Efectos de Wnt3A sobre el transcriptoma de fibroblastos colónicos humanos: regulación y acción de PKP2/Plakofilina-2

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 02-12-2016Esta tesis tiene embargado el acceso al texto completo hasta el 02-06-2018El cáncer colorrectal es el resultado de la transformación maligna de las células epiteliales del colon. Los fibroblastos estromales son el principal componente del microambiente tumoral y juegan un papel importante en la progresión de ésta y otras neoplasias. La señalización a través de la vía Wnt/β-catenina es esencial para la homeostasis del colon, pero su activación constitutiva es un rasgo característico del cáncer colorrectal. En esta Tesis doctoral mostramos el primer estudio transcriptómico del efecto de un factor Wnt sobre una línea celular de fibroblastos humanos de colon. Wnt3A regula la expresión de 1.136 genes, de los cuales 662 son inducidos y 474 inhibidos en células CCD-18Co. Entre ellos, destacan un grupo de genes inducidos por Wnt3A que codifican inhibidores de la propia vía Wnt/β-catenina, sugiriendo un mecanismo de retroinhibición. También mostramos que el gen PKP2, que codifica la proteína desmosomal Plakofilina-2, es una nueva diana transcripcional directa de Wnt/β-catenina en fibroblastos normales y asociados al cáncer de colon. PKP2 es inducido por β-catenina/TCF a través de tres sitios de unión en el promotor del gen y de un sitio adicional situado en una región enhancer, situada a unas 20 kb del sitio de inicio de transcripción. Por otro lado, Plakofilina-2 antagoniza la actividad transcripcional de Wnt/β-catenina en células HEK-293T, lo que sugiere que podría actuar como un inhibidor intracelular de la vía. Nuestros resultados demuestran que los fibroblastos estromales responden a la señalización Wnt canónica y que Plakofilina-2 juega un papel en la retroinhibición de este efecto.Colorectal cancer results from malignant transformation of colonic epithelial cells. Stromal fibroblasts are the major component of tumour microenvironment and play an important role in the progression of this and other neoplasias. Wnt/β-catenin signaling 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 established human colonic fibroblasts. Wnt3A regulates the expression of 1136 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 suggesting 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 the transcription start site. Moreover, Plakophilin-2 antagonizes Wnt/β-catenin transcriptional activity in HEK-293T cells suggesting that it may act as an intracelular inhibitor of the Wnt/β-catenin pathway. Our results demonstrate that stromal fibroblasts respond to canonical Wnt signaling and a role of Plakophilin-2 in the feedback control of this effect

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

Nuclear DICKKOPF-1 as a biomarker of chemoresistance and poor clinical outcome in colorectal cancer

Sporadic colorectal cancer (CRC) insurgence and progression depend on the activation of Wnt/β-catenin signaling. Dickkopf (DKK)-1 is an extracellular inhibitor of Wnt/β-catenin signaling that also has undefined β-catenin-independent actions. Here we report for the first time that a proportion of DKK-1 locates within the nucleus of healthy small intestine and colon mucosa, and of CRC cells at specific chromatin sites of active transcription. Moreover, we show that DKK-1 regulates several cancer-related genes including the cancer stem cell marker aldehyde dehydrogenase 1A1 (ALDH1A1) and Ral-binding protein 1-associated Eps domain-containing 2 (REPS2), which are involved in detoxification of chemotherapeutic agents. Nuclear DKK-1 expression is lost along CRC progression; however, it remains high in a subset (15%) of CRC patients (n = 699) and associates with decreased progression-free survival (PFS) after chemotherapy administration and overall survival (OS) [adjusted HR, 1.65; 95% confidence interval (CI), 1.23-2.21; P = 0.002)]. Overexpression of ALDH1A1 and REPS2 associates with nuclear DKK-1 expression in tumors and correlates with decreased OS (P = 0.001 and 0.014) and PFS. In summary, our findings demonstrate a novel location of DKK-1 within the cell nucleus and support a role of nuclear DKK-1 as a predictive biomarker of chemoresistance in colorectal cancer

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

Gene regulatory and phenotypic effects of calcitriol and canonical WNT in human colon fibroblasts

Trabajo presentado en el 42nd Congress of the Spanish Society of Biochemistry and Molecular Biology, celebrado en Madrid (España), 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 pathwaypromoting 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

Gene regulatory and phenotypic effects of calcitriol and canonical WNT in human colon fibroblasts

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

Milicias y tropas negras de Buenos Aires. Afro argentinos armados para defender a sus amos

