MYCN-induced calcium and magnesium signaling regulates Neuroblastoma progression

描述（由申请人提供）：神经母细胞瘤（NB）是一种颅外儿科癌症。原癌基因MYCN的扩增与晚期、高风险NB和不良预后相关。正在研究用于MYCN扩增的晚期NB患者的新药和替代治疗。然而，找到一种有效的治疗策略，晚期NB仍然难以捉摸。为了解决这一局限性，该项目研究了钙和镁信号对具有不同MYCN状态的NB细胞中细胞迁移和侵袭的影响。初步数据表明，MYCN启动钙信号传导的变化，伴随着钙通道（即Orai 1，Orai 3和TRPM 7）转录调节的变化。此外，MYCN诱导钙依赖性NB细胞迁移。这些观察结果支持以下假设，即MYCN通过涉及钙介导的整联蛋白信号转导和粘着斑周转调节以及钙和镁渗透性离子通道的机制，在细胞迁移和侵袭过程中改变钙和镁的稳态。本研究的具体目标如下：（1）研究MYCN对NB细胞内[Ca ~（2+）]和[Mg ~（2+）]稳态的影响。具有不同MYCN状态的NB细胞将在具有不同细胞外[Ca 2 +]/[Mg 2 +]比率的外部培养基中培养。将测量细胞内钙和镁，并确定这些细胞的迁移和侵袭率。(2)阐明MYCN调节钙和镁促进NB迁移和侵袭的机制。将在主动迁移的NB细胞中检查钙和镁对粘着斑激酶和整联蛋白的表达和磷酸化、粘着斑转换率（粘着斑蛋白的组装、拆卸和再循环）以及肌动蛋白细胞骨架重组的影响。(3)鉴定调节NB迁移和侵袭的分子组分。钙渗透性奥赖通道以及钙和镁渗透性TRPM 7通道蛋白将直接或使用分子方法（RNAi或瞬时过表达）调节，并且将在NB细胞中确定这些变化对细胞迁移和侵袭、粘着斑转换（粘着斑蛋白的组装、分解和再循环）和细胞内[Ca 2 +]的影响。这项研究可能会发现新的生物标志物为晚期NB，并揭示新的目标，为开发更有效的化疗药物。此外，这项研究可能会导致一种新的NB进展调节模型，其中包括监测预后标志物MYCN的状态以及钙和镁的饮食摄入，代谢和细胞调节。这种新模型可能适用于MYC扩增的其他癌症（例如乳腺癌和前列腺癌以及淋巴瘤等）。

DESCRIPTION (provided by applicant): Neuroblastoma (NB) is an extra-cranial pediatric cancer. Amplification of the proto-oncogene MYCN is associated with advanced stage, high risk NB and poor prognosis. Novel drugs and alternative treatments are being investigated for patients with advanced NB with MYCN amplification. However, finding an effective treatment strategy for advanced stage NB remains elusive. In order to address this limitation, the proposed project examines the effects of calcium and magnesium signaling on cell migration and invasion in NB cells with different MYCN status. The preliminary data indicate that MYCN initiates a change in calcium signaling, accompanied by a change in the transcriptional regulation of calcium channels, namely Orai1, Orai3 and TRPM7. In addition, MYCN induces calcium-dependent NB cell migration. These observations support the hypothesis that MYCN alters calcium and magnesium homeostasis during cell migration and invasion through a mechanism that involves calcium-mediated regulation of integrin signaling and focal adhesion turnover, and calcium- and magnesium-permeable ion channels. The research plan has the following specific aims: (1) Examine the effect of MYCN on [Ca2+] and [Mg2+] homeostasis in NB cells. NB cells with different MYCN status will be cultured in external media with different extracellular [Ca2+]/[Mg2+] ratios. The intracellular calcium and magnesium will be measured, and migration and invasion rates will be determined in these cells. (2) Elucidate the mechanism by which MYCN regulation of calcium and magnesium promotes NB migration and invasion. The effect of calcium and magnesium on the expression and phosphorylation of focal adhesion kinase and integrin, focal adhesion turnover rate (assembly, disassembly and recycling of focal adhesion proteins), and actin cytoskeletal re-organization will be examined in NB cells that are actively migrating. (3) Identify the molecular components regulating NB migration and invasion. The calcium- permeable ORAI channels and the calcium- and magnesium-permeable TRPM7 channel protein will be modulated pharmacologically or using molecular methods (RNAi or transient over-expression), and the effect of these changes on cell migration and invasion, focal adhesion turnover (assembly, disassembly, and recycling of focal adhesion proteins), and intracellular [Ca2+] will be determined in NB cells. This study may identify new biomarkers for advanced stage NB, and reveal novel targets for the development of more effective chemotherapeutic drugs. In addition, this study may lead to a new model of the regulation of NB progression, which includes monitoring the status of the prognostic marker MYCN as well as the dietary intake, metabolism and cellular regulation of calcium and magnesium. This new model may apply to other cancers with MYC amplification (e.g. breast and prostate cancers, and lymphoma, etc).