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Trabajo presentado en FEBS Open Bio, celebrado en Lisboa (Portugal) del 09 al 14 de julio de 2022., Many studies indicate an association between vitamin D deficiency and increased colorectal cancer risk and, specially, mortality. Accordingly, the active vitamin D metabolite 1a,25-dihydroxyvitamin D3 (calcitriol) inhibits the proliferation and promotes the differentiation of colon carcinoma cells and of other tumour cell types, and also has antitumour effects in animal models of colon cancer. These results prompted us to analyse the effects of calcitriol on human colon normal and cancer stem cells. To this end, we established a living biobank of patient-derived colon organoids generated from the tumour mass and from the adjacent healthy tissue obtained from surgical biopsies. Organoids are a three-dimensional culture system of normal or cancer stem cells and their progeny with a self-organized multicellular structure. By immunohistochemistry and RNAscope in situ hybridization, we found that vitamin D receptor is expressed in LGR5+ colon stem cells in human tissue and in normal and tumour organoid cultures. RNA-sequencing assays showed that both organoid types respond differentially to calcitriol with profound and contrasting changes in their transcriptomic profiles. This was confirmed in an independent series of patient-derived organoids by RT-qPCR assays. In normal organoids, calcitriol upregulates stemness-related genes and inhibits cell proliferation. In contrast, in tumour organoids calcitriol has little effect on stemnessrelated genes, while it induces differentiation-associated genes, and variably reduces cell proliferation. Concordantly, electron microscopy analyses showed that calcitriol does not affect the blastic cell phenotype in normal organoids, but it induces a series of differentiated features in tumour organoids. These results indicate that calcitriol maintains the undifferentiated phenotype of human normal colon stem cells (homeostatic action), while it promotes the differentiation of colon cancer stem cells (anticancer action). *

Trabajo presentado en el 6th Symposium on Biomedical Research >Advances and Perspectives in Molecular Endocrinology>, celebrado en Madrid (España) el 31 de mayo de 2019., Vitamin D3 (cholecalciferol) is synthesized in the skin by action of solar UV radiation or incorporated via diet, and is the inactive precursor of 1α,25-dihydroxyvitamin D3 (calcitriol), a major pleiotropic hormone in the human organism. By binding and activation of vitamin D receptor (VDR), a member of the superfamily of nuclear receptors, calcitriol regulates the expression of hundreds of genes in a tissue- and cell-type specific manner by transcriptional and posttranscriptional mechanisms. Human WNT factors are a family of 19 members of secreted proteins that regulate crucial processes in many tissues and organs during development and adult life. Some (canonical) WNTs act by modulating the transcriptional activity of β-catenin whereas other (non-canonical) WNTs affect different signal transducers (Ca2+, Rho, JNK...) independently of β-catenin. Colorectal cancer (CRC) is one of the most important neoplasias worldwide in terms of incidence and mortality. The key role of the constitutive activation of the WNT/β-catenin signaling pathway promoting CRC and the protective effect of VDR agonists, in part by antagonizing the WNT/β-catenin pathway, have been widely described. However, the effects of calcitriol and canonical WNTs on stromal fibroblasts, which are important players in CRC with protumorigenic action, remain mostly unknown. We have studied the effect of calcitriol and WNT3A on the human CCD-18Co colonic fibroblast cell line and on primary colon fibroblasts isolated from CRC patients. Data from global transcriptomic analyses by RNA-sequencing and also functional assays (cell proliferation, migration...) indicate that calcitriol and WNT3A exert a wide regulatory action on the gene expression and phenotype of colonic fibroblasts. Remarkably, a complex interplay exists between the two agents: while calcitriol and WNT3A act cooperatively in some effects, antagonistic actions are found in others. Our results show that calcitriol and WNT3A are important modulators of human colon fibroblasts, with potential implications in colon homeostasis and neoplastic transformation., Ministerio de Ciencia, Innovación y Universidades (SAF2016-76377-R, Nurcamein2) and Instituto de Salud Carlos III (CIBERONC) of Spain - Fondo Europeo de Desarrollo Regional.

