RESUMO
Neuroblastoma (NB) is one of the primary causes of death for pediatric malignancies. Given the high heterogeneity in NB's mutation landscape, optimizing individualized therapies is still challenging. In the context of genomic alterations, MYCN amplification is the most correlated event with poor outcomes. MYCN is involved in the regulation of several cellular mechanisms, including cell cycle. Thus, studying the influence of MYCN overexpression in the G1/S transition checkpoint of the cell cycle may unveil novel druggable targets for the development of personalized therapeutical approaches. Here, we show that high expression of E2F3 and MYCN correlate with poor prognosis in NB despite the RB1 mRNA levels. Moreover, we demonstrate through luciferase reporter assays that MYCN bypasses RB function by incrementing E2F3-responsive promoter activity. We showed that MYCN overexpression leads to RB inactivation by inducing RB hyperphosphorylation during the G1 phase through cell cycle synchronization experiments. Moreover, we generated two MYCN-amplified NB cell lines conditionally knockdown (cKD) for the RB1 gene through a CRISPRi approach. Indeed, RB KD did not affect cell proliferation, whereas cell proliferation was strongly influenced when a non-phosphorylatable RB mutant was expressed. This finding revealed the dispensable role of RB in regulating MYCN-amplified NB's cell cycle. The described genetic interaction between MYCN and RB1 provides the rationale for using cyclin/CDK complexes inhibitors in NBs carrying MYCN amplification and relatively high levels of RB1 expression.
Assuntos
Neuroblastoma , Criança , Humanos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Neuroblastoma (NB) is one of the most frequently occurring neurogenic extracranial solid cancers in childhood and infancy. Over the years, many pieces of evidence suggested that NB development is controlled by gene expression dysregulation. These unleashed programs that outline NB cancer cells make them highly dependent on specific tuning of gene expression, which can act co-operatively to define the differentiation state, cell identity, and specialized functions. The peculiar regulation is mainly caused by genetic and epigenetic alterations, resulting in the dependency on a small set of key master transcriptional regulators as the convergence point of multiple signalling pathways. In this review, we provide a comprehensive blueprint of transcriptional regulation bearing NB initiation and progression, unveiling the complexity of novel oncogenic and tumour suppressive regulatory networks of this pathology. Furthermore, we underline the significance of multi-target therapies against these hallmarks, showing how novel approaches, together with chemotherapy, surgery, or radiotherapy, can have substantial antineoplastic effects, disrupting a wide variety of tumorigenic pathways through combinations of different treatments.
Assuntos
Regulação Neoplásica da Expressão Gênica , Genes myc , Terapia de Alvo Molecular , Neuroblastoma/genética , Animais , Instabilidade Cromossômica , Epigênese Genética , Redes Reguladoras de Genes , Predisposição Genética para Doença , Humanos , Neuroblastoma/tratamento farmacológicoRESUMO
MYCN oncogene amplification is frequently observed in aggressive childhood neuroblastoma. Using an unbiased large-scale mutagenesis screen in neuroblastoma-prone transgenic mice, we identify a single germline point mutation in the transcriptional corepressor Runx1t1, which abolishes MYCN-driven tumorigenesis. This loss-of-function mutation disrupts a highly conserved zinc finger domain within Runx1t1. Deletion of one Runx1t1 allele in an independent Runx1t1 knockout mouse model is also sufficient to prevent MYCN-driven neuroblastoma development, and reverse ganglia hyperplasia, a known pre-requisite for tumorigenesis. Silencing RUNX1T1 in human neuroblastoma cells decreases colony formation in vitro, and inhibits tumor growth in vivo. Moreover, RUNX1T1 knockdown inhibits the viability of PAX3-FOXO1 fusion-driven rhabdomyosarcoma and MYC-driven small cell lung cancer cells. Despite the role of Runx1t1 in MYCN-driven tumorigenesis neither gene directly regulates the other. We show RUNX1T1 forms part of a transcriptional LSD1-CoREST3-HDAC repressive complex recruited by HAND2 to enhancer regions to regulate chromatin accessibility and cell-fate pathway genes.
