RESUMEN
The gastrulation process relies on complex interactions between developmental signaling pathways that are not completely understood. Here, we interrogated the contribution of the Hippo signaling effector YAP1 to the formation of the three germ layers by analyzing human embryonic stem cell (hESC)-derived 2D-micropatterned gastruloids. YAP1 knockout gastruloids display a reduced ectoderm layer and enlarged mesoderm and endoderm layers compared with wild type. Furthermore, our epigenome and transcriptome analysis revealed that YAP1 attenuates Nodal signaling by directly repressing the chromatin accessibility and transcription of key genes in the Nodal pathway, including the NODAL and FOXH1 genes. Hence, in the absence of YAP1, hyperactive Nodal signaling retains SMAD2/3 in the nuclei, impeding ectoderm differentiation of hESCs. Thus, our work revealed that YAP1 is a master regulator of Nodal signaling, essential for instructing germ layer fate patterning in human gastruloids.
Asunto(s)
Estómago/citología , Proteínas Señalizadoras YAP/metabolismo , Proteína Morfogenética Ósea 4/farmacología , Diferenciación Celular , Ensamble y Desensamble de Cromatina , Ectodermo/citología , Ectodermo/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Microscopía Fluorescente , Modelos Biológicos , Proteína Nodal/antagonistas & inhibidores , Proteína Nodal/genética , Proteína Nodal/metabolismo , Transducción de Señal , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Estómago/metabolismo , Proteínas Señalizadoras YAP/deficiencia , Proteínas Señalizadoras YAP/genéticaRESUMEN
The transforming growth factor beta (TGF-ß) signaling is fundamental for correct embryonic development. However, alterations of this pathway have been correlated with oncogenesis, tumor progression and sustaining of cancer stem cells (CSCs). Cripto-1 (CR-1) and Nodal are two embryonic proteins involved in TGF-ß signaling. Their expression is almost undetectable in terminally differentiated cells, but they are often re-expressed in tumor cells, especially in CSCs. Moreover, cancer cells that show high levels of CR-1 and/or Nodal display more aggressive phenotypes in vitro, while in vivo their expression correlates with a worse prognosis in several human cancers. The ability to target CSCs still represents an unmet medical need for the complete eradication of certain types of tumors. Given the prognostic role and the selective expression of CR-1 and Nodal on cancer cells, they represent archetypes for targeted therapy. The aim of this review is to clarify the role of CR-1 and Nodal in cancer stem populations and to summarize the current therapeutic strategy to target CSCs using monoclonal antibodies (mAbs) or other molecular tools to interfere with these two proteins.
Asunto(s)
Antineoplásicos/uso terapéutico , Proteínas Ligadas a GPI/antagonistas & inhibidores , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Proteína Nodal/antagonistas & inhibidores , Animales , Humanos , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
Following implantation, the human embryo undergoes major morphogenetic transformations that establish the future body plan. While the molecular events underpinning this process are established in mice, they remain unknown in humans. Here we characterise key events of human embryo morphogenesis, in the period between implantation and gastrulation, using single-cell analyses and functional studies. First, the embryonic epiblast cells transition through different pluripotent states and act as a source of FGF signals that ensure proliferation of both embryonic and extra-embryonic tissues. In a subset of embryos, we identify a group of asymmetrically positioned extra-embryonic hypoblast cells expressing inhibitors of BMP, NODAL and WNT signalling pathways. We suggest that this group of cells can act as the anterior singalling centre to pattern the epiblast. These results provide insights into pluripotency state transitions, the role of FGF signalling and the specification of anterior-posterior axis during human embryo development.
