RESUMEN
T memory stem cells (TSCM) display increased self-renewal and prolonged survival capabilities, thus preventing T cell exhaustion and promoting effective anti-tumor T cell responses. TSCM cells can be expanded by Urolithin A (UA), which is produced by the commensal gut microbiome from foods rich in ellagitannins and is known to improve mitochondrial health. Oral UA administration to tumor-bearing mice conferred strong anti-tumor CD8+ T cell immunity, whereas ex vivo UA pre-treated T cells displayed improved anti-tumor function upon adoptive cell transfer. UA-induced TSCM formation depended on Pink1-mediated mitophagy triggering cytosolic release of the mitochondrial phosphatase Pgam5. Cytosolic Pgam5 dephosphorylated ß-catenin, which drove Wnt signaling and compensatory mitochondrial biogenesis. Collectively, we unravel a critical signaling pathway linking mitophagy to TSCM formation and suggest that the well-tolerated metabolic compound UA represents an attractive option to improve immune therapy.
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Cumarinas , Mitofagia , Ratones , Animales , Cumarinas/farmacología , Vía de Señalización Wnt , Células Madre , Memoria InmunológicaRESUMEN
The EMT-transcription factor ZEB1 is heterogeneously expressed in tumor cells and in cancer-associated fibroblasts (CAFs) in colorectal cancer (CRC). While ZEB1 in tumor cells regulates metastasis and therapy resistance, its role in CAFs is largely unknown. Combining fibroblast-specific Zeb1 deletion with immunocompetent mouse models of CRC, we observe that inflammation-driven tumorigenesis is accelerated, whereas invasion and metastasis in sporadic cancers are reduced. Single-cell transcriptomics, histological characterization, and in vitro modeling reveal a crucial role of ZEB1 in CAF polarization, promoting myofibroblastic features by restricting inflammatory activation. Zeb1 deficiency impairs collagen deposition and CAF barrier function but increases NFκB-mediated cytokine production, jointly promoting lymphocyte recruitment and immune checkpoint activation. Strikingly, the Zeb1-deficient CAF repertoire sensitizes to immune checkpoint inhibition, offering a therapeutic opportunity of targeting ZEB1 in CAFs and its usage as a prognostic biomarker. Collectively, we demonstrate that ZEB1-dependent plasticity of CAFs suppresses anti-tumor immunity and promotes metastasis.
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Fibroblastos Asociados al Cáncer , Neoplasias Colorrectales , Inmunoterapia , Inflamación , Homeobox 1 de Unión a la E-Box con Dedos de Zinc , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/terapia , Neoplasias Colorrectales/inmunología , Animales , Ratones , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Humanos , Inflamación/metabolismo , Inflamación/genética , Inflamación/patología , Inmunoterapia/métodos , Regulación Neoplásica de la Expresión Génica , Fibroblastos/metabolismo , Línea Celular Tumoral , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Transición Epitelial-Mesenquimal/genéticaRESUMEN
BACKGROUND & AIMS: Epigenetic processes regulating gene expression contribute markedly to epithelial cell plasticity in colorectal carcinogenesis. The lysine methyltransferase SUV420H2 comprises an important regulator of epithelial plasticity and is primarily responsible for trimethylation of H4K20 (H4K20me3). Loss of H4K20me3 has been suggested as a hallmark of human cancer due to its interaction with DNMT1. However, the role of Suv4-20h2 in colorectal cancer is unknown. METHODS: We examined the alterations in histone modifications in patient-derived colorectal cancer organoids. Patient-derived colorectal cancer organoids and mouse intestinal organoids were genetically manipulated for functional studies in patient-derived xenograft and orthotopic transplantation. Gene expression profiling, micrococcal nuclease assay, and chromatin immunoprecipitation were performed to understand epigenetic regulation of chromatin states and gene expression in patient-derived and mouse intestinal organoids. RESULTS: We found that reduced H4K20me3 levels occurred predominantly in right-sided patient-derived colorectal cancer organoids, which were associated with increased chromatin accessibility. Re-compaction of chromatin by methylstat, a histone demethylase inhibitor, resulted in reduced growth selectively in subcutaneously grown tumors derived from right-sided cancers. Using mouse intestinal organoids, we confirmed that Suv4-20h2-mediated H4K20me3 is required for maintaining heterochromatin compaction and to prevent R-loop formation. Cross-species comparison of Suv4-20h2-depleted murine organoids with right-sided colorectal cancer organoids revealed a large overlap of gene signatures involved in chromatin silencing, DNA methylation, and stemness/Wnt signaling. CONCLUSIONS: Loss of Suv4-20h2-mediated H4K20me3 drives right-sided colorectal tumorigenesis through an epigenetically controlled mechanism of chromatin compaction. Our findings unravel a conceptually novel approach for subtype-specific therapy of this aggressive form of colorectal cancer.
