Many studies in different biological systems have revealed that 1?,25-dihydroxyvitamin D3 (1?,25(OH)2D3) modulates signaling pathways triggered at the plasma membrane by agents such as Wnt, transforming growth factor (TGF)-?, epidermal growth factor (EGF), and others. In addition, 1?,25(OH)2D3 may affect gene expression by paracrine mechanisms that involve the regulation of cytokine or growth factor secretion by neighboring cells. Moreover, post-transcriptional and post-translational effects of 1?,25(OH)2D3 add to or overlap with its classical modulation of gene transcription rate. Together, these findings show that vitamin D receptor (VDR) cannot be considered only as a nuclear-acting, ligand-modulated transcription factor that binds to and controls the transcription of target genes. Instead, available data support the view that much of the complex biological activity of 1?,25(OH)2D3 resides in its capacity to interact with membrane-based signaling pathways and to modulate the expression and secretion of paracrine factors. Therefore, we propose that future research in the vitamin D field should focus on the interplay between 1?,25(OH)2D3 and agents that act at the plasma membrane, and on the analysis of intercellular communication. Global analyses such as RNA-Seq, transcriptomic arrays, and genome-wide ChIP are expected to dissect the interactions at the gene and molecular levels.
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.
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 1?,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 ?-catenin and the inhibition of ?-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.
1?,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and other vitamin D compounds are promising molecules in the prevention and therapy of colon cancer and other neoplasias. To study the mechanism of action of 1,25(OH)(2)D(3) in colon cancer cells, we carried out a comparative proteomic analysis of the nuclear fractions of SW480-ADH cells treated with 1,25(OH)(2)D(3) or vehicle during 8 or 48h. 2D-DIGE analysis combined with MALDI-TOF-TOF mass spectrometry interrogation led to the identification of 59 differentially expressed unique proteins. Most identified proteins were nuclear, but several cytoskeleton-associated proteins were also detected. A good concordance between changes in expression at protein and RNA levels was observed for the validated proteins. A large group of identified proteins, such as SFPQ, SMARCE, KHSRP, TARDBP and PARP1, were involved in RNA processing or modification and have been ascribed to the spliceosome compartment of human cells. In addition, a smaller group of proteins (ERM (Ezrin, Radixin, Moesin) family, VCL, CORO1C, ACTB) were cytoskeleton-associated and played a role in cell adhesion and morphology. These results confirm the induction of epithelial phenotype by 1,25(OH)(2)D(3) and suggest a role for vitamin D compounds in the regulation of the spliceosome and thus, in alternative splicing and possibly microRNA synthesis in colon cancer cells.
Aberrant activation of the Wnt/?-catenin pathway is critical for the initiation and progression of most colon cancers. This activation provokes the accumulation of nuclear ?-catenin and the induction of its target genes. Apc(min/+) mice are the most commonly used model for colon cancer. They harbor a mutated Apc allele and develop intestinal adenomas and carcinomas during the first months of life. This phenotype is caused by the mutation of the second Apc allele and the consequent accumulation of nuclear ?-catenin in the affected cells. Here we describe that vitamin D receptor (VDR) is a crucial modulator of nuclear ?-catenin levels in colon cancer in vivo. By appropriate breeding of Apc(min/+) mice and Vdr(+/-) mice we have generated animals expressing a mutated Apc allele and two, one, or none Vdr wild type alleles. Lack of Vdr increased the number of colonic Aberrant Crypt Foci (ACF) but not that of adenomas or carcinomas in either small intestine or colon. Importantly, colon ACF and tumors of Apc(min/+)Vdr(-/-) mice had increased nuclear ?-catenin and the tumors reached a larger size than those of Apc(min/+)Vdr(+/+). Both ACF and carcinomas in Apc(min/+)Vdr(-/-) mice showed higher expression of ?-catenin/TCF target genes. In line with this, VDR knock-down in cultured human colon cancer cells enhanced ?-catenin nuclear content and target gene expression. Consistently, VDR depletion abrogated the capacity of 1,25(OH)(2)D(3) to promote the relocation of ?-catenin from the nucleus to the plasma membrane and to inhibit ?-catenin/TCF target genes. In conclusion, VDR controls the level of nuclear ?-catenin in colon cancer cells and can therefore attenuate the impact of oncogenic mutations that activate the Wnt/?-catenin pathway.
