Cosimo Commisso

Human Pancreatic Cancer Tumors Are Nutrient Poor and Tumor Cells Actively Scavenge Extracellular Protein

Glucose and amino acids are key nutrients supporting cell growth. Amino acids are imported as monomers, but an alternative route induced by oncogenic KRAS involves uptake of extracellular proteins via macropinocytosis and subsequent lysosomal degradation of these proteins as a source of amino acids. In this study, we examined the metabolism of pancreatic ductal adenocarcinoma (PDAC), a poorly vascularized lethal KRAS-driven malignancy. Metabolomic comparisons of human PDAC and benign adjacent tissue revealed that tumor tissue was low in glucose, upper glycolytic intermediates, creatine phosphate, and the amino acids glutamine and serine, two major metabolic substrates. Surprisingly, PDAC accumulated essential amino acids. Such accumulation could arise from extracellular proteins being degraded through macropinocytosis in quantities necessary to meet glutamine requirements, which in turn produces excess of most other amino acids. Consistent with this hypothesis, active macropinocytosis is observed in primary human PDAC specimens. Moreover, in the presence of physiologic albumin, we found that cultured murine PDAC cells grow indefinitely in media lacking single essential amino acids and replicate once in the absence of free amino acids. Growth under these conditions was characterized by simultaneous glutamine depletion and essential amino acid accumulation. Overall, our findings argue that the scavenging of extracellular proteins is an important mode of nutrient uptake in PDAC.

Molecular Pathways: Targeting the Dependence of Mutant RAS Cancers on the DNA Damage Response

Of the genes mutated in cancer, RAS remains the most elusive to target. Recent technological advances and discoveries have greatly expanded our knowledge of the biology of oncogenic Ras and its role in cancer. As such, it has become apparent that a property that intimately accompanies RAS-driven tumorigenesis is the dependence of RAS-mutant cells on a number of nononcogenic signaling pathways. These dependencies arise as a means of adaptation to Ras-driven intracellular stresses and represent unique vulnerabilities of mutant RAS cancers. A number of studies have highlighted the dependence of mutant RAS cancers on the DNA damage response and identified the molecular pathways that mediate this process, including signaling from wild-type Ras isoforms, ATR/Chk1, and DNA damage repair pathways. Here, we review these findings, and we discuss the combinatorial use of DNA-damaging chemotherapy with blockade of wild-type H- and N-Ras signaling by farnesyltransferase inhibitors, Chk1 inhibitors, or small-molecule targeting DNA damage repair as potential strategies through which the dependence of RAS cancers on the DNA damage response can be harnessed for therapeutic intervention. Clin Cancer Res; 21(6); 1243–7. ©2014 AACR.

Abstract PR08: Pulling out all the stops: Exploiting macropinocytosis inhibition for the treatment of pancreatic cancer

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.

Abstract PR12: Bringing the outside in: Macropinocytosis and cancer therapeutics

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. Citation Format: Cosimo Commisso, Venugopal Chenna, Elda Grabocka, Craig Ramirez, Maitra Anirban, Dafna Bar-Sagi. Bringing the outside in: Macropinocytosis and cancer therapeutics. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR12.

Abstract PR5: Macropinocytosis as a mechanism of amino acid supply.

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 structures called macropinosomes. To date, the functional significance of Ras-dependent macropinocytosis has not been explored. We have found that pancreatic cancer cells harboring oncogenic K-Ras utilize macropinocytosis to internalize extracellular albumin, a major constituent of extracellular fluids and a potentially rich source of amino acids. Furthermore, we have established that tumor cells harboring oncogenic Ras mutations display robust macropinocytosis in vivo. Utilizing cell biological approaches and amino acid quantification, we determined that extracellular albumin that is internalized via oncogenic K-Ras-stimulated macropinosomes is targeted for degradation leading to the intracellular production of albumin-derived amino acids. Cancer cells display a heightened dependence on glutamine due to their need to sustain rapid proliferation, bioenergetics, and macromolecular biosynthesis. We have observed that in cells expressing oncogenic K-Ras, the adverse effects of glutamine deprivation on cell growth can be rescued by macropinocytosismediated uptake of extracellular albumin. Moreover, the capacity of macropinocytic inhibition to diminish this growth advantage is dependent on both glutamine and glutamine-derived metabolites. These results indicate that macropinocytosis may serve as a glutamine supply mechanism that is particularly critical under conditions where free glutamine is limiting. Together, these data implicate oncogenic K-Ras-mediated macropinocytosis in the regulation of cancer cell metabolism and point to the possibility of exploiting this process in the design of anticancer therapies. This proffered talk is also presented as Poster B67. Citation Format: Cosimo Commisso, Shawn M. Davidson, Rengin G. Soydaner-Azeloglu, Seth J. Parker, Christian M. Metallo, Matthew G. Vander Heiden, Dafna Bar-Sagi. Macropinocytosis as a mechanism of amino acid supply. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr PR5.