Interactions and Functions of p14ARF in Cancer

p14ARF 在癌症中的相互作用和功能

• 批准号: 6858441
• 负责人: RUTH A GJERSET
• 金额: $ 39.07万
• 依托单位: SIDNEY KIMMEL CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6858441
• 关键词: SDS polyacrylamide gel electrophoresis; athymic mouse; crosslink; mass spectrometry; neoplasm /cancer genetics; neoplastic transformation; oncoproteins; p53 gene /protein; polymerase chain reaction; prostate neoplasms; protein protein interaction; protein structure function; tumor suppressor genes; western blottings; yeast two hybrid system

DESCRIPTION (provided by applicant): The broad, long-term objective of this study is to establish a rationale for improved therapies for cancer based on pl4ARF (ARF), whose gene is lost or silenced in some 40% of cancers. ARF plays a central role in cancer, attributable in part to its interaction with mdm2 and participation in the p53/mdm2 feedback mechanism, a DNA damage and stress response mechanism that is disrupted in most cancers. Loss of ARF destabilizes p53 as a result of unopposed mdm2-mediated degradation of p53, and impairs p53-mediated growth arrest or apoptosis. Loss of ARF may therefore compromise the outcome of p53-based therapies as well as conventional chemotherapies that use the p53 pathway to trigger apoptosis. In addition to this role in stabilizing p53, there is growing evidence for a broader array of activities and interactions of ARF that are less well understood, but that could be equally important for ARF function. For example, this laboratory has found that ectopic overexpression of ARF contributes to p53 protein accumulation through dual effects on protein synthesis and stabilization, and has observed that ARF can act independently of p53 in human tumor cells. This activity is enhanced by the presence of ARF C-terminal sequences, a region that is dispensable for mdm2 binding. The importance of ARF C-terminal sequences is further underscored by findings that cancer-associated C-terminal mutants of ARF can have altered physical properties and activities. It is therefore likely that important activities of ARF, relevant to the mechanism of cancer and to its treatment, lie outside of the p53/mdm2 feedback loop, and involve interactions with proteins other than mdm2, some of which require the ARF C-terminus. These additional activities and interactions, together with the involvement of ARF in the p53/mdm2 feedback loop, could be particularly relevant to cancers that frequently retain expression of wild-type p53 and may lose or deregulate ARF, such as prostate cancer, which is one focus of this study. These predictions will be tested through four Specific Aims designed to (1) identify protein-protein interactions of ARF or ARF 1b in prostate cancer cell lines and normal prostate epithelial cells, and identify possible changes accompanying cellular transformation using immobilized bacterial fusion proteins to select binding partners, followed by mass spectrometry, (2) elucidate the role of ARF in regulating translation of proteins other than p53, including the requirement for mdm2 and the ARF C-terminus. (3) Establish how ARF and ARF1b, alone and together with p53, contribute to growth suppression and to the therapy response of prostate epithelial cells (before and after cellular transformation), prostate cancer cells, and cancer cells of other origins, and the requirement for the ARF C-terminus, and (4) evaluate in nude mouse models of human and murine prostate cancer the anti-tumor activity of ARF, alone and together with p53 and chemotherapy. Through these studies the project will define the full range of ARF interactions and activities and their possible disruptions following cellular transformation, and will provide a rigorous pre-clinical evaluation in prostate cancer of the therapeutic potential of ARF-based therapies.

描述（由申请人提供）：本研究的广泛、长期目标是建立基于pl4ARF (ARF)的改进癌症疗法的基本原理，其基因在约40%的癌症中丢失或沉默。 ARF 在癌症中发挥着核心作用，部分原因在于它与 mdm2 相互作用并参与 p53/mdm2 反馈机制，这是一种在大多数癌症中被破坏的 DNA 损伤和应激反应机制。由于 mdm2 介导的 p53 降解不受阻碍，ARF 的缺失会破坏 p53 的稳定性，并损害 p53 介导的生长停滞或细胞凋亡。因此，ARF 的缺失可能会损害基于 p53 的疗法以及使用 p53 途径触发细胞凋亡的传统化疗的结果。除了稳定 p53 的作用之外，越来越多的证据表明 ARF 还具有更广泛的活性和相互作用，这些活性和相互作用尚不清楚，但这对于 ARF 功能可能同样重要。例如，该实验室发现ARF的异位过度表达通过对蛋白质合成和稳定的双重作用促进p53蛋白质积累，并观察到ARF可以在人类肿瘤细胞中独立于p53发挥作用。 ARF C 端序列的存在增强了这种活性，该区域对于 mdm2 结合来说是可有可无的。与癌症相关的 ARF C 端突变体可能改变物理特性和活性，这一发现进一步强调了 ARF C 端序列的重要性。因此，与癌症机制及其治疗相关的 ARF 重要活性可能位于 p53/mdm2 反馈环之外，并且涉及与 mdm2 以外的蛋白质的相互作用，其中一些蛋白质需要 ARF C 末端。这些额外的活性和相互作用，再加上 ARF 在 p53/mdm2 反馈环中的参与，可能与经常保留野生型 p53 表达并可能丢失或放松 ARF 的癌症特别相关，例如前列腺癌，这是一种本研究的重点。这些预测将通过四个具体目标进行测试，这些目标旨在 (1) 识别前列腺癌细胞系和正常前列腺上皮细胞中 ARF 或 ARF 1b 的蛋白质-蛋白质相互作用，并使用固定化细菌融合蛋白来选择结合来识别伴随细胞转化的可能变化合作伙伴，随后进行质谱分析，(2) 阐明 ARF 在调节 p53 以外的蛋白质翻译中的作用，包括对 mdm2 和 ARF C 末端的需求。 (3) 确定 ARF 和 ARF1b 单独以及与 p53 一起如何有助于前列腺上皮细胞（细胞转化之前和之后）、前列腺癌细胞和其他来源的癌细胞的生长抑制和治疗反应，以及要求(4)在人和鼠前列腺癌的裸鼠模型中评估ARF单独以及与p53和化疗一起的抗肿瘤活性。通过这些研究，该项目将定义 ARF 相互作用和活动的全部范围，以及它们在细胞转化后可能受到的干扰，并将对基于 ARF 的疗法的治疗潜力在前列腺癌中提供严格的临床前评估。