Assuntos
Carcinogênese , Proteína Proto-Oncogênica N-Myc , Neuroblastoma , Animais , Humanos , Camundongos , Carcinogênese/genética , Linhagem Celular Tumoral , Proteínas Correpressoras/metabolismo , Proteínas Correpressoras/genética , Regulação Neoplásica da Expressão Gênica , Histona Desmetilases/metabolismo , Histona Desmetilases/genética , Camundongos Knockout , Camundongos Transgênicos , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
The molecular mechanisms and gene regulatory networks sustaining cell proliferation in neuroblastoma (NBL) cells are still not fully understood. In this tumor context, it has been proposed that anti-proliferative drugs, such as the pan-HDAC inhibitor panobinostat, could be tested to mitigate tumor progression. Here, we set out to investigate the effects of panobinostat treatment at the unprecedented resolution offered by single-cell sequencing. We identified a global senescence signature paired with reduction in proliferation in treated Kelly cells and more isolated transcriptional responses compatible with early neuronal differentiation. Using master regulator analysis, we identified BAZ1A, HCFC1, MAZ, and ZNF146 as the transcriptional regulators most significantly repressed by panobinostat. Experimental silencing of these transcription factors (TFs) confirmed their role in sustaining NBL cell proliferation in vitro.
Assuntos
Ácidos Hidroxâmicos , Neuroblastoma , Humanos , Panobinostat/farmacologia , Panobinostat/uso terapêutico , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Indóis/farmacologia , Indóis/uso terapêutico , Apoptose , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Proteínas Cromossômicas não HistonaRESUMO
Autism Spectrum Disorder (ASD) is a highly heterogeneous neuropsychiatric disorder with a strong genetic component. The genetic architecture is complex, consisting of a combination of common low-risk and more penetrant rare variants. Voltage-gated calcium channels (VGCCs or Cav) genes have been implicated as high-confidence susceptibility genes for ASD, in accordance with the relevant role of calcium signaling in neuronal function. In order to further investigate the involvement of VGCCs rare variants in ASD susceptibility, we performed whole genome sequencing analysis in a cohort of 105 families, composed of 124 ASD individuals, 210 parents and 58 unaffected siblings. We identified 53 rare inherited damaging variants in Cav genes, including genes coding for the principal subunit and genes coding for the auxiliary subunits, in 40 ASD families. Interestingly, biallelic rare damaging missense variants were detected in the CACNA1H gene, coding for the T-type Cav3.2 channel, in ASD probands from two different families. Thus, to clarify the role of these CACNA1H variants on calcium channel activity we performed electrophysiological analysis using whole-cell patch clamp technology. Three out of four tested variants were shown to mildly affect Cav3.2 channel current density and activation properties, possibly leading to a dysregulation of intracellular Ca2+ ions homeostasis, thus altering calcium-dependent neuronal processes and contributing to ASD etiology in these families. Our results provide further support for the role of CACNA1H in neurodevelopmental disorders and suggest that rare CACNA1H variants may be involved in ASD development, providing a high-risk genetic background.
RESUMO
Neuroblastoma (NB) is a childhood cancer arising from sympatho-adrenal neural crest cells. MYCN amplification is found in half of high-risk NB patients; however, no available therapies directly target MYCN. Using multi-dimensional metabolic profiling in MYCN expression systems and primary patient tumors, we comprehensively characterized the metabolic landscape driven by MYCN in NB. MYCN amplification leads to glycerolipid accumulation by promoting fatty acid (FA) uptake and biosynthesis. We found that cells expressing amplified MYCN depend highly on FA uptake for survival. Mechanistically, MYCN directly upregulates FA transport protein 2 (FATP2), encoded by SLC27A2. Genetic depletion of SLC27A2 impairs NB survival, and pharmacological SLC27A2 inhibition selectively suppresses tumor growth, prolongs animal survival, and exerts synergistic anti-tumor effects when combined with conventional chemotherapies in multiple preclinical NB models. This study identifies FA uptake as a critical metabolic dependency for MYCN-amplified tumors. Inhibiting FA uptake is an effective approach for improving current treatment regimens.
