Prohibitin Promotes Androgen Receptor Activation In ER-positive Breast Cancer

Background:Breast cancer is the most common cancer in women in China.ER-positive breast cancer endocrine therapy resistance is a serious clinical topic,and it is particularly important to explore meaningful therapeutic and prognostic targets.Prohibitin(PHB)is an evolutionary conserved protein with multiple functions in both normal and cancer cells,the role of PHB in cancer cells is controversial.Androgen receptor(AR)is highly expressed in breast cancer and plays a different role in the ER-positive and ER-negative breast cancer.It was reported that PHB could repress AR activity and androgen-stimulated growth of prostate cancer cells.However,little is known regarding the role of PHB and whether PHB could regulate AR expression in the ER-positive breast cancer.So far,no relevant literature has been reported.Objectives:To determine the expression and clinical outcomes of PHB in breast cancer,and to investigate the role of PHB in the regulation of AR expression in ER-positive breast cancer.Methods:We determined the expression status of PHB in 121 breast cancer tissues and corresponding noncancerous tissues(NCTs)by immunohistochemical(IHC)staining.Relationship between the expression of PHB and clinicopathological features of 121 breast cancer patients was determined.The correlation between PHB and AR expression was also analyzed.To investigate the role of PHB and AR in the ER-positive breast cancer clinical outcomes,we performed meta-analyses using Kaplan-Meier plotter online breast cancer survival analysis.Followed by in vitro experiments,the plasmids encoding PHB were transfected into ER-positive breast cancer cell lines MCF-7 and T47D.The expression of PHB mRNA and PHB protein were detected to verify whether the PHB plasmids were successfully transfected.Cell proliferation was measured by MTT assay 24,48,or 72 h following transfection of plasmids encoding PHB into MCF-7 and T47D cell lines.Flow cytometry was used to analyze changes of cell cycle and apoptosis of ER-positive breast cancer cells after transfection.Quantitative real-time PCR and Western blotting were used to detect the mRNA and protein level of AR in ER-positive breast cancer cells after transfected with PHB plasmids,respectively.Finally,immunofluorescence(IF)staining was used to verify protein expression and examine the subcellular localization of PHB and AR.Results:We found the expression of PHB and AR were low in the human breast cancer tissue's.PHB expression was associated with lymph nodes metastasis(P=0.034),ER expression(P<0.001),PR expression(P<0.001)and HER-2 expression(P=).009),respectively.AR expression was associated with ages(P=0.014),ER expression(P=0.016)and E-cadherin expression(P=0.043),respectively.A significant positive correlation between PHB and AR expression was identified in the 121 breast cancer tissues(r = 0.521,P<0.001).Low expression of PHB and AR positively correlated with reduced overall survival time and distant metastasis-free survival time in the ER-positive breast cancer patients.After PHB plasmids were transiently transfected into MCF-7 and T47D cell lines,PHB was detected to be overexpressed at mRNA and protein levels,indicated successful transfection.The proliferation of MCF-7 and T47D cell lines transfected with PHB plasmids were slower than that of the control cell lines,cell cycle were arrested in S/G2 phase and cell apoptosis was significantly increased.PHB plasmids transfection led to increased AR expression in mRNA and protein levels compared with the controls in the MCF-7 and T47D cells(P<0.05).Immunofluorescence was performed to determine the cellular localization of AR in the cell lines stimulated with PHB plasmids for 48h,shown that AR staining was predominantly nuclear and the expression of AR was significantly increased in the nuclear levels in treated ER-positive breast cancer cells(P<0.05).Conclusion:The expression of PHB was significantly low in breast cancer samples,and low PHB expression positively correlated with poor prognosis of breast cancer.There was a significant positive correlation between PHB and AR expression in breast cancer patient tissues.Overexpression of PHB could inhibit breast cancer cell proliferation,change cell cycle distribution and promote cell apoptosis in the ER-positive breast cancer cells.PHB could significantly increase AR expression in the ER-positive breast cancer cells.Our results indicated that PHB could promote AR activation in ER-positive breast cancer,making PHB and AR potential molecular targets for ER-positive breast cancer therapy.To the best of our knowledge,this is the first report revealing that the association of PHB and AR expression in the breast cancer.However,additional larger studies and more detailed investigations of the regulatory mechanisms underlying the influence of PHB on the expression of AR in the ER-positive breast cancer are needed to validate our findings.