The Effect And Mechanism Of EPAC1 Downregulation Inhibiting Proliferation Of Ovarian Cancer Cells

Objective:Ovarian cancer is a common malignant tumor of gynecology, and is the most primary cause of death of patients with gynecology malignant tumor. Among different types of ovarian cancer, epithelial ovarian cancer is the most common and most deadly. Furthermore, serous ovarian carcinoma is the most common and most aggressive subtype of epithelial ovarian cancer. Most of the patients have been at an advanced stage when diagnosed, because women with ovarian cancer may have no specific symptoms and there are no effective diagnostic methods in early stage. Despite recent advances in surgical resections and systemic radiotherapies and chemotherapies, most cancer patients with advanced-stage will ultimately relapse and become incurable, and the 5-year survival rate is less than 30%. Many researchrs have found that some molecules are involved in the development of ovarian cancer, and have a great significance to the prognosis of ovarian cancer. Therefore, it is very necessary to explore specific, sensitive and new biological targets for diagnosing and treating ovarian cancer earlier, improving survival rate and prolonging survival period efficiently.cAMP is an important second messenger that participates in many physiological and pathological processes of cells. In the beginning, PKA has been assumed as the only downstream target of cAMP. But, along with the deeper research, the researchers found another signaling pathway:the Epac (exchange protein directly activated by cAMP) pathway.As the newly found cAMP-activated downstream signaling molecule, Epac (also known as cyclic AMP-activated guanine nucleotide exchange factors) can active Ras-like GTPases. Two isoforms of Epac, namely Epacl (also known as cAMP-GEF-1) and Epac2 (also known as cAMP-GEF-Ⅱ), have been identified so far. Epacl is a multi-domain protein consisting of an N-terminal regulatory region and a C-terminal catalytic region. Epacl mRNA is expressed ubiquitously in human. Meanwhile, Epacl plays important roles in regulating the biological activities, such as secretion, integrin-mediated cell adhesion, formation of cell-cell junctions, cellular calcium handling, apoptosis, cardiac hypertrophy, cell proliferation, cell differentiation and gene expression. Once Epacl was activated by an upstream cAMP, it can cause many changes of some signaling downstream pathways, including MAPK, mTOR and PI3K/AKT, and then regulate some activities, such as calcium handling, cell proliferation, survival, inflammation, learning, memory and so on. It has been reported that Epacl playes important roles in the proliferation of prostate cancer, migration and invasion of pancreatic cancer and migration of melanoma. However, there is no report about the effect of Epacl on ovarian cancer.In this research, we aim to study the effect and mechanism of Epacl in the most common gynecology malignant tumor:ovarian cancer. Firstly, we detected the expression levels of Epacl in three ovarian cancer cell lines:SKOV3,OVCAR3 and CAOV3. Then, we down regulated the Epacl expression in ovarian cancer cells SKOV3 and OVCAR3 and detected the effect of Epacl knockdown on the proliferation of ovarian cancer cells. Thirdly, we explore the possible mechanism:1. the MAPK pathway; 2. the AKT/CyclinDl/CDK4 pathway. At last, we verified our results using xenograft nude mouse model.Methods:1. The expression of Epacl in ovarian cancer cell lines SKOV3, OVCAR3 and CAOV3.We detected Epacl mRNA and protein expression in ovarian cancer cell lines SKOV3,OVCAR3 and CAOV3 by real time RT-PCR and western blot respectively.2. The effect of Epac1 knockdown on the proliferation and colony formation ability of ovarian cancer cells.(1) We chose ovarian cancer cell lines SKOV3 and OVCAR3 which have higher levels of Epac1 than CAOV3. After transfection of Epac1 siRNA or control siRNA, the efficiency of Epac1 knockdown was identified by RT-PCR.(2) After Epac1 downregulation, CCK8 assay was used to detect cell viability in SKOV3 and OVCAR3 cells.(3) After Epacl knockdown, colony formation assay was used to detect cell colony formation capacity of SKOV3 and OVCAR3 cells.3. The effect of Epac1 knockdown on the cell cycle and apoptosis of ovarian cancer cells.(1) After Epacl knockdown, the cell cycle of SKOV3 and OVCAR3 cells was detected by flow cytometry.(2) Flow cytometry was used to determine the apoptosis of SKOV3 and OVCAR3 cells after Epacl downregulation.4. The effect of Epacl knockdown on the proliferation-related pathways.(1) Western blot was used to detect the effect of Epac1 downregulation on MAPK pathway molecules (p-ERK, ERK, p-JNK, JNK, p-P38 and P38) in SKOV3 and OVCAR3 cells.(2) Western bot was used to detect the effect of Epac1 knockdown on AKT pathway molecules (p-AKT and AKT), as well as cell cycle-related proteins CyclinDl and CDK4 in SKOV3 and OVCAR3 cells.5. The effect of Epac1 knockdown on the proliferation of ovarian cancer cells and the cell proliferation-related pathways in tumor-bearing nude mice.(1) We chose SKOV3 for tumorigenesis in vivo. Twenty-seven 6-8-week-old female BALB/c nude mice were randomly assigned to three groups:the NS group (n =9), shControl group (n=9), and shEpac1 group (n=9). They were injected subcutaneously in the left flank with 7×106 SKOV3 cells (prepared in NS at a density of 7×107/ml), respectively.