Vitamin D is implicated as a protective factor in human colon carcinogenesis. Anti-tumor activity of vitamin D on colonocytes is thought to be mediated by 1,25 (OH)2D3, the active hormonal metabolite of vitamin D. We examined the role of 1,25 (OH)2D3on a putative VDR/β-catenin interaction. We first sought to confirm that VDR associates physically with β-catenin regulating critical Wnt signaling events in colon tumorigenesis. Second, we assessed the role of the intact and mutant adenomatous polyposis coli (APC) gene, commonly dysregulated in human colon cancers, as a modulator of the VDR/β-catenin interaction. Glutathione-S-transferase (GST) pulldown assays and an in-vitro transcription/translation system to produce β-catenin protein support an interaction between β-catenin and VDR that appears independent of 1,25 (OH)2D3. Further evaluation of this interaction within the cell using a mammalian two hybrid system in transfected HT29-APC, a cell line which allows inducible wildtype APC protein, suggest that APC positively influences the interaction between β-catenin and VDR, and that this association is greatly enhanced in the presence of 1,25 (OH)2D3. Co-immunoprecipitation of HT29-APC cellular extracts was also used to assess the hormone dependence of the interaction and role of APC. Co-IP studies demonstrated further that binding of β-catenin and VDR is observed in the absence of 1,25 (OH)2D3 but that the addition of APC and 1,25 (OH)2D3 similarly enhanced levels of protein-protein interaction. To determine the functional significance of VDR and 1,25 (OH)2D3 effect on the transcriptional activity of β-catenin , a TOPflash assay is being implemented in HT-29 cells. Our results support the presence of a VDR and β-catenin interaction that occurs independent of 1,25 (OH)2D3 and that appears to be significantly enhanced in the presence of both the hormone and intact APC. These data suggest an APC/VDR binary or ternary complex with β-catenin with early results implicating degradation of the protumorigenic β-catenin as a consequence. This represents a novel crosstalk mechanism whereby 1,25 (OH)2D3-VDR and APC could negatively regulate the Wnt signaling pathway to restrict tumorigenesis.
Sixth AACR International Conference on Frontiers in Cancer Prevention Research-- Dec 5-8, 2007; Philadelphia, PA