Expression and sequence analysis of candidates for the 1p36.31 tumor suppressor gene deleted in neuroblastomas

Neuroblastomas are characterized by 1p deletions, suggesting that a tumor suppressor gene (TSG) resides in this region. We have mapped the smallest region of deletion (SRD) to a 2 Mb region of 1p36.31 using microsatellite and single nucleotide polymorphisms. We have identified 23 genes in this region, and we have analysed these genes for mutations and RNA expression patterns to identify candidate TSGs. We sequenced the coding exons of these genes in 30 neuroblastoma cell lines. Although rare mutations were found in 10 of the 23 genes, none showed a pattern of genetic change consistent with homozygous inactivation. We examined the expression of these 23 genes in 20 neuroblastoma cell lines, and most showed readily detectable expression, and no correlation with 1p deletion. However, 7 genes showed uniformly low expression in the lines, and 2 genes (CHD5, RNF207) had virtually absent expression, consistent with the expected pattern for a TSG. Our mutation and expression analysis in neuroblastoma cell lines, combined with expression analysis in normal tissues, putative function and prior implication in neuroblastoma pathogenesis, suggests that the most promising TSG deleted from the 1p36 SRD is CHD5, but TNFRSF25, CAMTA1 and AJAP1 are also viable candidates.     

Homozygous deletion of CDKN2A (p16INK4a/p14ARF) but not within 1p36 or at other tumor suppressor loci in neuroblastoma

Loss of heterozygosity of several specific genomic regions is frequently observed in neuroblastoma tumors and cell lines, but homozygous deletion (HD) is rare, and no neuroblastoma tumor suppressor gene (TSG) has yet been identified. We performed a systematic search for HD, indicative of a disrupted TSG, in a panel of 46 neuroblastoma cell lines. An initial search focused on a well-characterized consensus region of hemizygous deletion at 1p36.3, which occurs in 35% of primary neuroblastomas. Each cell line was screened with 162 1p36 markers, for a resolution of 13 kb within the consensus 1p36.3 deletion region and 350 kb throughout the remainder of 1p36. No HDs were detected. This approach was expanded to survey 21 known TSGs, specifically targeting intragenic regions frequently inactivated in other malignancies. HD was detected only at the CDKN2A (p16INK4a/p14ARF) gene at 9p21 and was observed in 4 of 46 cell lines. The observed region of HD included all exons of both CDKN2A and the closely linked CDKN2B (p15INK4b) gene for cell lines LA-N-6 and CHLA-174, all exons of CDKN2A but none of CDKN2B for CHLA-179, and only 104 bp within CDKN2A exon 2 for CHLA-101. All four deletions are predicted to inactivate the coding regions of both p16INK4a and p14ARF. HD was observed in corresponding primary tumor samples for CHLA-101 and CHLA-174 but was not present in constitutional samples. These results suggest that for neuroblastoma, large HDs do not occur within 1p36, most known TSGs are not homozygously deleted, and biallelic inactivation of CDKN2A may contribute to tumorigenicity in a subset of cases.     

No evidence for the presence of an imprinted neuroblastoma suppressor gene within chromosome sub-band 1p36.3

Deletion of the distal short arm of chromosome 1 occurs in 35% of primary neuroblastomas (NBs). These deletions tend to be large and extend to the telomere, but a common region within sub-band 1p36.3 is consistently lost. Despite intensive investigation, no candidate tumor suppressor gene within this region has been shown to undergo tumor-specific mutation consistent with biallelic inactivation. In addition, initial studies demonstrated preferential loss of the maternally inherited 1p homologue in NBs with 1p loss of heterozygosity (LOH) without MYCN amplification. This has led to the widely accepted hypothesis that a genomically imprinted NB suppressor gene is the target of 1p deletion in this subset. To test this hypothesis we have studied 293 primary NBs for LOH within 1p36.3 and determined the parental origin of the deleted 1p homologue. LOH within 1p36.3 was demonstrated in 55 NBs (19%). Of these, 29 occurred in tumors without MYCN amplification: 13 had deletion of the maternally inherited 1p, whereas 16 had deletion of the paternally inherited 1p (P = 0.58). These data strongly refute a parent-of-origin effect for 1p deletions in NB and exclude the existence of an imprinted NB suppressor locus in this region.     

