Regulation of Intratumor Androgen/Androgen Receptor signaling in Prostrate Cancer

描述(由申请人提供)： 前列腺癌管理中的挑战来自1)无法区分惰性前列腺癌和具有转移潜能的侵袭性恶性肿瘤；2)复发、耐阉割的前列腺癌(CRPC)的治疗选择有限。CRPC中雄激素受体(AR)的重新激活是由于AR的表达和活性增加，并增加了DHEA的从头合成雄激素。抗AR/抗雄激素生物合成药物对化疗后癌症的抑制作用可持续几个月，但反应并不普遍。寻找癌症进展的新预测因素和新的药物靶点是高度优先的目标。我们将检查雄激素激活的AR信号的一个新的方面，这是从我们的研究中揭示的。这包括：1)通过前列腺表达的SULT2B调节雄激素的动态平衡，SULT2B是一种介导胆固醇和DHEA硫酸盐化的磺基转移酶，以及II)增强甲基化AR的活性。我们发现：1)临床标本中SULT2B基因和蛋白水平显著降低，组织芯片免疫组化染色显示肿瘤组织中SULT2B基因显著缺失(p&lt；0.001)；2)SULT2B沉默导致前列腺癌细胞增殖增加；3)维生素D受体(VDR)和氧化甾醇诱导的肝X受体(LXR)可以诱导SULT2B基因(SULT2B1)；4)AR通过SET9赖氨酸甲基转移酶在其铰链结构域的赖氨酸残基上单甲基化。SET9缺失降低了野生型AR的活性，但没有甲基化位点突变的AR活性；5)组蛋白赖氨酸去甲基酶LSD1可以将甲基化的AR转化为未甲基化的AR；6)LXR-？是编码SET9和LSD1的基因的潜在调节因子，因为在电子分析中这两个基因中存在LXR响应的顺式元件。我们开发了一种抗体，通过Western blotting可以特异性地识别赖氨酸甲基化的AR。我们假设晚期前列腺癌与SULT2B减少和甲基修饰AR增加相关，LXR-？通过防止SULT2B丢失和甲基化AR增加而部分抑制前列腺癌，VDR协同LXR效应。这一假说是基于我们自己的结果和报告建立的，这些报告如下：i)LXR--/-小鼠显示出腹侧前列腺对雄激素的敏感性增加，但AR水平没有变化；ii)对抗DHEA硫化的类固醇硫酸酯酶(Sts)在LXR--/-小鼠的前列腺中被诱导，而在LXR--/-小鼠中被抑制；iii)LXR--/-小鼠抑制异种移植瘤中的前列腺癌。我们建议确定：目的1)SULT2B基因缺失在前列腺癌中的意义，以及DNA甲基化在该缺失中的作用。我们将检查i)癌症组织中低SULT2B与i)DHT水平和AR靶基因表达的关系，ii)AKR1C3和SRD5A1酶的水平，以及iii)SULT2B1基因的甲基化状态。将探索临床变量(包括RACE的作用)与上述参数的统计关联。目的2)SULT2B调节的雄激素稳态与LXR信号在前列腺癌中的动态变化，与VDR的可能协同作用及其与前列腺癌的关系。我们将研究：i)SULT2B1中的LXR响应DNA顺式元件(S)及其与LXR-、辅调节因子、染色质修饰剂的相互作用；ii)LXR-与VDR在SULT2B诱导和前列腺癌抑制中的协同作用；iii)在体外和体内LXR沉默的前列腺癌细胞中的DHT水平，以及在LXR缺失的小鼠前列腺中的DHT水平以及骨化三醇逆转LXR沉默的效果；iv)临床标本中低SULT2B与LXR和LXR靶向致脂基因表达减弱之间的联系。目的3)AR甲基化在前列腺癌中的意义及其与LXR-β的相互作用。我们将检测：i)LXR中的甲基化AR水平--体外和体内被击倒的癌细胞；ii)LXR-在SET9和LSD1表达中的作用及其与AR的关联；iii)临床标本甲基化AR水平升高(相对于总AR)。方法：免疫组化、组织芯片、激光共聚焦显微镜、蛋白质印迹、焦磷酸测序、硫酸盐修饰DNA、启动子分析、芯片、慢病毒、si RNAs、LC-MS/MS等。将使用多核。意义：这项研究可能揭示新的预测标记物和新的干预靶点来预防CRPC。

DESCRIPTION (provided by applicant): Challenges in prostate cancer management stem from 1) inability to distinguish indolent prostate cancer from aggressive malignancy with metastatic potential; 2) limited treatment options for recurrent, castration-resistant prostate cancer (CRPC). Reactivated androgen receptor (AR) in CRPC results from elevated expression and activity of AR, and increased de novo androgen biosynthesis from DHEA. Anti-AR/anti-androgen biosynthesis drugs inhibit post chemotherapy cancer for a few months, but response is not universal. Finding new predictors for cancer progression and new drug targets are high-priority goals. We will examine a novel aspect of androgen-activated AR signaling that was revealed from our study. This entails i) regulation of androgen homeostasis by the prostate-expressed SULT2B, a sulfotransferase which mediates sulfation of cholesterol and DHEA, and ii) enhanced activity of methylated AR. We show 1) SULT2B mRNA and protein levels are markedly reduced in clinical specimens and IHC staining of tissue microarrays revealed highly statistically significant (p<0.001) loss of SULT2B in cancer tissue; 2) SULT2B silencing caused increased proliferation of prostate cancer cells; 3) Vitamin D receptor (VDR) and the oxysterol-inducible liver X receptor (LXR) can induce the SULT2B gene (SULT2B1); 4) AR is monomethylated at a lysine residue in its hinge domain by the SET9 lysine methyltransferase. SET9 depletion reduced wild type but not methylation-site-mutant AR activity; 5) LSD1, a histone lysine demethylase can convert methylated AR to unmethylated AR; 6) LXR-¿ is a potential regulator of genes encoding SET9 and LSD1, since LXR-responsive cis elements are present in these two genes based on in silico analysis. We have developed an antibody that can specifically recognize lysine- methylated AR by western blotting. We hypothesize that advanced prostate cancer associates with reduced SULT2B and elevated methyl-modified AR, LXR-¿ inhibits prostate cancer in part by preventing loss of SULT2B and elevation of methylated AR, and VDR synergizes LXR effects. The hypothesis is built from our own results and from reports that i) LXR- -/- mice show heightened androgen sensitivity in ventral prostates, but no change in AR levels; ii) steroid sulfatase (Sts) which counters DHEA sulfation, was induced in prostates of Lxr- -/- mice and suppressed in LXR- -activated mice; iii) LXR inhibited prostate cancer in xenograft tumors. We propose to determine: Aim 1) significance of SULT2B loss in prostate cancer, and a role for DNA methylation in this loss. We will examine i) diverse specimens for association of low SULT2B in cancer tissue with i) DHT levels and AR target gene expression, ii) levels of the enzymes AKR1C3 & SRD5A1, and iii) methylation status of the SULT2B1 gene. Statistical association of clinical variables (including role of race) with above parameters will be explored. Aim 2) the dynamics of LXR- signaling with SULT2B-regulated androgen homeostasis in prostate, possible synergy with VDR and their relevance in prostate cancer. We will investigate i) LXR-responsive DNA cis element(s) in SULT2B1 and its interaction with LXR- , coregulators, chromatin modifiers; ii) Synergy of LXR- with VDR in SULT2B induction and prostate cancer inhibition; iii) DHT levels in LXR- -silenced prostate cancer cells in vitro & in vivo, and in Lxr- -null mouse prostates and reversal of the effect of LXR silencing by calcitriol; iv) a link in clinical specimes between low SULT2B and attenuated expression of LXR- and LXR- -targeted lipogenic genes. Aim 3) significance of AR methylation in prostate cancer and its interplay with LXR-¿. We will examine i) methylated AR levels in LXR- -knocked down cancer cells in vitro & in vivo; ii) a role for LXR- in SET9 & LSD1 expression and in their association with AR iii) clinical specimens for elevated methylated AR levels (relative to total AR). Methods: IHC, tissue microarray, LCM, western blot, pyrosequencing, bisulfate-modified DNA, promoter assay, ChIP,, lentivirus, si RNAs, LC- MS/MS. Molecular biologists, pathologists and biostatisticians will collaborate. Multiple cores will be used. Significance: This study may reveal new predictive markers and new intervention targets to prevent CRPC.