Dissecting the role of serine metabolism in tumor initiating stem cells

剖析丝氨酸代谢在肿瘤起始干细胞中的作用

• 批准号: 9789652
• 负责人: Sanjeethan Baksh
• 金额: $ 5万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-18 至 2022-09-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9789652
• 关键词: Activities of Daily Living; Adult; Amino Acids; Anabolism; Automobile Driving; Branched-Chain Amino Acids; CD34 gene; CD44 gene; CRISPR/Cas technology; Cancer Intervention; Catabolism; Cell Proliferation; Cell physiology; Cells; Cellular Metabolic Process; Cellular Stress; Complex; Conflict (Psychology); Data; Diet; Engineering; FRAP1 gene; Feeds; Flow Cytometry; Frequencies; Gene Expression Regulation; Genes; Glucose; Glutamates; Glutamine; Glycine; Growth; Growth Factor; Hematoxylin and Eosin Staining Method; Immunofluorescence Immunologic; In Vitro; Integrin alpha6; Isotope Labeling; K-18 conjugate; Label; Life; Light; Link; Literature; Malignant Epithelial Cell; Malignant Neoplasms; Measures; Metabolic; Metabolic Pathway; Metabolism; Modeling; Monitor; Mus; Nitrogen; Nude Mice; Oncogenic; Output; Pathway interactions; Patients; Phenotype; Phosphoglycerate dehydrogenase; Phosphorylation; Phosphoserine aminotransferase; Population; Prophylactic treatment; Regulation; Repression; Risk; Role; Serine; Signal Pathway; Signal Transduction; Skin; Squamous cell carcinoma; Stem cells; Stress; Testing; Tissues; Transaminases; Translations; Tumor Initiators; Withdrawal; Work; adult stem cell; base; branched-chain-amino-acid transaminase; cancer cell; cancer stem cell; cell behavior; deprivation; experience; feeding; in vivo; insight; interest; knock-down; metabolomics; overexpression; programs; stem cell population; stemness; therapeutic target; transcription factor; tumor; tumor growth; tumor initiation; tumor progression; tumorigenesis; tumorigenic; uptake

Project Summary Serine metabolism is frequently dysregulated in cancer, but the underlying mechanisms remain unclear. Serine can be generated de novo or taken up exogenously. These pathways often do not compensate for one another, suggesting that flux through the synthesis pathway has serine independent effects. Studies are conflicting regarding the role of serine in regulating the mechanistic target of rapamycin complex 1 (mTORC1), a central integrator of metabolism, growth factor signaling and translational output during tumorigenesis. Even less is known about how this metabolic axis contributes to tumor initiation, which increasingly appears to be driven by adult stem cells. Previous work from the Fuchs lab has demonstrated that ectopic activation of the Sox2 gene in epidermal stem cells (EpdSCs) induces oncogenic stress. SOX2 is expressed by and required for the tumor- initiating stem cells of squamous cell carcinomas (SCCs), among the most common and life-threatening cancers. Interestingly, SOX2+ epidermal stem cells globally repress translation and activate an alternative translational program that enables them to drive tumorigenesis. Given that mTORC1 has been implicated in both stress translation and oncogenic serine catabolism, I am specifically interested in using this model to probe how serine metabolism converges on stem cell oncogenesis. Using primary cultured stem cells, I have discovered that SOX2+ EpdSCs downregulate de novo synthesis in favor of exogenous serine uptake. Moreover, repressing serine synthesis is critical to sustain mTORC1 signaling in SOX2+ cells, establishing a previously unappreciated potential explanation underlying why synthesis and uptake are not interchangeable. Thus, based upon this data, my central hypothesis that exogenous serine is necessary for mTORC1 dependent gene regulation during stem cell driven tumorigenesis. In this study, I will (1) dissect the functional role of exogenous serine on tumor initiation and cancer stem cell function in vivo, and (2) interrogate the mechanism by which serine synthesis regulates mTORC1. To test the importance of serine uptake, I will measure tumorigenic growth when mice are fed serine free diet, tracking stem cell proliferation, phenotype and functional ability via immunofluorescence, flow cytometry and serial limiting dilution studies. To establish a mechanistic link between de novo synthesis repression and mTORC1, I will use CRISPR-Cas9 to generate cells deficient in serine synthesis and perform steady state and heavy isotope labeling metabolomics on these cells with and without serine and glycine. I will focus on the ability of de novo serine synthesis to limit branched chain amino acid synthesis, which directly feeds into mTORC1. Subsequently, I will use CRISPR-Cas9 to ablate branched chain amino acid synthesis in SCC cells deficient for serine synthesis and track tumorigenesis and mTORC1 signaling. These studies will provide insights into how metabolism might regulate stemness during tumor initiation and implicate serine as a therapeutically targetable metabolic liability of cancer stem cells. !

项目摘要 丝氨酸代谢在癌症中经常失调，但其潜在机制仍不清楚。丝氨酸 可以从头产生或外源吸收。这些途径往往不能相互补偿， 这表明通过合成途径的通量具有不依赖于丝氨酸的作用。研究结果相互矛盾 关于丝氨酸在调节雷帕霉素复合物1（mTORC 1）的机制靶点中的作用， 肿瘤发生过程中代谢、生长因子信号传导和翻译输出的整合者。更不是 已知这种代谢轴如何促进肿瘤的发生，这似乎越来越多地受到以下因素的驱动： 成体干细胞Fuchs实验室以前的工作已经证明，在人乳腺癌中Sox 2基因的异位激活， 表皮干细胞（EpdSC）诱导致癌应激。SOX 2是肿瘤表达和需要的- 鳞状细胞癌（SCC）是最常见和威胁生命的癌症之一。 有趣的是，SOX 2+表皮干细胞全面抑制翻译，并激活替代翻译。 使它们能够驱动肿瘤发生的程序。鉴于mTORC 1与两种压力都有关系， 翻译和致癌丝氨酸催化剂，我特别感兴趣的是使用这个模型来探测丝氨酸 代谢集中于干细胞肿瘤发生。使用原代培养的干细胞，我发现， SOX 2 + EpdSC下调从头合成，有利于外源丝氨酸摄取。此外，抑制 丝氨酸合成对于维持SOX 2+细胞中的mTORC 1信号传导至关重要，建立了以前未被认识到的 可能解释为什么合成和吸收是不可互换的。因此，根据这些数据， 我的中心假设是，外源丝氨酸是mTORC 1依赖性基因调控所必需的 在干细胞驱动的肿瘤发生过程中。在本研究中，我将（1）剖析外源丝氨酸的功能作用， 对肿瘤发生和癌症干细胞功能的影响，以及（2）询问丝氨酸 合成调节mTORC 1。为了测试丝氨酸摄取的重要性，我将测量肿瘤生长， 给小鼠喂食不含丝氨酸的饮食，跟踪干细胞增殖、表型和功能能力， 免疫荧光、流式细胞术和连续有限稀释研究。在…之间建立机械联系 在新合成抑制和mTORC 1的情况下，我将使用CRISPR-Cas9来产生丝氨酸缺陷的细胞， 合成并在这些细胞上进行稳态和重同位素标记代谢组学， 丝氨酸和甘氨酸。我将集中在能力从头丝氨酸合成限制支链氨基酸 合成，其直接馈送到mTORC 1。随后，我将使用CRISPR-Cas9来消融支链 丝氨酸合成缺陷的SCC细胞中的氨基酸合成，并跟踪肿瘤发生和mTORC 1信号传导。 这些研究将提供有关代谢如何在肿瘤发生期间调节干性的见解， 表明丝氨酸是癌症干细胞治疗靶向代谢倾向。 !