Asunto(s)
Implantación del Embrión/genética , Desarrollo Embrionario , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica/genética , Estratos Germinativos/metabolismo , Análisis de la Célula Individual/métodos , Vía de Señalización Wnt , Proteína Morfogenética Ósea 1/antagonistas & inhibidores , Linaje de la Célula , Células Cultivadas , Implantación del Embrión/fisiología , Embrión de Mamíferos , Factores de Crecimiento de Fibroblastos/metabolismo , Gastrulación/fisiología , Estratos Germinativos/citología , Humanos , Procesamiento de Imagen Asistido por Computador , Familia de Multigenes , Proteína Nodal/antagonistas & inhibidores , RNA-Seq , Análisis Espacio-TemporalRESUMEN
The first differentiation event in mammalian embryos is the formation of the trophectoderm, which is the progenitor of the outer epithelial component of the placenta and supports the fetus during intrauterine life. Our understanding of these events is limited, particularly in human, because of ethical and legal restrictions and availability of adequate in vitro models would be very advantageous. Here we describe a method that converts human fibroblasts into trophoblast-like cells, combining the use of 5-azacytidine-CR (5-aza-CR) to erase the original cell phenotype and a cocktail containing bone morphogenetic protein 4 (BMP4) with inhibitors of the Activin/Nodal/ERK signaling pathways, to drive erased fibroblasts into the trophoblastic differentiation. This innovative method uses very easily accessible cells to derive trophoblast-like cells and it can be useful to study embryo implantation disorders related to aging.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Fibroblastos/citología , Trofoblastos/citología , Activinas/antagonistas & inhibidores , Animales , Azacitidina/farmacología , Proteína Morfogenética Ósea 4/metabolismo , Proteína Morfogenética Ósea 4/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Implantación del Embrión , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/citología , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Proteína Nodal/antagonistas & inhibidores , Placenta/citología , Embarazo , Transducción de Señal , Piel/citología , Piel/crecimiento & desarrolloRESUMEN
Pancreatic cancer remains a grueling disease that is projected to become the second-deadliest cancer in the next decade. Standard treatment of pancreatic cancer is chemotherapy, which mainly targets the differentiated population of tumor cells; however, it paradoxically sets the roots of tumor relapse by the selective enrichment of intrinsically chemoresistant pancreatic cancer stem cells that are equipped with an indefinite capacity for self-renewal and differentiation, resulting in tumor regeneration and an overall anemic response to chemotherapy. Crosstalk between pancreatic tumor cells and the surrounding stromal microenvironment is also involved in the development of chemoresistance by creating a supportive niche, which enhances the stemness features and tumorigenicity of pancreatic cancer cells. In addition, the desmoplastic nature of the tumor-associated stroma acts as a physical barrier, which limits the intratumoral delivery of chemotherapeutics. In this review, we mainly focus on the transforming growth factor beta 1 (TGFB1)/inhibin subunit beta A (INHBA) homodimer/Nodal-SMAD2/3 signaling network in pancreatic cancer as a pivotal central node that regulates multiple key mechanisms involved in the development of chemoresistance, including enhancement of the stem cell-like properties and tumorigenicity of pancreatic cancer cells, mediating cooperative interactions between pancreatic cancer cells and the surrounding stroma, as well as regulating the deposition of extracellular matrix proteins within the tumor microenvironment.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Terapia Molecular Dirigida , Neoplasias Pancreáticas/tratamiento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Humanos , Subunidades beta de Inhibinas/antagonistas & inhibidores , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Proteína Nodal/antagonistas & inhibidores , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Transducción de Señal , Proteína Smad2/antagonistas & inhibidores , Proteína smad3/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Microambiente TumoralRESUMEN
E protein transcription factors are crucial for many cell fate decisions. However, the roles of E proteins in the germ-layer specification of human embryonic stem cells (hESCs) are poorly understood. We disrupted the TCF3 gene locus to delete the E protein E2A in hESCs. E2A knockout (KO) hESCs retained key features of pluripotency, but displayed decreased neural ectoderm coupled with enhanced mesoendoderm outcomes. Genome-wide analyses showed that E2A directly regulates neural ectoderm and Nodal pathway genes. Accordingly, inhibition of Nodal or E2A overexpression partially rescued the neural ectoderm defect in E2A KO hESCs. Loss of E2A had little impact on the epigenetic landscape of hESCs, whereas E2A KO neural precursors displayed increased accessibility of the gene locus encoding the Nodal agonist CRIPTO. Double-deletion of both E2A and HEB (TCF12) resulted in a more severe neural ectoderm defect. Therefore, this study reveals critical context-dependent functions for E2A in human neural ectoderm fate specification.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas Ligadas a GPI/genética , Células Madre Embrionarias Humanas/citología , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de Neoplasias/genética , Proteína Nodal/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Diferenciación Celular/genética , Linaje de la Célula/genética , Ectodermo/crecimiento & desarrollo , Ectodermo/metabolismo , Epigénesis Genética/genética , Regulación del Desarrollo de la Expresión Génica/genética , Genoma Humano/genética , Células Madre Embrionarias Humanas/metabolismo , Humanos , Células-Madre Neurales/citología , Proteína Nodal/antagonistas & inhibidores , Transducción de Señal/genéticaRESUMEN
The roles of cancer-associated fibroblasts (CAFs) in progression of gastric cancer (GC) are far from well illustration. Here, we show that CAFs can trigger the proliferation and decrease the doxorubicin (Dox) sensitivity of GC cells via secretion of Nodal, one embryonic morphogen that can promote malignancy of various cancers. The neutralization antibody of Nodal can attenuate CAFs-induced cell proliferation. Further, CAFs can activate the Smad2/3 signal, which further increase the phosphorylation and nuclear localization of Akt, in GC cells. While anti-Nodal can abolish the CAFs-induced activation of Smad2/3/Akt signals. Further, both inhibitors of Smad2/3 and Akt can attenuate CAFs-induced proliferation of GC cells. All these data suggest that CAFs can increase the malignancy of GC cells via Nodal-induced activation of Smad2/3/Akt signals. It indicates that CAFs/Nodal signals might be a potential new target of clinical interventions for GC patients. SIGNIFICANCE OF THE STUDY: The roles about CAFs in progression of GC are not well illustrated. Our present study reveals that CAFs can increase the proliferation and decrease the Dox sensitivity of GC cells via secretion of Nodal. The secreted Nodal further activated Samd2/3/Akt signals to trigger the GC progression. It suggests that targeted inhibition CAFs/Nodal might be a potential approach for GC therapy.
