Efectos de la 1a,25-dihidroxivitamina D3 en fibroblastos estromales de cáncer colon

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 25-11-2016Esta tesis tiene embargado el acceso al texto completo hasta el 25-05-2018El 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

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

The Wnt/β-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/β-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 CRCThe work in the authors’ laboratories is supported by the Spanish Ministerio de Ciencia, Innovación y Universidades - Fondo Europeo de Desarrollo Regional (FEDER) (SAF2016-76377-R, SAF2017-90604-REDT), Consejo Superior de Investigaciones Científicas (201820I058), and Instituto de Salud Carlos III - FEDER (CIBERONC, CB16/12/00273; CIBERES, CB15/00037

Vitamin D effects on human colon normal and tumour organoids

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).

Cystatin D locates in the nucleus at sites of active transcription and modulates gene and protein expression

Cystatin D is an inhibitor of lysosomal and secreted cysteine proteases. Strikingly, cystatin D has been found to inhibit proliferation, migration, and invasion of colon carcinoma cells indicating tumor suppressor activity that is unrelated to protease inhibition. Here, we demonstrate that a proportion of cystatin D locates within the cell nucleus at specific transcriptionally active chromatin sites. Consistently, transcriptomic analysis show that cystatin D alters gene expression, including that of genes encoding transcription factors such as RUNX1, RUNX2, and MEF2C in HCT116 cells. In concordance with transcriptomic data, quantitative proteomic analysis identified 292 proteins differentially expressed in cystatin D-expressing cells involved in cell adhesion, cytoskeleton, and RNA synthesis and processing. Furthermore, using cytokine arrays we found that cystatin D reduces the secretion of several protumor cytokines such as fibroblast growth factor-4, CX3CL1/fractalkine, neurotrophin 4 oncostatin-M, pulmonary and activation-regulated chemokine/CCL18, and transforming growth factor B3. These results support an unanticipated role of cystatin D in the cell nucleus, controlling the transcription of specific genes involved in crucial cellular functions, which may mediate its protective action in colon cancer

Efectos de la 1alpha,25-dihidroxivitamina D3 en fibroblastos estromales de cáncer de colon

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.Peer Reviewe

Efectos de la vitamina D y WNT en fibroblastos colónicos humanos

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

Efecto de vitamina D y Wnt en fibroblastos colónicos humanos

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.Peer Reviewe

Vitamin D is a multilevel repressor of Wnt/β-catenin signaling in cancer cells

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license.The Wnt/β-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 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) inhibits Wnt/β-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/β-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/beta-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/β-catenin pathway genes and targets in cancer patients.The work in the authors’ laboratory is supported by Ministerio de Economía y Competitividad of Spain (SAF2010-18302, BFU2010-19659), Fondo Europeo de Desarrollo Regional-Instituto de Salud Carlos III (RD12/0036/0021) and Comunidad de Madrid (S2010/BMD-2344, Colomics2).Peer reviewe

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/β-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.The work in the authors' laboratories is supported by the Ministerio de Economía y Competitividad of Spain-Fondo Europeo de Desarrollo Regional (FEDER) (SAF2016-76377-R, SAF2017-90604-REDT/Nurcamein) and the Instituto de Salud Carlos III-FEDER (CIBERONC, CB16/12/00273 and CB16/12/00436).Peer reviewe