Colon cancer is one of the most common forms of cancer in the Western world. The predominant initial genetic events leading to colorectal tumors are mutations in either the adenomatous polyposis coli (APC) tumor suppressor gene or beta-catenin. These mutations result in the stabilization of cytosolic beta-catenin and subsequent association with the LEF/TCF transcription factors, leading to changes in gene expression. Several studies validate TCF mediated transcription as a potential target for colon cancer treatment. The objective of this proposal is to identify compounds that block LEF/TCF mediated transcription using a high-throughput cell-based assay. We intend to use computer-aided rational drug design to screen for compounds with the potential to interact with the Armadillo repeat region of beta-catenin, the site at which TCF binds to form a transcriptional activator. Following assay development, optimization and validation, the assay will be used to screen the putative beta-catenin- interacting compounds, as well as a pilot screen of the NCI Natural Products Repository. The high-throughput assay will utilize the HT-29 cell line, a human colon carcinoma in which both APC alleles are truncated. We will generate a line of HT-29 cells expressing destabilized green fluorescent protein variants dECFP and dEYFP, under the control of TCF dependent and TCF independent promoters. This cell line will be used to optimize conditions for monitoring the fluorescent proteins in live cells using a 96-well microplate reader. The dECFP and dEYFP proteins have short half-lives and can be detected and quantified independently in the same cell. This will allow us to screen for compounds that specifically turn off TCF-dependent transcription and eliminate those that have no effect, cause a general down-regulation of transcription, or are toxic. The assay has the potential to detect compounds that can disrupt TCF-specific transcription at multiple sites, including blocking beta-catenin/TCF interaction, interfering with the transcriptional activation domain, as well as increasing beta-catenin turnover. A variety of tumors are caused by mutations resulting in the stabilization of beta-catenin, including melanoma, esophageal, gastric, hepatoblastoma and medulloblastoma, as well as others. The lead compounds identified using the above assay, could potentially be useful in treating these other tumors as well as colon cancer.
| Sussman, Daniel J (2002) LEF/TCF-dependent, fluorescence-based reporter gene assay for Wnt signaling. Biotechniques 32:1000, 1002 |