Asunto(s)
Fibroblastos Asociados al Cáncer/metabolismo , Proteína Nodal/metabolismo , Transducción de Señal , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Antibióticos Antineoplásicos/farmacología , Fibroblastos Asociados al Cáncer/efectos de los fármacos , Fibroblastos Asociados al Cáncer/patología , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Doxorrubicina/farmacología , Humanos , Proteína Nodal/antagonistas & inhibidores , Proteína Nodal/genética , Transducción de Señal/efectos de los fármacos , Neoplasias Gástricas/tratamiento farmacológico , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Several studies have unraveled the negative role of Akt1 in advanced cancers, including metastatic prostate cancer (mPCa). Hence, understanding the consequences of targeting Akt1 in the mPCa and identifying its downstream novel targets is essential. We studied how Akt1 deletion in PC3 and DU145 cells activates the Nodal pathway and promotes PCa epithelial-to-mesenchymal transition (EMT) and metastasis. Here we show that Akt1 loss increases Nodal expression in PCa cells accompanied by activation of FoxO1/3a, and EMT markers Snail and N-cadherin as well as loss of epithelial marker E-cadherin. Treatment with FoxO inhibitor AS1842856 abrogated the Nodal expression in Akt1 deleted PCa cells. Akt1 deficient PCa cells exhibited enhanced cell migration and invasion in vitro and lung metastasis in vivo, which were attenuated by treatment with Nodal pathway inhibitor SB505124. Interestingly, Nodal mRNA analysis from two genomic studies in cBioportal showed a positive correlation between Nodal expression and Gleason score indicating the positive role of Nodal in human mPCa. Collectively, our data demonstrate Akt1-FoxO3a-Nodal pathway as an important mediator of PCa metastasis and present Nodal as a potential target to treat mPCa patients.
Asunto(s)
Transición Epitelial-Mesenquimal/genética , Neoplasias Pulmonares/secundario , Proteína Nodal/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Animales , Benzodioxoles/farmacología , Benzodioxoles/uso terapéutico , Movimiento Celular/genética , Supervivencia Celular/genética , Proteína Forkhead Box O3/antagonistas & inhibidores , Proteína Forkhead Box O3/metabolismo , Silenciador del Gen , Humanos , Imidazoles/farmacología , Imidazoles/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Masculino , Ratones , Ratones Desnudos , Metástasis de la Neoplasia/genética , Proteína Nodal/antagonistas & inhibidores , Células PC-3 , Piridinas/farmacología , Piridinas/uso terapéutico , Quinolonas/farmacología , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Fibroblasts become cancer-associated fibroblasts (CAFs) in the tumor microenvironment after activation by transforming growth factor-ß (TGF-ß) and are critically involved in cancer progression. However, it is unknown whether the TGF superfamily member Nodal, which is expressed in various tumors but not expressed in normal adult tissue, influences the fibroblast to CAF conversion. Here, we report that Nodal has a positive correlation with α-smooth muscle actin (α-SMA) in clinical melanoma and colorectal cancer (CRC) tissues. We show the Nodal converts normal fibroblasts to CAFs, together with Snail and TGF-ß signaling pathway activation in fibroblasts. Activated CAFs promote cancer growth in vitro and tumor-bearing mouse models in vivo. These results demonstrate that intercellular crosstalk between cancer cells and fibroblasts is mediated by Nodal, which controls tumor growth, providing potential targets for the prevention and treatment of tumors.
