Formononetin Influences Proliferation,migration,and Invasion Of ER-negative Breast Cancer Cells Through ER-α36-dependent EGFR Signaling Pathway

Objective: To investigate the role of ER-α36/EGFR signal pathway in formononetin-induced inhibition of proliferation,migration and invasion in ER-negative breast cancer cells.Methods: Two breast cancer cell lines MDA-MB-231 and SK-BR-3 were involved in this study and treated with different concentrations of formononetin,followed by detection of cell viability by MTT assay.Based on the MTT assay results,the suitable concentrations used here were 0,16,32 and 64 μM.Then the MTT assay,transwell migration assay and wound healing assay were respectively performed to evaluate the proliferative,metastatic and invasive abilities of breast cancer cells with treatment of formononetin.To identify the underlying mechanisms,we overexpressed and knockdowned ER-α36 in breast cancer cells,which was confirmed by Western Blot analysis.Transfected cells were treated with or without 64 μM formononetin,based on the different growth rates on each cell lines,MDA-MB-231 cells were treated with fomononetin for 48 h while SK-BR-3 cells were treated with 72 h,and subsequently the cell proliferation,migration and invasion assays were performed.Meanwhile,the expression levels of ER-α36 and EGFR were detected by Western Blot assay and RT-PCR,while the phosphorylation of EGFR downstream targets(MEK1/2 and ERK1/2)detected by Western Blot assay.Here,empty vector-transfected cells and untransfected cells treated with formononetin served as control.Results: When breast cancer cells MDA-MB-231 and SK-BR-3 was treated with increased concentrations of formononetin(0～64 μM),it was found that the ability of proliferation,migration and invasion of the two cell lines both gradually decreased(p<0.05).Moreover,formononetin significantly reduced expression levels of ER-α36 in MDA-MB-231 and SK-BR-3 cells(p<0.05).As expected,up-regulation of ER-α36 promoted cell growth and metastasis,which obviously inhibited the anti-cancer effect of formononetin in breast cancer cells(p<0.05).The opposite results were obtained by knockdown of ER-α36 in the two cell lines.In addition,the findings showed that ER-α36 could trigger the expression of EGFR and phosphorylation of MEK1/2 and ERK1/2 in ER-negative breast cancer cells.Therefore,ER-α36 overexpression led to increased expression of EGFR,p-MEK1/2 and p-ERK1/2,whereas ER-α36 knockdown led to decreased levels of EGFR,p-MEK1/2 and p-ERK1/2.Otherwise,formononetin was demonstrated to inhibit expression of ER-α36 and EGFR,as well as the phosphorylation of MEK1/2 and ERK1/2.However,these inhibitory effects were blocked by ER-α36 up-regulation and enhanced by ER-α36down-regulation.Conclusions: In ER-negative breast cancer cells,ER-α36 may play an important role in regulation of cell growth and metastasis via activation of EGFR pathway.By inactivating this ER-α36-dependent EGFR signaling,formononetin could inhibit the proliferation,migration and invasion of ER-negative breast cancer cells.