Trabajo presentado en los Seminarios del Departamento de Biología del Cáncer, celebrados en Madrid (Espala) el 21 de enero de 2019., Trabajo presentado en los Seminarios del Departamento de Biología del Cáncer, celebrados en Madrid (España) el 21 de enero de 2019.

Resumen del póster presentado al 42nd Congress of the Spanish Society of Biochemistry and Molecular Biology (SEBBM), celebrado en Madrid del 16 al 19 de julio de 2019., Vitamin D3 (cholecalciferol) is synthesized in the skin by action of solar UV radiation or incorporated via diet, and is the inactive precursor of 1α,25-dihydroxyvitamin D3 (calcitriol), a major pleiotropic hormone in the human organism. By binding and activation of vitamin D receptor (VDR), a member of the superfamily of nuclear receptors, calcitriol regulates the expression of hundreds of genes in a tissue- and cell-type specific manner by transcriptional and posttranscriptional mechanisms. Human WNT factors are a family of 19 members of secreted proteins that regulate crucial processes in many tissues and organs during development and adult life. Some (canonical) WNTs act by modulating the transcriptional activity of β-catenin whereas other (non-canonical) WNTs affect different signal transducers (Ca2+, Rho, JNK...) independently of β-catenin. Colorectal cancer (CRC) is one of the most important neoplasias worldwide in terms of incidence and mortality. The key role of the constitutive activation of the WNT/β-catenin signaling pathway promoting CRC and the protective effect of VDR agonists, in part by antagonizing the WNT/β-catenin pathway, have been widely described. However, the effects of calcitriol and canonical WNTs on stromal fibroblasts, which are important players in CRC with protumorigenic action, remain mostly unknown. We have studied the effect of calcitriol and WNT3A on the human CCD-18Co colonic fibroblast cell line and on primary colon fibroblasts isolated from CRC patients. Data from global transcriptomic analyses by RNA-sequencing and also functional assays (cell proliferation, migration...) indicate that calcitriol and WNT3A exert a wide regulatory action on the gene expression and phenotype of colonic fibroblasts. Remarkably, a complex interplay exists between the two agents: while calcitriol and WNT3A act cooperatively in some effects, antagonistic actions are found in others. Our results show that calcitriol and WNT3A are important modulators of human colon fibroblasts, with potential implications in colon homeostasis and neoplastic transformation.

Resumen del trabajo presentado al 41 Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Santander del 10 al 13 de septiembre de 2018., Vitamin D deficiency is associated with a higher risk of colorectal cancer (CRC). Accordingly, calcitriol, the most active vitamin D metabolite, inhibits the proliferation and promotes the epithelial differentiation of human colon carcinoma cell lines. Calcitriol action is mediated by the vitamin D receptor (VDR), a member of the superfamily of nuclear receptors that upon ligand binding regulates the transcription of target genes. Recent data indicate that fibroblasts are the principal cellular component of tumour stroma and contribute to tumourigenesis by numerous mechanisms. Thus, we have investigated calcitriol effects on primary cultures of CRC patient-derived colon normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs). NFs and CAFs express VDR and respond to calcitriol. Remarkably, calcitriol inhibits two fibroblast protumoural properties: the ability to reorganize the extracellular matrix and the capacity to paracrinally promote the migration of CRC cells. Moreover, global transcriptomic analyses show that calcitriol regulates the gene expression profile of NFs and CAFs, and imposes in CAFs a gene signature that correlates with a better outcome of CRC patients. We have also analysed the expression of VDR and of two calcitriol target genes (CD82, upregulated; and S100A4, downregulated) in 658 metastatic CRC patients. Importantly, high VDR expression in tumour stromal fibroblasts is associated with better prognosis. In addition, the expression of CD82 and S100A4 in these cells is associated directly and inversely, respectively, with that of VDR and with CRC patient clinical outcome. In summary, our results indicate that the antitumoural action of calcitriol on CRC is mediated not only by its direct action on carcinoma cells, but also by inhibiting the protumoural properties of CAFs. We propose that treatment of CRC patients with VDR agonists could be explored in those patients that express VDR in tumour stromal fibroblasts even in the absence of VDR expression in carcinoma cells.

