Reverse Engineering the Neuroblastoma Regulatory Network Uncovers MAX as One of the Master Regulators of Tumor Progression

Neuroblastoma is the most common extracranial tumor and a major cause of infant cancer mortality worldwide. Despite its importance, little is known about its molecular mechanisms. A striking feature of this tumor is its clinical heterogeneity. Possible outcomes range from aggressive invasion to other tissues, causing patient death, to spontaneous disease regression or differentiation into benign ganglioneuromas. Several efforts have been made in order to find tumor progression markers. In this work, we have reconstructed the neuroblastoma regulatory network using an information-theoretic approach in order to find genes involved in tumor progression and that could be used as outcome predictors or as therapeutic targets. We have queried the reconstructed neuroblastoma regulatory network using an aggressive neuroblastoma metastasis gene signature in order to find its master regulators (MRs). MRs expression profiles were then investigated in other neuroblastoma datasets so as to detect possible clinical significance. Our analysis pointed MAX as one of the MRs of neuroblastoma progression. We have found that higher MAX expression correlated with favorable patient outcomes. We have also found that MAX expression and protein levels were increased during neuroblastoma SH-SY5Y cells differentiation. We propose that MAX is involved in neuroblastoma progression, possibly increasing cell differentiation by means of regulating the availability of MYC:MAX heterodimers. This mechanism is consistent with the results found in our SH-SY5Y differentiation protocol, suggesting that MAX has a more central role in these cells differentiation than previously reported. Overexpression of MAX has been identified as anti-tumorigenic in other works, but, to our knowledge, this is the first time that the link between the expression of this gene and malignancy was verified under physiological conditions.神经母细胞瘤（Neuroblastoma）是全球范围内最常见的颅外肿瘤，也是婴儿癌症死亡的主要诱因。尽管该肿瘤具有重要研究价值，学界对其分子机制仍知之甚少。该肿瘤的一个显著特征是临床异质性：其临床转归跨度极大，既可侵袭周边组织导致患者死亡，也可发生自发性疾病消退，或分化为良性神经节细胞瘤。此前已有多项研究致力于筛选肿瘤进展标志物。本研究采用信息论方法重构了神经母细胞瘤的基因调控网络，旨在筛选参与肿瘤进展、可作为预后预测指标或治疗靶点的基因。我们使用侵袭性神经母细胞瘤转移基因特征对重构得到的调控网络进行查询，以识别其主调控因子（master regulators, MRs）。随后，我们在其他神经母细胞瘤数据集内分析了主调控因子的表达谱，以探究其潜在临床价值。分析结果显示，MAX是神经母细胞瘤进展的核心主调控因子之一。研究发现，MAX的高表达与患者良好的预后显著相关。此外，我们观察到在神经母细胞瘤SH-SY5Y细胞的分化过程中，MAX的表达及蛋白水平均显著升高。我们推测MAX参与了神经母细胞瘤的进展过程，可能通过调控MYC:MAX异二聚体的可用性来促进细胞分化。这一机制与我们在SH-SY5Y细胞分化实验中得到的结果相符，表明MAX在该细胞的分化过程中发挥着比此前报道更为核心的作用。已有研究证实MAX过表达具有抗肿瘤活性，但据我们所知，本研究首次在生理条件下验证了该基因表达与肿瘤恶性程度之间的关联。