Publications (23) View all
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Article: Functional MYCN signature predicts outcome of neuroblastoma irrespective of MYCN amplification.
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ABSTRACT: Neuroblastoma is a pediatric tumor of the sympathetic nervous system. MYCN (V-myc myelocytomatosis viral-related oncogene, neuroblastoma derived [avian]) is amplified in 20% of neuroblastomas, and these tumors carry a poor prognosis. However, tumors without MYCN amplification also may have a poor outcome. Here, we identified downstream targets of MYCN by shRNA-mediated silencing MYCN in neuroblastoma cells. From these targets, 157 genes showed an expression profile correlating with MYCN mRNA levels in NB88, a series of 88 neuroblastoma tumors, and therefore represent in vivo relevant MYCN pathway genes. This 157-gene signature identified very poor prognosis tumors in NB88 and independent neuroblastoma cohorts and was more powerful than MYCN amplification or MYCN expression alone. Remarkably, this signature also identified poor outcome of a group of tumors without MYCN amplification. Most of these tumors have low MYCN mRNA levels but high nuclear MYCN protein levels, suggesting stabilization of MYCN at the protein level. One tumor has an MYC amplification and high MYC expression. Chip-on-chip analyses showed that most genes in this signature are directly regulated by MYCN. MYCN induces genes functioning in cell cycle and DNA repair while repressing neuronal differentiation genes. The functional MYCN-157 signature recognizes classical neuroblastoma with MYCN amplification, as well as a newly identified group marked by MYCN protein stabilization.Proceedings of the National Academy of Sciences 10/2012; · 9.68 Impact Factor -
Article: Identification of BIRC6 as a novel intervention target for neuroblastoma therapy.
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ABSTRACT: BACKGROUND: Neuroblastoma are pediatric tumors of the sympathetic nervous system with a poor prognosis. Apoptosis is often deregulated in cancer cells, but only a few defects in apoptotic routes have been identified in neuroblastoma. METHODS: Here we investigated genomic aberrations affecting genes of the intrinsic apoptotic pathway in neuroblastoma. We analyzed DNA profiling data (CGH and SNP arrays) and mRNA expression data of 31 genes of the intrinsic apoptotic pathway in a dataset of 88 neuroblastoma tumors using the R2 bioinformatic platform (http://r2.amc.nl). BIRC6 was selected for further analysis as a tumor driving gene. Knockdown experiments were performed using BIRC6 lentiviral shRNA and phenotype responses were analyzed by western blot and MTT-assays. In addition, DIABLO levels and interactions were investigated with immunofluorescence and co-immunoprecipitation. RESULTS: We observed frequent gain of the BIRC6 gene on chromosome 2, which resulted in increased mRNA expression. BIRC6 is an inhibitor of apoptosis protein (IAP), that can bind and degrade the cytoplasmic fraction of the pro-apoptotic protein DIABLO. DIABLO mRNA expression was exceptionally high in neuroblastoma but the protein was only detected in the mitochondria. Upon silencing of BIRC6 by shRNA, DIABLO protein levels increased and cells went into apoptosis. Co-immunoprecipitation confirmed direct interaction between DIABLO and BIRC6 in neuroblastoma cell lines. CONCLUSION: Our findings indicate that BIRC6 may have a potential oncogenic role in neuroblastoma by inactivating cytoplasmic DIABLO. BIRC6 inhibition may therefore provide a means for therapeutic intervention in neuroblastoma.BMC Cancer 07/2012; 12(1):285. · 3.01 Impact Factor -
Article: Oncogenic activation of FOXR1 by 11q23 intrachromosomal deletion-fusions in neuroblastoma.
