RESUMEN
Hedgehog signaling is essential for tissue development and stemness, and its deregulation has been observed in many tumors. Aberrant activation of Hedgehog signaling is the result of genetic mutations of pathway components or other Smo-dependent or independent mechanisms, all triggering the downstream effector Gli1. For this reason, understanding the poorly elucidated mechanism of Gli1-mediated transcription allows to identify novel molecules blocking the pathway at a downstream level, representing a critical goal in tumor biology. Here, we clarify the structural requirements of the pathway effector Gli1 for binding to DNA and identify Glabrescione B as the first small molecule binding to Gli1 zinc finger and impairing Gli1 activity by interfering with its interaction with DNA. Remarkably, as a consequence of its robust inhibitory effect on Gli1 activity, Glabrescione B inhibited the growth of Hedgehog-dependent tumor cells in vitro and in vivo as well as the self-renewal ability and clonogenicity of tumor-derived stem cells. The identification of the structural requirements of Gli1/DNA interaction highlights their relevance for pharmacologic interference of Gli signaling.
Asunto(s)
ADN/metabolismo , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Isoflavonas/farmacología , Factores de Transcripción de Tipo Kruppel/metabolismo , Receptores de Superficie Celular/fisiología , Transducción de Señal/efectos de los fármacos , Animales , Cerebelo/citología , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , ADN/efectos de los fármacos , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Glioblastoma/metabolismo , Factores de Transcripción de Tipo Kruppel/química , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Mutación/genética , Receptores Patched , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened , Proteína con Dedos de Zinc GLI1RESUMEN
The transcription factor Nanog plays a critical role in the self-renewal of embryonic stem cells as well as in neural stem cells (NSCs). microRNAs (miRNAs) are also involved in stemness regulation. However, the miRNA network downstream of Nanog is still poorly understood. High-throughput screening of miRNA expression profiles in response to modulated levels of Nanog in postnatal NSCs identifies miR-17-92 cluster as a direct target of Nanog. Nanog controls miR-17-92 cluster by binding to the upstream regulatory region and maintaining high levels of transcription in NSCs, whereas Nanog/promoter association and cluster miRNAs expression are lost alongside differentiation. The two miR-17 family members of miR-17-92 cluster, namely miR-17 and miR-20a, target Trp53inp1, a downstream component of p53 pathway. To support a functional role, the presence of miR-17/20a or the loss of Trp53inp1 is required for the Nanog-induced enhancement of self-renewal of NSCs. We unveil an arm of the Nanog/p53 pathway, which regulates stemness in postnatal NSCs, wherein Nanog counteracts p53 signals through miR-17/20a-mediated repression of Trp53inp1.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Homeodominio/metabolismo , MicroARNs/metabolismo , Células-Madre Neurales/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/genética , Ciclo Celular , Proliferación Celular , Células Cultivadas , Cerebelo/citología , Proteínas de Choque Térmico/genética , Proteínas de Homeodominio/genética , Ratones , MicroARNs/genética , Proteína Homeótica Nanog , Células-Madre Neurales/citologíaRESUMEN
Hedgehog (Hh) pathway has a pivotal function in development and tumorigenesis, processes sustained by stem cells (SCs). The transcription factor Nanog controls stemness acting as a key determinant of both embryonic SC self-renewal and differentiated somatic cells reprogramming to pluripotency, in concert with the loss of the oncosuppressor p53. How Nanog is regulated by microenvironmental signals in postnatal SC niches has been poorly investigated. Here, we show that Nanog is highly expressed in SCs from postnatal cerebellum and medulloblastoma, and acts as a critical mediator of Hh-driven self-renewal. Indeed, the downstream effectors of Hh activity, Gli1 and Gli2, bind to Nanog-specific cis-regulatory sequences both in mouse and human SCs. Loss of p53, a key event promoting cell stemness, activates Hh signalling, thereby contributing to Nanog upregulation. Conversely, Hh downregulates p53 but does not require p53 to control Nanog. Our data reveal a mechanism for the function of Hh in the control of stemness that represents a crucial component of an integrated circuitry determining cell fate decision and involved in the maintenance of cancer SCs.
