Transcriptomic Analyses of MYCN-Regulated Genes in Anaplastic Wilms' Tumour Cell Lines Reveals Oncogenic Pathways and Potential Therapeutic Vulnerabilities.

Abstract

Simple SummaryWilms’ tumour (WT) is one of the most common solid paediatric malignancies and arises in the developing kidney. Treatment of most WTs is relatively successful, with the notable exception of tumours with anaplastic histology. Anaplastic WT survival rates can be as low as 30–50%, emphasising the need for a better understanding of their molecular aetiology, in order to facilitate the development of novel therapeutics for poor-prognosis WT. Previous studies have implicated increases in MYCN at the DNA and RNA level in WTs with anaplasia, although the biological functions of MYCN protein have not been reported. In this study, we define the expression pattern of MYCN protein in WTs and also modulate MYCN protein levels in anaplastic WT cell-lines to define the functions of MYCN in WT. We show that MYCN depletion leads to decreased cell growth and identify MYCN-regulated genes and pathways that may represent therapeutic vulnerabilities in poor-prognosis WT.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 overexpression of MYCN mRNA, as well as focal amplifications, copy number gains and presumptive change of function mutations of MYCN in Wilms’ tumours with poorer outcomes, including tumours with diffuse anaplasia. Surprisingly, 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 including 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 RE1-Silencing Transcription factor (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.