The Wnt–β-catenin pathway is aberrantly activated in most colon cancers. DICKKOPF-1 (DKK-1) gene encodes an extracellular Wnt inhibitor that blocks the formation of signalling receptor complexes at the plasma membrane. We report that 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], the most active vitamin D metabolite, increases the level of DKK-1 RNA and protein in human SW480-ADH colon cancer cells. This effect is dose dependent, slow and depends on the presence of a transcription-competent nuclear vitamin D receptor (VDR). Accordingly, 1,25(OH)₂D₃ activates a 2300 bp fragment of the human DKK-1 gene promoter. Chromatin immunoprecipitation assays revealed that 1,25(OH)₂D₃ treatment induced a pattern of histone modifications which is compatible with transcriptionally active chromatin. DKK-1 is expressed at high level in colon cancer cell lines with a differentiated phenotype such as Caco-2 or HT-29. Exogenous expression of E-cadherin into SW480-ADH cells results in a strong adhesive phenotype and a 17-fold increase in DKK-1 RNA. In contrast, an E-cadherin blocking antibody inhibits 1,25(OH)₂D₃-induced differentiation of SW480-ADH cells and DKK-1 gene expression. Remarkably, in vivo treatment with the vitamin D analogue EB1089 induced DKK-1 protein expression in SW480-ADH cells xenografted in immunodeficient mice, and a correlation was observed in the expression of VDR and DKK-1 RNA in a series of 32 human colorectal tumours. These data indicate that 1,25(OH)₂D₃ activates the transcription of the DKK-1 gene, probably in an indirect way that is associated to the promotion of a differentiated phenotype. DKK-1 gene induction constitutes a novel mechanism of inhibition of Wnt signalling and antitumour action by 1,25(OH)₂D₃.

Objective: Colorectal cancer (CRC) is a major health concern. Vitamin D deficiency is associated with high CRC incidence and mortality, suggesting a protective effect of vitamin D against this disease. Given the strong influence of tumour stroma on cancer progression, we investigated the potential effects of the active vitamin D metabolite 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) on CRC stroma. Design: Expression of vitamin D receptor (VDR) and two 1,25(OH)₂D₃ target genes was analysed in 658 patients with CRC with prolonged clinical follow-up. 1,25(OH)₂D₃ effects on primary cultures of patient-derived colon normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs) were studied using collagen gel contraction and migration assays and global gene expression analyses. Publicly available data sets (n=877) were used to correlate the 1,25(OH)₂D₃-associated gene signature in CAFs with CRC outcome. Results: High VDR expression in tumour stromal fibroblasts was associated with better overall survival (OS) and progression-free survival in CRC, independently of its expression in carcinoma cells. 1,25(OH)₂D₃ inhibited the protumoural activation of NFs and CAFs and imposed in CAFs a 1,25(OH)₂D₃-associated gene signature that correlated with longer OS and disease-free survival in CRC. Furthermore, expression of two genes from the signature, CD82 and S100A4, correlated with stromal VDR expression and clinical outcome in our cohort of patients with CRC. Conclusions: 1,25(OH)₂D₃ has protective effects against CRC through the regulation of stromal fibroblasts. Accordingly, expression of VDR and 1,25(OH)₂D₃-associated gene signature in stromal fibroblasts predicts a favourable clinical outcome in CRC. Therefore, treatment of patients with CRC with VDR agonists could be explored even in the absence of VDR expression in carcinoma cells.

KDM6B/JMJD3 is a histone H3 lysine demethylase with an important gene regulatory role in development and physiology. Here, we show that human JMJD3 expression is induced by the active vitamin D metabolite 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) and that JMJD3 modulates the gene regulatory action of this hormone. 1,25(OH)₂D₃ activates the JMJD3 gene promoter and increases the level of JMJD3 RNA in human cancer cells. JMJD3 upregulation was strictly dependent on vitamin D receptor (VDR) expression and was abolished by cycloheximide. In SW480-ADH colon cancer cells, JMJD3 knockdown or expression of an inactive mutant JMJD3 fragment decreased the induction by 1,25(OH)₂D₃ of several target genes and of an epithelial adhesive phenotype. Moreover, JMJD3 knockdown upregulated the epithelial-to-mesenchymal transition inducers SNAIL1 and ZEB1 and the mesenchymal markers fibronectin and LEF1, while it downregulated the epithelial proteins E-cadherin, Claudin-1 and Claudin-7. Additionally, JMJD3 knockdown abolished the nuclear export of β-catenin and the inhibition of β-catenin transcriptional activity caused by 1,25(OH)₂D₃. Importantly, the expression of JMJD3 correlated directly with that of VDR and inversely with that of SNAI1 in a series of 96 human colon tumours. Our results indicate for the first time that an epigenetic gene coding for a histone demethylase such as JMJD3 is a VDR co-target that partially mediates the effects of 1,25(OH)₂D₃ on human colon.

The Wnt/b-catenin signaling pathway is abnormally activated in most colorectal cancers and in a proportion of other neoplasias. This activation initiates or contributes to carcinogenesis by regulating the expression of a large number of genes in tumor cells. The active vitamin D metabolite 1a,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) inhibits Wnt/b-catenin signaling by several mechanisms at different points along the pathway. Additionally, paracrine actions of 1,25(OH)₂D₃ on stromal cells may also repress this pathway in neighbouring tumor cells. Here we review the molecular basis for the various mechanisms by which 1,25(OH)₂D₃ antagonizes Wnt/b-catenin signaling, preferentially in human colon carcinoma cells, and the consequences of this inhibition for the phenotype and proliferation rate. The effect of the vitamin D system on Wnt/b-catenin signaling and tumor growth in animal models will also be commented in detail. Finally, we revise existing data on the relation between vitamin D receptor expression and vitamin D status and the expression of Wnt/b-catenin pathway genes and targets in cancer patients.

Vitamin D from the diet or synthesized in the skin upon UV-B irradiation is converted in the organism into the active metabolite 1α,25- dihydroxyvitamin D 3 [1,25(OH) 2D 3, calcitriol], a pleiotropic hormone with wide regulatory actions. The classical model of 1,25(OH)2D3 action implies the activation of the vitamin D receptor, which binds specific DNA sequences in its target genes and modulates their transcription rate. We have recently shown that 1,25(OH) 2D 3 induces the expression of the JMJD3 gene coding for a histone demethylase that is involved in epigenetic regulation. JMJD3 mediates the effects of 1,25(OH) 2D 3 on a subset of target genes and affects the expression of ZEB1, ZEB2 and SNAI1, inducers of epithelial-mesenchymal transition. Novel data indicate that 1,25(OH) 2D 3 has an unanticipated wide regulatory action on the expression of genes coding for histone demethylases of the Jumonji C (JmjC) domain and lysine-specific demethylase (LSD) families. Moreover, JMJD3 knockdown decreases the expression of miR‑200b and miR‑200c, two microRNAs targeting ZEB1 RNA. This may explain the upregulation of this transcription factor found in JMJD3-depleted cells. Thus, 1,25(OH) 2D 3 exerts an ample regulatory effect on the expression of histone-modifying enzymes involved in epigenetic regulation that may mediate its actions on gene transcription and cell phenotype.