RESUMEN
In Rspondin-based 3D cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. We report the establishment of tumor organoid cultures from 20 consecutive colorectal carcinoma (CRC) patients. For most, organoids were also generated from adjacent normal tissue. Organoids closely recapitulate several properties of the original tumor. The spectrum of genetic changes within the "living biobank" agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43, rather than in APC. Organoid technology may fill the gap between cancer genetics and patient trials, complement cell-line- and xenograft-based drug studies, and allow personalized therapy design. PAPERCLIP.
Asunto(s)
Bancos de Muestras Biológicas , Neoplasias Colorrectales/patología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Organoides , Neoplasias Colorrectales/tratamiento farmacológico , Proteínas de Unión al ADN/metabolismo , Humanos , Proteínas Oncogénicas/metabolismo , Técnicas de Cultivo de Órganos , Organoides/efectos de los fármacos , Medicina de Precisión , Ubiquitina-Proteína LigasasRESUMEN
Neuroblastoma is a childhood tumour of the peripheral sympathetic nervous system. The pathogenesis has for a long time been quite enigmatic, as only very few gene defects were identified in this often lethal tumour. Frequently detected gene alterations are limited to MYCN amplification (20%) and ALK activations (7%). Here we present a whole-genome sequence analysis of 87 neuroblastoma of all stages. Few recurrent amino-acid-changing mutations were found. In contrast, analysis of structural defects identified a local shredding of chromosomes, known as chromothripsis, in 18% of high-stage neuroblastoma. These tumours are associated with a poor outcome. Structural alterations recurrently affected ODZ3, PTPRD and CSMD1, which are involved in neuronal growth cone stabilization. In addition, ATRX, TIAM1 and a series of regulators of the Rac/Rho pathway were mutated, further implicating defects in neuritogenesis in neuroblastoma. Most tumours with defects in these genes were aggressive high-stage neuroblastomas, but did not carry MYCN amplifications. The genomic landscape of neuroblastoma therefore reveals two novel molecular defects, chromothripsis and neuritogenesis gene alterations, which frequently occur in high-risk tumours.
Asunto(s)
Cromosomas Humanos/genética , Neuritas/metabolismo , Neuroblastoma/genética , Neuroblastoma/patología , Envejecimiento/genética , Análisis por Conglomerados , ADN Helicasas/genética , Análisis Mutacional de ADN , Regulación Neoplásica de la Expresión Génica , Genoma Humano/genética , Conos de Crecimiento/metabolismo , Conos de Crecimiento/patología , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Mutación , Estadificación de Neoplasias , Neuroblastoma/diagnóstico , Neuroblastoma/metabolismo , Proteínas Nucleares/genética , Pronóstico , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Proteína Nuclear Ligada al Cromosoma X , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Medulloblastoma is an aggressively growing tumour, arising in the cerebellum or medulla/brain stem. It is the most common malignant brain tumour in children, and shows tremendous biological and clinical heterogeneity. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from their disease. Those who survive often have a significantly reduced quality of life. Four tumour subgroups with distinct clinical, biological and genetic profiles are currently identified. WNT tumours, showing activated wingless pathway signalling, carry a favourable prognosis under current treatment regimens. SHH tumours show hedgehog pathway activation, and have an intermediate prognosis. Group 3 and 4 tumours are molecularly less well characterized, and also present the greatest clinical challenges. The full repertoire of genetic events driving this distinction, however, remains unclear. Here we describe an integrative deep-sequencing analysis of 125 tumour-normal pairs, conducted as part of the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. Tetraploidy was identified as a frequent early event in Group 3 and 4 tumours, and a positive correlation between patient age and mutation rate was observed. Several recurrent mutations were identified, both in known medulloblastoma-related genes (CTNNB1, PTCH1, MLL2, SMARCA4) and in genes not previously linked to this tumour (DDX3X, CTDNEP1, KDM6A, TBR1), often in subgroup-specific patterns. RNA sequencing confirmed these alterations, and revealed the expression of what are, to our knowledge, the first medulloblastoma fusion genes identified. Chromatin modifiers were frequently altered across all subgroups. These findings enhance our understanding of the genomic complexity and heterogeneity underlying medulloblastoma, and provide several potential targets for new therapeutics, especially for Group 3 and 4 patients.
