Etomoxir (ETO) is a widely used small-molecule inhibitor of fatty acid oxidation (FAO) through its irreversible inhibitory effects on the carnitine palmitoyl-transferase 1a (CPT1a). We used this compound to evaluate the role of fatty acid oxidation in rapidly proliferating T cells following costimulation through the CD28 receptor. We show that ETO has a moderate effect on T cell proliferation with no observable effect on memory differentiation, but a marked effect on oxidative metabolism. We show that this oxidative metabolism is primarily dependent upon glutamine rather than FAO. Using an shRNA approach to reduce CPT1a in T cells, we further demonstrate that the inhibition of oxidative metabolism in T cells by ETO is independent of its effects on FAO at concentrations exceeding 5 μM. Concentrations of ETO above 5 μM induce acute production of ROS with associated evidence of severe oxidative stress in proliferating T cells. In aggregate, these data indicate that ETO lacks specificity for CTP1a above 5 μM, and caution should be used when employing this compound for studies in cells due to its non-specific effects on oxidative metabolism and cellular redox.

Chemotherapy remains the standard of care for most cancers worldwide, however development of chemoresistance due to the presence of the drug-effluxing ATP binding cassette (ABC) transporters remains a significant problem. The development of safe and effective means to overcome chemoresistance is critical for achieving durable remissions in many cancer patients. We have investigated the energetic demands of ABC transporters in the context of the metabolic adaptations of chemoresistant cancer cells. Here we show that ABC transporters use mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer cells. We further demonstrate that the loss of methylation-controlled J protein (MCJ) (also named DnaJC15), an endogenous negative regulator of mitochondrial respiration, in chemoresistant cancer cells boosts their ability to produce ATP from mitochondria and fuel ABC transporters. We have developed MCJ mimetics that can attenuate mitochondrial respiration and safely overcome chemoresistance in vitro and in vivo. Administration of MCJ mimetics in combination with standard chemotherapeutic drugs could therefore become an alternative strategy for treatment of multiple cancers.

Introduction Aggressive cancers commonly ferment glucose to lactic acid at high rates, even in the presence of oxygen. This is known as aerobic glycolysis, or the “Warburg Effect.” It is widely assumed that this is a consequence of the upregulation of glycolytic enzymes. Oncogenic drivers can increase the expression of most proteins in the glycolytic pathway, including the terminal step of exporting H+ equivalents from the cytoplasm. Proton exporters maintain an alkaline cytoplasmic pH, which can enhance all glycolytic enzyme activities, even in the absence of oncogene-related expression changes. Based on this observation, we hypothesized that increased uptake and fermentative metabolism of glucose could be driven by the expulsion of H+ equivalents from the cell. Results To test this hypothesis, we stably transfected lowly glycolytic MCF-7, U2-OS, and glycolytic HEK293 cells to express proton-exporting systems: either PMA1 (plasma membrane ATPase 1, a yeast H+-ATPase) or CA-IX (carbonic anhydrase 9). The expression of either exporter in vitro enhanced aerobic glycolysis as measured by glucose consumption, lactate production, and extracellular acidification rate. This resulted in an increased intracellular pH, and metabolomic analyses indicated that this was associated with an increased flux of all glycolytic enzymes upstream of pyruvate kinase. These cells also demonstrated increased migratory and invasive phenotypes in vitro, and these were recapitulated in vivo by more aggressive behavior, whereby the acid-producing cells formed higher-grade tumors with higher rates of metastases. Neutralizing tumor acidity with oral buffers reduced the metastatic burden. Conclusions Therefore, cancer cells which increase export of H+ equivalents subsequently increase intracellular alkalization, even without oncogenic driver mutations, and this is sufficient to alter cancer metabolism towards an upregulation of aerobic glycolysis, a Warburg phenotype. Overall, we have shown that the traditional understanding of cancer cells favoring glycolysis and the subsequent extracellular acidification is not always linear. Cells which can, independent of metabolism, acidify through proton exporter activity can sufficiently drive their metabolism towards glycolysis providing an important fitness advantage for survival.

Fast proliferating cells require tight regulation to achieve a balance between the use of nutrients for ATP production (through glycolysis and oxidative phosphorylation) and the use of intermediate metabolites to sustain the increased biosynthetic activity. Cancer cells, but also high proliferative non-transformed cells exhibit high glycolytic activity during rapid proliferation even in the presence of normal oxygen concentrations in culture. However, despite the high glycolytic activity, the role of glycolysis is not necessary as a major contributor of ATP but to allow nutrient assimilation into biosynthetic precursors. Using Agilent Seahorse extracellular flux analysis, we have developed a cell-based assay which allows simultaneous measurement of the two-main cellular metabolic pathways to calculate the total rate of cellular ATP production as well as the fractional contribution from each pathway. The assay allows for real time changes in total ATP production rate to be quantified, and also the relative source of that ATP after exposure to drugs or changes in extracellular fuel supply. When we applied this new assay to a panel of 20 cancer and highly proliferative cell lines, we found that even in cell lines considered highly glycolytic, ATP production from glycolysis never represents more than 65% of total energy production and between 30-50% for most of the cell lines analyzed. The correlation between glycolytic ATP contribution to total ATP production and other cell phenotypes such as proliferation rate and motility was also analyzed. The use of this assay will allow for improved characterization of the bioenergetic profile of cancer cell variants, discrimination between normal and cancer cell types, and allow researchers to better understand the role of aerobic glycolysis in cell proliferation. Citation Format: Natalia Romero, Pamela M. Swain, Yoonseok Kam, George Rogers, Brian P. Dranka. Bioenergetic profiling of cancer cell lines: Quantifying the impact of glycolysis on cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3487.

The cellular metabolism of macrophages is an emerging element regulating inflammatory macrophages which are a critical component of tumor microenvironment. The inflammatory macrophage with highly glycolytic phenotype is also known to elevate the glycolytic activity upon pathogenic stimulation such as lipopolysaccharide (LPS). In this study, the dynamic changes in glycolysis were traced in a real-time manner by measuring proton efflux rates (PERs) and oxygen consumption rates (OCR) after an in-situ activation using Seahorse XFe96 analyzer. The PER of human peripheral blood monocyte (PBMC) derived M1 macrophages was increased within an hour after injection of LPS, which corresponding to cytokine release, tumor necrosis factor α (TNFα) and interleukin 1β (IL-1β). In contrast to PBMC-derived M1 macrophage activation, macrophage cell lines of RAW264.7 and J774.A1 required co-stimulation with interferon γ (IFNγ) for the full activation. Interestingly, the LPS and IFNγ co-stimulation modulates glycolytic rates in a bi-phasic manner which was identified only in long term (> 6 hr) monitoring. A series of long term XF analysis using in situ activation revealed that the immediate early glycolytic response fully relies on LPS stimulation while the secondary elevation in PER depends on IFNγ stimulus, which turns on inducible nitric oxide synthase (iNOS) signaling and in turn suppresses mitochondrial respiration. The TNFα production is closely related to the immediate early glycolytic elevation, but independent from IFNγ-induced second elevation. The IFNγ-dependent second glycolysis increase was totally abolished by iNOS inhibitors whereas the immediate early glycolysis elevation was not affected at all. These data imply a temporal orchestration mechanism of LPS and IFNγ signaling in the metabolic regulation and activation of inflammatory macrophages. Citation Format: Yoonseok Kam, Pamela M. Swain, Brian P. Dranka. Bi-phasic metabolic responses to in situ macrophage activation [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A67.