Modulation of neuroblastoma disease pathogenesis by an extensive network of epigenetically regulated microRNAs.

MicroRNAs (miRNAs) contribute to the pathogenesis of many forms of cancer, including the pediatric cancer neuroblastoma, but the underlying mechanisms leading to altered miRNA expression are often unknown. Here, a novel integrated approach for analyzing DNA methylation coupled with miRNA and mRNA expression data sets identified 67 epigenetically regulated miRNA in neuroblastoma. A large proportion (42%) of these miRNAs was associated with poor patient survival when underexpressed in tumors. Moreover, we demonstrate that this panel of epigenetically silenced miRNAs targets a large set of genes that are overexpressed in tumors from patients with poor survival in a highly redundant manner. The genes targeted by the epigenetically regulated miRNAs are enriched for a number of biological processes, including regulation of cell differentiation. Functional studies involving ectopic overexpression of several of the epigenetically silenced miRNAs had a negative impact on neuroblastoma cell viability, providing further support to the concept that inactivation of these miRNAs is important for neuroblastoma disease pathogenesis. One locus, miR-340, induced either differentiation or apoptosis in a cell context dependent manner, indicating a tumor suppressive function for this miRNA. Intriguingly, it was determined that miR-340 is upregulated by demethylation of an upstream genomic region that occurs during the process of neuroblastoma cell differentiation induced by all-trans retinoic acid (ATRA). Further biological studies of miR-340 revealed that it directly represses the SOX2 transcription factor by targeting of its 3'-untranslated region, explaining the mechanism by which SOX2 is downregulated by ATRA. Although SOX2 contributes to the maintenance of stem cells in an undifferentiated state, we demonstrate that miR-340-mediated downregulation of SOX2 is not required for ATRA induced differentiation to occur. In summary, our results exemplify the dynamic nature of the miRNA epigenome and identify a remarkable network of miRNA/mRNA interactions that significantly contribute to neuroblastoma disease pathogenesis.

Oncogenic activation of FOXR1 by 11q23 intrachromosomal deletion-fusions in neuroblastoma.

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.

Three chromosomal rearrangements in neuroblastoma cluster within a 300-kb region on 1p36.1.

Deletions in the short arm of chromosome 1 (1p36) and MYCN amplification are common in neuroblastoma. Previously we showed evidence of at least two different neuroblastoma tumor-suppressor loci on 1p. One is associated with MYCN single-copy tumors and maps distal on 1p36.3. A second, more proximal locus maps to 1p36.1 and is deleted in about 90% of neuroblastomas with MYCN amplification. The cell line UHG-NP has the smallest 1p36 deletion of all neuroblastoma cell lines with MYCN amplifications. We assume that the more proximal locus maps within this deletion, close to its proximal border. Here we present the exact localization of the 1p deletion breakpoint of UHG-NP. A 600-kb PAC contig spanning the breakpoint was analyzed for genes and aberrations. Two more neuroblastoma-associated aberrations were mapped within 150 kb of the UHG-NP breakpoint. Within the contig, we identified nine genes expressed in neuroblastoma cells. One of these genes, AML2, maps 200 kb distal to the UHG-NP breakpoint but is expressed only rarely in neuroblastoma and showed no mutations.

N-myc enhances the expression of a large set of genes functioning in ribosome biogenesis and protein synthesis.

The myc oncogenes are frequently activated in human tumors, but there is no comprehensive insight into the target genes and downstream cellular pathways of these transcription factors. We applied serial analysis of gene expression (SAGE) to identify targets of N-myc in neuroblastomas. Analysis of 42,000 mRNA transcript tags in SAGE libraries of N-myc- transfected and control neuroblastoma cells revealed 114 up-regulated genes. The majority of these genes have a role in ribosome assembly and activity. Northern blot analysis confirmed up-regulation of all tested transcripts. Induction was complete within 4 h after N-myc expression. The large majority of the ribosomal proteins were induced, as well as genes controlling rRNA maturation. Cellular rRNA content was 45% induced. SAGE libraries and northern blot analysis confirmed up-regulation of many of these genes in N-myc-amplified neuroblastomas. As N-myc can functionally replace c-myc, we analyzed whether N-myc targets were induced by c-myc as well. Approximately 40% of these N-myc targets were up-regulated in a c-myc-transfected melanoma cell line. These data suggest that myc genes function as major regulators of the protein synthesis machinery.

The MEIS1 oncogene is highly expressed in neuroblastoma and amplified in cell line IMR32.

