Effect And Mechanism Of AR-PDEF Pathway On ER–negative Breast Cancer

ObjectiveAndrogen receptor(AR)is expressed in 60%~70%oestrogen receptor(ER)-negative breast cancer(BC)cases and promotes the growth of this cancer subtype.Expression of prostate-derived Ets factor(PDEF),a transcription factor,is highly restricted to epithelial cells in hormone-regulated tissues.It has been demonstrated that PDEF expression is associated with AR in prostate cancer.However,the relationship between AR and PDEF and the function of PDEF in ER–negative BC proliferation are unclear.In this research,we aimed to investigate the relationship between PDEF and AR in ER–negative BC.Methods1.We immunohistochemically evaluated the correlation between PDEF and AR expression in 246 cases of ER–negative invasive BC.Statistical analysis was performed using SPSS 20.0 statistical software.Associations between AR and PDEF expression and the status of some clinicopathological factors were evaluated by Chi-squared tests.The correlation between PDEF and AR expression was determined by Chi-squared tests and Spearman correlation method,and the survival analysis was determined by Kaplan-Meier analyses.2.The qRT-PCR and western blot samples were obtained from Breast Cancer Pathology and Research Laboratory Department(N=37),which included randomly selected cases(n=15+8),AR+cases(n=7)and AR–cases(n=7).Furthermore,their normal breast tissue was used as the control group.3.ER–negative and AR-positive BC cell lines MDA-MB-453 and SKBR-3 were treated with DHT to promote AR expression or were infected with an AR-shRNA-expressing lentiviral vector to inhibit AR expression and analysed the PDEF expression after upregulated or downregulated AR expression.And performed immunofluorescence staining to reassess AR and PDEF protein levels.A time-course analysis of MDA-MB-453 and SKBR-3 cells AR and PDEF expression after treatment with increasing DHT doses.Co-immunoprecipitation and chromatin immunoprecipitation assays were performed to validate the regulation of AR–PDEF axis.4.To determine whether PDEF expression affected cellular growth,SKBR-3 cells were infected with a PDEF-expressing lentiviral vector to promote PDEF expression and MDA-MB-453 cells were infected with a PDEF-shRNA-expressing lentiviral vector to inhibit PDEF expression.CCK-8 assay,cell cycle analy and cell migration and invasion assays to determine whether PDEF expression affected cellular growth,migration and invasion.5.To determine the molecular mechanism underlying the oncogenic effect of PDEF on ER–negative BC cell proliferation.PDEF overexpression or downregulation in SKBR-3 or MDA-MB-453 cells,respectively,and AR downstream intracellular signalling components were determined by western blotting.Co-immunoprecipitation and chromatin immunoprecipitation assays were performed to validate the regulation of PDEF–MAD1-MYC axis.6.Because PDEF represses MAD1 expression,upregulation of MAD1 expression may suppress PDEF-mediated growth of ER–negative BC cells.To verify this hypothesis,we analysed PDEF-upregulated,simultaneous PDEF-and MAD1-upregulated and control SKBR-3 cells.Western blot assay,CCK-8 assay,cell cycle analy and cell migration and invasion assays to determine whether upregulation of MAD1 expression may suppress PDEF-mediated growth of ER–negative BC cells.7.We investigated whether simultaneous inhibition of AR and PDEF expression further suppressed tumour proliferation compared with the inhibition of AR alone.For this,we analysed AR-downregulated(AR-deprived),simultaneous AR-and PDEF-downregulated(AR-/PDEF-deprived)and control MDA-MB-453 cell clones.Western blot assay,CCK-8 assay,cell cycle analy and cell migration and invasion assays to determine whether inhibit AR-PDEF pathway may suppressed tumor growth.Results1.Among the 246 cases,150(150/246,61%)cases of the tumours showed a nuclear positive expression for PDEF,and 168(168/246,68%)showed positive expression for AR.PDEF expression was associated with tumour grade(P=0.009),pTNM stage(P=0.001),lymphatic metastasis(P<0.001)and HER2(P=0.032).Moreover,there was a positive association between PDEF expression and AR(P<0.001),and correlation analysis showed that high PDEF expression was associated with AR+(r=0.404,P<0.001).The AR~+PDEF~+(126/246,51.2%)samples were associated with tumour grade(P=0.024),pTNM stage(P=0.022)and lymphatic metastasis(P<0.001).In the 246 ER–patient series,we found an association between higher PDEF expression and shorter OS(P=0.006),but PDEF had no association with DFS(P=0.051).Multivariate analysis confirmed that PDEF expression(P=0.028)was a significant independent prognostic variable