The MYCN proto-oncogene is deregulated in many cancers, most notably in neuroblastoma where MYCN gene amplification identifies a clinical subset with very poor prognosis. Gene expression and DNA analyses have also demonstrated over-expression of MYCN mRNA, as well as focal amplifications, copy number gains and presumptive change of function mutations of MYCN in Wilms tumours with poorer outcome, including tumours with diffuse anaplasia. Surpisingly, however, the expression and functions of the MYCN protein in Wilms tumours still remain obscure. In this study, we assessed MYCN protein expression in primary Wilms tumours using immunohistochemistry of tissue microarrays. We found MYCN protein to be expressed in tumour blastemal cells, and absent in stromal and epithelial components. For functional studies, we used two anaplastic Wilms tumour cell-lines, WiT49 and 17.94, to study the biological and transcriptomic effects of MYCN depletion. We found that MYCN knockdown consistently led to growth suppression but not cell death. RNA sequencing identified 561 MYCN-regulated genes shared by WiT49 and 17.94 cell-lines. As expected, numerous cellular processes were downstream of MYCN. MYCN positively regulated the miRNA regulator and known Wilms tumour oncogene LIN28B, the genes encoding methylosome proteins PRMT1, PRMT5 and WDR77, and the mitochondrial translocase genes TOMM20 and TIMM50. MYCN repressed genes included the developmental signalling receptor ROBO1 and the stromal marker COL1A1. Importantly, we found that MYCN also repressed the presumptive Wilms tumour suppressor gene REST, with MYCN knockdown resulting in increased REST protein and concomitant repression of REST target genes. Together, our study identifies regulatory axes that interact with MYCN, providing novel pathways for potential targeted therapeutics for poor prognosis Wilms tumour.

中文翻译：

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对间变性Wilms肿瘤细胞系中MYCN调控基因的转录组学分析揭示了致癌途径和潜在的治疗脆弱性

MYCN原癌基因在许多癌症中均失控，最明显的是在神经母细胞瘤中，MYCN基因扩增可鉴定出临床预后很差的亚型。基因表达和DNA分析还表明，在结果较差的Wilms肿瘤（包括弥漫性发育不良的肿瘤）中，MYCN mRNA的过度表达以及MYCN的病灶扩增，拷贝数增加和功能突变的推测变化。然而，令人惊讶的是，MYCN蛋白在Wilms肿瘤中的表达和功能仍然不清楚。在这项研究中，我们使用组织微阵列的免疫组织化学评估了原发性Wilms肿瘤中MYCN蛋白的表达。我们发现MYCN蛋白在肿瘤母细胞中表达，在基质和上皮成分中不存在。对于功能研究，我们使用了两种变性的Wilms肿瘤细胞系WiT49和17.94来研究MYCN耗竭的生物学和转录组学效应。我们发现，MYCN基因敲低始终导致生长受到抑制，但没有导致细胞死亡。RNA测序鉴定了WiT49和17.94细胞株共有的561个MYCN调控基因。不出所料，MYCN的下游有许多细胞过程。MYCN积极调节miRNA调节剂和已知的Wilms肿瘤癌基因LIN28B，编码甲基脂质体蛋白PRMT1，PRMT5和WDR77的基因以及线粒体转位酶基因TOMM20和TIMM50。MYCN抑制的基因包括发育信号受体ROBO1和基质标志物COL1A1。重要的是，我们发现MYCN还抑制了假定的Wilms抑癌基因REST，MYCN敲低导致REST蛋白增加，并同时抑制REST目标基因。总之，我们的研究确定了与MYCN相互作用的调控轴，为预后不良的Wilms肿瘤提供了潜在靶向治疗的新途径。