Vitamin D opposes multilineage cell differentiation induced by Notch inhibition and BMP4 pathway activation in human colon organoids.

Understanding the mechanisms involved in colonic epithelial differentiation is key to unraveling the alterations causing inflammatory conditions and cancer. Organoid cultures provide an unique tool to address these questions but studies are scarce. We report a differentiation system toward enterocytes and goblet cells, the two major colonic epithelial cell lineages, using colon organoids generated from healthy tissue of colorectal cancer patients. Culture of these organoids in medium lacking stemness agents resulted in a modest ultrastructural differentiation phenotype with low-level expression of enterocyte (KLF4, KRT20, CA1, FABP2) and goblet cell (TFF2, TFF3, AGR2) lineage markers. BMP pathway activation through depletion of Noggin and addition of BMP4 resulted in enterocyte-biased differentiation. Contrarily, blockade of the Notch pathway using the γ-secretase inhibitor dibenzazepine (DBZ) favored goblet cell differentiation. Combination treatment with BMP4 and DBZ caused a balanced strong induction of both lineages. In contrast, colon tumor organoids responded poorly to BMP4 showing only weak signals of cell differentiation, and were unresponsive to DBZ. We also investigated the effects of 1α,25-dihydroxyvitamin D3 (calcitriol) on differentiation. Calcitriol attenuated the effects of BMP4 and DBZ on colon normal organoids, with reduced expression of differentiation genes and phenotype. Consistently, in normal organoids, calcitriol inhibited early signaling by BMP4 as assessed by reduction of the level of phospho-SMAD1/5/8. Our results show that BMP and Notch signaling play key roles in human colon stem cell differentiation to the enterocytic and goblet cell lineages and that calcitriol modulates these processes favoring stemness features.

From molecular basis to clinical insights: a challenging future for the vitamin D endocrine system in colorectal cancer.

Colorectal cancer (CRC) is one of the most life-threatening neoplasias in terms of incidence and mortality worldwide. Vitamin D deficiency has been associated with an increased risk of CRC. 1α,25-Dihydroxyvitamin D3 [1,25(OH)2 D3 ], the most active vitamin D metabolite, is a pleiotropic hormone that, through its binding to a transcription factor of the nuclear receptor superfamily, is a major regulator of the human genome. 1,25(OH)2 D3 acts on colon carcinoma and stromal cells and displays tumor protective actions. Here, we review the variety of molecular mechanisms underlying the effects of 1,25(OH)2 D3 in CRC, which affect multiple processes that are dysregulated during tumor initiation and progression. Additionally, we discuss the epidemiological data that associate vitamin D deficiency and CRC, and the most relevant randomized controlled trials of vitamin D3 supplementation conducted in both healthy individuals and CRC patients.

Vitamin D induces SIRT1 activation through K610 deacetylation in colon cancer.

Posttranslational modifications of epigenetic modifiers provide a flexible and timely mechanism for rapid adaptations to the dynamic environment of cancer cells. SIRT1 is an NAD+-dependent epigenetic modifier whose activity is classically associated with healthy aging and longevity, but its function in cancer is not well understood. Here, we reveal that 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3, calcitriol), the active metabolite of vitamin D (VD), promotes SIRT1 activation through auto-deacetylation in human colon carcinoma cells, and identify lysine 610 as an essential driver of SIRT1 activity. Remarkably, our data show that the post-translational control of SIRT1 activity mediates the antiproliferative action of 1,25(OH)2D3. This effect is reproduced by the SIRT1 activator SRT1720, suggesting that SIRT1 activators may offer new therapeutic possibilities for colon cancer patients who are VD deficient or unresponsive. Moreover, this might be extrapolated to inflammation and other VD deficiency-associated and highly prevalent diseases in which SIRT1 plays a prominent role.

Vitamin D Receptor Deficiency Upregulates Pulmonary Artery Kv7 Channel Activity.