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Neoplasias del Colon , N-Metiltransferasa de Histona-Lisina , Animales , Humanos , Ratones , Transformación Celular Neoplásica/genética , Cromatina/genética , Neoplasias del Colon/genética , Neoplasias Colorrectales/genética , Epigénesis Genética , Histonas/metabolismo , Xenoinjertos , N-Metiltransferasa de Histona-Lisina/metabolismoRESUMEN
BACKGROUND: The actin cytoskeleton has a crucial role in the maintenance of the immune homeostasis by controlling various cellular processes, including cell migration. Mutations in TTC7A have been described as the cause of a primary immunodeficiency associated to different degrees of gut involvement and alterations in the actin cytoskeleton dynamics. OBJECTIVES: This study investigates the impact of TTC7A deficiency in immune homeostasis. In particular, the role of the TTC7A/phosphatidylinositol 4 kinase type III α pathway in the control of leukocyte migration and actin dynamics. METHODS: Microfabricated devices were leveraged to study cell migration and actin dynamics of murine and patient-derived leukocytes under confinement at the single-cell level. RESULTS: We show that TTC7A-deficient lymphocytes exhibit an altered cell migration and reduced capacity to deform through narrow gaps. Mechanistically, TTC7A-deficient phenotype resulted from impaired phosphoinositide signaling, leading to the downregulation of the phosphoinositide 3-kinase/AKT/RHOA regulatory axis and imbalanced actin cytoskeleton dynamics. TTC7A-associated phenotype resulted in impaired cell motility, accumulation of DNA damage, and increased cell death in dense 3-dimensional gels in the presence of chemokines. CONCLUSIONS: These results highlight a novel role of TTC7A as a critical regulator of lymphocyte migration. Impairment of this cellular function is likely to contribute to the pathophysiology underlying progressive immunodeficiency in patients.
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Actinas , Fosfatidilinositol 3-Quinasas , Humanos , Animales , Ratones , Muerte Celular , Mutación , Movimiento Celular/genética , Daño del ADN , Proteínas , 1-Fosfatidilinositol 4-QuinasaRESUMEN
Immunotherapy using chimeric antigen receptor (CAR)-engineered lymphocytes has shown impressive results in leukemia. However, for solid tumors such as colorectal cancer (CRC), new preclinical models are needed that allow to test CAR-mediated cytotoxicity in a tissue-like environment. Here, we developed a platform to study CAR cell cytotoxicity against 3-dimensional (3D) patient-derived colon organoids. Luciferase-based measurement served as a quantitative read-out for target cell viability. Additionally, we set up a confocal live imaging protocol to monitor effector cell recruitment and cytolytic activity at a single organoid level. As proof of principle, we demonstrated efficient targeting in diverse organoid models using CAR-engineered NK-92 cells directed toward a ubiquitous epithelial antigen (EPCAM). Tumor antigen-specific cytotoxicity was studied with CAR-NK-92 cells targeting organoids expressing EGFRvIII, a neoantigen found in several cancers. Finally, we tested a novel CAR strategy targeting FRIZZLED receptors that show increased expression in a subgroup of CRC tumors. Here, comparative killing assays with normal organoids failed to show tumor-specific activity. Taken together, we report a sensitive in vitro platform to evaluate CAR efficacy and tumor specificity in a personalized manner.