Trabajo presentado a la 1ª Reunión NuRCaMein (Red española de grupos de investigación en receptores nucleares), celebrada en el Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) en Madrid del 21 al 22 de noviembre de 2018.

Trabajo presentado 21st Workshop on Vitamin D, celebrado en Barcelona (España) del 16 al 19 de mayo de 2018.

Trabajo presentado al Joint Congress, celebrado en Gijón (España) del 24 al 27 de octubre de 2017.

Trabajo presentado a la XXI Reunión Nacional de la Fundación Asociación Española de Coloproctología, celebrada en Almeria del 17 al 19 de mayo de 2017.

Trabajo presentado a las XI Jornadas de Jovenes Investigadores: Simposio - Novedades en la Investigación del Cáncer en España, celebradas en Albacete del 18 al 20 de octubre de 2017.

Resumen del póster presentado al 8th European Multidisciplinary Colorectal Cancer Congress, celbrado en Amsterdam (Netherlands) del 11 al 13 de diciembre de 2016., [Objective]: Colorectal cancer (CRC) is the neoplasia most strongly associated with vitamin D deficiency. Many epidemiological studies indicate that vitamin D deficiency increases the risk of CRC and thus, suggest that vitamin D has a protective effect on CRC. This is supported by preclinical studies using vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3, the most active vitamin D metabolite) or analogues in cultured cells and experimental animals. 1,25(OH)2D3 inhibits the proliferation and promotes the differentiation of colon carcinoma cells by mechanisms that include cell cycle arrest at G0/G1, the blockade of the Wnt/β-catenin pathway and the induction of E-cadherin and other epithelial adhesion proteins. 1,25(OH)2D3 action is mediated by the vitamin D receptor (VDR), a member of the superfamily of nuclear receptors that upon ligand biding regulates the transcription rate of hundreds of genes. Consequently, VDR expression is the main determinant of cell responsiveness to 1,25(OH)2D3. Fibroblasts are the main cellular component of tumour stroma and upon activation by the tumour microenvironment they contribute to tumorigenesis by several mechanisms. In this study we explored VDR expression and 1,25(OH)2D3 action on CRC stromal fibroblasts. [Methods]: The expression of VDR and of two 1,25(OH)2D3 target genes was analysed in 658 metastatic CRC patients with prolonged clinical follow-up. Primary cultures of patient-derived colon normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs) were set up to study 1,25(OH)2D3 effects on gene expression and on two fibroblast protumoural properties: collagen gel contraction and induction of carcinoma cell migration. Publically available datasets were used to correlate the expression of the gene signature imposed by 1,25(OH)2D3 in CAFs with CRC patient clinical outcome. [Results]: High VDR expression in tumour stromal fibroblasts is associated with better overall survival and progression-free survival in CRC. Patient-derived NFs and CAFs express VDR and respond to 1,25(OH)2D3. 1,25(OH)2D3 inhibits the ability to reorganize collagen fibres and contract collagen gels of NF and CAF primary cultures. In addition, 1,25(OH)2D3 reduces the capacity of NF and CAF cultures to paracrinally promote the migration of human colon carcinoma cells in Transwell-mediated coculture assays. Remarkably, global transcriptomic analyses show that 1,25(OH)2D3 imposes in CAFs a gene signature that correlates with longer overall survival and disease-free survival of CRC patients. Moreover, expression of two 1,25(OH)2D3 target genes CD82 (upregulated) and S100A4 (downregulated) is associated directly and inversely, respectively, with stromal VDR expression and CRC patient clinical outcome. [Conclusions]: Our results indicate that CRC stromal fibroblasts express VDR and that their gene expression and physiology is modulated by 1,25(OH)2D3 in a way that may contribute to the antitumoural action of 1,25(OH)2D3 against this neoplasia. High expression of VDR and of the 1,25(OH)2D3-associated gene signature in stromal fibroblasts predicts a favourable clinical outcome in CRC. Thus, treatment of CRC patients with VDR agonists could be explored even in the absence of VDR expression in carcinoma cells.

