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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). *

[Background]: The rising incidence of colorectal cancer (CRC) among young patients is alarming. We aim to characterize the clinico-pathological features and outcomes of patients with early-onset CRC (EOCRC)., [Methods]: We included all of the patients with pathologically confirmed diagnosis of CRC at Hospital Universitario La Paz from October 2016 to September 2020. EOCRC age cut-off was 50 years. All statistical analyses were carried out using SPSS v.25. [Results]: A total of 1152 patients were diagnosed with CRC, fifty-nine (5,1%) of them were After a median follow-up of 24 months, 279 patients have died. Median overall survival (OS) was not reached in either group (p ¼ 0,06). Three-year OS was 80% (95% CI: 73-87) and 67 (95%CI: 65-69) in the younger and older group, respectively. In patients with localized disease that underwent surgery or other antineoplastic treatment ( n ¼ 856), 159 events for disease-free survival (DFS) were observed. Median DFS was not reached in either group (p ¼0,144). Three-year DFS was 86% (95%CI: 79-93) and 73% (95%CI: 71-75, respectively). In patients with metastatic disease (n ¼ 332; synchronous or metachronic), median OS was not reach in the EOCRC group vs 18,1 (95%CI: 13,8-22,4), p ¼ 0,05). In those patients with metastatic EOCRC with mutational status assessed (n ¼23), no difference in OS according to RAS was observed (p ¼ 0,55)., [Conclusions]: Patients with EOCRC are diagnosed at a more advanced stage and display distinct biological features (more prevalence of dMMR and WT tumors among others). Studies focusing on screening in this population and deeper molecular profiling are needed.

Resistance to antitumor treatments is one of the most important problems faced by clinicians in the management of colorectal cancer (CRC) patients. Cancer-Associated Fibroblasts (CAFs) are the main producers and remodelers of the extracellular matrix (ECM), which is directly involved in drug resistance mechanisms. Primary Normal Fibroblasts (NFs) and CAFs and cell lines (fibroblasts and tumor cells), were used to generate ECM and to identify its role in the oxaliplatin and cetuximab chemoresistance processes of CRC cells mediated by SNAI1-expressing fibroblasts. Matrices generated by Snai1 KO MEFs (Knockout Mouse Embryonic Fibroblasts) confer less resistance on oxaliplatin and cetuximab than wild-type MEF-derived matrices. Similarly, matrices derived from CAFs cause greater survival of colorectal cancer cells than NF-derived matrices, in a similar way to Snai1 expression levels. In addition, Snail1 expression in fibroblasts regulates drug resistance and metabolism gene expression in tumor cells mediated by ECM. Finally, a series of 531 patients (TCGA) with CRC was used to assess the role of SNAI1 expression in patients' prognosis indicating an association between tumor SNAI1 expression and overall survival in colon cancer patients but not in rectal cancer patients. SNAI1 expression in CRC cancer patients, together with in vitro experimentation, suggests the possible use of SNAI1 expression in tumor-associated fibroblasts as a predictive biomarker of response to oxaliplatin and cetuximab treatments in patients with CRC., This research is supported by PI17/01847 and PI20/00602 from the Instituto de Salud Carlos III and co-financed by the European Development Regional Fund (FEDER) “A way to achieve Europe” (ERDF); by “CIBER de Cáncer”, CB16/12/00273 and CB16/12/00446 from the Instituto de Salud Carlos III FEDER “A way to achieve Europe” (ERDF). Manuel Collado is funding by the Instituto de Salud Carlos III and co-financed by the European Development Regional Fund (FEDER) “A way to achieve Europe” (ERDF), FI21/00132.

