RESUMEN
The systemic and resistant nature of metastatic neuroblastoma renders it largely incurable with current multimodal treatment. Clinical progression stems mainly from the increasing burden of metastatic colonization. Therapeutically inhibiting the migration-invasion-metastasis cascade would be of great benefit, but the mechanisms driving this cycle are as yet poorly understood. In-depth transcriptome analyses and ChIP-qPCR identified the cell surface glycoprotein, CD9, as a major downstream player and direct target of the recently described GRHL1 tumor suppressor. CD9 is known to block or facilitate cancer cell motility and metastasis dependent upon entity. High-level CD9 expression in primary neuroblastomas correlated with patient survival and established markers for favorable disease. Low-level CD9 expression was an independent risk factor for adverse outcome. MYCN and HDAC5 colocalized to the CD9 promoter and repressed transcription. CD9 expression diminished with progressive tumor development in the TH-MYCN transgenic mouse model for neuroblastoma, and CD9 expression in neuroblastic tumors was far below that in ganglia from wildtype mice. Primary neuroblastomas lacking MYCN amplifications displayed differential CD9 promoter methylation in methyl-CpG-binding domain sequencing analyses, and high-level methylation was associated with advanced stage disease, supporting epigenetic regulation. Inducing CD9 expression in a SH-EP cell model inhibited migration and invasion in Boyden chamber assays. Enforced CD9 expression in neuroblastoma cells transplanted onto chicken chorioallantoic membranes strongly reduced metastasis to embryonic bone marrow. Combined treatment of neuroblastoma cells with HDAC/DNA methyltransferase inhibitors synergistically induced CD9 expression despite hypoxic, metabolic or cytotoxic stress. Our results show CD9 is a critical and indirectly druggable suppressor of the invasion-metastasis cycle in neuroblastoma.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/fisiología , Histona Desacetilasas/metabolismo , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/patología , Tetraspanina 29/biosíntesis , Animales , Histona Desacetilasas/genética , Humanos , Ratones , Ratones Transgénicos , Proteína Proto-Oncogénica N-Myc/genética , Invasividad Neoplásica/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Tetraspanina 29/genéticaRESUMEN
PURPOSE: To optimize neuroblastoma treatment stratification, we aimed at developing a novel risk estimation system by integrating gene expression-based classification and established prognostic markers. EXPERIMENTAL DESIGN: Gene expression profiles were generated from 709 neuroblastoma specimens using customized 4 × 44 K microarrays. Classification models were built using 75 tumors with contrasting courses of disease. Validation was performed in an independent test set (n = 634) by Kaplan-Meier estimates and Cox regression analyses. RESULTS: The best-performing classifier predicted patient outcome with an accuracy of 0.95 (sensitivity, 0.93; specificity, 0.97) in the validation cohort. The highest potential clinical value of this predictor was observed for current low-risk patients [5-year event-free survival (EFS), 0.84 ± 0.02 vs. 0.29 ± 0.10; 5-year overall survival (OS), 0.99 ± 0.01 vs. 0.76 ± 0.11; both P < 0.001] and intermediate-risk patients (5-year EFS, 0.88 ± 0.06 vs. 0.41 ± 0.10; 5-year OS, 1.0 vs. 0.70 ± 0.09; both P < 0.001). In multivariate Cox regression models for low-risk/intermediate-risk patients, the classifier outperformed risk assessment of the current German trial NB2004 [EFS: hazard ratio (HR), 5.07; 95% confidence interval (CI), 3.20-8.02; OS: HR, 25.54; 95% CI, 8.40-77.66; both P < 0.001]. On the basis of these findings, we propose to integrate the classifier into a revised risk stratification system for low-risk/intermediate-risk patients. According to this system, we identified novel subgroups with poor outcome (5-year EFS, 0.19 ± 0.08; 5-year OS, 0.59 ± 0.1), for whom we propose intensified treatment, and with beneficial outcome (5-year EFS, 0.87 ± 0.05; 5-year OS, 1.0), who may benefit from treatment de-escalation. CONCLUSIONS: Combination of gene expression-based classification and established prognostic markers improves risk estimation of patients with low-risk/intermediate-risk neuroblastoma. We propose to implement our revised treatment stratification system in a prospective clinical trial.
