Differential role of different NAD+ kinase Isoforms in melanoma metastasis

项目摘要 转移是导致超过90%的癌症患者死亡的原因，但没有特异性的治疗方法， 靶向转移性疾病。目前许多体外转移模型都集中在转移的分子机制上 迁移，入侵和/或生存的失巢凋亡，但不能概括的复杂环境中， 转移发生在体内。相反，在转移的小鼠模型中，很难检查肿瘤的转移。 分子机制，使细胞能够通过每个不同的步骤转移，由于有限的 材料可以被隔离，并且在这些模型中很少发生转移事件。由于这些原因， 了解体内转移细胞面临的挑战，以及如何克服这些挑战。我们先前已经 建立了一个临床相关的黑色素瘤转移模型，使用患者来源的异种移植物（PDX）， 免疫受损小鼠，其在小鼠中概括患者疾病的结果，以解剖 转移级联成不同的步骤。利用这个模型，我们已经证明转移性黑色素瘤细胞 经历可逆的代谢适应，部分通过增加依赖性来抵抗氧化应激， 对一碳途径中的NADPH生成酶的影响。我们的初步数据还显示， 与皮下肿瘤相比，转移性结节中的NADP+水平，表明新发肿瘤增加。 NADP+合成。NADP+由NAD+通过NAD+激酶（NADK）产生。我们观察到较高水平的NADK 与皮下肿瘤相比，转移性结节表达 NADK具有最高活性，而皮下肿瘤没有。我们将检验转移性肿瘤 黑色素瘤细胞上调NADK的特异性亚型以增加NADP+的产生，增加氧化应激， 在转移级联的不同阶段的抵抗和存活。 使用黑色素瘤细胞系和PDX肿瘤细胞，Aim 1将确定不同的NADK亚型的作用， 在氧化应激抗性中，Aim 2将定义NADK同种型的转录调节机制， 目的3将确定不同NADK同种型作为体内转移驱动因子的作用。此外，Aim 3将 测试不同细胞器中氧化应激的扰动如何影响转移。这项工作将 对我们理解一种新的代谢可塑性机制有重要贡献， 特定NADK亚型的研究，并将细胞器特异性代谢途径确定为新型治疗靶点 黑色素瘤转移的脆弱性。

PROJECT SUMMARY Metastasis is responsible for more than 90% of cancer patient mortality yet there are no therapies that specifically target metastatic disease. Many of the current in vitro models of metastasis focus on the molecular mechanisms of migration, invasion and/or surviving anoikis, but cannot recapitulate the complexity of the environment in which metastasis occurs in vivo. Conversely, in mouse models of metastasis, it has been difficult to examine the molecular mechanisms that enable cells to proceed through each distinct step of metastasis due to limited material that can be isolated and infrequency of metastatic events in these models. For these reasons little is known about the challenges facing metastasizing cells in vivo, and how they are overcome. We have previously established a clinically relevant model of melanoma metastasis, using patient-derived xenografts (PDX) in immunocompromised mice, that recapitulates the outcome of the disease of the patient in mice, to dissect the metastatic cascade into distinct steps. Using this model, we have shown that metastasizing melanoma cells undergo reversible metabolic adaptations to withstand oxidative stress in part through an increased dependence on NADPH-generating enzymes in the one-carbon pathway. Our preliminary data also show an increase in NADP+ levels in metastatic nodules compared to subcutaneous tumors, suggesting an increase in de novo NADP+ synthesis. NADP+ is generated from NAD+ by NAD+ kinase (NADK). We observe higher levels of NADK in metastatic nodules compared to subcutaneous tumors, where metastatic nodules express the isoform of NADK with the highest activity, while subcutaneous tumors do not. We will test the hypothesis that metastasizing melanoma cells upregulate a specific isoform of NADK to increase NADP+ production, increase oxidative stress resistance and survival at different steps of the metastatic cascade. Using both melanoma cell lines and PDX tumor cells, Aim 1 will determine the role of different NADK isoforms in oxidative stress resistance, Aim 2 will define the mechanism of transcriptional regulation of NADK isoforms, and Aim 3 will establish the role of different NADK isoforms as metastatic drivers in vivo. In addition, Aim 3 will test how perturbation of oxidative stress in different organelles impacts metastasis. Together this work will significantly contribute to our understanding of a novel mechanism of metabolic plasticity through upregulation of a specific NADK isoform and identify organelle-specific metabolic pathways as novel therapeutically targetable vulnerabilities in melanoma metastasis.