A model system to study the tumor suppressor APC and its role in Wnt regulation.

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DESCRIPTION (provided by applicant): To properly orchestrate the dramatic events that occur during animal development, it is essential that cells communicate with their neighbors through the use of cell signaling pathways. While signaling pathways play critical roles in normal developmental processes, inappropriate activation of these pathways frequently results in cancer. The Wnt signaling pathway provides a paradigm for how misregulation of cell signaling pathways can contribute to oncogenesis. Loss-of-function mutations in the tumor suppressor protein, Adenomatous Polyposis Coli (APC), inappropriately activate the pathway and frequently initiate cancer progression. Mutations in APC are responsible for over 80% of all colon cancer cases, thus it is clear that understanding APC biology is crucial to our efforts to treat the disease. To truly understand how inactivation of APC initiates the oncogenic phenotype, it is first crucial to understand how APC contributes to normal Wnt regulation. In the current model of Wnt signaling, APC participates in a multi-protein complex (called the destruction complex) that targets the key effector protein ¿-catenin for ubiquitin-mediated proteolysis. In the absence of APC, the destruction complex is inactivated by an unknown mechanism, and ¿-catenin protein levels hyper- accumulate. Together with its nuclear partners (members of the TCF/LEF family), ¿-catenin functions as a bi- partite transcription factor, promoting expression of Wnt target genes such as c-myc and cyclinD1 that drive cell proliferation. While it is clear that APC participates in the ¿-catenin destruction complex, its mechanistic role in this complex has remained elusive. In the past decade, several models have been proposed to explain APC's mechanistic function in the ¿- catenin destruction complex. While these models are based on convincing biochemical data, they remain to be rigorously tested using functional studies. During my postdoctoral research, I began to address some models of APC function by performing an in vivo structure/function analysis of APC. This study uncovered several surprising findings, including the identification of two largely uncharacterized regions of APC essential for its function in Wnt regulation. Overall, this structure/function approach provided important insight into the destruction complex, and suggested an alternative model of its inner workings. Here, I expand upon these initial findings and propose experiments that will directly evaluate three prominent biochemical models of APC function, and are informed by my prior structure/function study. For these experiments, I propose to use Drosophila APC2 as a model for human APC, as members of the Wnt pathway are highly conserved throughout evolution. Moreover, I have previously shown that Drosophila APC2 works as effectively as human APC in functional studies when expressed in human colon cancer cell lines. Thus we can take advantage of the speed of this simpler genetic model system to investigate questions important to the progression and treatment of human colon cancer.