Asunto(s)
Diferenciación Celular , Neoplasias Colorrectales/patología , Melanoma/patología , Proteína Nodal/metabolismo , Actinas/metabolismo , Animales , Fibroblastos Asociados al Cáncer/citología , Fibroblastos Asociados al Cáncer/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Neoplasias Colorrectales/metabolismo , Femenino , Humanos , Melanoma/metabolismo , Ratones , Ratones Desnudos , Proteína Nodal/antagonistas & inhibidores , Proteína Nodal/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/farmacología , Trasplante HeterólogoRESUMEN
The number of molecules identified to be involved in communication between placenta and decidua is fast expanding. Previously, we showed that NODAL expressed in maternal endometrial stromal cells is able to affect NODAL and STOX1 expression in placental extravillous trophoblasts. The effect of maternal NODAL on placental NODAL expression is achieved via Activin A, while preliminary data suggests that maternal NODAL affects STOX1 expression in trophoblasts potentially via IGF1. In the current study, T-HESC endometrial stromal cells were treated with siRNAs against NODAL after which IGF1 mRNA expression was determined by quantitative RT-PCR, while IGF1 secretion was measured by ELISA. Recombinant IGF1 and inhibitors of the MAPK and PI3K/AKT pathways were added to SGHPL-5 extravillous trophoblasts after which the effects on STOX1 mRNA and STOX1 protein expression were determined. The effect of IGF1 and the MAPK and PI3K/AKT inhibitors on the invasive capacity of SGHPL-5 cells was investigated by performing invasion assays. We found that T-HESC cells treated with NODAL siRNAs showed significant upregulation of IGF1 mRNA expression and IGF1 protein secretion. Addition of IGF1 to SGHPL-5 cell media significantly upregulated STOX1 mRNA and protein expression. Using inhibitors of the PI3K/AKT and MAPK pathway showed that the effect of IGF1 on STOX1 expression is accomplished via MAPK signaling. Secondly, PI3K inhibition independently leads to reduced STOX1 expression which can be rescued by adding IGF1. IGF1 was unable to influence the invasive capacity of SGHPL-5 cells, while inhibiting the PI3K/AKT pathway did reduce the invasion of these cells. To conclude, here we show that downregulated NODAL expression in endometrial stromal cells, previously associated with pre-eclampsia like symptoms in mice, increases IGF1 secretion. Increased levels of IGF1 lead to increased expression levels of STOX1 in extravillous trophoblasts via the MAPK pathway, hereby identifying a novel signaling cascade involved in maternal-fetal communication.
Asunto(s)
Proteínas Portadoras/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Proteína Nodal/metabolismo , Trofoblastos/metabolismo , Línea Celular , Endometrio/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Nodal/antagonistas & inhibidores , Proteína Nodal/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Células del Estroma/metabolismoRESUMEN
Adult rudiment formation in some temnopleurids begins with the formation of a cell mass that is pinched off the left ectoderm in early larval development. The cell mass forms the adult rudiment with the left coelomic pouch of the mesodermal region. However, details of the mechanisms to establish position of the cell mass are still unknown. We analyzed the inhibiting effect of Nodal, a factor for morphogenesis of the oral region and right side, for location of the cell mass, in four temnopleurids. Pulse inhibition, at least 5 min inhibition, during coelomic pouch formation allowed a cell mass to form on both sides, whereas treatments after that period did not. These results indicate that Nodal signaling controls the oral-aboral axis before gastrulation and then affects the position of the cell mass and adult rudiment up to coelomic pouch formation. They also indicate that the position of the adult rudiment under Nodal signaling pathways is conserved in temnopleurids, as adult rudiment formation is dependent on the cell mass.
