Targeting aspartate biosynthesis in pancreatic tumors

胰腺导管腺癌(PDAC)是世界上第二大癌症死亡原因。 一旦扩散到远处的器官，存活率为3%。肿瘤内的PDAC细胞 由于它们的高增殖率和血管系统不足，经常缺乏氧气。这一程度 肿瘤中的缺氧触发癌细胞的强烈新陈代谢适应，使其存活并 扩散。在这些适应中，主要营养物质的吸收和利用发生了变化，如谷氨酰胺。 然而，胰腺癌代谢适应缺氧的确切机制，以及 这可能会被用于治疗，但目前尚不清楚。 在初步研究中，我们发现，当胰腺癌和肺癌暴露在低水平的 氧，蛋白质和核苷酸合成所需的氨基酸天冬氨酸，变得有限。简单 通过表达质膜天冬氨酸转运蛋白增加外源天冬氨酸的摄取 有力地促进了癌细胞在低氧和肿瘤中的生长速度，并增强了其 转移潜能。这些发现提供了天冬氨酸是一种限制癌症生长的代谢物的证据。 在活体内。此外，使用靶向限速代谢的sgRNAs文库的CRISPR/Cas9筛选 酶揭示了GOT2，从头合成细胞天冬氨酸的两种酶之一 谷氨酰胺是KRAS/TP53突变型PDAC细胞在低氧条件下体外增殖所必需的。建房 根据这些结果，我建议测试以天冬氨酸从头合成为目标的假设可能具有 胰腺肿瘤在原发肿瘤生长和转移水平的治疗潜力。 在本奖项的初始阶段，我们将定义GOT2介导的天冬氨酸的重要性 一组PDAC细胞系在暴露于低氧压下时的合成，重要的是，当 以肿瘤的形式生长，并在远处器官的殖民期间生长。此外，我们将确定捐款和 谷氨酰胺转化为天冬氨酸的两种不同代谢途径对肿瘤增殖的影响： 氧化和还原代谢。在已有成果的基础上，我们将在 临床前患者来源的模型和KRAS/TP53突变小鼠模型以及使用同位素标记 代谢组学分析，我们将在体内显示胰腺肿瘤使用哪条天冬氨酸合成途径。 最后，通过使用体内代谢组学和原发和转移性肿瘤的线粒体下拉图，我 建议定义低氧引发的代谢重连是否是肿瘤转移潜能的决定因素 PDAC细胞。这些分析将确定PDAC细胞在转移过程中经历了哪些代谢变化， 揭示可能有针对性的潜在责任，以减少PDAC向远处器官的扩散。 总之，拟议的实验将确定天冬氨酸合成在pdac肿瘤生长中的作用。 并将测试PDAC细胞是否需要任何其他代谢变化才能转移。

Pancreatic ductal adenocarcinomas (PDACs) are the second leading cause of cancer death in the United States, with a survival rate of 3% once it spreads to distant organs. PDAC cells within a tumor are frequently starved for oxygen due to their high proliferation rate and insufficient vasculature. This degree of hypoxia in the tumor triggers strong metabolic adaptations on cancer cells that allow their survival and proliferation. Among these adaptations are altered uptake and utilization of major nutrients, such as glutamine. However, the precise mechanisms through which pancreas cancer metabolism adapts to hypoxia, and whether this could be exploited for therapy, remain unknown. In preliminary studies, we found that, when pancreatic and lung cancers are exposed to low levels of oxygen, the amino acid aspartate, required for protein and nucleotide synthesis, becomes limiting. Simply increasing the uptake of exogenous aspartate by expression of a plasma membrane aspartate transporter strongly promoted the growth rate of cancer cells under hypoxia and in tumors, as well as enhanced their metastatic potential. These findings provide evidence that aspartate can be a cancer growth-limiting metabolite in vivo. Furthermore, a CRISPR/Cas9 screen using a library of sgRNAs targeting rate-limiting metabolic enzymes revealed that GOT2, one of the two enzymes that de novo synthesizes cellular aspartate from glutamine, is essential for in vitro proliferation under hypoxia of a KRAS/TP53 mutant PDAC cell line. Building upon these results, I propose to test the hypothesis that targeting de novo aspartate synthesis may have therapeutic potential in pancreatic tumors at the level of primary tumor growth and metastasis. Throughout the initial phase of this award, we will define the essentiality of GOT2-mediated aspartate synthesis in a panel of PDAC cell lines upon being exposed to low tensions of oxygen and, importantly, when grown as tumors and during colonization of distant organs. Additionally, we will determine the contribution and impact on cancer proliferation of the two divergent metabolic routes of glutamine conversion into aspartate: oxidative and reductive metabolism. Building upon the obtained results, we will target aspartate synthesis in pre-clinical patient-derived models and KRAS/TP53 mutant mouse models and, by using isotope-labeling metabolomic analysis, we will show which aspartate synthesis route is used by pancreatic tumors in vivo. Finally, by using in vivo metabolomics and mitochondrial pull-downs in primary and metastatic tumors, I propose to define whether hypoxia-triggered metabolic rewiring is a determinant of the metastatic potential of PDAC cells. These analysis will identify which metabolic changes PDAC cells undergo during metastasis, unveiling potential liabilities that could be targeted in order to decrease spread of PDAC to distant organs. Altogether, the proposed experiments will define the role of aspartate synthesis in PDAC tumor growth and metastasis, and will test whether any other metabolic changes are required for PDAC cells to metastasize.