Asunto(s)
Biomarcadores de Tumor/genética , Perfilación de la Expresión Génica , Neuroblastoma/genética , Neuroblastoma/mortalidad , Análisis por Conglomerados , Femenino , Estudios de Seguimiento , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Masculino , Neuroblastoma/diagnóstico , Neuroblastoma/terapia , Pronóstico , Análisis de Regresión , Reproducibilidad de los Resultados , Medición de Riesgo , Factores de RiesgoRESUMEN
Specific patterns of genomic aberrations have been associated with different types of malignancies. In colorectal cancer, losses of chromosome arm 8p and gains of chromosome arm 8q are among the most common chromosomal rearrangements, suggesting that the centromeric portion of chromosome 8 is particularly sensitive to breakage. Genomic alterations frequently occur in the early stages of tumorigenesis at specific genomic regions known as common fragile sites (cFSs). CFSs represent parts of the normal chromosome structure that are prone to breakage under replication stress. In this study, we identified the genomic location of FRA8I, spanning 530 kb at 8q11.21 and assessed the composition of the fragile DNA sequence. FRA8I encompasses KIAA0146, a large protein-coding gene with yet unknown function, as well as CEBPD and part of PRKDC, two genes encoding proteins involved in tumorigenesis in a variety of cancers. We show that FRA8I is unstable in lymphocytes and epithelial cells, displaying similar expression rates. We examined copy number alteration patterns within FRA8I in a panel of 25 colorectal cancer cell lines and surveyed publically available profiles of 56 additional colorectal cancer cell lines. Combining these data shows that focal recombination events disrupt the genomic integrity of KIAA0146 and neighboring cFS genes in 12.3% of colorectal cancer cell lines. Moreover, data analysis revealed evidence that KIAA0146 is a translocation partner of the immunoglobulin heavy chain gene in recurrent t(8;14)(q11;q32) translocations in a subset of patients with B-cell precursor acute lymphoblastic leukemia. Our data molecularly describe a region of enhanced chromosomal instability in the human genome and point to a role of the KIAA0146 gene in tumorigenesis.
Asunto(s)
Proteína delta de Unión al Potenciador CCAAT/genética , Sitios Frágiles del Cromosoma , Cromosomas Humanos Par 8 , Neoplasias Colorrectales/genética , Proteína Quinasa Activada por ADN/genética , Proteínas Nucleares/genética , Proteínas/genética , Línea Celular Tumoral , Inestabilidad Cromosómica , Aberraciones Cromosómicas , Cromosomas Artificiales Bacterianos/metabolismo , Neoplasias Colorrectales/metabolismo , Proteínas de Unión al ADN , Genoma Humano , Humanos , Hibridación Fluorescente in Situ , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Translocación GenéticaRESUMEN
OBJECTIVE: The CpG island methylator phenotype is strongly associated with poor survival in neuroblastomas. Neuroblastomas with the CpG island methylator phenotype include almost all neuroblastomas with MYCN amplification, and, even among neuroblastomas without MYCN amplification, have worse prognosis. At the same time, methylation of individual tumor-suppressor genes is also reported to be associated with poor survival. The purpose of this study was to compare the prognostic power of the CpG island methylator phenotype with that of methylation of individual genes. METHODS: Methylation-specific polymerase chain reaction was performed for five individual genes (CASP8, EMP3, HOXA9, NR1I2 and CD44) in 140 Japanese and 152 German neuroblastomas. Kaplan-Meier analysis and log-rank tests were conducted to compare the survival between groups defined by methylation status. RESULTS: Among the five individual genes, only CASP8 methylation had a significant association with poor overall survival both in Japanese (hazard ratio = 3.1; 95% confidence interval = 1.5-6.4; P = 0.002) and German (hazard ratio = 4.8; 95% confidence interval = 2.1-11; P = 0.0002) neuroblastomas. HOXA9 and NR1I2 methylation were associated with poor survival only in German neuroblastomas. On the other hand, the CpG island methylator phenotype had a strong and consistent association in Japanese (hazard ratio = 22; 95% confidence interval = 5.3-93; P = 1.5 × 10(-5)) and German (hazard ratio = 9.5; 95% confidence interval = 3.2-28; P = 4.7 × 10(-5)) neuroblastomas. CONCLUSION: The CpG island methylator phenotype is likely to have stronger prognostic power than methylation of individual genes in neuroblastomas.
