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

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.人类癌症基因组具有高度复杂性，这使得识别癌症发生、进展及肿瘤维持的特定驱动因素极具挑战。为突破这一障碍，我们将阵列比较基因组杂交（array CGH）技术应用于斑马鱼胚胎性横纹肌肉瘤（ERMS），并通过跨物种比较快速鉴定人类疾病中的基因组拷贝数变异及新型候选癌基因。斑马鱼ERMS存在小型局灶性低拷贝扩增区域，而这些区域在人类疾病中也常出现扩增。例如，斑马鱼ERMS中鉴定出的19处染色体增益中，有16处在人类疾病中同样存在局灶性低拷贝增益。我们对扩增基因组区域内的基因在人类和斑马鱼ERMS中促进肿瘤持续生长的功能作用进行了评估，从而鉴定出与肿瘤维持相关的关键基因。基因敲低实验证实，细胞周期蛋白D2（Cyclin D2，CCND2）、同源盒蛋白C6（HOXC6）以及丛蛋白A1（PLXNA1）在人类ERMS细胞增殖中发挥重要作用。敲低PLXNA1还可促进细胞分化、降低细胞迁移能力，并改变其非锚定依赖性生长特性。与之形成对照的是，对血管内皮生长因子（VEGF）信号通路进行化学抑制，可减少携带ERMS的斑马鱼体内的血管生成并缩小肿瘤体积。值得注意的是，ERMS患者体内血管内皮生长因子A（VEGFA）的表达水平与不良临床结局显著相关，这表明VEGF通路抑制剂有望成为改善患者生存的潜在治疗手段。本研究结果证实，阵列比较基因组杂交技术与跨物种比较相结合，可有效鉴定人类癌症发病机制所必需的候选癌基因。