Inactivation of NONO by Auranofin or RNA Interference Triggers Lethal Oxidative Stress in Neuroblastoma Cells.

BACKGROUND

Neuroblastoma (NB), a transcriptionally driven pediatric malignancy, exhibits a remarkable clinical and biological heterogeneity. Two major subtypes, adrenergic and mesenchymal, are differentially governed by distinct subsets of transcription factors that constitute the core regulatory circuitry (CRC). The adrenergic subtype is often associated with MYCN oncogene amplification and is particularly aggressive and therapy-resistant, underscoring the need for novel therapeutic targets.

METHODS

Gene knockdown with siRNAs, qRT-PCR, flow cytometry-assisted measurements of intracellular oxidation and cell death parameters, immunoblotting, cytotoxicity assays (MTT, colony formation).

RESULTS

We identified the multifunctional non-POU domain-containing octamer-binding (NONO) protein as a guardian of individual CRC genes, thereby promoting the survival of NB cells with different MYCN copy numbers. In the MYCN-amplified Kelly cell line, intracellular oxidation induced by auranofin, an inhibitor of thioredoxin reductase 1 (TrxR1), rapidly down-regulated NONO mRNA and protein levels. Conversely, NONO knockdown by RNA interference (siNONO) also triggered intracellular oxidation. These effects were less pronounced in the SK-N-AS cell line carrying a single MYCN copy, as well as in non-malignant HS5 fibroblasts. In Kelly and IMR-32 cells, siNONO attenuated auranofin-induced activation of CRC genes, namely, heart and neural crest derivatives expressed 2 (HAND2) and paired-like homeobox 2B (PHOX2B). Furthermore, the Kelly cells were more sensitive to combinations of sublethal auranofin concentrations and siNONO than the counterparts with single MYCN copy. Importantly, MYCN-amplified cells demonstrated a significantly suppressed clonogenic survival 14 days after transient exposure to these combinations compared with each agent alone; HS5 fibroblasts were largely spared.

CONCLUSIONS

Our findings reveal a new role for the transcriptional regulator NONO in maintenance of the cellular redox balance and justify the strategy of therapeutic targeting of MYCN-amplified tumors vulnerable to oxidative stress.