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Neoplasias Colorrectales/patología , Neoplasias Colorrectales/terapia , Citotoxicidad Inmunológica , Modelos Biológicos , Organoides/patología , Receptores Quiméricos de Antígenos/uso terapéutico , Técnicas de Cultivo de Tejidos/métodos , Células Cultivadas , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Citotoxicidad Inmunológica/efectos de los fármacos , Citotoxicidad Inmunológica/genética , Terapia Genética/métodos , Células HEK293 , Humanos , Inmunoterapia Adoptiva/métodos , Cultivo Primario de Células/métodos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/uso terapéutico , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Andamios del Tejido/químicaRESUMEN
Mammalian Wnt proteins are believed to act as short-range signals, yet have not been previously visualized in vivo. Self-renewal, proliferation and differentiation are coordinated along a putative Wnt gradient in the intestinal crypt. Wnt3 is produced specifically by Paneth cells. Here we have generated an epitope-tagged, functional Wnt3 knock-in allele. Wnt3 covers basolateral membranes of neighbouring stem cells. In intestinal organoids, Wnt3-transfer involves direct contact between Paneth cells and stem cells. Plasma membrane localization requires surface expression of Frizzled receptors, which in turn is regulated by the transmembrane E3 ligases Rnf43/Znrf3 and their antagonists Lgr4-5/R-spondin. By manipulating Wnt3 secretion and by arresting stem-cell proliferation, we demonstrate that Wnt3 mainly travels away from its source in a cell-bound manner through cell division, and not through diffusion. We conclude that stem-cell membranes constitute a reservoir for Wnt proteins, while Frizzled receptor turnover and 'plasma membrane dilution' through cell division shape the epithelial Wnt3 gradient.
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Membrana Celular/metabolismo , Mucosa Intestinal/citología , Nicho de Células Madre , Células Madre/citología , Células Madre/metabolismo , Vía de Señalización Wnt , Proteína Wnt3/metabolismo , Alelos , Animales , Adhesión Celular , División Celular , Difusión , Femenino , Receptores Frizzled/metabolismo , Técnicas de Sustitución del Gen , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Ratones , Organoides/citología , Organoides/metabolismo , Células de Paneth/citología , Células de Paneth/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Wnt3/genéticaRESUMEN
The small GTPases H, K, and NRAS are molecular switches indispensable for proper regulation of cellular proliferation and growth. Several mutations in the genes encoding members of this protein family are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered variants of the Ras-binding domain (RBD) of the C-Raf proto-oncogene, Ser/Thr kinase (CRAF). These variants bound with high affinity with the effector-binding site of Ras in an active conformation. Structural characterization disclosed how the newly identified RBD mutations cooperate and thereby enhance affinity with the effector-binding site in Ras compared with WT RBD. The engineered RBD variants closely mimicked the interaction mode of naturally occurring Ras effectors and acted as dominant-negative affinity reagents that block Ras signal transduction. Experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling, reducing cell growth and inducing apoptosis. Using these optimized RBD variants, we stratified patient-derived colorectal cancer organoids with known Ras mutational status according to their response to Ras inhibition. These results revealed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition, suggesting that not all of these tumors required Ras signaling for proliferation. In summary, by engineering the Ras/Raf interface of the CRAF-RBD, we identified potent and selective inhibitors of Ras in its active conformation that outcompete binding of Ras-signaling effectors.