Asunto(s)
Proteínas Hedgehog/metabolismo , Proteínas de Homeodominio/metabolismo , Neuronas/metabolismo , Células Madre/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Secuencia de Bases , Proliferación Celular , Células Cultivadas , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Meduloblastoma/metabolismo , Ratones , Datos de Secuencia Molecular , Proteína Homeótica Nanog , Células Madre Neoplásicas/metabolismo , Neuronas/citología , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Alineación de Secuencia , Células Madre/citología , Transactivadores/genética , Transactivadores/metabolismo , Transcripción Genética , Proteína p53 Supresora de Tumor/genética , Proteína con Dedos de Zinc GLI1RESUMEN
Hereditary breast and ovarian cancer are mainly linked to mutations in BRCA1 and BRCA2 genes which confer a similar cumulative risk of developing breast cancer. Importantly, while BRCA2 mutation carriers generally have a lower cumulative risk for ovarian cancer, mutations clustered in the central portion of BRCA2 are associated with a higher proportion of ovarian compared with breast cancer cases. The boundaries of this ovarian cancer cluster region (OCCR) have been tentatively defined within a 3.3 kb region of BRCA2 exon 11, and herein, we reassessed these boundaries using our series of Italian breast/ovarian cancer families. We used direct sequencing to investigate BRCA mutations in 367 breast/ovarian cancer families. We also studied the association between the location of the mutations and the ovarian cancer phenotype in our cohort of BRCA2-mutated families. We observed the novel c.7309_7309delA frameshift mutation and the c.7007G>A deleterious mutation in BRCA2 exons 14 and 13, respectively, in five independent Italian families characterized by a high proportion of ovarian cancer cases. Of note, a significantly higher proportion of ovarian versus breast cancer cases was associated not only with mutations in the previously defined OCCR (OR = 5.91; p = 0.004), but also with the exon 13-14 region (OR = 7.37; p = 0.001) in our BRCA2-mutated families. Our data provide initial evidence for a novel putative OCCR in BRCA2 exons 13-14.
Asunto(s)
Proteína BRCA2/genética , Neoplasias de la Mama Masculina/genética , Neoplasias Ováricas/genética , Adulto , Anciano , Neoplasias de la Mama/epidemiología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama Masculina/epidemiología , Exones , Femenino , Mutación de Línea Germinal , Humanos , Italia , Masculino , Persona de Mediana Edad , Mutación , Neoplasias Ováricas/epidemiología , Neoplasias Ováricas/patología , LinajeRESUMEN
BACKGROUND: Extraneural metastases (ENM) rarely occur in medulloblastoma (MBL) patients and only few cases of subcutaneous localizations have been described. ENM indicate an aggressive disease associated with a worse prognosis. The characterization of metastatic tumours might be useful to understand their pathogenesis and to identify the most appropriate therapeutic strategies. CASE PRESENTATION: We present the case of a child with Large Cell Anaplastic (LC/A) MBL, who developed multiple subcutaneous metastases in the scalp area after a ventriculo-peritoneal shunting procedure. The disease rapidly progressed and the child died despite chemotherapy and primary tumour surgical debulking.We molecularly classified the tumour as a group 3 MBL; in addition, we derived stem-like cells (SLC) from a metastatic lesion. Primary tumour, metastases and SLC were further analysed, particularly focusing on features linked to the cutaneous dissemination. Indeed, molecules involved in angiogenesis, cell invasion and epidermal growth factor signalling resulted highly expressed. CONCLUSIONS: The present report describes a very rare case of subcutaneous metastatic MBL. The tumour, metastases and SLC have been clinically, pathologically and molecularly characterized. Our case is an example of multidisciplinary approach aiming to characterize MBL aggressive behaviour.
Asunto(s)
Meduloblastoma/genética , Metástasis de la Neoplasia/genética , Células Madre Neoplásicas/patología , Preescolar , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Meduloblastoma/diagnóstico , Meduloblastoma/patología , Metástasis de la Neoplasia/patología , PronósticoRESUMEN
Reactivation of the HMGA1 protoncogene is very frequent in human cancer, but still very little is known on the molecular mechanisms leading to this event. Prompted by the finding of putative E2F binding sites in the human HMGA1 promoter and by the frequent deregulation of the RB/E2F1 pathway in human carcinogenesis, we investigated whether E2F1 might contribute to the regulation of HMGA1 gene expression. Here we report that E2F1 induces HMGA1 by interacting with a 193 bp region of the HMGA1 promoter containing an E2F binding site surrounded by three putative Sp1 binding sites. Both gain and loss of function experiments indicate that Sp1 functionally interacts with E2F1 to promote HMGA1 expression. However, while Sp1 constitutively binds HMGA1 promoter, it is the balance between different E2F family members that tunes the levels of HMGA1 expression between quiescence and proliferation. Finally, we found increased HMGA1 expression in pituitary and thyroid tumors developed in Rb(+/-) mice, supporting the hypothesis that E2F1 is a novel important regulator of HMGA1 expression and that deregulation of the RB/E2F1 path might significantly contribute to HMGA1 deregulation in cancer.