Asunto(s)
Islas de CpG , Metilación de ADN , Neuroblastoma/genética , Neuroblastoma/mortalidad , Pueblo Asiatico/genética , Caspasa 8/genética , Intervalos de Confianza , Proteínas de Homeodominio/genética , Humanos , Receptores de Hialuranos/genética , Estimación de Kaplan-Meier , Glicoproteínas de Membrana/genética , Valor Predictivo de las Pruebas , Receptor X de Pregnano , Pronóstico , Regiones Promotoras Genéticas , Receptores de Esteroides/genéticaRESUMEN
The TP53 tumor suppressor pathway is abrogated by TP53 mutations in the majority of human cancers. Increased levels of wild-type TP53 in aggressive neuroblastomas appear paradox but are tolerated by tumor cells due to co-activation of the TP53 ubiquitin ligase, MDM2. The role of the MDM2 antagonist, p14(ARF), in controlling the TP53-MDM2 balance in neuroblastoma is unresolved. In the present study, we show that conditional p14(ARF) expression substantially suppresses viability, clonogenicity and anchorage-independent growth in p14(ARF)-deficient or MYCN-amplified neuroblastoma cell lines. Furthermore, ectopic 14(ARF) expression induced accumulation of cells in the G1 phase and apoptosis, which was paralleled by accumulation of TP53 and its targets. Comparative genomic hybridization analysis of 193 primary neuroblastomas detected one homozygous deletion of CDKN2A (encoding both p14(ARF) and p16(INK4A)) and heterozygous loss of CDKN2A in 22% of tumors. Co-expression analysis of p14(ARF) and its transactivator, E2F1, in a set of 68 primary tumors revealed only a weak correlation, suggesting that further regulatory mechanisms govern p14(ARF) expression in neuroblastomas. Intriguingly, analyses utilizing chromatin immunoprecipitation revealed different histone mark-defined epigenetic activity states of p14(ARF) in neuroblastoma cell lines that correlated with endogenous p14(ARF) expression but not with episomal p14(ARF) promoter reporter activity, indicating that the native chromatin context serves to epigenetically repress p14(ARF) in neuroblastoma cells. Collectively, the data pinpoint p14(ARF) as a critical factor for efficient TP53 response in neuroblastoma cells and assign p14(ARF) as a neuroblastoma suppressor candidate that is impaired by genomic loss and epigenetic repression.
Asunto(s)
Apoptosis , Represión Epigenética , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Histonas/genética , Neuroblastoma/patología , Proteína p14ARF Supresora de Tumor/genética , Línea Celular Tumoral , Hibridación Genómica Comparativa , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Femenino , Eliminación de Gen , Expresión Génica , Histonas/metabolismo , Humanos , Pérdida de Heterocigocidad , Masculino , Neuroblastoma/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p14ARF Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
A broad range of human malignancies is associated with nonrandom 1p36 deletions, suggesting the existence of tumor suppressors encoded in this region. Evidence for tumor-specific inactivation of 1p36 genes in the classic "two-hit" manner is scarce; however, many tumor suppressors do not require complete inactivation but contribute to tumorigenesis by partial impairment. We discuss recent data derived from both human tumors and functional cancer models indicating that the 1p36 genes CHD5, CAMTA1, KIF1B, CASZ1, and miR-34a contribute to cancer development when reduced in dosage by genomic copy number loss or other mechanisms. We explore potential interactions among these candidates and propose a model where heterozygous 1p36 deletion impairs oncosuppressive pathways via simultaneous downregulation of several dosage-dependent tumor suppressor genes.
Asunto(s)
Trastornos de los Cromosomas/genética , Neoplasias/genética , Animales , Deleción Cromosómica , Cromosomas Humanos Par 1/genética , Dosificación de Gen , Genes Supresores de Tumor , HumanosRESUMEN
Common fragile sites (cFSs) are non-random chromosomal regions that are prone to breakage under conditions of replication stress. DNA damage and chromosomal alterations at cFSs appear to be critical events in the development of various human diseases, especially carcinogenesis. Despite the growing interest in understanding the nature of cFS instability, only a few cFSs have been molecularly characterised. In this study, we fine-mapped the location of FRA2H using six-colour fluorescence in situ hybridisation and showed that it is one of the most active cFSs in the human genome. FRA2H encompasses approximately 530 kb of a gene-poor region containing a novel large intergenic non-coding RNA gene (AC097500.2). Using custom-designed array comparative genomic hybridisation, we detected gross and submicroscopic chromosomal rearrangements involving FRA2H in a panel of 54 neuroblastoma, colon and breast cancer cell lines. The genomic alterations frequently involved different classes of long terminal repeats and long interspersed nuclear elements. An analysis of breakpoint junction sequence motifs predominantly revealed signatures of microhomology-mediated non-homologous recombination events. Our data provide insight into the molecular structure of cFSs and sequence motifs affected by their activation in cancer. Identifying cFS sequences will accelerate the search for DNA biomarkers and targets for individualised therapies.