Asunto(s)
Neoplasias Cerebelosas/genética , Genoma Humano/genética , Meduloblastoma/genética , Envejecimiento/genética , Secuencia de Aminoácidos , Transformación Celular Neoplásica , Neoplasias Cerebelosas/clasificación , Neoplasias Cerebelosas/diagnóstico , Neoplasias Cerebelosas/patología , Niño , Cromatina/metabolismo , Cromosomas Humanos/genética , ARN Helicasas DEAD-box/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Genómica , Proteínas Hedgehog/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Histona Demetilasas/genética , Humanos , Meduloblastoma/clasificación , Meduloblastoma/diagnóstico , Meduloblastoma/patología , Metilación , Mutación/genética , Tasa de Mutación , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/genética , Receptores Patched , Receptor Patched-1 , Fosfoproteínas Fosfatasas/genética , Poliploidía , Receptores de Superficie Celular/genética , Análisis de Secuencia de ARN , Transducción de Señal , Proteínas de Dominio T Box/genética , Factores de Transcripción/genética , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMEN
Neuroblastoma is a pediatric tumor of the sympathetic nervous system. MYCN (V-myc myelocytomatosis viral-related oncogene, neuroblastoma derived [avian]) is amplified in 20% of neuroblastomas, and these tumors carry a poor prognosis. However, tumors without MYCN amplification also may have a poor outcome. Here, we identified downstream targets of MYCN by shRNA-mediated silencing MYCN in neuroblastoma cells. From these targets, 157 genes showed an expression profile correlating with MYCN mRNA levels in NB88, a series of 88 neuroblastoma tumors, and therefore represent in vivo relevant MYCN pathway genes. This 157-gene signature identified very poor prognosis tumors in NB88 and independent neuroblastoma cohorts and was more powerful than MYCN amplification or MYCN expression alone. Remarkably, this signature also identified poor outcome of a group of tumors without MYCN amplification. Most of these tumors have low MYCN mRNA levels but high nuclear MYCN protein levels, suggesting stabilization of MYCN at the protein level. One tumor has an MYC amplification and high MYC expression. Chip-on-chip analyses showed that most genes in this signature are directly regulated by MYCN. MYCN induces genes functioning in cell cycle and DNA repair while repressing neuronal differentiation genes. The functional MYCN-157 signature recognizes classical neuroblastoma with MYCN amplification, as well as a newly identified group marked by MYCN protein stabilization.
Asunto(s)
Amplificación de Genes/genética , Perfilación de la Expresión Génica , Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Diferenciación Celular/genética , Análisis por Conglomerados , Reparación del ADN/genética , Regulación Neoplásica de la Expresión Génica , Genes Relacionados con las Neoplasias/genética , Humanos , Proteína Proto-Oncogénica N-Myc , Neuroblastoma/patología , Neuronas/patología , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/metabolismo , Pronóstico , Regiones Promotoras Genéticas/genética , Unión Proteica/genética , Resultado del Tratamiento , Regulación hacia Arriba/genéticaRESUMEN
The tightly controlled network of cell cycle genes consists of a core of cyclin dependent kinases (CDKs) that are activated by periodically expressed cyclins. The activity of the cyclin-CDK complexes is regulated by cyclin dependent kinase inhibitors (CDKIs) and multiple signal transduction routes that converge on the cell cycle. Neuroblastoma are pediatric tumors that belong to the group of small round blue cell tumors, characterized by a fast proliferation. Here, we present high throughput analyses of cell cycle regulating genes in neuroblastoma. We analyzed a series of 82 neuroblastomas by comparative genomic hybridization arrays, single nucleotide polymorphism arrays, and Affymetrix expression arrays and analyzed the datasets in parallel with the R2 bioinformatic tool (http://r2.amc.nl). About 30% of the tumors had genomic amplifications, gains, or losses with shortest regions of overlap that suggested implication of a series of G1 cell cycle regulating genes. CCND1 (cyclin D1) and CDK4 were amplified or gained and the chromosomal regions containing the CDKN2 (INK4) group of CDKIs were frequently deleted. Cluster analysis showed that tumors with genomic aberrations in G1 regulating genes over-expressed E2F target genes, which regulate S and G2/M phase progression. These tumors have a poor prognosis. Our findings suggest that pharmacological inhibition of cell cycle genes might bear therapeutic promises for patients with high risk neuroblastoma.
