CA125 Promotes Ovarian Cancer Cells Migration And Tumor Metastasis By Binding With MSLN To Reduce DKK1 Expression And Activate SGK3/FOXO3 Pathway

Background: Ovarian cancer(OC)is a highly aggressive malignant tumor,ranks the first for mortality in gynecological malignant tumors,and the fifth cause of cancerrelated death in women.Due to the lack of typical symptoms and effective diagnosis methods at the early stage of OC,more than 70% patients were not diagnosed before the cancer developed to stage III(51%)or stage IV(29%).The 5-year relative survival rate was 92% when cancer localized,however,the rate decreased to 75% once the tumor spread.Distant metastasis occurs at late stage with the 5-year survival rate of 29%.Therefore,the status of cell migration and tumor metastasis affects the disease prognosis in the development of ovarian cancer.Mucin-16(MUC16)is expressed on the surface of ovarian cancer cells.The extracellular domain of MUC16 is shed to the serum and known as cancer antigen 125(CA125).It is observed that the serum CA125 levels elevated in 83% of patients diagnosed with ovarian cancer.Thus,CA125 has been widely used in the diagnosis and prognosis of ovarian cancer.In recent years,it is proved that CA125 promotes the migration and invasion in ovarian cancer cells.However,DKK1 reverses the ability of migration promoting by CA125 in ovarian cancer cells.Based on this fact,this study was designed to go step to the mechanism of CA125 on the metastasis of ovarian cancer.The effect of CA125 on the metastasis in ovarian cancer was studied in xenograft mice model,and biotherapeutics inhibiting the migration and metastasis caused by CA125 was conducted.Objectives: 1.To analysis the expression levels of DKK1 in ovarian cancer and normal tissues by the online databases.2.To explore the relationship between CA125 promoted OC cells migration and down-regulated DKK1 expression.3.To screen the differentially expressed genes(DEGs)in ovarian cancer cells with CA125 stimulation or not,and to perform the pathway analysis of DEGs.4.To confirm the results of pathway analysis.To explore the relationship between the activation of pathway and the depression of DKK1 with CA125 stimulation.5.To block the binding sites between CA125 and the cell membrane.6.To explore the effect of CA125 on metastasis to ovarian cancer tumors in xenograft mice models,and to treat the mice by the biological agent.Methods: 1.The expression levels of DKK1 in ovarian cancer tissues were compared with that in normal tissues using the Oncomine and CSIOVDB databases.2.The expression levels of DKK1 in ovarian cancer cells stimulated by CA125 was detected by quantitative real-time PCR(q-PCR)method and enzyme-linked immunosorbent assay(ELISA).Transwells were used to detect the ovarian cancer cell migration ability with the conversion of DKK1 expression and the stimulation of CA125.3.Nextgeneration sequencing technology was used to detect the DEGs of ovarian cancer cells stimulated by CA125 and the pathway analyses of DEGs were conducted.4.The results of pathway analyses were verified by Western Blot.The activation of the pathway with DKK1 transfection and CA125 stimulation in ovarian cancer cells were detected.5.ELISA were used to detect the expression levels of DKK1 with CA125 and Antimesothelin antibody(Anti-MSLN)stimulation.Annexin-V-FITC/PI dual staining experiments were used to detect the changes of the apoptosis in ovarian cancer cells with CA125 and Anti-MSLN stimulation or not.6.Xenograft mice models were used to detect the effect of CA125 on ovarian cancer metastasis and the therapeutic effect of Anti-MSLN on ovarian cancer tumor formation and metastasis.Results: 1.In the sub-data sets Bonome and Yoshihara and the CSIOVDB database,DKK1 expression in ovarian cancer tissues was significantly lower than that in normal tissues,and were correlated with FIGO stage and grade.The overall survival and disease-free survival of patients with lower DKK1 expression were poorer.2.The expression levels of DKK1 in ovarian cancer cells were decreased with CA125 stimulation,and si DKK1 increased the promotional effect on migration caused by CA125 in ovarian cancer cells,and overexpress DKK1 reversed this effect.3.Forty one DEGs(Fold Change>1,FDR≤0.001)were identified in CA125 stimulated group compared with the control group.Pathway analyses of the DEGs revealed that the SGK3/FOXO3 pathway was activated significantly.4.The expression levels of FOXO3 in nuleus were decreased while SGK3 were unchanged under CA125 stimulation.The activation of SGK3/FOXO3 pathway was enhanced by downregulating of DKK1 expression and CA125 stimulation,and the pathway was suppressed by over-expressed DKK1.5.Anti-MSLN blocked the changes of DKK1 expression induced by CA125 stimulation.Moreover,the necrosis and apoptosis cells were increased by adding Anti-MSLN compared with CA125 stimulation group.6.CA125 promoted ovarian cancer metastasis in xenograft mice model.Anti-MSLN decreased the tumor formation and metastasis.Conclusions: 1.The expression levels of DKK1 were decreased in ovarian cancer tissues.CA125 reduced the DKK1 expression of ovarian cancer cells,which was related to the effect of CA125 promotes cell migration.2.CA125 stimulation caused SGK3/FOXO3 pathway activated in ovarian cancer cells.The expression levels of DKK1 were related to the activation of SGK3/FOXO3 pathway.The decreasion of DKK1 further activated the pathway.3.Anti-MSLN could inhibit the effect of CA125 on reducing DKK1 expression in cells,and increased the apoptosis rate of ovarian cancer cells under CA125 stimulation.4.CA125 accelerated the metastasis of ovarian cancer in the xenograft mice models.Anti-MSLN reduced the tumor formation and metastasis with the CA125 stimulation.The antibody targeting mesothelin is a potential biological agent for the treatment of ovarian cancer in the future.