Toward Selective Androgen Deprivation by Targeting Androgen Activation of SRF

项目概要/摘要 尽管雄激素受体（AR）在前列腺癌（CaP）进展中起着中心作用，但对特定的 AR控制驱动CaP进展的细胞过程的分子机制仍然主要是 难以捉摸。缺乏这方面的知识是一个重要的问题，因为没有它，新的治疗方法的发展， 特异性靶向致死表型的AR依赖性事件的方法是不可能的。很长的- 长期目标是了解AR如何控制临床相关基因表达的机制， CaP细胞可以被操纵用于治疗干预。这里的目的是确定治疗 破坏AR-应答PKN1-SRF相互作用的可能性。核心假设是，靶向PKN 1 将阻止由AR响应SRF作用介导的CaP进展。这一假设是根据以下内容提出的： 申请人实验室的初步工作。拟议研究的理由是，一旦 了解CaP中SRF作用的AR调节如何发生，选择与以下相关的AR作用靶向 疾病进展是可能的。通过追求3个具体目标来检验中心假设：1）确定 PKN1在AR反应性SRF转录复合物中的作用; 2）确定PKN1激酶的底物 在AR依赖性SRF转录复合物中的作用;和3）确定靶向AR-1的治疗潜力。 响应PKN1-SRF相互作用。目的1将确定遗传或药理学失活的影响 PKN1对靶基因处AR依赖性SRF复合物的组成和功能的影响， RNA-Seq、ChIP-Seq和ATAC-Seq测定。在目标2中，最先进的基于生物素的邻位连接测定法 在表达野生型或激酶死亡PKN1或PKN1作用被抑制的CaP模型中， SRF转录抑制将定义PKN1依赖的SRF磷酸化， 复合体及其染色质环境。目的3：确定抑制PKN1-SRF的治疗潜力 在临床相关的PDX和从患者获得的CaP组织的新鲜离体外植体中的相互作用。临床 将使用基因表达和邻近连接（proximityligation 测定。拟议的研究是创新的，因为它专注于一种完全不同的目标AR方法 在CaP中的作用：解开并靶向控制CaP的AR下游的AR依赖性信号通路。 与CaP进展相关的基因的表达。这一贡献意义重大，因为它是第一个 这是一个连续的研究步骤，预计将导致开发新的治疗方式， 特异性靶向作为致死CaP表型基础的AR介导的基因表达。相对于 预期的结果，拟议的研究将确定PKN1引入一个新的蛋白质的分子机制。 AR-PKN1-SRF信号级联中的治疗脆弱性，该信号级联引起侵袭性CaP 表型。这些结果将产生重要的积极影响，因为它们将从根本上推进 一般而言，关于CaP中AR作用的知识，并具体地为CaP治疗提供新靶点。

Project summary/Abstract Despite the central role of androgen receptor (AR) in prostate cancer (CaP) progression, insights into the specific molecular mechanisms by which AR controls the cellular processes that drive CaP progression remain largely elusive. Lack of this knowledge is an important problem, because without it, development of novel therapeutic approaches that target specifically AR-dependent events underlying the lethal phenotype is unlikely. The long- term goal is to understand how the mechanism(s) by which AR controls clinically relevant gene expression in CaP cells can be manipulated for therapeutic intervention. The objective here is to determine the therapeutic potential of disrupting AR-responsive PKN1-SRF interactions. The central hypothesis is that targeting PKN1 will prevent CaP progression mediated by AR-responsive SRF action. This hypothesis is formulated based on preliminary work produced in the applicant's laboratory. The rationale for the proposed studies is that, once it is understood how AR regulation of SRF action in CaP occurs, select targeting of AR action that is relevant to disease progression will be possible. The central hypothesis is tested by pursuing 3 specific aims: 1) determine the role of PKN1 in the AR-responsive SRF transcriptional complex; 2) define the substrate for PKN1's kinase action in the AR-dependent SRF transcriptional complex; and 3) determine therapeutic potential of targeting AR- responsive PKN1-SRF interactions. Aim 1 will determine the impact of genetic or pharmacological inactivation of PKN1 on the composition and function of AR-dependent SRF complex at target genes using a combination of RNA-Seq, ChIP-Seq and ATAC-Seq assays. In Aim 2, state-of-the-art biotin-based proximity ligation assays and mass spectrometry in CaP models that express wild-type or kinase-dead PKN1 or in which PKN1 action is pharmacologically inhibited will define PKN1-dependent phosphorylation of SRF, the SRF transcriptional complex and its chromatin environment. Aim 3 will determine the therapeutic potential of inhibiting PKN1-SRF interactions in clinically relevant PDXs and fresh ex vivo explants of CaP tissue obtained from patients. Clinically applicable biomarkers of response to such treatment will be sought using gene expression and proximity ligation assays. The proposed research is innovative because it focuses on an entirely different approach to target AR action in CaP: unraveling and targeting an AR-dependent signaling pathway downstream of AR that controls the expression of genes that are relevant for CaP progression. This contribution is significant because is it the first step in a continuum of research that is expected to lead to the development of novel treatment modalities that target specifically AR-mediated gene expression that underlies the lethal CaP phenotype. With respect to expected outcomes, the proposed studies will identify the molecular mechanisms by which PKN1 introduces a therapeutic vulnerability in the AR-PKN1-SRF signaling cascade that gives rise to the aggressive CaP phenotype. These results will have an important positive impact because they will fundamentally advance knowledge about AR action in CaP, in general, and provide new targets for CaP therapy, specifically.