ARD1-dependent Androgen Receptor Signaling in Prostate Cancer

 描述（由申请人提供）：雄激素依赖性和去势抵抗性前列腺癌（PCa）的生长和存活由雄激素受体（AR）信号传导驱动，该信号传导在很大程度上由AR辅激活剂调节。AR共激活物的失活正在成为PCa的有希望的疗法。我们报道了一种乙酰转移酶，Arrest-defective-1蛋白（ARD 1）是一种新型AR共激活剂，它使AR乙酰化以激活AR和PCa;然而，ARD 1调节AR在前列腺肿瘤发生中的临床相关性和机制尚不清楚。在本申请中，我们的目的是确定AR信号的ARD 1调节机制，AR乙酰化对前列腺肿瘤发生的病理影响，以及ARD 1过表达在PCa中的临床意义。我们对一小群前列腺癌患者的初步研究表明，ARD 1过表达与前列腺癌Gleason评分相关。此外，我们还收集了707例前列腺癌，并制备了肿瘤组织微阵列进行分析。此外，我们缩小了赖氨酸残基乙酰化的ARD 1到一个22个氨基酸的基序位于DNA结合域（DBD）的AR。此外，我们表明，ARD 1促进AR核转位。根据这些数据，我们假设ARD 1过表达可能与Gleason评分和PCa的其他临床特征相关，通过乙酰化AR中的特异性赖氨酸进行AR核转位、靶基因表达和前列腺肿瘤发生。在这项研究中，我们提出了以下具体目标。1.确定ARD 1乙酰化的AR中的赖氨酸及其对AR活化和前列腺肿瘤发生的影响。我们将首先通过定点诱变、体外乙酰化测定和质谱分析确定22个氨基酸基序中的三个赖氨酸中的哪一个被ARD 1乙酰化。然后，我们将使用组成性乙酰化或非乙酰化AR突变体来确定乙酰化残基对AR转录活性、PCa细胞增殖和异种移植肿瘤生长的生物学和病理学影响。2.确定ARD 1-乙酰化AR在前列腺肿瘤发生中的机制。初步数据显示，ARD 1促进AR核转位。我们将使用组成型乙酰化或非乙酰化AR突变体和染色质免疫沉淀（ChIP）测定来确定ARD 1乙酰化的赖氨酸是否是AR核转位和AR靶向启动子结合所需的。3.确定ARD 1表达水平与PCa特征之间的临床相关性。我们将使用ARD 1抗体对707例PCa组织微阵列进行免疫组织化学分析。将在病理学家和生物统计学家的帮助下确定ARD 1水平与PCa临床特征的相关性。这些实验将增强我们对ARD 1过表达在PCa进展中的临床意义以及ARD 1在AR信号传导和前列腺肿瘤发生中的机制的理解，这对于未来开发ARD 1-AR信号传导依赖性治疗方法来管理和治疗PCa至关重要。

 DESCRIPTION (provided by applicant): The growth and survival of androgen-dependent and castration-resistant prostate cancer (PCa) are driven by androgen receptor (AR) signaling which is largely modulated by AR co-activators. Inactivation of AR co- activators is emerging as a promising therapy for PCa. We reported Arrest-defective-1 protein (ARD1), an acetyltransferase, is a novel AR co-activator that acetylates AR for AR activation and PCa development; however, the clinical relevance and mechanisms of ARD1 regulation of AR in prostate tumorigenesis are unknown. In this application, we aim to determine the mechanisms of ARD1 regulation of AR signaling, the pathological impacts of AR acetylation on prostate tumorigenesis, and the clinical significance of ARD1 overexpression in PCa. Our preliminary study of a small cohort of PCa indicates that ARD1 overexpression correlates with PCa Gleason score. In addition, we have collected 707 PCa and prepared tumor tissue microarrays for this analysis. Moreover, we narrowed down the lysine residue acetylated by ARD1 to a 22- amino-acid motif located in the DNA binding domain (DBD) of AR. Furthermore, we show that ARD1 promotes AR nuclear translocation. In light of these data, we hypothesize that ARD1 overexpression may correlate with Gleason score and other clinical features of PCa by acetylating the specific lysine in AR for AR nuclear translocation, target gene expression, and prostate tumorigenesis. In this study, we proposed the following Specific Aims. 1. Determine the lysine in AR acetylated by ARD1 and its impact on AR activation and prostate tumorigenesis. We will first determine which one of the three lysines in the 22-amino-acid motif is acetylated by ARD1 by site-directed mutagenesis, in vitro acetylation assays, and mass spectrometry analysis. Then, we will determine the biological and pathological impacts of the acetylated residue on AR transcriptional activity, PCa cell proliferation, and xenograft tumor growth using constitutively acetylated or non-acetylated AR mutants. 2. Determine the mechanism of ARD1-acetylated AR in prostate tumorigenesis. Preliminary data shows that ARD1 promotes AR nuclear translocation. We will determine if the lysine acetylated by ARD1 is required for AR nuclear translocation and AR-targeted promoter binding using the constitutively acetylated or non-acetylated AR mutants and chromatin-immunoprecipitation (ChIP) assays. 3. Determine the clinical correlation between ARD1 expression levels and PCa characteristics. We will perform immunohistochemistry analysis of the 707 PCa tissue microarrays using an exclusive ARD1 antibody. The correlation of ARD1 levels with PCa clinical features will be determined with assistance from a pathologist and a biostatistician. These experiments will enhance our understanding of the clinical significance of ARD1 overexpression in PCa progression and the mechanisms of ARD1 in AR signaling and prostate tumorigenesis which will be critical for future development of ARD1-AR signaling-dependent therapeutic approaches to manage and treat PCa.