Regulation and Function of Stromal Macropinocytosis in Pancreatic Ductal Adenocarcinoma

项目总结 近年来，人们认识到新陈代谢适应在保证生存中所起的作用。 对于遇到肿瘤微环境中严酷的营养贫乏条件的细胞来说，这是一种优势。的 与这一提议特别相关的是现在被广泛接受的一个概念，即巨饮细胞症，一种内吞 液体相摄取的机制，在肿瘤中作为氨基酸供应途径。通过刺激吸收 胞外蛋白并将其定位于溶酶体降解，巨噬细胞吞噬为细胞提供了 蛋白质衍生氨基酸的来源，使肿瘤避免氨基酸耗尽并存活于营养 压力。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物质， 支持肿瘤生长。我们在PDAC细胞上发表的工作已经证实，谷氨酰胺耗竭具有 调节巨噬细胞吞噬的能力--根据需要向上或向下调节过程。有趣的是，我们的 这项提案中提出的初步数据首次表明，谷氨酰胺的缺乏也可以 刺激肿瘤相关成纤维细胞(CAF)的巨噬细胞吞噬。从机制上讲，我们已经将 谷氨酰胺应激诱导CAF巨噬细胞摄取CAMKK2-AMPK信号，从而导致rac1- 巨噬细胞吞噬所需的依赖性肌动蛋白细胞骨架动力学。AMPK是一种生物能量应激 最常在葡萄糖饥饿的情况下研究的传感器，与谷氨酰胺耗竭不同的是， 而不是促进CAF巨噬细胞增多。值得注意的是，对AMPK的激活和功能在 谷氨酰胺耗竭。我们的初步研究表明，巨噬细胞增多在CAFS中具有双重作用。 可以维持CAF的活性和功能，并能提供分泌的氨基酸来滋养肿瘤。 细胞。重要的是，我们在体内和体外对正常成纤维细胞巨噬细胞吞噬作用的检测，以及在 来自其他肿瘤类型的CAF表明，谷氨酰胺耗竭诱导的间质摄取是 胰腺CAF。基于这些数据，我们的中心假设是谷氨酰胺的缺乏选择性地推动 CAF中依赖于CAMKK2-AMPK的巨噬细胞吞噬作用，间质巨噬细胞吞噬是一个 可用于PDAC治疗。在这项提案中，我们将1)研究推动 谷氨酰胺在CAF巨噬细胞增多中的选择性作用，2)肾小管间质巨噬细胞增多的功能特征 PDAC，以及3)确定巨噬细胞吞噬抑制所发生的间质重组是否可以 用于PDAC治疗。这个项目将具有重大的意义、新颖性和影响力，它将 首次评估巨噬细胞吞噬在PDAC肿瘤间质中的作用，并首次分析 选择性地将谷氨酰胺饥饿与CAMKK2-AMPK信号联系起来。此外，因为巨噬细胞增多症 对肿瘤细胞和间质都很重要，我们的工作可能会对发展产生巨大的影响 对PDAC的新的治疗方式。

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.