Metabolic changes accompanying durable, hypoxia-driven EMT in pancreas cancer

项目摘要 本申请是为了响应被标识为NOT-CA的特别利益通知（NOSI）而提交的- 23-045. 胰腺导管腺癌（PDAC）肿瘤表现出低氧张力（缺氧）区域， 典型的血管形成不良。Lazzara实验室最近表明，缺氧会导致一种特别严重的 在转化的PDAC导管细胞中的持久上皮-间质转化（EMT）可持续数周， 相比之下，当EMT由生长因子驱动时，持续时间只有几天。机制 导致持久EMT涉及组蛋白甲基化和MAP激酶激活之间的正反馈 这是由组蛋白脱甲基酶活性在低氧环境中的丧失引发的。因为新陈代谢的重组 伴随EMT在某些癌症背景下，我们提出了新的假设，缺氧促进 尤其是PDAC细胞中持久的代谢变化，其在低氧驱动力被缓解后持续很长时间。 这些代谢状态的持久变化可能通过重新配置细胞内的蛋白质来促进疾病进展。 能量需求和增加免疫抑制代谢物的丰度。我们将探讨这一点 使用缺氧介导的PDAC细胞EMT和代谢组学分析的多种模型的新假设。的 这项工作将由美国国家癌症研究所癌症系统的两名主要研究员合作进行 生物学联盟的成员分布在不同的学术机构。在第一阶段，我们会 使用三种不同的模型系统来创建PDAC细胞系的样品，这些细胞系被驱动以响应于 缺氧或生长因子。模型系统包括建立的人PDAC系、来源于人PDAC的细胞系。 患者来源的异种移植物，以及来源于在以下谱系中产生的自发性PDAC肿瘤的细胞系- 追踪本地小鼠模型。在去除EMT驱动力（缺氧或缺氧）后的几个时间点， 生长因子），将细胞流式分选成间充质和上皮细胞群，并冷冻细胞样品 将被生成。在第二阶段，将通过液相色谱-质谱法分析样品 代谢组学，以探测代谢组学程序的变化和缺氧相关的 免疫抑制代谢物。将应用还原技术鉴别代谢物和 代谢途径的变化，最大限度地解释样本之间的差异。在第3阶段，代谢 将检测样本间方差分析指定的途径控制EMT的能力 持久性。这些研究将揭示持久缺氧驱动的EMT的程度， 伴随着持久的代谢变化，以及这些变化如何改变间充质细胞的表型， PDAC导管细胞。了解这些问题将最终揭示新的可药物漏洞， 用于治疗PDAC。

PROJECT SUMMARY This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA- 23-045. Pancreatic ductal adenocarcinoma (PDAC) tumors exhibit regions of low oxygen tension (hypoxia) due to characteristically poor vascularization. The Lazzara lab recently showed that hypoxia drives an especially durable epithelial-mesenchymal transition (EMT) in transformed PDAC ductal cells that can last for weeks, compared to a persistence timescale of just days when EMT is driven by growth factors. The mechanism leading to durable EMT involves a positive feedback between histone methylation and MAP kinase activation that is initiated by loss of histone demethylase activity in low-oxygen settings. Because metabolic rewiring accompanies EMT in some cancer contexts, we propose the novel hypothesis that hypoxia promotes especially durable metabolic changes in PDAC cells that persist long after the hypoxic driving force is relieved. These durable changes to the metabolic state may promote disease progression by reconfiguring cellular energetic needs and by increasing the abundance of immune-suppressive metabolites. We will explore this novel hypothesis using multiple models of hypoxia-mediated PDAC cell EMT and metabolomic profiling. The work will be undertaken collaboratively by two principal investigator members of the NCI Cancer Systems Biology Consortium who are located at different academic institutions. In Phase I of the proposed work, we will use three different model systems to create samples of PDAC cell lines driven to undergo EMT in response to hypoxia or growth factors. The model systems include established human PDAC lines, cell lines derived from patient-derived xenografts, and cell lines derived from spontaneous PDAC tumors generated in a lineage- traced autochthonous mouse model. At several time points after removal of the EMT driving force (hypoxia or growth factors), cells will be flow-sorted into mesenchymal and epithelial populations, and frozen cell samples will be generated. In Phase 2, samples will be analyzed by liquid chromatography-mass spectrometry metabolomics to probe for changes in metabolomic programs and accumulation of hypoxia-associated immunosuppressive metabolites. Dimensionality reduction techniques will be applied to identify metabolite and metabolic pathway changes that maximally explain variance among the samples. In Phase 3, metabolic pathways nominated by the analysis of variance among samples will be tested for their ability to control EMT persistence. These investigations will uncover the degree to which durable hypoxia-driven EMT is accompanied by durable metabolic changes and how such changes may alter the phenotypes of mesenchymal PDAC ductal cells. Understanding these issues will ultimately reveal new druggable vulnerabilities that can be exploited for treating PDAC.