The city of Buenos Aires was founded, for the second time 1580 essentially as an outpost of the Spanish Empire to be protected from Indian attacks and possible foreign incursion, basically Portuguese in origin. Thus, from the beginnings of the city's founding, African slaves were used for the defense of the city. They acted in the urban militias, and were outstanding in the defense and re-conquest of the city when the English attacked in 1806- 1807. Innumerable prizes and eulogies were followed by integration of segregated divisions with a white officer corps. After 1813, a series of decrees ordered owners to exchange slaves for use in the Wars of Independence. Thus, property owners were obliged to sell to the State a number of their slaves according to the work performed by the slaves. The slaves would enter the armies as free persons and would serve five years as 'front line" soldiers in order to obtain their freedom. This article studies the participation of the Afro-Argentines in the distinct 'fronts" of the armies of liberation. The paper also reflects on the opinions of the generals who commanded Afro-Argentine troops.La ciudad de Buenos Aires fue fundada, por segunda vez, en 1580 esencialmente como un puesto de avanzada para que los españoles se protegieran de los indígenas y de una posible incursión extranjera de origen portugués. Por lo tanto desde la fundación de la ciudad los esclavos africanos fueron empleados para su defensa. Los esclavos actuaban en milicias urbanas y sus acciones fueron decisivas para la defensa y reconquista de Buenos Aires cuando los ingleses la atacaron en 1806-1807. Innumerables premios y elogios fueron seguidos por la integración de las divisiones separadas, con un cuerpo de oficiales blancos. Después de 1813, una serie de decretos ordenaba a los dueños entregar a sus esclavos para las guerras de Independencia . De esta manera los esclavos entraron al ejército como personas libres y debían servir por cinco años en el frente para obtener su libertad definitiva. Este artículo estudia la participación de afro-argentinos en distintos frentes de los ejércitos de liberación. El ensayo también refleja las opiniones de los generales que comandaban las tropas afro-argentinas

Nuclear DICKKOPF-1 as a biomarker of chemoresistance and poor clinical outcome in colorectal cancer

This work is licensed under a Creative Commons Attribution 3.0 License.-- et al.Sporadic colorectal cancer (CRC) insurgence and progression depend on the activation of Wnt/β-catenin signaling. Dickkopf (DKK)-1 is an extracellular inhibitor of Wnt/β-catenin signaling that also has undefined β-catenin-independent actions. Here we report for the first time that a proportion of DKK-1 locates within the nucleus of healthy small intestine and colon mucosa, and of CRC cells at specific chromatin sites of active transcription. Moreover, we show that DKK-1 regulates several cancerrelated genes including the cancer stem cell marker aldehyde dehydrogenase 1A1 (ALDH1A1) and Ral-binding protein 1-associated Eps domain-containing 2 (REPS2), which are involved in detoxification of chemotherapeutic agents. Nuclear DKK-1 expression is lost along CRC progression; however, it remains high in a subset (15%) of CRC patients (n = 699) and associates with decreased progression-free survival (PFS) after chemotherapy administration and overall survival (OS) [adjusted HR, 1.65; 95% confidence interval (CI), 1.23-2.21; P = 0.002)]. Overexpression of ALDH1A1 antiand REPS2 associates with nuclear DKK-1 expression in tumors and correlates with decreased OS (P = 0.001 and 0.014) and PFS. In summary, our findings demonstrate a novel location of DKK-1 within the cell nucleus and support a role of nuclear DKK-1 as a predictive biomarker of chemoresistance in colorectal cancer.This work was supported by grants from the Ministerio de Economía y Competitividad of Spain and Fondo Europeo de Desarrollo Regional (FEDER) (SAF2013-43468-R, A.M.; BFU2010-19659, J.M.G-S.; SAF2010-19256, B.J.; SAF2011-23089, C.L-O.), Comunidad de Madrid (S2010/BMD-2344 Colomics2, A.M., F.R. and J.M.G-S.) and FEDER-Instituto de Salud Carlos III (RD12/0036/0021, A.M.; RD12/0036/0051, PT13/0010/0012 and PI12/01552, F.R.; ProteoRed Networked Proteomics Platform, F.C.).Peer Reviewe

The human PKP2/plakophilin-2 gene is induced by Wnt/β-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/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.This work was supported by the Spanish Ministerio de Economía y Competitividad and the European Regional Development Fund (Fondos FEDER) (SAF2013-43468‐R, and SAF2014-53819‐R), the Agencia Estatal de Investigación‐Fondos FEDER (SAF2016-76377‐R, AEI/FEDER, UE), the Comunidad de Madrid (S2010/BMD‐2344 Colomics2), the Instituto de Salud Carlos III-Fondos FEDER (RD12/0036/0021, and CIBERONC‐CB16/12/00273) and the Nuclear Receptors Network (Nurcamein, SAF2015-71878‐REDT)