Neuroblastoma is an embryonal tumor originating from neural crest-derived cells. Here we present the serendipitous cloning of amplified sequences of chromosome 2p15 in neuroblastoma cell line IMR32. The amplified region was analyzed for oncogene activation using a SAGE (serial analysis of gene expression) library of IMR32. SAGE permits a quantitative analysis of all transcripts of a tissue or cell line. The expression of genes and ESTs mapping within a 30-cR region covering the amplicon was compared to 4 additional SAGE libraries of neuroblastomas and 12 SAGE libraries of other tissues in the CGAP databases. The IMR32 SAGE database revealed increased expression of the MEIS1 oncogene, whereas other SAGE libraries showed little or no MEIS1 expression. MEIS1 turned out to be highly amplified and overexpressed in IMR32. Analysis of 24 neuroblastoma cell lines and 22 tumors showed high-level expression in about 25% of the cases. The MEIS1 homeobox protein forms a complex with the HOXA9 and PBX proteins that are implicated in human leukemia. MEIS1 is a target of retroviral insertion in murine leukemia. This is the first report of a MEIS1 amplification and high expression levels in human cancer and the first time that identification of a candidate target of amplification is facilitated by high-throughput mRNA expression profiling.

Chromosome bands 1p35-36 contain two distinct neuroblastoma tumor suppressor loci, one of which is imprinted.

A previous loss of heterozygosity (LOH) study of a series of 91 neuroblastomas suggested that the 1p35-36 region encodes at least two tumor suppressor genes (TSGs) of importance in neuroblastoma development. Here we present the results of a study including 205 neuroblastomas that were analyzed for LOH at chromosome 1 and MYCN amplification. The results corroborate the existence of two TSGs on 1p. Distinct 1p loci seem to be involved in MYCN single copy vs. MYCN amplified neuroblastoma, as these tumors display a different type of shortest region of overlap (SRO). About 15% of MYCN single copy neuroblastomas show 1p deletions of variable length with an SRO of 47 cR at 1p36.3. The lost alleles are preferentially of maternal origin (P = 0.0002), suggesting parental imprinting of the locus. MYCN amplified neuroblastomas have a contrasting pattern of 1p loss. These tumors display much larger deletions of at least 89 cR comprising the region from 1p36.1 to the telomere. LOH of 1p is detected in 86% of the cases. The lost alleles are of random parental origin, suggesting inactivation of a non-imprinted TSG.

An integrated 5-Mb physical, genetic, and radiation hybrid map of a 1p36.1 region implicated in neuroblastoma pathogenesis.

Common genetic aberrations of neuroblastoma are deletions of the short arm of chromosome 1 (1p36) and MYCN amplification. Our deletion analysis of 25 tumor cell lines and 171 tumors strongly suggests that 1p harbors several tumor suppressor loci. Distinct loci are involved in MYCN single-copy versus MYCN-amplified neuroblastoma. Deletions in MYCN single-copy tumors have a shortest region of overlap (SRO) of 20 cM at 1p36.3. MYCN-amplified tumors have large deletions with an SRO of about 60 cM, from 1p36.1 to the telomere. This SRO is defined by D1S7 (1p36.1), which was the most distal locus retained. Therefore, a suppressor gene associated with MYCN-amplified tumors probably maps within a few megabases distal of D1S7. In order to map this locus, we further refined this SRO. We mapped the breakpoint of the MYCN-amplified neuroblastoma with the smallest 1p deletion between 56.6 and 57.2 cM from 1pter. Pulsed-field gel electrophoresis and radiation hybrid mapping were used to construct a 5-Mb physical map of this region. The map includes the region from 82.73 till 92.89 cR from 1pter. About half of it was isolated in P1 and PAC clones. The region harbors the genes FGR, SLC9A1, HMG17, EXTL1, AML2, RH, OP18, four ESTs, and a newly identified gene with a transcript size of approximately 7 Kb. Several of the mapped genes have a putative role in cell growth, differentiation, and morphogenesis. Genes Chromosomes Cancer 27:143-152, 2000.

Allelic loss of the short arm of chromosome 4 in neuroblastoma suggests a novel tumour suppressor gene locus.

Neuroblastoma is a childhood neural crest tumour, genetically characterized by frequent deletions of the short arm of chromosome 1 and amplification of N-myc. Here we report the first evidence for a neuroblastoma tumour suppressor locus on 4pter. Cytogenetically we demonstrated rearrangements of 4p in 7 out of 26 evaluable tumours (27%). Subsequent analysis of loss of heterozygosity (LOH) by Southern blotting revealed allelic loss of 4p in 16/82 (19.5%) informative neuroblastomas. Taken together cytogenetic and Southern blot analyses showed loss of 4p in 20/86 neuroblastomas analysed (23%). The common deleted region was bordered by the probe D4S123 and encompassed the distal 34 cM of 4p. We found no evidence for genomic imprinting of the 4p locus as the 4p alleles lost in the tumours were of random maternal and paternal origin. LOH4p was found at all disease stages and in every age group. Furthermore LOH4p was present both in cases with and without LOH1p and amplification of N-myc.

Allelic loss of chromosome 1p as a predictor of unfavorable outcome in patients with neuroblastoma.

