Dissecting the role of serine metabolism in tumor initiating stem cells

项目摘要 丝氨酸代谢在癌症中经常被失调，但其潜在的机制仍不清楚。丝氨酸 可以从头产生，也可以从外部吸收。这些途径往往不能相互弥补， 这表明通过合成途径的通量具有丝氨酸不依赖的作用。研究相互矛盾 关于丝氨酸在调节雷帕霉素复合体1(MTORC1)机制靶点中的作用， 肿瘤发生过程中代谢、生长因子信号和翻译输出的整合因子。更少的是 知道这个代谢轴如何促进肿瘤的发生，而肿瘤的发生似乎越来越多地由 成体干细胞。福克斯实验室之前的工作已经证明，Sox2基因的异位激活 表皮干细胞(EPDSCs)诱导致癌压力。SOX2由肿瘤表达，并且是肿瘤所必需的- 鳞状细胞癌(SCCs)的启动干细胞，是最常见和威胁生命的癌症之一。 有趣的是，SOX2+表皮干细胞在全球范围内抑制翻译并激活另一种翻译 使他们能够推动肿瘤发生的计划。鉴于mTORC1与这两种压力有关 翻译和致癌丝氨酸分解代谢，我特别感兴趣的是用这个模型来探索丝氨酸是如何 新陈代谢集中在干细胞的致癌作用上。使用原代培养的干细胞，我发现 SOX2+EPdSCs下调从头合成，有利于外源性丝氨酸摄取。此外，压制 丝氨酸合成对维持SOX2+细胞中的mTORC1信号至关重要，建立了以前未被认识到的 潜在的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性丝氨酸是mTORC1依赖的基因调控所必需的 在干细胞驱动的肿瘤形成过程中。在本研究中，我将(1)剖析外源丝氨酸的功能作用 关于体内肿瘤启动和肿瘤干细胞功能的研究；(2)丝氨酸的作用机制 合成调节mTORC1。为了测试丝氨酸摄取的重要性，我将在以下情况下测量肿瘤生长 给予小鼠无丝氨酸饮食，跟踪干细胞的增殖、表型和功能 免疫荧光、流式细胞术和系列限制稀释研究。在两国之间建立机械联系 从头合成抑制和mTORC1，我将使用CRISPR-Cas9来产生缺乏丝氨酸的细胞 合成并在有或无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。我将重点介绍从头合成丝氨酸来限制支链氨基酸的能力。 合成，它直接馈送到mTORC1。随后，我将使用CRISPR-Cas9来消融支链 鳞状细胞癌细胞的氨基酸合成缺乏丝氨酸合成，跟踪肿瘤发生和mTORC1信号转导。 这些研究将为新陈代谢如何在肿瘤发生和发展过程中调节干细胞提供深入的见解。 暗示丝氨酸是癌症干细胞的一种治疗靶向代谢易感性。 好了！

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. !