AR intracellular trafficking in prostate cancer

前列腺癌中的 AR 细胞内转运

• 批准号: 6916128
• 负责人: Zhou Wang
• 金额: $ 26.12万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6916128
• 关键词: SDS polyacrylamide gel electrophoresis; androgen receptor; binding proteins; biological signal transduction; cell growth regulation; chimeric proteins; green fluorescent proteins; heat shock proteins; immunocytochemistry; immunoprecipitation; intracellular transport; laboratory mouse; microinjections; molecular cloning; neoplastic growth; nuclear transport; prostate neoplasms; protein protein interaction; tissue /cell culture; transfection /expression vector; western blottings; yeast two hybrid system

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.

描述（由申请人提供）：我们的长期目标是阐明前列腺癌不依赖雄激素的生长机制。雄激素受体（AR）的配体独立核定位对于雄激素非依赖性生长至关重要。在雄激素敏感的前列腺癌细胞中，在没有配体的情况下，AR定位于细胞质，在存在配体的情况下易位到细胞核。相反，在雄激素难治性前列腺癌细胞中，即使在没有配体的情况下，AR也定位于细胞核。这些观察结果导致我们假设，在雄激素难治性前列腺癌细胞中缺乏配体时，核输出信号（NES-AR）不活跃或不再比核输入信号（NLS）NL1占主导地位。我们的初步研究已经确定了配体结合域（LBD）中的核输出信号（NES-AR）。在没有配体的情况下，NES-AR 比位于 DMA 结合域 (DBD) 和铰链区的 NL1 占主导地位。雄激素与 LBD 结合抑制 NES-AR。有趣的是，NES-AR 介导的核输出是由热休克蛋白 90 (HSP90) 调节的。提出了五个具体目标来进一步表征 NES-AR。 1.确认NES导致核输出。 GST-GFP-NES-AR融合蛋白将被显微注射到细胞核中，以直接测试NES-AR是核输出信号而不是细胞质保留信号。 2. 鉴定 NES-AR 中在没有配体的情况下核输出和/或抑制 NL1 所必需的氨基酸残基。将采用删除和接头扫描取代诱变来定义 NES 中功能上重要的氨基酸残基。 3. 检验以下假设：在雄激素难治性前列腺癌细胞中缺乏配体时，NES-AR 失活或不再比 NL1 占主导地位。培养中的 AR 阳性雄激素难治性前列腺癌细胞和异种移植肿瘤将用作模型。 4. 鉴定并表征与 NES-AR 结合的蛋白质。人类前列腺 cDNA 文库的酵母 2 杂交筛选已鉴定出 4 种候选 NES-AR 结合蛋白。将进行功能获得和丧失分析以确定它们在 NES-AR 介导的核输出中的作用。 5. 描述 HSP90 在 NES-AR 行动中的作用。我们将测试 HSP90 是否直接与 NES-AR 相互作用和/或影响核输出机制。