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Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas ras/metabolismo , Apoptosis , Sitios de Unión , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Simulación de Dinámica Molecular , Mutagénesis , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica , Dominios Proteicos , Estructura Terciaria de Proteína , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Transducción de Señal , Proteínas ras/antagonistas & inhibidores , Proteínas ras/genéticaRESUMEN
OBJECTIVE: Cancer-associated fibroblasts (CAFs) influence the tumour microenvironment and tumour growth. However, the role of CAFs in colorectal cancer (CRC) development is incompletely understood. DESIGN: We quantified phosphorylation of STAT3 (pSTAT3) expression in CAFs of human colon cancer tissue using a tissue microarray (TMA) of 375 patients, immunofluorescence staining and digital pathology. To investigate the functional role of CAFs in CRC, we took advantage of two murine models of colorectal neoplasia and advanced imaging technologies. In loss-of-function and gain-of-function experiments, using genetically modified mice with collagen type VI (COLVI)-specific signal transducer and activator of transcription 3 (STAT3) targeting, we evaluated STAT3 signalling in fibroblasts during colorectal tumour development. We performed a comparative gene expression profiling by whole genome RNA-sequencing of fibroblast subpopulations (COLVI+ vs COLVI-) on STAT3 activation (IL-6 vs IL-11). RESULTS: The analysis of pSTAT3 expression in CAFs of human TMAs revealed a negative correlation of increased stromal pSTAT3 expression with the survival of colon cancer patients. In the loss-of-function and gain-of-function approach, we found a critical role of STAT3 activation in fibroblasts in driving colorectal tumourigenesis in vivo. With different imaging technologies, we detected an expansion of activated fibroblasts in colorectal neoplasias. Comparative gene expression profiling of fibroblast subpopulations on STAT3 activation revealed the regulation of transcriptional patterns associated with angiogenesis. Finally, the blockade of proangiogenic signalling significantly reduced colorectal tumour growth in mice with constitutive STAT3 activation in COLVI+ fibroblasts. CONCLUSION: Altogether our work demonstrates a critical role of STAT3 activation in CAFs in CRC development.
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Neoplasias Colorrectales/etiología , Interleucina-11/metabolismo , Interleucina-6/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Colon/metabolismo , Neoplasias Colorrectales/diagnóstico , Fibroblastos/metabolismo , Humanos , Ratones , Fosforilación , Pronóstico , Análisis de Matrices Tisulares , TranscriptomaRESUMEN
Loss of tumor suppressor adenomatous polyposis coli (APC) activates ß-catenin to initiate colorectal tumorigenesis. However, ß-catenin (CTNNB1) activating mutations rarely occur in human colorectal cancer (CRC). We found that APC loss also results in up-regulation of IL-6 signal transducer (IL-6ST/gp130), thereby activating Src family kinases (SFKs), YAP, and STAT3, which are simultaneously up-regulated in the majority of human CRC. Although, initial YAP activation, which stimulates IL6ST gene transcription, may be caused by reduced serine phosphorylation, sustained YAP activation depends on tyrosine phosphorylation by SFKs, whose inhibition, along with STAT3-activating JAK kinases, causes regression of established colorectal tumors. These results explain why APC loss is a more potent initiating event than the mere activation of CTNNB1.
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Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Neoplasias Colorrectales/metabolismo , Receptor gp130 de Citocinas/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Adulto , Anciano , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Receptor gp130 de Citocinas/genética , Femenino , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Persona de Mediana Edad , Mutación , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Although Lgr5(+) intestinal stem cells have been expanded in vitro as organoids, homogeneous culture of these cells has not been possible thus far. Here we show that two small molecules, CHIR99021 and valproic acid, synergistically maintain self-renewal of mouse Lgr5(+) intestinal stem cells, resulting in nearly homogeneous cultures. The colony-forming efficiency of cells from these cultures is ~100-fold greater than that of cells cultured in the absence of CHIR99021 and valproic acid, and multilineage differentiation ability is preserved. We made use of these homogeneous cultures to identify conditions employing simultaneous modulation of Wnt and Notch signaling to direct lineage differentiation into mature enterocytes, goblet cells and Paneth cells. Expansion in these culture conditions may be feasible for Lgr5(+) cells from the mouse stomach and colon and from the human small intestine. These methods provide new tools for the study and application of multiple intestinal epithelial cell types.
