Original articleHigh-resolution analysis of 3p deletion in neuroblastoma and differential methylation of the SEMA3B tumor suppressor gene
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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2016, Drug Resistance UpdatesCitation Excerpt :Sema3A also inhibited the anchorage-independent proliferation of triple negative breast cancer cells as did additional class-3 semaphorins such as sema3D and sema3F but not sema3E (Kigel et al., 2008). The sema3B gene was identified along with sema3F as a tumor suppressor whose function is lost in small cell lung carcinoma cells by a variety of mechanisms that include promoter methylation and loss of heterozygosity (Tomizawa et al., 2001; Kuroki et al., 2003; Nair et al., 2007; Campioni et al., 2008; Chen et al., 2014; Loginov et al., 2015; Gao et al., 2015). Single nucleotide polymorphisms in the sema3B gene were also found to be associated with poor prognosis of prostate cancer (Beuten et al., 2009).
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2013, Experimental Cell ResearchCitation Excerpt :It is known that Semas regulate the immune system [87] and modulate the function of TAMs, contributing to the regulation of tumor angiogenesis. For instance, Sema3B, considered potential tumor suppressor and down-modulated in many cancers [53,54], surprisingly, can promote metastasis formation by recruiting TAMs to the tumors. Indeed, Sema3B induces the production of IL-8 in cancer cells that contribute to the recruitment of TAMs that sustain tumor angiogenesis and progression [88].
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2013, Seminars in Cell and Developmental BiologyCitation Excerpt :Moreover, there are recurrent reports of the methylation of CpG islands upstream of TSS of Sema3B in lung cancer, glioma and malignant liver tumors, although it is not yet been associated with early or late stages of the disease [53–56]. Sema3B promoter hypermethylation is furthermore observed in neuroblastoma and renal cell carcinoma [54,57]. In liver cancer Sema3B promoter hypermethylation is found in advanced metastatic stages [49].