Asunto(s)
Factores de Transcripción E2F/metabolismo , Fase G1/genética , Dosificación de Gen , Genes cdc , Neuroblastoma/genética , Aberraciones Cromosómicas , Análisis por Conglomerados , Ciclina D1/genética , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/genética , Quinasas Ciclina-Dependientes/genética , Expresión Génica , Humanos , Neuroblastoma/diagnóstico , Pronóstico , ARN Mensajero/genéticaRESUMEN
Embryonal tumor with multilayered rosettes (ETMR, previously known as ETANTR) is a highly aggressive embryonal CNS tumor, which almost exclusively affects infants and is associated with a dismal prognosis. Accurate diagnosis is of critical clinical importance because of its poor response to current treatment protocols and its distinct biology. Amplification of the miRNA cluster at 19q13.42 has been identified previously as a genetic hallmark for ETMR, but an immunohistochemistry-based assay for clinical routine diagnostics [such as INI-1 for atypical teratoid rhabdoid tumor (AT/RT)] is still lacking. In this study, we screened for an ETMR-specific marker using a gene-expression profiling dataset of more than 1,400 brain tumors and identified LIN28A as a highly specific marker for ETMR. The encoded protein binds small RNA and has been implicated in stem cell pluripotency, metabolism and tumorigenesis. Using an LIN28A specific antibody, we carried out immunohistochemical analysis of LIN28A in more than 800 childhood brain-tumor samples and confirmed its high specificity for ETMR. Strong LIN28A immunoexpression was found in all 37 ETMR samples tested, whereas focal reactivity was only present in a small (6/50) proportion of AT/RT samples. All other pediatric brain tumors were completely LIN28A-negative. In summary, we established LIN28A immunohistochemistry as a highly sensitive and specific, rapid, inexpensive diagnostic tool for routine pathological verification of ETMR.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Encefálicas/genética , Proteínas de Unión al ADN/genética , Neoplasias de Células Germinales y Embrionarias/genética , Neurópilo/metabolismo , Adolescente , Neoplasias Encefálicas/patología , Niño , Preescolar , Diagnóstico Diferencial , Perfilación de la Expresión Génica/métodos , Humanos , MicroARNs/genética , Neoplasias de Células Germinales y Embrionarias/patología , Tumores Neuroectodérmicos Primitivos/genética , Tumores Neuroectodérmicos Primitivos/patología , Neurópilo/patología , Proteínas de Unión al ARN , Tumor Rabdoide/genética , Tumor Rabdoide/patologíaRESUMEN
BACKGROUND: Neuroblastoma are pediatric tumors of the sympathetic nervous system with a poor prognosis. Apoptosis is often deregulated in cancer cells, but only a few defects in apoptotic routes have been identified in neuroblastoma. METHODS: Here we investigated genomic aberrations affecting genes of the intrinsic apoptotic pathway in neuroblastoma. We analyzed DNA profiling data (CGH and SNP arrays) and mRNA expression data of 31 genes of the intrinsic apoptotic pathway in a dataset of 88 neuroblastoma tumors using the R2 bioinformatic platform ( http://r2.amc.nl). BIRC6 was selected for further analysis as a tumor driving gene. Knockdown experiments were performed using BIRC6 lentiviral shRNA and phenotype responses were analyzed by Western blot and MTT-assays. In addition, DIABLO levels and interactions were investigated with immunofluorescence and co-immunoprecipitation. RESULTS: We observed frequent gain of the BIRC6 gene on chromosome 2, which resulted in increased mRNA expression. BIRC6 is an inhibitor of apoptosis protein (IAP), that can bind and degrade the cytoplasmic fraction of the pro-apoptotic protein DIABLO. DIABLO mRNA expression was exceptionally high in neuroblastoma but the protein was only detected in the mitochondria. Upon silencing of BIRC6 by shRNA, DIABLO protein levels increased and cells went into apoptosis. Co-immunoprecipitation confirmed direct interaction between DIABLO and BIRC6 in neuroblastoma cell lines. CONCLUSION: Our findings indicate that BIRC6 may have a potential oncogenic role in neuroblastoma by inactivating cytoplasmic DIABLO. BIRC6 inhibition may therefore provide a means for therapeutic intervention in neuroblastoma.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/genética , Neuroblastoma/genética , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis , Caspasa 9/genética , Hibridación Genómica Comparativa , Citoplasma/metabolismo , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Terapia Molecular Dirigida/métodos , Neuroblastoma/metabolismo , Polimorfismo de Nucleótido Simple , ARN Interferente Pequeño/genética , SurvivinRESUMEN
