A High-Content Imaging Screen For Cellular Regulators Of -Catenin Protein Abundance

Abnormal accumulation of -catenin protein, a key transcriptional activator required for Wnt signaling, is the hallmark of many tumor types, including colon cancer. In normal cells, -catenin protein level is tightly controlled by a multiprotein complex through the proteosome pathway. Mutations in the components of the -catenin degradation complex, such as adenomatous polyposis coli (APC) and Axin, lead to -catenin stabilization and the constitutive activation of target genes. Since the signal transduction of Wnt/-catenin is mainly mediated by protein-protein interactions, this pathway has been particularly refractory to conventional target-based small-molecule screening. Here we designed a cellular high-content imaging assay to detect -catenin protein through immunofluorescent staining in the SW480 colon cancer cell line, which has elevated -catenin endogenously. We demonstrate that the assay is robust and specific to screen a focused biologically diverse chemical library set against known targets that play diverse cellular functions. We identified a number of hits that reduce -catenin levels without causing cell death. These hits may serve as tools to understand the dynamics of -catenin degradation. This study demonstrates that detecting cell-based -catenin protein stability is a viable approach to identifying novel mechanisms of -catenin regulation as well as small molecules of therapeutic potential.