RESUMEN
The Hippo transducers YAP/TAZ have been shown to play positive, as well as negative, roles in Wnt signaling, but the underlying mechanisms remain unclear. Here, we provide biochemical, functional, and genetic evidence that YAP and TAZ are integral components of the ß-catenin destruction complex that serves as cytoplasmic sink for YAP/TAZ. In Wnt-ON cells, YAP/TAZ are physically dislodged from the destruction complex, allowing their nuclear accumulation and activation of Wnt/YAP/TAZ-dependent biological effects. YAP/TAZ are required for intestinal crypt overgrowth induced by APC deficiency and for crypt regeneration ex vivo. In Wnt-OFF cells, YAP/TAZ are essential for ß-TrCP recruitment to the complex and ß-catenin inactivation. In Wnt-ON cells, release of YAP/TAZ from the complex is instrumental for Wnt/ß-catenin signaling. In line, the ß-catenin-dependent maintenance of ES cells in an undifferentiated state is sustained by loss of YAP/TAZ. This work reveals an unprecedented signaling framework relevant for organ size control, regeneration, and tumor suppression.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Fosfoproteínas/metabolismo , Factores de Transcripción/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Aciltransferasas , Animales , Proteínas de Ciclo Celular , Línea Celular , Células Madre Embrionarias/metabolismo , Células HEK293 , Humanos , Ratones , Modelos Biológicos , Proteínas Señalizadoras YAPRESUMEN
Cancer stem cells (CSCs) are proposed to drive tumor initiation and progression. Yet, our understanding of the cellular and molecular mechanisms that underlie CSC properties is limited. Here we show that the activity of TAZ, a transducer of the Hippo pathway, is required to sustain self-renewal and tumor-initiation capacities in breast CSCs. TAZ protein levels and activity are elevated in prospective CSCs and in poorly differentiated human tumors and have prognostic value. Gain of TAZ endows self-renewal capacity to non-CSCs. In epithelial cells, TAZ forms a complex with the cell-polarity determinant Scribble, and loss of Scribble--or induction of the epithelial-mesenchymal transition (EMT)--disrupts the inhibitory association of TAZ with the core Hippo kinases MST and LATS. This study links the CSC concept to the Hippo pathway in breast cancer and reveals a mechanistic basis of the control of Hippo kinases by cell polarity.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Células Madre Neoplásicas/patología , Transducción de Señal , Factores de Transcripción/metabolismo , Aciltransferasas , Polaridad Celular , Transición Epitelial-Mesenquimal , Femenino , Humanos , Proteínas de la Membrana/metabolismo , Metástasis de la Neoplasia/patología , Células Madre Neoplásicas/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Despite substantial progress in treatment of T-cell acute lymphoblastic leukemia (T-ALL), mortality remains relatively high, mainly due to primary or acquired resistance to chemotherapy. Further improvements in survival demand better understanding of T-ALL biology and development of new therapeutic strategies. The Notch pathway has been involved in the pathogenesis of this disease and various therapeutic strategies are currently under development, including selective targeting of NOTCH receptors by inhibitory antibodies. We previously demonstrated that the NOTCH1-specific neutralizing antibody OMP52M51 prolongs survival in TALL patient-derived xenografts bearing NOTCH1/FBW7 mutations. However, acquired resistance to OMP52M51 eventually developed and we used patient-derived xenografts models to investigate this phenomenon. Multi-level molecular characterization of T-ALL cells resistant to NOTCH1 blockade and serial transplantation experiments uncovered heterogeneous types of resistance, not previously reported with other Notch inhibitors. In one model, resistance appeared after 156 days of treatment, it was stable and associated with loss of Notch inhibition, reduced mutational load and acquired NOTCH1 mutations potentially affecting the stability of the heterodimerization domain. Conversely, in another model resistance developed after only 43 days of treatment despite persistent down-regulation of Notch signaling and it was accompanied by modulation of lipid metabolism and reduced surface expression of NOTCH1. Our findings shed light on heterogeneous mechanisms adopted by the tumor to evade NOTCH1 blockade and support clinical implementation of antibody-based target therapy for Notch-addicted tumors.
