Neuroblastoma Tumor Suppressor Gene Research Articles

MiR-1271-5p promotes the growth and migration of neuroblastoma cells by regulating ACY-1.

Aminoacylase 1 (ACY-1) has been found to be a tumor suppressor gene in neuroblastoma (NB). This study aimed to identify and verify the microRNAs (miRNAs) that may regulate ACY-1 through database prediction analysis, and verify the mutual regulatory effect of miRNA and ACY-1 in NB through cell experiments. The miRNAs that might bind ACY-1 were predicted and selected by TargetScan, miRTarBase and four other databases, the expression of the predicted miRNAs and ACY-1 was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in four groups of clinical samples, and the differentially expressed miRNAs were screened. Then, luciferase vector was constructed according to the ACY-1 gene sequence to detect whether ACY-1 binds to the selected miRNA. Then, miR-1271-5p expression was silenced to detect miR-1271-5p function in the growth and migration of NB. Finally, ACY-1 and miR-1271-5p were silenced to change ACY-1 expression, and ACY-1 function in NB and the regulatory role of miR-1271-5p were explored. ACY-1 was downregulated in NB, miR-1271-5p was upregulated in NB, and miR-1271-5p could be targeted to ACY-1. Silencing miR-1271-5p expression can reduce cell viability and inhibit tumor progression. After interfering with ACY-1 expression in cells, cell viability was enhanced, apoptosis was significantly decreased, and migration and invasion were enhanced. After partially restoring ACY-1 expression, the effect of si-ACY-1 on cells was weakened. In SK-N-SH and SH-SY-5Y cells, the miR-1271-5p inhibitor restored ACY-1 expression and improved ACY-1 function. MiR-1271-5p can promote the growth and migration of tumor cells by inhibiting ACY-1 expression in NB.

MOXD1 is a lineage-specific gene and a tumor suppressor in neuroblastoma.

Neuroblastoma is a childhood developmental cancer; however, its embryonic origins remain poorly understood. Moreover, in-depth studies of early tumor-driving events are limited because of the lack of appropriate models. Herein, we analyzed RNA sequencing data obtained from human neuroblastoma samples and found that loss of expression of trunk neural crest-enriched gene MOXD1 associates with advanced disease and worse outcome. Further, by using single-cell RNA sequencing data of human neuroblastoma cells and fetal adrenal glands and creating in vivo models of zebrafish, chick, and mouse, we show that MOXD1 is a determinate of tumor development. In addition, we found that MOXD1 expression is highly conserved and restricted to mesenchymal neuroblastoma cells and Schwann cell precursors during healthy development. Our findings identify MOXD1 as a lineage-restricted tumor-suppressor gene in neuroblastoma, potentiating further stratification of these tumors and development of novel therapeutic interventions.

Exosomes from Plasma of Neuroblastoma Patients Contain Doublestranded DNA Reflecting the Mutational Status of Parental Tumor Cells.

Neuroblastoma (NB) is an aggressive infancy tumor, leading cause of death among preschool age diseases. Here we focused on characterization of exosomal DNA (exo-DNA) isolated from plasma cell-derived exosomes of neuroblastoma patients, and its potential use for detection of somatic mutations present in the parental tumor cells. Exosomes are small extracellular membrane vesicles secreted by most cells, playing an important role in intercellular communications. Using an enzymatic method, we provided evidence for the presence of double-stranded DNA in the NB exosomes. Moreover, by whole exome sequencing, we demonstrated that NB exo-DNA represents the entire exome and that it carries tumor-specific genetic mutations, including those occurring on known oncogenes and tumor suppressor genes in neuroblastoma (ALK, CHD5, SHANK2, PHOX2B, TERT, FGFR1, and BRAF). NB exo-DNA can be useful to identify variants responsible for acquired resistance, such as mutations of ALK, TP53, and RAS/MAPK genes that appear in relapsed patients. The possibility to isolate and to enrich NB derived exosomes from plasma using surface markers, and the quick and easy extraction of exo-DNA, gives this methodology a translational potential in the clinic. Exo-DNA can be an attractive non-invasive biomarker for NB molecular diagnostic, especially when tissue biopsy cannot be easily available.