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Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Colon/citología , Intestino Delgado/citología , Receptores Acoplados a Proteínas G/metabolismo , Células Madre/citología , Animales , Diferenciación Celular , Células Cultivadas , Cromosomas/ultraestructura , Citometría de Flujo , Proteínas Fluorescentes Verdes/metabolismo , Heterocigoto , Cariotipificación , Ratones , Microscopía Confocal , Células de Paneth/citología , Piridinas/química , Pirimidinas/química , Transducción de Señal , Estómago/citología , Ácido Valproico/químicaRESUMEN
Initiation of cardiac excitation depends on a specialized group of cardiomyocytes at the venous pole of the heart, the sinoatrial node (SAN). The T-box transcription factor gene Tbx18 is expressed in the SAN myocardium and is required for formation of a large portion of the pacemaker. Previous studies suggested that Tbx18 is also sufficient to reprogram ventricular cardiomyocytes into SAN cells in rat, guinea-pig and pig hearts. To evaluate the consequences of misexpression of Tbx18 for imposing a nodal phenotype onto chamber myocardial cells in fetal mice, we used two independent conditional approaches with chamber-specific cre driver lines and an Hprt(Tbx18) misexpression allele. Myh6-Cre/+;Hprt(Tbx18/y) mice developed dilated atria with thickened walls, reduced right ventricles and septal defects that resulted in reduced embryonic and post-natal survival. Tagln-Cre/+;Hprt(Tbx18/y) mice exhibited slightly smaller hearts with rounded trabeculae that supported normal embryonic survival. Molecular analyses showed that the SAN gap junction and ion channel profile was not ectopically induced in chamber myocardium but the working myocardial gene program was partially inhibited in atria and ventricles of both misexpression models. Left atrial expression of Pitx2 was strongly repressed in Myh6-Cre/+;Hprt(Tbx18/y) embryos. We conclude that exclusion of Tbx18 expression from the developing atria and (right) ventricle is important to achieve normal cardiac left-right patterning and myocardial differentiation, and that Tbx18 is not sufficient to induce full SAN differentiation of chamber cardiomyocytes in fetal mice.
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Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Miocardio/metabolismo , Nodo Sinoatrial/metabolismo , Proteínas de Dominio T Box/genética , Transcriptoma , Animales , Biomarcadores , Análisis por Conglomerados , Femenino , Feto , Perfilación de la Expresión Génica , Genes Letales , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Masculino , Ratones , Ratones Transgénicos , Miocardio/patologíaRESUMEN
Heritable genetic variants can significantly affect the lifetime risk of developing cancer, including polyposis and colorectal cancer (CRC). Variants in genes currently known to be associated with a high risk for polyposis or CRC, however, explain only a limited number of hereditary cases. The identification of additional genetic causes is, therefore, crucial to improve CRC prevention, detection and treatment. We have performed genome-wide and targeted DNA copy number profiling and resequencing in early-onset and familial polyposis/CRC patients, and show that deletions affecting the open reading frame of the tumour suppressor gene FOCAD are recurrent and significantly enriched in CRC patients compared with unaffected controls. All patients carrying FOCAD deletions exhibited a personal or family history of polyposis. RNA in situ hybridization revealed FOCAD expression in epithelial cells in the colonic crypt, the site of tumour initiation, as well as in colonic tumours and organoids. Our data suggest that monoallelic germline deletions in the tumour suppressor gene FOCAD underlie moderate genetic predisposition to the development of polyposis and CRC.
