Functional Study Of Cav-1 In The Migration And Invasion Of Breast Cancer Cells Induced By Ectopic ATP5B

Breast cancer is the most common malignancy in women,and has become the leading cause of cancer-related death in women.Although distant metastasis in patients diagnosed with early breast cancer accounts for only 5%-10%,the risk of metastasis remains quite high after primary surgical resection and adjuvant radiotherapy.The metastasis of breast cancer brings clinical and economic burden.Therefore,it's urgent to identify the metastasis-associated proteins of breast cancer.Our preliminary work showed that prostate cancer metastasis-associated short peptide B04 can specifically bind to ATP5 B expressed on the plasma membrane of PC-3M,and inhibit the migration and invasion of PC-3M.The migration and invasion promotional functions of ectopic ATP5 B in prostate cancer were further confirmed.As the ? subunits of F1 Fo ATPase,ATP5 B was once thought to be strictly localized on the inner membrane of mitochondrial.Recently studies shows that ATP5 B can ectopically expressed on the plasma membrane of many types of normal and tumor cells.Ectopic ATP5 B is usually located in caveolae.Cav-1 is an essential component of caveolae and is implicated in numerous signaling pathways.Ectopic ATP5 B can activate endothelial cells by binding to Cav-1.This study intends to investigate the role of plasma membrane ATP5 B in breast cancer migration and invasion and the function of Cav-1.Methods:1.Effect of plasma membrane ATP5 B on the migration and invasion of breast cancer cellsMDA-MB-231 cells were exposed to cholesterol for different times and the membrane proteins were extracted.The ectopic expression of ATP5 B on the cell membrane was detected by Western blot.Transwell chamber migration/invasion assaywas used to confirm the involvement of plasma membrane ATP5 B in the migration and invasion of breast cancer induced by cholesterol.2.The role of Cav-1 in the migration and invasion of breast cancer cells induced by ectopic ATP5BThe positional relationship of plasma membrane ATP5 B and Cav-1 in MDA-MB-231 was determined by immunofluorescence double staining technique.Then the membrane proteins were extracted.Co-immunoprecipitation assay was used to further confirm the interaction between the two proteins.The plasmid encoding sh RNA targeting Cav-1 gene as well as expressing GFP was transfected into the breast cancer cell line MDA-MB-231.Puromycin was used to screen stable Cav-1 knocking down cells.The cells were further identified by RT-PCR and Western blot.Western blot was performed to detect the effect of Cav-1 knocking down on the expression of cell membrane Cav-1.The interaction of ectopic ATP5 B and Cav-1 while knocking down Cav-1 was determined by Co-immunoprecipitation.Transwell chamber migration and invasion assay were used to compare the effect of ATP5B-Cav-1 interaction on the migration and invasion ability of breast cancer cells.Results:1.Effect of plasma membrane ATP5 B on migration and invasion of breast cancer cellsThe cytoplasmic membrane proteins were extracted after exposed to cholesterol for different times and the ectopic expression of ATP5 B was determined by Western blot.Relative to control,the level of ectopic ATP5 B was significantly up-regulated following treatment with cholesterol for 12 h,but not the group exposed to cholesterol for 24 h.Transwell chamber migration and invasion assays showed that the number of migration and invasion cells in the short peptide B04 treated group was significantly lower than that in control.The number of migration and invasion cells of the group up-regulated plasma membrane ATP5 B increased significantly induced by cholesterol compared with control.However,pretreated with short peptide B04 before exposed to cholesterol,the number of migration and invasion cells had no difference with the group pretreated with B04.The above results suggest that ectopic ATP5 B expressed on plasma membrane can promote the migration and invasion of breast cancer cells.Cholesterol loading increases the ectopic expression of ATP5 B on breast cancer cell membrane,but the effect is not persistent.Cholesterol promotes the migration and invasion of breast cancer cells via increasing the expression of ectopic ATP5 B.2.The role of Cav-1 in the migration and invasion of breast cancer cells induced by ectopic ATP5BImmunofluorescence double staining ascertained the colocalization of ATP5 B and Cav-1 on the surface of MDA-MB-231 cell membrane,and Immunoprecipitation assay further confirms the binding of ATP5 B and Cav-1?.The cell lines selected by puromycin were identified by RT-PCR and Western blot.Both of the Cav-1 m RNA and protein level of the Cav-1 sh RNA1(plasmid 15806-1 transfection)group are significantly down-regulated compared with the scramble.However,relative to scramble,the Cav-1 sh RNA2(plasmid 18201-1 transfection)group had no significant changes in Cav-1 protein and m RNA.We successfully obtained stable knock-down Cav-1 cells by plasmid 15806-1 which uniformly represented by Cav-1 sh RNA.The plasma membrane proteins were detected by Western blot.The expression of cell membrane Cav-1 was down-regulated in the Cav-1 sh RNA group compared with the scramble group.The membrane protein of the Cav-1 sh RNA group was immunoprecipitated with antibody against ATP5 B and no isoforms of Cav-1 was detected.The results of Transwell chamber migration and invasion assay were consistent.The number of migration and invasion cells in the Cav-1 sh RNA group was significant lower than the scramble group.And the migration and invasion of MDA-MB-231 cells showed no difference whether exposed to cholesterol or not.The above results suggest that ATP5 B binds to Cav-1? on the surface of breast cancer cells.Ectopic ATP5 B promotes the migration and invasion of breast cancer cells by binding to Cav-1.Conclusion:1.Ectopic ATP5 B on plasma membrane promotes the migration and invasion of breast cancer cells MDA-MB-231.2.Cholesterol loading promotes the migration and invasion of breast cancer cells MDA-MB-231 via increasing ectopic ATP5 B.3.ATP5 B binds to Cav-1? on the plasma membrane of MDA-MB-231.4.Ectopic ATP5 B on plasma membrane promotes the migration and invasion of breast cancer cells MDA-MB-231 through binding to Cav-1.