Cancer therapy frequently fails due to the emergence of resistance. Many tumors include phenotypically immature tumor cells, which have been implicated in therapy resistance. Neuroblastoma cells can adopt a lineage-committed adrenergic (ADRN) or an immature mesenchymal (MES) state. They differ in epigenetic landscape and transcription factors, and MES cells are more resistant to chemotherapy. Here we analyzed the response of MES cells to targeted drugs. Activating anaplastic lymphoma kinase (ALK) mutations are frequently found in neuroblastoma and ALK inhibitors (ALKi) are in clinical trials. ALKi treatment of ADRN neuroblastoma cells with a tumor-driving ALK mutation induced cell death. Conversely, MES cells did not express either mutant or wild-type ALK and were resistant to ALKi, and MES cells formed tumors that progressed under ALKi therapy. In assessing the role of MES cells in relapse development, TRAIL was identified to specifically induce apoptosis in MES cells and to suppress MES tumor growth. Addition of TRAIL to ALKi treatment of neuroblastoma xenografts delayed relapses in a subset of the animals, suggesting a role for MES cells in relapse formation. While ADRN cells resembled normal embryonal neuroblasts, MES cells resembled immature precursor cells, which also lacked ALK expression. Resistance to targeted drugs can therefore be an intrinsic property of immature cancer cells based on their resemblance to developmental precursors. SIGNIFICANCE: In neuroblastoma, mesenchymal tumor cells lack expression of the tumor-driving ALK oncogene and are resistant to ALKi, but dual treatment with ALKi and mesenchymal cell-targeting TRAIL delays tumor relapse.
Asunto(s)
Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Neuroblastoma/genética , Línea Celular Tumoral , Humanos , Neuroblastoma/patologíaRESUMEN
Amplification of the oncogenic transcription factor MYCN plays a major role in the pathogenesis of several pediatric cancers, including neuroblastoma, medulloblastoma, and rhabodomyosarcoma. For neuroblastoma, MYCN amplification is the most powerful genetic predictor of poor patient survival, yet the mechanism by which MYCN drives tumorigenesis is only partially understood. To gain an insight into the distribution of MYCN binding and to identify clinically relevant MYCN target genes, we performed an integrated analysis of MYCN ChIP-chip and mRNA expression using the MYCN repressible SHEP-21N neuroblastoma cell line. We hypothesized that genes exclusively MYCN bound in SHEP-21N cells over-expressing MYCN would be enriched for direct targets which contribute to the process of disease progression. Integrated analysis revealed that MYCN drives tumorigenesis predominantly as a positive regulator of target gene transcription. A high proportion of genes (24%) that are MYCN bound and up-regulated in the SHEP-21N model are significantly associated with poor overall patient survival (OS) in a set of 88 tumors. In contrast, the proportion of genes down-regulated when bound by MYCN in the SHEP-21N model and which are significantly associated with poor overall patient survival when under-expressed in primary tumors was significantly lower (5%). Gene ontology analysis determined a highly statistically significant enrichment for cell cycle related genes within the over-expressed MYCN target group which were also associated with poor OS. We conclude that the over-expression of MYCN leads to aberrant binding and over-expression of genes associated with cell cycle regulation which are significantly correlated with poor OS and MYCN amplification.
Asunto(s)
Biomarcadores de Tumor/genética , Redes Reguladoras de Genes/genética , Genes cdc/fisiología , Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Proteína Proto-Oncogénica N-Myc , Neuroblastoma/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Transition between differentiation states in development occurs swift but the mechanisms leading to epigenetic and transcriptional reprogramming are poorly understood. The pediatric cancer neuroblastoma includes adrenergic (ADRN) and mesenchymal (MES) tumor cell types, which differ in phenotype, super-enhancers (SEs) and core regulatory circuitries. These cell types can spontaneously interconvert, but the mechanism remains largely unknown. Here, we unravel how a NOTCH3 intracellular domain reprogrammed the ADRN transcriptional landscape towards a MES state. A transcriptional feed-forward circuitry of NOTCH-family transcription factors amplifies the NOTCH signaling levels, explaining the swift transition between two semi-stable cellular states. This transition induces genome-wide remodeling of the H3K27ac landscape and a switch from ADRN SEs to MES SEs. Once established, the NOTCH feed-forward loop maintains the induced MES state. In vivo reprogramming of ADRN cells shows that MES and ADRN cells are equally oncogenic. Our results elucidate a swift transdifferentiation between two semi-stable epigenetic cellular states.
