CHD5, A Tumor Suppressor Gene Deleted From 1p36.31 in Neuroblastomas

Tomoyuki Fujita; Jun Igarashi; Erin R. Okawa; Takahiro Gotoh; Jayanthi Manne; Venkatadri Kolla; Jessica Kim; Huaqing Zhao; Bruce R. Pawel; Wendy B. London; John M. Maris; Peter S. White; Garrett M. Brodeur

Disclosures

J Natl Cancer Inst. 2008;100(13):940-949. 

In This Article

Abstract and Introduction

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.

Neuroblastoma, a tumor of the sympathetic nervous system, is the most common childhood extracranial solid tumor, accounting for 8%–10% of childhood cancers and 15% of childhood cancer deaths.[1] Neuroblastomas demonstrate clinical heterogeneity, from spontaneous regression to relentless progression. We and others have identified different patterns of genetic change that underlie these disparate clinical behaviors.[2] One of the most characteristic genetic changes in neuroblastomas is deletion of the short arm of chromosome 1 (1p).[3,4] We have found the 1p deletion in approximately 35% of all neuroblastomas and in 70%–80% of high-risk tumors,[5,6,7,8,9,10,11] suggesting the loss of a tumor suppressor gene from this region. We analyzed more than 1200 neuroblastomas and mapped the smallest region of consistent deletion (SRD) to 1p36.31, from D1S2660 distally to D1S214 proximally (approximately 2 Mb).[10,12] The SRD that has been identified by most other groups who have mapped 1p deletions in neuroblastomas overlaps this region.[13,14,15,16,17,18,19,20,21] Furthermore, the proximal and distal boundaries of this SRD are defined by three patients each,[10,12] clearly establishing that this region contains at least one neuroblastoma tumor suppressor gene that is deleted from these tumors.

In a previous study,[12] we mapped 23 genes to the maximal SRD we defined on 1p36.31. We analyzed 30 neuroblastoma cell lines for mutations in these genes but found no examples of mutational inactivation (ie, deletion, frameshift, or stop codon). We analyzed the expression of these 23 genes in neuroblastoma cell lines: seven had very low expression, two (CHD5, RNF207) had virtually absent expression, five had preferential expression in the nervous system, and seven had been previously associated with cancer pathogenesis (two with neuroblastoma). Only CHD5 had all of these features.[12,22]

In the current study, we hypothesized that CHD5 was the best candidate tumor suppressor gene deleted from 1p36.31 in neuroblastomas. We assessed the methylation status of the promoter and determined whether methylation status was associated with the presence or absence of 1p deletion in neuroblastoma cell lines. We transfected CHD5 into neuroblastoma cell lines and assessed the effect on both colony formation in soft agar (clonogenicity) and growth of xenograft tumors in athymic mice. We also compared the expression of 12 genes from the 1p36 SRD we defined with clinical and biologic risk factors in 101 neuroblastomas. Our goal was to determine whether CHD5 is a bona fide tumor suppressor gene and the likely target of the 1p36.31 deletions that characterize neuroblastoma.

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