Androgen Receptor Coregulators in Prostate Cancer

描述（由申请人提供） 雄激素受体（AR）对基因表达的调节涉及到 关联和转录共调节蛋白的作用。虽然 已经鉴定了过多的AR相互作用蛋白，生理和 这些因子在AR介导的信号通路中所起的病理作用 仍然知之甚少。该应用程序的目标是调查和 表征正常过程中特定AR共调节因子的参与， 前列腺生长和前列腺肿瘤发生的进展。鉴于 前列腺癌细胞最初是雄激素依赖性的， 雄激素非依赖性细胞，我们假设AR中的肿瘤性变化 信号通路和/或AR蛋白本身可以异常影响 与受体结合的特定类型的共调节蛋白复合物。到 为了解决这些问题，我们将产生稳定的FLAG表位标记的AR（f：AR） 表达来自永生化原代前列腺细胞的细胞系（正常和 恶性）和转移性前列腺肿瘤细胞。这些线路将作为 我们将用免疫亲和法从激素中纯化f：AR的生物工具， 处理（和未处理）的细胞，随后检查和表征 AR相关蛋白的研究。我们的具体 目标是：（1）确定不同类型的转录 辅调节蛋白与正常对照组和正常对照组中的f：AR差异相关。 恶性前列腺细胞稳定的表达f：AR的前列腺系将是 在存在（或不存在）不同雄激素和抗雄激素的情况下培养; f：AR-辅因子复合物将被纯化并通过银染色表征， 蛋白质印迹和质谱。(2)确定是否 雄激素非依赖性信号通路诱导f：AR-辅因子复合物组装 在正常和恶性前列腺细胞中。这些研究将探讨是否 特异性受体酪氨酸激酶的激活（先前与 前列腺癌和雄激素非依赖性生长）可以触发特异性F： 在不存在AR配体的情况下AR-辅因子复合物的形成。(3)确定 AR基因中的病理相关突变/多态性是否影响 f：AR-辅因子组装。将对f：AR cDNA进行定点扩增， 诱变并随后稳定地引入前列腺细胞中。突变的 f：从配体处理的细胞和相关辅因子中纯化AR 识别和表征。总之，这里概述的研究应该 增加我们对共调节因子在以下方面的作用的基本理解： AR介导的信号通路，并可能识别和定义新的靶点 用于治疗前列腺癌的治疗剂。

DESCRIPTION (Provided by the applicant) Regulation of gene expression by the androgen receptor (AR) involves the association and action of transcriptional coregulatory proteins. Although a plethora of AR-interacting proteins have been identified, the physiological and pathological roles fulfilled by these factors in AR-mediated signaling pathways remain poorly understood. The goal of this application is to investigate and characterize the involvement of specific AR-coregulatory factors during normal prostate growth and the progression of prostate tumorigenesis. Given that prostate cancer cells are initially androgen-dependent and eventually progress into androgen-independent cells, we hypothesize that neoplastic changes in AR signaling pathways and/or the AR protein itself can abnormally affect the specific types of coregulatory protein complexes that bind to the receptor. To address these issues, we will generate stable FLAG epitope-tagged AR (f:AR) expressing cell lines from immortalized primary prostate cells (normal and malignant) and from metastatic prostate tumor cells. The lines will serve as biological tools with which we will immunoaffinity purify f:AR from hormone treated (and untreated) cells and subsequently examine and characterize the AR-associated proteins using a number of biochemical techniques. Our specific goals are to: (1) Determine whether distinct types of transcriptional coregulatory proteins are differentially associated with f:AR in normal versus malignant prostate cells. Stable f:AR-expressing prostate lines will be cultured in the presence (or absence) of distinct androgens and anti-androgens; f:AR-cofactor complexes will be purified and characterized by silver stain, Western blotting and mass spectrometry. (2) Determine whether androgen-independent signaling pathways induce f:AR-cofactor complex assembly in normal and malignant prostate cells. These studies will examine whether activation of specific receptor tyrosine kinases (previously implicated in prostate cancer and androgen-independent growth) can trigger specific f: AR-cofactor complex formation in the absence of AR ligands. (3) Determine whether pathologically associated mutations/polymorphisms in the AR gene affect f:AR-cofactor assembly. The f:AR cDNA will be subjected to site-directed mutagenesis and subsequently stably introduced into prostate cells. The mutated f:AR will be purified from ligand-treated cells and the associated cofactors identified and characterized. In summary, the studies outlined here should increase our fundamental understanding of the role of coregulatory factors in AR-mediated signaling pathways and potentially identify and define new targets for therapeutic agents in the treatment of prostate cancer.