Abstract 1412: Cross-species array comparative genomic hybridization identifies novel driver genes in embryonal rhabdomyosarcoma.
Authors: Chen Eleanor , Dobrinski Kimberly P. , Brown Kim H. , Clagg Ryan , Edelman Elena , Ignatius Myron , Brockmann Jillian , Nielsen G. Petur , Ramaswamy Sridhar , Keller Charles , Lee Charles , Langenau David M. 
Published: August 2014

Human cancers are often associated with large chromosomal amplifications and deletions comprising megabases of sequence - making it challenging to identify drivers of cancer growth and progression. To bypass this obstacle, we have applied array comparative genomic hybridization (aCGH) to zebrafish embryonal rhabdomyosaroma (ERMS) and utilized cross-species comparison to identify novel collaborating oncogenes important to human disease. Because zebrafish ERMS predominantly contain small, focal amplifications that span only 50-100kb of sequence, candidate oncogenes were easily identified by cross-species comparisons with human ERMS. Remarkably, 16 of 19 chromosomal gains identified in zebrafish ERMS also exhibited focal, copy gains in human disease. Candidate oncogenes found in these regions were also over-expressed in a majority of human ERMS as assessed by microarray gene expression analysis and immunohistochemical stain of primary human ERMS and normal fetal muscle. siRNA and shRNA knockdown studies identified important roles for candidate ocogenes in regulating human ERMS growth. For example, knockdown of Cyclin D2, Homeobox Protein C6, and Plexin A1 (PLXNA1) resulted in reduced proliferation in both human RD and SMS-CTR cell lines. PLXNA1 knockdown also resulted in impaired migratory potential in scratch and transwell migration assays. Moreover, PLXNA1 loss resulted in increased numbers of terminally-differentiated myosin-expressing myocytes, suggesting that dysregulated expression of PLXNA1 plays a key role in the differentiation arrest of human ERMS. Finally, vascular endothelial growth factor (VEGF) was also amplified and over-expressed in a subset of human and zebrafish ERMS. Chemical inhibition of VEGF in ERMS-bearing zebrafish led to significantly reduced tumor growth which was associated with decreased tumor neovascularization. Analysis of human microarray gene expression data revealed that increased VEGFA expression correlated with poor clinical outcome in patients with ERMS independent of tumor grade and stage, indicating high VEGFA expression as an independent prognostic indicator and implicating inhibitors of the VEGF pathway as a promising therapy for improving patient survival. In total, our results demonstrate the utility of aCGH and cross-species comparisons to rapidly identify candidate genes essential for the pathogenesis of human cancer.

Citation Format: Eleanor Chen, Kimberly P. Dobrinski, Kim H. Brown, Ryan Clagg, Elena Edelman, Myron Ignatius, Jillian Brockmann, G. Petur Nielsen, Sridhar Ramaswamy, Charles Keller, Charles Lee, David M. Langenau. Cross-species array comparative genomic hybridization identifies novel driver genes in embryonal rhabdomyosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1412. doi:10.1158/1538-7445.AM2013-1412