N-myc modulates expression of p73 in neuroblastoma

The human p73 gene is a homolog of p53, which has been localized to chromosome 1p36 in a region that is frequently deleted in neuroblastoma. Transfection of the p73 gene into neuroblastoma cells that lack detectable p73 protein has been shown to result in growth suppression and to induce neuronal differentiation. In this study, we have identified by means of restriction landmark genome scanning (RLGS) a genomic fragment that was frequently reduced in intensity in neuroblastomas. The cloned fragment contained exon 1 of p73 as well as intronic and promoter sequences. We investigated the genomic and expression status of p73 and N-myc in 34 neuroblastoma tumors and 12 neuroblastoma cell lines. Approximately a third of neuroblastomas in our series exhibited deletion of p73. Most tumors analyzed exhibited reduced expression of p73, as determined by quantitative RT-PCR, in the absence of detectable p73 gene deletion. The reduced expression of p73 correlated with overexpression of N-myc in a statistically significant manner. The N-myc gene was transfected into two neuroblastoma cell lines that lacked N-myc amplification to determine its effect on p73 RNA levels. p73 was detectable at low level by RT-PCR in untransfected SK-N-AS cells and became undetectable following N-myc transfection, whereas in SH-EP1 cells, p73 levels were substantially reduced following transfection but remained detectable. Our data suggest that the N-myc gene modulates expression of p73, allowing neuroblastoma cells to escape the growth suppressing properties of p73.     

Identification of a novel recurrent 1q42.2-1qter deletion in high risk MYCN single copy 11q deleted neuroblastomas

Neuroblastoma is an aggressive embryonal tumor that accounts for ～15% of childhood cancer deaths. Hitherto, despite the availability of comprehensive genomic data on DNA copy number changes in neuroblastoma, relatively little is known about the genes driving neuroblastoma tumorigenesis. In this study, high resolution array comparative genome hybridization (CGH) was performed on 188 primary neuroblastoma tumors and 33 neuroblastoma cell lines to search for previously undetected recurrent DNA copy number gains and losses. A new recurrent distal chromosome 1q deletion (del(1)(q42.2qter)) was detected in seven cases. Further analysis of available array CGH datasets revealed 13 additional similar distal 1q deletions. The majority of all detected 1q deletions was found in high risk 11q deleted tumors without MYCN amplification (Fisher exact test p = 5.61 × 10(-5) ). Using ultra-high resolution (～115 bp resolution) custom arrays covering the breakpoints on 1q for 11 samples, clustering of nine breakpoints was observed within a 12.5-kb region, of which eight were found in a 7-kb copy number variable region, whereas the remaining two breakpoints were colocated 1.4-Mb proximal. The commonly deleted region contains one miRNA (hsa-mir-1537), four transcribed ultra conserved region elements (uc.43-uc.46) and 130 protein coding genes including at least two bona fide tumor suppressor genes, EGLN1 (or PHD2) and FH. This finding further contributes to the delineation of the genomic profile of aggressive neuroblastoma, offers perspectives for the identification of genes contributing to the disease phenotype and may be relevant in the light of assessment of response to new molecular treatments.     

Molecular analysis of the putative tumour-suppressor gene EXTL1 in neuroblastoma patients and cell lines

Although neuroblastoma is the most common extracranial solid tumour of childhood, little is known about its aetiology. Together with MYCN amplification and chromosome 17q gain, chromosome 1p deletion is one of the most frequently occurring genetic abnormalities in neuroblastoma. Based upon mapping of deletion breakpoints, putative tumour suppressor gene loci have been assigned to the distal part of the short arm of chromosome 1. Recently, the EXTL1 gene was suggested as a candidate neuroblastoma-suppressor gene and to evaluate this hypothesis, we performed 1p deletion analysis and mutation screening of the EXTL1-coding region on DNA from 22 primary neuroblastomas and 21 neuroblastoma cell lines. Deletions of the chromosome region 1p36.1, including the EXTL1 gene, were detected in several neuroblastoma cell lines and primary tumours. EXTL1 mutation screening resulted in the detection of one unclassified variant (Ser28Cys) but could not provide additional evidence of EXTL1 being involved in the aetiology of neuroblastoma.     

Analysis of location and integrity of the human PITSLRE (p58(cdc2L1)) genes in neuroblastoma cell genomes

The family of PITSLRE kinase genes, located in chromosome 1p36, has recently been associated with neuroblastoma tumorigenesis. In order to evaluate the role of these genes as putative tumor suppressor genes, we have analyzed the integrity of the coding region in primary tumors and its location relative to a neuroblastoma consensus deletion. A subset of aggressive neuroblastoma tumors with allelic loss of different parts of chromosome 1p were investigated. Single-strand conformation polymorphism (SSCP), heteroduplex (HD) and sequencing analysis of tumor DNA did not reveal any significant changes in the coding region. In particular, a primary tumor with an interstitial allelic deletion in 1p36 did not reveal concomitant loss of heterozygosity of the PITSLRE gene region when analyzed with a C/T DNA sequence polymorphism in exon 5 of PITSLRE1. FISH analysis on neuroblastoma cell lines with small interstitial deletions and with a balanced translocation in 1p36 revealed that the PITSLRE gene cluster was localized distal to the neuroblastoma consensus deletion. against an involvement of the PITSLRE genes in neuroblastoma tumorigenesis.     

