RESUMEN
Neuroblastoma is a childhood cancer of the sympathetic nervous system that accounts for approximately 10% of all paediatric oncology deaths. To identify genetic risk factors for neuroblastoma, we performed a genome-wide association study (GWAS) on 2,251 patients and 6,097 control subjects of European ancestry from four case series. Here we report a significant association within LIM domain only 1 (LMO1) at 11p15.4 (rs110419, combined P = 5.2 × 10(-16), odds ratio of risk allele = 1.34 (95% confidence interval 1.25-1.44)). The signal was enriched in the subset of patients with the most aggressive form of the disease. LMO1 encodes a cysteine-rich transcriptional regulator, and its paralogues (LMO2, LMO3 and LMO4) have each been previously implicated in cancer. In parallel, we analysed genome-wide DNA copy number alterations in 701 primary tumours. We found that the LMO1 locus was aberrant in 12.4% through a duplication event, and that this event was associated with more advanced disease (P < 0.0001) and survival (P = 0.041). The germline single nucleotide polymorphism (SNP) risk alleles and somatic copy number gains were associated with increased LMO1 expression in neuroblastoma cell lines and primary tumours, consistent with a gain-of-function role in tumorigenesis. Short hairpin RNA (shRNA)-mediated depletion of LMO1 inhibited growth of neuroblastoma cells with high LMO1 expression, whereas forced expression of LMO1 in neuroblastoma cells with low LMO1 expression enhanced proliferation. These data show that common polymorphisms at the LMO1 locus are strongly associated with susceptibility to developing neuroblastoma, but also may influence the likelihood of further somatic alterations at this locus, leading to malignant progression.
Asunto(s)
Proteínas de Unión al ADN/genética , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo , Neuroblastoma/genética , Oncogenes/genética , Factores de Transcripción/genética , Alelos , Línea Celular Tumoral , Proliferación Celular , Cromosomas Humanos Par 11/genética , Variaciones en el Número de Copia de ADN/genética , Progresión de la Enfermedad , Europa (Continente)/etnología , Duplicación de Gen/genética , Regulación Neoplásica de la Expresión Génica/genética , Genoma Humano/genética , Genómica , Genotipo , Humanos , Proteínas con Dominio LIM , Neuroblastoma/patología , Oportunidad Relativa , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Tasa de SupervivenciaRESUMEN
Neuroblastoma is a childhood cancer that is often fatal despite intense multimodality therapy. In an effort to identify therapeutic targets for this disease, we performed a comprehensive loss-of-function screen of the protein kinome. Thirty kinases showed significant cellular cytotoxicity when depleted, with loss of the cell cycle checkpoint kinase 1 (CHK1/CHEK1) being the most potent. CHK1 mRNA expression was higher in MYC-Neuroblastoma-related (MYCN)-amplified (P < 0.0001) and high-risk (P = 0.03) tumors. Western blotting revealed that CHK1 was constitutively phosphorylated at the ataxia telangiectasia response kinase target site Ser345 and the autophosphorylation site Ser296 in neuroblastoma cell lines. This pattern was also seen in six of eight high-risk primary tumors but not in control nonneuroblastoma cell lines or in seven of eight low-risk primary tumors. Neuroblastoma cells were sensitive to the two CHK1 inhibitors SB21807 and TCS2312, with median IC(50) values of 564 nM and 548 nM, respectively. In contrast, the control lines had high micromolar IC(50) values, indicating a strong correlation between CHK1 phosphorylation and CHK1 inhibitor sensitivity (P = 0.0004). Furthermore, cell cycle analysis revealed that CHK1 inhibition in neuroblastoma cells caused apoptosis during S-phase, consistent with its role in replication fork progression. CHK1 inhibitor sensitivity correlated with total MYC(N) protein levels, and inducing MYCN in retinal pigmented epithelial cells resulted in CHK1 phosphorylation, which caused growth inhibition when inhibited. These data show the power of a functional RNAi screen to identify tractable therapeutical targets in neuroblastoma and support CHK1 inhibition strategies in this disease.