Asunto(s)
Sitios Frágiles del Cromosoma , Reordenamiento Génico , Recombinación Genética , Secuencia de Bases , Línea Celular Tumoral , Cromosomas Artificiales Bacterianos , Cromosomas Humanos Par 2 , Hibridación Genómica Comparativa , Cartilla de ADN , Humanos , Hibridación Fluorescente in Situ , Elementos de Nucleótido Esparcido Largo , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Although the prognostic value of the ATP-binding cassette, subfamily C (ABCC) transporters in childhood neuroblastoma is usually attributed to their role in cytotoxic drug efflux, certain observations have suggested that these multidrug transporters might contribute to the malignant phenotype independent of cytotoxic drug efflux. METHODS: A v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN)-driven transgenic mouse neuroblastoma model was crossed with an Abcc1-deficient mouse strain (658 hMYCN(1/-), 205 hMYCN(+/1) mice) or, alternatively, treated with the ABCC1 inhibitor, Reversan (n = 20). ABCC genes were suppressed using short interfering RNA or overexpressed by stable transfection in neuroblastoma cell lines BE(2)-C, SH-EP, and SH-SY5Y, which were then assessed for wound closure ability, clonogenic capacity, morphological differentiation, and cell growth. Real-time quantitative polymerase chain reaction was used to examine the clinical significance of ABCC family gene expression in a large prospectively accrued cohort of patients (n = 209) with primary neuroblastomas. Kaplan-Meier survival analysis and Cox regression were used to test for associations with event-free and overall survival. Except where noted, all statistical tests were two-sided. RESULTS: Inhibition of ABCC1 statistically significantly inhibited neuroblastoma development in hMYCN transgenic mice (mean age for palpable tumor: treated mice, 47.2 days; control mice, 41.9 days; hazard ratio [HR] = 9.3, 95% confidence interval [CI] = 2.65 to 32; P < .001). Suppression of ABCC1 in vitro inhibited wound closure (P < .001) and clonogenicity (P = .006); suppression of ABCC4 enhanced morphological differentiation (P < .001) and inhibited cell growth (P < .001). Analysis of 209 neuroblastoma patient tumors revealed that, in contrast with ABCC1 and ABCC4, low rather than high ABCC3 expression was associated with reduced event-free survival (HR of recurrence or death = 2.4, 95% CI = 1.4 to 4.2; P = .001), with 23 of 53 patients with low ABCC3 expression experiencing recurrence or death compared with 31 of 155 patients with high ABCC3. Moreover, overexpression of ABCC3 in vitro inhibited neuroblastoma cell migration (P < .001) and clonogenicity (P = .03). The combined expression of ABCC1, ABCC3, and ABCC4 was associated with patients having an adverse event, such that of the 12 patients with the "poor prognosis" expression pattern, 10 experienced recurrence or death (HR of recurrence or death = 12.3, 95% CI = 6 to 27; P < .001). CONCLUSION: ABCC transporters can affect neuroblastoma biology independently of their role in chemotherapeutic drug efflux, enhancing their potential as targets for therapeutic intervention.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/metabolismo , Pirazoles/farmacología , Pirimidinas/farmacología , Adolescente , Animales , Western Blotting , Diferenciación Celular , Línea Celular Tumoral , Movimiento Celular , Niño , Preescolar , Modelos Animales de Enfermedad , Supervivencia sin Enfermedad , Regulación hacia Abajo , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Humanos , Lactante , Estimación de Kaplan-Meier , Masculino , Ratones , Ratones Transgénicos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteína Proto-Oncogénica N-Myc , Proteínas Nucleares/metabolismo , Oportunidad Relativa , Proteínas Oncogénicas/metabolismo , Reacción en Cadena de la Polimerasa , Valor Predictivo de las Pruebas , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Prospectivos , ARN Interferente Pequeño/metabolismo , Recurrencia , Factores de Tiempo , Transfección , Regulación hacia Arriba , Adulto JovenRESUMEN
A distal portion of human chromosome 1p is often deleted in neuroblastomas and other cancers and it is generally assumed that this region harbors one or more tumor suppressor genes. In neuroblastoma, a 261 kb region at 1p36.3 that encompasses the smallest region of consistent deletion pinpoints the locus for calmodulin binding transcription activator 1 (CAMTA1). Low CAMTA1 expression is an independent predictor of poor outcome in multivariate survival analysis, but its potential functionality in neuroblastoma has not been explored. In this study, we used inducible cell models to analyze the impact of CAMTA1 on neuroblastoma biology. In neuroblastoma cells that expressed little endogenous CAMTA1, its ectopic expression slowed cell proliferation, increasing the relative proportion of cells in G(1)/G(0) phases of the cell cycle, inhibited anchorage-independent colony formation, and suppressed the growth of tumor xenografts. CAMTA1 also induced neurite-like processes and markers of neuronal differentiation in neuroblastoma cells. Further, retinoic acid and other differentiation- inducing stimuli upregulated CAMTA1 expression in neuroblastoma cells. Transciptome analysis revealed 683 genes regulated on CAMTA1 induction and gene ontology analysis identified genes consistent with CAMTA1-induced phenotypes, with a significant enrichment for genes involved in neuronal function and differentiation. Our findings define properties of CAMTA1 in growth suppression and neuronal differentiation that support its assignment as a 1p36 tumor suppressor gene in neuroblastoma.
