Regulation and Function of Stromal Macropinocytosis in Pancreatic Ductal Adenocarcinoma

项目摘要 近年来，人们已经认识到代谢适应在生存中的作用 这对遇到肿瘤微环境的苛刻营养不良条件的细胞有利。的 与此建议特别相关的是现在广泛接受的概念，即巨胞饮作用，一种内吞作用， 液相摄取机制，在肿瘤中作为氨基酸供应途径发挥作用。通过刺激摄取 细胞外蛋白质和靶向它的溶酶体降解，巨胞饮作用提供细胞， 蛋白质衍生氨基酸的来源，使肿瘤能够绕过氨基酸耗尽并在营养中生存 应力胰腺导管腺癌（PDAC）肿瘤缺乏谷氨酰胺，谷氨酰胺是一种重要的营养素， 支持肿瘤生长。我们在PDAC细胞中发表的工作已经确定，谷氨酰胺缺失具有 调节巨胞饮作用的能力-根据需要上调或下调该过程。有趣的是，我们 该提案中提出的初步数据首次表明，谷氨酰胺缺乏也可能 刺激癌症相关成纤维细胞（CAF）中巨胞饮作用。机械地说，我们认为 谷氨酰胺应激诱导CAFs中巨胞饮细胞摄取CAMKK 2-AMPK信号，导致Rac 1- 依赖肌动蛋白的细胞骨架动力学所需的巨胞饮。AMPK是一种生物能量应激 最常在葡萄糖饥饿的背景下研究的传感器，与谷氨酰胺耗尽不同， 不增加CAF巨胞饮作用。值得注意的是，人们对AMPK的激活和功能知之甚少。 谷氨酰胺耗竭。我们的初步研究表明，巨胞饮在CAFs中具有双重作用， 可以维持CAF的活力和功能，并且可以提供分泌的氨基酸来滋养肿瘤 细胞重要的是，我们在正常成纤维细胞中的巨胞饮作用的体内和体外检查，以及在 来自其他肿瘤类型的CAFs表明，谷氨酰胺耗竭诱导的基质摄取是CAFs特有的。 胰腺CAF。基于这些数据，我们的中心假设是，谷氨酰胺的稀缺选择性地驱动 CAF中CAMKK 2-AMPK依赖的巨胞饮作用，基质巨胞饮作用是一个过程， 可以在PDAC治疗中加以利用。在这个提议中，我们将1）研究驱动 谷氨酰胺在CAF巨胞饮作用中的选择性作用，2）功能性表征CAF中基质巨胞饮作用， PDAC，和3）确定是否与巨胞饮抑制发生的基质重组， 用于PDAC治疗。该项目将具有重要意义、新奇和影响力， 构成了对PDAC肿瘤间质中巨胞饮作用的首次评价和对PDAC肿瘤间质中巨胞饮作用的首次分析。 选择性地将谷氨酰胺饥饿与CAMKK 2-AMPK信号传导联系起来。此外，由于巨胞饮作用是 在肿瘤细胞和间质中都很重要，我们的工作可能对肿瘤细胞的发展产生巨大影响。 PDAC的新型治疗方法。

PROJECT SUMMARY Recent years have witnessed an appreciation of the role that metabolic adaptation plays in conferring survival advantages to cells that encounter the harsh nutrient-poor conditions of the tumor microenvironment. Of particular relevance to this proposal is the now widely accepted notion that macropinocytosis, an endocytic mechanism of fluid-phase uptake, functions in tumors as an amino acid supply route. By stimulating the uptake of extracellular protein and targeting it for lysosomal degradation, macropinocytosis provides cells with a source of protein-derived amino acids, allowing tumors to circumvent amino acid depletion and survive nutrient stress. Pancreatic ductal adenocarcinoma (PDAC) tumors are deficient in glutamine, a vital nutrient that supports tumor growth. Our published work in PDAC cells has established that glutamine depletion has the capacity to modulate macropinocytosis – dialing the process up or down as required. Interestingly, our preliminary data presented in this proposal demonstrate for the first time that glutamine scarcity can also stimulate macropinocytosis in cancer-associated fibroblasts (CAFs). Mechanistically, we have attributed glutamine stress-induced macropinocytic uptake in CAFs to a CAMKK2-AMPK signal that leads to the Rac1- dependent actin cytoskeleton dynamics that are required for macropinocytosis. AMPK is a bioenergetic stress sensor that is most often studied in the context of glucose starvation, which unlike glutamine depletion, does not boost CAF macropinocytosis. Notably, not much is known about AMPK activation and function during glutamine depletion. Our preliminary studies suggest that macropinocytosis has a dual purpose in CAFs – it can serve to sustain CAF viability and function, and it can provide secreted amino acids to nourish the tumor cells. Importantly, our in vivo and in vitro examinations of macropinocytosis in normal fibroblasts, as well as in CAFs originating from other tumor types, suggest that glutamine depletion-induced stromal uptake is unique to pancreatic CAFs. Based on these data, our central hypothesis is that glutamine scarcity selectively drives CAMKK2-AMPK-dependent macropinocytosis in CAFs, and that stromal macropinocytosis is a process that can be harnessed in PDAC therapy. In this proposal, we will 1) examine the molecular mechanisms driving the selective role of glutamine in CAF macropinocytosis, 2) functionally characterize stromal macropinocytosis in PDAC, and 3) determine whether the stromal reorganization that occurs with macropinocytosis inhibition can be leveraged for PDAC therapy. This project will be of great significance, novelty and impact, as it will constitute the first evaluation of the role of macropinocytosis in the PDAC tumor stroma and the first analysis to selectively link glutamine starvation to CAMKK2-AMPK signaling. Moreover, because macropinocytosis is important in both the tumor cells and the stroma, our work could have tremendous impact on the development of novel therapeutic modalities for PDAC.