Asunto(s)
Neuronas Adrenérgicas/patología , Reprogramación Celular/genética , Células Madre Mesenquimatosas/patología , Neuroblastoma/patología , Receptor Notch3/fisiología , Neuronas Adrenérgicas/metabolismo , Línea Celular Tumoral , Epigénesis Genética , Retroalimentación Fisiológica , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Células Madre Mesenquimatosas/metabolismo , Neuroblastoma/metabolismo , Receptor Notch3/genética , Receptor Notch3/metabolismoRESUMEN
Neuroblastoma and ganglioneuroma are neuroblastic tumors originating from the developing sympathetic peripheral nervous system. Ganglioneuromas are usually benign, while neuroblastomas have a variable prognosis and include very aggressive tumors. Examples exist of neuroblastomas regressing to ganglioneuromas and ganglioneuromas progressing to neuroblastomas. Little is known of the molecular differences between the tumor types. Here we report that Dickkopf-3 (DKK3), a putative extra cellular inhibitor of the Wnt/beta-catenin pathway, showed a strongly differential expression between neuroblastoma and ganglioneuroma. Microarray analyses of 109 neuroblastic tumors revealed that DKK3 is strongly expressed in ganglioneuroma but only weakly in neuroblastoma. Low DKK3 expression in neuroblastoma correlated with a poor prognosis. The expression of DKK3 in the tumor series and in neuroblastoma cell lines was inversely correlated with the expression of the MYCN oncogene. Analysis of 2 neuroblastoma cell lines with inducible activity of MYCN showed that DKK3 is down-regulated by MYCN. We subsequently generated cell lines with inducible expression of DKK3, which revealed an inhibitory effect of DKK3 on proliferation. High DKK3 expression in the benign ganglioneuromas and down-regulation of DKK3 by MYCN in neuroblastoma might contribute to the strongly different clinical behavior of both neuroblastic tumor types.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Ganglioneuroma/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neuroblastoma/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Northern Blotting , Western Blotting , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Quimiocinas , Progresión de la Enfermedad , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunoprecipitación , Estimación de Kaplan-Meier , Proteína Proto-Oncogénica N-Myc , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Transducción de SeñalRESUMEN
Mutations affecting the RAS-MAPK pathway frequently occur in relapsed neuroblastoma tumors, which suggests that activation of this pathway is associated with a more aggressive phenotype. To explore this hypothesis, we generated several model systems to define a neuroblastoma RAS-MAPK pathway signature. Activation of this pathway in primary tumors indeed correlated with poor survival and was associated with known activating mutations in ALK and other RAS-MAPK pathway genes. Integrative analysis showed that mutations in PHOX2B, CIC, and DMD were also associated with an activated RAS-MAPK pathway. Mutation of PHOX2B and deletion of CIC in neuroblastoma cell lines induced activation of the RAS-MAPK pathway. This activation was independent of phosphorylated ERK in CIC knockout systems. Furthermore, deletion of CIC caused a significant increase in tumor growth in vivo These results show that the RAS-MAPK pathway is involved in tumor progression and establish CIC as a powerful tumor suppressor that functions downstream of this pathway in neuroblastoma.Significance: This work identifies CIC as a powerful tumor suppressor affecting the RAS-MAPK pathway in neuroblastoma and reinforces the importance of mutation-driven activation of this pathway in cancer. Cancer Res; 78(21); 6297-307. ©2018 AACR.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Neuroblastoma/genética , Proteínas Represoras/genética , Animales , Línea Celular Tumoral , Análisis por Conglomerados , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes ras , Genoma Humano , Genómica , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Ratones Noqueados , Ratones Desnudos , Mutación , Recurrencia Local de Neoplasia/genética , Trasplante de Neoplasias , Neuroblastoma/patología , Fenotipo , Fosforilación , Pronóstico , Proteínas Represoras/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Resultado del TratamientoRESUMEN