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Leucemia-Linfoma Linfoblástico de Células T Precursoras , Animales , Humanos , Ratones Endogámicos NOD , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptor Notch1/genética , Transducción de Señal , Linfocitos T , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pediatric T-acute lymphoblastic leukemia (T-ALL) patients often display resistance to glucocorticoid (GC) treatment. These patients, classified as prednisone poor responders (PPR), have poorer outcome than do the other pediatric T-ALL patients receiving a high-risk adapted therapy. Because glucocorticoids are administered to ALL patients during all the different phases of therapy, GC resistance represents an important challenge to improving the outcome for these patients. Mechanisms underlying resistance are not yet fully unraveled; thus our research focused on the identification of deregulated signaling pathways to point out new targeted approaches. We first identified, by reverse-phase protein arrays, the lymphocyte cell-specific protein-tyrosine kinase (LCK) as aberrantly activated in PPR patients. We showed that LCK inhibitors, such as dasatinib, bosutinib, nintedanib, and WH-4-023, are able to induce cell death in GC-resistant T-ALL cells, and remarkably, cotreatment with dexamethasone is able to reverse GC resistance, even at therapeutic drug concentrations. This was confirmed by specific LCK gene silencing and ex vivo combined treatment of cells from PPR patient-derived xenografts. Moreover, we observed that LCK hyperactivation in PPR patients upregulates the calcineurin/nuclear factor of activated T cells signaling triggering to interleukin-4 (IL-4) overexpression. GC-sensitive cells cultured with IL-4 display an increased resistance to dexamethasone, whereas the inhibition of IL-4 signaling could increase GC-induced apoptosis in resistant cells. Treatment with dexamethasone and dasatinib also impaired engraftment of leukemia cells in vivo. Our results suggest a quickly actionable approach to supporting conventional therapies and overcoming GC resistance in pediatric T-ALL patients.
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Resistencia a Antineoplásicos/efectos de los fármacos , Glucocorticoides/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Niño , Dasatinib/farmacología , Dexametasona/farmacología , Xenoinjertos , Humanos , Interleucina-4/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos/enzimología , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Prednisona/farmacologíaRESUMEN
Cytochrome c oxidase (COX), complex IV of the mitochondrial respiratory chain, is comprised of 14 structural subunits, several prosthetic groups and metal cofactors, among which copper. Its biosynthesis involves a number of ancillary proteins, encoded by the COX-assembly genes that are required for the stabilization and membrane insertion of the nascent polypeptides, the synthesis of the prosthetic groups, and the delivery of the metal cofactors, in particular of copper. Recently, a modular model for COX assembly has been proposed, based on the sequential incorporation of different assembly modules formed by specific subunits. We have cloned and characterized the human homologue of yeast COX16. We show that human COX16 encodes a small mitochondrial transmembrane protein that faces the intermembrane space and is highly expressed in skeletal and cardiac muscle. Its knockdown in C. elegans produces COX deficiency, and its ablation in HEK293 cells impairs COX assembly. Interestingly, COX16 knockout cells retain significant COX activity, suggesting that the function of COX16 is partially redundant. Analysis of steady-state levels of COX subunits and of assembly intermediates by Blue-Native gels shows a pattern similar to that reported in cells lacking COX18, suggesting that COX16 is required for the formation of the COX2 subassembly module. Moreover, COX16 co-immunoprecipitates with COX2. Finally, we found that copper supplementation increases COX activity and restores normal steady state levels of COX subunits in COX16 knockout cells, indicating that, even in the absence of a canonical copper binding motif, COX16 could be involved in copper delivery to COX2.
