Differential role of different NAD+ kinase Isoforms in melanoma metastasis

项目总结 转移是癌症患者死亡的90%以上的原因，但目前还没有专门的治疗方法 瞄准转移的疾病。目前许多肿瘤转移的体外模型都集中在分子机制上。 关于迁徙、入侵和/或幸存的阿尼基人，但不能概括所处环境的复杂性 转移发生在体内。相反，在转移的小鼠模型中，很难检查 分子机制，使细胞能够通过每个不同的转移步骤，由于有限的 在这些模型中，可以分离的材料和转移事件的频率很低。出于这些原因，几乎没有 了解体内转移细胞面临的挑战，以及如何克服这些挑战。我们之前已经 建立了临床相关的黑色素瘤转移模型，使用患者来源的异种移植(PDX)在 免疫功能低下的小鼠，它概括了小鼠的疾病结局，以解剖 转移性肿瘤层叠成不同的步骤。使用这个模型，我们已经证明了转移的黑色素瘤细胞 经历可逆的代谢适应，部分通过增加依赖性来抵御氧化应激 一碳途径中NADPH产生酶的研究。我们的初步数据还显示， 转移结节中NADP+水平与皮下肿瘤相比，提示新生血管增加 NADP+合成。NADP+由NAD+激酶(NADK)由NAD+产生。我们观察到NADK水平较高 转移性结节与皮下肿瘤相比，在皮下肿瘤中，转移性结节表达异构体 NADK活性最高，而皮下肿瘤则不是。我们将检验转移的假说 黑色素瘤细胞上调一种特定的NADK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耐药和存活。 使用黑色素瘤细胞系和PDX肿瘤细胞，Aim 1将确定不同NADK亚型的作用 在氧化应激抗性中，目标2将定义NADK亚型转录调控的机制， 目标3将确定不同的NADK亚型在体内作为转移驱动因素的作用。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。这项工作将共同努力 有助于我们理解一种通过上调代谢可塑性的新机制 一种特定的NADK亚型，并鉴定细胞器特异性代谢途径为新的治疗靶点 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.