Recent evidence suggests that vitamin D is involved in the development of pulmonary arterial hypertension (PAH). The aim of this study was to analyze the electrophysiological and contractile properties of pulmonary arteries (PAs) in vitamin D receptor knockout mice (Vdr-/-). PAs were dissected and mounted in a wire myograph. Potassium membrane currents were recorded in freshly isolated PA smooth muscle cells (PASMCs) using the conventional whole-cell configuration of the patch-clamp technique. Potential vitamin D response elements (VDREs) in Kv7 channels coding genes were studied, and their protein expression was analyzed. Vdr-/- mice did not show a pulmonary hypertensive phenotype, as neither right ventricular hypertrophy nor endothelial dysfunction was apparent. However, resistance PA from these mice exhibited increased response to retigabine, a Kv7 activator, compared to controls and heterozygous mice. Furthermore, the current sensitive to XE991, a Kv7 inhibitor, was also higher in PASMCs from knockout mice. A possible VDRE was found in the gene coding for KCNE4, the regulatory subunit of Kv7.4. Accordingly, Vdr-/- mice showed an increased expression of KCNE4 in the lungs, with no changes in Kv7.1 and Kv7.4. These results indicate that the absence of Vdr in mice, as occurred with vitamin D deficient rats, is not sufficient to induce PAH. However, the contribution of Kv7 channel currents to the regulation of PA tone is increased in Vdr-/- mice, resembling animals and humans suffering from PAH.

Organoids and Colorectal Cancer.

Organoids were first established as a three-dimensional cell culture system from mouse small intestine. Subsequent development has made organoids a key system to study many human physiological and pathological processes that affect a variety of tissues and organs. In particular, organoids are becoming very useful tools to dissect colorectal cancer (CRC) by allowing the circumvention of classical problems and limitations, such as the impossibility of long-term culture of normal intestinal epithelial cells and the lack of good animal models for CRC. In this review, we describe the features and current knowledge of intestinal organoids and how they are largely contributing to our better understanding of intestinal cell biology and CRC genetics. Moreover, recent data show that organoids are appropriate systems for antitumoral drug testing and for the personalized treatment of CRC patients.

Wnt and Vitamin D at the Crossroads in Solid Cancer.

Abnormal activation of the Wnt/ß-catenin pathway is common in many types of solid cancers. Likewise, a large proportion of cancer patients have vitamin D deficiency. In line with these observations, Wnt/ß-catenin signaling and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active vitamin D metabolite, usually have opposite effects on cancer cell proliferation and phenotype. In recent years, an increasing number of studies performed in a variety of cancer types have revealed a complex crosstalk between Wnt/ß-catenin signaling and 1,25(OH)2D3. Here we review the mechanisms by which 1,25(OH)2D3 inhibits Wnt/ß-catenin signaling and, conversely, how the activated Wnt/ß-catenin pathway may abrogate vitamin D action. The available data suggest that interaction between Wnt/ß-catenin signaling and the vitamin D system is at the crossroads in solid cancers and may have therapeutic applications.

Vitamin D Effects on Cell Differentiation and Stemness in Cancer.

Vitamin D3 is the precursor of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), a pleiotropic hormone that is a major regulator of the human genome. 1,25(OH)2D3 modulates the phenotype and physiology of many cell types by controlling the expression of hundreds of genes in a tissue- and cell-specific fashion. Vitamin D deficiency is common among cancer patients and numerous studies have reported that 1,25(OH)2D3 promotes the differentiation of a wide panel of cultured carcinoma cells, frequently associated with a reduction in cell proliferation and survival. A major mechanism of this action is inhibition of the epithelial-mesenchymal transition, which in turn is largely based on antagonism of the Wnt/ß-catenin, TGF-ß and EGF signaling pathways. In addition, 1,25(OH)2D3 controls the gene expression profile and phenotype of cancer-associated fibroblasts (CAFs), which are important players in the tumorigenic process. Moreover, recent data suggest a regulatory role of 1,25(OH)2D3 in the biology of normal and cancer stem cells (CSCs). Here, we revise the current knowledge of the molecular and genetic basis of the regulation by 1,25(OH)2D3 of the differentiation and stemness of human carcinoma cells, CAFs and CSCs. These effects support a homeostatic non-cytotoxic anticancer action of 1,25(OH)2D3 based on reprogramming of the phenotype of several cell types.

Vitamin D differentially regulates colon stem cells in patient-derived normal and tumor organoids.