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ABSTRACT: Neuroblastoma tumors frequently show loss of heterozygosity of chromosome 11q with a shortest region of overlap in the 11q23 region. These deletions are thought to cause inactivation of tumor suppressor genes leading to haploinsufficiency. Alternatively, micro-deletions could lead to gene fusion products that are tumor driving. To identify such events we analyzed a series of neuroblastomas by comparative genomic hybridization and single-nucleotide polymorphism arrays and integrated these data with Affymetrix mRNA profiling data with the bioinformatic tool R2 (http://r2.amc.nl). We identified three neuroblastoma samples with small interstitial deletions at 11q23, upstream of the forkhead-box R1 transcription factor (FOXR1). Genes at the proximal side of the deletion were fused to FOXR1, resulting in fusion transcripts of MLL-FOXR1 and PAFAH1B2-FOXR1. FOXR1 expression has only been detected in early embryogenesis. Affymetrix microarray analysis showed high FOXR1 mRNA expression exclusively in the neuroblastomas with micro-deletions and rare cases of other tumor types, including osteosarcoma cell line HOS. RNAi silencing of FOXR1 strongly inhibited proliferation of HOS cells and triggered apoptosis. Expression profiling of these cells and reporter assays suggested that FOXR1 is a negative regulator of fork-head box factor-mediated transcription. The neural crest stem cell line JoMa1 proliferates in culture conditional to activity of a MYC-ER transgene. Over-expression of the wild-type FOXR1 could functionally replace MYC and drive proliferation of JoMa1. We conclude that FOXR1 is recurrently activated in neuroblastoma by intrachromosomal deletion/fusion events, resulting in overexpression of fusion transcripts. Forkhead-box transcription factors have not been previously implicated in neuroblastoma pathogenesis. Furthermore, this is the first identification of intrachromosomal fusion genes in neuroblastoma.Oncogene 08/2011; 31(12):1571-81. · 6.37 Impact Factor -
Article: Cyclin D1 is a direct transcriptional target of GATA3 in neuroblastoma tumor cells.
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ABSTRACT: Almost all neuroblastoma tumors express excess levels of Cyclin D1 (CCND1) compared to normal tissues and other tumor types. Only a small percentage of these neuroblastoma tumors have high-level amplification of the Cyclin D1 gene. The other neuroblastoma tumors have equally high Cyclin D1 expression without amplification. Silencing of Cyclin D1 expression was previously found to trigger differentiation of neuroblastoma cells. Overexpression of Cyclin D1 is therefore one of the most frequent mechanisms with a postulated function in neuroblastoma pathogenesis. The cause for the Cyclin D1 overexpression is unknown. Here we show that Cyclin D1 overexpression results from transcriptional upregulation. To identify upstream regulators, we searched in mRNA profiles of neuroblastoma tumor series for transcription factors with expression patterns correlating to Cyclin D1. GATA3 most consistently correlated to Cyclin D1 in four independent data sets. We identified a highly conserved GATA3 binding site 27 bp upstream of the Cyclin D1 transcriptional start. Chromatin immune precipitation confirmed binding of GATA3 to the Cyclin D1 promoter. Overexpression of GATA3 induced Cyclin D1 promoter activity, which decreased after site-directed mutagenesis of the GATA3 binding site in the Cyclin D1 promoter. Silencing of GATA3 resulted in reduced Cyclin D1 promoter activity and reduced Cyclin D1 mRNA and protein levels. Moreover, GATA3 silencing caused differentiation that was similar to that caused by Cyclin D1 inhibition. These finding implicate GATA3 in Cyclin D1 overexpression in neuroblastoma.Oncogene 02/2010; 29(18):2739-45. · 6.37 Impact Factor -
Article: LIN28B induces neuroblastoma and enhances MYCN levels via let-7 suppression.
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ABSTRACT: LIN28B regulates developmental processes by modulating microRNAs (miRNAs) of the let-7 family. A role for LIN28B in cancer has been proposed but has not been established in vivo. Here, we report that LIN28B showed genomic aberrations and extensive overexpression in high-risk neuroblastoma compared to several other tumor entities and normal tissues. High LIN28B expression was an independent risk factor for adverse outcome in neuroblastoma. LIN28B signaled through repression of the let-7 miRNAs and consequently resulted in elevated MYCN protein expression in neuroblastoma cells. LIN28B-let-7-MYCN signaling blocked differentiation of normal neuroblasts and neuroblastoma cells. These findings were fully recapitulated in a mouse model in which LIN28B expression in the sympathetic adrenergic lineage induced development of neuroblastomas marked by low let-7 miRNA levels and high MYCN protein expression. Interference with this pathway might offer therapeutic perspectives.Nature Genetics 10/2012; · 35.53 Impact Factor