Trabajo presentado en el IV Young Researchers Meeting CIBERONC_BBN, celebrado en Barcelona (España) del 13 al 14 de diciembre de 2021., Introduction: Resistance to antitumor treatments is one of the most important problems faced by clinicians in the management of colon cancer (CC) patients. Cancer Associated Fibroblasts (CAFs) are the main producers and remodelers of extracellular matrix (ECM). The ECM conditions tumor growth, favoring angiogenesis and conditioning the migration of malignant cells, as well as participating in drug resistance mechanisms.Objective: Our group determined the role of SNAI1 in the regulation and remodeling of ECM. We aim to identify the role of the ECM in the chemoresistance processes of CC cells mediated by SNAI1-expressing fibroblasts. Moreover, we propose CAF-Snai1 expression as a potential biomarker to identify a group of patients that could present treatment resistances.Material and Methods:Primary NFs and CAFs were obtained from colon tissue samples from patients under surgery at Hospital Ramón y Cajal. ECM were generated with wild-type MEF, Snai1 knock-out MEFs and primary NFs and CAFs. Survival of drug-treated tumor cells seeded on ECM was tested by fluorescence. PCR Array of genes involved in drug resistance and cancer (PAHS-004Z, Qiagen) was used to identify Snai1 regulated genes. SNAIL gene expression information was obtained from TCGA in a CC cohort (N=436). Results:Matrices generated by Snai1 KO MEFs confer less resistance to oxaliplatin and cetuximab than wild-type MEFs derived matrices. Matrices derived from CAFs induce higher survival to colon cancer cells than NFs derived matrices in a related manner to Snai1 expression levels. In addition, Snail1 expression in fibroblasts regulate drug resistance and metabolism gene expression in tumor cells mediated by ECM. Finally, an association between tumor SNAI1 expression and overall survival is observed in advanced stages CC patient. Conclusions:SNAI1 expression in fibroblasts conditions the resistance of CC cells to oxaliplatin and cetuximab, by the ECM communication. It is important to point out the prognostic value of SNAI1 expression in patients with advanced stages CC, to whom different pharmacological treatments are provided as standard, which, together with in vitro experimentation, suggests the possible use of SNAI1 expression in tumor-associated fibroblasts as a predictive biomarker of response to oxaliplatin and cetuximab treatments in patients with CC.

Trabajo presentado en el XIX Congreso de la Sociedad Española de Biología Celular, celebrado en Boadilla del Monte (Madrid) del 26 al 29 de octubre de 2021.

Trabajo presentado en el 43rd Annual Meeting of the SEBBM, celebrado en Barcelona (España) del 19 al 22 de julio de 2021., Worldwide, colorectal cancer (CRC) is the third most common malignant neoplasm and the second leading cause of cancer-associated mortality, with an estimated increase in global prevalence of 60% by 2030 (1,2). Mutational inactivation of adenomatous polyposis coli (APC) is the hallmark of CRC and leads to an overactivation of WNT signaling that favors the development and progression of CRC (3). Large epidemiological studies suggest that the diabetic population is at increased risk for site-specific cancers, including CRC (4). Our laboratory has shown that hyperglycemia induces the accumulation of ROS in CRC but not healthy cells, driving the activation of a newly described ROS/AMPK/EP300 axis that enhances Wnt/b-catenin signaling. Increased EP300 leads to increased acetylation of β-catenin at K354, a requirement for nuclear accumulation and transcriptional activation of WNT target genes (5,6). The critical role driven by ROS suggest a possible involvement of the NADPH oxidases (NOX family, as a source of ROS. Specifically, NOX 1 and NOX 4 are expressed in colon epithelial cells, and their overexpression in CRC cells promotes cell proliferation and invasiveness (7,8,9,10). Our results indicate that hyperglycemia significantly increases NOX1 levels, in correlation with increased ROS production in CRC cells, suggesting a possible regulation of the ROS/ AMPK/EP300 axis by NOX1. Antioxidant mechanisms dealing with NOX1-induced ROS should be effective against CRC. Vitamin D (1α, 25-dihydroxyvitamin D3) is a powerful antioxidant that inhibits proliferation and promotes differentiation of CRC cells at least partially through inhibition of Wnt/β-catenin signalling. Consequently, vitamin D deficiency is associated with poor survival to CRC (11,12). Our results indicate that vitamin D causes a reduction in the levels and / or activity of some members of the NOX family by turning off the ROS/AMPK/EP300/β-catenin axis and its proliferative and tumorigenic effects. The data suggest a new antitumor mechanism of vitamin D linked to its anti-oxidant action. Our results integrate independent epidemiological links between vitamin D deficiency, diabetes and cancer in one overarching and unifying mechanism.

Trabajo presentado en el XIX Congreso de la Sociedad Española de Biología Celular, celebrado en Boadilla del Monte (Madrid) del 26 al 29 de octubre de 2021.

Trabajo presentado en Keystone eSymposia Meeting Vitamin D Workshop, celebrado en modalidad virtual del 13 al 14 de octubre de 2021.

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 el Simposio "Nuclear receptors and cancer", celebrado en Madrid (España) del 3 al 4 de diciembre 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.

Trabajo presentado en el Ciclo de seminarios de Innovación Biomédica, celebrado en Madrid (España) el 25 de julio de 2019.

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

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.