The Regulatory Mechanism Of Slug-c-Met And The Therapeutic Implications In Metastasized Ovarian Cancer

Objectives:Ovarian cancer belongs to the most deadly form of gynecologic malignant neoplasms.One of the reasons which made it the most lethiferous is the highly percentages of metastasis.On the other hand,the limited clinical used anti-metastatic drugs and strategies greatly hinder the improvement of treatment of ovarian cancer patients.Focusing on the anti-metastasis studies is a crucial important project in ovarian cancer therapeutics.Therefore,the exploration of the mechanisms by which the ovarian cancer cells obtain the high motility and metastatic potential will aid in identification of molecular targets and strategies to improve the treatment of metastatic ovarian cancer.Mounting evidences indicate that the epithelial-mesenchymal transition,an early stage through which cancer cells could gain enhanced mobility,migration abilities and lower adhesive capacity,is the most promising program that could be interfered to prevent the metastasis from the initiative period,thus open opportunities to achieve better therapeutic effects.In the present study,we established a cell line SKOV3/T4 through enriching the cells from parental SKOV3 cells in the Transwell lower chamber,then we detected the motile ability and EMT related protein markers expression compared with the parental SKOV3 cells to explore the signaling molecules and pathways involved in metastasis of ovarian cancer,so as to provide new strategies in metastatic ovarian carcinoma therapy.Methods:(1)Wound-healing assay and Transwell assay were conducted to detect the motility and migration ability of SKOV3/T4 cells and parental SKOV3 cells,chorioallantoic membrane model was used to compare the diffusion activity between SKOV3/T4 cells and SKOV3 cells;(2)The protein levels of EMT-related marker and c-Met,EGFR,VEGFR signaling pathways were determined by western blot or immunofluorescence;(3)The mRNA expression levels of EMT related proteins and c-Met signaling pathway components were detected by real-time PCR;(4)Immunohistochemical staining was performed to detect the Slug and p-Met expression level with correlation analysis in clinical samples;(5)Enzyme-linked immuno-sorbent assay(Elisa)was used to exam the HGF secretion level in culture supernatant of SKOV3/T4 cells and SKOV3 cells;(6)Slug,IntegrinaV or Fibronectin was silenced by small interfering RNA and the alteration of p-Met protein expression level was determined by western blot;(7)Over expressing Slug,the p-Met protein expression level,the EMT related protein marker expression level and the cell mobility changes were examined by western blot and Transwell assay respectively;(8)SRB assay was performed to exam the drug susceptibility of SKOV3/T4 cells and parental SKOV3 cells,with the drug susceptibility changes after disturbing the Slug and c-Met signal pathway;(9)The mice xenograft model was constructed and used to evaluate the drug susceptibility to ADR and XL184 in vivo of SKOV3/T4 cells and SKOV3 cells.Results:1.Metastasized ovarian cancer cell line SKOV3/T4 enrichment and mobility detection.We collected the cells in the lower chamber in the Transwell assay and reseeded them into the upper compartment,then the rest can be done in the same manner for four circles to get SKOV3/T4 cells.Transwell and wound-healing assay were performed to uncover that SKOV3/T4 cells gained enhanced motility and migration ability compared with parental SKOV3 cells,and chorioallantoic membrane assay showed that SKOV3/T4 cells were more efficient in spread out of the blood vessels.2.EMT was triggered in SKOV3/T4 cells and Slug was involved in this progress.Subsequently,western blot,immunofluorescence were used to detect the important protein markers expression levels involved in EMT progress and the results implicated the EMT-like properties of SKOV3/T4 cells.RT-PCR results uncovered that transcription factor Slug was up-regulated in SKOV3/T4 cells compared with SKOV3 cells while other transcription factors showed no significant differences between these two cell lines.Meanwhile,exogenous over-expression of Slug could induce EMT in SKOV3 cells accompanied with the enhanced migration ability.3.c-Met signaling pathway was activated in SKOV3/T4 cells and the phosphorylation level of c-Met was positively correlated with Slug expression level.Given the close relationship between tyrosine kinase pathways and EMT,we detected several tyrosine kinase pathways,and found only c-Met signaling pathway was activated in SKOV3/T4 cells,and when we using siRNA to specifically interfering with or over-expressing Slug,the phosphorylation level of c-Met were down-regulated or up-regulated,respectively.In immunohistochemical assay,the clinical samples analyses showed that the phosphorylation level of c-Met was correlated positively with the expression of Slug.4.Transcription factor Slug could up-regulated Fibronectin,through which binding to IntegrinaV and activated c-Met signaling pathway.Western blot and RT-PCR results showed that the protein and mRNA expression levels of HGF and c-Met had no significant differences between SKOV3/T4 cells and SKOV3 cells.And the secretion level of HGF showed no significant differences.Thus c-Met signaling pathway was activated in SKOV3/T4 cells in a HGF-independent manner.While the phosphorylation level of c-Met of SKOV3/T4 cells decreased upon the transfection with small interfering RNA targeting IntegrinaV or Fibronectin,respectively.Similar observation was also achieved in those SKOV3/T4 cells with exogenous Slug transfection followed by IntegrinaV siRNA.Together with the immunofluorescence assay demonstrating that Fibronectin was up-regulated by over-expressing Slug,these results implicated that Slug could regulate the phosphorylation level of c-Met through modulating Fibronectin expression and the subsequent binding to IntegrinaV5.c-Met inhibitor XL 184 could counteract against the enhanced mobility induced by over-expressing Slug in SKOV3 cells.Western blot and wound-healing assay implied that c-Met inhibitor XL 184 could abrogate the mobility enhanced by over-expressed Slug in SKOV3 cells with the inhibitory effects on the phosphorylation of c-Met.6.c-Met inhibitor exerts better anti-cancer efficacy in SKOV3/T4 and Slug over-expressed SKOV3 cells which were resistant to most chemotherapeutics agents.SKOV3/T4 cells and Slug over-expressed SKOV3 cells exhibited a decrease in drug sensitivity to many chemotherapeutics agents than the parental SKOV3 cells,however these cells were exclusively susceptible to c-Met inhibitor.And Slug expression levels and IC50 values of c-Met inhibitor XL 184 in eight different cancer cell lines were negatively correlated with the correlation value of 0.66.In addition,disturbing the Slug and c-Met signaling pathway through silencing Slug or Integrin?V could up-regulate drug sensitivity of Slug over-expressed cells.Conclusion:In the present study,the established SKOV3/T4 cells exhibited higher motility and EMT-like characteristics.With higher expression level of mRNA and protein in SKOV3/T4 cells,the key EMT transcription factor Slug increase the expression of Fibronectin to activate c-Met signaling pathway in a HGF-independent manner by binding to IntegrinaV and enhance the migration ability of ovarian cancer cells;Meanwhile,the Slug regulated c-Met pathway also involved in the resistance to chemotherapeutic agents of SKOV3/T4 cells.The interferences of the signal transduction with siRNA or inhibitor could suppress the migration and drug resistance.Hence,blocking the key molecules in this pathway could be applied in overcoming the metastasis and metastasis-related drug resistance in metastatic ovarian cancer therapeutics in clinical as new strategies.