Abstract 3084: Constitutively active RAS signaling reduces 1,25 dihydroxyvitamin D-mediated gene transcription by suppressing vitamin D receptor gene expression in mouse colon epithelial cells

Abstract

Abstract The active metabolite of vitamin D, 1,25 dihydroxyvitamin D (1,25(OH)2D), binds to the vitamin D receptor (VDR) and stimulates anti-cancer transcriptional events in many cell lines including colon cancer cells. However, we found that tumors which form in the distal colon of mice with an APC allele deletion had significantly lower VDR mRNA levels compared to RNA isolated from the distal colon of wild-type mice (-92%, p≤0.05). These data suggest that colon carcinogenesis may impair 1,25(OH)2D-regulated chemoprevention in colon epithelial cells. APC inactivation and KRAS activation are early events in the development of distal colon that may suppress VDR gene expression. Compared to immortalized colonic epithelial cells derived from the “Immorto” mouse (YAMC), cells from a cross between APCmin and “Immorto” mice (IMCE), and YAMC cells overexpressing the v-Ha-RAS gene (YAMC-RAS) have significantly lower VDR mRNA (-80%; p≤0.05) expression. Previous research shows that constitutively active (ca) RAS signaling suppresses 1,25(OH)2D action in many cell lines so we conducted additional studies on the impact of this mutation. YAMC-RAS cells had a significant reduction in 1,25(OH)2D-induced CYP24 mRNA expression (-40%; p≤0.05). Consistent with lower VDR mRNA levels, YAMC-RAS cells also had lower VDR protein levels than YAMC cells and the suppression of 1,25(OH)2D-induced CYP24 expression in YAMC-RAS was overcome with VDR over-expression. The mechanism for RAS-mediated suppression of VDR mRNA levels was further explored. First we hypothesized that caRAS signaling expression reduced VDR mRNA stability, however, YAMC and YAMC-RAS cells treated with 4 µg/ml of the transcription inhibitor actinomycin D for up to 12 h had no significant difference in the VDR mRNA half-life (about 8 h). Next we examined whether caRAS signaling reduced accessibility within chromosomal regions in the VDR gene previously identified as necessary for VDR gene expression. Using a DNAse I hypersensitivity assay we found that in YAMC-RAS cells there was a significant reduction (p≤0.05) in chromosome accessibility on the proximal promoter (-23%, 300 bp upstream of exon 1), an enhancer region located 6 kb upstream of the transcriptional start site (-56%), and an enhancer region located in exon 2 (-100%) compared to the YAMC cells. These data show that caRAS signaling suppresses 1,25(OH)2D-mediated gene transcription in colon epithelial cells by reducing VDR gene transcription. Supported by NIDDK Award DK54111and AICR Award 09A098 to JCF Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3084. doi:1538-7445.AM2012-3084