Asunto(s)
Proteína Nodal/metabolismo , Erizos de Mar/crecimiento & desarrollo , Animales , Benzamidas/farmacología , Tipificación del Cuerpo , Dioxoles/farmacología , Regulación del Desarrollo de la Expresión Génica , Proteína Nodal/antagonistas & inhibidores , Erizos de Mar/clasificación , Erizos de Mar/genéticaRESUMEN
The encouraging response and improved survival of acute promyelocytic leukemia patients following retinoic acid treatment has rendered differentiation therapy an attractive option in cancer treatment. Given that terminal differentiation represents a considerable barrier in retinoblastoma tumorigenesis and that retinoblastoma has a significantly higher spontaneous degeneration rate compared with other tumors (1,000-fold change), differentiation therapy represents a promising alternative in the treatment of retinoblastoma. However, the full differentiation potential of retinoblastoma still unknown. The present study was designed to investigate the extend differentiation of the classical retinoblastoma cell line WERI-Rb-1 (W-RBCs). Several critical cell signaling pathways and key genes related to cell proliferation and differentiation were comprehensively regulated to control the fate of W-RBCs. Various strategies were applied to optimize simple and time-saving methods to induce W-RBCs into different types of retinal neuron-like cells (RNLCs) in vitro. Further, the tumorigenesis of these differentiated W-RBCs was tested in nude mice in vivo. W-RBCs were found to inherently express both retinal progenitor cell- and embryonic stem cell-related genes or proteins. Moreover, the addition of antagonists of critical cell signals (Wnt, Nodal, BMP4 and Notch), even without atonal bHLH transcription factor 7 gene transfection, could directly induce W-RBCs into RNLCs, and especially into photoreceptor-like and retinal ganglion-like cells. Interestingly, the differentiated cells showed remarkably poorer tumorigenesis in vivo. These findings may offer new insights on the oriented differentiation of W-RBCs into RNLCs with low tumorigenicity and provide potential targets for retinoblastoma differentiation therapy.
Asunto(s)
Diferenciación Celular/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/genética , Neoplasias de la Retina/terapia , Retinoblastoma/terapia , Animales , Proteína Morfogenética Ósea 4/antagonistas & inhibidores , Proteína Morfogenética Ósea 4/genética , Proteína Morfogenética Ósea 4/metabolismo , Línea Celular Tumoral , Proliferación Celular , Medios de Cultivo/farmacología , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Proteína Nodal/antagonistas & inhibidores , Proteína Nodal/genética , Proteína Nodal/metabolismo , Receptores Notch/antagonistas & inhibidores , Receptores Notch/genética , Receptores Notch/metabolismo , Neoplasias de la Retina/genética , Neoplasias de la Retina/metabolismo , Neoplasias de la Retina/patología , Neuronas Retinianas/citología , Neuronas Retinianas/metabolismo , Retinoblastoma/genética , Retinoblastoma/metabolismo , Retinoblastoma/patología , Transducción de Señal , Proteínas Wnt/antagonistas & inhibidores , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
Metastatic melanoma is a highly aggressive skin cancer with a poor prognosis. It is the leading cause of skin cancer deaths with a median overall survival for advanced-stage metastatic disease of <6 months. Despite advances in the field with conventional and targeted therapies, the heterogeneity of melanoma poses the greatest ongoing challenge, ultimately leading to relapse and progression to a more drug-resistant tumor in most patients. Particularly noteworthy are recent findings, indicating that these therapies exert selective pressure on tumors resulting in the activation of pathways associated with cancer stem cells that are unresponsive to current therapy. Our previous studies have shown how Nodal, an embryonic morphogen of the transforming growth factor-beta superfamily, is one of these critical factors that is reactivated in aggressive melanoma and resistant to conventional chemotherapy, such as dacarbazine. In the current study, we sought to determine whether BRAF inhibitor (BRAFi) therapy targeted Nodal-expressing tumor cells in uniquely matched unresectable stage III and IV melanoma patient samples before and after therapy that preceded their eventual death due to disease. The results demonstrate that BRAFi treatment failed to affect Nodal levels in melanoma tissues. Accompanying experiments in soft agar and in nude mice showed the advantage of using combinatorial treatment with BRAFi plus anti-Nodal monoclonal antibody to suppress tumor growth and metastasis. These data provide a promising new approach using front-line therapy combined with targeting a cancer stem cell-associated molecule-producing a more efficacious response than monotherapy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Melanoma/tratamiento farmacológico , Proteína Nodal/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/administración & dosificación , Western Blotting , Línea Celular Tumoral , Femenino , Humanos , Imidazoles/administración & dosificación , Inmunohistoquímica , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Melanoma/genética , Melanoma/metabolismo , Ratones Desnudos , Terapia Molecular Dirigida/métodos , Mutación , Proteína Nodal/inmunología , Proteína Nodal/metabolismo , Oximas/administración & dosificación , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Piridonas/administración & dosificación , Pirimidinonas/administración & dosificación , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Resultado del Tratamiento , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
BACKGROUND/AIMS: The previous study in our lab showed that Nodal molecule on bronchial epithelial cells (BECs) was modulated by all kinds of lung microbes. The present study was designed to determine the effects of Nodal on proliferation of BECs and BECs-induced differentiation of T-helper (Th) cells. The epigenetic mechanisms of Nodal expression following treatments of different lung microbes were also identified. METHODS: Real-time polymerization chain reaction (PCR) and western blot were used to determine the expression of Nodal. Flow cytometry was used to observe the effects of proliferation of BECs and subsequent BECs-induced differentiation of Th cells. Methylation levels of CpG islands in Nodal promoters were also analyzed by time of flight mass spectrometry. RESULTS: The results showed that Nodal promoted proliferation of BECs and BECs-induced differentiation of Th cell from Th1 to Th2 and Th17. Nodal promoter showed a hyper-methylation in normal BECs. Through methylation modification in the promoter, P. aeruginosa or A.baumanni inhibited the expression of Nodal while RSV promoted the expression of Nodal. CONCLUSIONS: Our data showed that Nodal promoted Th2 and Th17 differentiation and inhibited Th1 differentiation which may cause imbalance of airway microenvironment. P. aeruginosa or A.baumanni may be hopeful for the treatment of airway hyperresponsveness by inhibition Nodal expression through DNA methylation modification in the promoter.