Asunto(s)
Factor de Transcripción E2F1/metabolismo , Proteína HMGA1a/genética , Neoplasias Hipofisarias/metabolismo , Factor de Transcripción Sp1/metabolismo , Neoplasias de la Tiroides/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Western Blotting , Inmunoprecipitación de Cromatina , Factor de Transcripción E2F1/genética , Proteína HMGA1a/metabolismo , Humanos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación/genética , Neoplasias Hipofisarias/genética , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína de Retinoblastoma/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción Sp1/genética , Neoplasias de la Tiroides/genética , Activación TranscripcionalRESUMEN
The developmental protein Numb is a major determinant of binary cell fates. It is also required for the differentiation of cerebellar granule cell progenitors (GCPs) at a stage of development responsive to the morphogenic glycoprotein Hedehog. Hedgehog signalling is crucial for the physiological maintenance and self-renewal of neural stem cells and its deregulation is responsible for their progression towards tumorigenesis. The mechanisms that inhibit this pathway during the differentiation stage are poorly understood. Here, we identify Numb as a Hedgehog-pathway inhibitor that is downregulated in early GCPs and GCP-derived cancer cells. We demonstrate that the Hedgehog transcription factor Gli1 is targeted by Numb for Itch-dependent ubiquitination, which suppresses Hedgehog signals, thus arresting growth and promoting cell differentiation. This novel Numb-dependent regulatory loop may limit the extent and duration of Hedgehog signalling during neural-progenitor differentiation, and its subversion may be a relevant event in brain tumorigenesis.
Asunto(s)
Proteínas Hedgehog/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinas/metabolismo , Animales , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Cerebelo/citología , Cerebelo/patología , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Meduloblastoma/genética , Ratones , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Células Madre/citología , Factores de Transcripción/genética , Proteína con Dedos de Zinc GLI1RESUMEN
Notch3 overexpression has been previously shown to positively regulate the generation and function of naturally occurring regulatory T cells and the expression of Foxp3, in cooperation with the pTα/pre-TCR pathway. In this study, we show that Notch3 triggers the trans activation of Foxp3 promoter depending on the T cell developmental stage. Moreover, we discovered a novel CSL/NF-κB overlapping binding site within the Foxp3 promoter, and we demonstrate that the activation of NF-κB, mainly represented by p65-dependent canonical pathway, plays a positive role in Notch3-dependent regulation of Foxp3 transcription. Accordingly, the deletion of protein kinase C, which mediates canonical NF-κB activation, markedly reduces regulatory T cell number and per cell Foxp3 expression in transgenic mice with a constitutive activation of Notch3 signaling. Collectively, our data indicate that the cooperation among Notch3, protein kinase C, and p65/NF-κB subunit modulates Foxp3 expression, adding new insights in the understanding of the molecular mechanisms involved in regulatory T cell homeostasis and function.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , FN-kappa B/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Animales , Línea Celular , Células Cultivadas , Citometría de Flujo , Factores de Transcripción Forkhead/genética , Immunoblotting , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteína Quinasa C-theta , Receptor Notch3 , Receptores Notch/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Reguladores/metabolismo , Timo/citología , Timo/metabolismo , Factores de Tiempo , Factor de Transcripción ReIA/metabolismo , Transcripción Genética , Activación TranscripcionalRESUMEN
Histone deacetylases (HDACs) play a crucial role in several physiological and pathological cell functions, including cell development and cancer, by deacetylating both histones and others proteins. HDACs belong to a large family of enzymes including Class I, II and IV as well as Class III or sirtuins subfamilies, that undergo a complex transcriptional and post-translational regulation. In current years, antitumor therapy is attempting to exploit several chemical classes of inhibitors that target HDACs, frequently reported to be misregulated in cancer. Nevertheless, the identity of gene products directly involved in tumorigenesis and preventing HDAC misregulation in cancer is still poorly understood. Recent evidence has demonstrated that the tumor suppressors HIC1 and DBC1 induce direct repression of Sirt1 function, whereas Chfr and REN(KCTD11/KASH family) downregulate HDAC1, by inducing its ubiquitin-dependent degradation. Loss of these gene products leads to imbalanced enhancement of HDAC activity and subsequently to oncogenesis. All these genes are frequently deleted or silenced in human cancers, highlighting the role of endogenous HDAC inhibitors to counteracts HDAC-mediated tumorigenesis. Thus, endogenous HDAC inhibitors represent a promising class of "antitumor agents" thanks to which oncogenic addiction pathways may be selectively therapeutically targeted.
