Mechanisms Of EMT Elicited By Rac1 In Epithelial Ovarian Cancer

Epithelial ovarian cancer(EOC), which is the most common type of ovarian cancer, is characterized by the leading cause of death from gynecologic malignancies. Worldwide, nearly 204,000 women are diagnosed, and 125,000 die due to ovarian cancer each year. Due to the absence of specific symptoms, more than 70% of EOC patients are diagnosed with FIGO stage III or IV tumors, with a five year survival rate of about 35% or 20% respectively. Known as a highly metastatic tumor, EOC spreads within the peritoneal cavity. Despite the huge progress on elucidating factors involved in EOC carcinogenesis, the understanding of the complex nature of its development and the unusual mechanism(s) of disease progression still remains poor. Hence, an explanation of the molecular changes associated with EOC metastasis could lead to the identification of targets for novel therapeutic interventions.Commonly, carcinoma spreads by nodal extension and hematogenous dissemination, but EOC forges its own path. The characteristics of EOC spreading are as follows:(1) EOC spreads within the peritoneal cavity long before lymphovascular dissemination, rarely in hematogenous dissemination. (2) To facilitate the interaction with the peritoneal stroma and mesothelium, EOC cells undergo morphological and molecular changes during the transition called epithelial mesenchymal transition (EMT). Epithelial-mesenchymal transition (EMT) describes as epithelial cells exhibit reduced cell-cell adhesion with the down regulation of E-cadherin and acquire fibroblastlike properties and increased motility, which facilitates the escape of tumor cells from primary tumors.Our previous study have suggested overexpression of WAVE1 correlated with a poor prognosis and might be an important predictor for EOC metastasis. We also have found c-Abl, a major activator of WAVE1, predicted an unfavorable outcome of EOC. Evidences have shown rac1 is another activator of WAVE1, rac1/WAVE1 signaling pathway is crucial for cell motility. Ras-related C3 botulinum toxin substrate 1 (racl) is a member of Rho family of small GTPases. Human rac1 gene comprises 7 exons over a length of 29 kb and is localized to chromosome 7p22. Rac1 has two transcripts,1.2 and 2.5 kb in size. Rac GTPases are small GTP-binding proteins (GTPases) that range between 20 and 25 KDa in size. Rac GTPases work as sensitive molecular switches existing either in an inactive, GDP-bound form or an active GTP-bound form by GTP nucleotide binding and hydrolyzation and play different roles by activating different downstream effectors. Rac1 is reported to be highly expressed in various tumors including gastric cancer, breast cancer, lung cancer, pancreatic cancer and colon cancer. Rac1 is implicated in the regulation of cell motility, proliferation, apoptosis and metastasis and plays an important role in cell EMT. Despite the above and other findings, research on the role of racl in EOC EMT remains deficient. The present study aims to examine the expression of rac1 in human EOC tissues and analyze the correlation of rac1 expression with clinicopathological features and prognosis of patients. Furthermore, the roles and possible mechanisms of rac1 in EMT of ovarian cancer cells were investigated.PART ONETHE EXPRESSION AND CLINICAL SIGNIFICATION OF RAC1 IN EPITHELIAL OVARIAN CANCERObjectivesTo determine the expression of racl and analyze the correlation of rac1 expression with clinicopathological features and prognosis of EOC patients.Methods1. Rac1 protein level was examined in 150 EOC tissues,21 borderline and 41 benign tumor tissues, and 24 normal ovaries by immunohistochemical staining, and the association of rac1 protein expression with clinicopathogic features in 150 EOC patients was investigated.2. Rac1 protein level was examined in 43 EOC tissues,11 borderline and 28 benign tumor tissues, and 18 normal ovaries by Western blot analysis, and the association of rac1 protein expression with clinicopathogic features in 43 EOC patients was investigated.3. Kaplan-Meier method was performed to assess the correlation between racl expression and survival rate.4. QRT-PCR and Western blot analysis was used to examine the mRNA and protein level of rac1 in ovarian cancer cell lines. Immunofluorescence was also used to local the expression of rac1 in the SKOV3 and 3AO cell lines.Results1.Immunohistochemical staining revealed rac1 protein expression was significantly higher in EOC tissues than in borderline tumors, benign tumors, and normal ovaries. Rac1 immunoreactivity was significantly associated with serum Ca-125, advanced tumor stages (i.e., stage III or IV), tumor grade (grades 2 and 3) and residual tumor size (size≥1 cm).2.Western blot analysis revealed racl protein expression was significantly higher in EOC tissues than in borderline tumors, benign tumors, and normal ovaries (1.810±0.390,0.543±0.277,0.389±0.188, 0.384±0.150)(P<0.05).3.Log-rank test showed that patients with low rac1 expression levels exhibited a longer overall survival rate and disease free survival than patients with high rac1 expression levels (P<0.001). By multivariate analysis, high rac1 expression, poor grade and advanced tumor stage remained independent prognostic factors of poor overall survival.4.The mRNA levels of rac1 in four ovarian cancer cell lines (SKOV3,OVCAR3,3AO,ES-2) were 2.207±0.251,1.870±0.207,0.983 ±0.097,0.818±0.150. The protein levels of racl in SKOV3, OVCAR3, 3AO, ES-2 were 1.256±0.156,1.370±0.060,0.724±0.0802,0.850± 0.142, respectively. Racl was localized predominantly in both the cytoplasm and the cytomembrane.ConclusionsOur present study identifies that