11q Deletion or ALK Activity Curbs DLG2 Expression to Maintain an Undifferentiated State in Neuroblastoma

High-risk neuroblastomas typically display an undifferentiated or poorly differentiated morphology. It is therefore vital to understand molecular mechanisms that block the differentiation process. We identify an important role for oncogenic ALK-ERK1/2-SP1 signaling in the maintenance of undifferentiated neural crest-derived progenitors through the repression of DLG2, a candidate tumor suppressor gene in neuroblastoma. DLG2 is expressed in the murine "bridge signature" that represents the transcriptional transition state when neural crest cells or Schwann cell precursors differentiate to chromaffin cells of the adrenal gland. We show that the restoration of DLG2 expression spontaneously drives neuroblastoma cell differentiation, highlighting the importance of DLG2 in this process. These findings are supported by genetic analyses of high-risk 11q deletion neuroblastomas, which identified genetic lesions in the DLG2 gene. Our data also suggest that further exploration of other bridge genes may help elucidate the mechanisms underlying the differentiation of NC-derived progenitors and their contribution to neuroblastomas.

Genome‐wide DNA methylation analysis identifies MEGF10 as a novel epigenetically repressed candidate tumor suppressor gene in neuroblastoma

Neuroblastoma is a childhood cancer in which many children still have poor outcomes, emphasising the need to better understand its pathogenesis. Despite recent genome‐wide mutation analyses, many primary neuroblastomas do not contain recognizable driver mutations, implicating alternate molecular pathologies such as epigenetic alterations. To discover genes that become epigenetically deregulated during neuroblastoma tumorigenesis, we took the novel approach of comparing neuroblastomas to neural crest precursor cells, using genome‐wide DNA methylation analysis. We identified 93 genes that were significantly differentially methylated of which 26 (28%) were hypermethylated and 67 (72%) were hypomethylated. Concentrating on hypermethylated genes to identify candidate tumor suppressor loci, we found the cell engulfment and adhesion factor gene MEGF10 to be epigenetically repressed by DNA hypermethylation or by H3K27/K9 methylation in neuroblastoma cell lines. MEGF10 showed significantly down‐regulated expression in neuroblastoma tumor samples; furthermore patients with the lowest‐expressing tumors had reduced relapse‐free survival. Our functional studies showed that knock‐down of MEGF10 expression in neuroblastoma cell lines promoted cell growth, consistent with MEGF10 acting as a clinically relevant, epigenetically deregulated neuroblastoma tumor suppressor gene. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.

Altered expression of miRNAs and methylation of their promoters are correlated in neuroblastoma.

Neuroblastoma is the most common human extracranial solid tumor during infancy. Involvement of several miRNAs in its pathogenesis has been ascertained. Interestingly, most of their encoding genes reside in hypermethylated genomic regions: thus, their tumor suppressor function is normally disallowed in these tumors. To date, the therapeutic role of the demethylating agent 5′-Aza-2 deoxycytidine (5'-AZA) and its effects on miRNAome modulation in neuroblastoma have not been satisfactorily explored. Starting from a high-throughput expression profiling of 754 miRNAs and based on a proper selection, we focused on miR-29a-3p, miR-34b-3p, miR-181c-5p and miR-517a-3p as candidate miRNAs for our analysis. They resulted downregulated in four neuroblastoma cell lines with respect to normal adrenal gland. MiRNAs 29a-3p and 34b-3p also resulted downregulated in vivo in a murine neuroblastoma progression model. Unlike the amount of methylation of their encoding gene promoters, all these miRNAs were significantly overexpressed following treatment with 5′-AZA. Transfection with candidate miRNAs mimics significantly decreased neuroblastoma cells proliferation rate. A lower expression of miR-181c was significantly associated to a worse overall survival in a public dataset of 498 neuroblastoma samples (http://r2.amc.nl). Our data strongly suggest that CDK6, DNMT3A, DNMT3B are targets of miR-29a-3p, while CCNE2 and E2F3 are targets of miR-34b-3p. Based on all these data, we propose that miR-29a-3p, miR-34b-3p, miR-181c-5p and miR-517a-3p are disallowed tumor suppressor genes in neuroblastoma and suggest them as new therapeutic targets in neuroblastoma.

MiR-137 Modulates a Tumor Suppressor Network-Inducing Senescence in Pancreatic Cancer Cells.

