Regulation and Function of Stromal Macropinocytosis in Pancreatic Ductal Adenocarcinoma

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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.