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

Discussion

We previously identified 23 genes in the SRD on 1p36[10,12] and sequenced every coding exon of these genes in 30 neuroblastoma cell lines (21 with 1p deletion). None of the genes showed evidence of homozygous genetic inactivation by deletion, frameshift, or inactivating mutation.[12] However, expression analyses and functional considerations suggested that CHD5 was the most promising tumor suppressor gene candidate. CHD5 was the only gene that had preferential expression in the nervous system,[12] virtually absent expression in neuroblastomas with 1p deletion, and prior implication in neuroblastoma pathogenesis.[22] We transfected CHD5 into several neuroblastoma cell lines, and there was a limited effect in vitro, yet we observed dramatic suppression of clonogenicity and tumorigenicity. This result suggests that CHD5 functions as a tumor suppressor gene and may be particularly important for anchorage-independent growth. Indeed, the phenomenon of a limited effect on in vitro growth yet suppression of clonogenicity and in vivo tumorigenicity has been seen at times even with classical tumor suppressor genes, such as RB1 and TP53.[30,31,32,33] Furthermore, the chromatin remodeling function of CHD5 is consistent with other tumor suppressor genes in human cancers, including SMARCB1 (formerly known as INI1/SNF5),[34,35,36]SMARCA4 (BRG1/hBRM),[37,38] and BMI1.[39,40,41,42] Thus, based on these analyses, CHD5 emerged as the lead tumor suppressor gene candidate from the 1p36 SRD in neuroblastomas.

To provide additional evidence to support CHD5 as a tumor suppressor gene, we analyzed the relationship of gene expression with prognostic variables, event-free survival, and overall survival for 12 of the 23 genes from the SRD in 101 neuroblastomas. Only six genes showed statistically significant associations with three or more risk factors. CHD5 expression showed the strongest association with all eight clinical and biologic variables. High CHD5 expression was also strongly associated with favorable outcome. Indeed, using Cox regression analysis, 1p deletion was no longer associated with outcome after correction for CHD5 expression. Conversely, the association between CHD5 expression and outcome was retained even after correction for both MYCN amplification and 1p deletion (P = .027). These data suggest that CHD5 expression is a potent prognostic variable, that it likely plays a role in neuroblastoma pathogenesis, and that it may play a role even in some tumors without apparent 1p deletion. Our studies also demonstrate that CHD5 expression is not just a surrogate marker for 1p deletion because it was by far most strongly associated with outcome, and the expression of other genes tested from the SRD was weakly or not at all associated with outcome. More detailed studies will be required to determine whether addition of CHD5 expression as a prognostic marker may more rigorously defined neuroblastoma risk groups.

Although we did not find homozygous genetic inactivation of CHD5,[12] we did find strong promoter methylation and transcriptional silencing of the remaining allele in 1p-deleted neuroblastoma lines compared with lines without 1p deletion or with fetal brain tissue. This epigenetic mechanism could inactivate expression or lower it sufficiently to functionally silence the gene. Indeed, there is precedent for transcriptional silencing by methylation to inactivate the second allele of other tumor suppressor genes, including RASSF1A[43,44] and OPCML.[45] Also, CHD5 could be re-expressed by growing these cells in 5-aza-2-deoxycytidine, so this approach may have utility in treating neuroblastomas with low or absent CHD5 expression.

We now have convincing evidence that CHD5 is the tumor suppressor gene deleted from the 1p36.31 SRD in neuroblastomas. It is difficult to prove unequivocally that no other gene from the SRD or elsewhere on 1p36 contributes to the pathogenesis of neuroblastomas. However, within this SRD, CHD5 is the only gene that fulfills all tumor suppressor gene criteria. We first described CHD5 as a novel member of this chromatin remodeling gene family that was consistently deleted from 1p36 in neuroblastomas and as a candidate tumor suppressor gene.[22] Recently, Bagchi et al.[46] took an independent approach using chromosome engineering to generate mouse models with gain and loss of a region corresponding to the region of human 1p36 that contained mouse Chd5. They identified mChd5 as a gene that controls proliferation, apoptosis, and senescence via the p19Arf/p53 pathway. Their results also suggest that gene dosage is tightly regulated and that homozygous deletion of Chd5 in tumors is rarely seen, which is consistent with our finding that homozygous genetic inactivation of CHD5 is rare in human neuroblastomas. Furthermore, their independent identification of Chd5 as a tumor suppressor gene in a mouse model supports our findings in human neuroblastomas and suggests that CHD5 may have a role in the pathogenesis of other human cancers characterized by 1p36 deletions. Future studies will determine whether the primary consequence of mouse Chd5 inactivation is through p19Arf/p53 and/or other pathways in these tumors.

This study has potential limitations. Only 12 of the 23 genes from the SRD were represented on the expression profiling microarray so we were unable to assess associations between clinical outcomes with expression of 11 of the genes in the SRD. Nevertheless, the four genes that were the most promising candidates, including CHD5, were all included on the microarray. It was beyond the scope of this project to transfect full-length cDNAs of all 23 genes from the SRD into neuroblastoma cell lines with and without 1p deletion and to assess effects on growth, clonogenicity, and tumorigenicity. However, 10 of the genes from this region were tested in the study by Bagchi et al.,[46] and only CHD5 was able to restore growth control.

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