Asunto(s)
Caenorhabditis elegans/enzimología , Coenzimas/metabolismo , Cobre/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animales , Sistemas CRISPR-Cas , Caenorhabditis elegans/genética , Cationes Bivalentes , Clonación Molecular , Transporte de Electrón/fisiología , Complejo IV de Transporte de Electrones/genética , Expresión Génica , Técnicas de Inactivación de Genes , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Transporte Iónico , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , Músculo Esquelético/enzimología , Miocardio/enzimología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
A sexual dimorphism in liver inflammation and repair was previously demonstrated. Its cellular dissection in the course of acute liver injury (ALI) was explored. BALB/c mice were treated with carbon tetrachloride (CCl4) by intraperitoneal injection and killed after 3, 5, and 8 days. Histological and hepatic cell population analyses were performed. The correlation between androgen receptor (AR) expression and liver recruited inflammatory cells was investigated by treatment with the AR antagonist flutamide. Additionally, patients with a diagnosis of drug induced liver injury (DILI) were included in the study, with a particular focus on gender dimorphism in circulating monocytes. A delayed resolution of necrotic damage and a higher expression of proinflammatory cytokines were apparent in male mice along with a slower recruitment of inflammatory monocytes. F4/80+CD11b+ macrophages and CD11bhighGr-1high monocytes expressed AR and were recruited later in male compared with female livers after CCl4 treatment. Moreover, CD11bhighAR+Gr-1high recruitment was negatively modulated by flutamide in males. Analysis of DILI patients showed overall a significant reduction in circulating mature monocytes compared with healthy subjects. More interestingly, male patients had higher numbers of immature monocytes compared with female patients.A stronger cytotoxic tissue response was correlated with an impaired recruitment of CD11bhighAR+Gr-1high cells and F4/80+CD11b+ macrophages in the early inflammatory phase under AR signaling. During DILI, a dimorphic immune response was apparent, characterized by a massive recruitment of monocytes to the liver both in males and females, but only in males was this recruitment sustained by a turnover of immature monocytes.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Modelos Animales de Enfermedad , Regeneración Hepática/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Animales , Tetracloruro de Carbono/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Femenino , Expresión Génica/inmunología , Hepatocitos/inmunología , Hepatocitos/metabolismo , Hígado/inmunología , Hígado/metabolismo , Macrófagos/metabolismo , Masculino , Ratones Endogámicos BALB C , Monocitos/metabolismo , Factores Sexuales , Factores de TiempoRESUMEN
Lab-on-a-chip systems with molecular motor driven transport of analytes attached to cytoskeletal filament shuttles (actin filaments, microtubules) circumvent challenges with nanoscale liquid transport. However, the filaments have limited cargo-carrying capacity and limitations either in transportation speed (microtubules) or control over motility direction (actin). To overcome these constraints we here report incorporation of covalently attached antibodies into self-propelled actin bundles (nanocarriers) formed by cross-linking antibody conjugated actin filaments via fascin, a natural actin-bundling protein. We demonstrate high maximum antigen binding activity and propulsion by surface adsorbed myosin motors. Analyte transport capacity is tested using both protein antigens and microvesicles, a novel class of diagnostic markers. Increased incubation concentration with protein antigen in the 0.1-100 nM range (1 min) reduces the fraction of motile bundles and their velocity but maximum transportation capacity of >1 antigen per nm of bundle length is feasible. At sub-nanomolar protein analyte concentration, motility is very well preserved opening for orders of magnitude improved limit of detection using motor driven concentration on nanoscale sensors. Microvesicle-complexing to monoclonal antibodies on the nanocarriers compromises motility but nanocarrier aggregation via microvesicles shows unique potential in label-free detection with the aggregates themselves as non-toxic reporter elements.
