AR intracellular trafficking in prostate cancer

描述(申请人提供)：我们的长期目标是阐明前列腺癌雄激素非依赖性生长的机制。雄激素受体(AR)的配体非依赖性核定位在雄激素非依赖性生长中是必不可少的。在雄激素敏感的前列腺癌细胞中，AR在没有配体的情况下定位于细胞质，在有配体的情况下转移到细胞核。相反，在雄激素耐药的前列腺癌细胞中，AR即使在没有配体的情况下也定位于细胞核。这些观察导致了我们的假设，即在雄激素耐药的前列腺癌细胞中，在缺乏配体的情况下，核输出信号(NES-AR)不活跃或不再主导于核输入信号(NLS)NL1。我们的初步研究已经在配体结合域(LBD)中发现了核输出信号(NES-AR)。在缺乏配体的情况下，NES-AR比NL1占优势，NL1位于DMA结合区(DBD)和铰链区。雄激素与LBD结合抑制NES-AR。有趣的是，NES-AR介导的核输出受热休克蛋白90(HSP90)的调节。为了进一步描述NES-AR，本文提出了五个具体目标。1.确认核出口是NES造成的。将GST-GFP-NES-AR融合蛋白显微注射到细胞核内，直接检测NES-AR是核输出信号而不是细胞质滞留信号。 2.确定NES-AR中对于核输出和/或在没有配体的情况下抑制NL1所必需的氨基酸残基。缺失和连接扫描替换突变将被用来确定NES中具有重要功能的氨基酸残基。 3.验证在雄激素耐药的前列腺癌细胞中，在缺乏配体的情况下，NES-AR不活跃或不再对NL1显性的假设。AR阳性的雄激素耐药的前列腺癌细胞和异种移植瘤将被用作模型。4.鉴定和鉴定与NES-AR结合的蛋白质。酵母双杂交筛选人前列腺cDNA文库，鉴定出4个候选NES-AR结合蛋白。将进行功能得失分析，以确定它们在NES-AR介导的核出口中的作用。5.研究HSP90在NES-AR作用中的作用。我们将测试HSP90是否直接与NES-AR相互作用和/或影响核出口机制。

DESCRIPTION (provided by applicant): Our long-term objective is to elucidate the mechanism of androgen-independent growth of prostate cancer. Ligand-independent nuclear localization of the androgen receptor (AR) is essential in androgen-independent growth. In androgen-sensitive prostate cancer cells, AR is localized to the cytoplasm in the absence of ligand and translocates to the nucleus in the presence of ligand. In contrast, in androgen-refractory prostate cancer cells, AR is localized to the nucleus even in the absence of ligand. These observations led to our hypothesis that the nuclear export signal (NES-AR) is inactive or no longer dominant over the nuclear import signal (NLS), NL1, in the absence of ligand in androgen-refractory prostate cancer cells. Our preliminary studies have identified nuclear export signal (NES-AR) in the ligand-binding domain (LBD). NES-AR is dominant over NL1, located in the DMA-binding domain (DBD) and hinge region, in the absence of ligand. Androgen binding to LBD represses NES-AR. Interestingly, NES-AR-mediated nuclear export is modulated by heat shock protein 90 (HSP90). Five specific aims are proposed to further characterize NES-AR. 1. Confirm that NES causes nuclear export. GST-GFP-NES-AR fusion protein will be microinjected into the nuclei to test directly that NES-AR is a nuclear export signal instead of a cytoplasmic retention signal. 2. Identify amino acid residues in NES-AR that are necessary for the nuclear export and/or inhibition of the NL1 in the absence of ligand. Deletion and linker-scanning substitution mutagenesis will be employed to define the functionally important amino acid residues in NES. 3. Test the hypothesis that NES-AR is inactive or no longer dominant over NL1 in the absence of ligand in androgen-refractory prostate cancer cells. AR-positive androgen-refractory prostate cancer cells in culture and xenograft tumors will be used as models. 4. Identify and characterize proteins that bind to NES-AR. Yeast 2-hybrid screening of a human prostate cDNA library has identified 4 candidate NES-AR-binding proteins. Gain- and loss-of-function analysis will be carried out to determine their roles in the NES-AR-mediated nuclear export. 5. Characterize the role of HSP90 in NES-AR action. We will test if HSP90 directly interacts with NES-AR and/or influences the nuclear export machinery.