Activating K-Ras mutations occurs frequently in pancreatic cancers and is implicated in their development. Cancer-initiating events, such as oncogenic Ras activation, lead to the induction of cellular senescence, a tumor suppressor response. During senescence, the decreased levels of KDM4A lysine demethylase contribute to p53 activation, however, the mechanism by which KDM4A is downregulated is unknown. We show that miR-137 targets KDM4A mRNA during Ras-induced senescence and activates both p53 and retinoblastoma (pRb) tumor suppressor pathways. Restoring the KDM4A expression contributed to bypass of miR-137-induced senescence and inhibition of endogenous miR-137 with an miRNA sponge-compromised Ras-induced senescence. miR-137 levels are significantly reduced in human pancreatic tumors, consistent with previous studies revealing a defective senescence response in this cancer type. Restoration of miR-137 expression inhibited proliferation and promoted senescence of pancreatic cancer cells. These results suggest that modulating levels of miR-137 may be important for triggering tumor suppressor networks in pancreatic cancer.

Correlation between expression profile of Wilms tumor 1 gene isoforms and neuroblastoma grade malignancy

Wilms tumor 1 gene (WT1) is expressed in neuroblastoma (NB) which represents the most aggressive extracranial pediatric tumor. This latter may transform into a more benign form such as ganglioneuroblastoma and ganglioneuroma or progress into a highly aggressive metastatic cancer with a poor survival rate. WT1 acts as tumor suppressor gene in NB by inducing the maturation in a less invasive mass. To date, it has been identified 13 mRNA WT1 variants encoding 13 proteins, however, most of the studies have focused their attention exclusively on isoform of ~49 kDa molecular weight (1, 2). In the present study, we have analyzed, the expression profile of WT1 isoforms, in undifferentiated and all-trans retinoic acid (RA) differentiated NB cells in order to evaluate their involvement in tumor malignancy. Results have shown that different isoforms are expressed both in untreated and RA treated NB cells. Their expression is significantly increased in RA treated cells, suggesting that WT1 isoforms are inversely related to NB malignancy grade. In accord to this hypothesis, WT1 isoforms and nestin expression are inversely related in undifferentiated and RA treated cells. Furthermore, the inhibition of the two signalling pathways specifically involved in differentiation of NB, PI3K/Akt and MAPK/ERK respectively, trigger an overexpression of all WT1 isoforms. In conclusion, these data suggest that overexpression of WT1 isoforms might promote trans-differentiation of NB into a more benign tumor such as ganglioneuroblastoma or ganglioneuroma.

Unlikely Suspects Identified in Neuroblastoma Conspiracy

KIF1B is a candidate tumor-suppressor gene in neuroblastoma whose function is to mediate apoptosis when nerve growth factor becomes limiting in the developing nervous system. Chen and colleagues now provide mechanistic insight into how one of the protein products of this locus, KIF1Bβ, induces apoptosis.

Abstract 2630: Tumor suppressor CASZ1 inhibits normal myoblast and rhabdomyosarcoma (RMS) migration.

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, and is thought to arise from primitive mesenchymal cells committed to skeletal muscle differentiation. Less than 30% of patients with metastatic disease survive even with the most intensive treatment regimens. Approximately 50% of patients with RMS have intermediate risk and the event-free survival of the intermediate risk patients is approximately 70%. However, even within this group, the survival of patients with metastatic embryonal tumors is much poorer (∼50%). Thus understanding the molecular mechanism of RMS metastasis and the development of therapeutic strategies to inhibit RMS metastasis is critical for these patients. We have reported that zinc finger transcription factor CASZ1 functions as a tumor suppressor gene in neuroblastoma. In these studies we found that CASZ1 regulates genes involved in cell movement and many pertaining to skeletal/muscle system development. To understand whether the induction of muscle developmental genes by CASZ1 was an anomaly associated with pathology, we evaluated the expression and regulation of CASZ1 in normal myoblasts. CASZ1 expression significantly increased when the C2C12 murine myoblast cell line differentiated upon serum withdrawal. Knockdown of CASZ1 in myoblasts causes a 30% increase in cell migration (p<0.02) as determined using an Essen IncuCyte scratch-wound healing assay. Furthermore knock-down of CASZ1 in C2C12 cells leads to a 50% decrease in expression of the skeletal muscle development gene myogenin while over-expression of CASZ1 leads to 2-fold increase in myogenin mRNA. Since CASZ1 regulates aspects of normal muscle development, we hypothesized that its dysregulation in RMS may impact tumor progression. To test this hypothesis, we first evaluated the expression of CASZ1 in primary RMS tumors. We found that CASZ1 mRNA level in embryonal RMS patients’ samples is ∼1.5 lower than in normal muscle or alveolar RMS (p<0.001). To investigate the role of CASZ1 in embryonal RMS cell growth and migration, we developed a Tet-On CASZ1 expression system using the RD RMS cell line. Overexpression of CASZ1 does not affect RD cell proliferation based on MTS and IncuCyte cell confluence assays. Over-expression of CASZ reduces cell motility by 20% (p<0.01) using an Essen IncuCyte scratch-wound healing assay. Knockdown of CASZ1 in RD cells increases cell migration by 40% (p<0.01) but does not affect cell proliferation. However when CASZ1 is expressed in the presence of a muscle inducing differentiation cue, 12-O-tetradecanoylphorbol-13-acetate, TPA, the proliferation of RD cells is suppressed an additional 40% (p<0.01). Taken together, our results suggest that regulation of myogenin expression and cell migration by CASZ1 in C2C12 maybe integral to proper myogenic differentiation, and dysregulation of CASZ1 during early myogenesis may contribute to embryonal RMS tumorigenesis and metastasis. Citation Format: Zhihui Liu, Wilson Chan, Carol J. Thiele. Tumor suppressor CASZ1 inhibits normal myoblast and rhabdomyosarcoma (RMS) migration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2630. doi:10.1158/1538-7445.AM2013-2630 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

