| Wnt/beta-catenin signaling regulates many aspects of metazoan development and adult homeostasis. Aberrant Wnt signaling has been linked to numerous pathological conditions. A thorough understanding of the Wnt/beta-catenin signal transduction events is crucial to the development of therapies that may be therapeutic in these pathological conditions. In the last few years, high-throughput screening of both chemicals and siRNAs has identified several novel protein regulators of Wnt/beta-catenin signaling. The research outlined in this dissertation describes the construction and characterization of a robust reporter of Wnt/beta-catenin signaling and its application in both high-throughput small molecule and siRNA screens to identify, Bruton's tyrosine kinase (BTK), metabotropic glutamate receptor 1 (GRM1), and the mevalonate synthesis pathway as regulators of Wnt/beta-catenin signaling.;After introducing various aspects of Wnt/beta-catenin signaling in Chapter 1, I describe, in Chapter 2, the construction and detailed application of a new Wnt/beta-catenin reporter system, beta-catenin Activated Reporter (BAR). This reporter system has set the stage for numerous high-throughput screens in several disease relevant cell types and uncovered several novel regulators of Wnt/beta-catenin signaling. In Chapter 3, I describe an integrated screening approach that combined small molecule, siRNA, and proteomics screen data to identify BTK (mutated in X-linked agammaglobulinemia (XLA)) as a negative regulator of Wnt/beta-catenin signaling. Proteomic analysis identified CDC73, a know regulator of Wnt/beta-catenin signaling, as a BTK interacting protein. We then show that BTK regulates the stability of CDC73 and that CDC73 protein levels are significantly reduced in XLA patient beta-cells. In Chapter 4. I describe a screen of human experienced drugs for small-molecule regulators of Wnt/beta-catenin signaling. We identified several small molecule enhancers and characterize riluzole and the mevalonate synthesis pathway inhibitors lovastatin, fluvastatin, and alendronate in the context of Wnt/beta-catenin signaling in melanoma. We show that these drugs enhance the effects of Wnt/beta-catenin signaling to upregulate markers of melanocyte differentiation and those lost during melanoma progression, decreases proliferation of melanoma cells and reduces metastatic disease in a mouse model. In Chapter 5, I discuss the future directions of this work. |
