The Relationship Between The Androgen Receptor And MTA1?HDAC6 In ER-negative Breast Cancer

Objective:To explore whether there is interaction between the AR?HER2 and MTA1 in ER-negative breast cancer cell lines and the effect of MTA1 on cell proliferation and migration.To verify the relationship between the three in vivo.To explore the relationship between AR and HDAC6 and the impact of HDAC6 on the prognosis of breast cancer patients.Method: 1.The expression of HER2 and AR in ER-breast cancer cells were analysed by Q-RT-PCR and Western-blot.Localization of MTA1 in the breast cancer cells were observed by Immunofluorescence.2.MTA1 plasmid were transfected into ER(-)breast cancer cells,MTT and wound healing assay were used to test the growth and migration of breast cancer cells.3.AR shRNA were transfected into ER-/Her2+ breast cancer cells,the expression of HER2 in ER-breast cancer cells were analysed by Q-RT-PCR and Western-blot.HER2 shRNA were transfected into ER-/AR+ breast cancer cells,the expression of AR in ER-breast cancer cells were analysed by Q-RT-PCR and Western-blot.4.AR shRNA were transfected into ER-/Her2+ breast cancer cells,the expression of MTA1 in ER-breast cancer cells were analysed by Q-RT-PCR and Western-blot.As the same time ER-/Her2+ breast cancer cells act as a control group,to future confirme the reliability of the experiment,shHER2/AR plasmid were transfected into ER-/Her2+AR+ breast cancer cells,the expression of MTA1 in ER-breast cancer cells were analysed by Q-RT-PCR and Western-blot.5.MTA1 shRNA plasmid were transfected into AR+Her2+ breast cancer cells,the expression of AR and HER2 in ER-breast cancer cells were analysed by Q-RT-PCR and Western-blot.6.We injected AR+Her2+ breast cancer cells with MTA1 siRNA or control cells into subcutaneous of female nude mice.And we devided 10 nude mice into two groups randomly,The expression of HER2,AR and Ki67 in xenograft tumor was detected by using immunohistochemistry.7.We randomly obtained tumor tissues of 230 breast cancer patients with invasive breast carcinoma to analyze the expression of HDAC6 and AR by immunohistochemistry and collected follow-up data by making phone calls and checking medical records to analyze prognosis of breast cancer.8.In ER-negative samples,we analyzed the expression of HDAC6 and AR by immunohistochemistry and prognosis.We divided 228 patients into MABC group andnon-MABC group to analyze prognosis.Results 1.The results showed that the expression of AR and HER2 in MDA-MB-453 was AR-/HER2+;SK-BR-3 was AR-/HER2+;MDA-MB-231 was AR-/HER2-by Q-RT-PCR and Western-blot.The location of MTA1 in breast cancer cells was nucleus.2.MTT assays showed that decreased expression of MTA1 reduced growth of breast cancer cells,compared with control group.Wound healing assays showed decreased expression of MTA1 reduced the migration of breast cancer cells,compared with control group.3.Up-regulation of AR in SK-BR-3 and MDA-MB-453 breast cancer cells,the Q-RT-PCR and Western-blot results showed the HER2 protein and mRNA level was increased,respectively.Down-regulation of AR,the expression of HER2 was decreased.Then HER2 were transfected into MDA-MB-453 breast cancer cells,the AR protein and mRNA level was increased,Down-regulation of HER2,AR protein and mRNA were also decreased.4.Up-regulation of AR in SK-BR-3 and MDA-MB-453 breast cancer cells,the Q-RT-PCR and Western-blot results showed the MTA1 protein and mRNA level was increased in MDA-MB-453;Howere,there was no change in SK-BR-3,compared to the control group.Then we transferred AR into shHER2 MDA-MB-453,the results showed there was no change.5.MTA1 shRNA plasmid were transfected into AR+Her2+ breast cancer cells,the resuts of Q-RT-PCR and Western-blot showed the AR and HER2 protein and mRNA level was decreased.6.The results showed tumor growth was slow which mices injected with MDA-MB-453 which transfected with AR,compared to control cells.Q-RT-PCR showed that the protein expression of MTA1 were increased,and there were lung metastases in AR group.There were no liver and lung metastases in control group.7.We randomly obtained tumor tissues of 230 breast cancer patients with invasive breast carcinoma to analyze the expression of HDAC6 and AR by immunohistochemistry and collected follow-up data by making phone calls and checking medical records to analyze prognosis of breast cancer.Positive and negative AR expression levels were found in 67%(152/228)and 33%(75/228)of patients respectively,and 58.8%(134/228)of specimens had high expression of HDAC6.8.Among 98 ER-negative specimens,tumors with high HDAC6 expression were more likely exhibit AR positivity(P < 0.001).AR and HDAC6 expression was significantly correlated with OS(P = 0.015 and P = 0.042,respectively).It seems that patients with high HDAC6 expression and AR positivity in the ER-negative group had worse outcomes.We divided 228 patients into MABC(ER-PR-AR+,51/228,22.3%)group and non-MABC(all samples not ER-PR-AR+,177/228,77.7%)group.High HDAC6 expression was more commonly found in the MABC group than in the non-MABC group(P<0.001).We also used Kaplan–Meier analysis to determine OS and DFS within the MABC and non-MABC groups.The MABC group had a significantly worse outcome(P<0.001).Conclusion Our study showed that there was a cross-talk between AR and HER2,both AR and HER2 can affect the expression of MTA1,MTA1 can not be changed with either of them.Understand the regulatory mechanism of MTA1 might provide a new opportunity for breast cancer therapy.MTA1 is a member of NuRD family,Another important member of the NuRD family is HDACs,and our study also showed the expression levels of HDAC6 and AR are correlated in breast cancer The expression of HDAC6 occurred at a high frequency in ER-negative breast cancer and the MABC subgroup.Thus,we propose that HDAC6 is a marker that represents a worse outcome in the ER-negative subgroup.HDAC6 might provide a new opportunity for breast cancer therapy,especially in the ER-negative subgroup.