Asunto(s)
Actinas/química , Anticuerpos Monoclonales/química , Proteínas Portadoras/química , Micropartículas Derivadas de Células/química , Inmunoglobulina G/análisis , Proteínas de Microfilamentos/química , Subfragmentos de Miosina/química , Animales , Línea Celular Tumoral , Colorantes Fluorescentes/química , Humanos , Inmunoglobulina G/inmunología , Antígenos Comunes de Leucocito/inmunología , Microscopía Fluorescente , Movimiento (Física) , Conejos , Rodaminas/químicaRESUMEN
5-aminolevulinic acid (5-ALA) introduction in the surgical management of Glioblastoma (GBM) enables the intra-operatively identification of cancer cells in the mass by means of fluorescence. Here, we analyzed the phenotype of GBM cells isolated from distinct tumour areas determined by 5-ALA (tumour core, 5-ALA intense and vague layers) and the potency of 5-ALA labelling in identifying GBM cells and cancer stem cells (CSCs) in the mass. 5-ALA identified distinct layers in the mass, with less differentiated cells residing in the core of the tumour. 5-ALA was able to stain up to 68.5% of CD133(+) cells in the 5-ALA intense layer and, although 5-ALA(+) cells retrieved from different tumour areas contained a similar proportion of CD133(+) cells (range 27.5-35.6%), those from the vague layer displayed the lowest ability to self-renew. In conclusion, our data demonstrate that a substantial amount of GBM cells and CSCs in the mass are able to avoid 5-ALA labelling and support the presence of heterogenic CSC populations in the GBM mass.
Asunto(s)
Ácido Aminolevulínico , Neoplasias Encefálicas/patología , Glioblastoma/patología , Células Madre Neoplásicas/patología , Fármacos Fotosensibilizantes , Antígeno AC133 , Ácido Aminolevulínico/metabolismo , Antígenos CD/metabolismo , Biopsia , Neoplasias Encefálicas/cirugía , Citometría de Flujo , Glioblastoma/cirugía , Glicoproteínas/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Péptidos/metabolismo , Fármacos Fotosensibilizantes/metabolismoRESUMEN
5-Aminolevulinic acid (5-ALA) fluorescence has been proved advantageous in glioma surgery. Conflicting results have been reported by few studies published in literature about intra-operative 5-ALA-induced fluorescence of medulloblastoma (MDB). The aim of this study is to verify if these conflicting results could be explained by intra-tumoral histological and phenotypic differences. In the present case of a 45-year-old patient affected by a cerebellar MDB, histological analysis of cell phenotype and 5-ALA and CD133 correlation were performed in multiple samples according to different fluorescence patterns. Intra-operatively, the tumor appeared unevenly fluorescent under blue-violet light. Histologically, 5-ALA-intense biopsies from inner areas were characterized by a significant amount of cancer cells, whereas 5-ALA faint regions from peripheral areas displayed normal cerebellar features, with MDB cells infiltrating healthy tissues. Presenting our findings, we show the correlation between different 5-ALA fluorescence patterns of medulloblastoma with specific histological and phenotypical features. Thus, we hypothesize that a distinct relationship between CD133 expression and fluorescence accumulation presented in our study could partially explain the divergent results published in literature.
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Ácido Aminolevulínico , Antígenos CD/biosíntesis , Neoplasias Cerebelosas/metabolismo , Colorantes Fluorescentes , Glicoproteínas/biosíntesis , Meduloblastoma/metabolismo , Procedimientos Neuroquirúrgicos/métodos , Antígeno AC133 , Neoplasias Cerebelosas/cirugía , Humanos , Masculino , Meduloblastoma/cirugía , Persona de Mediana Edad , Péptidos , FenotipoRESUMEN
Ontogenesis of T cells in the thymus is a complex process whose molecular control is poorly understood. The present study investigated microRNAs involved in human thymocyte differentiation by comparing the microRNA expression profiles of thymocytes at the double-positive, single-positive CD4(+) and single-positive CD8(+) maturation stages. Microarray analysis showed that each thymocyte population displays a distinct microRNA expression profile that reflects their developmental relationships. Moreover, analysis of small-RNA libraries generated from human unsorted and double-positive thymocytes and from mature peripheral CD4(+) and CD8(+) T lymphocytes, together with the microarray data, indicated a trend toward up-regulation of microRNA expression during T-cell maturation after the double-positive stage and revealed a group of microRNAs regulated during normal T-cell development, including miR-150, which is strongly up-regulated as maturation progresses. We showed that miR-150 targets NOTCH3, a member of the Notch receptor family that plays important roles both in T-cell differentiation and leukemogenesis. Forced expression of miR-150 reduces NOTCH3 levels in T-cell lines and has adverse effects on their proliferation and survival. Overall, these findings suggest that control of the Notch pathway through miR-150 may have an important impact on T-cell development and physiology.