Asunto(s)
Proteínas de Unión al Calcio/genética , Cromosomas Humanos Par 1 , Neuroblastoma/genética , Transactivadores/genética , Secuencia de Aminoácidos , Animales , Proteínas de Unión al Calcio/biosíntesis , Diferenciación Celular/genética , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , Genes Supresores de Tumor , Humanos , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Neuroblastoma/metabolismo , Neuroblastoma/patología , Transactivadores/biosíntesis , Regulación hacia ArribaRESUMEN
Common fragile sites (cFS) represent chromosomal regions that are prone to breakage after partial inhibition of DNA synthesis. Activation of cFS is associated with various forms of DNA instability in cancer cells, and is thought to be an initiating event in the generation of DNA damage in early-stage tumorigenesis. Only a few cFS have been fully characterized despite the growing interest in cFS instability in cancer genomes. In this study, six-color fluorescence in situ hybridization revealed that FRA2C consists of two cFS spanning 747 kb FRA2Ctel and 746 kb FRA2Ccen at 2p24.3 and 2p24.2, respectively. Both cFS are separated by a 2.8 Mb non-fragile region containing MYCN. Fine-tiling array comparative genomic hybridization of MYCN amplicons from neuroblastoma (NB) cell lines and primary tumors revealed that 56.5% of the amplicons cluster in FRA2C. MYCN amplicons are either organized as double minutes or as homogeneously stained regions in addition to the single copy of MYCN retained at 2p24. We suggest that MYCN amplicons arise from extra replication rounds of unbroken DNA secondary structures that accumulate at FRA2C. This hypothesis implicates cFS in high-level gene amplification in cancer cells. Complex genomic rearrangements, including deletions, duplications and translocations, which originate from double-strand breaks, were detected at FRA2C in different cancers. These data propose a dual role for cFS in the generation of gross chromosomal rearrangements either after DNA breakage or by inducing extra replication rounds, and provide new insights into the highly recombinogenic nature of cFS in the human cancer genome.
Asunto(s)
Neoplasias de la Mama/genética , Cromosomas Humanos Par 2/genética , Neoplasias del Colon/genética , Amplificación de Genes , Neuroblastoma/genética , Secuencia de Bases , Línea Celular Tumoral , Sitios Frágiles del Cromosoma , Mapeo Cromosómico , Cromosomas Humanos Par 2/ultraestructura , Hibridación Genómica Comparativa , Roturas del ADN , Femenino , Dosificación de Gen , Reordenamiento Génico , Humanos , Hibridación Fluorescente in Situ , Secuencias Repetidas TerminalesRESUMEN
PURPOSE: To evaluate the impact of a predefined gene expression-based classifier for clinical risk estimation and cytotoxic treatment decision making in neuroblastoma patients. PATIENTS AND METHODS: Gene expression profiles of 440 internationally collected neuroblastoma specimens were investigated by microarray analysis, 125 of which were examined prospectively. Patients were classified as either favorable or unfavorable by a 144-gene prediction analysis for microarrays (PAM) classifier established previously on a separate set of 77 patients. PAM classification results were compared with those of current prognostic markers and risk estimation strategies. RESULTS: The PAM classifier reliably distinguished patients with contrasting clinical courses (favorable [n = 249] and unfavorable [n = 191]; 5-year event free survival [EFS] 0.84 +/- 0.03 v 0.38 +/- 0.04; 5-year overall survival [OS] 0.98 +/- 0.01 v 0.56 +/- 0.05, respectively; both P < .001). Moreover, patients with divergent outcome were robustly discriminated in both German and international cohorts and in prospectively analyzed samples (P Asunto(s)
Perfilación de la Expresión Génica
, Neuroblastoma/clasificación
, Adolescente
, Adulto
, Niño
, Preescolar
, Humanos
, Lactante
, Recién Nacido
, Neuroblastoma/genética
, Neuroblastoma/mortalidad
, Pronóstico
, Modelos de Riesgos Proporcionales
RESUMEN
The cell cycle regulator, SKP2, is overexpressed in various cancers and plays a key role in p27 degradation, which is involved in tumor cell dedifferentiation. Little is known about the mechanisms leading to impaired SKP2 transcriptional control in tumor cells. We used neuroblastoma as a model to study SKP2 regulation because SKP2 transcript levels gradually increase with aggressiveness of neuroblastoma subtypes. The highest SKP2 levels are found in neuroblastomas with amplified MYCN. Accordingly, we found 5.5-fold (range, 2-9.5) higher SKP2 core promoter activity in MYCN-amplified cells. Higher SKP2 core promoter activity in MYCN-amplified cells is mediated through a defined region at the transcriptional start site. This region includes a specific E2F-binding site that makes SKP2 activation largely independent of mitogenic signals integrated through the SP1/ELK-1 site. We show by chromatin immunoprecipitation that SKP2 activation through the transcriptional start site in MYCN-amplified cells is associated with the low abundance of pRB-E2F1 complexes bound to the SKP2 promoter. Transcriptional control of SKP2 through this regulatory mechanism can be reestablished in MYCN-amplified cells by restoring pRB activity using selective small compound inhibitors of CDK4. In contrast, doxorubicin or nutlin-3 treatment-both leading to p53-p21 activation-or CDK2 inhibition had no effect on SKP2 regulation in MYCN-amplified cells. Together, this implies that deregulated MYCN protein levels in MYCN-amplified neuroblastoma cells activate SKP2 through CDK4 induction, abrogating repressive pRB-E2F1 complexes bound to the SKP2 promoter.