Assuntos
Ácidos Graxos , Neuroblastoma , Animais , Linhagem Celular Tumoral , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/metabolismoRESUMO
MYCN activation is a hallmark of advanced neuroblastoma (NB) and a known master regulator of metabolic reprogramming, favoring NB adaptation to its microenvironment. We found that the expression of the main regulators of the molecular clock loops is profoundly disrupted in MYCN-amplified NB patients, and this disruption independently predicts poor clinical outcome. MYCN induces the expression of clock repressors and downregulates the one of clock activators by directly binding to their promoters. Ultimately, MYCN attenuates the molecular clock by suppressing BMAL1 expression and oscillation, thereby promoting cell survival. Reestablishment of the activity of the clock activator RORα via its genetic overexpression and its stimulation through the agonist SR1078, restores BMAL1 expression and oscillation, effectively blocks MYCN-mediated tumor growth and de novo lipogenesis, and sensitizes NB tumors to conventional chemotherapy. In conclusion, reactivation of RORα could serve as a therapeutic strategy for MYCN-amplified NBs by blocking the dysregulation of molecular clock and cell metabolism mediated by MYCN.
Assuntos
Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Fatores de Transcrição ARNTL/metabolismo , Animais , Antineoplásicos/uso terapêutico , Benzamidas/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Humanos , Lipogênese/fisiologia , Camundongos , Regiões Promotoras Genéticas/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neuroblastoma (NB) arises from oncogenic disruption of neural crest (NC) differentiation. Treatment with retinoic acid (RA) to induce differentiation has improved survival in some NB patients, but not all patients respond, and most NBs eventually develop resistance to RA. Loss of the chromatin modifier chromatin assembly factor 1 subunit p150 (CHAF1A) promotes NB cell differentiation; however, the mechanism by which CHAF1A drives NB oncogenesis has remained unexplored. This study shows that CHAF1A gain-of-function supports cell malignancy, blocks neuronal differentiation in three models (zebrafish NC, human NC, and human NB), and promotes NB oncogenesis. Mechanistically, CHAF1A upregulates polyamine metabolism, which blocks neuronal differentiation and promotes cell cycle progression. Targeting polyamine synthesis promotes NB differentiation and enhances the anti-tumor activity of RA. The authors' results provide insight into the mechanisms that drive NB oncogenesis and suggest a rapidly translatable therapeutic approach (DFMO plus RA) to enhance the clinical efficacy of differentiation therapy in NB patients.
Assuntos
Carcinogênese/metabolismo , Diferenciação Celular/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Neuroblastoma/metabolismo , Neurônios/metabolismo , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Fator 1 de Modelagem da Cromatina/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Camundongos Nus , Neuroblastoma/genética , Peixe-ZebraRESUMO
Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1, results in genotypes wildtype GG, and variants AG/AA. CRISPR-cas9 technology was used to investigate the effects of AG clones from wildtype MYCN-amplified SK-N-BE(2)-C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and preferentially bound the G allele over the A. Two neuroblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with improved survival, while poor prognosis was associated with the GG genotype and AG/GG genotypes in MYCN-amplified and non-amplified patients, respectively. These effects were lost in the GWAS cohort. We have demonstrated that the ODC1 G316A polymorphism has functional significance in neuroblastoma and is subject to allele-specific regulation by the MYCN oncoprotein.