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Poliposis Adenomatosa del Colon/genética , Neoplasias Colorrectales/genética , Eliminación de Gen , Mutación de Línea Germinal/genética , Proteínas Supresoras de Tumor/genética , Poliposis Adenomatosa del Colon/metabolismo , Adulto , Estudios de Casos y Controles , Cromosomas Humanos Par 9/genética , Neoplasias Colorrectales/metabolismo , Variaciones en el Número de Copia de ADN/genética , Células Epiteliales/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/genética , Sistemas de Lectura Abierta/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Vertebrate limb outgrowth is driven by a positive feedback loop that involves Sonic hedgehog (Shh) and Gremlin1 (Grem1) in the posterior limb bud mesenchyme and Fibroblast growth factors (Fgfs) in the overlying epithelium. Proper spatio-temporal control of these signaling activities is required to avoid limb malformations such as polydactyly. Here we show that, in Tbx2-deficient hindlimbs, Shh/Fgf4 signaling is prolonged, resulting in increased limb bud size and duplication of digit 4. In turn, limb-specific Tbx2 overexpression leads to premature termination of this signaling loop with smaller limbs and reduced digit number as phenotypic manifestation. We show that Tbx2 directly represses Grem1 in distal regions of the posterior limb mesenchyme allowing Bone morphogenetic protein (Bmp) signaling to abrogate Fgf4/9/17 expression in the overlying epithelium. Since Tbx2 itself is a target of Bmp signaling, our data identify a growth-inhibiting positive feedback loop (Bmp/Tbx2/Grem1). We propose that proliferative expansion of Tbx2-expressing cells mediates self-termination of limb bud outgrowth due to their refractoriness to Grem1 induction.
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Factores de Crecimiento de Fibroblastos/genética , Proteínas Hedgehog/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Esbozos de los Miembros/crecimiento & desarrollo , Proteínas de Dominio T Box/genética , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Citocinas , Epitelio/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Esbozos de los Miembros/metabolismo , Mesodermo/crecimiento & desarrollo , Mesodermo/metabolismo , Ratones , Fenotipo , Transducción de Señal , Proteínas de Dominio T Box/metabolismoRESUMEN
Vertebrate organ development relies on the precise spatiotemporal orchestration of proliferation rates and differentiation patterns in adjacent tissue compartments. The underlying integration of patterning and cell cycle control during organogenesis is insufficiently understood. Here, we have investigated the function of the patterning T-box transcription factor gene Tbx2 in lung development. We show that lungs of Tbx2-deficient mice are markedly hypoplastic and exhibit reduced branching morphogenesis. Mesenchymal proliferation was severely decreased, while mesenchymal differentiation into fibrocytes was prematurely induced. In the epithelial compartment, proliferation was reduced and differentiation of alveolar epithelial cells type 1 was compromised. Prior to the observed cellular changes, canonical Wnt signaling was downregulated, and Cdkn1a (p21) and Cdkn1b (p27) (two members of the Cip/Kip family of cell cycle inhibitors) were strongly induced in the Tbx2-deficient lung mesenchyme. Deletion of both Cdkn1a and Cdkn1b rescued, to a large degree, the growth deficits of Tbx2-deficient lungs. Prolongation of Tbx2 expression into adulthood led to hyperproliferation and maintenance of mesenchymal progenitor cells, with branching morphogenesis remaining unaffected. Expression of Cdkn1a and Cdkn1b was ablated from the lung mesenchyme in this gain-of-function setting. We further show by ChIP experiments that Tbx2 directly binds to Cdkn1a and Cdkn1b loci in vivo, defining these two genes as direct targets of Tbx2 repressive activity in the lung mesenchyme. We conclude that Tbx2-mediated regulation of Cdkn1a and Cdkn1b represents a crucial node in the network integrating patterning information and cell cycle regulation that underlies growth, differentiation, and branching morphogenesis of this organ.
