MYCN-induced calcium and magnesium signaling regulates Neuroblastoma progression

描述（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扩增与晚期、高风险NB和不良预后相关。对于MYCN扩增的晚期NB患者，正在研究新的药物和替代治疗方法。然而，找到一个有效的治疗策略晚期NB仍然是难以捉摸的。为了解决这一限制，该项目研究了钙和镁信号在不同MYCN状态的NB细胞中对细胞迁移和侵袭的影响。初步数据表明，MYCN启动钙信号通路的改变，同时伴随着钙通道Orai1、Orai3和TRPM7转录调控的改变。此外，MYCN诱导钙依赖性NB细胞迁移。这些观察结果支持了MYCN在细胞迁移和侵袭过程中改变钙和镁稳态的假设，其机制涉及钙介导的整合素信号调节和局灶黏附转换，以及钙和镁透性离子通道。本研究计划有以下具体目的：(1)研究MYCN对NB细胞[Ca2+]和[Mg2+]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[Ca2+]/[Mg2+]比例的外培养基中培养。将测量细胞内钙和镁的含量，并测定这些细胞的迁移和侵袭率。(2)阐明MYCN调控钙、镁促进NB迁移侵袭的机制。钙和镁对局灶黏附激酶和整合素的表达和磷酸化、局灶黏附周转率（局灶黏附蛋白的组装、拆卸和再循环）和肌动蛋白细胞骨架重组的影响将在积极迁移的NB细胞中进行研究。(3)确定调控NB迁移和入侵的分子成分。钙渗透性ORAI通道和钙、镁渗透性TRPM7通道蛋白将通过药理学或分子方法（RNAi或瞬时过表达）进行调节，这些变化对NB细胞迁移和侵袭、局灶黏附转换（局灶黏附蛋白的组装、拆卸和再循环）和细胞内[Ca2+]的影响将被确定。这项研究可能会发现晚期NB的新生物标志物，并为开发更有效的化疗药物提供新的靶点。此外，本研究可能会导致NB进展调节的新模型，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。这个新模型可能适用于其他MYC扩增的癌症（如乳腺癌、前列腺癌、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).