Vitamin D and APC modulate molecular crosstalk between β-catenin/Wnt signaling and the vitamin D receptor in colon cancer cells

A151

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.