RESUMO
Histone deacetylases (HDACs) are evolutionary conserved enzymes which operate by removing acetyl groups from histones and other protein regulatory factors, with functional consequences on chromatin remodeling and gene expression profiles. We provide here a review on the recent knowledge accrued on the zinc-dependent HDAC protein family across different species, tissues, and human pathologies, specifically focusing on the role of HDAC inhibitors as anti-cancer agents. We will investigate the chemical specificity of different HDACs and discuss their role in the human interactome as members of chromatin-binding and regulatory complexes.
Assuntos
Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/genética , Neoplasias/genética , Fatores de Transcrição/genética , Antineoplásicos/uso terapêutico , Histonas/genética , Humanos , Neoplasias/terapia , Especificidade por Substrato/genéticaRESUMO
Chromosome 17q21-ter is commonly gained in neuroblastoma, but it is unclear which gene in the region is important for tumorigenesis. The JMJD6 gene at 17q21-ter activates gene transcription. Here we show that JMJD6 forms protein complexes with N-Myc and BRD4, and is important for E2F2, N-Myc and c-Myc transcription. Knocking down JMJD6 reduces neuroblastoma cell proliferation and survival in vitro and tumor progression in mice, and high levels of JMJD6 expression in human neuroblastoma tissues independently predict poor patient prognosis. In addition, JMJD6 gene is associated with transcriptional super-enhancers. Combination therapy with the CDK7/super-enhancer inhibitor THZ1 and the histone deacetylase inhibitor panobinostat synergistically reduces JMJD6, E2F2, N-Myc, c-Myc expression, induces apoptosis in vitro and leads to neuroblastoma tumor regression in mice, which are significantly reversed by forced JMJD6 over-expression. Our findings therefore identify JMJD6 as a neuroblastoma tumorigenesis factor, and the combination therapy as a treatment strategy.
Assuntos
Histona Desmetilases com o Domínio Jumonji/metabolismo , Neuroblastoma/tratamento farmacológico , Receptores de Superfície Celular/metabolismo , Animais , Apoptose/efeitos dos fármacos , Carcinogênese , Proliferação de Células/efeitos dos fármacos , Fator de Transcrição E2F2/genética , Fator de Transcrição E2F2/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases/administração & dosagem , Humanos , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/fisiopatologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genéticaRESUMO
MYCN is a major driver for the childhood cancer, neuroblastoma, however, there are no inhibitors of this target. Enhanced MYCN protein stability is a key component of MYCN oncogenesis and is maintained by multiple feedforward expression loops involving MYCN transactivation target genes. Here, we reveal the oncogenic role of a novel MYCN target and binding protein, proliferation-associated 2AG4 (PA2G4). Chromatin immunoprecipitation studies demonstrated that MYCN occupies the PA2G4 gene promoter, stimulating transcription. Direct binding of PA2G4 to MYCN protein blocked proteolysis of MYCN and enhanced colony formation in a MYCN-dependent manner. Using molecular modeling, surface plasmon resonance, and mutagenesis studies, we mapped the MYCN-PA2G4 interaction site to a 14 amino acid MYCN sequence and a surface crevice of PA2G4. Competitive chemical inhibition of the MYCN-PA2G4 protein-protein interface had potent inhibitory effects on neuroblastoma tumorigenesis in vivo. Treated tumors showed reduced levels of both MYCN and PA2G4. Our findings demonstrate a critical role for PA2G4 as a cofactor in MYCN-driven neuroblastoma and highlight competitive inhibition of the PA2G4-MYCN protein binding as a novel therapeutic strategy in the disease. SIGNIFICANCE: Competitive chemical inhibition of the PA2G4-MYCN protein interface provides a basis for drug design of small molecules targeting MYC and MYCN-binding partners in malignancies driven by MYC family oncoproteins.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína Proto-Oncogênica N-Myc/genética , Proteínas Oncogênicas/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais/genética , Animais , Animais Geneticamente Modificados , Carcinogênese/genética , Linhagem Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina/métodos , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neuroblastoma/genética , Peixe-ZebraRESUMO
