| Objective:Breast cancer is one of the malignant tumors that seriously threaten women’s health,and its incidence has been increasing in recent years.Multidrug resistance(MDR)and the production of cancer stem cells(CSCs)are two important factors affecting the prognosis of breast cancer.Breast cancer resistance protein(BCRP)is a newly discovered membrane transporter of the ABC(ATP binding cassette)family,which has been confirmed as one of CSCs markers.Epithelial mesenchymal transition(EMT)is the main cause of tumor metastasis,and it has been confirmed in recent years that it is closely related to CSCs and BCRP-mediated MDR.Epithelial cell adhesion molecule(EpCAM)is a newly established tumor-associated antigen.Recent studies indicated that EpCAM induced the EMT process.This project aims to investigate whether EpCAM promotes BCRP-mediated MDR and stemness of breast cancer by inducing EMT,and then affects the prognosis of breast cancer.Methods:1.MCF-7 cells were treated with mitoxantrone(MX,0.04,0.08,0.16 nM)for 1 week,and the expression of BCRP,EpCAM,CSCs markers and EMT-related proteins were measured using Western blot.2.Western blot was used to determine the expression of EpCAM,BCRP,CSCs markers and EMT-related proteins in MCF-7 and MCF-7/MX cells.3.MCF-7 cells were treated with TGF-β1(10 ng/ml)and/or TNF-α(10 ng/ml)for 6 days.The expressions of EMT-related proteins,BCRP,EpCAM and stemness markers were measured using Western blot.4.The expression of EpCAM in MCF-7/MX cells was interfered using siRNA,and the interference efficiency of EpCAM expression was detected by Real-time PCR and Western blot.MCF-7/MX,negative controls MCF-7/MX/siNC,and two cell lines with interference efficiency greater than 50%,MCF-7/MX/siEpCAM-RNA1 and-RNA2 were selected for subsequent experiments.5.Western blot was used to detect the expression of BCRP,PCNA,stemness markers and EMT-related proteins in all above cell lines.6.The sensitivity of all above cells to MX was measured by CCK8.7.The migration and invasion ability of all above cells were measured using Transwell.8.MCF-7/MX cells were treated with ICG-001(5 μM and 10 μM),a specific Wnt pathway inhibitor,for 24 h,and the expression of Wnt pathway-related proteins was measured using Western blot.Results:1.The expression of EpCAM was related to BCRP-mediated MDR and stemness in breast cancer cells.After MCF-7 cells were treated with MX(0.04,0.08,0.16 nM)for 1 week,the expression of BCRP,CSCs markers,and EpCAM protein increased.Compared with MCF-7 cells,MCF-7/MX cells showed increased BCRP-mediated MDR and stemness,and EpCAM expression levels increased(P < 0.001).2.EpCAM expression was inhibited using siRNA.The results of Real-time PCR and Western blotting reveald a significant downregulation of EpCAM expression in MCF-7/MX/siEpCAM-RNA1 and-RNA2 cells(P < 0.05),while approximately equal amounts of EpCAM protein were observed in MCF-7/MX/siNC and MCF-7/MX cells(P > 0.05).MCF-7/MX,MCF-7/MX/siNC,MCF-7/MX/siEpCAM-RNA1 and-RNA2 were selected for subsequent experiments.3.Knockdown of EpCAM expression reversed BCRP-mediated MDR in MCF-7/MX cells.A significant decreased BCRP expression and resistance to MX was observed in MCF-7/MX/siEpCAM-RNA1 and-RNA2 cells(P < 0.05,P < 0.001),while approximately equal amounts of BCRP protein and sensitivity to MX were observed in MCF-7/MX and MCF-7/MX/siNC cells(P > 0.05)..4.Knockdown of EpCAM expression inhibited the stemness of MCF-7/MX cells.A significant decreased expression of PCNA and CSCs markers was observed in MCF-7/MX/siEpCAM-RNA1 and-RNA2 cells(P < 0.05,P < 0.001),while approximately equal amounts of the above proteins were observed in MCF-7/MX and MCF-7/MX/siNC cells(P > 0.05).5.Up-regulation of EpCAM promoted EMT that enhances BCRP-mediated MDR and stemness in breast cancer.When MCF-7 cells were treated with TGF-β1(10 ng/ml)and/or TNF-α(10 ng/ml)for 6 days to induce the EMT process,a significant increased EpCAM expression was observed in MCF-7 cells(P < 0.05,P < 0.01).Treatment with TGF-β1(10 ng/ml)and/or TNF-α(10 ng/ml)also resulted in increased expression of BCRP and CSCs markers in MCF-7 cells(P < 0.001,P < 0.0001).6.Knockdown of EpCAM expression reduced the migration and invasion capabilities of MCF-7/MX cells,and reversed the EMT process.Significant reduced migration and invasion abilities(P < 0.01,P < 0.001),increased E-cadherin levels(P < 0.01,P < 0.001),and decreased N-cadherin and vimentin expression(P < 0.01,P < 0.001)were observed in MCF-7/MX/siEpCAM-RNA1 and-RNA2 cells(P < 0.05,P < 0.001),while approximately equal migration and invasion abilities and amounts of the above proteins were observed in MCF-7/MX and MCF-7/MX/siNC cells(P > 0.05).7.Knockdown of EpCAM expression reversed EMT and stemness.EpCAM expression was inhibited using siRNA in MCF-7/siCK18-3C cells with EMT phenotypeconstructed by our team.Significant increased E-cadherin(P < 0.001)and decreased N-cadhein epression(P < 0.01)was observed MCF-7/siCK18-3C/siEpCAM cells(P < 0.05,P < 0.001),while approximately equal amounts of the above proteins were observed in MCF-7/siCK18-3C and MCF-7/siCK18-3C/siNC cells(P > 0.05).8.Up-regulation of EpCAM activated the Wnt/β-catenin pathway,and inhibition of the Wnt pathway reduced the expression of EpCAM in MCF-7/MX cells.Compared with MCF-7,the expression of EpCAM(P < 0.001)and β-catenin(P < 0.001)increased significantly in MCF-7/MX cells.ICG-001,a specific inhibitior of Wnt signaling pathway,significantly inhibited the expression of β-catenin in MCF-7/MX cells(P < 0.01),and EpCAM and CSCs marker proteins expressions decreased at the same time(P < 0.01).Conclusions:1.EpCAM promotes BCRP-mediated MDR and stemness in breast cancer cells.2.EpCAM promotes MDR and stemness of breast cancer cells by inducing EMT.3.EpCAM enhances BCRP-mediated MDR and stemness of breast cancer cells partly via the Wnt/β-catenin signaling pathway. |
