Genetic Analysis of Beta-Catenin Function in Human CA

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Oncogenic activation of g-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 13-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 13-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 fS-catenin genes. Initial functional studies have shed light on several aspects of the function of 13-catenin in human cancer. In particular, we have shown that activated 13-catenin is necessary for TCF-mediated transcriptional activation in human cancer cells. We have also demonstrated that deletion of activated g-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 13-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 13-catenin, as detected by Western Blot and immunocytochemistry analysis. Finally, we have identified and characterized BMP-4, a TGF-f3 homolog, as a putative effector of activated 13-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 13-catenin in the pathogenesis of human cancer. In particular, we plan to: 1) Determine the cell biological and pathobiologieal phenotypes of activated 13-catenin in human cancer cells and tissues, 2) Determine whether oncogenic activation of/3-catenin is required for maintenance of tumorigenicity in human cancer cells containing multiple genetic hits, 3) Determine the biochemical mechanism(s) of g-catenin activation in human cancer, and 4) Identify and characterize genes regulated by the activated B-catenin transcription factor.