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.