Asunto(s)
Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Proteínas Quinasas Asociadas a Fase-S/genética , Línea Celular Tumoral , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/metabolismo , Factor de Transcripción E2F1/metabolismo , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Humanos , Proteína Proto-Oncogénica N-Myc , Neuroblastoma/metabolismo , Proteínas Nucleares/biosíntesis , Proteínas Oncogénicas/biosíntesis , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína de Retinoblastoma/metabolismo , Proteínas Quinasas Asociadas a Fase-S/biosíntesis , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
Increased expression of specific ATP-binding cassette (ABC) transporters is known to mediate the efflux of chemotherapeutic agents from cancer cells. Therefore, establishing how ABC transporter genes are controlled at their transcription level may help provide insight into the role of these multifaceted transporters in the malignant phenotype. We have investigated ABC transporter gene expression in a large neuroblastoma data set of 251 tumor samples. Clustering analysis demonstrated a strong association between differential ABC gene expression patterns in tumor samples and amplification of the MYCN oncogene, suggesting a correlation with MYCN function. Using expression profiling and chromatin immunoprecipitation studies, we show that MYCN oncoprotein coordinately regulates transcription of specific ABC transporter genes, by acting as either an activator or a repressor. Finally, we extend these notions to c-MYC showing that it can also regulate the same set of ABC transporter genes in other tumor cells through similar dynamics. Overall our findings provide insight into MYC-driven molecular mechanisms that contribute to coordinate transcriptional regulation of a large set of ABC transporter genes, thus affecting global drug efflux.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Resistencia a Antineoplásicos , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción/metabolismo , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Perfilación de la Expresión Génica , Humanos , Concentración 50 Inhibidora , Modelos Genéticos , Fenotipo , Retinoblastoma/metabolismo , Transcripción GenéticaRESUMEN
We analysed the expression of BIRC5 and BIRC5-2B in primary neuroblastoma (NB) tumors and NB model systems. In tumors, overexpression of BIRC5 correlated closely with its isoform BIRC5-2B. Expression of both transcripts was stage-dependent, associated with poor prognosis and with the expression of the transcription factor E2F1. In cell culture, we identified BIRC5 as a direct transcriptional target of activating E2Fs, primarily when p21(Cip1) and p27(Kip1), two other E2F1 targets, are strongly suppressed. Deregulated MYCN indirectly induces BIRC5 through suppression of CDKN1A/p21(Cip1) and induction of Skp2, which in turn favors the degradation of p27(Kip1). In addition, increased BIRC5 protein stability via phosphorylation is mediated by expression of E2F targets such as CDC2. In line with this, selective knock down of CDC2 inhibited BIRC5 abundance and suppressed its anti-apoptotic activities. We conclude that BIRC5 is induced via a functional cooperation between MYCN and E2F1.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neuroblastoma/metabolismo , Apoptosis , Biomarcadores de Tumor/genética , Proteína Quinasa CDC2 , Línea Celular Tumoral , Ciclina B/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes , Factor de Transcripción E2F1/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Inhibidoras de la Apoptosis , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteína Proto-Oncogénica N-Myc , Estadificación de Neoplasias , Neuroblastoma/genética , Neuroblastoma/mortalidad , Neuroblastoma/patología , Neuroblastoma/terapia , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/metabolismo , Fosforilación , Isoformas de Proteínas , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Interferencia de ARN , ARN Mensajero/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Survivin , Factores de Tiempo , Transfección , Resultado del TratamientoRESUMEN
PURPOSE: MYCN amplification is an important therapy-stratifying marker in neuroblastoma. Fluorescence in situ hybridization with signal detection on the single-cell level allows a critical judgement of MYCN intratumoral heterogeneity. EXPERIMENTAL DESIGN: The MYCN status was investigated by fluorescence in situ hybridization at diagnosis and relapse. Heterogeneity was defined as the simultaneous presence of amplified cells (>/=5 cells per slide) and nonamplified cells within one tumor or sequential change of the amplification status during the course of the disease. Likewise, heterogeneity can be detected between primary tumor and metastasis. RESULTS: From 1,341 patients analyzed, 1,071 showed no amplification, 250 showed homogeneous amplification, and 20 patients showed MYCN heterogeneity. Of the patients with heterogeneity, 12 of 20 had clusters