Fábio Pereira

Snail2 cooperates with Snail1 in the repression of vitamin D receptor in colon cancer

Vitamin D receptor (VDR) mediates the antitumoral action of the active vitamin D metabolite 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). VDR expression is lost during colon cancer progression causing unresponsiveness to 1,25(OH)(2)D(3) and its analogs. Previously, Snail1, an inducer of epithelial-to-mesenchymal transition (EMT), was reported to inhibit VDR expression. Here, we show that Snail2/Slug, but not other EMT inducers such as Zeb1, Zeb2, E47 or Twist1, represses VDR gene promoter. Moreover, Snail2 and Snail1 show additive repressing effect on VDR promoter. Snail2 inhibits VDR RNA and protein and blocks the induction of E-cadherin and an adhesive phenotype by 1,25(OH)(2)D(3). Snail2 reduces the ligand-induced VDR transcriptional activation of a consensus response element and of the CYP24 promoter. Concordantly, Snail2 inhibits the induction of CYP24 RNA and p21(CIP1), filamin A and vinculin proteins and the repression of c-MYC by 1,25(OH)(2)D(3). Additionally, Snail2 abrogates beta-catenin nuclear export and the antagonism of the transcriptional activity of beta-catenin-T-cell factor complexes by 1,25(OH)(2)D(3). SNAI2 expression is upregulated in 58% of colorectal tumors and correlates inversely with that of VDR. However, VDR downregulation is higher in tumors coexpressing SNAI2 and SNAI1 than in those expressing only one of these genes. Together, these data indicate that Snail2 and Snail1 cooperate for VDR repression in colon cancer.

KDM6B/JMJD3 histone demethylase is induced by vitamin D and modulates its effects in colon cancer 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 D(3) (1,25(OH)(2)D(3)) and that JMJD3 modulates the gene regulatory action of this hormone. 1,25(OH)(2)D(3) 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)(2)D(3) 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)(2)D(3). 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)(2)D(3) on human colon.

Vitamin D has wide regulatory effects on histone demethylase genes

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 D3 [1,25(OH)2D3, 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)2D3 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)2D3 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)2D3 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)2D3 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.

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

SPROUTY-2 (SPRY2) regulates receptor tyrosine kinase signalling and therefore cell growth and differentiation. In this study, we show that SPRY2 expression in colon cancer cells is inhibited by the active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) through E-cadherin-dependent and -independent mechanisms. In turn, SPRY2 represses both basal and 1,25(OH)2D3-induced E-cadherin expression. In line with this, SPRY2 induces ZEB1 RNA and protein, but not that of other epithelial-to-mesenchymal transition inducers that repress the CDH1/E-cadherin promoter. Consistently, SPRY2 and E-cadherin protein levels inversely correlate in colon cancer cell lines and xenografted tumours. Moreover, SPRY2 knockdown by small hairpin RNA increases CDH1/E-cadherin expression and, reciprocally, CDH1/E-cadherin knockdown increases that of SPRY2. In colon cancer patients, SPRY2 is upregulated in undifferentiated high-grade tumours and at the invasive front of low-grade carcinomas. Quantification of protein expression in 34 tumours confirmed an inverse correlation between SPRY2 and E-cadherin. Our data demonstrate a tumourigenic action of SPRY2 that is based on the repression of E-cadherin, probably by the induction of ZEB1, and a reciprocal regulation of SPRY2 and E-cadherin that dictates cell phenotype. We propose SPRY2 as a candidate novel marker for high-grade tumours and a target of therapeutic intervention in colon cancer.

Hybrid Cluster Proteins and Flavodiiron Proteins Afford Protection to Desulfovibrio vulgaris upon Macrophage Infection

Desulfovibrio species are Gram-negative anaerobic sulfate-reducing bacteria that colonize the human gut. Recently, Desulfovibrio spp. have been implicated in gastrointestinal diseases and shown to stimulate the epithelial immune response, leading to increased production of inflammatory cytokines by macrophages. Activated macrophages are key cells of the immune system that impose nitrosative stress during phagocytosis. Hence, we have analyzed the in vitro and in vivo responses of Desulfovibrio vulgaris Hildenborough to nitric oxide (NO) and the role of the hybrid cluster proteins (HCP1 and HCP2) and rubredoxin oxygen oxidoreductases (ROO1 and ROO2) in NO protection. Among the four genes, hcp2 was the gene most highly induced by NO, and the hcp2 transposon mutant exhibited the lowest viability under conditions of NO stress. Studies in murine macrophages revealed that D. vulgaris survives incubation with these phagocytes and triggers NO production at levels similar to those stimulated by the cytokine gamma interferon (IFN-γ). Furthermore, D. vulgaris hcp and roo mutants exhibited reduced viability when incubated with macrophages, revealing that these gene products contribute to the survival of D. vulgaris during macrophage infection.

