Investigation Of The Effects Of Fulvestrant On The EMT Phenotype And Its Signaling Mechanism In Breast Cancer Cells

Objective: The effects of fulvestrant(ICI182780,ICI)on the EMT phenotype and its signaling mechanism in breast cancer cells were studied with the aim of providing experimental basis for exploring an effective therapy for breast cancer.Methods: Human breast cancer cell lines MCF-7(ER+,GPER+)and MDA-MB-468(ER-,GPER+)were employed as a model system.Cells were treated with ICI(with estradiol,E2 as control)and pretreated with GPER inhibitor(G15)where needed.Wound-healing assay was applied to study cell migration ability.Cell invasion was determined by matrigel coated transwell assay.Western blotting assay was performed to observe protein expression.Sh-RNA-CANP2 was transfected into the cells to knockdown the gene CANP2 and the stable cell lines were screened with puromycin.Results:(1)Treatment of model cells with ICI(10μM)or E2(50nM)caused dramatically increased migration in MCF-7 cells(P <0.05 vs DMSO),and enhanced invasion potential(P<0.01 vs DMSO);In MDA-MB-468 cells;the potentials of migration and invasion were also increased(P <0.01 vs DMSO).(2)In MCF-7 cells,treatment with ICI or E2 upregulated the expression of fibronectin(P<0.05,P<0.01 vs control,respectively.),but down-regulated E-cadherin(P<0.01,P<0.05 vs DMSO).Similar results were oberved in MDA-MB-468 cells(P<0.05 vs control).(3)Pretreatment with GPER specific inhibitor G15(10 μM)inhibited ICI-or E2-induced migration in MCF-7 cells(P<0.05,P<0.01 vs ICI or E2)and cell invasion(P<0.01,P<0.05 vs ICI or E2).In MDA-MB-468 cells,migration rate was reduced(P<0.05,P<0.01 vs ICI or E2),and cell invasion rate reduced(P<0.05 vs ICI or E2).(4)G15 could significantly inhibit the upregulation of FN induced by ICI or E2 in model cells(P<0.01 vs ICI or E2).G15 could inhibit the down-regulation of E-cadinduced by ICI or E2 in MDA-MB-468 cells(P<0.05 vs ICI or E2),but had no significant impact on MCF-7 cells(P>0.05 vs ICI or E2)(5)Sh-RNA transfection silencing CANP2 could significantly reduce the migration and invasion in model cells(P<0.05 vs NCSH).(6)Silencing of CANP2 down-regulated FN expression in both model cells(P<0.01,P<0.05 vs NCSH),yet up-regulatiated E-cad level(P<0.01 vs NCSH).(7)knockdown of CANP2 significantly inhibited cell migration and invasion induced by ICI or E2.The migration rate and invasion rate of MCF-7 cells decreased simultaneously(P <0.05 vs NCSH).The migration rate of MDA-MB-468 cells decreased(P<0.05 vs NCSH),and the invasion rate decreased(P<0.01 vs NCSH).(8)Silencing CANP2 with Sh-RNA transfection reversed the changes of EMT phenotype induced by ICI or E2.The up-regulation of FN and down-regulation of E-cad expression in model cells induced by E2 or ICI were significantly inhibited by knockdown of calpain2 gene(P<0.05 vs NCSH).Conclusion: ICI triggers transition of EMT phenotype from wild subtype to a complete or incomplete subtype in breast cancer cells,and these alterations are accompanied by enhanced potential of migration and invasion.The ICI-induced altered EMT phenotype may be mediated via a GPER-CANP2 signaling pathway.