Amplification of the MYCN oncogene is associated with an aggressive phenotype and poor outcome in childhood neuroblastoma. Polyamines are highly regulated essential cations that are frequently elevated in cancer cells, and the rate-limiting enzyme in polyamine synthesis, ornithine decarboxylase 1 (ODC1), is a direct transcriptional target of MYCN. Treatment of neuroblastoma cells with the ODC1 inhibitor difluoromethylornithine (DFMO), although a promising therapeutic strategy, is only partially effective at impeding neuroblastoma cell growth due to activation of compensatory mechanisms resulting in increased polyamine uptake from the surrounding microenvironment. In this study, we identified solute carrier family 3 member 2 (SLC3A2) as the key transporter involved in polyamine uptake in neuroblastoma. Knockdown of SLC3A2 in neuroblastoma cells reduced the uptake of the radiolabeled polyamine spermidine, and DFMO treatment increased SLC3A2 protein. In addition, MYCN directly increased polyamine synthesis and promoted neuroblastoma cell proliferation by regulating SLC3A2 and other regulatory components of the polyamine pathway. Inhibiting polyamine uptake with the small-molecule drug AMXT 1501, in combination with DFMO, prevented or delayed tumor development in neuroblastoma-prone mice and extended survival in rodent models of established tumors. Our findings suggest that combining AMXT 1501 and DFMO with standard chemotherapy might be an effective strategy for treating neuroblastoma.
Assuntos
Progressão da Doença , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Poliaminas/metabolismo , Animais , Vias Biossintéticas/genética , Linhagem Celular Tumoral , Estudos de Coortes , Modelos Animais de Doenças , Amplificação de Genes , Regulação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Análise Multivariada , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Prognóstico , Modelos de Riscos Proporcionais , Análise de Sobrevida , Resultado do TratamentoRESUMO
The majority of patients with neuroblastoma due to MYCN oncogene amplification and consequent N-Myc oncoprotein over-expression die of the disease. Here our analyses of RNA sequencing data identify the long noncoding RNA lncNB1 as one of the transcripts most over-expressed in MYCN-amplified, compared with MYCN-non-amplified, human neuroblastoma cells and also the most over-expressed in neuroblastoma compared with all other cancers. lncNB1 binds to the ribosomal protein RPL35 to enhance E2F1 protein synthesis, leading to DEPDC1B gene transcription. The GTPase-activating protein DEPDC1B induces ERK protein phosphorylation and N-Myc protein stabilization. Importantly, lncNB1 knockdown abolishes neuroblastoma cell clonogenic capacity in vitro and leads to neuroblastoma tumor regression in mice, while high levels of lncNB1 and RPL35 in human neuroblastoma tissues predict poor patient prognosis. This study therefore identifies lncNB1 and its binding protein RPL35 as key factors for promoting E2F1 protein synthesis, N-Myc protein stability and N-Myc-driven oncogenesis, and as therapeutic targets.
Assuntos
Carcinogênese/genética , RNA Longo não Codificante/metabolismo , Proteínas Ribossômicas/metabolismo , Animais , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Fator de Transcrição E2F1/metabolismo , Feminino , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/genética , Neuroblastoma/patologia , Prognóstico , Biossíntese de Proteínas , Estabilidade Proteica , RNA Longo não Codificante/genética , Transcrição Gênica , Regulação para Cima/genéticaRESUMO
[This corrects the article DOI: 10.18632/oncotarget.24859.].
