Original article
High-resolution analysis of 3p deletion in neuroblastoma and differential methylation of the SEMA3B tumor suppressor gene

https://doi.org/10.1016/j.cancergencyto.2006.11.017Get rights and content

Abstract

Large-scale hemizygous loss of chromosome 3p is a common event in neuroblastoma, occurring preferentially in tumors that exhibit loss of chromosome 11q and lack MYCN amplification. Although numerous tumor suppressor genes (TSG) have been mapped to the 3p region, the gene or genes contributing to neuroblastoma pathogenesis have remained elusive. High-resolution oligonucleotide array CGH mapping of chromosome 3p breakpoints relative to the positions of known TSGs indicates that more than one gene may contribute to neuroblastoma pathogenesis. We evaluated the methylation status of semaphorin 3B (SEMA3B), one of the chromosome 3p TSGs, in neuroblastoma tumors with (n = 12) and without (n = 32) 3p deletions. A significantly higher percentage of methylated CpG sites in the SEMA3B promoter was detected in tumors exhibiting 3p loss (95%), relative to tumors without loss (52%), suggestive of a two-hit mechanism of allele inactivation. The involvement of methylation in the control of SEMA3B expression was confirmed by treatment of neuroblastoma cell lines with the demethylating agent 5-aza-2-deoxycytidine. Transcriptional regulation of this locus is complex, however; low levels of SEMA3B expression were also seen in tumors with unmethylated SEMA3B promoters (n = 4). SEMA3B is known to play an important role in the development of normal sympathetic neurons, and interestingly, we found higher levels of SEMA3B expression in differentiated tumors with favorable histopathology (n = 19) than in tumors with unfavorable histology (n = 22). Furthermore, SEMA3B was upregulated in the SK-N-BE neuroblastoma cell line following induction of differentiation with retinoic acid. The association of SEMA3B expression with neuroblastoma differentiation suggests that this TSG may play a role in neuroblastoma pathobiology.

Introduction

Neuroblastoma is remarkable for its broad spectrum of clinical behavior, ranging from spontaneous regression to rapidly progressive disease and death, despite intensive multimodal therapy. This clinical heterogeneity is correlated, to some extent, with genetic abnormalities found in the tumors. At least three major genetic subtypes of the disease exist, including hyperdiploid tumors, which generally are associated with favorable outcomes; MYCN-amplified tumors, which have poor prognosis; and tumors characterized by hemizygous loss of chromosome 11q material, which are associated with poor survival. (For a review, see reference [1].) Each of these genetic subtypes is characterized by significantly different global gene expression profiles [2]. The 11q− genetic subtype is preferentially associated with loss of chromosome 3p, which occurs in ∼50% of the 11q− tumors [3], [4], [5]. Loss of 3p likely occurs secondarily to loss of 11q [6] and is preferentially associated with tumors from older children [5], [7].

Although a number of tumor suppressor genes (TSGs) mapping to the 3p region have been identified in various forms of cancer [8], [9], [10], [11], [12], direct evidence for the involvement of any 3p tumor suppressor gene in neuroblastoma pathogenesis has remained elusive. Semaphorin 3B (SEMA3B), a TSG that maps to chromosome 3p21 [11], has a number of features that make it an intriguing candidate TSG in neuroblastoma. First, this gene plays an important role in the development of sympathetic neurons (axon guidance) [13], and a class III semaphorin is involved with mediating the early stages of neuronal apoptosis during development [14]. SEMA3B also significantly suppresses tumorigenicity when exogenously introduced into ovarian and lung cancer cell lines by the induction of apoptosis [11], [15]. Second, we have demonstrated that SEMA3B, even though highly expressed in ganglioneuroma (a differentiated benign neuroblastic tumor) is underexpressed in malignant neuroblastoma [2]. The association of SEMA3B expression with a differentiated phenotype, and absence of expression with malignancy, is consistent with the hypothesis that SEMA3B plays a role in neuroblastoma pathobiology.

In the present study, we examined patterns of methylation of CpG dinucleotides in the SEMA3B promoter region, along with quantitative expression analysis and high-resolution mapping of 3p deletion breakpoints, to further assess the possibility that inactivation of this gene is important for neuroblastoma pathogenesis.

Section snippets

Cell lines and tissues

Methylation studies were performed on genomic DNA isolated from 44 primary neuroblastoma tumor tissues, 6 neuroblastoma cell lines [SK-N-AS, SK-N-BE, Kelly (N206), IMR32, NGP, NB69], 1 neuroepithelioma cell line (SK-N-MC), and 2 human fibroblast cell lines (MJ90 and IMR90). Primary tumors were obtained from tumor banks either at Children's Oncology Group (Philadelphia, PA) or Our Lady's Hospital for Sick Children (Dublin, Ireland). Some primary tumors and cell lines used in this study have been

High-resolution mapping of chromosome 3p deletion breakpoints

We performed high-resolution oligonucleotide array CGH (oaCGH) to precisely map breakpoints leading to hemizygous loss of chromosome 3p material, and to identify potential cryptic homozygous deletions that were missed on lower resolution analyses. A total of 19 primary tumors and 3 cell lines known to have large scale hemizygous loss of chromosome 3p material were analyzed. Each breakpoint could be mapped to either a 25 kb or 50 kb interval, as documented in Table 1 and illustrated in Figure 1.

Discussion

The chromosome 3p deletion mapping data presented in this report, along with data from the literature, indicate that more than one TSG on chromosome 3p may contribute to neuroblastoma pathogenesis since a single shortest region of loss could not be identified. Homozygous deletions, which have helped identify TSGs in other forms of cancer, were not evident on chromosome 3p for any of the tumors studied here. The only homozygous deletion of chromosome 3p in neuroblastoma is an 8 Mb region between

Acknowledgments

We gratefully acknowledge Dr. John Maris and the COG for providing nucleic acids from some of the tumors used in this study, as well as the expertise of the UTHSCSA DNA Sequencing Core Facility. We thank Drs. Sharon Murphy and Vivian Rebel for critical reading of the manuscript.

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