Neuroblastoma (NB) is a common and deadly tumor of childhood. We identified deletion of 1p as the most characteristic genetic change in NBs. We mapped the region of consistent loss to 2 Mb on 1p36.31 and have identified 23 candidate tumor suppressor genes (TSGs) in this region. Based on genetic, epigenetic and functional evidence, we hypothesize that CHD5 is the best candidate TSG from this region. CHD5 encodes a novel chromodomain-helicase-DNA-binding (CHD) protein. Evidence that CHD5 is a NB TSG includes: 1) CHD5 expression is very low or absent in tumors with 1p deletion;2) forced expression of CHD5 in NBs with 1p deletion suppresses clonogenicity and tumorigenicity 3) the CHD5 promoter is methylated in tumors with 1p deletion may silence the second allele;4) CHD5 expression is highly correlated with favorable prognostic features and outcome;5) the chromatin-remodeling function is consistent with other TSGs;and 6) CHD5 is expressed preferentially in neural tissues.
Aim 1. We will determine the frequency and prognostic significance of CHD5 inactivation in primary NBs. We are extending our preliminary findings to 915 highly annotated NBs to test the predictive value of CHD5 in a multivariable model. We will analyze 194 NBs (with and without 1p deletion) for mutations in CHD5 and 100 for methylation of the promoter to assess the prevalence and significance of CHD5 inactivation by these mechanisms.
Aim 2. We will develop specific antibodies to CHD5 and determine if CHD5 associates with other NuRD complex proteins and functions as an ATP-dependent chromatin remodeling protein. We will also characterize the temporal and spatial patterns of CHD5 expression during development.
Aim 3. We will explore the mechanisms involved in CHD5-mediated tumor suppression in NBs. We will create CHD5 inactive mutants as a control in our tumorigenicity studies (Aim 3) and protein complex studies (Aim 2). We will map the CHD5 promoter, including sites of methylation to identify critical regulatory domains. We will perform microarray expression profiling and ChIP-chop analysis with an inducible CHD5 expression construct to identify genes regulated by CHD5 that may be responsible for its function as a TSG.
Aim 4. We will knock out (KO) the Chd5 gene in embryonic stem (ES) cells and develop mice that are heterozygous and homozygous for this inactivated gene. We will then analyze heterozygous and homozygous knockouts for tumor predisposition and for developmental defects. We will use a Cre-LoxP system and the tyrosine hydroxylase promoter to selectively inactivate this gene in adrenergic cells. We will also cross CHD5 KO mice with TH-MYCN transgenic mice to determine if NB tumorigenicity is accelerated. Relevance: Our data strongly suggest that somatically acquired inactivation of CHD5 contributes to the development of high-risk NBs, and possibly other pediatric and adult tumors as well. The successful completion of these studies should clarify the role CHD5 plays in normal development and in the pathogenesis of NB, which may assist in the diagnosis, prognosis and management of children with NB, and perhaps other cancers characterized by 1p36 deletions.

Public Health Relevance

Neuroblastoma is a common and lethal childhood cancer, and the most aggressive forms have specific genetic changes. The most common of these is deletion of chromosome 1p, which presumably represents deletion of a tumor suppressor gene (TSG). We have identified CHD5 as the strongest candidate for the 1p36 TSG, and our proposal will clarify the normal function of this gene and protein, as well as the role that CHD5 deletion plays in neuroblastoma development, progression or predisposition.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA039771-24
Application #
8223222
Study Section
Cancer Molecular Pathobiology Study Section (CAMP)
Program Officer
Mietz, Judy
Project Start
1985-05-01
Project End
2014-02-28
Budget Start
2012-03-01
Budget End
2013-02-28
Support Year
24
Fiscal Year
2012
Total Cost
$377,150
Indirect Cost
$147,879
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
073757627
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Okawa, E R; Gotoh, T; Manne, J et al. (2008) Expression and sequence analysis of candidates for the 1p36.31 tumor suppressor gene deleted in neuroblastomas. Oncogene 27:803-10

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