MYCN is recruited to the RASSF1A promoter but is not critical for DNA hypermethylation in neuroblastoma

Tumor suppressor genes such as RASSF1A are often epigenetically repressed by DNA hypermethylation in neuroblastoma, where the MYCN proto-oncogene is frequently amplified. MYC has been shown to associate with DNA methyltransferases, thereby inducing transcriptional repression of target genes, which suggested that MYCN might play a similar mechanistic role in the hypermethylation of tumor suppressor genes in neuroblastoma. This study tested that hypothesis by using co-immunoprecipitation and ChIP to investigate MYCN-DNA methyltransferase interactions, together with MYCN knock-down and over-expression systems to examine the effect of MYCN expression changes on gene methylation, employing both candidate gene and genome-wide assays. We show that MYCN interacts with DNA methyltransferases and is recruited to the promoter region of RASSF1A. However, using four model systems, we showed that long-term silencing of MYCN induces only a small loss of DNA methylation at the RASSF1A promoter in MYCN amplified neuroblastoma cell lines and over-expression of MYCN does not induce any DNA methylation, suggesting that MYCN is not critical for DNA hypermethylation in neuroblastoma.

Identification and characterization of FoxP1 as a candidate tumor suppressor gene in neuroblastoma

To identify genes differenzially expressed between favourable and unfavourable neuroblastomas (NB), microarray profiles of 476 NB specimens were generated. High expression of FOXP1 (forkhead box P1) was found in favourable NBs. FOXP1 maps to a tumour suppressor locus frequently deleted aggressive NB (3p14) and belongs to a family of winged-helix transcription factors that are involved in processes of cell proliferation, differentiation and neoplastic transformation.

CAMTA1, a 1p36 Tumor Suppressor Candidate, Inhibits Growth and Activates Differentiation Programs in Neuroblastoma Cells

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.

Abstract 4880: CASZ1, a neuroblastoma tumor suppressor gene, is epigenetically silenced by EZH2

Abstract Casz1 is a C2H2 Zn-finger transcriptional factor that localizes to a region of LOH on Chr1p36.22 in tumors of Neuroblastoma patients who have a poor prognosis. Moreover, high CASZ1 expression is found in tumors of patients with a good prognosis while tumors from patients with a poor prognosis express low levels of CASZ1 (58 patients, p= 2.07×10-6). Studies indicate that CASZ1 functions as a tumor suppressor gene. Our finding that treatment of NB cells with HDAC inhibitors results in a 10-fold increase in CASZ1 expression suggests that epigenetic silencing contributes to the functional loss of CASZ1 expression from the remaining Chr1p36.22 allele. In studying the mechanism of CASZ1 epigenetic silencing, we found a bivalent chromatin modification region around the CASZ1 Transcription Start Site (TSS) that contains both a H3K27 methylation mark as well as an H3K4 methylation mark. The H3K27 methylation is thought to be a gene repressive marker, which is catalyzed by Polycomb repressor complex 2 (PRC2) proteins. PRC2 contains three core components, EZH2, EED and SUZ12, in which EZH2 (enhancer of zeste 2) is a methyltransferase catalyzing H3K27 methylation. Using Chromatin Immunoprecipitation (ChIP) we identified a 5-fold decrease in binding of EZH2, EED and SUZ12 to the bivalent region after HDAC inhibitor treatment. Consistent with decreased PRC2 binding is the finding of decreased tri-methylation of H3K27 and an increase in H3K4me3, a histone mark associated with transcriptionally active genes. The changes in histone modifications are associated with the activation of the CASZ1 gene. RNA silencing of EZH2 also leads to increases in CASZ1 expression. Consistent with this, CASZ1 expression is also up-regulated (3- fold) in EZH2 knockout MEFs compared to EZH2 wild type MEFs. This indicates that CASZ1 expression on the remaining allele is silenced by epigenetic modifications mediated by EZH2. High expression of EZH2 and EED are also found in tumor samples of neuroblastoma patients with a poor prognosis (58 patients, p=2.6×10-3 for EED and p=5.87×10-4 for EZH2). This is consistent with a model in which aberrant epigenetic gene silencing of the tumor suppressor gene CASZ1 contributes to neuroblastoma tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4880.

