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).

DNA methylation and Yin Yang-1 repress adenosine A2A receptor levels in human brain

Adenosine A2A receptors (A2ARs) are G-protein coupled receptors that stimulate adenylyl cyclase activity. The most A 2ARs-enriched brain region is the striatum, in which A2ARs are largely restricted to GABAergic neurons of the indirect pathway. We recently described how DNA methylation controls basal A2AR expression levels in human cell lines. The present report provides clues about the molecular mechanisms that promote human brain region-specific A2AR gene (ADORA2A) basal expression. The transcription factors ZBP-89 and Yin Yang-1 (YY1) have been characterized as regulators of ADORA2A in SH-SY5Y cells by means of specific expression vectors/siRNAs transient transfection and chromatin immunoprecipitation assay. ZBP-89 plays a role as an activator and YY1 as a repressor. No differences were found in ZBP-89 levels with western blot between the putamen and cerebellum of human postmortem brains. However, increased YY1 levels and DNA methylation percentage in the 5′ untranslated region of ADORA2A, using SEQUENOM MassArray, were found in the cerebellum with respect to the putamen of human brains, showing an inverse relationship with A 2AR levels in the two cerebral regions. © 2010 International Society for Neurochemistry.This study was funded by grants from the Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III (PI05/1631 and CP08/00095) to M.B., and from the European Union through the Marie-Curie Research Training Network PRAIRIES (Contract MRTN-CT-2006-035810), the Consejerı́a de Educación y Ciencia (PCI08-0125), the Consejerı́a de Sanidad-FISCAM (PI-2007/50 and G-2007-C/13) of the Junta de Comunidades de Castilla-La Mancha and the Ministerio de Ciencia e Innovación (BFU2008-00138) to M.M.Peer Reviewe

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.The studies were funded by Networks of Excellence from Spanish Ministry of Science, Innovation and Universities (MICINN) SAF2016‐76377‐R, ‘Nuclear Receptors in Cancer, Metabolism and Inflammation’ (NuRCaMeIn) SAF2017‐90604‐REDT to A.M., ISCIII‐Biomedical Research Networking Centres‐Oncology (CIBERONC) CB16/12/00273 to A.M. and A.B.; CB16/12/00453 to F.X.R.; CB16/12/00342 to E.B. and CB16/12/00241 to F.R. ISCIII‐Biomedical Research Networking Centres‐Respiratory Diseases (CIBERES) CB15/00037 to L.dP, and ISCIII‐FEDER PI15/00934 to F.R

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.The studies were funded by Networks of Excellence from Spanish Ministry of Science, Innovation and Universities (MICINN) SAF2016‐76377‐R, ‘Nuclear Receptors in Cancer, Metabolism and Inflammation’ (NuRCaMeIn) SAF2017‐90604‐REDT to A.M., ISCIII‐Biomedical Research Networking Centres‐Oncology (CIBERONC) CB16/12/00273 to A.M. and A.B.; CB16/12/00453 to F.X.R.; CB16/12/00342 to E.B. and CB16/12/00241 to F.R. ISCIII‐Biomedical Research Networking Centres‐Respiratory Diseases (CIBERES) CB15/00037 to L.dP, and ISCIII‐FEDER PI15/00934 to F.R