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Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Pulmón , Proteínas de Dominio T Box , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Células Epiteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Pulmón/crecimiento & desarrollo , Pulmón/metabolismo , Mesodermo , Ratones , Morfogénesis , Transducción de Señal , Proteínas de Dominio T Box/deficiencia , Proteínas de Dominio T Box/genéticaAsunto(s)
Síndrome Linfoproliferativo Autoinmune/genética , Síndrome Linfoproliferativo Autoinmune/inmunología , Caspasa 8/genética , Colitis Ulcerosa/genética , Colitis Ulcerosa/inmunología , Células Epiteliales/inmunología , Intestinos/inmunología , Linfocitos/inmunología , Edad de Inicio , Animales , Apoptosis , Síndrome Linfoproliferativo Autoinmune/terapia , Biopsia , Colitis Ulcerosa/patología , Colitis Ulcerosa/terapia , Endoscopía Gastrointestinal , Células Epiteliales/patología , Predisposición Genética a la Enfermedad , Herencia , Humanos , Inflamasomas/inmunología , Mediadores de Inflamación/inmunología , Intestinos/patología , Linfocitos/patología , Ratones Noqueados , Mutación , FenotipoRESUMEN
Smooth muscle cells (SMCs) are a key component of many visceral organs, including the ureter, yet the molecular pathways that regulate their development from mesenchymal precursors are insufficiently understood. Here, we identified epithelial Wnt7b and Wnt9b as possible ligands of Fzd1-mediated ß-catenin (Ctnnb1)-dependent (canonical) Wnt signaling in the adjacent undifferentiated ureteric mesenchyme. Mice with a conditional deletion of Ctnnb1 in the ureteric mesenchyme exhibited hydroureter and hydronephrosis at newborn stages due to functional obstruction of the ureter. Histological analysis revealed that the layer of undifferentiated mesenchymal cells directly adjacent to the ureteric epithelium did not undergo characteristic cell shape changes, exhibited reduced proliferation and failed to differentiate into SMCs. Molecular markers for prospective SMCs were lost, whereas markers of the outer layer of the ureteric mesenchyme fated to become adventitial fibroblasts were expanded to the inner layer. Conditional misexpression of a stabilized form of Ctnnb1 in the prospective ureteric mesenchyme resulted in the formation of a large domain of cells that exhibited histological and molecular features of prospective SMCs and differentiated along this lineage. Our analysis suggests that Wnt signals from the ureteric epithelium pattern the ureteric mesenchyme in a radial fashion by suppressing adventitial fibroblast differentiation and initiating smooth muscle precursor development in the innermost layer of mesenchymal cells.
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Hipoxantina Fosforribosiltransferasa/genética , Mioblastos del Músculo Liso/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Uréter/embriología , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismo , Animales , Diferenciación Celular/fisiología , Cruzamientos Genéticos , Fluorescencia , Técnicas de Sustitución del Gen , Hibridación in Situ , Ratones , Mioblastos del Músculo Liso/metabolismo , Uréter/citología , Uréter/metabolismo , beta Catenina/deficienciaRESUMEN
BACKGROUND: Inflammatory bowel disease (IBD) is one of the most common chronic gastrointestinal diseases, but the underlying molecular mechanisms remain largely unknown. Studies of monogenic diseases can provide insight into the pathogenesis of IBD. OBJECTIVE: We thought to determine the underlying molecular causes of IBD occurring in 2 unrelated families in association with an immune deficiency. METHODS: We performed genetic linkage analysis and candidate gene sequencing on 13 patients from a large consanguineous family affected by early-onset IBD, progressive immune deficiency, and, in some cases, autoimmunity and alopecia, a condition we named enteropathy-lymphocytopenia-alopecia. The candidate gene was also sequenced in an unrelated patient with a similar phenotype. We performed histologic analysis of patients' intestinal biopsy specimens and carried out functional assays on PBMCs. Gut organoids derived from a patient's biopsy specimen were analyzed. RESULTS: We identified biallelic missense mutations in tetratricopeptide repeat domain 7A (TTC7A) in all patients from both families. The resulting TTC7A depletion modified the proliferation, adhesion, and migratory capacities of lymphocytes through inappropriate activation of the RhoA signaling pathway. Normal function was restored by wild-type TTC7A expression or addition of a RhoA kinase inhibitor. The growth and polarity of gut epithelial organoids were also found to be dependent on the RhoA signaling pathway. CONCLUSIONS: We show that TTC7A regulates the actin cytoskeleton dynamics in lymphocytes through the RhoA signaling pathway and is required in both lymphocytes and epithelial cells for maintaining equilibrium between cell proliferation, migration, polarization, and cell death. Our study highlights variability in the phenotypic expression resulting from TTC7A deficiency and outlines that impairment of both epithelial cells and lymphocytes cooperatively causes IBD.
