The Wnt/wingless (wg) pathway is an evolutionarily conserved cell-signaling pathway that regulates many aspects of metazoan development. Dysregulation of the Wnt pathway has been associated with tumorigenesis of the liver, colon, breast and skin. One of the most important effectors of the Wnt pathway is encoded by the transcription factor, ?-catenin (?-cat). Since Catenin Responsive Transcription (CRT) has been implicated in the genesis of many cancers, it makes a good target for developing therapeutics that could modulate the nuclear activity of ?-cat. Recently, we employed an innovative RNAi-based targeted chemical genetic high-throughput-screen (HTS) to identify novel and specific compound modulators of CRT in Drosophila and human cell lines. Objective/Hypothesis: We hypothesize that our primary screening strategy specifically targets CRT and that the novel compound modulators of the Wnt pathway could serve as effective therapeutic reagents in Wnt- relevant disease and developmental models. Specific Aims: 1) Determine (and improve) the specificity as well as efficacy of candidate small molecules in blocking CRT-induced/dependent phenotypes in cell-based assays; 2) Determine the molecular mechanisms by which candidate small molecules identified in the primary screen impact CRT and identify their protein targets; 3) Test the ability/efficacy of lead inhibitory compounds in blocking Wnt/CRT-dependent phenotypes in xenograft models, as well as in mouse models of Wnt-relevant cancers. Study design: The goal of the primary screen was to identify novel inhibitors of nuclear ?-cat activity that act downstream of the Axin-mediated degradation complex. In order to determine the mechanism of candidate small molecules, we will test their ability to alter ?-cat's interaction with its known protein interaction partners, or their ability to alter the DNA binding properties of ?-cat/TCF-transcriptional complex using co- immunoprecipitation, immunolocalization, and EMSA assays. We will utilize ELISA, and pull-down assays (with purified proteins), together with Surface Plasmon Resonance assays to determine direct binding of candidate compounds to purified ?-cat or other target proteins. We will validate the inhibitory effect of candidate compounds from our pilot screen in a variety of Wnt-responsive mammalian and cancer cell lines, including, HEK293 cells, C57mg mouse mammary epithelial cells, MCF7 human breast adenocarcinoma cell line, and the HCT116 & HT29 colon cancer cell lines. We will assess the effect of these compounds in blocking Wnt/CRT-induced tumor establishment and metastasis models in mouse xenografts, in vivo. We will also perform SAR studies coupled with in silico docking models to improve the efficacy/potency of the novel class of compounds identified in the primary screen. The improved candidate compounds will subsequently be validated empirically using the already optimized cell-based and in vivo assays for Wnt/CRT activity. PUBLIC HEALTH RELEVANCE: The overall goal of this proposal is to develop and implement innovative screening technologies for the identification of novel compound modulators of the Wnt/Wingless signaling pathway. Results from this study will have a broad impact in the development of targeted therapeutics for the treatment of Wnt-induced carcinogenesis.