of MYCN amplifications, 3 of 20 had amplified single cells, 3 of 20 showed MYCN amplifications in the bone marrow but not in the primary tumor, and 2 of 20 acquired MYCN amplification during the course of the disease. All stage 4 patients were treated according to high-risk protocols; 7 of 8 later progressed. Four patients with localized disease were treated according to high-risk protocol because of MYCN-amplified clusters; 1 of 4 later progressed. One patient treated with mild chemotherapy experienced progression. Seven patients with localized/4S disease underwent no chemotherapy: 4 of 5 patients with MYCN heterogeneity at diagnosis remained disease-free, and 1 of 5 experienced local progression. Two patients had normal MYCN status at diagnosis but acquired MYCN amplification during the course of the disease. CONCLUSION: MYCN heterogeneity is rare. Our results suggest that small amounts of MYCN-amplified cells are not correlated to adverse outcomes. More patients with heterogeneity are warranted to clarify the role of MYCN heterogeneity for risk classification.
Asunto(s)
Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Oncogenes , Amplificación de Genes , Humanos , Proteína Proto-Oncogénica N-Myc , Estadificación de Neoplasias , Neuroblastoma/patologíaRESUMEN
BACKGROUND: Amplified MYCN oncogene resulting in deregulated MYCN transcriptional activity is observed in 20% of neuroblastomas and identifies a highly aggressive subtype. In MYCN single-copy neuroblastomas, elevated MYCN mRNA and protein levels are paradoxically associated with a more favorable clinical phenotype, including disseminated tumors that subsequently regress spontaneously (stage 4s-non-amplified). In this study, we asked whether distinct transcriptional MYCN or c-MYC activities are associated with specific neuroblastoma phenotypes. RESULTS: We defined a core set of direct MYCN/c-MYC target genes by applying gene expression profiling and chromatin immunoprecipitation (ChIP, ChIP-chip) in neuroblastoma cells that allow conditional regulation of MYCN and c-MYC. Their transcript levels were analyzed in 251 primary neuroblastomas. Compared to localized-non-amplified neuroblastomas, MYCN/c-MYC target gene expression gradually increases from stage 4s-non-amplified through stage 4-non-amplified to MYCN amplified tumors. This was associated with MYCN activation in stage 4s-non-amplified and predominantly c-MYC activation in stage 4-non-amplified tumors. A defined set of MYCN/c-MYC target genes was induced in stage 4-non-amplified but not in stage 4s-non-amplified neuroblastomas. In line with this, high expression of a subset of MYCN/c-MYC target genes identifies a patient subtype with poor overall survival independent of the established risk markers amplified MYCN, disease stage, and age at diagnosis. CONCLUSIONS: High MYCN/c-MYC target gene expression is a hallmark of malignant neuroblastoma progression, which is predominantly driven by c-MYC in stage 4-non-amplified tumors. In contrast, moderate MYCN function gain in stage 4s-non-amplified tumors induces only a restricted set of target genes that is still compatible with spontaneous regression.
Asunto(s)
Neuroblastoma/genética , Neuroblastoma/patología , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transcripción Genética , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Genes myc , Proteína Proto-Oncogénica N-Myc , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Análisis de Supervivencia , Células Tumorales CultivadasRESUMEN
AFAR genes play a key role in the detoxification of the carcinogen Aflatoxin B(1) (AFB(1)). In the rat, Afar1 induction can prevent AFB(1)-induced liver cancer. It has been proposed that AFAR enzymes can metabolise endogenous diketones and dialdehydes that may be cytotoxic and/or genotoxic. Furthermore, human AFAR1 catalyses the rate limiting step in the synthesis of the neuromodulator gamma-hydroxybutyrate (GHB) and was found elevated in neurodegenerative diseases such as Alzheimer's and dementia with Lewy bodies (DLB). The human AFAR gene family maps to a genomic region in 1p36 of frequent hemizygous deletions in various human cancers. To investigate, if genetic variation of AFAR1 and AFAR2 exists that may alter protein detoxification capabilities and confer susceptibility to cancer, we have analysed a spectrum of human tumours and tumour cell lines for genetic heterogeneity. From 110 DNA samples, we identified nine different amino acid changes; two were in AFAR1 and seven in AFAR2. In AFAR1, we found genetic variation in the proposed substrate-binding amino acid 113, encoding Ala(113) or Thr(113). An AFAR2 variant had a Glu(55) substituted by Lys(55) at a position that is conserved among many aldo-keto reductases. This polarity change may have an effect on the proposed substrate binding amino acids nearby (Met(47), Tyr(48), Asp(50)). Further population analyses and functional studies of the nine variants detected may show if these variants are disease-related.