The c-Myc and MYCN oncogenes strongly induce cell proliferation. Although a limited series of cell cycle genes were found to be induced by the myc transcription factors, it is still unclear how they mediate the proliferative phenotype. We therefore analysed a neuroblastoma cell line with inducible MYCN expression. We found that all members of the minichromosome maintenance complex (MCM2-7) and MCM8 and MCM10 were up-regulated by MYCN. Expression profiling of 110 neuroblastoma tumours revealed that these genes strongly correlated with MYCN expression in vivo. Extensive chromatin immunoprecipitation experiments were performed to investigate whether the MCM genes were primary MYCN targets. MYCN was bound to the proximal promoters of the MCM2 to -8 genes. These data suggest that MYCN stimulates the expression of not only MCM7, which is a well defined MYCN target gene, but also of the complete minichromosome maintenance complex.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Genes myc , Neuroblastoma/genética , Proteínas de Ciclo Celular/biosíntesis , Diferenciación Celular , Proliferación Celular , Proteínas de Unión al ADN/biosíntesis , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Neuroblastoma/metabolismo , Proteínas NuclearesRESUMEN
Neuroblastoma and other pediatric tumors show a paucity of gene mutations, which has sparked an interest in their epigenetic regulation. Several tumor types include phenotypically divergent cells, resembling cells from different lineage development stages. It has been proposed that super-enhancer-associated transcription factor (TF) networks underlie lineage identity, but the role of these enhancers in intratumoral heterogeneity is unknown. Here we show that most neuroblastomas include two types of tumor cells with divergent gene expression profiles. Undifferentiated mesenchymal cells and committed adrenergic cells can interconvert and resemble cells from different lineage differentiation stages. ChIP-seq analysis of isogenic pairs of mesenchymal and adrenergic cells identified a distinct super-enhancer landscape and super-enhancer-associated TF network for each cell type. Expression of the mesenchymal TF PRRX1 could reprogram the super-enhancer and mRNA landscapes of adrenergic cells toward a mesenchymal state. Mesenchymal cells were more chemoresistant in vitro and were enriched in post-therapy and relapse tumors. Two super-enhancer-associated TF networks, which probably mediate lineage control in normal development, thus dominate epigenetic control of neuroblastoma and shape intratumoral heterogeneity.
Asunto(s)
Diferenciación Celular/genética , Epigénesis Genética , Neuroblastoma/genética , Neuroblastoma/patología , Antígeno AC133/genética , Neuronas Adrenérgicas/citología , Línea Celular Tumoral , Linaje de la Célula , Proteínas de Homeodominio/genética , Humanos , Mesodermo/citología , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
OTX1 and OTX2 are transcription factors with an essential role in the development of the cerebellum. We previously described a high OTX2 expression in medulloblastoma. Here, we analyzed amplification and mRNA expression of OTX1 and OTX2 in a series of human medulloblastomas. In addition, OTX2 protein expression was analyzed on tissue arrays. The OTX2 gene was amplified in the medulloblastoma cell line D425 and mRNA and protein data showed expression in 114 of 152 medulloblastomas (75%), but not in postnatal cerebellum. Northern blot (n = 10) and reverse transcriptase-polymerase chain reaction (n = 45) analyses demonstrated that virtually all medulloblastomas expressed OTX1, OTX2, or both. OTX2 mRNA expression correlated with a classic medulloblastoma histology (29 of 34 cases), whereas expression of OTX1 mRNA only was correlated with a nodular/desmoplastic histology (9 of 11 cases). Immunohistochemical analysis of a series of classic medulloblastomas detected OTX2 protein expression in 83 of 107 (78%) cases. The OTX2-positive tumors of this series were preferentially localized in the vermis of the cerebellum, whereas OTX2-negative tumors more frequently occurred in the hemispheres of the cerebellum. In addition, OTX2-positive tumors were mainly found in children, but OTX2-negative tumors occurred in 2 patient groups: very young patients (<5 years) and adults (>20 years). Nodular/desmoplastic medulloblastomas are thought to arise from the external granular layer (EGL). However, it is unclear whether classic medulloblastomas also originate from the EGL or from the ventricular matrix. Analysis of human fetal brain showed OTX2 protein expression in a small number of presumptive neuronal precursor cells of the EGL, but not in precursor cells of the ventricular matrix. Combined with data from rodents, our results therefore suggest that both nodular/desmoplastic and at least part of the classic medulloblastomas originate from cells of the EGL, albeit from different regions.