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 25-11-2016, Esta tesis tiene embargado el acceso al texto completo hasta el 25-05-2018, El cáncer colorrectal (CRC) es una de las neoplasias de mayor incidencia y mortalidad. Estudios epidemiológicos indican que la deficiencia en vitamina D se asocia a un mayor riesgo de padecer CRC. De acuerdo con esto, la 1α,25‐dihidroxivitamina D3 (1,25(OH)2D3), el metabolito más activo de la vitamina D, inhibe la proliferación y promueve la diferenciación de células de CRC a través de su unión al receptor de vitamina D (VDR), un factor de transcripción de la superfamilia de los receptores nucleares que regula la expresión de centenares de genes. VDR es el principal determinante de la sensibilidad celular a 1,25(OH)2D3 y su expresión se pierde durante la tumorogénesis colónica, lo que generaría resistencia a una posible terapia con 1,25(OH)2D3. En los últimos años se ha puesto de manifiesto la relevancia del estroma tumoral en la progresión del cáncer. Los fibroblastos son el principal componente celular de dicho estroma y tras su activación por el microambiente tumoral contribuyen a la tumorogénesis por varios mecanismos. Sin embargo, los posibles efectos de la 1,25(OH)2D3 sobre los fibroblastos estromales de los tumores colorrectales no se conocen. En esta Tesis Doctoral hemos establecido cultivos primarios de fibroblastos normales (NFs) y de fibroblastos asociados a tumores (CAFs) a partir de biopsias procedentes de pacientes con CRC, y hemos estudiado los efectos de la 1,25(OH)2D3 sobre ellos. Los resultados muestran que los NFs y los CAFs de colon expresan VDR y responden a 1,25(OH)2D3. La 1,25(OH)2D3 inhibe en ellos dos propiedades protumorales características de los fibroblastos: la capacidad de contraer geles de colágeno y la de inducir la migración de las células de carcinoma. Asimismo, la 1,25(OH)2D3 reduce las acciones protumorales de fibroblastos de otros orígenes. Los análisis de expresión génica global revelan que la 1,25(OH)2D3 modula el patrón de expresión génica de NFs y CAFs, y nos han permitido caracterizar que la inhibición de la expresión de las quimioquinas CCL2, CCL11 y CCL13 por 1,25(OH)2D3 es necesaria para el efecto de ésta sobre la capacidad de los fibroblastos colónicos de contraer geles de colágeno. Por otro lado, hemos analizado la expresión de VDR y de las proteínas codificadas por los genes diana de 1,25(OH)2D3 CD82 y S100A4 en tumores procedentes de una serie amplia de pacientes con CRC metastásico. Los resultados indican que una alta expresión de VDR en los fibroblastos estromales de dichos tumores correlaciona con una mayor supervivencia de los pacientes. Asimismo, la expresión de CD82 y de S100A4 en dichos fibroblastos se asocia con la de VDR y con el pronóstico clínico de los pacientes. Los resultados obtenidos indican que la 1,25(OH)2D3 ejerce su acción antitumoral en CRC actuando no solo sobre las células de carcinoma, sino también sobre los fibroblastos estromales y que, por tanto, el tratamiento de pacientes con CRC con 1,25(OH)2D3 puede ser explorado incluso si las células de carcinoma carecen de expresión de VDR., Colorectal cancer (CRC) is one of the most frequent and lethal neoplasias. Epidemiological studies indicate that vitamin D deficiency is associated with a higher risk of CRC. In agreement with this, 1α,25‐dihydroxyvitamin D3 (1,25(OH)2D3), the most active metabolite of vitamin D, inhibits the proliferation and induces the differentiation of CRC cells by binding the vitamin D receptor (VDR), a transcription factor of the nuclear receptor superfamily that regulates the expression of hundreds of genes. VDR is the main determinant of cellular sensibility to 1,25(OH)2D3 and its expression is lost during colon tumorigenesis, leading to resistance to 1,25(OH)2D3. In recent years, the relevance of tumor stroma on cancer progression has emerged. Fibroblasts are the main cellular component of cancer stroma and upon activation by the tumor microenvironment they contribute to the tumorigenesis by several mechanisms. Despite all this, the possible effects of 1,25(OH)2D3 on stromal fibroblasts of colorectal tumors are unknown. In this Doctoral Thesis, we have established primary cultures of normal fibroblasts (NFs) and cancer‐associated fibroblasts (CAFs) from CRC patient biopsies, and have studied the effects of 1,25(OH)2D3 on them. The results show that colon NFs and CAFs express VDR and respond to 1,25(OH)2D3. 1,25(OH)2D3 inhibits two fibroblast protumoral characteristics in these cells: the capacity to contract collagen gels and to induce the migration of carcinoma cells. Likewise, 1,25(OH)2D3 reduces the protumoral actions of fibroblasts from different origins. Global gene expression analyses show that 1,25(OH)2D3 modulates the gene expression profile of NFs and CAFs, and have led us to characterize that the inhibition of the expression of the chemokines CCL2, CCL11 and CCL13 by 1,25(OH)2D3 is required for 1,25(OH)2D3 effect on the capacity to contract collagen gels of colonic fibroblasts. Moreover, we have analyzed the expression of VDR and of the proteins encoded by the 1,25(OH)2D3 target genes CD82 and S100A4 in tumors from a large cohort of metastatic CRC patients. Our results indicate that high VDR expression in stromal fibroblasts correlates with longer patient survival. In addition, CD82 and S100A4 expression in those fibroblasts is associated with that of VDR and with clinical outcome of CRC patients. Our results indicate that 1,25(OH)2D3 exerts its antitumoral effects in CRC by acting not only on carcinoma cells, but also on stromal fibroblasts. Therefore, treatment of CRC patients with 1,25(OH)2D3 should be explored even if carcinoma cells lack VDR expression.