(2) After two weeks, palpable tumors developed in the nude mice. Then the mice were treated with 100ul NS or 30ug shControl plasmid or 30ug shEpacl plasmid by intra-tumor injection every two days. In the meantime, measure the size of the tumors. We measured the length, width and width of the tumors every day. Tumor volume was assessed and calculated as follows:length×width xwidth x 0.5.(3) After nine days treatment, all mice were sacrificed and the tumors were removed for measurement of volume and weight. Then the part of tumors was fixed in formalin and embedded in paraffin for IHC analysis, and the rest was frozen at -80℃ for western blot detection.Results:1. The expression of Epacl mRNA and protein was higher in ovarian cancer cells SKOV3 and OVCAR3.Epacl mRNA and protein expression was higher in SKOV3 and OVCAR3 cells, but low in CAOV3 cells.2. The specific siRNAs targeting Epacl could effectively downregulate the expression of Epacl in SKOV3 and OVCAR3 cells.We detected the interfering efficiency by RT-PCR. The results revealed that the two different siRNAs were both efficient, SKOV3 and OVCAR3 cells transfected with Epacl siRNA showed lower expression of Epacl mRNA than the cells in untreated group or negative control group.3. Epacl knockdown inhibited cell proliferation and colony formation ability in ovarian cancer cell lines.(1) CCK-8 assay revealed that the downregulation of Epac1 led to a significant reduction of the proliferation rate at 48h and 72h in both SKOV3 and OVCAR3 cells.(2) In SKOV3 and OVCAR3 cells, Epacl knockdown markedly reduced the ability of colony formation compared with siControl and untreated groups.4. Epacl knockdown induced Gl phase arrest of ovarian cancer cells SKOV3 and OVCAR3, but had no effect on apoptosis.(1) Epacl knockdown resulted in a significant increase of cell number in G1 phase compared with the controls in SKOV3 and OVCAR3 ovarian cancer cells.(2) To further elucidate the effect mechanism of Epac1 knockdown in ovarian cancer cells, we measured apoptosis in Epacl siRNA-transfected SK0V3 and OVCAR3 cells using Annexin V-FITC/PI double staining assay.The results showed that there were no significant differences in the rate of apoptosis between siEpacl cells and control cells.5. Epacl knockdown decreased the expression of p-AKT, CyclinDl and CDK4 in SKOV3 and OVCAR3 cells, but had no effect on the MAPK pathway.(1) We measured the expression of p-ERK, ERK, p-JNK, JNK, p-P38 and P38 in SKOV3 and OVCAR3 cells after the downregulation of Epacl expression. Epacl knockdown had no effect on the phosphorylated ERK, JNK and P38 proteins in SKOV3 and OVCAR3 cells.(2) To determine the effect of Epacl knockdown on PI3K/AKT pathway, we measured the expression of AKT and p-AKT in Epacl-silenced SKOV3 and OVCAR3 cells using western blot. We found the downregulation of Epacl markedly decreased the expression of p-AKT. PI3K/AKT pathway could regulate the expression of G1 phase checkpoint CyclinDl and CDK4, therefore, we then measured the expression of CyclinDl and CDK4. The results showed the silence of Epacl could obviously decrease the expression of CyclinDl and CDK4, which indicated that Epacl knockdown may induce G1 arrest by inactivation of the AKT/CyclinDl/CDK4 pathways.6. Epacl knockdown significantly inhibited the proliferation of ovarian cancer cells in tumor-bearing nude mice and decreased the expression of p-AKT, CyclinDl and CDK4.(1) shEpac1 could effectively downregulate the expression of Epacl in SKOV3 cells.(2) We found that the growth of tumor in shEpacl treatment group was obviously inhibited compared with shControl and NS groups.(3) The tumor volume and weight in shEpacl treatment group were also significantly lower than those in shControl and NS groups.(4) Epacl protein expression in shEpacl treatment group was lower than that in shControl and NS groups detected by IHC and the downregulation of Epacl could also markedly decrease the expression of p-AKT, CyclinD1 and CDK4, which was in consistent with the results in vitro.Conclusion:1. Epacl expression was significantly increased in ovarian cancer cells SKOV3 and OVCAR3.2. The downregulation of Epacl expression could inhibit the proliferation, colony forming ability and arrest cell cycle of ovarian cancer cells, but has no effect on the apoptosis in vitro and in vivo.3. Decreased Epacl expression could inhibit the expression of p-AKT, CyclinDl and CDK4, suggesting that Epacl may play important roles by affecting cell proliferation-related signaling pathway in vitro and in vivo.Originality:As the newly found cAMP-activated downstream signaling molecule, Epac1 playes important roles in the proliferation of some cancers. But, there is no report about the effect of Epacl on ovarian cancer. Our study is the first time to report that Epacl expression was significantly increased in ovarian cancer cells SKOV3 and OVCAR3. Meanwhile, it is also the first to demonstrate that Epacl knockdown can inhibit the proliferation of ovarian cancer cells by AKT/CyclinDl/CDK4 pathway in vitro and in vivo. The results suggest that Epacl can regulate the expression of p-AKT, CyclinDl and CDK4, induce the changes of cell cycle, and then promote proliferation of ovarian cancer cells. Our results may lay the foundation of further exploring the effect and mechanism of Epacl in ovarian cancer, and provide a new target for diagnosis and treatment of ovarian cancer in the future.