RESUMO
Naphthenic acids (NAs) are an important group of toxic organic compounds naturally occurring in hydrocarbon deposits. This work shows that Rhodococcus aetherivorans BCP1 cells not only utilize a mixture of eight different NAs (8XNAs) for growth but they are also capable of marked degradation of two model NAs, cyclohexanecarboxylic acid (CHCA) and cyclopentanecarboxylic acid (CPCA) when supplied at concentrations from 50 to 500 mgL-1. The growth curves of BCP1 on 8XNAs, CHCA, and CPCA showed an initial lag phase not present in growth on glucose, which presumably was related to the toxic effects of NAs on the cell membrane permeability. BCP1 cell adaptation responses that allowed survival on NAs included changes in cell morphology, production of intracellular bodies and changes in fatty acid composition. Transmission electron microscopy (TEM) analysis of BCP1 cells grown on CHCA or CPCA showed a slight reduction in the cell size, the production of EPS-like material and intracellular electron-transparent and electron-dense inclusion bodies. The electron-transparent inclusions increased in the amount and size in NA-grown BCP1 cells under nitrogen limiting conditions and contained storage lipids as suggested by cell staining with the lipophilic Nile Blue A dye. Lipidomic analyses revealed significant changes with increases of methyl-branched (MBFA) and polyunsaturated fatty acids (PUFA) examining the fatty acid composition of NAs-growing BCP1 cells. PUFA biosynthesis is not usual in bacteria and, together with MBFA, can influence structural and functional processes with resulting effects on cell vitality. Finally, through the use of RT (Reverse Transcription)-qPCR, a gene cluster (chcpca) was found to be transcriptionally induced during the growth on CHCA and CPCA. Based on the expression and bioinformatics results, the predicted products of the chcpca gene cluster are proposed to be involved in aerobic NA degradation in R. aetherivorans BCP1. This study provides first insights into the genetic and metabolic mechanisms allowing a Rhodococcus strain to aerobically degrade NAs.
RESUMO
The MYC oncogenes and p53 have opposing yet interrelated roles in normal development and tumorigenesis. How MYCN expression alters the biology and clinical responsiveness of pediatric neuroblastoma remains poorly defined. Neuroblastoma is p53 wild type at diagnosis and repression of p53 signaling is required for tumorigenesis. Here, we tested the hypothesis that MYCN amplification alters p53 transcriptional activity in neuroblastoma. Interestingly, we found that MYCN directly binds to the tetrameric form of p53 at its C-terminal domain, and this interaction is independent of MYCN/MAX heterodimer formation. Chromatin analysis of MYCN and p53 targets reveals dramatic changes in binding, as well as co-localization of the MYCN-p53 complex at p53-REs and E-boxes of genes critical to DNA damage responses and cell cycle progression. RNA sequencing studies show that MYCN-p53 co-localization significantly modulated the expression of p53 target genes. Furthermore, MYCN-p53 interaction leads to regulation of alternative p53 targets not regulated in the presence of low MYCN levels. These novel targets include a number of genes involved in lipid metabolism, DNA repair, and apoptosis. Taken together, our findings demonstrate a novel oncogenic role of MYCN as a transcriptional co-regulator of p53 in high-risk MYCN amplified neuroblastoma. Targeting this novel oncogenic function of MYCN may enhance p53-mediated responses and sensitize MYCN amplified tumors to chemotherapy.