Resumen del trabajo presentado al 15th International Congress of the ASociación Española de Investigación sobre el CAncer (ASEICA), celebrado en Sevilla del 21 al 23 de octubre de 2015., Colorectal cancer (CRC) is one of the most frequent and mortal neoplasias worldwide. Despite improvements in CRC patient management and treatment in the last twenty years, no satisfactory therapy exists when surgery is not curative. There is thus a clear need for increased prevention, early diagnosis, novel treatments and better knowledge of this disease. The active vitamin D derivative 1α.25-dihydroxyvitamin D3 (calcitriol or 1,25(OH)2D3) is a pleiotropic hormone that, in addition to its classical effects on the regulation of calcium and phosphate homeostasis and bone biology, modulates proliferation, survival, differentiation and metabolism, and potentiates the antitumoral activity of certain chemotherapeutic agents, in a cell-type- and -context-dependent manner in many ce lis in the organism. These effects rely on the activation of vitamin D receptor (VDR), a member of the superfamily of nuclear receptors that regulares the transcription rate of hundreds of human genes. Many epidemiological studies suggest that vitamin D deficiency is associated with increased colorectal cancer risk and mortality. Additionally, high circulating 1,25(OH)2D3 levels at diagnosis associate with better outcome in CRC. However, definitive clinical evidence of the protective action of vitamin D awaits for the results of ongoing prospective intervention trials. 1,25(OH)2D3 inhibits proliferation and prometes epithelial differentiation of human colon carcinoma ce lis. We have reported that 1,25(OH)2D3 induces the expression of the key intercellular adhesion molecule and invasion suppressor E-cadherin and antagonizes the Wnt/β-catenin signaling pathway, whose alteration initiates and fuels colon tumorigenesis. Moreover, we have reported the regulation by 1,25(OH)2D3 of several genes that variably affect CRC such as SPROUTY(SPRY-2, DICKKOPF(DKK)-1, CST5/Cystatin D or KDM6BIJMJD3. In vivo, 1,25(OH)2D3 reduces intestinal tumorigenesis inApcM> mice and in other mouse models of colon cancer.Aiso,VDR deficiency in mice (Ydr'·) provokes colonic hyperplasia and a strong oxidative stress, and we and others have shown that it in creases tumor load in ApcMin mice. We have also shown that SNAI and SNAI2 genes encoding the epithelial-to-mesenchymal inducers SNAILI and SNAIL2/SLUG repress VDR and that an inverse relation between the expression of these genes exist in human colon tumors. Today it is accepted that both tumor stroma and the alteration of tissue stem cells play crucial roles in cancer. Fibroblasts are the major stromal cellular component and u pon activation by the tumor microenvironment contribute to tumorigenesis. Recendy, we have started the study of the action of 1,25(OH)2D3 on CRC patient-derived normal and cancer-associated fibroblasts and on normal and cancer stem cells.lnitial data of the effects of 1,25(OH)2D3 on these two key tumor compartments will be presented.