Asunto(s)
Diferenciación Celular , Metilación de ADN , Proteína Nodal/metabolismo , Linfocitos T Colaboradores-Inductores/citología , Acinetobacter baumannii/fisiología , Bronquios/citología , Linfocitos T CD4-Positivos/citología , Línea Celular , Técnicas de Cocultivo , Islas de CpG , Epigénesis Genética , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Proteína Nodal/antagonistas & inhibidores , Proteína Nodal/genética , Regiones Promotoras Genéticas , Pseudomonas aeruginosa/fisiología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Virus Sincitiales Respiratorios/patogenicidad , Espectrometría de Masa por Ionización de Electrospray , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
Nodal is highly expressed in various human malignancies, thus supporting the rationale for exploring Nodal as a therapeutic target. Here, we describe the effects of a novel monoclonal antibody (mAb), 3D1, raised against human Nodal. In vitro treatment of C8161 human melanoma cells with 3D1 mAb shows reductions in anchorage-independent growth and vasculogenic network formation. 3D1 treated cells also show decreases of Nodal and downstream signaling molecules, P-Smad2 and P-ERK and of P-H3 and CyclinB1, with an increase in p27. Similar effects were previously reported in human breast cancer cells where Nodal expression was generally down-regulated; following 3D1 mAb treatment, both Nodal and P-H3 levels are reduced. Noteworthy is the reduced growth of human melanoma xenografts in Nude mice treated with 3D1 mAb, where immunostaining of representative tumor sections show diminished P-Smad2 expression. Similar effects both in vitro and in vivo were observed in 3D1 treated A375SM melanoma cells harboring the active BRAF(V600E) mutation compared to treatments with IgG control or a BRAF inhibitor, dabrafenib. Finally, we describe a 3D1-based ELISA for the detection of Nodal in serum samples from cancer patients. These data suggest the potential of 3D1 mAb for selecting and targeting Nodal expressing cancers.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Neoplasias de la Mama/patología , Melanoma/patología , Proteína Nodal/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Ciclina B1/biosíntesis , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Quinasas MAP Reguladas por Señal Extracelular/biosíntesis , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Imidazoles/farmacología , Ratones , Proteína Nodal/sangre , Proteína Nodal/inmunología , Oximas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Proteína Smad2/biosíntesis , Resonancia por Plasmón de SuperficieRESUMEN
The secreted glycoprotein sonic hedgehog (Shh) is expressed in the prechordal mesoderm, where it plays a crucial role in induction and patterning of the ventral forebrain. Currently little is known about how Shh is regulated in prechordal tissue. Here we show that in the embryonic chick, Shh is expressed transiently in prechordal mesoderm, and is governed by unprocessed Nodal. Exposure of prechordal mesoderm microcultures to Nodal-conditioned medium, the Nodal inhibitor CerS, or to an ALK4/5/7 inhibitor reveals that Nodal is required to maintain both Shh and Gsc expression, but whereas Gsc is largely maintained through canonical signalling, Nodal signals through a non-canonical route to maintain Shh. Further, Shh expression can be maintained by a recombinant Nodal cleavage mutant, proNodal, but not by purified mature Nodal. A number of lines of evidence suggest that proNodal acts via FGFR3. ProNodal and FGFR3 co-immunoprecipitate and proNodal increases FGFR3 tyrosine phosphorylation. In microcultures, soluble FGFR3 abolishes Shh without affecting Gsc expression. Further, prechordal mesoderm cells in which Fgfr3 expression is reduced by Fgfr3 siRNA fail to bind to proNodal. Finally, targeted electroporation of Fgfr3 siRNA to prechordal mesoderm in vivo results in premature Shh downregulation without affecting Gsc. We report an inverse correlation between proNodal-FGFR3 signalling and pSmad1/5/8, and show that proNodal-FGFR3 signalling antagonises BMP-mediated pSmad1/5/8 signalling, which is poised to downregulate Shh. Our studies suggest that proNodal/FGFR3 signalling governs Shh duration by repressing canonical BMP signalling, and that local BMPs rapidly silence Shh once endogenous Nodal-FGFR3 signalling is downregulated.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Hedgehog/metabolismo , Mesodermo/embriología , Proteína Nodal/metabolismo , Prosencéfalo/embriología , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/fisiología , Animales , Embrión de Pollo , Electroporación , Inmunohistoquímica , Inmunoprecipitación , Hibridación in Situ , Mesodermo/metabolismo , Proteína Nodal/antagonistas & inhibidores , ARN Interferente Pequeño/genética , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Proteínas Smad/metabolismoRESUMEN
