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Macropinocytosis is a highly conserved endocytic process by which extracellular fluid and its contents are internalized into cells through large, heterogeneous vesicles known asmacropinosomes.Oncogenic Ras proteins have been shown to stimulatemacropinocytosis but the functional contribution of this uptake mechanism to the transformed phenotype remains unknown^sup 1-3^. Here we show that Rastransformed cells use 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 nutrient uptake in tumours, its pharmacological inhibition compromises the growth of Ras-transformed pancreatic tumour 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 anticancer therapies.
To date, the induction of macropinocytosis by oncogenic Ras has been characterized in the setting of overexpressed proteins1-3. To determine whether stimulated macropinocytosis is a feature of cancer cells endogenously expressing oncogenic Ras, we analysed fluid-phase uptake in human pancreatic and urinary bladder cancer cell lines harbouring oncogenic Ras mutations and compared uptake to wild-type Ras-expressing cells originating from carcinomas of the same tissue type. Macropinosomes were visualized on the basis of the ability of cells to internalize extracellular mediumcontaining tetramethylrhodaminelabelled high-molecular-mass dextran (TMR-dextran), an established marker of macropinocytosis. Pancreatic adenocarcinoma-derived human MIA PaCa-2 cells, which are homozygous for the KRASG12C allele4, displayed appreciably higher levels of TMR-dextran uptake compared to BxPC-3 cells, which express wild-type KRAS5 (Fig. 1a, b). That the TMR-dextran labelling in the oncogenic Ras-expressing cells reflects uptake through macropinocytosis is indicated by the observation that uptake was inhibited in a dose-dependent manner by 5-(N-ethyl-Nisopropyl) amiloride (EIPA) (Fig. 1c, d), which has been shown to inhibit macropinosome formation without affecting other endocytic pathways6,7. Importantly, the knockdown of KRAS led to an attenuation of macropinocytosis, confirming the dependence of this uptake mechanism on oncogenic Ras expression (Supplementary Fig. 1a-d). This conclusion is further supported by the observation that bladder carcinoma-derived T24 cells, which are homozygous for the HRASG12V allele8, exhibit increased levels of macropinocytosis relative to 5637 cells, which...
