Methylation-Associated Silencing of the Heat Shock Protein 47 Gene in Human Neuroblastoma

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Abstract
Hypermethylation of gene promoter CpG islands is a frequent mechanism for gene inactivation in a variety of human cancers, including neuroblastoma (NB). We demonstrated recently that treatment with the demethylating agent 5′-aza-2′-deoxycytidine (5-Aza-dC) significantly inhibited NB growth in vivo. In an effort to identify the genes and biological pathways that are responsible for the impaired NB tumor growth observed after treatment with 5-Aza-dC, we performed genome-wide gene expression analysis of control and treated NBL-W-S NB cells. We found ≥3-fold changes in expression of 44 genes that play roles in angiogenesis, apoptosis, cell adhesion, transcriptional regulation, and signal transduction. The gene encoding heat shock protein 47 (Hsp47), a collagen-specific molecular chaperon, was up-regulated >80-fold after 5-Aza-dC treatment. Expression studies confirmed that Hsp47 is silenced in a subset of NB cell lines and tumors. We also show that silencing of Hsp47 in NB cells is associated with aberrant methylation of promoter CpG islands and that Hsp47 expression can be restored after treatment with 5-Aza-dC. A strong correlation between Hsp47 and collagen type I and IV expression was seen in NB cells. Interestingly, tumorigenicity was inversely correlated with the level of collagen expression in NB cell lines, and higher levels of collagen were detected in mature NB tumors that are associated with favorable outcome compared with undifferentiated, advanced-stage NBs. Our studies support a role for Hsp47 in the regulation of collagen type I and IV production in NB cells and suggest that the level of collagen expression may influence NB tumor phenotype.