RESUMEN
The paucity of recurrent mutations has hampered efforts to understand and treat neuroblastoma. Alternative splicing and splicing-dependent RNA-fusions represent mechanisms able to increase the gene product repertoire but their role in neuroblastoma remains largely unexplored. Here we investigate the presence and possible roles of aberrant splicing and splicing-dependent RNA-fusion transcripts in neuroblastoma. In addition, we attend to establish whether the spliceosome can be targeted to treat neuroblastoma. Through analysis of RNA-sequenced neuroblastoma we show that elevated expression of splicing factors is a strong predictor of poor clinical outcome. Furthermore, we identified >900 primarily intrachromosomal fusions containing canonical splicing sites. Fusions included transcripts from well-known oncogenes, were enriched for proximal genes and in chromosomal regions commonly gained or lost in neuroblastoma. As a proof-of-principle that these fusions can generate altered gene products, we characterized a ZNF451-BAG2 fusion, producing a truncated BAG2-protein which inhibited retinoic acid induced differentiation. Spliceosome inhibition impeded neuroblastoma fusion expression, induced apoptosis and inhibited xenograft tumor growth. Our findings elucidate a splicing-dependent mechanism generating altered gene products in neuroblastoma and show that the spliceosome is a potential target for clinical intervention.
Asunto(s)
Chaperonas Moleculares/genética , Proteínas Mutantes Quiméricas/genética , Neuroblastoma/genética , Empalme del ARN , Empalmosomas/efectos de los fármacos , Aminoaciltransferasas/metabolismo , Animales , Apoptosis , Diferenciación Celular , Línea Celular Tumoral , Femenino , Fusión Génica , Proteínas del Choque Térmico HSC70/metabolismo , Humanos , Ratones Desnudos , Chaperonas Moleculares/metabolismo , Proteínas Mutantes Quiméricas/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patología , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Eliminación de Secuencia , Factores de Transcripción/metabolismo , Proteínas tau/metabolismoRESUMEN
In the originally published version of this Article, financial support was not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to include support to Thomas Perlmann provided by Knut and Alice Wallenberg Foundation (grant 2013.0075) and Swedish Research Council (VR; grant 2016-02506).
RESUMEN
Glioblastoma (GBM) is regarded as the most common malignant brain tumor but treatment options are limited. Thus, there is an unmet clinical need for compounds and corresponding targets that could inhibit GBM growth. We screened a library of 80 dopaminergic ligands with the aim of identifying compounds capable of inhibiting GBM cell line proliferation and survival. Out of 45 active compounds, 8 were further validated. We found that the dopamine receptor D2 antagonist trifluoperazine 2HCl inhibits growth and proliferation of GBM cells in a dose dependent manner. Trifluoperazine's inhibition of GBM cells is cell line dependent and correlates with variations in dopamine receptor expression profile. We conclude that components of the dopamine receptor signaling pathways are potential targets for pharmacological interventions of GBM growth.
Asunto(s)
Evaluación Preclínica de Medicamentos , Glioblastoma/patología , Trifluoperazina/farmacología , Recuento de Células , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Dopamina/metabolismo , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/genética , Humanos , Ligandos , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/metabolismo , Transducción de Señal/efectos de los fármacos , Trifluoperazina/químicaRESUMEN
The brain is composed of hundreds of different neuronal subtypes, which largely retain their identity throughout the lifespan of the organism. The mechanisms governing this stability are not fully understood, partly due to the diversity and limited size of clinically relevant neuronal populations, which constitute a technical challenge for analysis. Here, using a strategy that allows for ChIP-seq combined with RNA-seq in small neuronal populations in vivo, we present a comparative analysis of permissive and repressive histone modifications in adult midbrain dopaminergic neurons, raphe nuclei serotonergic neurons, and embryonic neural progenitors. Furthermore, we utilize the map generated by our analysis to show that the transcriptional response of midbrain dopaminergic neurons following 6-OHDA or methamphetamine injection is characterized by increased expression of genes with promoters dually marked by H3K4me3/H3K27me3. Our study provides an in vivo genome-wide analysis of permissive/repressive histone modifications coupled to gene expression in these rare neuronal subtypes.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Regulación de la Expresión Génica , Código de Histonas , Neuronas Serotoninérgicas/metabolismo , Animales , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Femenino , Expresión Génica , Silenciador del Gen , Genoma , Estudio de Asociación del Genoma Completo , Masculino , Ratones , Ratones Transgénicos , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Estrés FisiológicoRESUMEN
The Notch signaling pathway is a key regulator of stem cells during development, and its deregulated activity is linked to developmental defects and cancer. Transcriptional activation of Notch target genes requires cleavage of the Notch receptor in response to ligand binding, production of the Notch intracellular domain (NICD1), NICD1 migration into the nucleus, and assembly of a transcriptional complex. Post-translational modifications of Notch regulate its trafficking, turnover, and transcriptional activity. Here, we show that NICD1 is modified by small ubiquitin-like modifier (SUMO) in a stress-inducible manner. Sumoylation occurs in the nucleus where NICD1 is sumoylated in the RBPJ-associated molecule (RAM) domain. Although stress and sumoylation enhance nuclear localization of NICD1, its transcriptional activity is attenuated. Molecular modeling indicates that sumoylation can occur within the DNA-bound ternary transcriptional complex, consisting of NICD1, the transcription factor Suppressor of Hairless (CSL), and the co-activator Mastermind-like (MAML) without its disruption. Mechanistically, sumoylation of NICD1 facilitates the recruitment of histone deacetylase 4 (HDAC4) to the Notch transcriptional complex to suppress Notch target gene expression. Stress-induced sumoylation decreases the NICD1-mediated induction of Notch target genes, which was abrogated by expressing a sumoylation-defected mutant in cells and in the developing central nervous system of the chick in vivo. Our findings of the stress-inducible sumoylation of NICD1 reveal a novel context-dependent regulatory mechanism of Notch target gene expression.