Reduced levels of miR‐34a in neuroblastoma are not caused by mutations in the TP53 binding site

Neuroblastoma (NB) is the most common extracranial solid tumor in children below the age of 5 years. miR-34a, located in chromosome band 1p36, has been recently implicated as a tumor suppressor gene in NB. In addition, it has been shown that miR-34a is activated by TP53 by binding to a TP53 binding site upstream to the mature miR-34a. We studied NB tumors from 57 patients for miR-34a expression levels, 1p status, mutations in the TP53 coding region and mutations of the TP53 binding site. Reduced expression levels of miR-34a were identified in tumors harboring 1p36.3 Loss (P = 0.028). No mutations were identified in the coding region of TP53, or in the TP53 binding site. Thus, mutations in the binding site are not an additional mechanism for the inactivation of miR-34a in NB. Other regulatory mechanisms controlling miR-34a expression and its relationship to TP53 should be further explored.

Aberrant methylation of candidate tumor suppressor genes in neuroblastoma

CpG island hypermethylation has been recognized as an alternative mechanism for tumor suppressor gene inactivation. In this study, we performed methylation-specific PCR (MSP) to investigate the methylation status of 10 selected tumor suppressor genes in neuroblastoma. Seven of the investigated genes ( CD44, RASSF1A, CASP8, PTEN, ZMYND10, CDH1, PRDM2) showed high frequencies (⩾30%) of methylation in 33 neuroblastoma cell lines. In 42 primary neuroblastoma tumors, the frequencies of methylation were 69%, CD44; 71%, RASSF1A; 56%, CASP8; 25%, PTEN; 15%, ZMYND10; 8%, CDH1; and 0%, PRDM2. Furthermore, CASP8 and CDH1 hypermethylation was significantly associated with poor event-free survival. Meta-analysis of 115 neuroblastoma tumors demonstrated a significant correlation between CASP8 methylation and MYCN amplification. In addition, there was a correlation between ZMYND10 methylation and MYCN amplification. The MSP data, together with optimized mRNA re-expression experiments (in terms of concentration and time of treatment and use of proper reference genes) further strengthen the notion that epigenetic alterations could play a significant role in NB oncogenesis. This study thus warrants the need for a global profiling of gene promoter hypermethylation to identify genome-wide aberrantly methylated genes in order to further understand neuroblastoma pathogenesis and to identify prognostic methylation markers.

A Functional Screen Identifies miR-34a as a Candidate Neuroblastoma Tumor Suppressor Gene

MicroRNAs are small noncoding RNAs that have critical roles in regulating a number of cellular functions through transcriptional silencing. They have been implicated as oncogenes and tumor suppressor genes (oncomirs) in several human neoplasms. We used an integrated genomics and functional screening strategy to identify potential oncomirs in the pediatric neoplasm neuroblastoma. We first identified microRNAs that map within chromosomal regions that we and others have defined as frequently deleted (1p36, 3p22, and 11q23-24) or gained (17q23) in high-risk neuroblastoma. We then transiently transfected microRNA precursor mimics or inhibitors into a panel of six neuroblastoma cell lines that we characterized for these genomic aberrations. The majority of transfections showed no phenotypic effect, but the miR-34a (1p36) and miR-34c (11q23) mimics showed dramatic growth inhibition in cell lines with 1p36 hemizygous deletion. In contrast, there was no growth inhibition by these mimics in cell lines without 1p36 deletions. Quantitative reverse transcription-PCR showed a perfect correlation of absent miR-34a expression in cell lines with a 1p36 aberration and phenotypic effect after mimetic add-back. Expression of miR-34a was also decreased in primary tumors (n = 54) with 1p36 deletion (P = 0.009), but no mutations were discovered in resequencing of the miR-34a locus in 30 neuroblastoma cell lines. Flow cytometric time series analyses showed that the likely mechanism of miR-34a growth inhibition is through cell cycle arrest followed by apoptosis. BCL2 and MYCN were identified as miR-34a targets and likely mediators of the tumor suppressor phenotypic effect. These data support miR-34a as a tumor suppressor gene in human neuroblastoma.