Resumen del póster presentado al 15th International Congress of the ASociación Española de Investigación sobre el CAncer (ASEICA), celebrado en Sevilla del 21 al 23 de octubre de 2015., Cystatin D is a member of the cystatin superfamily of inhibitors of cathepsins, cysteine proteases that degrade multiple targets including adhesion proteins, matrix components and other proteases. Cystatins play multiple roles in physiology and pathology, including tumorigenesis and neurodegenerative disorders, and a number of cathepsins are thought to be involved in cancer and other diseases as regulators of a variety of biochemical processes. Preferential attention has been paid to the deregulation and imbalance between cathepsins and cystatins in invasion and metastasis of several neoplasias. We have previously reportad that cystatin D prometes cell adhesion and decreases proliferation. migration and invasion of colon carcinoma cells. lt is downregulated during human colon carcinogenesis, and considerad as a candidate tumor suppressor gene that is transcriptionally induced by 1,25(OH)2D3, the most active metabolite of vitamin D, mediating its protective effects against this neoplasia (Álvarez-Díaz et al., J. Clin. lnvest., 2009). The finding that mutant forms of cystatin D with no protease inhibitory activity lack the antimigratory but not the antiproliferative effect on colon cancer cells indicates that cystatin D has cathepsin-independent mechanism(s) of action. Cathepsins have traditionally been considerad endosomal/lysosomal or secreted proteases; however, new evidences support their localization in other cellular compartments. Recent studies have reported the activity of cathepsin L. a cystatin D target, within the cell nucleus. Analogously, a few cystatins and other protease inhibitors have been found to act in the nuclear compartment. Taken together, these findings prompted us to investigate in depth the mechanism of action of cystatin D in colon carcinoma cells.