Asunto(s)
Regulación de la Expresión Génica , Receptor Notch1/química , Receptor Notch1/metabolismo , Estrés Fisiológico , Sumoilación , Animales , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Estrés Oxidativo , Transducción de SeñalRESUMEN
Glioblastoma (GBM) is regarded as the most common malignant brain tumor but treatment options are limited. Thus, there is an unmet clinical need for compounds and corresponding targets that could inhibit GBM growth. We screened a library of 80 dopaminergic ligands with the aim of identifying compounds capable of inhibiting GBM cell line proliferation and survival. Out of 45 active compounds, 8 were further validated. We found that the dopamine receptor D2 antagonist trifluoperazine 2HCl inhibits growth and proliferation of GBM cells in a dose dependent manner. Trifluoperazine's inhibition of GBM cells is cell line dependent and correlates with variations in dopamine receptor expression profile. We conclude that components of the dopamine receptor signaling pathways are potential targets for pharmacological interventions of GBM growth.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Trifluoperazina/administración & dosificación , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Antagonistas de Dopamina/administración & dosificación , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas/métodos , Glioblastoma/metabolismo , Humanos , Receptores Dopaminérgicos/metabolismoRESUMEN
We recently identified pathogenic KIF1Bß mutations in sympathetic nervous system malignancies that are defective in developmental apoptosis. Here we deleted KIF1Bß in the mouse sympathetic nervous system and observed impaired sympathetic nervous function and misexpression of genes required for sympathoadrenal lineage differentiation. We discovered that KIF1Bß is required for nerve growth factor (NGF)-dependent neuronal differentiation through anterograde transport of the NGF receptor TRKA. Moreover, pathogenic KIF1Bß mutations identified in neuroblastoma impair TRKA transport. Expression of neuronal differentiation markers is ablated in both KIF1Bß-deficient mouse neuroblasts and human neuroblastomas that lack KIF1Bß. Transcriptomic analyses show that unfavorable neuroblastomas resemble mouse sympathetic neuroblasts lacking KIF1Bß independent of MYCN amplification and the loss of genes neighboring KIF1B on chromosome 1p36. Thus, defective precursor cell differentiation, a common trait of aggressive childhood malignancies, is a pathogenic effect of KIF1Bß loss in neuroblastomas. Furthermore, neuropathy-associated KIF1Bß mutations impede cargo transport, providing a direct link between neuroblastomas and neurodegeneration.
Asunto(s)
Diferenciación Celular/genética , Cinesinas/genética , Cinesinas/metabolismo , Neuroblastoma/genética , Neuronas/citología , Receptor trkA/metabolismo , Animales , Apoptosis/genética , Línea Celular Tumoral , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Mutación , Neuroblastoma/fisiopatología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/fisiopatología , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Células PC12 , Ratas , Transducción de Señal/genética , Sistema Nervioso Simpático/citología , Proteínas ras/genéticaRESUMEN
Glioma cells recruit and exploit microglia (the resident immune cells of the brain) for their proliferation and invasion ability. The underlying molecular mechanism used by glioma cells to transform microglia into a tumor-supporting phenotype has remained elusive. We found that glioma-induced microglia conversion was coupled to a reduction in the basal activity of microglial caspase-3 and increased S-nitrosylation of mitochondria-associated caspase-3 through inhibition of thioredoxin-2 activity, and that inhibition of caspase-3 regulated microglial tumor-supporting function. Furthermore, we identified the activity of nitric oxide synthase 2 (NOS2, also known as iNOS) originating from the glioma cells as a driving stimulus in the control of microglial caspase-3 activity. Repression of glioma NOS2 expression in vivo led to a reduction in both microglia recruitment and tumor expansion, whereas depletion of microglial caspase-3 gene promoted tumor growth. Our results provide evidence that inhibition of the denitrosylation of S-nitrosylated procaspase-3 mediated by the redox protein Trx2 is a part of the microglial pro-tumoral activation pathway initiated by glioma cancer cells.
