Lin28A Can Promote Development Of ER-/Her2+ Breast Cancer By Activating Androgen Receptor

Objectives The aim of this study was to examine the expression of Lin28 A and androgen receptor(AR)in ER-/Her2+ breast cancer,and research the association of Lin28 A and AR co-expression status with patients prognosis.And we tested the hypothesis that Lin28 A can activate AR and promote growth of ER-/Her2+ breast cancer.Methods The expression of Lin28 A and AR in formalin-fixed and paraffin-embedded(FFPE)surgical sections from 305 patients with ER-/Her2+ breast cancer was analysed by immunohistochemistry(IHC)and the co-expression patterns in breast cancer cells were investigated by immunofluorescent(IF)staining.The expression of Lin28 A and AR in ER-/Her2+ breast cancer cells were analysed by Q-RT-PCR and Western-blot.The impact of the expression of Lin28 A and AR in prognosis was also assessed using the Kaplan-Meier,univariate and multivariate logistic regression models.The Lin28 A si RNAs and Lin28 A plasmid were chemically synthesized by Genechem(Shanghai,China).c-myc sh RNA oligonucleotides for short hairpin RNAs were provided by Key Laboratory of Cancer Prevention and Therapy of Tianjin.The expression of AR and c-myc were examined after Lin28 A si RNA and Lin28 A plasmid were transfected into ER-/Her2+ breast cancer cells.Chromatin immune-precipitation(Ch IP)analysis and Luciferase Assays were used to evaluate the effect of Lin28 A and c-myc on AR promoter activity.MTT assays,Boyden chamber invasion assays and colony formation assays were performed.The effect of Lin28 A in cellular cycle and apoptosis were examined by flow cytometry analysis.ER-/Her2+ breast cancer cells which transfected with Lin28 A si RNAs and Lin28 A plasmid were injected into nude mice,and tumorigenesis was monitored.Results 1.This study included 305 cases ER-/Her2+ breast cancer patients.IHC showed that Lin28 A and AR were expressed in 240 cases(78.7%)and 220 cases(72.1%),respectively.Lin28 A tended to be higher in AR positive patients(75.0%).Lin28 A and AR co-expression(Lin28+/AR+)was significantly associated with high tumor grade(G3)(P=0.023)and high Ki67 index(P=0.020).In univariate analysis,Lin28A+/AR+ was significant risk factors associated with unfavorable OS(P=0.049)and RFS(P=0.019).Kaplan–Meier analysis showed that Lin28A+/AR+ expression showed lower RFS rates compared with Lin28A-/AR+(P=0.043)and Lin28A-/ARpatients(P=0.019).Multivariate cox model showed that Lin28A+/AR+ remained an independent negative prognostic factor for RFS.2.Lin28 A and AR were co-expressed in ER-/Her2+ breast cancer cells by immunofluorescence.The m RNA and protein expression level of Lin28 A and AR were higher in MDA-MB-453 cells(ER-/Her2+)than in the MDA-MB-231 cells(ER-/Her2-).3.Down-regulation of Lin28 A can decrease AR m RNA and c-myc m RNA level 3.5-fold and 3-fold,respectively,in MDA-MB-453 cells.The AR and c-myc protein level were also decreased in MDA-MB-453 cells when Lin28 A was down-regulated.Over-expression of Lin28 A increased AR m RNA and c-myc m RNA level 2.7-fold and 2-fold,respectively,in SK-BR-3 cells.The AR and c-myc protein level were also increased in SK-BR-3 cells when Lin28 A was up-regulated.4.Dual luciferase reporter gene showed that down-regulation of Lin28 A expression by Lin28 Asi RNA decreased,whereas overexpression of Lin28 A increased,the activation of the AR promoter.Down-regulation of c-myc by c-myc sh RNA reduced AR promoter activity,whereas overexpression of c-myc enhanced AR promoter activity.Lin28 A increased AR promoter activity,which was reversed by suppression of c-myc.Ch IP analysis showed that overexpression of Lin28 A increased the recruitment of c-myc to the AR promoter.5.MTT assay showed that decreased expression of Lin28 A reduced growth of MDA-MB-453 cells,and SK-BR-3 cells expressing Lin28 A exhibited a higher rate of growth compared with control group.Boyden chamber invasion assays showed that the number of Lin28 A si RNA MDA-MB-453 cells invading through Matrigel in FBS-containing medium was 31±4,whereas the number of control cells invading through Matrigel was 91±5.The number of Lin28 A SK-BR-3 cells invading through Matrigel in FBS-containing medium was 55±4,whereas the number of control cells was 18±4.The clonogenic assays showed that the number of colonies formed by Lin28A si RNA MDA-MB-453 cells was 45±5,whereas the number of colonies formed by control MDA-MB-453 cells was 110±10.Similarly,the number of colonies formed by SK-BR-3 cells expressing Lin28 A was 77±5,whereas the number of colonies formed by control SK-BR-3 cells was 24±4.Flow cytometry showed that the number of MDA-MB-453 cells in G1 phase increased,while the number of cells in S phase decreased in the Lin28 A si RNA group compared to the control group.The number of SK-BR-3 cells in G1 phase decreased,while the number of cells in S phase increased in the Lin28 A group compared to the control group.The percentage of apoptotic cells was higher in the Lin28 A si RNA group compared to the control group in MDA-MB-453 cells? 6.Mices injected with Lin28 A si RNA MDA-MB-453 cells exhibited significantly lower rates tumor growth compared to control cells.Mices injected with Lin28 A SK-BR-3 cells exhibited significantly higher tumors growth compared to control cells.Q-RT-PCR data showed that Lin28 A was correlated positively with expression levels of ARm RNA and c-mycm RNA in vivo.Conclusions Our study showed that Lin28 A and AR co-expressed in ER-/Her-2+ breast cancer and correlated with poor prognosis.Our study demonstrates that Lin28 A can activates androgen receptor via regulation of c-myc and promotes malignancy of ER-/Her2+ breast cancer.Our findings underline a novel role for Lin28 A in breast cancer development and activation of the AR axis.