Abstract
BackgroundEpigenetic aberrations and a CpG island methylator phenotype have been shown to be associated with poor outcomes in children with neuroblastoma (NB). Seven cancer related genes (THBS-1, CASP8, HIN-1, TIG-1, BLU, SPARC, and HIC-1) that have been shown to have epigenetic changes in adult cancers and play important roles in the regulation of angiogenesis, tumor growth, and apoptosis were analyzed to investigate the role epigenetic alterations play in determining NB phenotype.MethodsTwo NB cell lines (tumorigenic LA1-55n and non-tumorigenic LA1-5s) that differ in their ability to form colonies in soft agar and tumors in nude mice were used. Quantitative RNA expression analyses were performed on seven genes in LA1-5s, LA1-55n and 5-Aza-dC treated LA1-55n NB cell lines. The methylation status around THBS-1, HIN-1, TIG-1 and CASP8 promoters was examined using methylation specific PCR. Chromatin immunoprecipitation assay was used to examine histone modifications along the THBS-1 promoter. Luciferase assay was used to determine THBS-1 promoter activity. Cell proliferation assay was used to examine the effect of 5-Aza-dC on NB cell growth. The soft agar assay was used to determine the tumorigenicity.ResultsPromoter methylation values for THBS-1, HIN-1, TIG-1, and CASP8 were higher in LA1-55n cells compared to LA1-5s cells. Consistent with the promoter methylation status, lower levels of gene expression were detected in the LA1-55n cells. Histone marks associated with repressive chromatin states (H3K9Me3, H3K27Me3, and H3K4Me3) were identified in the THBS-1 promoter region in the LA1-55n cells, but not the LA1-5s cells. In contrast, the three histone codes associated with an active chromatin state (acetyl H3, acetyl H4, and H3K4Me3) were present in the THBS-1 promoter region in LA1-5s cells, but not the LA1-55n cells, suggesting that an accessible chromatin structure is important for THBS-1 expression. We also show that 5-Aza-dC treatment of LA1-55n cells alters the DNA methylation status and the histone code in the THBS-1 promoter modifies cell morphology, and inhibits their ability to form colonies in soft agar.ConclusionOur results suggest that epigenetic aberrations contribute to NB phenotype, and that tumorigenic properties can be inhibited by reversing the epigenetic changes with 5-Aza-dC.
Highlights
Epigenetic aberrations and a CpG island methylator phenotype have been shown to be associated with poor outcomes in children with neuroblastoma (NB)
Expression analysis of cancer related genes To investigate the relationship between epigenetic changes and NB phenotype, we analyzed the level of expression of seven genes with tumor suppressor function that are epigenetically silenced in a variety of adult and pediatric cancers (THBS-1, HIC-1, High in Normal 1 (HIN-1) Tazarotene-induced gene 1 (TIG-1), CASP8, BLU, and Secreted protein (SPARC)), in a tumorigenic N-type NB cell line (LA1-55n) and a non-tumorigenic S-type cell line (LA1-5s)
Lower levels of THBS-1, SPARC, HIC-1, HIN-1, TIG-1 and CASP8 expression were detected in the tumorigenic LA1-55n cells compared to non-tumorigenic LA1-5s cells (Figure 1A)
Summary
Epigenetic aberrations and a CpG island methylator phenotype have been shown to be associated with poor outcomes in children with neuroblastoma (NB). Biologic differences are commonly seen in NB cell lines established from human NB tumors, and morphologically distinct cell types [neuroblastic (N-type) and substrate-adherent (S-type)] have been extensively characterized. N-type cells have small, rounded, loosely adherent cell bodies with numerous neurite-like processes and express neuronal markers, such as tyrosine hydroxylase. S-type cells are substrate-adherent large, flat cells that resemble epithelial cells or fibroblasts and lack neuronal markers [7,8]. Purified N-type and S-type NB cells can spontaneously interconvert from one cell type to the other, suggesting that epigenetic changes that are reversible may play a role in determining phenotype
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