Metabolic Recycling and Compartmentalization in Tumor Progression

项目总结 癌细胞促进营养物质的分解代谢，导致代谢产物过剩和堆积 废物，尤指乳酸和氨水。利用稳定同位素示踪和质谱分析，我们发现 乳腺癌细胞清除氨基酸分解代谢产生的氨作为一种再利用的氮 生物量来源(Spinelli等人。科学，2017)。氨循环加速3D细胞增殖 培养和体内小鼠异种移植模型。这些研究引出了两个后续问题，将是 在本提案的具体目标1(F99阶段)中处理。第一(目标1A)，通过什么机制 氨会刺激乳腺癌的增殖吗？初步数据支持亚细胞的假设 氨代谢对增殖率的影响需要进行分区。使用新陈代谢 追踪和快速免疫沉淀，我们追踪了氨同化到线粒体的定位 而随后代谢产物外流到胞浆部分，显示了氨刺激的作用 扩散。第二(目标1B)，这种新的氨循环途径对以下反应有什么影响 治疗？初步数据显示，氨同化可以避开谷氨酰胺酶(GLS)的作用 通过谷氨酸脱氢酶(GDH)补充谷氨酸水平。我们建议的研究 将确定GDH和GLS抑制在体内和在原发性乳腺癌肿瘤中是否协同作用 阐明乳腺癌细胞抵抗GLS抑制的机制。 除了氨以外，线粒体还通过大量代谢促进肿瘤的生长和增殖 小路。然而，营养物质穿越线粒体内膜的机制却很少。 研究，特别是因为45%的线粒体营养转运蛋白家族没有特征。因此， 利用我在代谢物追踪和质谱学方面的专业知识，以及在遗传学方面获得的新技能 (CRISPR)筛查和生物信息学，拟议的研究(目标2，K00阶段)将系统地评估 线粒体营养转运体在肿瘤生长和存活中的作用。这些数据将是第一批 对含有致癌驱动因素的癌细胞进行代谢区划的重要性进行全球评估 如KRAS、IDH和PI3K突变。此外，本研究还将阐明未特定化的功能 线粒体营养转运体。 除了拟议的研究外，研究金培训计划还包括获得以下方面的经验 指导，参加负责任的研究、团队管理和预算方面的课程，以及 参加科学会议，如肿瘤代谢关键会议，以发展一个网络 科学合作者。这项拟议的研究将在哈佛医学院和布罗德研究所进行， 拥有具备设备、技术、核心设施、协作潜力和 完成拟议培训计划所需的研究和职业发展资源。

PROJECT SUMMARY Cancer cells elevate nutrient catabolism, causing excess production and accumulation of metabolic waste, especially lactate and ammonia. Using stable isotope tracing and mass spectrometry, we discovered that breast cancer cells scavenged ammonia generated by amino acid catabolism as a re-purposed nitrogen source for biomass (Spinelli et al. Science, 2017). Ammonia recycling accelerated proliferation in 3D cell culture and in vivo mouse xenograft models. These studies lead to two follow-up questions that will be addressed in Specific Aim 1 (F99 phase) of this proposal. First (Aim 1A), what is the mechanism by which ammonia stimulates breast cancer proliferation? Preliminary data supports the hypothesis that subcellular compartmentalization of ammonia metabolism is required for its effect on proliferation rate. Using metabolic tracing and rapid immunoprecipiation, we tracked the localization of ammonia assimilation to the mitochondria and the subsequent efflux of metabolites to the cytosolic fraction, showing a role in ammonia-stimulated proliferation. Second (Aim 1B), what is the effect of this novel ammonia-recycling pathway on response to therapy? Preliminary data shows that ammonia assimilation circumvents the effect of glutaminase (GLS) inhibitors through replenishing glutamate levels via glutamate dehydrogenase (GDH). Our proposed studies will determine if GDH and GLS inhibition are synergistic in vivo and in primary breast cancer tumors to elucidate a mechanism by which breast cancer cells are resistant to GLS inhibition. Beyond ammonia, mitochondria promote tumor growth and proliferation through numerous metabolic pathways. However, the mechanisms by which nutrients traverse the inner mitochondrial membrane are little studied, particularly because 45% of the mitochondria nutrient transporter family is uncharacterized. Therefore, using my expertise in metabolite tracing and mass spectrometry in addition to new skills gained in genetic (CRISPR) screening and bioinformatics, the proposed studies (Aim 2, K00 phase) will systematically assess the role of mitochondrial nutrient transporters in tumor growth and survival. These data will be the first to globally evaluate the essentiality of metabolic compartmentalization in cancer cells harboring oncogenic drivers such as KRAS, IDH and PI3K mutations. Furthermore, this study will elucidate the function of uncharacterized mitochondrial nutrient transporters. In addition to the proposed studies, the fellowship training plan includes gaining experience with mentorship, taking courses on responsible conduct of research, team management, and budgeting, and attending scientific conferences such as the Tumor Metabolism Keystone meeting to develop a network of scientific collaborators. The proposed studies will occur at Harvard Medical School and The Broad Institute, which have environments with the equipment, technology, core facilities, potential for collaboration, and the resources for research and career development needed to complete the proposed training plan.