Functional Interconnection Among AR,AREG And EGFR In Invasive Breast Cancer

Objective In androgen-sensitive prostate cancer,the synthesis of amphiregulin(AREG)is induced by androgenic stimulation.However,there are few studies on the co-expression of AREG and androgen receptor(AR)and their role on prognosis in invasive breast cancer.Moreover,the interaction among AR,AREG and epidermal growth factor receptor(EGFR)signalling in ER-negative breast cancer are unclear.In this study,we aimed to investigate the expression of AREG oncogene and AR/AREG and to assess the prognostic values of AREG and AR/AREG expression in invasive breast cancer,especially in ER-negative breast cancer;to analyse the interaction among AR,AREG and EGFR signalling pathways in ER-negative breast cancer;to investigate the expression features of AR in special subtypes of breast cancer.Methods 1.The expression of AREG and AR in 298 invasive breast carcinomas was detected by immunohistochemical(IHC)method.The prognostic role of AREG and AR/AREG expression in invasive breast carcinoma was performed by survival analysis.2.The ER-negative breast cancer cell lines MDA-MB-453,SK-BR-3 and MDA-MB-231 were chosen for this study.The expression of AR,AREG and EGFR in ER-negative breast cancer cells was analysed using Western blotting,q PCR analysis,immunofluorescence and co-immunoprecipitation assays,with dihydrotestosterone(DHT)and/or AR up-or down-regulation.The effect of AREG on ER-negative breast cancer cells was determined using MTT,clonogenic,Transwell and wound-healing assays.Finally,the functional interconnection among AR,AREG and EGFR was verified using a xenograft tumour model in vivo.3.To determine the features of AR in special subtypes of breast cancer,we collected the clinicopathological data of 718 special subtypes of breast cancer from Tianjin Medical University Cancer Institute and Hospital from March 1,2018 to December 31,2018.Results 1.Immunohistochemically,AREG was 46.0%(137/298)with cytoplasmic staining in invasive breast cancer.AREGhigh correlated with lymph node involvement(P=0.002)and molecular phenotypes(P=0.001).AR/AREG co-expression patients accounted for 62.4%(186/298)of cases.Both AREGhigh and AR+/AREGhigh decreased patients’ overall survival(OS,P<0.05)and disease-free survival(DFS,P<0.05).In Cox models,AR+/AREGhigh remained an independent prognostic indicator for OS and DFS(hazard ratio,0.591 and 0.449;95% confidence interval,0.407-0.859 and 0.236-0.853,P<0.05,respectively).In ER-negative tumours,high levels of AREG decreased patients’ survival(P<0.05).AREG in combination with AR also reduced participants’ survival outcomes(P<0.05).2.Western blot and q PCR analysis showed that AR was highly expressed in MDA-MB-453 cells,but slightly lower levels in SK-BR-3 and MDA-MB-231 cells;EGFR was highly expressed in MDA-MB-453 and MDA-MB-231 cells,but lower levels in SK-BR-3 cells.In comparison,AREG was highly expressed in SK-BR-3 cells,but lower levels in MDA-MB-453 and MDA-MB-231 cells.A significant increased of the level of EGFR protein in both MDA-MB-453 and SK-BR-3 cells in the presence of DHT stimulation.3.Immunofluorescence(IF)illustrated that AR was localized in the nucleus of MDA-MB-453 and SK-BR-3 cells.In contrast,EGFR was localized in the membrane of MDA-MB-453 and SK-BR-3 cells.The protein expression intensity of AR and EGFR was enhanced with DHT stimulation.The interaction between AR and EGFR was verified through a co-immunoprecipitation assay showing that AR co-immunoprecipitated with EGFR.4.AREG was more highly expressed in breast tumours than their non-tumour counterparts,as shown by both m RNA and protein levels.DHT stimulation induced an upregulation of AREG expression in SK-BR-3 cells,and the induced overexpression of AR led to an increase in the expression of AREG and EGFR in SK-BR-3 cells.We transfected sh-AR into MDA-MB-453 cells,and the effect of down-regulated AR expression decreased the expression of AREG and EGFR.5.We selected SK-BR-3 cells to down-regulate AREG and MDA-MB-231 cells to up-regulate AREG to explore its biological function in ER-negative breast cancer.Results showed that AREG promoted the proliferation,invasion and migration of ER-negative breast cancer cells.6.Stable AR knockdown(KD),stable synchronous,AR KD/AREG-upregulated and control SK-BR-3 cell clones were successfully constructed.Results showed that the overexpression of AREG partially reversed the effects of AR inhibition on the proliferation and invasion of SK-BR-3 cells.7.Female nude mice were injected with stable AR KD,AR KD/AREG and control SK-BR-3 cell clones.We found that the volume of anti-androgen was suppressed.However,the AR KD/AREG group partially reversed the phenomenon after AR inhibition.Female nude mice in the AR KD/AREG group developed a liver metastasis.AR KD/AREG-upregulated had higher AREG and EGFR expression,and the inhibition of AR reduced the protein expression of AREG and EGFR.8.The expression features of AR in special subtypes of breast cancer and invasive carcinoma of no special type(IC-NST)are different.The positive expression rate of AR in carcinoma with apocrine differentiation is beyond 95%,and the coloration intensity is stronger than that of other subtypes.The positive rate of AR is the lowest in metaplastic carcinoma.The positive rate of AR in invasive lobular carcinoma(ILC),invasive micropapillary carcinoma(IMPC),invasive papillary carcinoma(IPC)and mucinous carcinoma is about 70.0%-80.0%.Conclusions This study demonstrated the co-expression between AREG and AR in invasivebreast cancer.AR,AREG and EGFR are interconnected and cooperatively promotethe progression of ER-negative breast cancer.Inhibition of AR,AREG and EGFRalone or the synergistic inhibition may be an effective intervention for ER-negativebreast cancer.In addition,the expression of AR varies in different pathological typesof breast cancer,and to some extent,AR may be a useful biomarker for clinicaldiagnosis of special subtypes of breast cancer.