Asunto(s)
Inhibidores de Histona Desacetilasas/uso terapéutico , Histona Desacetilasas/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Animales , Humanos , Neoplasias/patologíaRESUMEN
MicroRNAs (miRNA) are crucial post-transcriptional regulators of gene expression and control cell differentiation and proliferation. However, little is known about their targeting of specific developmental pathways. Hedgehog (Hh) signalling controls cerebellar granule cell progenitor development and a subversion of this pathway leads to neoplastic transformation into medulloblastoma (MB). Using a miRNA high-throughput profile screening, we identify here a downregulated miRNA signature in human MBs with high Hh signalling. Specifically, we identify miR-125b and miR-326 as suppressors of the pathway activator Smoothened together with miR-324-5p, which also targets the downstream transcription factor Gli1. Downregulation of these miRNAs allows high levels of Hh-dependent gene expression leading to tumour cell proliferation. Interestingly, the downregulation of miR-324-5p is genetically determined by MB-associated deletion of chromosome 17p. We also report that whereas miRNA expression is downregulated in cerebellar neuronal progenitors, it increases alongside differentiation, thereby allowing cell maturation and growth inhibition. These findings identify a novel regulatory circuitry of the Hh signalling and suggest that misregulation of specific miRNAs, leading to its aberrant activation, sustain cancer development.
Asunto(s)
Cerebelo/citología , Proteínas Hedgehog/metabolismo , Meduloblastoma/metabolismo , MicroARNs/metabolismo , Neuronas/fisiología , Transducción de Señal/fisiología , Células Madre/fisiología , Adulto , Anciano , Animales , Secuencia de Bases , Diferenciación Celular , Proliferación Celular , Cromosomas Humanos Par 17/genética , Perfilación de la Expresión Génica , Proteínas Hedgehog/genética , Humanos , Meduloblastoma/genética , Meduloblastoma/patología , Ratones , MicroARNs/genética , Análisis por Micromatrices , Persona de Mediana Edad , Datos de Secuencia Molecular , Neuronas/citología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened , Células Madre/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína con Dedos de Zinc GLI1RESUMEN
Glucocorticoids (GCs) play a critical role in neural development; however, their prenatal or neonatal therapeutic use can have detrimental effects on the developing brain. In this issue of the JCI, Heine and Rowitch report that the molecular mechanisms underlying these detrimental effects involve the sonic hedgehog (Shh) signaling pathway, a crucial regulator of brain development and neural stem/progenitor cells (see the related study beginning on page 267). They show that GCs suppress Shh-induced proliferation of cerebellar progenitor cells in postnatal mice and that, conversely, Shh signaling is protective against GC-induced neonatal cerebellar injury by inducing the enzyme 11betaHSD2, which inactivates the GCs corticosterone and prednisolone, but not dexamethasone. The data provide a rationale for the therapeutic use of 11betaHSD2-sensitive GCs, but not dexamethasone, or for the exploitation of the neuroprotective effect of Shh agonists to prevent GC-induced pre- or neonatal brain injury.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/fisiología , Encéfalo/efectos de los fármacos , Glucocorticoides/toxicidad , Proteínas Hedgehog/fisiología , Animales , Animales Recién Nacidos , RatonesRESUMEN
We identify a novel alternative TrkA splice variant, TrkAIII, with deletion of exons 6, 7, and 9 and functional extracellular IG-C1 and N-glycosylation domains, that exhibits expression restricted to undifferentiated early neural progenitors, human neuroblastomas (NBs), and a subset of other neural crest-derived tumors. This NGF-unresponsive isoform is oncogenic in NIH3T3 cells and promotes tumorigenic NB cell behavior in vitro and in vivo (cell survival, xenograft growth, angiogenesis) resulting from spontaneous tyrosine kinase activity and IP3K/Akt/NF-kappaB but not Ras/MAPK signaling. TrkAIII antagonizes NGF/TrkAI signaling, which is responsible for NB growth arrest and differentiation through Ras/MAPK, and its expression is promoted by hypoxia at the expense of NGF-responsive receptors, providing a mechanism for converting NGF/TrkA/Ras/MAPK antioncogenic signals to TrkAIII/IP3K/Akt/NF-kappaB tumor-promoting signals during tumor progression.
