Journal of the National Cancer Institute Advance Access originally published online on June 24, 2008
JNCI Journal of the National Cancer Institute 2008 100(13):940-949; doi:10.1093/jnci/djn176
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ARTICLES |
CHD5, a Tumor Suppressor Gene Deleted From 1p36.31 in Neuroblastomas
Affiliations of authors: Division of Oncology (TF, JI, ERO, TG, JM, VK, JK, JMM, PSW, GMB), Biostatistics and Data Management Core, Department of Pediatrics (HZ), and Department of Pathology (BRP), The Children's Hospital of Philadelphia, The University of Pennsylvania, Philadelphia, PA; Children's Oncology Group Statistics and Data Center, University of Florida, Gainesville, FL (WBL)
Correspondence to: Garrett M. Brodeur, MD, Division of Oncology, The Children's Hospital of Philadelphia, 3615 Civic Center Blvd, ARC Rm 902D, Philadelphia, PA 19104-4318 (e-mail: brodeur{at}chop.edu).
Background: Neuroblastomas are characterized by hemizygous 1p deletions, suggesting that a tumor suppressor gene resides in this region. We previously mapped the smallest region of consistent deletion to a 2-Mb region of 1p36.31 that encodes 23 genes. Based on mutation analysis, expression pattern, and putative function, we identified CHD5 as the best tumor suppressor gene candidate.
Methods: We determined the methylation status of the CHD5 gene promoter in NLF and IMR5 (with 1p deletion) and SK-N-SH and SK-N-FI neuroblastoma cell lines using methylation-specific sequencing and measured CHD5 mRNA expression by reverse transcription polymerase chain reaction in cells treated with or without 5-aza-2-deoxycytidine, an inhibitor of DNA methylation. We transfected the cells with CHD5 and antisense (AS) CHD5 DNA to assess the effect of CHD5 overexpression and suppression, respectively, on colony formation in soft agar and growth of xenograft tumors in athymic mice. We also analyzed the association of CDH5 expression with outcomes of 99 neuroblastoma patients. Statistical tests were two-sided.
Results: CHD5 expression was very low or absent in neuroblastoma cell lines. The CHD5 promoter was highly methylated in NLF and IMR5 lines, and CHD5 expression increased after treatment with 5-aza-2-deoxycytidine. Clonogenicity and tumor growth were abrogated in NLF and IMR5 cells overexpressing CHD5 compared with antisense CHD5 (clonogenicity: mean no. of colonies per plate, NLF-CHD5, 43 colonies, 95% confidence interval [CI] = 35 to 51 colonies, vs NLF-CHD5-AS, 74 colonies, 95% CI = 62 to 86 colonies, P < .001; IMR5-CHD5, 11 colonies, 95% CI = 2 to 20 colonies, vs IMR5-CHD5-AS, 39 colonies, 95% CI = 17 to 60 colonies, P = .01; tumor growth, n = 10 mice per group: mean tumor size at 5 weeks, NLF-CHD5, 0.36 cm3, 95% CI = 0.17 to 0.44 cm3, vs NLF-CHD5-AS, 1.65 cm3, 95% CI = 0.83 to 2.46 cm3, P = .002; IMR5-CHD5, 0.28 cm3, 95% CI = 0.18 to 0.38 cm3, vs IMR5-CHD5-AS, 1.15 cm3, 95% CI = 0.43 to 1.87 cm3; P = .01). High CHD5 expression was strongly associated with favorable event-free and overall survival (P < .001), even after correction for MYCN amplification and 1p deletion (P = .027).
Conclusions: CHD5 is the strongest candidate tumor suppressor gene that is deleted from 1p36.31 in neuroblastomas, and inactivation of the second allele may occur by an epigenetic mechanism.
| CONTEXT AND CAVEATS Prior knowledge Neuroblastoma is a childhood cancer that is characterized as having genomic deletions at chromosome 1p. A neuroblastoma tumor suppressor gene may lie in this region, and based on previous studies, CHD5 is a candidate. Study design CHD5 promoter methylation and expression in human neuroblastoma cell lines and effects of CHD5 overexpression on tumor growth in mouse models were assayed. Associations between CHD5 expression and clinical outcomes of 99 neuroblastoma patients were determined. Contributions CHD5 expression was low in the cell lines, and the CHD5 promoter was highly methylated. Overexpression of CHD5 slowed tumor growth in mouse models. CHD5 expression was strongly associated with increased event-free and overall survival of neuroblastoma patients. Implications CHD5 may be a neuroblastoma tumor suppressor gene, and its expression may be inhibited by promoter methylation. Limitations Other genes that are located in the region of the 1p deletions still need to be studied.
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T. Fujita and J. Igarashi contributed equally to this work.
The sponsors had no role in the study design, data collection and analysis, interpretation of the results, the preparation of the manuscript, or the decision to submit the manuscript for publication.
Manuscript received December 4, 2007; revised April 25, 2008; accepted April 30, 2008.
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