Asunto(s)
Diferenciación Celular , Regulación de la Expresión Génica , MicroARNs/metabolismo , Receptores Notch/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo , Regiones no Traducidas 3' , Adulto , Apoptosis , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Células Cultivadas , Preescolar , Perfilación de la Expresión Génica , Genes Reporteros , Humanos , Lactante , Recién Nacido , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , ARN Mensajero/metabolismo , Receptor Notch3 , Receptores Notch/antagonistas & inhibidores , Receptores Notch/genética , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismo , Timo/citología , Timo/metabolismoRESUMEN
Juvenile myelomonocytic leukemia (JMML) is a rare clonal stem cell disorder that occurs in early childhood and is characterized by the hyperactivation of the RAS pathway in 95% of the patients. JMML is characterized by a hyperproliferation of granulocytes and monocytes, and little is known about the heterogeneous nature of leukemia-initiating cells, as well as of the cellular hierarchy of the JMML bone marrow. In this study, we report the generation and characterization of a novel patient-derived three-dimensional (3D) in vitro JMML model, called patient-derived JMML Atypical Organoid (pd-JAO), sustaining the long-term proliferation of JMML cells with stem cell features and patient-specific hallmarks. JMML cells brewed in a 3D model under different microenvironmental conditions acquired proliferative and survival advantages when placed under low oxygen tension. Transcriptomic and microscopic analyses revealed the activation of specific metabolic energy pathways and the inactivation of processes leading to cell death. Furthermore, we demonstrated the pd-JAO-derived cells' migratory, propagation, and self-renewal capacities. Our study contributes to the development of a robust JMML 3D in vitro model for studying and defining the impact of microenvironmental stimuli on JMML disease and the molecular mechanisms that regulate JMML initiating and propagating cells. Pd-JAO may become a promising model for compound tests focusing on new therapeutic interventions aimed at eradicating JMML progenitors and controlling JMML disease.
Asunto(s)
Leucemia Mielomonocítica Juvenil , Humanos , Preescolar , Leucemia Mielomonocítica Juvenil/terapia , Médula Ósea , Granulocitos , Proliferación CelularRESUMEN
Interleukin (IL)-23 is a proinflammatory cytokine belonging to the IL-12 superfamily. The antitumor activity of IL-23 is controversial, and it is unknown whether or not the cytokine can act directly on tumor cells. The aim of this study was to investigate the potential direct antitumor activity of IL-23 in pediatric B-acute lymphoblastic leukemia (B-ALL) cells and to unravel the molecular mechanisms involved. Here, we show, for the first time, that IL-23R is up-regulated in primary B-ALL cells, compared with normal early B lymphocytes, and that IL-23 dampens directly tumor growth in vitro and in vivo through the inhibition of tumor cell proliferation and induction of apoptosis. The latter finding is related to IL-23-induced up-regulation of miR15a expression and the consequent down-regulation of BCL-2 protein expression in pediatric B-ALL cells. This study demonstrates that IL-23 possesses antileukemic activity and unravels the underlying mechanisms. Thus, IL-23 may be a candidate novel drug for the treatment of B-ALL patients unresponsive to current therapeutic standards.