Abstract 291: Vitamin D induces KDM6B/JMJD3 histone demethylase gene in human colorectal cancer cells

Epidemiological and preclinical studies have shown that the active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) has protective effects against colorectal cancer (CRC). Histone tails are subjected to a variety of post-translational modifications, including methylation, which play an important gene regulatory role in development and physiology. Misregulation of histone modifications alters gene expression and leads to perturbations of the cellular identity, which may contribute to cancer initiation, progression and/or metastasis. We have found that 1,25(OH)2D3 regulates the expression of JMJD3 gene (also known as KDM6B) that encodes for a histone H3 lysine 27 demethylase with an important gene regulatory role in development and physiology. 1,25(OH)2D3 induces JMJD3 RNA expression in a time- and dose-dependent manner, activates the JMJD3 gene promoter and increases the level of JMJD3 RNA in human cancer cells of colon, breast and skin origin. JMJD3 upregulation is strictly dependent on vitamin D receptor (VDR) expression and is abolished by the protein synthesis inhibitor cycloheximide. We have also shown that JMJD3 modulates the gene regulatory action of 1,25(OH)2D3. In SW480-ADH CRC cells, JMJD3 knockdown by short-hairpin RNA or expression of an inactive mutant JMJD3 fragment decreases the induction by 1,25(OH)2D3 of CYP24A1, CDH1/E-cadherin and CST5/cystatin D genes and of an epithelial adhesive phenotype. Moreover, JMJD3 knockdown upregulates the epithelial-mesenchymal transition inducers SNAI1, ZEB1 and ZEB2 and the mesenchymal markers FN1/fibronectin 1 and LEF1, while it downregulates the epithelial genes CDH1/E-cadherin, CLDN1/claudin-1 and CLDN7/claudin-7. Additionally, JMJD3 knockdown blunts the nuclear export of β-catenin and abolishes the inhibition of β-catenin transcriptional activity caused by 1,25(OH)2D3. In line with data in cultured cells, the expression level of JMJD3 RNA is lower in tumour than in normal tissue in 56% of the CRC patients studied and correlates directly with VDR and inversely with SNAI1 RNA expression. Together, our results indicate that an epigenetic gene coding for a histone demethylase such as JMJD3 is a VDR target that partially mediate the effects of 1,25(OH)2D3 on human CRC cells. 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 291. doi:1538-7445.AM2012-291

Abstract 1067: SPROUTY-2 represses E-cadherin expression and vitamin D action in colorectal cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Colorectal cancer (CRC) is one of the highest in prevalence and mortality worldwide. Epidemiological and preclinical studies indicate that 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3), the most active metabolite of vitamin D, and several analogs have protective effects against CRC. Previously we have reported that 1,25(OH)2D3 inhibits the proliferation and promotes the differentiation of human colon cancer cells at least in part by the induction of the adhesion molecule E-cadherin and the antagonism of the Wnt/β-catenin pathway (Palmer et al., J. Cell Biol., 2001; Aguilera et al., Carcinogenesis, 2007). A transcriptomic analysis revealed a decrease in the RNA level of SPROUTY-2 (SPRY2), a gene that modulates signaling mediated by several tyrosine kinase receptors following treatment of human SW480-ADH cells with 1,25(OH)2D3 (5.6-fold at 4 h post-treatment; Palmer et al., Cancer Res., 2003). Methods: Protein expression in cell lines were done by Western blot analysis. RNA expression were performed by quantitative RT-PCR. To knock-down gene expression, cells were infected with lentiviral particles containing a U6 promoter driving an shRNA targeting the respective RNA. Protein expression in human colon tumors were performed by immunofluorescence. Cell proliferation was measure by MTT assay. Results: In this study, we show that the expression of SPRY2 is inhibited by 1,25(OH)2D3 in colon cancer cells by E-cadherin-dependent and -independent mechanisms. In turn, ectopic expression of SPRY2 in SW480-ADH cells inhibits both basal and 1,25(OH)2D3-induced E-cadherin expression. SPRY2 induces ZEB-1 RNA and protein, a transcriptional repressor of CDH1/E-cadherin, and prevents the induction of an adhesive phenotype by 1,25(OH)2D3. A study of the mutual repression between SPRY2 and E-cadherin proteins revealed that their expression levels are inversely correlated in colon cancer cell lines and in xenografted tumors. In colon cancer patients, SPRY2 expression correlates also inversely with that of E-cadherin and is upregulated in invasive areas and high grade tumors. In addition, the ectopic expression of SPRY2 precludes other 1,25(OH)2D3 actions such as the antagonism of Wnt/beta-catenin signaling and the inhibition of cell proliferation. Conclusions: Our data indicate that SPRY2 is a negative regulator of E-cadherin and 1,25(OH)2D3 action and has a putative tumorigenic action in colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1067. doi:10.1158/1538-7445.AM2011-1067