Asunto(s)
Neuroblastoma/tratamiento farmacológico , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , ARN Interferente Pequeño/farmacología , Apoptosis/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos , Humanos , Proteína Proto-Oncogénica N-Myc , Neuroblastoma/patología , Proteínas Nucleares/análisis , Proteínas Oncogénicas/análisis , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero , Fase S/efectos de los fármacosRESUMEN
Neuroblastoma is remarkable for its clinical heterogeneity and is characterized by genomic alterations that are strongly correlated with tumor behavior. The specific genes that influence neuroblastoma biology and are targeted by genomic alterations remain largely unknown. We quantified mRNA expression in a highly annotated series of 101 prospectively collected diagnostic neuroblastoma primary tumors using an oligonucleotide-based microarray. Genomic copy number status at the prognostically relevant loci 1p36, 2p24 (MYCN), 11q23, and 17q23 was determined by PCR and was aberrant in 26, 20, 40, and 38 cases, respectively. In addition, 72 diagnostic neuroblastoma primary tumors assayed in a different laboratory were used as an independent validation set. Unsupervised hierarchical clustering showed that gene expression was highly correlated with genomic alterations and clinical markers of tumor behavior. The vast majority of samples with MYCN amplification and 1p36 loss of heterozygosity (LOH) clustered together on a terminal node of the sample dendrogram, whereas the majority of samples with 11q deletion clustered separately and both of these were largely distinct from the copy number neutral group of tumors. Genes involved in neurodevelopment were broadly overrepresented in the more benign tumors, whereas genes involved in RNA processing and cellular proliferation were highly represented in the most malignant cases. By combining transcriptomic and genomic data, we showed that LOH at 1p and 11q was associated with significantly decreased expression of 122 (61%) and 88 (27%) of the genes mapping to 1p35-36 and all of 11q, respectively, suggesting that multiple genes may be targeted by LOH events. A total of 71 of the 1p35-36 genes were also differentially expressed in the independent validation data set, providing a prioritized list of candidate neuroblastoma suppressor genes. Taken together, these data are consistent with the hypotheses that the neuroblastoma transcriptome is a sensitive marker of underlying tumor biology and that chromosomal deletion events in this cancer likely target multiple genes through alteration in mRNA dosage. Lead positional candidates for neuroblastoma suppressor genes can be inferred from these data, but the potential multiplicity of transcripts involved has significant implications for ongoing gene discovery strategies.
Asunto(s)
Neuroblastoma/genética , Aberraciones Cromosómicas , Dosificación de Gen , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Marcación de Gen , Genoma Humano , Genómica/métodos , Humanos , Lactante , Pérdida de Heterocigocidad , Neuroblastoma/clasificación , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Neuroblastoma is a cancer of the sympathetic nervous system that accounts for approximately 10% of all pediatric oncology deaths. Here, we report a genome-wide association study of 2,817 neuroblastoma cases and 7,473 controls. We identified two new associations at 6q16, the first within HACE1 (rs4336470; combined P=2.7×10(-11); odds ratio 1.26, 95% confidence interval (CI) 1.18-1.35) and the second within LIN28B (rs17065417; combined P=1.2×10(-8); odds ratio 1.38, 95% CI 1.23-1.54). Expression of LIN28B and let-7 miRNA correlated with rs17065417 genotype in neuroblastoma cell lines, and we observed significant growth inhibition upon depletion of LIN28B, specifically in neuroblastoma cells that were homozygous for the risk allele. Low HACE1 and high LIN28B expression in diagnostic primary neuroblastomas were associated with worse overall survival (P=0.008 and 0.014, respectively). Taken together, these data show that common variants in HACE1 and LIN28B influence neuroblastoma susceptibility and indicate that both genes likely have a role in disease progression.
Asunto(s)
Proteínas de Unión al ADN/genética , Neuroblastoma/genética , Polimorfismo de Nucleótido Simple , Ubiquitina-Proteína Ligasas/genética , Estudios de Casos y Controles , Línea Celular Tumoral , Proliferación Celular , Cromosomas Humanos Par 6 , Estudios de Cohortes , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Frecuencia de los Genes , Técnicas de Silenciamiento del Gen , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Lactante , Estimación de Kaplan-Meier , Desequilibrio de Ligamiento , Neuroblastoma/metabolismo , Neuroblastoma/mortalidad , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , Proteínas de Unión al ARN , Análisis de Secuencia de ADN , Transcriptoma , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The mechanisms underlying genetic susceptibility at loci discovered by genome-wide association study (GWAS) approaches in human cancer remain largely undefined. In this study, we characterized the high-risk neuroblastoma association at the BRCA1-related locus, BARD1, showing that disease-associated variations correlate with increased expression of the oncogenically activated isoform, BARD1ß. In neuroblastoma cells, silencing of BARD1ß showed genotype-specific cytotoxic effects, including decreased substrate-adherence, anchorage-independence, and foci growth. In established murine fibroblasts, overexpression of BARD1ß was sufficient for neoplastic transformation. BARD1ß stabilized the Aurora family of kinases in neuroblastoma cells, suggesting both a mechanism for the observed effect and a potential therapeutic strategy. Together, our findings identify BARD1ß as an oncogenic driver of high-risk neuroblastoma tumorigenesis, and more generally, they illustrate how robust GWAS signals offer genomic landmarks to identify molecular mechanisms involved in both tumor initiation and malignant progression. The interaction of BARD1ß with the Aurora family of kinases lends strong support to the ongoing work to develop Aurora kinase inhibitors for clinically aggressive neuroblastoma.
