The overall objective of the proposed work is to understand how the Wnt signal transduction pathway contributes to tumorigenesis. Their long term goal is to define how Wnt proteins and their downstream signaling components modulate cell growth. The general strategy will be to conduct biochemical and genetic analyses of the Wnt-signaling cascade in cultured mammary epithelial, colon, and other cell types. They will identify Wnt signaling components that induce cellular transformation and regulatory molecules that suppress Wnt-mediated transformation. Their focus is on disheveled, glycogen synthase kinase-3, beta-catenin, adenomatous polyposis coli (APC), axin (fused), TCF/Lef and on the potential interactions between Wnt and signaling cascades that include Akt and RhoA. Which of these signaling components are required for Wnt- mediated transformation will be evaluated by ectopically expressing their genes in cell lines that are sensitive to transformation by Wnt-1. The cell lines used in this study respond morphologically and mitogenically to Wnt-1 stimulation. Measurement of distinct biochemical activation steps will establish where in the signaling cascade novel components, like axin, function in Wnt signaling. The stability and signaling activity of cytosolic beta-catenin is tightly regulated by Wnt action. The applicant proposes to study the assembly and/or modification of beta-catenin complexes in the cytosol. These complexes will be analyzed in cells with Wnt signaling activated and compared to non-stimulated cells. The role of the newly discovered protein, axin, and the kinase Akt in Wnt signal transduction will be evaluated using both biological and biochemical assays. To define signal activation in human cancer we will use colon cancer cells with defined mutations in APC or beta-catenin to analyze how the suppression of GSK-3 kinase uncouples beta-catenin from APC-mediated turnover and how axin acts to regulate beta-catenin stability and/or function. Mutations in APC and beta-catenin lead to activation of Wnt signaling in colon cancer, melanoma, and possibly several other human cancers. Thus, the Wnt signaling pathway is linked to the pathology of human cancers. It clearly represents an important problem in cancer biology with increasing relevance to human disease.