Genetic Analysis of Beta-Catenin Function in Human CA

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DESCRIPTION (provided by applicant): Oncogenic activation of beta-catenin is common to the pathogenesis of a wide range of human cancers. As is the case for many cancer-related genes, some of the most valuable studies in beta-catenin biology have been undertaken in genetically tractable model organisms such as Drosophila melanogaster and Mus musculus. By exploiting recent technological advances in gene targeting it is now possible to undertake similar studies of gene function in human cancer cells themselves. To do this, we have created high-efficiency human promoterless beta-catenin gene-targeting vectors and employed them to create isogenic sets of human cancer cells that differ only in the presence or absence of their endogenous mutant and/or wild-type beta-catenin genes. Initial functional studies have shed light on several aspects of the function of beta-catenin in human cancer. In particular, we have shown that activated beta-catenin is necessary for TCF-mediated transcriptional activation in human cancer cells. We have also demonstrated that deletion of activated beta-catenin causes cancer cells to form dome-like structures, recapitulating a phenotype seen in differentiating Caco-2 cell monolayers. Pilot experiments have suggested that oncogenic beta-catenin is not required for the human colon cancer cells to form xenograft tumors in immunodeficient mice. Also surprisingly, we have detected no discernible differences in the steady state levels and intracellular localization of wild-type and mutant, activated beta-catenin, as detected by Western Blot and immunocytochemistry analysis. Finally, we have identified and characterized BMP-4, a TGF-beta homolog, as a putative effector of activated beta-catenin signaling in human cancer using Affymetrix Genechip analysis. We propose to further utilize these isogenic human cancer cell systems to study the functions of activated beta-catenin in the pathogenesis of human cancer. In particular, we plan to: 1) Determine the cell biological and pathobiological phenotypes of activated beta-catenin in human cancer cells and tissues, 2) Determine whether oncogenic activation of beta-catenin is required for maintenance of tumorigenicity in human cancer cells containing multiple genetic hits, 3) Determine the biochemical mechanism(s) of beta-catenin activation in human cancer, and 4) Identify and characterize genes regulated by the activated beta-catenin transcription factor.