Intestine is a major target of vitamin D and several studies indicate an association between vitamin D deficiency and inflammatory bowel diseases (IBD), but also increased colorectal cancer (CRC) risk and mortality. However, the putative effects of 1α,25-dihydroxyvitamin D3 (calcitriol), the active vitamin D metabolite, on human colonic stem cells are unknown. Here we show by immunohistochemistry and RNAscope in situ hybridization that vitamin D receptor (VDR) is unexpectedly expressed in LGR5+ colon stem cells in human tissue and in normal and tumor organoid cultures generated from patient biopsies. Interestingly, normal and tumor organoids respond differentially to calcitriol with profound and contrasting changes in their transcriptomic profiles. In normal organoids, calcitriol upregulates stemness-related genes, such as LGR5, SMOC2, LRIG1, MSI1, PTK7, and MEX3A, and inhibits cell proliferation. In contrast, in tumor organoids calcitriol has little effect on stemness-related genes while it induces a differentiated phenotype, and variably reduces cell proliferation. Concordantly, electron microscopy showed that calcitriol does not affect the blastic undifferentiated cell phenotype in normal organoids but it induces a series of differentiated features in tumor organoids. Our results constitute the first demonstration of a regulatory role of vitamin D on human colon stem cells, indicating a homeostatic effect on colon epithelium with relevant implications in IBD and CRC.

Vitamin D and Wnt3A have additive and partially overlapping modulatory effects on gene expression and phenotype in human colon fibroblasts.

The Wnt/ß-catenin signalling pathway is essential for intestinal epithelium homeostasis, but its aberrant activation is a hallmark of colorectal cancer (CRC). Several studies indicate that the bioactive vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) inhibits proliferation and promotes epithelial differentiation of colon carcinoma cells in part through antagonism of the Wnt/ß-catenin pathway. It is now accepted that stromal fibroblasts are crucial in healthy and pathologic intestine: pericryptal myofibroblasts are constituents of the stem cell niche and cancer-associated fibroblasts (CAFs) contribute to CRC progression. However, studies on the combined action of 1,25(OH)2D3 and Wnt factors in colon fibroblasts are lacking. Here we show by global transcriptomic studies that 1,25(OH)2D3 and Wnt3A have profound, additive, partially overlapping effects on the gene expression profile of CCD-18Co human colon myofibroblasts. Moreover, 1,25(OH)2D3 and Wnt3A inhibit CCD-18Co cell proliferation and migration, while 1,25(OH)2D3 reduces, but Wnt3A increases, their capacity to contract collagen gels (a marker of fibroblast activation). These data were largely confirmed in patient-derived primary colon normal fibroblasts and CAFs, and in fibroblasts from other origins. Our results indicate that 1,25(OH)2D3 and Wnt3A are strong regulators of colon fibroblast biology and contribute to a better knowledge of intestinal homeostasis and stromal fibroblast action in CRC.

Mechanisms of action of vitamin D in colon cancer.

Colorectal cancer (CRC) is the neoplasia that is most frequently associated with vitamin D deficiency in epidemiological and observational studies in terms of incidence and mortality. Many mechanistic studies show that the active vitamin D metabolite (1α,25-dihydroxyvitamin D3 or calcitriol) inhibits proliferation and promotes epithelial differentiation of human colon carcinoma cell lines that express vitamin D receptor (VDR) via the regulation of a high number of genes. A key action underlining this effect is the multilevel inhibition of the Wnt/ß-catenin signaling pathway, whose abnormal activation in colon epithelial cells initiates and promotes CRC. Recently, our group has shown that calcitriol modulates gene expression and inhibits protumoral properties of patient-derived colon cancer-associated fibroblasts (CAFs). Accordingly, high VDR expression in tumor stromal fibroblasts is associated with longer survival of CRC patients. Moreover, many types of immune cells express VDR and are regulated by calcitriol, which probably contributes to its action against CRC. Given the role attributed to the intestinal microbiota in CRC and the finding that it is altered by vitamin D deficiency, an indirect antitumoral effect of calcitriol is also plausible at this level. In summary, calcitriol has an array of potential protective effects against CRC by acting on carcinoma cells, CAFs, immune cells and probably also the gut microbiota.

