The β-catenin signaling pathway is deregulated in nearly all colon cancers. Nonhypercalcemic vitamin D3 (1α,25-dehydroxyvitamin D3) analogues are candidate drugs to treat this neoplasia. We show that these compounds promote the differentiation of human colon carcinoma SW480 cells expressing vitamin D receptors (VDRs) (SW480-ADH) but not that of a malignant subline (SW480-R) or metastasic derivative (SW620) cells lacking VDR. 1α,25(OH)2D3 induced the expression of E-cadherin and other adhesion proteins (occludin, Zonula occludens [ZO]-1, ZO-2, vinculin) and promoted the translocation of β-catenin, plakoglobin, and ZO-1 from the nucleus to the plasma membrane. Ligand-activated VDR competed with T cell transcription factor (TCF)-4 for β-catenin binding. Accordingly, 1α,25(OH)2D3 repressed β-catenin–TCF-4 transcriptional activity. Moreover, VDR activity was enhanced by ectopic β-catenin and reduced by TCF-4. Also, 1α,25(OH)2D3 inhibited expression of β-catenin–TCF-4-responsive genes, c-myc, peroxisome proliferator-activated receptor δ, Tcf-1, and CD44, whereas it induced expression of ZO-1. Our results show that 1α,25(OH)2D3 induces E-cadherin and modulates β-catenin–TCF-4 target genes in a manner opposite to that of β-catenin, promoting the differentiation of colon carcinoma cells.

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Colorectal cancer is a major cause of cancer death worldwide. A number of key oncogenes and tumor suppressor genes have been proposed to drive progression from healthy colonic epithelia to malignant tumors, including members of the Wnt/beta-catenin pathway. Recently, CpG island promoter hypermethylation was shown to cause inactivation of two extracellular Wnt inhibitors in colon cancer: secreted frizzled-related proteins (sFRPs) and Wnt inhibitory factor-1 (WIF-1). Here, we show for the first time that another extracellular Wnt inhibitor, the DICKKOPF-1 (DKK-1) gene, is transcriptionally silenced by CpG island promoter hypermethylation in colon cancer cell lines (n=9), whereas treatment with the DNA-demethylating agent 5-aza-2-deoxycytidine restored DKK-1 expression. Restoration of DKK-1 function in non-expressing cells bearing a truncated APC (Adenomatous Polyposis Coli) gene had no effect on beta-catenin/T-cell factor-dependent transcription, but induced tumor suppressor-like features such as reduced colony formation density and tumor growth inhibition in nude mice. These results suggest additional functions for DKK-1 other than inhibiting canonical Wnt signaling. In primary colorectal tumors, DKK-1 was found hypermethylated in 17% (nine of 54) of cases. Furthermore, while for both SFRP-1 and WIF-1 methylation-associated silencing occurred across the whole spectrum of colorectal tumorigenesis, DKK-1 promoter was selectively hypermethylated in advanced colorectal neoplasms (Duke's C and D tumors).

The activity of c-Jun, the major component of the transcription factor AP-1, is potentiated by amino-terminal phosphorylation on serines 63 and 73 (Ser-63/73). This phosphorylation is mediated by the Jun amino-terminal kinase (JNK) and required to recruit the transcriptional coactivator CREB-binding protein (CBP). AP-1 function is antagonized by activated members of the steroid/thyroid hormone receptor superfamily. Recently, a competition for CBP has been proposed as a mechanism for this antagonism. Here we present evidence that hormone-activated nuclear receptors prevent c-Jun phosphorylation on Ser-63/73 and, consequently, AP-1 activation, by blocking the induction of the JNK signaling cascade. Consistently, nuclear receptors also antagonize other JNK-activated transcription factors such as Elk-1 and ATF-2. Interference with the JNK signaling pathway represents a novel mechanism by which nuclear hormone receptors antagonize AP-1. This mechanism is based on the blockade of the AP-1 activation step, which is a requisite to interact with CBP. In addition to acting directly on gene transcription, regulation of the JNK cascade activity constitutes an alternative mode whereby steroids and retinoids may control cell fate and conduct their pharmacological actions as immunosupressive, anti-inflammatory, and antineoplastic agents. Lipophilic hormones such as steroids, retinoic acid (RA), thyroid hormone (T3), and vitamin D mediate most, if not all, of their actions through specific intracellular receptors that are members of the nuclear receptor superfamily (Mangelsdorf et al. 1995). Nuclear hormone receptors are ligand-regulated sequence-specific transcription factors that may activate or repress gene expression. Ligand-activated gene transcription is generally mediated by binding of nuclear receptors to their cognate DNA elements. Though negative binding elements have been described, repression is mainly conducted by interference with other transcription factors, of which AP-1 is one of the most representative (for review, see Saatcioglu et al. 1994).AP-1 is a sequence-specific transcription factor composed of either homo-or heterodimers among members within the Jun family (c-Jun, JunB, and JunD) or among proteins of the Jun and Fos (c-Fos, FosB, Fra1, and Fra2) families (for review, see Angel and Karin 1991). Among them, c-Jun is the major component of the AP-1 complex (Bohmann et al. 1987;Angel et al. 1988) and c-Fos is its best known partner. AP-1 is activated by mitogens, oncoproteins, cytokines, and stress agents such as ultraviolet (UV) light. AP-1 activation may be mediated by both transcriptionally independent and -dependent mechanisms, involving post-translational modifications of its components or increases in the expression of their corresponding genes, respectively (Angel and Karin 1991;Karin et al. 1997).c-Jun transcriptional activity is enhanced by aminoterminal phosphorylation on Ser-63/73 (Pulverer et al. 1991;Smeal et al. 1991). This inducible phosphorylation is mediated by members of the Jun amino-te...

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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.

Secreted signaling proteins of the Wnt family are known to regulate a diverse range of developmental processes, and their signaling pathway through b-catenin is frequently activated in cancer. The identification of both Frizzled and LRP5/6 (LRP: low-density lipoprotein receptor-related protein) proteins as components of cellsurface receptors for Wnt proteins has raised questions about their individual functions. We have investigated this issue through a structure-function analysis of Frizzled and LRP proteins that have been implicated in Wnt1 signaling. Consistent with other reports, we find that LRP6/Arrow proteins deleted for their extracellular domain are able to activate the Wnt/b-catenin signaling pathway. Importantly, our results demonstrate that this signaling from LRP6/Arrow derivatives can occur in a Frizzled-and ligand-independent manner. Furthermore, we show that the PPSP motifs within the intracellular domain of LRP6 are required for signaling. In contrast to results with LRP6, overexpression of Frizzled proteins did not activate the pathway. Based on evidence of ligand binding to both Frizzled and LRP6, current models suggest that both proteins are components of a Wnt receptor complex that signals to b-catenin. In light of these models, our data imply that LRP5/6/Arrow proteins constitute the distal signal-initiating component of these receptors. The results also support the notion that LRP5/ 6 are candidate oncogenes.