Caspase-9 and Apaf-1 are expressed and functionally active in human neuroblastoma tumor cell lines with 1p36 LOH and amplified MYCN

Important roles have been suggested for caspase-8, caspase-9 and Apaf-1 in controlling tumor development and their sensitivity to chemotherapeutic agents. Methylation and deletion of Apaf-1 and CASP8 results in the loss of their expression in melanoma and neuroblastoma, respectively, while CASP9 localization to 1p36.1 suggests it is a good candidate tumor suppressor. The status of CASP9 and Apaf-1 expression in numerous neuroblastoma cell lines with/without amplified MYCN and chromosome 1p36 loss-of-heterozygosity (LOH) was therefore examined to test the hypothesis that one or both of these genes are tumor suppressors in neuroblastoma. Although CASP9 is included in the region encompassing 1p36 LOH in all neuroblastoma cell lines examined, the remaining CASP9 allele(s) express a functional caspase-9 enzyme. Apaf-1 is also expressed in all neuroblastoma tumor cell lines examined. Thus, the CASP9 or Apaf-1 genes do not appear to function as tumor suppressors in MYCN amplified neuroblastomas. However, approximately 20% of the neuroblastoma cell lines with methylated CASP8 alleles are also highly resistant to staurosporine (STS)- and radiation-induced cell death, presumably because cytochrome c is not released from mitochondria. This suggests that a second, smaller sub-group of MYCN amplified neuroblastoma tumors exists with defect(s) in apoptotic signaling components upstream of caspase-9 and Apaf-1. Since no consistent differences in Bcl-2, Bcl-x(L) or Bax expression were seen in the STS- and radiation-resistant neuroblastomas, it suggests that a unique mitochondrial signaling factor(s) is responsible for the defect in cytochrome c release in this sub-group of tumors.     

Identification and characterization of a 500-kb homozygously deleted region at 1p36.2-p36.3 in a neuroblastoma cell line

Loss of heterozygosity of the distal region of chromosome 1p where tumor suppressor gene(s) might harbor is frequently observed in many human cancers including neuroblastoma (NBL) with MYCN amplification and poor prognosis. We have identified for the first time a homozygously deleted region at the marker D1S244 within the smallest region of overlap at 1p36.2-p36.3 in two NBL cell lines, NB-1 and NB-C201 (MASS-NB-SCH1), although our genotyping has suggested the possibility that both lines are derived from the same origin. The 800-kb PAC contig covering the entire region of homozygous deletion was made and partially sequenced (about 60%). The estimated length of the deleted region was 500 kb. We have, thus far, identified six genes within the region which include three known genes (DFF45, PGD, and CORT) as well as three other genes which have been reported during processing our present project for the last 3(1/2) years (HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14). They include the genes related to apoptosis, glucose metabolism, ubiquitin-proteasome pathway, a neuronal microtubule-associated motor molecule and biogenesis of peroxisome. At least three genes (HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14) were differentially expressed at high levels in favorable and at low levels in unfavorable subsets of primary neuroblastoma. Since the 1p distal region is reported to be imprinted, those differentially expressed genes could be the new members of the candidate NBL suppressor, although RT-PCR-SSCP analysis has demonstrated infrequent mutation of the genes so far identified. Full-sequencing and gene prediction for the region of homozygous deletion would elucidate more detailed structure of this region and might lead to discovery of additional candidate genes. Oncogene (2000) 19, 4302 - 4307     

Allelic variants of CAMTA1 and FLJ10737 within a commonly deleted region at 1p36 in neuroblastoma

Deletion of a distal portion of 1p is seen in a wide range of human malignancies, including neuroblastoma. Here, a 1p36.3 commonly deleted region of 216 kb has been defined encompassing two genes, CAMTA1 and FLJ10737. Low expression of CAMTA1 has been recently shown to be an independent predictor of poor outcome in neuroblastoma patients. The present study surveys CAMTA1 and FLJ10737 for genetic alterations by fluorescence-based single strand conformation polymorphism (SSCP) using a panel of DNAs from 88 neuroblastomas, their matching blood samples and 97 unaffected individuals. Nucleotide variants encoding amino acid substitutions were found in both genes. One CAMTA1 variant (T1336I) was not detected in 97 unaffected individuals, another (N1177K) resides in a conserved domain of the CAMTA1 protein and was found hemizygous in six neuroblastomas. We found no evidence for somatic mutations in FLJ10737 or CAMTA1. Further investigations are needed to address the functional impact of the identified variants and their possible significance for neuroblastoma.     

