Craig Ramirez, Ph.D.

Oncogenic activation of RAS is associated with the acquisition of a unique set of metabolic dependencies that contribute to tumour cell fitness. Cells that express oncogenic RAS are able to internalize and degrade extracellular protein via a fluid-phase uptake mechanism termed macropinocytosis. There is increasing recognition of the role of this RAS-dependent process in the generation of free amino acids that can be used to support tumour cell growth under nutrient-limiting conditions. However,… Show more

Oncogenic activation of RAS is associated with the acquisition of a unique set of metabolic dependencies that contribute to tumour cell fitness. Cells that express oncogenic RAS are able to internalize and degrade extracellular protein via a fluid-phase uptake mechanism termed macropinocytosis. There is increasing recognition of the role of this RAS-dependent process in the generation of free amino acids that can be used to support tumour cell growth under nutrient-limiting conditions. However, little is known about the molecular steps that mediate the induction of macropinocytosis by oncogenic RAS. Here we identify vacuolar ATPase (V-ATPase) as an essential regulator of RAS-induced macropinocytosis. Oncogenic RAS promotes the translocation of V-ATPase from intracellular membranes to the plasma membrane via a pathway that requires the activation of protein kinase A by a bicarbonate-dependent soluble adenylate cyclase. Accumulation of V-ATPase at the plasma membrane is necessary for the cholesterol-dependent plasma-membrane association of RAC1, a prerequisite for the stimulation of membrane ruffling and macropinocytosis. These observations establish a link between V-ATPase trafficking and nutrient supply by macropinocytosis that could be exploited to curtail the metabolic adaptation capacity of RAS-mutant tumour cells. Show less

The product of the KRAS oncogene, KRAS4B, promotes tumor growth when associated with the plasma membrane (PM). PM association is mediated, in part, by farnesylation of KRAS4B, but trafficking of nascent KRAS4B to the PM is incompletely understood. We performed a genome-wide screen to identify genes required for KRAS4B membrane association and identified a G protein–coupled receptor, GPR31. GPR31 associated with KRAS4B on cellular membranes in a farnesylation-dependent fashion, and retention of… Show more

The product of the KRAS oncogene, KRAS4B, promotes tumor growth when associated with the plasma membrane (PM). PM association is mediated, in part, by farnesylation of KRAS4B, but trafficking of nascent KRAS4B to the PM is incompletely understood. We performed a genome-wide screen to identify genes required for KRAS4B membrane association and identified a G protein–coupled receptor, GPR31. GPR31 associated with KRAS4B on cellular membranes in a farnesylation-dependent fashion, and retention of GPR31 on the endoplasmic reticulum inhibited delivery of KRAS4B to the PM. Silencing of GPR31 expression partially mislocalized KRAS4B, slowed the growth of KRAS-dependent tumor cells, and blocked KRAS-stimulated macropinocytosis. Our data suggest that GPR31 acts as a secretory pathway chaperone for KRAS4B. Show less

Uptake of nutrients, such as glucose and amino acids, is critical to support cell growth and is typically mediated by cell surface transporters. An alternative mechanism for the bulk uptake of nutrients from the extracellular space is macropinocytosis, a nonclathrin, and nonreceptor-mediated endocytic process, in which extracellular fluid is taken up into large intracellular vesicles called macropinosomes. Oncogenic transformation leads to the increased metabolic activity of tumor cells, and in… Show more

Uptake of nutrients, such as glucose and amino acids, is critical to support cell growth and is typically mediated by cell surface transporters. An alternative mechanism for the bulk uptake of nutrients from the extracellular space is macropinocytosis, a nonclathrin, and nonreceptor-mediated endocytic process, in which extracellular fluid is taken up into large intracellular vesicles called macropinosomes. Oncogenic transformation leads to the increased metabolic activity of tumor cells, and in the Ras-driven tumor part of this enhanced activity is the stimulation of macropinocytosis. To measure oncogene-dependent macropinocytosis, we used HeLa cells expressing oncogenic HRASG12D driven from a Tet-regulated promoter. Upon oncogenic HRAS expression, the cells undergo metabolic changes that include the elevation of macropinocytosis. We detected macropinocytosis through the uptake of lysine-fixable tetramethyl rhodamine (TMR)-Dextran (70 kDa) from the cell media into nascent intracellular macropinosomes. These macropinosomes were quantified by image-based high-content analysis, with the size, intensity, and position of macropinosomes measured. Using this model system, we ran a full genome-wide siRNA screen (siGenome™; GE) to identify genes involved in controlling oncogenic HRAS-dependent macropinocytosis. Hits from the primary screen were confirmed with siRNA reagents from a different library (GE, OTP), which allowed us to mitigate potential off-target effects. Candidate genes from this screen include known regulators of macropinocytosis as well as novel targets. Show less