Endothelial cell activation on 3D-matrices derived from PDGF-BB-stimulated fibroblasts is mediated by Snail1.

Carcinomas, such as colon cancer, initiate their invasion by rescuing the innate plasticity of both epithelial cells and stromal cells. Although Snail is a transcriptional factor involved in the Epithelial-Mesenchymal Transition, in recent years, many studies have also identified the major role of Snail in the activation of Cancer-Associated Fibroblast (CAF) cells and the remodeling of the extracellular matrix. In CAFs, Platelet-derived growth factor (PDGF) receptor signaling is a major functional determinant. High expression of both SNAI1 and PDGF receptors is associated with poor prognosis in cancer patients, but the mechanism(s) that underlie these connections are not understood. In this study, we demonstrate that PDGF-activated fibroblasts stimulate extracellular matrix (ECM) fiber remodeling and deposition. Furthermore, we describe how SNAI1, through the FAK pathway, is a necessary factor for ECM fiber organization. The parallel-oriented fibers are used by endothelial cells as "tracks", facilitating their activation and the creation of tubular structures mimicking in vivo capillary formation. Accordingly, Snail1 expression in fibroblasts was required for the co-adjuvant effect of these cells on matrix remodeling and neoangiogenesis when co-xenografted in nude mice. Finally, in tumor samples from colorectal cancer patients a direct association between stromal SNAI1 expression and the endothelial marker CD34 was observed. In summary, our results advance the understanding of PDGF/SNAI1-activated CAFs in matrix remodeling and angiogenesis stimulation.

Differential distribution and enrichment of non-coding RNAs in exosomes from normal and Cancer-associated fibroblasts in colorectal cancer.

Exosome production from cancer-associated fibroblasts seems to be an important driver of tumor progression. We report the first in-depth biotype characterization of ncRNAs, analyzed by Next Generation Sequencing and Bioinformatics, expressed in established primary human normal and cancer-associated fibroblasts (CAFs) from cancer and normal mucosa tissues from 9 colorectal cancer patients, and/or packaged in their derived exosomes. Differential representation and enrichment analyses based on these ncRNAs revealed a significant number of differences between the ncRNA content of exosomes and the expression patterns of the normal and cancer-associated fibroblast cells. ncRNA regulatory elements are specifically packaged in CAF-derived exosomes, supporting a specific cross-talk between CAFs and colon cancer cells and/or other stromal cells, mediated by exosomes. These sncRNAs are potential biomarkers present in cancer-associated fibroblast-derived exosomes, which should thereby contribute to developing new non-invasive diagnostic, prognostic and predictive methods for clinical applications in management of cancer patients.

The human PKP2/plakophilin-2 gene is induced by Wnt/ß-catenin in normal and colon cancer-associated fibroblasts.

Colorectal cancer results from the malignant transformation of colonic epithelial cells. Stromal fibroblasts are the main component of the tumour microenvironment, and play an important role in the progression of this and other neoplasias. Wnt/ß-catenin signalling is essential for colon homeostasis, but aberrant, constitutive activation of this pathway is a hallmark of colorectal cancer. Here we present the first transcriptomic study on the effect of a Wnt factor on human colonic myofibroblasts. Wnt3A regulates the expression of 1,136 genes, of which 662 are upregulated and 474 are downregulated in CCD-18Co cells. A set of genes encoding inhibitors of the Wnt/ß-catenin pathway stand out among those induced by Wnt3A, which suggests that there is a feedback inhibitory mechanism. We also show that the PKP2 gene encoding the desmosomal protein Plakophilin-2 is a novel direct transcriptional target of Wnt/ß-catenin in normal and colon cancer-associated fibroblasts. PKP2 is induced by ß-catenin/TCF through three binding sites in the gene promoter and one additional binding site located in an enhancer 20 kb upstream from the transcription start site. Moreover, Plakophilin-2 antagonizes Wnt/ß-catenin transcriptional activity in HEK-293T cells, which suggests that it may act as an intracellular inhibitor of the Wnt/ß-catenin pathway. Our results demonstrate that stromal fibroblasts respond to canonical Wnt signalling and that Plakophilin-2 plays a role in the feedback control of this effect suggesting that the response to Wnt factors in the stroma may modulate Wnt activity in the tumour cells.