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Alopecia , Enfermedades Inflamatorias del Intestino , Linfopenia , Proteínas/genética , Proteínas/inmunología , Adolescente , Adulto , Alopecia/genética , Alopecia/inmunología , Alopecia/patología , Niño , Preescolar , Colon/patología , Duodeno/patología , Femenino , Humanos , Lactante , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Linfopenia/genética , Linfopenia/inmunología , Linfopenia/patología , Masculino , Persona de Mediana Edad , Mutación Missense , Antro Pilórico/patología , Adulto Joven , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/inmunología , Proteína de Unión al GTP rhoA/inmunologíaRESUMEN
The study of gene function in endodermal epithelia such as of stomach, small intestine and colon relies heavily on transgenic approaches. Establishing such animal models is laborious, expensive and time-consuming. We present here a method based on Cre recombinase-inducible retrovirus vectors that allows the conditional manipulation of gene expression in primary mouse organoid culture systems.
Asunto(s)
Mucosa Intestinal/citología , Organoides/citología , Receptores Acoplados a Proteínas G/fisiología , Animales , Expresión Génica , Técnicas de Silenciamiento del Gen/métodos , Proteínas Fluorescentes Verdes/genética , Humanos , Integrasas/metabolismo , Ratones , Organoides/metabolismo , Receptores Notch/fisiología , Retroviridae/genética , Células Madre/virología , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Transducción Genética/métodosRESUMEN
The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKß independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.
Asunto(s)
Viroterapia Oncolítica , Virus Oncolíticos , Succinatos , Animales , Humanos , Viroterapia Oncolítica/métodos , Succinatos/farmacología , Ratones , Línea Celular Tumoral , Interferón Tipo I/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Neoplasias del Colon/terapia , Neoplasias del Colon/inmunología , Neoplasias del Colon/tratamiento farmacológico , Antivirales/farmacología , FN-kappa B/metabolismo , Quinasa I-kappa B/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Inflamación/tratamiento farmacológico , Femenino , Virus de la Estomatitis Vesicular Indiana/fisiología , Virus de la Estomatitis Vesicular Indiana/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND & AIMS: Wnt signaling regulates multiple aspects of intestinal physiology, including stem cell maintenance. Paneth cells support stem cells by secreting Wnt, but little is known about the exact sources and primary functions of individual Wnt family members. METHODS: We analyzed intestinal tissues and cultured epithelial cells from adult mice with conditional deletion of Wnt3 (Vil-CreERT2;Wnt3fl/fl mice). We also analyzed intestinal tissues and cells from Atoh1 mutant mice, which lack secretory cells. RESULTS: Unexpectedly, Wnt3 was dispensable for maintenance of intestinal stem cells in mice, indicating a redundancy of Wnt signals. By contrast, cultured crypt organoids required Paneth cell-derived Wnt3. Addition of exogenous Wnt, or coculture with mesenchymal cells, restored growth of Vil-CreERT2;Wnt3fl/fl crypt organoids. Intestinal organoids from Atoh1 mutant mice did not grow or form Paneth cells; addition of Wnt3 allowed growth in the absence of Paneth cells. Wnt signaling had a synergistic effect with the Lgr4/5 ligand R-spondin to induce formation of Paneth cells. Mosaic expression of Wnt3 in organoids using a retroviral vector promoted differentiation of Paneth cells in a cell-autonomous manner. CONCLUSIONS: Wnt is part of a signaling loop that affects homeostasis of intestinal stem and Paneth cells in mice. Wnt3 signaling is required for growth and development of organoid cultures, whereas nonepithelial Wnt signals could provide a secondary physiological source of Wnt.