Asunto(s)
Meduloblastoma , Factores de Transcripción Otx/metabolismo , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Cerebelo/crecimiento & desarrollo , Cerebelo/metabolismo , Cerebelo/patología , Niño , Preescolar , Femenino , Feto/anatomía & histología , Feto/fisiología , Humanos , Lactante , Recién Nacido , Masculino , Meduloblastoma/clasificación , Meduloblastoma/genética , Meduloblastoma/patología , Meduloblastoma/fisiopatología , Persona de Mediana Edad , Factores de Transcripción Otx/genética , ARN Mensajero/metabolismoRESUMEN
Atypical teratoid/rhabdoid tumor (ATRT) is one of the most common brain tumors in infants. Although the prognosis of ATRT patients is poor, some patients respond favorably to current treatments, suggesting molecular inter-tumor heterogeneity. To investigate this further, we genetically and epigenetically analyzed 192 ATRTs. Three distinct molecular subgroups of ATRTs, associated with differences in demographics, tumor location, and type of SMARCB1 alterations, were identified. Whole-genome DNA and RNA sequencing found no recurrent mutations in addition to SMARCB1 that would explain the differences between subgroups. Whole-genome bisulfite sequencing and H3K27Ac chromatin-immunoprecipitation sequencing of primary tumors, however, revealed clear differences, leading to the identification of subgroup-specific regulatory networks and potential therapeutic targets.
Asunto(s)
Epigénesis Genética/genética , Tumor Rabdoide/genética , Teratoma/genética , Neoplasias Encefálicas/genética , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Humanos , Mutación/genética , Proteína SMARCB1 , Factores de Transcripción/genéticaRESUMEN
Whole-genome sequencing detected structural rearrangements of TERT in 17 of 75 high-stage neuroblastomas, with five cases resulting from chromothripsis. Rearrangements were associated with increased TERT expression and targeted regions immediately up- and downstream of TERT, positioning a super-enhancer close to the breakpoints in seven cases. TERT rearrangements (23%), ATRX deletions (11%) and MYCN amplifications (37%) identify three almost non-overlapping groups of high-stage neuroblastoma, each associated with very poor prognosis.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Reordenamiento Génico , Neuroblastoma/genética , Neuroblastoma/patología , Telomerasa/genética , Telómero/genética , ADN Helicasas/genética , Amplificación de Genes , Eliminación de Gen , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Proteína Proto-Oncogénica N-Myc , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Proteína Nuclear Ligada al Cromosoma XRESUMEN
Blastemal histology in chemotherapy-treated pediatric Wilms tumors (nephroblastoma) is associated with adverse prognosis. To uncover the underlying tumor biology and find therapeutic leads for this subgroup, we analyzed 58 blastemal type Wilms tumors by exome and transcriptome sequencing and validated our findings in a large replication cohort. Recurrent mutations included a hotspot mutation (Q177R) in the homeo-domain of SIX1 and SIX2 in tumors with high proliferative potential (18.1% of blastemal cases); mutations in the DROSHA/DGCR8 microprocessor genes (18.2% of blastemal cases); mutations in DICER1 and DIS3L2; and alterations in IGF2, MYCN, and TP53, the latter being strongly associated with dismal outcome. DROSHA and DGCR8 mutations strongly altered miRNA expression patterns in tumors, which was functionally validated in cell lines expressing mutant DROSHA.
Asunto(s)
Proteínas de Homeodominio/genética , Proteínas del Tejido Nervioso/genética , Proteínas de Unión al ARN/genética , Ribonucleasa III/genética , Tumor de Wilms/genética , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/biosíntesis , Mutación , Proteínas de Neoplasias/biosíntesis , Transcriptoma , Tumor de Wilms/patologíaRESUMEN
Ependymal tumors across age groups are currently classified and graded solely by histopathology. It is, however, commonly accepted that this classification scheme has limited clinical utility based on its lack of reproducibility in predicting patients' outcome. We aimed at establishing a uniform molecular classification using DNA methylation profiling. Nine molecular subgroups were identified in a large cohort of 500 tumors, 3 in each anatomical compartment of the CNS, spine, posterior fossa, supratentorial. Two supratentorial subgroups are characterized by prototypic fusion genes involving RELA and YAP1, respectively. Regarding clinical associations, the molecular classification proposed herein outperforms the current histopathological classification and thus might serve as a basis for the next World Health Organization classification of CNS tumors.