Vitamin D receptor expression and associated gene signature in tumour stromal fibroblasts predict clinical outcome in colorectal cancer.

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.

The endocrine vitamin D system in the gut.

The active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) has important regulatory actions in the gut through endocrine and probably also intracrine, autocrine and paracrine mechanisms. By activating the vitamin D receptor (VDR), which is expressed at a high level in the small intestine and colon, 1,25(OH)2D3 regulates numerous genes that control gut physiology and homeostasis. 1,25(OH)2D3 is a major responsible for epithelial barrier function and calcium and phosphate absorption, and the host's defense against pathogens and the inflammatory response by several types of secretory and immune cells. Moreover, recent data suggest that 1,25(OH)2D3 has a regulatory effect on the gut microbiota and stromal fibroblasts. Many studies have linked vitamin D deficiency to inflammatory bowel diseases (ulcerative colitis and Crohn's disease) and to an increased risk of colorectal cancer, and the possible use of VDR agonists to prevent or treat these diseases is receiving increasing interest.

Vitamin D and the Epithelial to Mesenchymal Transition.

Several studies support reciprocal regulation between the active vitamin D derivative 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and the epithelial to mesenchymal transition (EMT). Thus, 1,25(OH)2D3 inhibits EMT via the induction of a variety of target genes that encode cell adhesion and polarity proteins responsible for the epithelial phenotype and through the repression of key EMT inducers. Both direct and indirect regulatory mechanisms mediate these effects. Conversely, certain master EMT inducers inhibit 1,25(OH)2D3 action by repressing the transcription of VDR gene encoding the high affinity vitamin D receptor that mediates 1,25(OH)2D3 effects. Consequently, the balance between the strength of 1,25(OH)2D3 signaling and the induction of EMT defines the cellular phenotype in each context. Here we review the current understanding of the genes and mechanisms involved in the interplay between 1,25(OH)2D3 and EMT.

Exosomes enriched in stemness/metastatic-related mRNAS promote oncogenic potential in breast cancer.

Cancer cells efficiently transfer exosome contents (essentially mRNAs and microRNAs) to other cell types, modifying immune responses, cell growth, angiogenesis and metastasis. Here we analyzed the exosomes release by breast tumor cells with different capacities of stemness/metastasis based on CXCR4 expression, and evaluated their capacity to generate oncogenic features in recipient cells. Breast cancer cells overexpressing CXCR4 showed an increase in stemness-related markers, and in proliferation, migration and invasion capacities. Furthermore, recipient cells treated with exosomes from CXCR4-cells showed increased in the same abilities. Moreover, inoculation of CXCR4-cell-derived exosomes in immunocompromised mice stimulated primary tumor growth and metastatic potential. Comparison of nucleic acids contained into exosomes isolated from patients revealed a "stemness and metastatic" signature in exosomes of patients with worse prognosis. Finally, our data supported the view that cancer cells with stem-like properties show concomitant metastatic behavior, and their exosomes stimulate tumor progression and metastasis. Exosomes-derived nucleic acids from plasma of breast cancer patients are suitable markers in the prognosis of such patients.

Interaction of vitamin D with membrane-based signaling pathways.

Many studies in different biological systems have revealed that 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) modulates signaling pathways triggered at the plasma membrane by agents such as Wnt, transforming growth factor (TGF)-ß, epidermal growth factor (EGF), and others. In addition, 1α,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 1α,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 1α,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 1α,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.

Vitamin D Is a Multilevel Repressor of Wnt/b-Catenin Signaling in Cancer Cells.

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.

Synthesis and biological evaluation of 1α,25-dihydroxyvitamin D3 analogues with a long side chain at C12 and short C17 side chains.

Structure-guided optimization was used to design new analogues of 1α,25-dihydroxyvitamin D3 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 D3 analogues bound less strongly to the VDR than 1α,25-dihydroxyvitamin D3 but had similar antiproliferative, pro-differentiating, and transcriptional activity as the native hormone. In vivo, the three analogues had markedly low calcemic effects.