Macropinocytosis is a highly conserved endocytic process by which extracellular fluid and its contents are internalized into cells via large, heterogeneous vesicles known as macropinosomes. Oncogenic Ras proteins have been shown to stimulate macropinocytosis but the functional contribution of this uptake mechanism to the transformed phenotype remains unknown1-3. Here we show that Ras-transformed cells utilize macropinocytosis to transport extracellular protein into the cell. The internalized protein undergoes proteolytic degradation, yielding amino acids including glutamine that can enter central carbon metabolism. Accordingly, the dependence of Ras-transformed cells on free extracellular glutamine for growth can be suppressed by the macropinocytic uptake of protein. Consistent with macropinocytosis representing an important route of tumor nutrient uptake, its pharmacological inhibition compromised the growth of Ras-transformed pancreatic tumor xenografts. These results identify macropinocytosis as a mechanism by which cancer cells support their unique metabolic needs and point to the possible exploitation of this process in the design of anti-cancer therapies.

Although the polycomb group protein Enhancer of Zeste Homolog 2 (EZH2) is well recognized for its role as a key regulator of cell differentiation, its involvement in tissue regeneration is largely unknown. Here we show that EZH2 is up-regulated following cerulein-induced pancreatic injury and is required for tissue repair by promoting the regenerative proliferation of progenitor cells. Loss of EZH2 results in impaired pancreatic regeneration and accelerates KRas G12D -driven neoplasia. Our findings implicate EZH2 in constraining neoplastic progression through homeostatic mechanisms that control pancreatic regeneration and provide insights into the documented link between chronic pancreatic injury and an increased risk for pancreatic cancer.

The N-terminal domain of L9 (NTL9) is a 56-residue mixed α-β protein that lacks disulfides, does not bind cofactors, and folds reversibly. NTL9 has been widely used as a model system for experimental and computational studies of protein folding and for investigations of the unfolded state. The role of side-chain interactions in the folding of NTL9 is probed by mutational analysis. ϕ-values, which represent the ratio of the change in the log of the folding rate upon mutation to the change in the log of the equilibrium constant for folding, are reported for 25 point mutations and 15 double mutants. All ϕ-values are small, with an average over all sites probed of only 0.19 and a largest value of 0.4. The effect of modulating unfolded-state interactions is studied by measuring ϕ-values in second- site mutants and under solvent conditions that perturb unfolded-state energetics in a defined way. Neither of these alterations significantly affects the distribution of ϕ-values. The results, combined with those of earlier studies that probe the role of hydrogen-bond formation in folding and the burial of surface area, reveal that the transition state for folding contains extensive backbone structure and buries a significant fraction of hydrophobic surface area, but lacks well developed side-chain-side-chain interactions. The folding transition state for NTL9 does not contain a specific "nucleus" consisting of a few key residues; rather, it involves extensive backbone hydrogen bonding and partially formed structure delocalized over almost the entire domain. The potential generality of these observations is discussed.

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Oncogenic Ras stimulates macropinocytosis, an endocytic mechanism of fluid-phase uptake that produces large intracellular vesicles known as macropinosomes. Recently, we have linked the macropinocytic uptake of extracellular albumin and its subsequent degradation to amino acid supply and proliferation in Ras-transformed cells. The ability of albumin to serve as a nutrient source in oncogenic Ras-expressing cells is blocked by inhibiting its internalization via treatment with 5-(N-Ethyl-N-isopropyl) amiloride (EIPA). We determined that EIPA treatment diminished the growth of pancreatic tumor xenografts and that this effect was selective for tumors with a high macropinocytic index. Currently, we are exploring the feasibility of employing macropinocytosis inhibition as an anticancer therapeutic modality utilizing an autochthonous mouse model of pancreatic cancer. In these autochthonous tumors, macropinocytosis is a prominent feature of pancreatic cells found in mid- to late-stage PanIN lesions, as well as in fibroblasts and immune cells residing within the tumor stroma. We have found that EIPA treatment results in a rapid and robust reduction in proliferative capacity both in tumor cells and the surrounding stromal cells. Intriguingly, our preliminary data indicates that EIPA treatment reduces the number of activated fibroblasts associated with PanIN lesions, decreases collagen deposition and results in an increase in blood vessel diameter. Studies have demonstrated that targeting components of the extracellular matrix within the tumor stroma can cause expansion of the vasculature, which can be harnessed to improve drug delivery and permeability to the tumor. Altogether, our findings suggest that macropinocytosis inhibition could be exploited not only to target the tumor cells, but also to target the tumor stroma and enhance the delivery of chemotherapeutics. This abstract is also presented as Poster B37. Citation Format: Cosimo Commisso, Craig Ramirez, Rengin Soydaner-Azeloglu, David L. Bajor, Robert H. Vonderheide, Dafna Bar-Sagi. Pulling out all the stops: Exploiting macropinocytosis inhibition for the treatment of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr PR08.