Asunto(s)
Transformación Celular Neoplásica/genética , Predisposición Genética a la Enfermedad/genética , Neuroblastoma/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Apoptosis/genética , Secuencia de Bases , Línea Celular Tumoral , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Immunoblotting , Inmunoprecipitación , Ratones , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Isoformas de Proteínas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Matrices TisularesRESUMEN
Cancer genomic studies that rely on analysis of biopsies from primary tumors may not fully identify the molecular events associated with tumor progression. We hypothesized that characterizing the transcriptome during tumor progression in the TH-MYCN transgenic model would identify oncogenic drivers that would be targetable therapeutically. We quantified expression of 32,381 murine genes in nine hyperplastic ganglia harvested at three time points and four tumor cohorts of progressively larger size in mice homozygous for the TH-MYCN transgene. We found 93 genes that showed a linearly increasing or decreasing pattern of expression from the preneoplastic ganglia to end stage tumors. Cross-species integration identified 24 genes that were highly expressed in human MYCN-amplified neuroblastomas. The genes prioritized were not exclusively driven by increasing Myc transactivation or proliferative rate. We prioritized three targets [centromere-associated protein E (Cenpe), Gpr49, and inosine monophosphate dehydrogenase type II] with previously determined roles in cancer. Using siRNA knockdown in human neuroblastoma cell lines, we further prioritized CENPE due to inhibition of cellular proliferation. Targeting CENPE with the small molecular inhibitor GSK923295 showed inhibition of in vitro proliferation of 19 neuroblastoma cell lines (median IC(50), 41 nmol/L; range, 27-266 nmol/L) and delayed tumor growth in three xenograft models (P values ranged from P < 0.0001 to P = 0.018). We provide preclinical validation that serial transcriptome analysis of a transgenic mouse model followed by cross-species integration is a useful method to identify therapeutic targets and identify CENPE as a novel therapeutic candidate in neuroblastoma.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Neuroblastoma/genética , Algoritmos , Animales , Procesos de Crecimiento Celular/genética , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Progresión de la Enfermedad , Expresión Génica , Humanos , Ratones , Ratones SCID , Ratones Transgénicos , Proteína Proto-Oncogénica N-Myc , Neuroblastoma/metabolismo , Neuroblastoma/patología , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Proteínas Oncogénicas/biosíntesis , Proteínas Oncogénicas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie , Activación TranscripcionalRESUMEN
We conducted a SNP-based genome-wide association study (GWAS) focused on the high-risk subset of neuroblastoma. As our previous unbiased GWAS showed strong association of common 6p22 SNP alleles with aggressive neuroblastoma, we restricted our analysis here to 397 high-risk cases compared to 2,043 controls. We detected new significant association of six SNPs at 2q35 within the BARD1 locus (P(allelic) = 2.35 x 10(-9)-2.25 x 10(-8)). We confirmed each SNP association in a second series of 189 high-risk cases and 1,178 controls (P(allelic) = 7.90 x 10(-7)-2.77 x 10(-4)). We also tested the two most significant SNPs (rs6435862, rs3768716) in two additional independent high-risk neuroblastoma case series, yielding combined allelic odds ratios of 1.68 each (P = 8.65 x 10(-18) and 2.74 x 10(-16), respectively). We also found significant association with known BARD1 nonsynonymous SNPs. These data show that common variation in BARD1 contributes to the etiology of the aggressive and most clinically relevant subset of human neuroblastoma.
Asunto(s)
Variación Genética , Neuroblastoma/genética , Polimorfismo de Nucleótido Simple/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Cromosomas Humanos Par 6/genética , Predisposición Genética a la Enfermedad , Genotipo , Heterocigoto , Humanos , Neuroblastoma/epidemiología , Oportunidad Relativa , Factores de RiesgoRESUMEN
Neuroblastoma is a heterogeneous neoplasm that has served as a paradigm for the clinical utility of somatically acquired genomic aberrations. DNA copy number alterations (CNA) are currently used to predict prognosis, including MYCN amplification and deletions at chromosome bands 1p36 and 11q23. We predicted that genome-wide assessment of DNA aberrations in neuroblastoma tumors would provide a more precise estimation of clinical phenotype, and could be used to predict outcome. We measured CNAs in a representative set of 82 diagnostic tumors on a customized high-resolution BAC array-based CGH platform supplemented with additional clones across 1p36, 2p24, 3p21-22, 11q14-24, and 16p12-13, and integrated these data with RNA expression data. We used an unbiased statistical method to define a set of minimal common regions (MCRs) of aberration. Unsupervised hierarchical clustering identified four distinct genomic subclasses. First, a subset of tumors with a clinically benign phenotype showed predominantly whole chromosome gains and losses. Second, tumors with MYCN amplification had a unique genomic signature of 1p deletion and 17q gain, but few other rearrangements. Third, tumors with an aggressive clinical phenotype without MYCN amplification, showed multiple structural rearrangements. Most notable were deletions of 3p, 4p, and 11q and gain of 1q, 2p, 12q, and 17q. Lastly, there was a subset of tumors with an aggressive clinical phenotype and no detectable DNA CNAs. The genomic subsets were highly correlated with patient outcome, and individual MCRs remained prognostic in a multivariable model. DNA signature patterns embed important prognostic information in diagnostic neuroblastoma samples, and can identify candidate cancer-related genes.