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Subunidad p19 de la Interleucina-23/inmunología , Subunidad p19 de la Interleucina-23/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/inmunología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Niño , Preescolar , Femenino , Expresión Génica/efectos de los fármacos , Genes bcl-2/efectos de los fármacos , Humanos , Técnicas In Vitro , Lactante , Subunidad p19 de la Interleucina-23/genética , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Transducción de Señal/inmunologíaRESUMEN
Glioblastoma multiforme (GBM) are highly proliferative tumors currently treated by surgical removal, followed by radiotherapy and chemotherapy, which are counteracted by intratumoral hypoxia. Here we exploited image guided surgery to sample multiple intratumoral areas to define potential cellular heterogeneity in correlation to the oxygen tension gradient within the GBM mass. Our results indicate that more immature cells are localized in the inner core and in the intermediate layer of the tumor mass, whereas more committed cells, expressing glial fibrillary acidic protein and beta-III-tubulin, are distributed along the peripheral and neo-vascularized area, where Smad1/5/8 and Stat3 result to be activated. Moreover, GBM stem cells, identified with the stem cell marker CD133, express high level of DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT) known to be involved in chemotherapy resistance and highly expressed in the inner core of the tumor mass. Importantly, these cells and, particularly, CD133(+) cells result to be resistant to temozolomide (TMZ), the most used oral alkylating agent for the treatment of GBM, which specifically causes apoptosis only in GBM cells derived from the peripheral layer of the tumor mass. These results indicate a correlation between the intratumoral hypoxic gradient, the tumor cell phenotype, and the tumor resistance to chemotherapy leading to a novel concentric model of tumor stem cell niche, which may be useful to define the real localization of the chemoresistant GBM tumor cells in order to design more effective treatment strategies.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Metilasas de Modificación del ADN/biosíntesis , Enzimas Reparadoras del ADN/biosíntesis , Glioblastoma/metabolismo , Glioblastoma/patología , Hipoxia/patología , Células Madre Neoplásicas/metabolismo , Proteínas Supresoras de Tumor/biosíntesis , Antígeno AC133 , Adulto , Antígenos CD/metabolismo , Neoplasias Encefálicas/enzimología , Desdiferenciación Celular/fisiología , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Glioblastoma/enzimología , Glicoproteínas/metabolismo , Humanos , Hipoxia/enzimología , Recién Nacido , Imagen por Resonancia Magnética , Persona de Mediana Edad , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Procedimientos Neuroquirúrgicos , Péptidos/metabolismo , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genéticaRESUMEN
Medulloblastoma (MDB) is the most common brain malignancy of childhood. It is currently thought that MDB arises from aberrantly functioning stem cells in the cerebellum that fail to maintain proper control of self-renewal. Additionally, it has been reported that MDB cells display higher endogenous Notch signaling activation, known to promote the survival and proliferation of neoplastic neural stem cells and to inhibit their differentiation. Although interaction between hypoxia-inducible factor-1α (HIF-1α) and Notch signaling is required to maintain normal neural precursors in an undifferentiated state, an interaction has not been identified in MDB. Here, we investigate whether hypoxia, through HIF-1α stabilization, modulates Notch1 signaling in primary MDB-derived cells. Our results indicate that MDB-derived precursor cells require hypoxic conditions for in vitro expansion, whereas acute exposure to 20% oxygen induces tumor cell differentiation and death through inhibition of Notch signaling. Importantly, stimulating Notch1 activation with its ligand Dll4 under hypoxic conditions leads to expansion of MDB-derived CD133(+) and nestin(+) precursors, suggesting a regulatory effect on stem cells. In contrast, MDB cells undergo neuronal differentiation when treated with γ-secretase inhibitor, which prevents Notch activation. These results suggest that hypoxia, by maintaining Notch1 in its active form, preserves MDB stem cell viability and expansion.