rac1 overexpression is associated with poor prognosis in EOC. Rac1 may play an important role in predicting EOC metastasis.PART TWOCONSTRUCTION OF SHRNA LENTIVIRAL VECTORS TARGETING RAC1 GENE AND SELECTION CONSTRUCTED-STABLY TRANSFECTED OVARIAN CANCER CELL LINESObjectivesTo construct shRNA lentiviral vector targeting rac1 gene and select a construct-stably transfected the human ovarian carcinoma cell lines.Methods1.Design and synthesis of two human rac1 specific shRNA sequences and a negative control sequence. To verify the recombinant plasmid, DNA sequencing and double restriction digestion were performed.2.The recombinant plasmid and construction of lentiviral vectors were co-transfected into the 293T cells, and then collected and purified.3.SKOV3 and OVCAR3 cells were transfected with lentivirus transfected cells were cultured for 72h and were harvested.4.Rea1-time PCR and western blot were used to verify the best inhibited effect of rac1 in the stably transfected human ovarian carcinoma cell lines.Results1.The DNA sequencing and double restriction digestion showed that two rac1 specific shRNA (rac1 shRNA-1, racl shRNA-2) sequences were designed and synthesized successfully.2.Stably transfected human ovarian carcinoma cell lines were harvested by puromycin.3.The mRNA and protein levels of racl were down-regulated in SKOV3 and OVCAR3 by real-time-PCR and Western blot. The SKOV3-Ril and OVCAR3-Ri2 were with the best inhibitory effects of rac1.ConclusionsTwo specific human racl shRNA sequences were successfully designed and synthesized. SKOV3-Ril and OVCAR3-Ri2 were harvested with the best inhibitory effects of racl.PART THREEIMPACTS OF RAC1 GENE SILENCING ON THE MALIGNANT BEHAVIOR OF OVARIAN CANCER CELLSObjectivesTo examine the impact of rac1 gene silencing on the adhesion, migration, invasion and proliferation of ovarian cancer cells.Methods1. Wound healing assay was used to evaluate the cell migration of SKOV3 and OVCAR3 before and after racl interference.2. Transwell assay was used to evaluate the cell invasion of SKOV3 and OVCAR3 before and after racl interference.3. Cell adhesion assay was used to evaluate the cell adhesion of SKOV3 and OVCAR3 before and after racl interference.4. Plate clone formation assay was used to evaluate the cell clone formation of SKOV3 and OVCAR3 before and after rac1 interference.5. BrdU assay was used to evaluate the cell proliferation of SKOV3 and OVCAR3 before and after rac1 interference.6. In nude mice mode we have measured the the tumor formation of SKOV3 before and after rac1 interference.Results1.Rac1 silencing reduces cell migration, invasion and adhesion of ovarian cancer cell linesThe results of wound healing and transwell assay showed compared with SKOV3-NC and OVCAR3-NC, SKOV3-Ri1 and OVCAR3-Ri2 showed significantly slower wound closure (P< 0.001 and P< 0.001, respectively) and reduced invasion through the Matrigel (P< 0.001 and P< 0.001, respectively).The results of cell adhesion assay showed the number of adherent cells in SKOV3-Ri1 and OVCAR-3-Ri2 cell models significantly decreased after 48 h compared with SKOV3-NC and OVCAR-3-NC (P< 0.001 and P< 0.001, respectively).2.Racl silencing reduces cell proliferation of ovarian cancer cell linesThe results of plate colony formation assay showed SKOV3-Ri1 and OVCAR3-Ri2 formed significantly fewer colonies than SKOV3-NC and OVCAR3-NC (P< 0.01 and P< 0.01, respectively).The results of BrdU assay showed SKOV3-Ri1 and OVCAR3-Ri2 displayed marked reduction of the number of cells in the S phase compared with SKOV3-NC and OVCAR3-NC (P< 0.001 and P< 0.001, respectively).3.Rac1 silencing suppresses ovarian tumor growth in vivoThe results of tumor formation in nude mice assay showed formation rates were 75.0% and 100.0% for SKOV3-Ril and SKOV3-NC, respectively. After five weeks, the tumor size of SKOV3-Ril was significantly smaller (P<0.001) than that of SKOV3-NC.ConclusionsOur present study demonstrated the racl silencing reduced cell migration, invasion, adhesion, proliferation in vitro and cell tumor formation in vivo.PART FOURIMPACT OF RAC1 GENE SILENCING ON THE MOLECULAR MECHANISM OF OVARIAN CANCER CELLSObjectivesTo determine the molecular mechanism of regulation of racl-induced invasion and proliferation in ovarian cancer and to provide evidence of molecular biology for ovarian cancer cell EMT mediated by rac1.Methods1. HE staining was performed to investigate the morphological changes of xenografts.2. Immunohistochemical staining was performed to investigate the levels of E-cadherin and vimentin in xenografts.3.Western blot analysis was performed to investigate the levels of E-cadherin and vimentin in ovarian cancer cells after racl interference.4.Western blot analysis was performed to investigate the levels of pak1、p-pak1、p38 MAPK、p-p38MAPK in ovarian cancer cells after racl interference.Results1. HE staining showed the morphology of SKOV3-Ril changed.2.The results of immunohistochemical staining showed a significant increase in E-cadherin and decrease in vimentin in SKOV3-Ri1 compared with SKOV3-NC in xenografts (P< 0.05).3.The results of western blot showed a significant increase in E-cadherin and decrease in vimentin in SKOV3-Ril and OVCAR3-Ri2 compared with the controls (P< 0.05).4.The results of western blot showed in ovarian cancer cells total pakl, phospho-pak1, and phospho-p38 protein levels were down-regulated in racl-silenced cells. By contrast, no rac1 silencing-induced changes were observed in total p38 levels.ConclusionsOur study demonstrated the effect of rac1 in regulating the malignant behavior of EOC might via racl/pakl and P38MAPK pathway and further proved ovarian cancer EMT was mediated by rac1.