Asunto(s)
Empalme Alternativo/genética , Neuroblastoma/metabolismo , Neuroblastoma/patología , Receptor trkA/genética , Receptor trkA/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Secuencia de Bases , Línea Celular , Clonación Molecular , Doxorrubicina/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Hipoxia/genética , Ratones , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Neovascularización Patológica , Factor de Crecimiento Nervioso/metabolismo , Factor de Crecimiento Nervioso/farmacología , Neuroblastoma/irrigación sanguínea , Neuroblastoma/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfolipasa C gamma , Unión Proteica , Receptor trkA/antagonistas & inhibidores , Receptor trkA/química , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Fosfolipasas de Tipo C/metabolismoRESUMEN
Regulation of gene expression in response to mitogenic stimuli is a critical aspect underlying many forms of human cancers. The AP-1 complex mediates the transcriptional response to mitogens, and its deregulation causes developmental defects and tumors. We report that the coactivator CRTC1 cyclic AMP response element-binding protein (CREB)-regulated transcription coactivator 1 is a potent and indispensable modulator of AP-1 function. After exposure of cells to the AP-1 agonist 12-O-tetradecanoylphorbol-13-acetate (TPA), CRTC1 is recruited to AP-1 target gene promoters and associates with c-Jun and c-Fos to activate transcription. CRTC1 consistently synergizes with the proto-oncogene c-Jun to promote cellular growth, whereas AP-1-dependent proliferation is abrogated in CRTC1-deficient cells. Remarkably, we demonstrate that CRTC1-Maml2 oncoprotein, which causes mucoepidermoid carcinomas, binds and activates both c-Jun and c-Fos. Consequently, ablation of AP-1 function disrupts the cellular transformation and proliferation mediated by this oncogene. Together, these data illustrate a novel mechanism required to couple mitogenic signals to the AP-1 gene regulatory program.
Asunto(s)
Proliferación Celular , Transformación Celular Neoplásica , Factor de Transcripción AP-1/fisiología , Factores de Transcripción/fisiología , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-fos/fisiología , Proteínas Proto-Oncogénicas c-jun/fisiología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Acetato de Tetradecanoilforbol/farmacología , Transactivadores , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
E2F1, a member of the E2F family of transcription factors, in addition to its established proliferative effect, has also been implicated in the induction of apoptosis through p53-dependent and p53-independent pathways. Several genes involved in the activation or execution of the apoptotic programme have recently been shown to be upregulated at the transcriptional level by E2F1 overexpression, including the genes encoding INK4a/ARF, Apaf-1, caspase 7 and p73 (refs 3-5). E2F1 is stabilized in response to DNA damage but it has not been established how this translates into the activation of specific subsets of E2F target genes. Here, we applied a chromatin immunoprecipitation approach to show that, in response to DNA damage, E2F1 is directed from cell cycle progression to apoptotic E2F target genes. We identify p73 as an important E2F1 apoptotic target gene in DNA damage response and we show that acetylation is required for E2F1 recruitment on the P1p73 promoter and for its transcriptional activation.