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… Show more

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. Show less

Oncogenic Ras mutations are prevalent in a variety of tumor types, including adenocarcinomas of the pancreas, colon, and lung. One of the most overt phenotypes associated with the expression of oncogenic Ras mutants is the stimulation of macropinocytosis, an endocytic process that involves extensive membrane remodeling and the internalization of extracellular fluid via large membrane-bound vesicles called macropinosomes. The functional consequences of this stimulation in oncogenic… Show more

Oncogenic Ras mutations are prevalent in a variety of tumor types, including adenocarcinomas of the pancreas, colon, and lung. One of the most overt phenotypes associated with the expression of oncogenic Ras mutants is the stimulation of macropinocytosis, an endocytic process that involves extensive membrane remodeling and the internalization of extracellular fluid via large membrane-bound vesicles called macropinosomes. The functional consequences of this stimulation in oncogenic Ras-expressing cancer cells were unknown prior to our recent work where we linked macropinocytic uptake to nutrient delivery and amino acid supply in tumor cells. We found that Ras-transformed cells utilize macropinocytosis to internalize extracellular albumin, which is then lysosomally degraded releasing the constituent amino acids intracellularly. These protein-derived amino acids have the capacity to enter central carbon metabolism and fuel tumor cell proliferation even in a nutrient-depleted environment. Of particular relevance is the finding that the pharmacological inhibition of macropinocytosis compromises the growth of Ras-driven pancreatic xenograft tumors. This discovery raises the question of whether the inhibition of macropinocytosis can be utilized as a therapeutic intervention in a subset of cancers. Moreover, because macropinocytosis is an established mechanism of drug delivery for nanoparticles, our work highlights the possibility of exploiting this feature of Ras-induced tumors for the effective delivery of nanoscale therapeutics. Our recent studies focused on identifying novel modulators of macropinocytosis and examining macropinocytosis as a delivery mechanism for nab-paclitaxel, an albumin-based nanoparticle therapy, will be discussed. Show less

Blood vessels deliver oxygen, nutrients, hormones and immunity factors throughout the body. To perform these vital functions, vascular cords branch, lumenize and interconnect. Yet, little is known about the cellular, molecular and physiological mechanisms that control how circulatory networks form and interconnect. Specifically, how circulatory networks merge by interconnecting in parallel along their boundaries remains unexplored. To examine this process we studied the formation and functional… Show more

Blood vessels deliver oxygen, nutrients, hormones and immunity factors throughout the body. To perform these vital functions, vascular cords branch, lumenize and interconnect. Yet, little is known about the cellular, molecular and physiological mechanisms that control how circulatory networks form and interconnect. Specifically, how circulatory networks merge by interconnecting in parallel along their boundaries remains unexplored. To examine this process we studied the formation and functional maturation of the plexus that forms between the Dorsal Longitudinal Anastomotic Vessels (DLAVs) in the zebrafish. We find that the migration and proliferation of endothelial cells within the DLAVs and their Segmental (Se) vessel precursors drives DLAV plexus formation. Remarkably, the presence of Se vessels containing only endothelial cells of the arterial lineage is sufficient for DLAV plexus morphogenesis, suggesting that endothelial cells from the venous lineage make a dispensable or null contribution to this process. The discovery of a circuit that integrates the inputs of circulatory flow and Vascular Endothelial Growth Factor (VEGF) signaling to modulate aortic arch angiogenesis, together with the expression of components of this circuit in the trunk vasculature, prompted us to interrogate the role of these inputs and their relationship during DLAV plexus formation. We find that circulatory flow and VEGF signaling make additive contributions to DLAV plexus morphogenesis, rather than acting as essential inputs with equivalent contributions as they do during aortic arch angiogenesis. Our observations underscore the existence of context-dependent differences in the integration of physiological stimuli and signaling cascades during vascular development. Show less

We continue our study of diffusion–reaction processes on finite aperiodic lattices, viz., the Penrose lattice and a Girih tiling. Focusing on bimolecular reactions, we mobilize the theory of finite Markov processes to document the effect of attractive forces on the reaction efficiency. Considering both a short-range square-well potential and a longer-range 1/rS (S = 4, 6) potential, we find that irreversible reactive encounters between reactants on a Girih platelet are kinetically advantaged… Show more

We continue our study of diffusion–reaction processes on finite aperiodic lattices, viz., the Penrose lattice and a Girih tiling. Focusing on bimolecular reactions, we mobilize the theory of finite Markov processes to document the effect of attractive forces on the reaction efficiency. Considering both a short-range square-well potential and a longer-range 1/rS (S = 4, 6) potential, we find that irreversible reactive encounters between reactants on a Girih platelet are kinetically advantaged relative to processes on a Penrose platelet. This result generalizes the conclusion reached in our earlier study [Roberto A. Garza-López, Aaron Kaufman, Reena Patel, Joseph Chang, Jack Brzezinski, John J. Kozak, Chem. Phys. Lett. 459 (2008) 137] where entropic factors (only) were assessed. Show less