Innovative Thiosemicarbazones Down-Regulate N-Myc Expression in Neuroblastoma Cells via Transcriptional and Post-Transcriptional Mechanisms

Neuroblastoma (NB) is an aggressive solid childhood tumor. A key oncogenic effector in NB is the transcription factor, N-Myc, whose genetic amplification leads to poor prognosis. Previous studies have indicated that iron-binding ligands such as desferrioxamine (DFO) effectively down-regulate N-myc expression. However, the mechanism of how this occurs remains unknown and could provide clues to elucidating potential therapeutic interventions. Herein, we mechanistically dissect the activity of a new class of iron-binding ligands that have entered clinical trials and show potent anti-proliferative activity against NB, namely the thiosemicarbazone, DpC. This was achieved using RNA sequencing (RNA-seq), where we have examined the effect of these ligands on gene expression in 3 NB cell-types with (Kelly and BE2-C) or without (SH-SY5Y) N-myc amplification. RNA-seq revealed that DpC mimicked the effect of DFO on gene expression at a 20-fold lower concentration, up-regulating key N-Myc targets such as the metastasis suppressor NDRG1, which translocates to the nucleus upon treatment. DFO and DpC significantly reduced N-Myc mRNA in p53-mutant NB cells (Kelly, BE(2)-C). In contrast, DFO increased N-Myc mRNA in wild-type p53 SH-SY5Y NB cells, where it was demonstrated that MDM2 stabilized N-Myc mRNA. Overall design: RNA-seq profiling of DFO or DpC-treated Neuroblastoma cells (SH-SY5Y, Kelly, BE(2)-C) related to untreated cells at 24 hours.

神经母细胞瘤（Neuroblastoma, NB）是一种侵袭性儿童实体肿瘤。NB中关键的致癌效应因子为转录因子N-Myc，其基因扩增会导致不良预后。既往研究表明，去铁胺（desferrioxamine, DFO）这类铁结合配体可有效下调N-myc的表达，但其具体作用机制尚未阐明，而该机制可为潜在治疗干预策略的开发提供线索。

本文对一类已进入临床试验、且对NB具有强效抗增殖活性的新型铁结合配体的活性进行了机制解析，这类配体即硫代半卡巴腙类化合物DpC。本研究通过RNA测序（RNA sequencing, RNA-seq）技术，在3种NB细胞系中检测了这类配体对基因表达的影响：分别为携带N-myc扩增的Kelly、BE2-C细胞，以及不携带该扩增的SH-SY5Y细胞。

RNA测序结果显示，DpC在浓度仅为DFO的1/20时，即可模拟DFO对基因表达的调控效果，上调包括转移抑制因子NDRG1在内的关键N-Myc靶基因；该因子在配体处理后会转位进入细胞核。DFO与DpC均可显著降低p53突变型NB细胞（Kelly、BE(2)-C）中的N-Myc mRNA水平；与之相反，DFO会升高野生型p53的SH-SY5Y NB细胞中的N-Myc mRNA水平，该现象已被证实与MDM2稳定N-Myc mRNA有关。

实验整体设计：对经DFO或DpC处理24小时的NB细胞（SH-SY5Y、Kelly、BE(2)-C）进行RNA测序分析，并以未处理细胞作为对照。