RASSF1A promoter region CpG island hypermethylation in phaeochromocytomas and neuroblastoma tumours.

Deletions of chromosome 3p are frequent in many types of neoplasia including neural crest tumours such as neuroblastoma (NB) and phaeochromocytoma. Recently we isolated several candidate tumour suppressor genes (TSGs) from a 120 kb critical interval at 3p21.3 defined by overlapping homozygous deletions in lung and breast tumour lines. Although mutation analysis of candidate TSGs in lung and breast cancers revealed only rare mutations, expression of one of the genes (RASSF1A) was absent in the majority of lung tumour cell lines analysed. Subsequently methylation of a CpG island in the promoter region of RASSF1A was demonstrated in a majority of small cell lung carcinomas and to a lesser extent in non-small cell lung carcinomas. To investigate the role of 3p TSGs in neural crest tumours, we (a) analysed phaeochromocytomas for 3p allele loss (n=41) and RASSF1A methylation (n=23) and (b) investigated 67 neuroblastomas for RASSF1A inactivation. 46% of phaeochromocytomas showed 3p allele loss (38.5% at 3p21.3). RASSF1A promoter region hypermethylation was found in 22% (5/23) of sporadic phaeochromocytomas and in 55% (37/67) of neuroblastomas analysed but RASSF1A mutations were not identified. In two neuroblastoma cell lines, methylation of RASSF1A correlated with loss of RASSF1A expression and RASSF1A expression was restored after treatment with the demethylating agent 5-azacytidine. As frequent methylation of the CASP8 gene has also been reported in neuroblastoma, we investigated whether RASSF1A and CASP8 methylation were independent or related events. CASP8 methylation was detected in 56% of neuroblastomas with RASSF1A methylation and 17% without RASSF1A methylation (P=0.0031). These results indicate that (a) RASSF1A inactivation by hypermethylation is a frequent event in neural crest tumorigenesis, particularly neuroblastoma, and that RASSF1A is a candidate 3p21.3 neuroblastoma TSG and (b) a subset of neuroblastomas may be characterized by a CpG island methylator phenotype.     

CHD5, a new member of the chromodomain gene family,is preferentially expressed in the nervous system

Chromatin remodeling is one of the mechanisms by which gene expression is regulated developmentally. Chromatin structure is controlled at least in part by post-translational modification of histones, as well as by chromodomain proteins. We have identified a novel gene encoding a protein with chromatin remodeling, helicase and DNA-binding motifs. This gene, called CHD5, is the fifth member of the CHD gene family identified in humans. This gene is most homologous to CHD3 and CHD4, which encode proteins that are part of the nucleosome remodeling and histone deacetylation (NuRD) complex. CHD5 is preferentially expressed in total brain, fetal brain, and cerebellum. It is also moderately expressed in the adrenal gland, but expression is undetectable in almost all other tissues examined. CHD5 maps within a small region of deletion on 1p36.3 in human neuroblastomas, a common pediatric tumor. We examined a panel of neuroblastoma cell lines for CHD5 expression, which was consistently low or undetectable in all these lines. Expression was also examined in a panel of 137 primary neuroblastomas, and low expression was highly correlated with 1p deletion, MYCN amplification, advanced stage, and unfavorable histology. These findings suggest that this gene may play a role in the development of the nervous system, and it may also play a role in the pathogenesis of neural tumors.     

Patched 2, located in 1p32-34, is not mutated in high stage neuroblastoma tumors

Neuroblastoma is a childhood malignancy originating from cells of the sympathetic nervous system, exhibiting a marked diversity in outcome, with spontaneous regression at one end of the spectrum and severe disease and death at the other end. Features associated with frequent recurrence, a poor prognosis, and high tumor stage are loss of heterozygosity in the distal region of chromosome 1p and amplification of the N-myc gene. Patched 2 is a novel homologue to the tumor suppressor gene Patched 1, and has been mapped to 1p32-34, a part of chromosome 1 frequently deleted in high stage neuroblastoma tumors. RT-PCR analysis of 9 neuroblastoma cell lines showed expression of both Patched 1 and 2. We analyzed 14, mainly high stage, neuroblastoma tumors for mutations in the Patched 2 gene with denaturing HPLC using the Wave DNA fragment analysis system. In four tumor samples variations were detected within the coding sequence, and two of them gave rise to amino-acid substitutions. These variations were, however, also detected in normal DNA from the respective patients. We conclude that Patched 2 is expressed, but not frequently mutated, in high stage neuroblastomas and is therefore not likely to be involved in the genesis of this tumor.     