Asunto(s)
Caspasa 3/metabolismo , Glioma/metabolismo , Glioma/patología , Microglía/metabolismo , Fenotipo , Animales , Línea Celular Tumoral , Movimiento Celular , Modelos Animales de Enfermedad , Activación Enzimática , Técnicas de Silenciamiento del Gen , Glioma/inmunología , Xenoinjertos , Humanos , Masculino , Ratones , Microglía/inmunología , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Tiorredoxinas/metabolismo , Carga TumoralRESUMEN
The zinc finger transcription factor Zac1 is expressed in dividing progenitors of the nervous system with expression levels negatively controlled by genomic imprinting. To explore the consequences of elevated ZAC1 levels during neurogenesis we overexpressed it in the developing CNS. Increased levels of ZAC1 rapidly promoted upregulation of CDK inhibitors P57 and P27 followed by cell cycle exit. Surprisingly this was accompanied by stalled neuronal differentiation. Genome wide expression analysis of cortical cells overexpressing Zac1 revealed a decrease in neuronal gene expression and an increased expression of imprinted genes, factors regulating mesoderm formation as well as features of differentiated muscle. In addition, we observed a rapid induction of several genes regulating pluripotency. Taken together, our data suggests that expression levels of Zac1 need to be kept under strict control to avoid premature cell cycle exit, disrupted neurogenesis and aberrant expression of non-neuronal genes including pluripotency associated factors.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Reprogramación Celular , Neurogénesis , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Tipificación del Cuerpo , Ciclo Celular , Diferenciación Celular , Proliferación Celular , Pollos , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes Supresores de Tumor , Genoma , Células HEK293 , Humanos , Ratones , Modelos Biológicos , Músculos/citología , Tubo Neural/embriología , Tubo Neural/metabolismo , Neuronas/citología , Células Madre Pluripotentes/metabolismo , Unión Proteica , Factores de Transcripción SOXB1RESUMEN
Activation of Notch signaling requires intracellular routing of the receptor, but the mechanisms controlling the distinct steps in the routing process is poorly understood. We identify PKCζ as a key regulator of Notch receptor intracellular routing. When PKCζ was inhibited in the developing chick central nervous system and in cultured myoblasts, Notch-stimulated cells were allowed to undergo differentiation. PKCζ phosphorylates membrane-tethered forms of Notch and regulates two distinct routing steps, depending on the Notch activation state. When Notch is activated, PKCζ promotes re-localization of Notch from late endosomes to the nucleus and enhances production of the Notch intracellular domain, which leads to increased Notch activity. In the non-activated state, PKCζ instead facilitates Notch receptor internalization, accompanied with increased ubiquitylation and interaction with the endosomal sorting protein Hrs. Collectively, these data identify PKCζ as a key regulator of Notch trafficking and demonstrate that distinct steps in intracellular routing are differentially modulated depending on Notch signaling status.
Asunto(s)
Proteína Quinasa C/fisiología , Receptor Notch1/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Embrión de Pollo , Células HEK293 , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Transporte de Proteínas , Receptor Notch1/genética , Homología de Secuencia de Aminoácido , Transducción de Señal/genéticaRESUMEN
The chromatin remodeler CHD5 is expressed in neural tissue and is frequently deleted in aggressive neuroblastoma. Very little is known about the function of CHD5 in the nervous system or its mechanism of action. Here we report that depletion of Chd5 in the developing neocortex blocks neuronal differentiation and leads to an accumulation of undifferentiated progenitors. CHD5 binds a large cohort of genes and is required for facilitating the activation of neuronal genes. It also binds a cohort of Polycomb targets and is required for the maintenance of H3K27me3 on these genes. Interestingly, the chromodomains of CHD5 directly bind H3K27me3 and are required for neuronal differentiation. In the absence of CHD5, a subgroup of Polycomb-repressed genes becomes aberrantly expressed. These findings provide insights into the regulatory role of CHD5 during neurogenesis and suggest how inactivation of this candidate tumor suppressor might contribute to neuroblastoma.