LSD1调控AR的稳定性的分子机制研究及在前列腺肿瘤中的作用

Prostate cancer is one of the most common malignant tumors in men and the number of prostate cancer cases is increasing rapidly in China. The androgen receptor (AR) and its signaling axis are critical to the occurrence and development of prostate cancers, and remain to be the main targets for prostate cancer therapy. Recent studies showed that histone demethylase LSD1 regulates the transcriptional activity of AR, but the underlying mechanisms are unclear. Our preliminary study shows that when knocking-down LSD1 expression by RNA interference in prostate cancer cell line LNCaP, not only the expression levels of AR target genes such as PSA and NKX3.1 decrease significantly, AR protein level also goes down dramatically. Furthermore, we find that the methyltransferase SET9 promotes AR degradation, and the expression of LSD1 is able to promote AR stability. Based on these results, in this project we propose to: 1) Identify the methylation sites of AR and the methyltransferases in addition to SET9 involved in AR methylation, the relationship between AR methylation and AR protein stability; 2) Test the hypothesis that LSD1 promote AR protein stability by demethylating AR; 3) Examine the expression pattern of LSD1 in prostate cancer tissues and determine if LSD1 overexpression correlates with the AR overexpression; 4) Study if inhibition of LSD1 demethylase activity via small molecule inhibitors would lead to AR degradation in prostate cancer cells. The results from this project will not only reveal the molecular mechanism on how LSD1 regulates AR signaling pathway, but also provide new insight on treatment of castration-resistant prostate cancer (CRPC).

前列腺癌是男性常见的恶性肿瘤之一，目前在我国呈快速增长之势。雄性激素受体（AR）及其介导的信号通路在前列腺癌的发生发展中起重要作用，也是前列腺癌治疗的主要靶点。近年来的研究显示组蛋白去甲基化酶LSD1调控 AR转录活性，但其作用的分子机制还不甚确定。我们的研究发现在前列腺癌细胞LNCaP中，如果用RNA干扰的方法降低LSD1的表达，不仅AR的靶基因如PSA和NKX3.1的表达大幅下降，AR的蛋白水平也大幅下降。进一步的研究表明甲基化酶SET9能催化AR甲基化并促进AR降解，而表达LSD1则能促进AR稳定性。在此基础上，本课题我们将研究1）AR甲基化修饰、甲基化修饰位点和甲基化修饰与AR稳定性的关系；2）LSD1是否通过催化AR去甲基化促进AR蛋白稳定性；3）LSD1在前列腺癌中过表达是否与AR过表达正相关；4）是否可以通过抑制LSD1活性促进AR在前列腺癌细胞中的降解。该课题的顺利实施不仅

UHRF1是一个重要的表观遗传调控因子，在维持DNA甲基化及促进细胞增殖方面具有重要作用。我们的工作发现去甲基化酶LSD1能够促进UHRF1的蛋白稳定性，并且对UHRF1蛋白稳定性的促进作用依赖于其去甲基化酶活性。在多种细胞系中过表达LSD1能增加UHRF1的蛋白稳定性，反之，敲低LSD1则降低UHRF1的蛋白水平，这种调控作用是在蛋白水平而不是转录水平上的；UHRF1与LSD1之间存在相互作用，说明LSD1调控UHRF1的蛋白稳定性有可能是通过直接调控UHRF1的甲基化修饰而起作用的。另外，我们发现甲基化酶SET8能够通过甲基化修饰UHRF1促进其泛素化修饰和蛋白降解，并且该作用也依赖于SET8的甲基化酶活性。进一步的实验结果显示SET8和LSD1通过甲基化和去甲基化修饰调控UHRF1的蛋白稳定性，进而调控细胞整体DNA甲基化水平。我们的工作揭示细胞可能存在一种通过甲基化修饰对UHRF1的稳定性调控来负反馈调控DNA甲基化的分子机制。这一工作有望于2017年发表。