Nodal is a potent embryonic morphogen belonging to the TGF-ß superfamily. Typically, it also binds to the ALK4/ActRIIB receptor complex in the presence of the co-receptor Cripto-1. Nodal expression is physiologically restricted to embryonic tissues and human embryonic stem cells, is absent in normal cells but re-emerges in several human cancers, including melanoma, breast, and colon cancer. Our aim was to obtain mAbs able to recognize Nodal on a major CBR (Cripto-Binding-Region) site and to block the Cripto-1-mediated signalling. To achieve this, antibodies were raised against hNodal(44-67) and mAbs generated by the hybridoma technology. We have selected one mAb, named 3D1, which strongly associates with full-length rhNodal (KD 1.4 nM) and recognizes the endogenous protein in a panel of human melanoma cell lines by western blot and FACS analyses. 3D1 inhibits the Nodal-Cripto-1 binding and blocks Smad2/3 phosphorylation. Data suggest that inhibition of the Nodal-Cripto-1 axis is a valid therapeutic approach against melanoma and 3D1 is a promising and interesting agent for blocking Nodal-Cripto mediated tumor development. These findings increase the interest for Nodal as both a diagnostic and prognostic marker and as a potential new target for therapeutic intervention.
Asunto(s)
Anticuerpos Monoclonales/química , Modelos Moleculares , Proteína Nodal/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Anticuerpos Monoclonales/farmacología , Mapeo Epitopo/métodos , Epítopos/química , Epítopos/metabolismo , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/metabolismo , Factores de Diferenciación de Crecimiento/química , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteína Nodal/antagonistas & inhibidores , Proteína Nodal/metabolismo , Péptidos/síntesis química , Péptidos/química , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Unión ProteicaRESUMEN
INTRODUCTION: Pre-eclampsia is a pregnancy-specific disorder and characterized by reduced trophoblast invasion and reduced spiral artery remodeling in the first trimester placenta. A polymorphism located in the promoter region of ACVR2A (rs1424954 (A > G)) has previously been shown to be significantly associated with pre-eclampsia. METHODS: The effects of this variant on ACVR2A expression and its function in the Activin-A signaling pathway were studied by transfections in SGHPL-5 extravillous trophoblasts followed by qRT-PCR. RESULTS: Here we show that the ACVR2A promoter susceptibility variant causes a downregulation of ACVR2A expression. We also provide evidence for transcription of a so-called PROMPT (PROMoter-uPstream-Transcript) in the opposite direction of ACVR2A, containing the polymorphism, and downregulated when the susceptibility allele is carried, which either shares the same promoter as ACVR2A or is a non-coding RNA that is able to enhance ACVR2A transcription. Furthermore, when the effect of the susceptibility variant is mimicked by knockdown of ACVR2A, physiologic concentrations of Activin-A cause a reduction in NODAL mRNA expression in the SGHPL-5 trophoblasts, indicative of a protective effect as reduction in NODAL expression is associated with an increase in trophoblast invasion. However, at pathologic levels of Activin-A, as found in pre-eclampsia, this effect is no longer seen, and we show this is potentially caused by a lack of downregulation of ACVR2B. DISCUSSION: The combined data suggest a double hit phenomenon in which the first hit, the promoter variant, together with the second hit, pathological levels of Activin-A, lead to high levels of NODAL, associated with reduced trophoblast invasion and observed in pre-eclamptic placentas.