High delta-like 1 expression in a subset of neuroblastoma cell lines corresponds to a differentiated chromaffin cell type

The childhood tumour neuroblastoma originates from neural crest-derived progenitors of the sympathetic nervous system. By Serial Analysis of Gene Expression (SAGE), we previously identified the Drosophila Delta homologue Dlk1 as one of the genes most highly expressed in the neuroblastoma cell line SK-N-FI. The Delta-Notch pathway controls many differentiation steps in Drosophila and man. We analysed expression of 21 genes of this pathway in 21 neuroblastoma cell lines. Dlk1 expression was very high in 5 cell lines, while another subset expressed Notch3. The imprinting of Dlk1 was faithfully preserved in neuroblastomas. The single paternal allele can therefore produce over 0.5% of all cellular mRNAs. Dlk1 maps to 14q32, a region that exhibited LOH in 31/170 (18%) tumours. The random parental origin of the deleted alleles excluded Dlk1 as target of the LOH. In addition, Dlk1 was not amplified, rearranged or mutated in neuroblastoma cell lines and tumours. We therefore analysed whether high Dlk1 expression marks a specific differentiation stage of the sympatho-adrenal lineage. Many neuroblastomas arise in the adrenal medulla, which predominantly consists of chromaffin cells. Normal adrenal medulla exhibited equally high Dlk1 levels as the SK-N-FI cell line. Chromaffin cells in young children are marked by noradrenalin production, which is mediated by dopamine-beta-hydroxylase (DBH). DBH expression in the neuroblastoma cell lines almost perfectly corresponded to Dlk1 expression. Neuroblastoma cell lines with high Dlk1 expression are therefore arrested in a relatively late stage of chromaffin lineage differentiation, while Notch3 expression might correspond to earlier precursor stages or to an alternative developmental fate.     

DLC-1 gene inhibits human breast cancer cell growth and in vivo tumorigenicity

The human DLC-1 (deleted in liver cancer 1) gene was cloned from a primary human hepatocellular carcinoma (HCC) and mapped to the chromosome 8p21-22 region frequently deleted in common human cancers and suspected to harbor tumor suppressor genes. DLC-1 was found to be deleted or downregulated in a significant number of HCCs. We expanded our investigations to other cancers with recurrent deletions of 8p22, and in this study examined alterations of DLC-1 in primary human breast tumors, human breast, colon, and prostate tumor cell lines. Genomic deletion of DLC-1 was observed in 40% of primary breast tumors, whereas reduced or undetectable levels of DLC-1 mRNA were seen in 70% of breast, 70% of colon, and 50% of prostate tumor cell lines To see whether DLC-1 expression affects cell growth and tumorigenicity, two breast carcinoma cell lines lacking the expression of endogenous gene were transfected with the DLC-1 cDNA. In both cell lines, DLC-1 transfection caused significant growth inhibition and reduction of colony formation. Furthermore, introduction of the DLC-1 cDNA abolished the in vivo tumorigenicity in nude mice, suggesting that the DLC-1 gene plays a role in breast cancer by acting as a bona fide tumor suppressor gene.     

Gene expression profiling of 1p35-36 genes in neuroblastoma

Deletion of the chromosome 1p36 region is a frequent abnormality in neuroblastoma. To gain further insights into the role of this alteration in oncogenesis, we have constructed a specific cDNA microarray representing most known genes and ESTs from the 1p35-36 region and analysed the expression profiles of 15 neuroblastoma cell lines and 28 neuroblastoma tumours. Hierarchical clustering using expression levels of 320 cDNAs from 1p35-36 separated localized or 4S cases without 1p deletion from advanced stages and cell lines. Supervised learning classification enabled to predict reliably the status of chromosome 1p according to its expression profile. Around 15% of the genes or ESTs presented a significantly decreased expression in samples with 1p deletion as compared to 1p-normal samples suggesting that 1p deletion results in a gene dosage effect on a subset of genes critical for the development of 1p-deleted neuroblastoma. Several genes presumed to have functions in neural differentiation (CDC42, VAMP3, CLSTN1), signal transduction in neural cells (GNB1) and cell cycle regulation (STMN1, RPA2, RBAF600, FBXO6, MAD2L2) exhibited a decreased expression in samples presenting 1p deletion. The identification of such genes provides baseline information for further studies to elucidate how these genes could individually or collectively play a critical role in neuroblastoma tumorigenesis.