TWIST1 is a transcription factor that belongs to the family of basic helix-loop-helix proteins involved in epithelial-to-mesenchymal transition and invasion processes. The TWIST1 protein possesses oncogenic, drug-resistant, angiogenic and invasive properties, and has been related with several human tumors and other pathologies. Colorectal cancer is one of the tumors in which TWIST1 is over-expressed, but its involvement in the clinical outcome of the disease is still unclear. We tested, by RT-PCR, the expression levels of TWIST1 in normal and tumor paired-sample tissues from a series of 151 colorectal cancer patients, in order to investigate its prognostic value as a tumor marker. TWIST1 expression was restricted to tumor tissues (86.1%) and correlated with lymph node metastasis (LNM). Adjusted analysis showed that the expression levels of TWIST1 correlated with overall survival (OS) and disease-free survival (DFS). Importantly, TWIST1 expression levels predicted OS specifically at stages I and II. Moreover, patients with stage II tumors and high TWIST1 levels showed even shorter survival than patients with stage III tumors. These results suggest that TWIST1 expression levels could be a tumor indicator in stage II patients and help select patients at greater risk of poor prognosis who might benefit from adjuvant chemotherapy.
An improved synthetic route to 1?,25-dihydroxyvitamin D(3) des-side chain analogues 2?a and 2?b with substituents at C18 is reported, along with their biological activity. These analogues display significant antiproliferative effects toward MCF-7 breast cancer cells and prodifferentiation activity toward SW480-ADH colon cancer cells; they are also characterized by a greatly decreased calcemic profile. The crystal structure of the human vitamin?D receptor (hVDR) complexed to one of these analogues, 20(17?18)-abeo-1?,25-dihydroxy-22-homo-21-norvitamin D(3) (2?a) reveals that the side chain introduced at position C18 adopts the same orientation in the ligand binding pocket as the side chain of 1?,25-dihydroxyvitamin D(3).
Metastases are frequently found during colorectal cancer diagnoses and are the main determinants of clinical outcome. The lack of reliable models of metastases has precluded their mechanistic understanding and our capacity to improve outcome. We studied the effect of E-cadherin and Snail1 expression on metastagenesis in a colorectal cancer model. We microinjected SW480-ADH human colorectal cancer cells, transfected with an empty vector (Mock) or overexpressing Snail1 (Snail1(OE)) or E-cadherin (E-cadherin(OE)), in the ceca of nude mice (eight per group) and analyzed tumor growth, dissemination, and Snail1, E-cadherin, ?-catenin, and Presenilin1 (PS1) expression in local tumors and/or metastatic foci. Snail1(OE) cells disseminated only to lymph nodes, whereas Mock or E-cadherin(OE) cells spread to lymph nodes and peritoneums. Peritoneal tumor foci developed by E-cadherin(OE) cells presented an increase in E-cadherin proteolysis and nuclear translocation, and enhanced expression of proteolytically active PS1, which was linked to increased tumor growth and shortened mouse survival. Interestingly, local and lymph node tumors in mice bearing E-cadherin(OE) cells overexpressed E-cadherin, but they did not show E-cadherin proteolysis or nuclear translocation. Remarkably, E-cadherin nuclear translocation and enhanced expression of active PS1 were found in a patient with colorectal signet-ring cell carcinoma. In conclusion, we have established a colorectal cancer metastasis model in which E-cadherin proteolyis and nuclear translocation associates with aggressive foci growth only in the peritoneal microenvironment.
The transcription factor Snail1 induces epithelial-to-mesenchymal transition (EMT), a process responsible for the acquisition of invasiveness during tumorigenesis. Several transcriptomic studies have reported Snail1-regulated genes in different cell types, many of them involved in cell adhesion. However, only a few studies have used proteomics as a tool for the characterization of proteins mediating EMT.