High resolution tiling‐path BAC array deletion mapping suggests commonly involved 3p21‐p22 tumor suppressor genes in neuroblastoma and more frequent tumors

The recurrent loss of 3p segments in neuroblastoma suggests the implication of 1 or more tumor suppressor genes but thus far few efforts have been made to pinpoint their detailed chromosomal position. To achieve this goal, array-based comparative genomic hybridization was performed on a panel of 23 neuroblastoma cell lines and 75 primary tumors using a tiling-path bacterial artificial chromosome array for chromosome 3p. A total of 45 chromosome 3 losses were detected, including whole chromosome losses, large terminal deletions and interstitial deletions. The latter, observed in cell lines as well as a number of distal deletions detected in primary tumors, allowed us to demarcate 3 minimal regions of loss of 3.6 Mb [3p21.31-p21.2, shortest regions of overlap (SRO)1], 1.4 Mb (3p22.3-3p22.2, SRO2) and 3.8 Mb (3p25.3-p25.1, SRO3) in size. The present data significantly extend previous findings and now firmly establish critical regions on 3p implicated in neuroblastoma. Interestingly, the 2 proximal regions coincide with previously defined SROs on 3p21.3 in more frequent tumors including lung and breast cancer. As such, similar tumor suppressor genes may play a critical role in development or progression of a variety of neoplasms, including neuroblastoma.

High-Resolution Analysis of Chromosomal Breakpoints and Genomic Instability Identifies PTPRD as a Candidate Tumor Suppressor Gene in Neuroblastoma

Although neuroblastoma is characterized by numerous recurrent, large-scale chromosomal imbalances, the genes targeted by such imbalances have remained elusive. We have applied whole-genome oligonucleotide array comparative genomic hybridization (median probe spacing 6 kb) to 56 neuroblastoma tumors and cell lines to identify genes involved with disease pathogenesis. This set of tumors was selected for having either 11q loss or MYCN amplification, abnormalities that define the two most common genetic subtypes of metastatic neuroblastoma. Our analyses have permitted us to map large-scale chromosomal imbalances and high-level amplifications at exon-level resolution and to identify novel microdeletions and duplications. Chromosomal breakpoints (n = 467) generating imbalances >2 Mb were mapped to intervals ranging between 6 and 50 kb in size, providing substantial information on each abnormality. For example, breakpoints leading to large-scale hemizygous loss of chromosome 11q were highly clustered and preferentially associated with segmental duplications. High-level amplifications of MYCN were extremely complex, often resulting in a series of discontinuous regions of amplification. Imbalances (n = 540) <2 Mb long were also detected. Although the majority (78%) of these imbalances mapped to segmentally duplicated regions and primarily reflect constitutional copy number polymorphisms, many subtle imbalances were detected that are likely somatically acquired alterations and include genes involved with tumorigenesis, apoptosis, or neural cell differentiation. The most frequent microdeletion involved the PTPRD locus, indicating a possible tumor suppressor function for this gene.

Homozygous deletion in a neuroblastoma cell line defined by a high-density STS map spanning human chromosome band 1p36.

Recent molecular studies have shown a relatively high rate of loss of heterozygosity (LOH) in neuroblastoma (NB) as well as other types of tumors in human chromosome band 1p36. To identify candidate tumor suppressor genes in NB, we searched for homozygous deletions in NB cell lines with PCR according to a high-density sequence tagged site (STS)-content map spanning 1p35-36. Among 25 NB cell lines examined, only one cell line, NB-1, showed no signal with 27 STSs in a 480 kb region in 1p36.2. The sequence analysis has revealed that the defective region included seven known genes (E4, KIF1B, SCYA5, PGD, Cortistatin, DFF45, and PEX14), nine expressed sequence tags (ESTs), and two microsatellite markers. These genes are related to apoptosis, an ubiquitin-proteasome pathway, a neuronal microtubule-associated motor molecule, and components of a common translocation machinery. The region between the DFF45 and KIF1B genes was defined as homozygous deletion by Southern blotting. The search in LOH regions with high-density STSs may be useful for the isolation and identification of tumor suppressor genes in other tumors as well as NBs.