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Neoplasias Encefálicas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Meduloblastoma/metabolismo , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/metabolismo , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Antígeno AC133 , Antígenos CD/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias Encefálicas/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proliferación Celular , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Glicoproteínas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Inmunohistoquímica , Proteínas de Filamentos Intermediarios/metabolismo , Meduloblastoma/genética , Proteínas del Tejido Nervioso/metabolismo , Nestina , Péptidos/metabolismo , Reacción en Cadena de la Polimerasa , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transducción de Señal/genética , Factor de Transcripción HES-1 , Células Tumorales CultivadasRESUMEN
BACKGROUND AIMS: Bone marrow (BM)-derived cells appear to be a promising therapeutic source for the treatment of acute myocardial infarction (AMI). However, the quantity and quality of the cells to be used, along with the appropriate time of administration, still need to be defined. We thus investigated the use of BM CD34(+)-derived cells as cells suitable for a cell therapy protocol (CTP) in the treatment of experimental AMI. METHODS: The need for a large number of cells was satisfied by the use of a previously established protocol allowing the expansion of human CD34(+) cells isolated from neonatal and adult hematopoietic tissues. We evaluated gene expression, endothelial differentiation potential and cytokine release by BM-derived cells during in vitro culture. Basal and expanded CD34(+) cells were used as a delivery product in a murine AMI model consisting of a coronary artery ligation (CAL). Cardiac function recovery was evaluated after injecting basal or expanded cells. RESULTS: Gene expression analysis of in vitro-expanded cells revealed that endothelial markers were up-regulated during culture. Moreover, expanded cells generated a CD14(+) subpopulation able to differentiate efficiently into VE-cadherin-expressing cells. In vivo, we observed a cardiac function recovery in mice sequentially treated with basal and expanded cells injected 4 h and 7 days after CAL, respectively. CONCLUSIONS: Our data suggest that combining basal and expanded BM-derived CD34(+) cells in a specific temporal pattern of administration might represent a promising strategy for a successful cell-based therapy.
Asunto(s)
Vasos Coronarios/cirugía , Ligadura , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Infarto del Miocardio/terapia , Animales , Antígenos CD/metabolismo , Antígenos CD34/biosíntesis , Médula Ósea/patología , Cadherinas/metabolismo , Línea Celular , Proliferación Celular , Modelos Animales de Enfermedad , Endotelio/metabolismo , Perfilación de la Expresión Génica , Humanos , Células Madre Mesenquimatosas/patología , Ratones , Ratones Endogámicos NOD , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Recuperación de la FunciónRESUMEN
Neuroblastoma is an embryonal malignancy of early childhood arising from the embryonic sympatho-adrenal lineage of the neural crest. About half of all cases are currently classified as high-risk of disease recurrence, with an overall survival rate of less than 40% at 5 years despite intensive therapy. Recent studies on matched primary tumours and at the relapse revealed downregulation of genes transcriptionally silenced by YAP as significant association with neuroblastoma relapse. Here, we evaluated the pharmacological targeting of YAP/TAZ with the YAP/TAZ-TEAD inhibitor Verteporfin (VP) in Tumour Initiating Cells (TICs) derived from High-Risk Neuroblastoma patients. VP treatment suppresses YAP/TAZ expression, induces apoptosis and causes the re-organization of the cytoskeleton reducing cells migration and clonogenic ability. Moreover, VP reduces the percentage of side population cells and ABC transporters involved in drug resistance, and the percentage of stem cell subpopulations CD133+ and CD44+ of TICs. Finally, we demonstrated that VP sensitizes TICs to the standard drugs used for neuroblastoma therapy etoposide and cis-platin opening the way to use VP as drug repositioning candidate for recurrent neuroblastoma.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Células Madre Neoplásicas/efectos de los fármacos , Neuroblastoma/tratamiento farmacológico , Células de Población Lateral/efectos de los fármacos , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Verteporfina/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Reposicionamiento de Medicamentos , Etopósido/farmacología , Humanos , Invasividad Neoplásica , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neuroblastoma/metabolismo , Neuroblastoma/patología , Células de Población Lateral/metabolismo , Células de Población Lateral/patología , Transducción de Señal , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAPRESUMEN
