Akt抑制剂MK-2206和Rapamycin联合应用治疗神经母细胞瘤产生协同效应的机制研究

Neuroblastoma (NB) is the most common extracranial solid tumor that derives from neural crest in children. MYCN is a well-known oncogene which is associated with unfavorable prognosis in NB. In our previous study, we found that a synergistic cytotoxic effect was induced by a combination of Akt inhibitor MK-2206 and mTOR inhibitor Rapamycin, compared to each individual treatment alone, in MYCN-amplified NB cells. The cell death induced by the combination treatment was caspase-independent and could be blocked by reactive oxygen species (ROS) inhibitor NAC. Similar observations were not found in NB cells without MYCN amplification. So far, the mechanisms of the different responses to the combination treatment of MK-2206 and Rapamycin in different NB cells, and the pathways that medicate the combination-induced cell death are not clarified. In the present study, we will use a tetracycline-regulated MYCN expressing system and MYCN siRNA silencing technique, to study the association of MYCN overexpression and synergistic cytotoxic effect induced by the combination of MK-2206 and Rapamycin. Also, based on our pioneering microarray analysis that was performed by treating MYCN amplified NB cells with MK-2206 and Rapamycin, either alone or in combination, we will investigate the possible targets (such as glutathione, glutathione reductase) that mediate the intracellular increases of ROS, as well as the possible mechanisms of caspase-independent cell death (such as autophagy or necroptosis) in NB cells. The purpose of this study is to illuminate the novel biological functions of MK-2206 and Rapamycin combination, and to look for more effective treatment for MYCN-amplified NB. Our study will provide theoretical evidence for the use of MK-2206 and Rapamycin in clinical trial in order to enhance the treatment efficacy and improve the prognosis of NB.

神经母细胞瘤(NB)是起源于神经嵴的小儿常见颅外恶性实体瘤。MYCN是公认与NB不良预后密切相关的癌基因。我们前期发现，Akt抑制剂MK-2206和mTOR抑制剂Rapamycin联合治疗比单独应用，在MYCN扩增NB中产生明显药物协同效应，细胞死亡是ROS介导和Caspase-非依赖性的；在无MYCN扩增NB中无协同效应。不同细胞治疗反应差异的原因及联合治疗诱导细胞死亡的机制目前尚不清楚。本项目利用四环素调节MYCN表达体系及siRNA沉默技术，研究NB细胞MYCN过表达与联合治疗协同效应的关系；同时结合我们前期进行的MK-2206和Rapamycin治疗后基因表达芯片结果，探索MK-2206和Rapamycin联合治疗引起ROS水平升高靶点及caspase-非依赖性细胞死亡机制。通过本研究寻找对MYCN扩增难治性NB治疗新方法，为提高NB患者的治疗效果和改善预后提供理论指导。

本项目是申请者在前期研究基础上提出科学问题并进行的深入研究。神经母细胞瘤是小儿常见颅外恶性实体肿瘤，低中危患者预后多较好，但高危组患者对常规治疗的低反应是影响患者整体预后的主要因素。我们前期发现Akt的小分子抑制剂MK-2206与mTOR抑制剂Rapamycin联合作用时在一些神经母细胞瘤细胞系中产生明显的协同作用。在本项目中我们采用药物联合处理后进行基因芯片分析，筛选可能的协同作用机制，并探寻具有哪些生物学特点的神经母细胞瘤会有协同作用产生。我们的研究发现，细胞程序性坏死和细胞自噬均参与了MK-2206/Rapamycin协同作用，相关标志性的分子有明显的变化，如程序性坏死相关分子RIP1和RIP3，细胞自噬相关分子如ATG5，ATG7，LC3A/B,Beeclin-1等；同时发现，这两种小分子抑制剂的协同作用是MYCN依赖性的，即在具有MYCN扩增的神经母细胞瘤细胞中MK-2206/Rapamycin会产生协同作用，在用小干扰RNA降低MYCN的表达后协同作用明显减弱；而在无MYCN扩增的细胞系中过表达MYCN后会出现MK-2206/Rapamycin的协同作用。进一步研究发现，MYCN过表达后神经母细胞瘤细胞中线粒体的功能受损。这一发现具有重要的理论价值和临床意义，因为MYCN是神经母细胞瘤中具有独立预后判断能力的指标，通常具有MYCN扩展的神经母细胞瘤对化疗的反应性差，容易出现耐药、复发及转移，预后多不良。我们的发现无疑为这种高危险性的神经母细胞瘤提供了新的治疗思路，为提高具有MYCN扩增的神经母细胞瘤的治愈率和改善预后提供了新策略。因此，本项研究具有重要的理论研究价值，更重要的是具有广阔的临床应用价值。