Asunto(s)
Apoptosis/fisiología , Proteínas de Ciclo Celular , Daño del ADN , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Proteínas Nucleares/genética , Factores de Transcripción/metabolismo , Acetilación/efectos de los fármacos , Apoptosis/efectos de los fármacos , Apoptosis/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/fisiología , Doxorrubicina/farmacología , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Etopósido/farmacología , Fibroblastos , Eliminación de Gen , Genes Reporteros , Genes Supresores de Tumor , Histonas/análisis , Humanos , Proteínas Nucleares/fisiología , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Activación Transcripcional , Células Tumorales Cultivadas , Proteína Tumoral p73 , Proteínas Supresoras de TumorRESUMEN
Numb is an evolutionary conserved protein that plays critical roles in cell fate determination. Mammalian Numb displays a higher degree of structural complexity compared to the Drosophila homolog based on the number of encoding genes (Numb and Numb-like) and of alternative spliced isoforms. Accordingly, Numb proteins display a complex pattern of functions such as the control of asymmetric cell division and cell fate choice, endocytosis, cell adhesion, cell migration, ubiquitination of specific substrates and a number of signaling pathways (i.e. Notch, Hedgehog, p53). Recent findings indicate that, besides controlling such physiologic developmental processes, subversion of the above Numb-dependent events plays a critical role in disease (e.g. cancer). We will review here the multiple functions of mNumb and their underlying molecular mechanisms in development and disease.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Isoformas de Proteínas/metabolismo , Animales , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Endocitosis/fisiología , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Neoplasias/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Transducción de Señal/fisiología , Relación Estructura-ActividadRESUMEN
A hallmark of several human cancers is loss of heterozygosity (LOH) of chromosome 17p13. The same chromosomal region is also frequently hypermethylated in cancer. Although loss of 17p13 has been often associated with p53 genetic alteration or Hypermethylated in Cancer 1 (HIC1) gene hypermethylation, other tumor suppressor genes (TSGs) located in this region have critical roles in tumorigenesis. A novel TSG mapping on human chromosome 17p13.2 is KCTD11REN (KCTD11). We have recently demonstrated that KCTD11 expression is frequently lost in human medulloblastoma (MB), in part by LOH and in part by uncharacterized epigenetic events. Using a panel of human 177 tumor samples and their normal matching samples representing 18 different types of cancer, we show here that the down-regulation of KCTD11 protein level is a specific and a diffusely common event in tumorigenesis. Additionally, in order to characterize the regulatory regions in KCTD11 promoter, we identified a CpG island and several Sp1 binding sites on this promoter, and demonstrated that Sp1 transcription factor and DNA methylation contribute, at least in part, to regulate KCTD11 expression. Our findings identify KCTD11 as a widely down-regulated gene in human cancers, and provide a basis to understand how its expression might be deregulated in tumor cells.
Asunto(s)
Metilación de ADN , Regulación Neoplásica de la Expresión Génica/fisiología , Canales de Potasio/genética , Factor de Transcripción Sp1/fisiología , Secuencia de Bases , Proteínas de Ciclo Celular , ADN , Humanos , Pérdida de Heterocigocidad , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , TransferasasRESUMEN
Dysregulated generation and/or function of naturally occurring 'CD4(+)CD25(+) regulatory T cells' (T(reg)s) play key role in the development of autoimmune diseases, including type 1 diabetes. Recent findings suggest that Notch3 signaling activation promotes thymic generation and peripheral expansion and in vivo function of naturally occurring T(reg)s, thus preventing autoimmune diabetes progression in mouse models. However, the mechanisms underlying these effects have remained elusive, thus far. Here, we show that the expression of pTalpha gene is up-regulated in naturally occurring T(reg)s, at both mRNA and protein levels. Moreover, by using double mutant mice, with T cell-targeted constitutive activation of Notch3 in a pTalpha(-/-) background, we demonstrate that pTalpha deletion significantly counteracts the Notch3-dependent expansion, the increased FoxP3 expression and the enhanced in vitro activity of naturally occurring T(reg)s. Notably, the absence of pTalpha also impairs the Notch3-dependent protection against experimentally induced autoimmune diabetes. Finally, by adoptive cell transfer experiments, we demonstrated that this failure is directly related to the impaired in vivo function of naturally occurring T(reg)s bearing pTalpha deletion. Collectively, our data suggest that pTalpha expression is required for the in vivo function of naturally occurring T(reg)s and that the activation of Notch3 signaling may positively regulate the function of this population, through the pTalpha/pre-T cell receptor pathway.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Glicoproteínas de Membrana/metabolismo , Células Precursoras de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores Notch/metabolismo , Linfocitos T Reguladores/metabolismo , Animales , Proliferación Celular , Factores de Transcripción Forkhead/biosíntesis , Tolerancia Inmunológica/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Células Precursoras de Linfocitos T/inmunología , Células Precursoras de Linfocitos T/patología , Receptor Notch3 , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores Notch/genética , Receptores Notch/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Activación TranscripcionalRESUMEN
