Survivin is a member of the Inhibitor of Apoptosis (IAP) gene family that has attracted attention for its dual role in cell division and cytoprotection, and its overexpression in nearly every human tumor. Translational targeting of the survivin pathway is being vigorously pursued for novel cancer therapeutics, and several modalities from gene therapy to small molecule antagonists have entered clinical trials. This prompts the need for a detailed understanding of the cellular and molecular requirements of survivin function in tumor cells, in vivo. Work carried out during the previous cycle of CA90917 identified a novel molecular circuitry of apoptosis regulation by survivin. This is centered on the dynamics of a mitochondrial pool of survivin that is rapidly released in the cytosol in response to cell death stimuli. In turn, mitochondria-derived survivin forms a complex with IAP family protein, XIAP, and this has broad cytoprotective consequences with intrinsic inhibition of apoptosis, activation of gene expression contributed by NFicB, and preservation of cell viability during the DNA damage response. Therefore, a unifying hypothesis can be formulated that cytoprotection by survivin directly contributes to tumor formation/progression, in vivo, and this will constitute the focus of the present continuation application. In the first specific aim, experiments will be carried out to map the post- translational requirements of the survivin-XlAP complex, which requires dynamic phosphorylation of survivin on Ser20 and Ser81 by protein kinase A. A quantitative structure-function analysis of the survivin-XlAP recognition will be obtained using recombinant protein-protein interactions, synthetic peptidyl mimicry and targeted mutagenesis. The second specific aim will dissect the role of survivin in the DNA damage response. Emphasis will be placed on the role of the checkpoint kinase Chk2 in the regulation of mitochondrial survivin dynamics with respect to p53 requirements, recruitment of proapoptotic Bcl-2 family proteins, and modulation of apical caspase activation. The third specific aim will take a combined cellular and genetic approach to map the relevance and contribution of survivin cytoprotection to tumor progression, in vivo. This will involve kinetic analysis of human tumor growth in immunocompromised animals reconstituted with genetically defined cell lines, and modulation of multistage tumorigenesis using a novel transgenic mouse model with conditional expression of survivin or its mutant in pancreatic islet p cells. The overall application is designed to elucidate the molecular underpinnings of how survivin counteracts cell death in human tumors, and the crossroads signaling pathways associated with its functions, in vivo. This work will provide a mechanistic foundation for the validation and further development of molecular antagonists of the survivin pathway for novel cancer therapeutics in humans. ? ? ?
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