Elucidating the mechanisms underlying tumor formation and response to therapy is necessary to develop more effective treatments for cancer patients. Defining the cellular context that promotes apoptosis in cancer cells is one of the most fundamental, yet incompletely understood, issues in oncology. ASPP2 (Apoptosis Stimulating Protein of p53 2) is a highly regulated member of a family of p53-binding proteins that enhance apoptosis through stimulation of p53-transactivation of pro-apoptotic target genes. However, the mechanisms underlying the important upstream pathways controlling ASPP2 remain unknown. Preliminary data suggests ASPP2 is an E2F target gene--implying that ASPP2 is a link between the p53 and Rb/E2F pathways. The broad long-term objectives are to understand how ASPP2 contributes to cancer development and resistance to therapy. The objective of this proposal is to characterize the upstream pathways regulating ASPP2 and how these affect ASPP2 biologic function. The central hypothesis of this proposal is that E2F regulates ASPP2 expression and that ASPP2 may function downstream of E2F to enhance p53- mediated apoptosis. The rationale for this proposed research is that by understanding the upstream pathways controlling ASPP2 expression, the cellular context in which ASPP2 promotes apoptosis will be revealed. The hypothesis will be tested by three SPECIFIC AIMS: (1). Determine how E2F modulates ASPP2 expression. An ASPP2 promoter-luciferase system will be interrogated, and the endogenous ASPP2 promoter manipulated, to reveal the mechanism of E2F-induced expression---with and without cellular damage. (2). Determine the extent to which ASPP2 mediates E2F activity using ASPP2 loss of function models. An ASPP2 mouse has been constructed and will be used to generate ASPP2-/- cells. ASPP2 will also be silenced with siRNA in human tumor cells with defined genotypes. These systems will be then be interrogated for resistance to E2F (and other)-induced apoptosis. (3). Determine the extent to which ASPP2 cooperates with the p53 tumor suppressor pathway in vivo. ASPP2 mice have been bred into the tumor prone p53 background and alterations in tumor formation latency and spectrum will be determined. We expect to demonstrate that ASPP2 is a downstream mediator of E2F apoptotic function, gain insight into the E2F-1-ASPP2 pathway mediating damage-induced apoptosis, and obtain in vivo evidence that ASPP2 cooperates with p53-dependent tumor suppression. These findings would be significant because they would demonstrate a new link between the p53 and Rb/E2F tumor suppressor pathways. ? ?
