Metabolic Responses In Cancer Cells With Differential Susceptibility To Glut1 Inhibition

Abstract Malignant cells are known for their accelerated metabolism, high energy requirements, and increased glucose uptake. They are characterized by high rates of glycolysis and metabolize glucose into lactate even under aerobic conditions, a hallmark of cancer known as the Warburg effect. Transport of glucose across the plasma membrane is the first and rate-limiting step for glucose metabolism and is mediated among others by facilitative glucose transporter (GLUT) proteins. Among the 14 GLUT proteins, GLUT1 and to a lesser degree GLUT3 are up regulated in many tumors. GLUT1 is also a prime target for the transcription factor hypoxia-inducible factor (HIF)-1α. Close relationships between GLUT1 expression, tumor development and progression, as well as poor overall survival have been described for several tumor entities. We have developed a GLUT1-selective, potent small molecule inhibitor, BAY-GLUT1, which inhibits both glucose uptake and ATP generation in GLUT1-expressing DLD-1 tumor cells in a glucose competitive way with IC50s of 4 and 3 nM, respectively. Selectivity was tested in either GLUT1-deficient DLD-1 cells or GLUT2/4-overexpressing recombinant CHO cells. In this study we report on the metabolic reaction of two adenocarcinoma cell lines, HeLa-MaTu (cervix) and DLD-1 (colon), to BAY-GLUT1 treatment. The two cell lines were treated with 30 nM BAY-GLUT1 for 6 and 24 hours under normoxic conditions and investigated by Metanomics Health's untargeted, broad metabolite profiling platform (MxP® Broad Profiling) to identify metabolic mechanisms explaining the differential susceptibility of cancer cells towards BAY-GLUT1. Hereby, two types of mass spectrometry (gas chromatography-mass spectrometry and liquid chromatography-MS/MS) analysis were applied to evaluate levels of 215 intracellular metabolites covering major metabolic pathways. Both inhibitor-treated cell lines showed dramatic decreases of glucose-6-phosphate and other glycolysis-related metabolites indicating efficient reduction in glycolytic activity. Subsequently, levels of several TCA cycle intermediates were also reduced. Our metabolic findings might suggest that DLD-1 preferentially uses glutamate and its precursor metabolites proline and 5-oxoproline to replenish the TCA cycle at the level of alpha-ketoglutarate. Interestingly, due to a gain-of-function mutation of isocitrate dehydrogenase, DLD-1 cells are able to reduce alpha-ketoglutarate into 2-hydroxyglutaric acid which might play an important role in controlling stability of transcription factor HIF-1α and in inducing metabolic reprogramming. Understanding the metabolic pathway changes upon GLUT inhibition could help to identify predictive conditions and/or biomarkers for treatment response. Citation Format: Sylvia Gruenewald, Ulrike Rennefahrt, Sandra G. Maldonado, Alexander Walter, Heike Petrul, Mélanie Héroult, Iring Heisler, Maria Quanz, Patrick Steigemann, Bernd Buchmann, Andrea Haegebarth. Metabolic responses in cancer cells with differential susceptibility to GLUT1 inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1164. doi:10.1158/1538-7445.AM2015-1164