Asunto(s)
Aldehído Reductasa/genética , Variación Genética , Neoplasias/genética , Polimorfismo Genético , Aflatoxina B1/toxicidad , Sustitución de Aminoácidos , Animales , Carcinógenos/toxicidad , Línea Celular Tumoral , Mapeo Cromosómico , Cromosomas Humanos Par 1 , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/genética , ADN/genética , ADN/aislamiento & purificación , Cartilla de ADN , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Humanos , Familia de Multigenes , Neoplasias/enzimología , Polimorfismo Conformacional Retorcido-Simple , RatasRESUMEN
Neuroblastoma (NB) is the most commonly occurring solid tumor in children. The disease usually arises in the adrenal medulla, and it is characterized by a remarkable heterogeneity in its progression. Most NB patients with an advanced disease have massive bone marrow infiltration at diagnosis. Lung metastasis represents a widely disseminated stage and is typically considered to be a terminal event. Much like other malignancies, NB progression is a complex, multistep process. The expression, function, and significance of the various factors involved in NB progression must be studied in relevant in vivo and in vitro models. Currently, models consisting of metastatic and nonmetastatic cell variants of the same genetic background exist for several types of cancer; however, none exists for NB. In the present study, we describe the generation of a NB metastasis model. SH-SY5Y and MHH-NB-11 NB cells were inoculated orthotopically into the adrenal glands of athymic nude mice. Neuroblastoma cells metastasizing to the lungs were isolated from mice bearing adrenal tumors. Lung metastatic variants were generated by repeated cycles of in vivo passage. Characterization of these variants included cellular morphology and immunophenotyping in vitro, aggressiveness in vivo, and various biologic parameters in vitro. The NB metastatic variant in each model displayed unique properties, and both metastatic variants demonstrated a metastatic phenotype in vivo. These reproducible models of human NB metastasis will serve as an unlimited source of transcriptomic and proteomic material. Such models can facilitate future studies on NB metastasis and the identification of novel NB biomarkers and targets for therapy.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/patología , Modelos Animales de Enfermedad , Neoplasias Pulmonares/secundario , Neoplasias Experimentales/secundario , Neuroblastoma/secundario , Neoplasias de las Glándulas Suprarrenales/tratamiento farmacológico , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Deferoxamina/farmacología , Doxorrubicina/uso terapéutico , Ensayos de Selección de Medicamentos Antitumorales , Citometría de Flujo , Humanos , Inmunofenotipificación , Cariotipificación , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Tasa de Supervivencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
High incidence of chemotherapy resistance is the primary cause of treatment failure in a subset of neuroblastomas with amplified MYCN. We have reported previously that ectopic MYCN expression promotes proliferation of neuroblastoma Tet21N cells and simultaneously sensitizes them to the drug-induced apoptosis. In search for genes that are involved in MYCN-dependent regulation of drug resistance, we used a function-based gene cloning approach and identified CTSD encoding for a lysosomal aspartyl protease cathepsin D. Downregulation of cathepsin D expression by RNA interference or inhibition of its enzymatic activity increased sensitivity of MYCN-expressing Tet21N cells to doxorubicin. Overexpression of cathepsin D in Tet21N cells attenuated doxorubicin-induced apoptosis. It was accompanied by activation of protein kinase B (Akt) and persistent antiapoptotic activity of Bcl-2. In primary neuroblastomas, high CTSD messenger RNA (mRNA) levels were associated with amplified MYCN, a strong predictive marker of adverse outcome. Chromatin immunoprecipitation and luciferase promoter assays revealed that MYCN protein binds to the CTSD promoter and activates its transcription, suggesting a direct link between deregulated MYCN and CTSD mRNA expression. We further show that neuroblastoma cells can secrete mitogenic procathepsin D and that MYCN expression and especially doxorubicin treatment promote procathepsin D secretion. Extracellular exogenous cathepsin D induces Akt-1 phosphorylation and doxorubicin resistance in sensitive cells. These results demonstrate an important role of cathepsin D in antiapoptotic signaling in neuroblastoma cells and suggest a novel mechanism for the development of chemotherapy resistance in neuroblastoma.