BACKGROUND: Neuroblastoma is a childhood tumor derived from cells of the neural crest, with a widely variable outcome. Differences in the behavior and prognosis of the tumor suggest that neuroblastoma can be divided into several biologic subgroups. We evaluated the most frequent genetic abnormalities in neuroblastoma to determine their prognostic value. METHODS: We used Southern blot analysis to study the allelic loss of chromosomes 1p, 4p, 11q, and 14q, the duplication of chromosome 17q, and the amplification of the N-myc oncogene in 89 neuroblastomas. We also determined the nuclear DNA content of the tumor cells. RESULTS: Allelic loss of chromosome 1p, N-myc amplification, and extra copies of chromosome 17q were significantly associated with unfavorable outcome. In a multivariate analysis, loss of chromosome 1p was the most powerful prognostic factor. It provided strong prognostic information when it was included in multivariate models containing the prognostic factors of age and stage or serum ferritin level and stage. Among the patients with stage I, II, or IVS disease, the mean (+/- SD) three-year event-free survival was 100 percent in those without allelic loss of chromosome 1p and 34 +/- 15 percent in those with such loss; the rates of three-year event-free survival among the patients with stage III and stage IV disease were 53 +/- 10 percent and 0 percent, respectively. CONCLUSIONS: The loss of chromosome 1p is a strong prognostic factor in patients with neuroblastoma, independently of age and stage. It reliably identifies patients at high risk in stages I, II, and IVS, which are otherwise clinically favorable. More intensive therapy may be considered in these patients. Patients in stages III and IV with allelic loss of chromosome 1p have a very poor outlook, whereas those without such loss are at moderate risk.

Evidence for two tumour suppressor loci on chromosomal bands 1p35-36 involved in neuroblastoma: one probably imprinted, another associated with N-myc amplification.

Previous reports on possible genomic imprinting of the neuroblastoma tumour suppressor gene on chromosome 1p36 have been conflicting. Here we report on the parental origin of 1p36 alleles lost in 47 neuroblastomas and on a detailed Southern blot analysis of the extent of the 1p deletions in 38 cases. The results are remarkably different for tumours with and without N-myc amplification. In the N-myc single copy tumours we show that the lost 1p36 alleles are of preferential maternal origin (16 of 17 cases) and that the commonly deleted region maps to 1p36.2-3. In contrast, all N-myc amplified neuroblastomas have larger 1p deletions, extending from the telomere to at least 1p35-36.1. These deletions are of random parental origin (18 of 30 maternal LOH). This strongly suggests that different suppressor genes on 1p are inactivated in these two types of neuroblastoma. Deletion of a more proximal suppressor gene is associated with N-myc amplification, while a distal, probably imprinted, suppressor can be deleted in N-myc single copy cases.

Recurrent 1;17 translocations in human neuroblastoma reveal nonhomologous mitotic recombination during the S/G2 phase as a novel mechanism for loss of heterozygosity.

Neuroblastomas often show loss of heterozygosity of the chromosomal region 1p36 (LOH 1p), probably reflecting loss of a tumor-suppressor gene. Here we describe three neuroblastoma tumors and two cell lines in which LOH 1p results from an unbalanced translocation between the p arm of chromosome 1 and the q arm of chromosome 17. Southern blot and cytogenetic analyses show that in all cases the chromosome 17 homologue from which the 1;17 translocation was derived is still present and intact. This suggests a model in which a translocation between the short arm of chromosome 1 and the long arm of chromosome 17 takes place in the S/G2 phase of the cell cycle and results in LOH 1p. Nonhomologous mitotic recombination in the S/G2 phase is a novel mechanism of LOH.

Allelic loss of chromosome 1p36 in neuroblastoma is of preferential maternal origin and correlates with N-myc amplification.

Neuroblastomas frequently have deletions of chromosome 1p and amplification of the N-myc oncogene. We analysed 53 neuroblastomas for the N-myc copy number, loss of heterozygosity (LOH) of chromosome 1p36 and the parental origin of the lost alleles. Allelic loss of 1p36 was found in 15 tumours. All N-myc amplified tumours belonged to this subset. In 13/15 tumours with LOH of 1p36 the lost allele was of maternal origin. This non-random distribution implies that the two alleles of the putative neuroblastoma suppressor gene on chromosome 1p36 are functionally different, depending on their parental origin. This is the first evidence as far as we know for genomic imprinting on chromosome 1p.

Inactivation of transforming Bacillus subtilis deoxyribonucleic acid by monoadducts of 4,5',8-trimethylpsoralen.

4,5',8-Trimethylpsoralen (TMP) monoadducts inactive transforming deoxyribonucleic acid (DNA) in Bacillus subtilis. Contrary to TMP diadducts (TMP cross-links), which severely inhibit entry of donor DNA (G. Venema and U. Canosi, Mol. Gen. Genet. 179:1--11), TMP monoadducts have only a slight effect on entry. Since reextracted TMP-monoadduct-containing transforming DNA is a differentially repaired by Uvr- and Uvr+ recipients and cross-linkable to the recipient strand in the heteroduplex recombinant DNA molecules, the monoadducts can be integrated along with the donor DNA into the recipient chromosome.