GLUT3 upregulation promotes metabolic reprogramming associated with antiangiogenic therapy resistance
Ruby Kuang, … , Suneil Koliwad, Manish K. Aghi
Ruby Kuang, … , Suneil Koliwad, Manish K. Aghi
Published January 26, 2017
Citation Information: JCI Insight. 2017;2(2):e88815. https://doi.org/10.1172/jci.insight.88815.
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Research Article Metabolism Oncology

GLUT3 upregulation promotes metabolic reprogramming associated with antiangiogenic therapy resistance

Abstract

Clinical trials revealed limited response duration of glioblastomas to VEGF-neutralizing antibody bevacizumab. Thriving in the devascularized microenvironment occurring after antiangiogenic therapy requires tumor cell adaptation to decreased glucose, with 50% less glucose identified in bevacizumab-treated xenografts. Compared with bevacizumab-responsive xenograft cells, resistant cells exhibited increased glucose uptake, glycolysis, 13C NMR pyruvate to lactate conversion, and survival in low glucose. Glucose transporter 3 (GLUT3) was upregulated in bevacizumab-resistant versus sensitive xenografts and patient specimens in a HIF-1α–dependent manner. Resistant versus sensitive cell mitochondria in oxidative phosphorylation–selective conditions produced less ATP. Despite unchanged mitochondrial numbers, normoxic resistant cells had lower mitochondrial membrane potential than sensitive cells, confirming poorer mitochondrial health, but avoided the mitochondrial dysfunction of hypoxic sensitive cells. Thin-layer chromatography revealed increased triglycerides in bevacizumab-resistant versus sensitive xenografts, a change driven by mitochondrial stress. A glycogen synthase kinase-3β inhibitor suppressing GLUT3 transcription caused greater cell death in bevacizumab-resistant than -responsive cells. Overexpressing GLUT3 in tumor cells recapitulated bevacizumab-resistant cell features: survival and proliferation in low glucose, increased glycolysis, impaired oxidative phosphorylation, and rapid in vivo proliferation only slowed by bevacizumab to that of untreated bevacizumab-responsive tumors. Targeting GLUT3 or the increased glycolysis reliance in resistant tumors could unlock the potential of antiangiogenic treatments.

Authors

Ruby Kuang, Arman Jahangiri, Smita Mascharak, Alan Nguyen, Ankush Chandra, Patrick M. Flanigan, Garima Yagnik, Jeffrey R. Wagner, Michael De Lay, Diego Carrera, Brandyn A. Castro, Josie Hayes, Maxim Sidorov, Jose Luiz Izquierdo Garcia, Pia Eriksson, Sabrina Ronen, Joanna Phillips, Annette Molinaro, Suneil Koliwad, Manish K. Aghi

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Figure 1

Increased glycolytic metabolism in a xenograft model of antiangiogenic therapy resistance.

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Increased glycolytic metabolism in a xenograft model of antiangiogenic t...
(A) Bevacizumab treatment (10 mg/kg twice per week until volumetric endpoint reached) lowered intratumoral glucose compared with IgG control antibody treatment of subcutaneous U87 xenografts. P = 0.03, Wilcoxon-Mann-Whitney test, n = 4 replicates/group. (B) Cultured bevacizumab-resistant glioma cell line–derived xenograft (U87-BevR) cells exhibited more glucose uptake than cultured bevacizumab-sensitive (U87-BevS) cells in normoxia (P = 0.03) and hypoxia (P = 0.03). Wilcoxon-Mann-Whitney test, n = 4 replicates/group. 2NBDG, fluorescently labeled deoxyglucose analog. (C) Cultured U87-BevR cells exhibited the same baseline extracellular acidification rate (ECAR) as U87-BevS cells (P = 0.1–0.2, Wilcoxon-Mann-Whitney test), but greater ECAR when treated with inhibitors of mitochondrial oxidative phosphorylation, consistent with greater stress-associated glycolysis (range: P < 0.001 to P = 0.03, Wilcoxon-Mann-Whitney test). n = 46 samples/cell type/time point. (D) Cultured U87-BevR cells produced more pyruvate than U87-BevS cells in hypoxia (P = 0.008) but not normoxia (P = 0.2). Wilcoxon-Mann-Whitney test, n = 5 replicates/group. (E) 13C NMR spectroscopic measurement of pyruvate to lactate conversion revealed greater conversion in U87-BevR relative to U87-BevS. (F) ATP production assessed by a chemiluminescence assay revealed U87-BevR to produce more ATP production than U87-BevS in normoxic cells in DMEM (P = 0.03), with the difference persisting in hypoxia (P = 0.03). Wilcoxon-Mann-Whitney test, n = 4 replicates/group. For ECAR curves, error bars represent SDs. For box-and-whisker plots, the horizontal line in the box is the median, while the box extends from the 25th to 75th percentile and the whiskers from minimum to maximum values. *P < 0.05 and ***P < 0.001.