RESUMO
Childhood neuroblastoma, a disease of the sympathetic nervous system, is the most common solid tumour of infancy, remarkably refractory to therapeutic treatments. One of the most powerful independent prognostic indicators for this disease is the amplification of the MYCN oncogene, which occurs at high levels in approximately 25% of neuroblastomas. Interestingly, amplification and not just expression of MYCN has a strong prognostic value, although this fact appears quite surprising as MYCN is a transcription factor that requires dimerising with its partner MAX, to exert its function. This observation greatly suggests that the role of MYCN in neuroblastoma should be examined in the context of MAX expression. In this report, we show that, in contrast to what is found in normal cells, MAX expression is significantly different among primary NBs, and that its level appears to correlate with the clinical outcome of the disease. Importantly, controlled modulation of MAX expression in neuroblastoma cells with different extents of MYCN amplification, demonstrates that MAX can instruct gene transcription programs that either reinforce or weaken the oncogenic process enacted by MYCN. In general, our work illustrates that it is the MAX to MYCN ratio that can account for tumour progression and clinical outcome in neuroblastoma and proposes that such a ratio should be considered as an important criterion to the design and development of anti-MYCN therapies.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Espaço Intracelular/metabolismo , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/patologia , Apoptose/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Células HEK293 , Humanos , Invasividade Neoplásica , Neuroblastoma/genética , Neurônios/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Resultado do TratamentoRESUMO
Myc oncoproteins exert tumorigenic effects by regulating expression of target oncogenes. Histone H3 lysine 79 (H3K79) methylation at Myc-responsive elements of target gene promoters is a strict prerequisite for Myc-induced transcriptional activation, and DOT1L is the only known histone methyltransferase that catalyzes H3K79 methylation. Here, we show that N-Myc upregulates DOT1L mRNA and protein expression by binding to the DOT1L gene promoter. shRNA-mediated depletion of DOT1L reduced mRNA and protein expression of N-Myc target genes ODC1 and E2F2 DOT1L bound to the Myc Box II domain of N-Myc protein, and knockdown of DOT1L reduced histone H3K79 methylation and N-Myc protein binding at the ODC1 and E2F2 gene promoters and reduced neuroblastoma cell proliferation. Treatment with the small-molecule DOT1L inhibitor SGC0946 reduced H3K79 methylation and proliferation of MYCN gene-amplified neuroblastoma cells. In mice xenografts of neuroblastoma cells stably expressing doxycycline-inducible DOT1L shRNA, ablating DOT1L expression with doxycycline significantly reduced ODC1 and E2F2 expression, reduced tumor progression, and improved overall survival. In addition, high levels of DOT1L gene expression in human neuroblastoma tissues correlated with high levels of MYCN, ODC1, and E2F2 gene expression and independently correlated with poor patient survival. Taken together, our results identify DOT1L as a novel cofactor in N-Myc-mediated transcriptional activation of target genes and neuroblastoma oncogenesis. Furthermore, they characterize DOT1L inhibitors as novel anticancer agents against MYCN-amplified neuroblastoma. Cancer Res; 77(9); 2522-33. ©2017 AACR.
Assuntos
Metiltransferases/genética , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Transcrição Gênica , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Inibidores Enzimáticos/administração & dosagem , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/genética , Humanos , Metiltransferases/antagonistas & inibidores , Camundongos , Proteína Proto-Oncogênica N-Myc/biossíntese , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Regiões Promotoras GenéticasRESUMO
Amplification of the MYCN oncogene, a member of the MYC family of transcriptional regulators, is one of the most powerful prognostic markers identified for poor outcome in neuroblastoma, the most common extracranial solid cancer in childhood. While MYCN has been established as a key driver of malignancy in neuroblastoma, the underlying molecular mechanisms are poorly understood. Transcription factor activating enhancer binding protein-4 (TFAP4) has been reported to be a direct transcriptional target of MYC. We show for the first time that high expression of TFAP4 in primary neuroblastoma patients is associated with poor clinical outcome. siRNA-mediated suppression of TFAP4 in MYCN-expressing neuroblastoma cells led to inhibition of cell proliferation and migration. Chromatin immunoprecipitation assay demonstrated that TFAP4 expression is positively regulated by MYCN. Microarray analysis identified genes regulated by both MYCN and TFAP4 in neuroblastoma cells, including Phosphoribosyl-pyrophosphate synthetase-2 (PRPS2) and Syndecan-1 (SDC1), which are involved in cancer cell proliferation and metastasis. Overall this study suggests a regulatory circuit in which MYCN by elevating TFAP4 expression, cooperates with it to control a specific set of genes involved in tumor progression. These findings highlight the existence of a MYCN-TFAP4 axis in MYCN-driven neuroblastoma as well as identifying potential therapeutic targets for aggressive forms of this disease.