Asunto(s)
Receptores de Activinas Tipo II/genética , Activinas/metabolismo , Regulación hacia Abajo , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Transducción de Señal , Trofoblastos/metabolismo , Receptores de Activinas Tipo II/antagonistas & inhibidores , Receptores de Activinas Tipo II/química , Receptores de Activinas Tipo II/metabolismo , Alelos , Línea Celular , Exones , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Proteína Nodal/antagonistas & inhibidores , Proteína Nodal/genética , Proteína Nodal/metabolismo , Fragmentos de Péptidos/agonistas , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Preeclampsia/genética , Preeclampsia/metabolismo , Embarazo , Interferencia de ARN , ARN Interferente Pequeño , Proteínas Recombinantes/metabolismoRESUMEN
Placentation is critical for establishing a healthy pregnancy. Trophoblasts mediate implantation and placentation and certain subtypes, most notably extravillous cytotrophoblast, are highly invasive. Trophoblast invasion is tightly regulated by microenvironmental cues that dictate placental morphology and depth. In choriocarcinomas, malignant trophoblast cells become hyperinvasive, breaching the myometrium and leading to major complications. Nodal, a member of the TGF-ß superfamily, is expressed throughout the endometrium during the peri-implantation period and in invasive trophoblast cells. Nodal promotes the invasion of numerous types of cancer cells. However, Nodal's role in trophoblast and choriocarcinoma cell invasion is unclear. Here we show that Nodal stimulates the invasion of both the non-malignant HTR-8SV/neo trophoblast and JAR choriocarcinoma cells in a dose-dependent manner. We found that endogenous ß-arrestins and Ral GTPases, key regulators of the cell cytoskeleton, are constitutively associated with Nodal receptors (ALK4 and ALK7) in trophoblast cells and that RalA is colocalized with ALK4 in endocytic vesicles. Nodal stimulates endogenous ß-arrestin2 to associate with phospho-ERK1/2, and knockdown of ß-arrestin or Ral proteins impairs Nodal-induced trophoblast and choriocarcinoma cell invasion. These results demonstrate, for the first time, that ß-arrestins and RalGTPases are important regulators of Nodal-induced invasion.
Asunto(s)
Arrestinas/metabolismo , Proteína Nodal/metabolismo , Transducción de Señal , Proteínas de Unión al GTP ral/metabolismo , Receptores de Activinas Tipo I/química , Receptores de Activinas Tipo I/metabolismo , Arrestinas/antagonistas & inhibidores , Arrestinas/genética , Línea Celular , Movimiento Celular/efectos de los fármacos , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Nodal/antagonistas & inhibidores , Proteína Nodal/genética , Fosforilación , Unión Proteica , Interferencia de ARN , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Transferrina/metabolismo , Trofoblastos/citología , Trofoblastos/metabolismo , beta-Arrestinas , Proteínas de Unión al GTP ral/antagonistas & inhibidores , Proteínas de Unión al GTP ral/genéticaRESUMEN
BACKGROUND: A shift in glucose metabolism from oxidative phosphorylation to anaerobic glycolysis is the biochemical hallmark of malignant cancer cells. METHODS: In the present study, we demonstrated that Nodal stimulated the expression of glycolytic enzymes and decreased reliance on mitochondrial oxidative phosphorylation in human glioma cancer cells. The shift in glucose metabolism was mediated by induction of the hypoxia-inducible factor (HIF). RESULTS: Nodal protein expression was shown to be correlated with expression levels of glucose transporter (Glut)-1, hexokinase (HK)-II, pyruvate dehydrogenase kinase (PDK)-1, the phosphorylation level of pyruvate dehydrogenase (PDH), glucose uptake, and lactate accumulation in human glioma cells. These effects were inversely correlated with mitochondrial oxygen consumption and ATP production. Knockdown of Nodal expression with specific small hairpin RNA reduced Glut-1, HK-II, and PDK-1 expressions and PDH phosphorylation. Nodal knockdown also reduced glucose uptake and lactate generation, which in turn increased mitochondrial membrane potential (Ψ), O2 utilization, and ATP synthesis. The ectopic expression of Nodal in low-expressing Nodal glioma cells resulted in the opposite results compared with those of Nodal knockdown glioma cells. Treatment of cells with recombinant Nodal increased HIF-1 expression, and this effect was regulated at the transcriptional level. Blockage of the Nodal receptor by a pharmacological inhibitor or Nodal knockdown in U87MG cells decreased HIF-1α expression. Furthermore, HIF-1α knockdown in U87MG cells decreased Glut-1, HK-II, and PDK-1 expressions and PDH phosphorylation, which were similar to results in Nodal knockdown cells. CONCLUSION: Taken together, these results suggest that Nodal affects energy metabolism through HIF-1α.