Cross-Species Array Comparative Genomic Hybridization Identifies Novel Oncogenic Events in Zebrafish and Human Embryonal Rhabdomyosarcoma

Abstract

Human cancer genomes are highly complex, making it challenging to identify specific drivers of cancer growth, progression, and tumor maintenance. To bypass this obstacle, we have applied array comparative genomic hybridization (array CGH) to zebrafish embryonal rhabdomyosaroma (ERMS) and utilized cross-species comparison to rapidly identify genomic copy number aberrations and novel candidate oncogenes in human disease. Zebrafish ERMS contain small, focal regions of low-copy amplification. These same regions were commonly amplified in human disease. For example, 16 of 19 chromosomal gains identified in zebrafish ERMS also exhibited focal, low-copy gains in human disease. Genes found in amplified genomic regions were assessed for functional roles in promoting continued tumor growth in human and zebrafish ERMS – identifying critical genes associated with tumor maintenance. Knockdown studies identified important roles for Cyclin D2 (CCND2), Homeobox Protein C6 (HOXC6) and PlexinA1 (PLXNA1) in human ERMS cell proliferation. PLXNA1 knockdown also enhanced differentiation, reduced migration, and altered anchorage-independent growth. By contrast, chemical inhibition of vascular endothelial growth factor (VEGF) signaling reduced angiogenesis and tumor size in ERMS-bearing zebrafish. Importantly, VEGFA expression correlated with poor clinical outcome in patients with ERMS, implicating inhibitors of the VEGF pathway as a promising therapy for improving patient survival. Our results demonstrate the utility of array CGH and cross-species comparisons to identify candidate oncogenes essential for the pathogenesis of human cancer.

Author Summary

Cancer is a complex genetic disease that is often associated with regional gains and losses of genomic DNA segments. These changes result in aberrant gene expression and drive continued tumor growth. Because amplified and deleted DNA segments tend to span large regions of chromosomes, it has been challenging to identify the genes that are required for continued tumor growth and progression. Array comparative genomic hybridization (array CGH) is an effective technology in identifying abnormal copy number variations in cancer genomes. In this study, array CGH was used in a zebrafish model of embryonal rhabdomyosarcoma - a pediatric muscle tumor. Our work shows that the zebrafish cancer genome contains a small number of recurrent DNA copy number changes, which are also commonly amplified in the human disease. Moreover, these chromosomal regions are small, facilitating rapid identification of candidate oncogenes. A subset of genes identified in zebrafish array CGH was prioritized for functional characterization in human ERMS, identifying evolutionarily conserved pathways that regulate proliferation, migration, differentiation, and neovascularization. Our results demonstrate the broad utility of cross-species array CGH comparisons of human and zebrafish cancer and provide a much needed discovery platform for identifying critical cancer-causing genes in a wide range of malignancies.