Androgen Receptor Coregulators in Prostate Cancer

前列腺癌中的雄激素受体共调节因子

• 批准号: 6644087
• 负责人: JOSEPH D FONDELL
• 金额: $ 25.54万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6644087
• 关键词: SDS polyacrylamide gel electrophoresis; androgen receptor; biological signal transduction; cell line; cofactor; epitope mapping; gel mobility shift assay; gene expression; gene induction /repression; gene mutation; genetic polymorphism; growth factor receptors; hormone related neoplasm /cancer; human tissue; immunoprecipitation; mass spectrometry; matrix assisted laser desorption ionization; neoplastic process; northern blottings; prostate; prostate neoplasms; receptor binding; reproductive development; site directed mutagenesis; tissue /cell culture; transcription factor; western blottings

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.

描述(由申请人提供) 雄激素受体(AR)对基因表达的调节涉及 转录共调节蛋白的结合和作用。尽管一个 已发现过多的AR相互作用蛋白，包括生理和 这些因子在AR介导的信号通路中的病理作用 人们对此仍然知之甚少。此应用程序的目标是调查和 表征正常过程中特定的AR共调节因子的参与 前列腺生长和前列腺癌发生的进展。考虑到 前列腺癌细胞最初是雄激素依赖性的，最终进展 进入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蛋白本身可以异常地影响 与受体结合的特定类型的共调节蛋白复合体。至 解决这些问题，我们将产生稳定的标志表位标记的AR(f：AR) 永生化的原代前列腺细胞(正常和 恶性)和转移性前列腺癌细胞。这些线路将作为 免疫亲和从激素中提纯F：Ar的生物工具 经过处理(和未处理)的细胞，然后检查和表征 利用多种生化技术研究AR相关蛋白质。我们的特定 目标是：(1)确定不同类型的转录 协同调节蛋白与F：AR在正常对照中存在差异 恶性前列腺细胞。稳定表达F：AR的前列腺癌株将 在存在(或不存在)不同的雄激素和抗雄激素的情况下培养； F：Ar-辅因子复合体将被提纯并用银染法进行表征， 蛋白质印迹和质谱分析。(2)确定是否 雄激素非依赖性信号通路诱导F：AR-辅因子复合体组装 在正常和恶性前列腺细胞中。这些研究将检验是否 特异性受体酪氨酸激酶的激活(先前涉及 前列腺癌和雄激素非依赖性生长)可以触发特定的f： 在没有AR配体的情况下形成AR辅因子复合体。(三)确定 AR基因的病理相关突变/多态是否会影响 F：AR辅因子组件。F：AR基因将受到位点定向的影响 诱变，随后稳定地导入前列腺细胞。突变的 F：AR将从配体处理的细胞和相关的辅助因子中提纯 确定和表征的。总而言之，这里概述的研究应该 加深我们对共同调节因素在 AR介导的信号通路，并可能识别和定义新的靶点 用于前列腺癌治疗的治疗剂。