BACKGROUND: The interplay between neoplastic cells and surrounding extracellular matrix (ECM) is one of the determinant elements for cancer growth. The remodeling of the ECM by cancer-associated fibroblasts (CAFs) shapes tumor microenvironment by depositing and digesting ECM proteins, hence promoting tumor growth and invasion. While for epithelial tumors CAFs are well characterized, little is known about the stroma composition of mesenchymal cancers, such as in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma during childhood and adolescence. The aim of this work is to identify the importance of CAFs in specifying RMS microenvironment and the role of these stromal cells in RMS growth. METHODS: We assessed in two dimensional (2D) and three dimensional (3D) systems the attraction between RMS cells and fibroblasts using epithelial colon cancer cell line as control. CAFs were studied in a xenogeneic mouse model of both tumor types and characterized in terms of fibroblast activation protein (FAP), mouse PDGFR expression, metalloproteases activation, and ECM gene and protein expression profiling. RESULTS: In 2D model, the rate of interaction between stromal and malignant cells was significantly lower in RMS with respect to colon cancer. Particularly, in 3D system, RMS spheroids tended to dismantle the compact aggregate when grown on the layer of stromal cells. In vivo, despite the well-formed tumor mass, murine CAFs were found in low percentage in RMS xenogeneic samples. CONCLUSIONS: Our findings support the evidence that, differently from epithelial cancers, RMS cells are directly involved in their own ECM remodeling, and less dependent on CAFs support for cancer cell growth.
RESUMEN
The RNA-binding protein LIN28B regulates developmental timing and determines stem cell identity by suppressing the let-7 family of microRNAs. Postembryonic reactivation of LIN28B impairs cell commitment to differentiation, prompting their transformation. In this study, we assessed the extent to which ectopic lin28b expression modulates the physiological behavior of neural crest cells (NCC) and governs their transformation in the trunk region of developing embryos. We provide evidence that the overexpression of lin28b inhibits sympathoadrenal cell differentiation and accelerates NCC migration in two vertebrate models, Xenopus leavis and Danio rerio. Our results highlight the relevance of ITGA5 and ITGA6 in the LIN28B-dependent regulation of the invasive motility of tumor cells. The results also establish that LIN28B overexpression supports neuroblastoma onset and the metastatic potential of malignant cells through let-7a-dependent and let-7a-independent mechanisms.
Asunto(s)
Movimiento Celular , Cresta Neural/citología , Proteínas de Unión al ARN/metabolismo , Células Madre/metabolismo , Sistema Simpatoadrenal/citología , Torso/fisiología , Proteínas de Xenopus/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Diferenciación Celular , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Adhesiones Focales/metabolismo , Humanos , Integrinas/metabolismo , Neuronas/citología , Neuronas/metabolismo , Fenotipo , Transducción de Señal , Xenopus laevis , Pez CebraRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Chronic HCV infection is characterized by several immunological alterations, such as the accumulation of suppressor cells and of hyperactivated T lymphocytes. However, it is unclear whether direct-acting antiviral (DAA)-mediated HCV clearance restores immune dysfunctions. We performed a phenotypic characterization by flow cytometry of different immune cell subsets, including monocytic myeloid-derived suppressor cells (M-MDSCs) and T lymphocytes in 168 patients with persistent HCV infection not treated, under DAA therapies and sustained virological responders. Chronic HCV infection prompted the accumulation of M-MDSCs independently of patient and clinical characteristics, and altered their metabolic properties. HCV RNA was undetectable in the majority of patients just after few weeks of DAA therapy, whereas M-MDSC levels normalized only 6 months after therapy. In addition, HCV infection deeply perturbed the T cell compartment since a re-distribution of memory CD4+ and CD8+ T cells was observed at the expenses of naïve cells, and memory T lymphocytes displayed increased activation. Notably, these features were only partially restored by DAA therapies in the CD4, but not in the CD8, compartment as high immune activation levels persisted in the terminally differentiated memory CD8+ T cells even more than 1 year after sustained virological response. Together, these results suggest that successful DAA therapies do not lead to full immunological reconstitution as fast as viral clearance.