ENGINEERING ORGANELLE FUNCTION TO REWIRE CANCER CELL METABOLISM

描述（由申请人提供）：细胞在其生命周期的每一秒内，在数百个不同的位置同时进行10E8次以上的生化反应。协调这些无数的过程是一项艰巨的任务，对每个组织和整个生物体的正常运作至关重要。为了应对不断变化的内部和外部信号，细胞已经进化出能够感知营养和能量状态的机制，并在相应的情况下，提示代谢活动的微调变化，以缓冲这些变化。我们最近的工作强调了一种细胞器的作用，溶酶体，作为代谢稳态的守门人。溶酶体可以感知细胞营养水平的变化，并将其传递给主要的生长调节蛋白激酶mTORC1。反过来，mTORC1控制溶酶体内的分解代谢反应，为细胞提供代谢构建块并帮助维持全球营养供应。重要的是，溶酶体功能失调是遗传性代谢紊乱的原因，并且正在成为一些侵袭性癌症进展的一个促成因素。目前在全细胞或细胞群中使用质谱仪或荧光生物传感器的技术无法到达溶酶体内部，以识别和测量随时间在其内部产生的数百种代谢物。为了建立一个关于溶酶体如何调节细胞代谢的全面的系统级模型，需要一种简化的方法来捕捉溶酶体功能的基本空间和时间特征。我们的目标是开发一种新的体外系统，使代谢的研究在单一细胞器水平。在目前的建议中，我们的系统将在试管或盖子表面重建溶酶体与细胞器（称为自噬体）的融合。在高空间和时间分辨率的成像中，我们将剖析溶酶体在自噬中的参与，自噬是一种细胞的“自我吞噬”过程，对正常细胞和癌细胞的稳态都是必不可少的。通过扩大这种制备，并将其与高通量代谢物分析相结合，我们将生成一个空间和时间的“代谢图”，描绘溶酶体内产生的数百种营养物质，描述它们积累和输出的时间过程，并确定将这些营养物质释放到细胞的运输机制。利用我们系统的空间和时间分辨率，我们将解决当今癌症研究中的一个主要挑战-高致死性胰腺导管腺癌（PDAC）如何利用自噬在营养贫乏的微环境中获得生长和生存优势。利用我们对溶酶体代谢组的“内部知识”，我们将测试自噬可能允许PDAC细胞通过利用大量细胞内营养物质来维持体内平衡的假设，我们将设计新的策略来消耗这些内部营养物质储存。该项目将产生新的工具来阐明代谢的亚细胞组织，并为创新方法重新连接癌细胞代谢奠定基础。

DESCRIPTION (provided by applicant): A cell executes well over 10E8 simultaneous biochemical reactions at hundreds of different locations during every second of its lifetime. Coordinating these innumerable processes is a formidable task that is essential to the correct functioning of each tissue and the entire organism. In response to ever changing internal and external cues, cells have evolved mechanisms that can sense nutrient and energy status and, in response, prompt fine-tuned changes in metabolic activity that buffer these variations. Our recent work has highlighted the role of one organelle, the lysosome, as a gate-keeper of metabolic homeostasis. The lysosome can sense and relay variations in cellular nutrient levels to the master growth regulatory protein kinase mTORC1. In turn, mTORC1 governs catabolic reactions within the lysosome that supply the cell with metabolic building blocks and help maintain global nutrient supply. Importantly, dysregulated lysosomal function is the cause of hereditary metabolic disorders, and is emerging as a contributing factor to the progression of some aggressive cancers. Current technologies that employ mass spectrometers or fluorescent biosensors in whole cells or cell populations cannot reach inside the lysosome to identify and measure the hundreds of metabolites that are generated inside it over time. In order to build a comprehensive, systems-level model of how the lysosome regulates cellular metabolism, a reductionist approach that captures essential spatial and temporal features of lysosomal function in a simplified context is needed. Our goal is to develop a novel in vitro system that wil enable the study of metabolism at the single organelle level. In the current proposal, our system will reconstitute the fusion of lysosomes with organelles known as autophagosomes in test tubes or on the surface of coverslips. Imaging at high spatial and temporal resolution, we will dissect the participation of the lysosome in autophagy, a cellular 'self-eat' process that is essential to the homeostasis of both normal and cancer cells. By scaling up this preparation, and coupling it to high throughput metabolite profiling, we will generate a spatial and temporal 'metabolic map' that profiles hundreds of nutrients generated within the lysosome, describes the time course of their buildup and export, and identifies the transport mechanisms that release these nutrients to the cell. Leveraging the spatial and temporal resolution of our system, we will address a major challenge in present-day cancer research- how highly lethal pancreatic ductal adenocarcinoma (PDAC) exploits autophagy to gain a growth and survival advantage in nutrient-poor microenvironments. Using our 'inside knowledge' of the lysosomal metabolome, we will test the hypothesis that autophagy may allow PDAC cells to maintain homeostasis by tapping into large intracellular reservoirs of nutrients, and we will devise novel strategies to deplete these internal nutrient stores. This project will generate novel tools to illuminate the subcellular organization of metabolism, and lay the foundations for innovative ways to rewire cancer cell metabolism.