Resumen del póster presentado al XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Granada del 9 al 12 de septiembre de 2014., Vitamin D3 is obtained from the diet or mainly synthesized in the skin and is converted into the active metabolite 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3). Epidemiological data suggest a protective role of vitamin D against several neoplasias, particularly colorectal cancer. 1,25(OH)2D3 is a major regulator of gene expression. Histones are subject to posttranslational modifications including methylation, acetylation and others. Misregulation of histone modifi cations alters gene expression and cell phenotype, which may contribute to cancer initiation, progression and/or metastasis. We found that 1,25(OH)2D3has a wide regulatory action on the expression of genes coding for histone demethylases of the Jumonji C domain and lysine-specifi c demethylase families (Pereira et al., 2011; 2012). Notably, 1,25(OH)2D3induces the expression of the histone demethylase JMJD3/KDM6B that specifi cally demethylates the lysine 27 of histone H3 (H3K27) and has tumour suppressor activity. We found that the induction of JMJD3 is necessary for the adequate gene regulatory, antiproliferative, and prodifferentiation actions of 1,25(OH)2D3 in human colon cancer cells. Conversely, 1,25(OH)2D3 inhibits the expression of several Polycomb group proteins that participate in the Polycomb repressive complexes (PRC) 1 and 2, which are responsible for H3K27 methylation and promote tumorigenesis. The inhibition of PRC2 activity by DZNep potentiates the gene regulatory action of 1,25(OH)2D3 and also its effects on colon cancer cell proliferation and differentiation. Thus, 1,25(OH)2D3 exerts an ample regulatory effect on the expression of histone modifying enzymes involved in epigenetic regulation that, in turn, mediate 1,25(OH)2D3 actions on gene expression and cell phenotype in colon cancer.

Resumen del trabajo presentado al "6th International EMT Meeting: Symposium VI. Cancer and EMT I" celebrado en Alicante (España) del 13 al 16 de noviembre de 2013., The active vitamin D metabolite 1alpha,25-dihydroxyvitamin D3 (calcitriol, 1,25(OH)2D3) is a pleiotropic secosteroid hormone with wide regulatory actions. 1,25(OH)2D3 acts via the vitamin D receptor (VDR), a member of the superfamily of nuclear receptors, which binds specific DNA sequences in its target genes and modulates their transcription rate. Additionally, 1,25(OH)2D3 activates rapid, non-genomic signaling pathways. We have reported that 1,25(OH)2D3 inhibits proliferation and induces differentiation of human colon carcinoma cells via the control of a high number of genes and the antagonism of the Wnt/beta-catenin pathway (Pálmer et al., J. Cell Biol., 2001; Cancer Res., 2003). 1,25(OH)2D3 increases the expression of CDH1/E-cadherin RNA and protein through the activation of a rapid non-genomic pathway (Ca2+-RhoA-ROCK-MSK1) and the subsequent increase in transcription (Ordóñez-Morán et al., J. Cell Biol., 2008). Additionally, it induces other proteins involved in epithelial adhesion structures such as the tight junctions components claudin-7, occludin and ZO-1. These effects are partially mediated by the induction of CST5/cystatin D, a protease inhibitor that regulates gene expression within the cell nucleus, and KDM6B/JMJD3 histone H3 lysine 27 demethylase, and by the repression of SPROUTY-2, a modulator of tyrosine kinase receptor signalling (Alvarez-Díaz et al., J. Clin. Invest., 2009; Barbáchano et al., Oncogene, 2010; Pereira et al., Human Mol. Genet., 2011). In addition, CST5/cystatin D and KDM6B/JMJD3 downregulate SNAI1, SNAI2, ZEB1 and ZEB2 genes. Moreover, SPROUTY-2 represses miR-200b/c leading to ZEB1 upregulation, and also several adhesion and polarity genes and ESRP1, an inhibitor of EMT. Remarkably, a reciprocal inhibitory loop exists between 1,25(OH)2D3 and EMT, as SNAIL1 and SNAIL2/SLUG repress VDR expression (Pálmer et al., Nat. Med., 2004; Larriba et al., Carcinogenesis, 2009). Altogether, our results show that 1,25(OH)2D3 represses EMT in human colon carcinoma cells through several mechanisms that affect the expression of EMT regulators and of epithelial adhesion and polarity genes. Conversely, the EMT inducers SNAIL1/2 inactivate the vitamin D system via VDR downregulation.

