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）的改进癌症治疗的基本原理，该基因在约40%的癌症中丢失或沉默。ARF在癌症中发挥着核心作用，部分原因是它与mdm2的相互作用以及参与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。由于未对抗mdm2介导的p53降解，ARF的丧失使p53不稳定，并损害p53介导的生长停滞或凋亡。因此，ARF的缺失可能会影响基于p53的治疗以及使用p53途径触发细胞凋亡的常规化疗的结果。除了稳定p53的作用外，越来越多的证据表明，ARF的一系列更广泛的活动和相互作用尚不清楚，但它们对ARF的功能可能同样重要。如本实验室发现异位过表达ARF通过对蛋白质合成和稳定的双重作用促进p53蛋白积累，并观察到在人肿瘤细胞中ARF可以独立于p53起作用。ARF c端序列的存在增强了这种活性，这是mdm2结合所必需的区域。癌症相关的ARF c端突变体可以改变物理性质和活性，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的重要活性可能位于p53/mdm2反馈回路之外，并涉及与mdm2以外的蛋白质的相互作用，其中一些蛋白质需要ARF的c端。这些额外的活动和相互作用，以及ARF在p53/mdm2反馈回路中的参与，可能与经常保留野生型p53表达并可能失去或解除ARF调控的癌症特别相关，例如前列腺癌，这是本研究的一个重点。这些预测将通过四个特定目标进行验证，旨在：(1)确定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-蛋白相互作用，并使用固定化细菌融合蛋白选择结合伙伴，随后使用质谱法确定细胞转化过程中可能发生的变化；(2)阐明ARF在调节p53以外蛋白翻译中的作用。包括对mdm2和ARF c端的要求。(3)确定ARF和ARF1b单独和联合p53如何促进前列腺上皮细胞（细胞转化前后）、前列腺癌细胞和其他来源的癌细胞的生长抑制和治疗应答，以及对ARF c -末端的需求；(4)在人、鼠前列腺癌裸鼠模型中评价ARF单独和联合p53及化疗的抗肿瘤活性。通过这些研究，该项目将确定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破坏，并将对基于ARF的治疗方法在前列腺癌中的治疗潜力进行严格的临床前评估。