MicroRNAs are a class of sophisticated regulators of gene expression, acting as post-transcriptional inhibitors that recognize their target mRNAs through base pairing with short regions along the 3'UTRs. Several microRNAs are tissue specific, suggesting a specialized role in tissue differentiation or maintenance, and quite a few are critically involved in tumorigenesis. We studied miR-128, a brain-enriched microRNA, in retinoic acid-differentiated neuroblastoma cells, and we found that this microRNA is up-regulated in treated cells, where it down-modulates the expression of two proteins involved in the migratory potential of neural cells: Reelin and DCX. Consistently, miR-128 ectopic overexpression suppressed Reelin and DCX, whereas the LNA antisense-mediated miR-128 knockdown caused the two proteins to increase. Ectopic miR-128 overexpression reduced neuroblastoma cell motility and invasiveness, and impaired cell growth. Finally, the analysis of a small series of primary human neuroblastomas showed an association between high levels of miR-128 expression and favorable features, such as favorable Shimada category or very young age at diagnosis. Thus, we provide evidence for a role for miR-128 in the molecular events modulating neuroblastoma progression and aggressiveness.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Regulación de la Expresión Génica/fisiología , MicroARNs/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuroblastoma/tratamiento farmacológico , Neuropéptidos/metabolismo , Serina Endopeptidasas/metabolismo , Secuencia de Bases , Moléculas de Adhesión Celular Neuronal/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Proteínas de la Matriz Extracelular/genética , Humanos , MicroARNs/genética , Proteínas Asociadas a Microtúbulos/genética , Datos de Secuencia Molecular , Invasividad Neoplásica , Proteínas del Tejido Nervioso/genética , Neuroblastoma/metabolismo , Neuronas/efectos de los fármacos , Neuronas/fisiología , Neuropéptidos/genética , Proteína Reelina , Serina Endopeptidasas/genética , Tretinoina/farmacologíaRESUMEN
Notch signaling pathway regulates a wide variety of cellular processes during development and it also plays a crucial role in human diseases. This important link is firmly established in cancer, since a rare T-ALL-associated genetic lesion has been initially reported to result in deletion of Notch1 ectodomain and constitutive activation of its intracellular region. Interestingly, the cellular response to Notch signaling can be extremely variable depending on the cell type and activation context. Notch signaling triggers signals implicated in promoting carcinogenesis and autoimmune diseases, whereas it can also sustain responses that are critical to suppress carcinogenesis and to negatively regulate immune response. However, Notch signaling induces all these effects via an apparently simple signal transduction pathway, diversified into a complex network along evolution from Drosophila to mammals. Indeed, an explanation of this paradox comes from a number of evidences accumulated during the last few years, which dissected the intrinsic canonical and non-canonical components of the Notch pathway as well as several modulatory extrinsic signaling events. The identification of these signals has shed light onto the mechanisms whereby Notch and other pathways collaborate to induce a particular cellular phenotype. In this article, we review the role of Notch signaling in cells as diverse as T lymphocytes and epithelial cells of the epidermis, with the main focus on understanding the mechanisms of Notch versatility.
Asunto(s)
Evolución Biológica , Enfermedad , Receptores Notch/metabolismo , Transducción de Señal , Animales , Autoinmunidad , Humanos , Neoplasias/metabolismoRESUMEN
Medulloblastoma is an aggressive brain malignancy with high incidence in childhood. Current treatment approaches have limited efficacy and severe side effects. Therefore, new risk-adapted therapeutic strategies based on molecular classification are required. MicroRNA expression analysis has emerged as a powerful tool to identify candidate molecules playing an important role in a large number of malignancies. However, no data are yet available on human primary medulloblastomas. A high throughput microRNA expression profiles was performed in human primary medulloblastoma specimens to investigate microRNA involvement in medulloblastoma carcinogenesis. We identified specific microRNA expression patterns which distinguish medulloblastoma differing in histotypes (anaplastic, classic and desmoplastic), in molecular features (ErbB2 or c-Myc overexpressing tumors) and in disease-risk stratification. MicroRNAs expression profile clearly differentiates medulloblastoma from either adult or fetal normal cerebellar tissues. Only a few microRNAs displayed upregulated expression, while most of them were downregulated in tumor samples, suggesting a tumor growth-inhibitory function. This property has been addressed for miR-9 and miR-125a, whose rescued expression promoted medulloblastoma cell growth arrest and apoptosis while targeting the proproliferative truncated TrkC isoform. In conclusion, misregulated microRNA expression profiles characterize human medulloblastomas, and may provide potential targets for novel therapeutic strategies.