The Wnt antagonist DICKKOPF-1 gene is induced by 1 ,25-dihydroxyvitamin D3 associated to the differentiation of human colon cancer cells

Abstract

The Wnt-beta-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 1alpha,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.

SPROUTY-2 and E-cadherin regulate reciprocally and dictate colon cancer cell tumourigenicity

“…To further investigate this, we studied SPRY2 expression in SW480-ADH cells stably expressing an exogenous E-cadherin gene (SW480-ADH-E-cadh). These cells contain high levels of E-cadherin and show strong adhesiveness (Aguilera et al, 2007). In the absence of 1,25(OH) 2 D 3 , the level of SPRY2 RNA and protein was lower in SW480-ADH-E-cadh cells than in Mock cells (Figures 4d and e).…”

“…SW480-ADH-E-cadh cells were previously described (Aguilera et al, 2007). All experiments using 1,25(OH) 2 D 3 (provided by Dr R Bouillon and A Verstuyf, Leuven, Belgium, and Dr JP van de Velde, Weesp, The Netherlands) were performed in medium supplemented with charcoal-treated serum.…”

“…We have reported that 1,25(OH) 2 D 3 inhibits the proliferation and promotes the differentiation of human colon cancer cells by the induction of the adhesion molecule E-cadherin and the antagonism of the Wnt/ b-catenin pathway (Pa´lmer et al, 2001;Aguilera et al, 2007)). A transcriptomic analysis revealed a decrease in the level of SPROUTY-2 (SPRY2) RNA on treatment of SW480-ADH cells with 1,25(OH) 2 D 3 (5.6-fold at 4 h) (Pa´lmer et al, 2003).…”

SPROUTY-2 represses the epithelial phenotype of colon carcinoma cells via upregulation of ZEB1 mediated by ETS1 and miR-200/miR-150

“…52 ZEB1 silencing in SPRY2-wt and Mock cells, and ETS1 silencing in SPRY2-wt cells was carried out using the lentiviral vector pGIPz containing different shRNA mir sequences: ZEB1, V3LHS_356186 (sh1), V3LHS_356187 (sh2) and V2LHS_226625 (sh3); ETS1, V2LHS_244928 (sh1) and V3LHS_301171 (sh2) (Dharmacon, GE Healthcare, Pittsburgh, PA, USA). Non-silencing shRNA mir sequence (NS), with no homology to known mammalian genes was used as control, cloned in pGIPz vector (Dharmacon).…”

Paricalcitol Enhances the Chemopreventive Efficacy of 5-Fluorouracil on an Intermediate-Term Model of Azoxymethane-Induced Colorectal Tumors in Rats

“…At this point, approximately 80% of all human colorectal cancers have aberrant overactivations in Wnt/b-catenin signal and its downstream components in the colorectal cells (25). On the other hand, the expression pattern of Dkk-1, an inhibitor of Wnt/b-catenin pathway, by blocking Wnt signaling receptor complexes and contributing to colon cancer suppression, is remarkably downregulated in the colonic biopsies of colorectal cancer patients, and its downregulation is disclosed as a biomarker of chemoresistance and poor clinical outcome (25)(26)(27)(28)(29). Although the overall available data still have discrepancies, Dkk-1 has been found to not only act as an inhibitor of Wnt/ b-catenin signaling but also has additional b-catenin-independent tumor suppressor, antiangiogenesis, and antimetastasis actions in colorectal cancer disease (27,41).…”