Interactions and Functions of p14ARF in Cancer

描述(申请人提供)：这项研究的广泛、长期目标是建立基于pl4ARF(ARF)的癌症改进治疗的理论基础，其基因在大约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相互作用，并参与了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。ARF的缺失破坏了p53的稳定性，这是由于非相反的MDM2介导的P53的降解，并损害了P53介导的生长停滞或凋亡。因此，ARF的丢失可能会影响基于P53的治疗以及使用P53途径触发细胞凋亡的传统化疗的结果。除了这种稳定p53的作用外，越来越多的证据表明ARF有更广泛的活动和相互作用，这些活动和相互作用还不太清楚，但这对ARF功能可能同样重要。例如，该实验室发现ARF的异位过表达通过对蛋白质合成和稳定的双重作用促进了P53蛋白的积累，并观察到在人类肿瘤细胞中ARF可以独立于P53发挥作用。这种活性因ARF C-末端序列的存在而增强，这是MDM2结合所必需的区域。ARF的癌症相关C末端突变体可以改变物理性质和活性的研究结果进一步强调了ARF C末端序列的重要性。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反馈环之外，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 C-末端。这些额外的活动和相互作用，加上ARF参与P53/MDM2反馈环，可能与经常保留野生型P53表达并可能丢失或取消调节ARF的癌症特别相关，例如前列腺癌，这是本研究的重点之一。这些预测将通过四个特定目标得到验证，这些目标旨在：(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的蛋白质间相互作用，并使用固定化细菌融合蛋白选择结合伙伴，确定伴随细胞转化的可能变化，然后进行质谱分析，(2)阐明ARF在调节除p53以外的蛋白质翻译中的作用，包括对MDM2和ARF C-端的要求。(3)确定ARF和ARF1b单独和与P53一起对前列腺上皮细胞(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反应的作用，以及对ARF C末端的要求，以及(4)在人和小鼠前列腺癌裸鼠模型上评价ARF单独和与P53和化疗一起的抗肿瘤活性。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干扰，并将在前列腺癌中对基于ARF的疗法的治疗潜力进行严格的临床前评估。

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.