Regulation Of AR-mediated Gene Transcription By BAP 18 And Its Role In ER Negative Breast Cancer

Objective: Endocrine therapy of breast cancer that acts on estrogen receptor has brought clinical benefits to patients.Similar to estrogen receptor(ER),androgen receptor(AR)is also a member of the steroid receptor family,and its positive rate in breast cancer is as high as 60% to 80%.AR is composed of N-Terminus domain?DNA-domain?flexible hinge and ligand-binding domain.After binding to androgen,AR transfer to nucleus and bind to the androgen response elements(AREs),and meanwhile recruit coregulators to mediate the transcription of AR target genes.Changes of coregulators and epigenetic mechanisms in the transcriptional regulation process have attracted more and more attention.ER negative breast cancer cannot get benefits from endocrine therapy,AR and coregulators involved in AR signal pathway may become potential drug targets in the treatment of this kind of breast cancer.MDA-MB-453 cell line is a widely used cell line for ER negative breast cancer research.Studies have shown that androgen stimulate the proliferation of this cell,while androgen receptor antagonists inhibit the proliferation of this cell.Therefore,some people speculate that AR drives gene transcription in a way independent of ER in ER negative breast cancer.BPTF associated protein(BAP18)is a member of MLL1-WDR5 complex and NURF/BPTF complex,it contains a SANT domain which carries a variety of chromatin remodeling factors.Studies have shown that BAP18 is an important coactivator of AR,which can up regulate AR mediated gene transcription and promote the proliferation of prostate cancer and castration resistant prostate cancer cells.In ER positive breast cancer,BAP18 and COMPASS complex recruit to the downstream target genes of ER and promote the transcription of target genes.However,the role of BAP18 in ER negative breast cancer remains to be studied.Histone acetylation promotes gene transcription,whereas histone deacetylation inhibits gene transcription.SIN3A/HDAC complex is a multiprotein complex,in which SIN3 A act like a scaffold and bring together HDAC1/2,SAP30 and SAP130 to act on the promoter region of target gene.This complex mainly inhibits transcription through histone deacetylation.Some studies have shown that in ER positive breast cancer SIN3A/HDAC complex can inhibit the transcription of important genes which mediate apoptosis and then stimulate cell proliferation,but the role of SIN3A/HDAC complex in ER negative breast cancer has not been reported.In this study,we aimed to(1)clarify the expression level of BAP18 in ER negative breast cancer tissues;(2)clarify the role of BAP18 in AR mediated regulation and its molecular biological mechanism;(3)analyze the biological function of BAP18,so as to find new targets for the treatment of ER negative breast cancer.Methods: In this study,20 ER negative breast cancer patients without preoperative treatment were selected,there expression level of BAP18 was analyzed by Western blot.Then the expression level of BAP18 in microarray was by immunohistochemistry(IHC).Kaplan-Meier method was used to analyze the effect of BAP18 expression level on overall survival.The relationship between the expression of BAP18 and clinicopathological factors and the factors affecting the prognosis of the patients were analyzed.2.Protein immunoprecipitation assay(Co-IP)was used to determine whether BAP18 and AR were combined in ER negative breast cancer cells;then fluorescence confocal microscope scanning technology was used to explore the localization of BAP18 and AR in cells,and explored whether they can be co localized after androgen stimulation;then explored the localization of GFP-tagged BAP18 and AR.3.Dual luciferase reporter gene assay was used to detect the regulatory effect of BAP18 on AR mediated gene transcription in different ER negative breast cancer cell lines,and the regulatory effect of BAP18 on ARAF-1 and ARAF-2.4.Western blot and real-time PCR were used to detect the expression level of AR downstream target genes after knockdown of BAP18 to verify whether the change of AR target genes was consistent with dual luciferase reporter gene assay results.5.In order to further explore the mechanism of BAP18 regulating AR mediated gene transcription,CO-IP experiments