Noncoding RNA elements within MYCN mRNA are potent autonomous drivers of oncogenesis.

Abstract

Neuroblastoma (NB) is a highly metastatic pediatric cancer arising from the neural crest lineage. Genetic amplification of the MYCN proto-oncogene is a defining feature of NB, present in about 20% of all cases. The let-7 tumor suppressor microRNA targets the 3 UTR of MYCN mRNA. We previously demonstrated that the 3 UTR of MYCN mRNA acquires the ability to sequester let-7 in MYCN-amplified (MA) disease, thus inhibiting its function. This work established that a noncoding element within an oncogenic mRNA can contribute independently to disease pathology and genetic patterning. To further investigate the roles of noncoding RNA elements within the MYCN mRNA, we engineered cells expressing either MYCN-ORF (MYCN open reading frame only), MYCN-GL (full-length MYCN mRNA from the intact genetic locus), and Null-GL (full-length MYCN mRNA variant where EGFP replaces MYCN protein). We observe that all constructs enhance growth compared to controls in vitro and in vivo. MYCN-GL-expressing cells displayed the most robust growth in vitro despite containing multiple regulatory RNA elements. Remarkably, the Null-GL construct induces cells to grow as fast or faster than MYCN-ORF-expressing cells. Animal studies further confirmed these observations, where the Null-GL-driven tumors had the highest incidence and lowest latency, followed by MYCN-GL and then MYCN-ORF. Further, through NGS analysis, let-7, miR-101, and miR-34a targets are enriched in both MYCN-GL and Null-GL expressing cells. Thus, the 3UTR of MYCN, which is also targeted by these microRNAs, may interact with them in MYCN-GL and Null-GL cells to deliver a protective effect for the mRNA targets of these microRNAs. We also observed more dynamic differential gene expression in the -GL constructs than in ORF and GFP-expressing cells. In addition, MYCN-GL and Null-GL expressing cells are similarly enriched in gene ontology pathways for cancer, RNA metabolism, and microRNA processing pathways as compared to ORF and GFP. Whole genome sequencing also revealed more similarities in copy number variation in MYCN-GL and Null-GL than in ORF and GFP, suggesting that these constructs may provide selective pressure to favor specific CNV patterns. These observations show that full-length MYCN mRNA containing noncoding regulatory elements are more robust drivers of cell growth and oncogenicity than MYCN protein alone and provide insights into the mechanisms of oncogenic contribution. These results open an exciting door for our understanding of NB pathology and genetic patterning and have broad implications for other oncogene-driven cancers.