)To further investigate the function of SOX3 in ovariancancer cells,cell adhesion abilities were measured using matrigel,laminin and BSA.Results showed that SOX3 decreased the cell adhesion,which might facilitate the migration and invasion of cancer cells.(6)To explore the oncogenic mechanism of SOX3,we tested its role in ovarian cancer cells by flow cytometry.The number of apoptotic HO8910-exSOX3 cells was less than the negative control and wild type cells.Conversely,a greater percentage of apoptotic SKOV3-siSOX3 and SKOV3-ip-siSOX3 cells was showed,compared with the negative control and wild type cells.These data suggest that SOX3 inhibits apoptosis and maintains the survival of ovarian cancer cells.Part IIISOX3 Enhances the Migration of Ovarian Cancer Cells via Src Kinase Objective: To investigate the role of SOX3 in EOC cell proliferation and invasion.And explore the relationship between SOX3 and Src kinase.Methods: We constructed the SOX3 over expression of HO8910 stable cell line,and studied the mechanism of promotion invasion and migration by SOX3 using western blot and transwell method.Results:(1)To investigate the molecular mechanism of SOX3-promoted cellularity changement,we discovered that elevated phosphorylation of FAK,SRC and p130-cas which were the related downstream cell signal molecules,was led to because of the overexpression of SOX3 in HO8910 cells.Furthermore,lower phosphorylation of FAK,SRC and p130-cas was observed after the inhibition of SOX3 expression inSKOV3 and SKOV3-ip cells using siRNA.(2)We knocked down the gene of Src using siRNA in HO8910-vector and HO8910-exSOX3 cells to test whether the activity of Src kinase is indispensable for the SOX3-mediated tyrosine phosphorylation of p130-cas.The interference efficiency was evaluated by qRT-PCR and western blotting.Transwell assays revealed that the knockdown of Src decreased the migration and invasion of HO8910-exSOX3 cells,whereas increasing the cell adhesion to matrigel or laminin,with the depression of tyrosine phosphorylation of p130-cas.