Many studies support a protective action of vitamin D against colon cancer. 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) exerts wide gene regulatory effects in human colon cancer cells. We previously reported that 1,25(OH)2D3 increases cytosolic Ca2+ concentration and transiently activates RhoA and its effector the Rho-associated coiled-kinase (ROCK), and later p38MAPK-MSK. We found that the inhibition of ROCK signaling by Y27632 or that of MSK by Ro318220 prevent the formation of epithelioid islands of SW480-ADH cells by 1,25(OH)2D3 and disrupts the adhesive phenotype of HT29 cells. ROCK and MSK inhibition also abrogates the induction of 1,25(OH)2D3 24-hydroxylase (CYP24), E-cadherin, and vinculin and the repression of cyclin D1 by 1,25(OH)2D3. Moreover, 1,25(OH)2D3 does not promote the localization of the tight junction protein occludin at the plasma membrane in cells expressing a dominant negative RhoA (N19-RhoA). In addition, 1,25(OH)2D3 specifically increases the level of the cysteine protease-inhibitor cystatin D, whereas that of cystatin SN is unaffected. The increase of cystatin D protein caused by 1,25(OH)2D3 is abrogated in N19-RhoA cells. Thus, activation of the RhoA-ROCK-p38MAPK-MSK signaling pathway is essential for the regulation of the phenotype and of the CST5/cystatin D candidate tumor suppressor and other target genes by 1,25(OH)2D3 in colon cancer cells.
Vitamin D receptor (VDR) mediates the antitumoral action of the active vitamin D metabolite 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). However, VDR expression is lost during colon cancer progression, possibly causing unresponsiveness to 1,25(OH)2D3. Although several mechanisms responsible for resistance to 1,25(OH)2D3 action in different types of cancer had been reported, none explained the loss of VDR expression. We have found that the transcription factors Snail1 and Snail2, known as inducers of epithelial-to-mesenchymal transition (EMT), inhibit VDR expression and block 1,25(OH)2D3 action in colon cancer cells. Snail1 and Snail2 have an additive repressing effect on VDR gene promoter. These effects are specific to the Snail family, as other transcription factors that function as EMT inducers do not inhibit VDR expression in colon cancer cells. Moreover, we also found that the RNA expression of SNAI1 and SNAI2 is upregulated in human colorectal tumors and inversely correlates with that of VDR. Our results suggest that high levels of SNAIL1 and SNAIL2 are a probable cause of VDR downregulation and 1,25(OH)2D3 unresponsiveness in colon cancer. In addition, they may contribute to the improvement of protocols for the clinical use of vitamin D compounds, as they indicate that advanced colon cancer patients overexpressing SNAIL1 and SNAIL2 are not suitable candidates for this therapy.
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.
Structure-guided optimization was used to design new analogues of 1?,25-dihydroxyvitamin D? bearing the main side chain at C12 and a shorter second hydroxylated chain at C17. The new compounds 5a-c were efficiently synthesized from ketone 9 (which is readily accessible from the Inhoffen-Lythgoe diol) with overall yields of 15%, 6%, and 3% for 5a, 5b, and 5c, respectively. The triene system was introduced by the Pd-catalyzed tandem cyclization-Suzuki coupling method. The new analogues were assayed against human colon and breast cancer cell lines and in mice. All new vitamin D? analogues bound less strongly to the VDR than 1?,25-dihydroxyvitamin D? but had similar antiproliferative, pro-differentiating, and transcriptional activity as the native hormone. In vivo, the three analogues had markedly low calcemic effects.
The most active vitamin D metabolite, 1?,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), is a pleiotropic hormone with wide regulatory actions. Classically, vitamin D deficiency was known to alter calcium and phosphate metabolism and bone biology. In addition, recent epidemiological and experimental studies support the association of vitamin D deficiency with a large variety of human diseases, and particularly with the high risk of colorectal cancer. By regulating the expression of many genes via several mechanisms, 1,25(OH)(2)D(3) induces differentiation, controls the detoxification metabolism and cell phenotype, sensitises cells to apoptosis and inhibits the proliferation of cultured human colon carcinoma cells. Consistently, 1,25(OH)(2)D(3) and several of its analogues decrease intestinal tumourigenesis in animal models. Molecular, genetic and clinical data in humans are scarce but they suggest that vitamin D is protective against colon cancer. Clearly, the available evidence warrants new, well-designed, large-scale trials to clarify the role of vitamin D in the prevention and/or therapy of this important neoplasia.
Vitamin D from the diet or synthesized in the skin upon UV-B irradiation is converted in the organism into the active metabolite 1?,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.
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