were carried out to determine whether there is interaction between BAP18,AR and SIN3A-HDAC1 complex members,and dual luciferase reporter gene assay were carried out to determine whether HDAC1 and its inhibitor TSA affect the regulation of BAP18 on AR mediated gene transcription.6.The effect of BAP18 on the recruitment of SIN3A-HDAC1 complex and histone modification to AR target gene AREs region was detected by chromatin immunoprecipitation assay(CHIP).7.The effect of BAP18 on cell proliferation was detected by clone formation assay and MTS assay;the effect of BAP18 on cell cycle was detected by flow cytometry.8.The effect of BAP18 on tumor formation and tumor growth of ER negative breast cancer cells in vivo were detected in tumor-bearing mice.Results: 1.The results of Western blot showed that the expression of BAP18 protein in ER negative breast cancer was significantly higher than that in adjacent normal tissues.Consistent with that the results of IHC in 96 ER negative breast cancer tissues and 56 adjacent normal tissues also showed that the expression of BAP18 protein in human breast cancer tissues was significantly higher than that in adjacent normal breast tissues.The high expression of BAP18 was associated with poor prognosis.Further analysis of the relationship between BAP18 and clinicopathological factors showed that the expression level of BAP18 was positively correlated with tumor size and clinical stage.There was no significant correlation between BAP18 and age,lymph node metastasis status,histological grade and Ki-67.These results suggesting that BAP18 may promote the occurrence and development of breast cancer.In addition,we analyzed the independent prognostic factors of these patients,the results showed that tumor size,histological grade and BAP18 expression level were independent prognostic factors.2.Co-IP assay showed that BAP18 interacted with AR in the classic AR positive ER negative breast cancer cell line MDA-MB-453,and the interaction was enhanced after androgen stimulation.Another ER negative breast cancer cell line MDA-MB-231 also confirmed this point.Fluorescence confocal microscopy showed that both endogenous and exogenous BAP18 co-localized with AR in MDA-MB-453 cells under androgen stimulation.3.Dual luciferase reporter gene assay showed that BAP18 could inhibit AR mediated gene transcription and significantly down regulate the ligand dependent domain mediated gene transcription at C-terminal.4.Real time RCR results showed that compared with the control group,the m RNA levels of P21,PTEN,KLLN,EAF2,PISD,KIBRA and MYC were significantly increased in the BAP18 knockout group under androgen stimulation.Western blot results also showed that the protein levels of AR downstream target genes were increased after BAP18 knockout.5.Co-IP assay showed that BAP18 and AR could combined to SAP130,HDAC1 and SIN3 A in MDA-MB-453 cells.Dual luciferase reporter gene assay showed that the degree of inhibition of AR mediated gene transcription activity by BAP18 was dose-dependent with HDAC1,and the inhibition was significantly restored after adding HDAC1 inhibitor TSA.6.CHIP assay showed that AR,BAP18,SAP130,HDAC1 and SIN3 A were recruited to the promoter region of AR target gene P21,and the recruitment was reduced when BAP18 was knocked down under androgen stimulation,the acetylation level of histone H3 and H4 in this region was significantly increased.7.The results of clone formation assay showed that the proliferation of cells was inhibited after knockdown BAP18,and MTS assay showed that the proliferation of cells was reduced after knockdown BAP18.Flow cytometry showed that the number of cells arrested in G1 phase increased after knockdown BAP18.8.The results of tumor-bearing mice showed that knockdown BAP18 could reduce the tumor formation and tumor growth ability of ER negative breast cancer cells in vivo.Conclusion: 1.The high expression of BAP18 in ER negative breast cancer is significantly correlated with clinical stage and prognosis.2.In ER negative breast cancer,BAP18 and AR co-located in the nucleus and interacted to inhibit AR mediated gene transcription.3.BAP18 can combined with SIN3A/HDAC1 complex to recruit to ARE,catalyze the deacetylation of histone H3 and H4.4.BAP18 can promote the growth of AR positive ER negative breast cancer cells.