Resumen del póster presentado al XXXVI Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Madrid del 3 al 6 de septiembre de 2013.-- et al., SPROUTY-2 (SPRY2) is a modulator of receptor tyrosine kinase signalling, and therefore of cell growth and differentiation, with cell type-dependent tumor promoting or suppressive effects. Previously we reported that SPRY2 expression in colon cancer cells is inhibited by the active vitamin D metabolite 1alpha, 25-dihydroxyvitamin D3 through E-cadherin-dependent and -independent mechanisms. In turn, SPRY2 represses both basal and 1alpha, 25-dihydroxyvitamin D3-induced E-cadherin expression. Recent data indicate that in human colon cancer cells SPRY2 expression is induced by beta-catenin in cooperation with the transcription factor FOXO3a instead of TCF/LEF1 proteins. In colon cancer patients, SPRY2 is upregulated in undifferentiated high grade tumors and at the invasive front of low grade carcinomas, and SPRY2 protein expression correlates with nuclear beta-catenin and FOXO3a colocalization. Importantly, the amount of SPRY2 protein correlates with shorter overall survival of colon cancer patients. We have found that SPRY2 dysregulates tight junction and epithelial polarity genes via microRNA-200-dependent upregulation of the transcriptional repressor ZEB1, which provides a mechanistic basis for the tumorigenic role of SPRY2 in colon cancer. In conclusion, our data reveal SPRY2 as a novel Wnt/beta-catenin and FOXO3a target gene indicative of poor prognosis in colon cancer.

Resumen del trabajo presentado al XXXIV Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Barcelona del 5 al 8 de septiembre de 2011., La 1α,25-dihidroxivitamina D3 (1,25(OH)2D3), el metabolito más activo de la vitamina D, es uno de los principales reguladores de la expresión génica en el organismo. La 1,25(OH)2D3 inhibe la proliferación y promueve la diferenciación de células humanas de cáncer de colon. Nuestros resultados indican que estas acciones se deben, al menos en parte, al antagonismo de la vía de señalización Wnt/β-catenina, cuya activación aberrante es el paso inicial en cáncer colorrectal, por tres mecanismos: a) la unión de VDR a β-catenina, que impide la formación de complejos β-catenina-TCF y la activación de sus genes diana; b) la inducción de la expresión de CDH1/Ecadherina, que codifica una proteína esencial en la unión intercelular en epitelios supresora de invasividad y favorece la relocalización de β-catenina desde núcleo a la membrana plasmática; y c) la inducción de la expresión de DICKKOPF(DKK)-1, que codifica un inhibidor de la vía Wnt/β-catenina con actividad supresora tumoral. La deficiencia de VDR en ratones Apcmin que expresan una forma mutada del gen supresor Apc contribuye a la activación de la vía Wnt/β-catenina y causa un aumento de la carga tumoral. La 1,25(OH)2D3 induce CST5/cistatina D, que codifica un inhibidor de proteasas con actividad antiproliferativa y antiinvasiva, y reprime SPROUTY-2, un modulador de la señalización por receptores tirosinaquinasa que favorece la tumorogénesis colorrectal. La regulación de E-cadherina, DKK-1, cistatina D y SPROUTY-2 debe contribuir al efecto protector de la 1,25(OH)2D3 en cáncer de colon.

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/β-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/β-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/β-catenin in normal and colon cancer-associated fibroblasts. PKP2 is induced by β-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/β-catenin transcriptional activity in HEK-293T cells, which suggests that it may act as an intracellular inhibitor of the Wnt/β-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., Grant sponsor: Consejería de Educación, Juventud y Deporte, Comunidad de Madrid; Grant number: S2010/BMD-2344 Colomics2; Grant sponsor: Spanish Ministerio de Economía y Competitividad-Fondos FEDER; Grant numbers: Nurcamein SAF-2015-71878-REDT, SAF2013- 43468-R, SAF2014-53819-R; Grant sponsor: Instituto de la Salud Carlos III - Fondos FEDER; Grant number: CB16/12/00273 CIBERONC, RD12/0036/0021; Grant sponsor: Agencia Estatal de Investigación - Fondos FEDER; Grant number: SAF2016-76377-R