SUMO E3连接酶介导雄激素受体的转录促进乳腺癌他莫昔芬耐药

SUMO E3 ligase mediates androgen receptor transcription to promote tamoxifen resistance of breast cancer

目的:探讨SUMO化的雄激素受体(androgen receptor,AR)参与他莫昔芬耐药的机制以及SUMO抑制剂银杏酸在耐药中的作用。方法:通过Real-Time PCR检测亲本细胞MCF-7W和耐药细胞MCF-7R中AR mRNA水平,Western blot检测MCF-7W和MCF-7R细胞中AR蛋白水平以及SUMO水平,免疫沉淀和染色质免疫沉淀(CB/IP)检测MCF-7R细胞染色质中AR与SUMO E3连接酶PIAS1、HSP27相互作用,荧光报告基因实验检测SUMO化AR的转录活性,CCK-8法、台盼蓝拒染法分别检测细胞活性和细胞活力。结果:MCF-7R细胞中AR的mRNA和蛋白表达水平表达量明显高于MCF-W7细胞(P<0.05),并且MCF-7R细胞中显示高SUMO化的AR;进一步研究发现MCF-7R细胞染色质中AR与SUMO E3连接酶PIAS1、HSP27存在明显相互作用,即SUMO化的AR是由E3连接酶修饰的;双荧光素酶报告基因试剂盒检测发现雄激素R1881呈浓度依赖性增强SUMO化的AR的转录活性;与单用银杏酸相比,10μmol/L银杏酸联合10μmol/L恩杂鲁胺治疗处理MCF-7R细胞3 d后,细胞数明显降低,细胞死亡数明显增加(P<0.05)。结论:他莫昔芬耐药机制可能是由于高活性SUMO化的AR使AR转录增强、活性增高,SUMO抑制剂银杏酸联合AR拮抗剂恩杂鲁胺可成为治疗他莫昔芬耐药的新策略。

AIM:To investigate the mechanism of the involvement of SUMO-ylated Androgen receptor(AR)in tamoxifen resistance and the role of SUMO inhibitor ginkgolic acid in resistance.METHODS:Real-Time PCR was used to detect AR mRNA levels in parental cells MCF-7W and drug-resistant cells MCF-7R,AR protein levels and SUMO levels in MCF-7W and MCF-7R cells was performed by western blot,and CB/IP was applied to detect AR interacts with SUMOE3 ligase PIAS1 and HSP27 in MCF-7R cell chromatin,the transcriptional activity of SUMO AR was also evaluated by the fluorescent reporter gene experiment,the CCK-8 method and the trypan blue exclusion method were used to detect cell viability and cell viability respectively.RESULTS:The mRNA and protein expression levels of AR in MCF-7R cells were significantly higher than those in MCF-7W cells(P<0.05),and there was highly SUMOylated AR in MCF-7R cells.Further research found that there had an obvious interaction between AR and SUMO E3 ligase PIAS1 and HSP27,that was,the SUMOylated AR was modified by E3 ligase.Moreover,androgen R1881 could enhance the transcriptional activity of the SUMOylated AR in a concentration-dependent manner.Compared with ginkgo acid alone,10μmol/L of ginkgolic acid combined with 10μmol/L of enzalutamide treated MCF-7R cells for 3 days,the cell number was significantly reduced,and the number of cell death increased significantly(P<0.05).CONCLUSION:The resistance mechanism of tamoxifen may be due to the enhanced AR transcription and activity increased by the hyperactive SUMOylated AR,SUMO inhibitor ginkgolic acid combined with AR antagonist enzalutamide can be a new strategy for the treatment of tamoxifen resistance.