influencing OS.2.In the mRNA and protein levels,we found higher PDEF expression in ER–breast tumour tissues through qRT-PCR.This was consistent with our IHC results.In addition,as AR expression is the major molecular determinant of breast tumours,PDEF is significantly overexpressed in AR+tumours.All of those studies suggested a co-expression relationship between AR and PDEF.3.ER–negative and AR-positive BC cell lines MDA-MB-453 and SKBR-3 were treated with 1 nM DHT for 48 h to promote AR expression or were infected with an AR-shRNA-expressing lentiviral vector to inhibit AR expression.Upregulated AR expression promoted PDEF mRNA and protein overexpression,whereas downregulated AR expression significantly inhibited PDEF mRNA and protein expression.A time-course analysis of MDA-MB-453 and SKBR-3 cells showed that treatment with increasing DHT doses strongly increased PDEF mRNA expression.We found that AR co-immunoprecipitated with PDEF in both MDA-MB-453 and SKBR-3 cells because of DHT-induced interaction between these proteins.Direct AR ChIP assay by using MDA-MB-453 cells showed DHT-induced recruitment of AR at the second enhancer of PDEF,thus confirming that PDEF was a direct target of AR.4.To determine whether PDEF expression affected cellular growth,SKBR-3 cells were infected with a PDEF-expressing lentiviral vector to promote PDEF expression and MDA-MB-453 cells were infected with a PDEF-shRNA-expressing lentiviral vector to inhibit PDEF expression.Results of the CCK-8 assay cell cycle analy and cell migration and invasion assays showed increased proliferation of PDEF-upregulated SKBR-3 cells and decreased proliferation of PDEF-downregulated MDA-MB-453 cells.5.PDEF overexpression or downregulation in SKBR-3 or MDA-MB-453 cells,respectively,did not alter the key signal transducers acting downstream of AR,indicating the absence of a reciprocal regulatory loop between PDEF and AR.However,we found that MYC expression was significantly upregulated in PDEF-overexpressing SKBR-3 cells and was downregulated in PDEF-downregulated MDA-MB-453 cells,indicating a positive role of PDEF in regulating MYC expression.PDEF co-immunoprecipitated with MYC in PDEF-upregulated SKBR-3cells,which are suggested to show no interaction between PDEF and MYC.Results of a direct ChIP-qPCR assay showed PDEF recruitment at the MAD1 promoter.These data suggest that PDEF upregulates MYC-mediated gene transcription by promoting MAD1 degradation.6.We analysed PDEF-upregulated,simultaneous PDEF-and MAD1-upregulated and control SKBR-3 cells by performing cell function experiments.PDEF overexpression increased cell proliferation,invasion and stem-like properties of tumour cells;however,MAD1 expression suppressed PDEF overexpression-induced cell proliferation,invasion and stem-like properties of tumour cells.To investigate the effect of MAD1 overexpression on PDEF,female nude mice were inoculated with stable PDEF-upregulated,stable simultaneous PDEF-and MAD1-upregulated and control SKBR-3 cell clones.We found that MAD1 overexpression dramatically inhibited PDEF-mediated growth of and reduced PDEF overexpression-induced Ki67and MYC expression in tumours isolated from the BC cell-inoculated nude mice.Moreover,compared with the mice inoculated with PDEF-overexpressing cells,the mice inoculated with MAD1-overexpressing cells did not show pulmonary metastasis.These findings indicate that upregulation of MAD1 expression can inhibit PDEF-induced proliferation of ER-negative BC cells.7.Results of the cell function experiments showed that the cell proliferation,invasion and stem-like properties of tumour cells was lower among AR-/PDEF-deprived MDA-MB-453 cells than among AR-deprived and control MDA-MB-453 cells.To examine whether simultaneous inhibition of AR and PDEF expression was sufficient for inhibiting tumour proliferation and formation,female nude mice were inoculated with stable AR-downregulated,stable simultaneous AR-and PDEF-downregulated and control MDA-MB-453 cell clones.We found that the simultaneous inhibition of AR and PDEF expression dramatically inhibited tumour growth and considerably reduced Ki67 and MYC expression.These results indicate that the simultaneous inhibition of AR and PDEF expression significantly suppresses the proliferation of ER-negative BC cells.ConclusionsOur findings provide evidence that PDEF is strongly correlated with AR expression in ER–breast cancer;it is a downstream target gene of AR and a potential prognostic factor in ER–BC.AR-PDEF pathway play important roles in the mechanism of the malignalt of ER–BC.AR and PDEF can be used as new clinical therapeutic target for treating ER–BC.