GLUT3 upregulation promotes metabolic reprogramming associated with antiangiogenic therapy resistance
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
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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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 9

GLUT3 upregulation drives the metabolic and therapeutic response changes associated with resistance to antiangiogenic therapy in a targetable manner.

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GLUT3 upregulation drives the metabolic and therapeutic response changes...
(A) Extracellular acidification rate (ECAR) over time, a marker of glycolysis, was higher in cultured bevacizumab-sensitive glioma cell line–derived xenograft cells transfected with GLUT3 (U87-BevS/GLUT3) cells vs. U87-BevS/empty vector (EV) cells treated with various oxidative phosphorylation inhibitors (range: P < 0.0001–0.02). Wilcoxon-Mann-Whitney test, n = 46/group. (B) Oxygen consumption rate (OCR) over time after treatment with 4 different mitochondrial inhibitors, as per the Seahorse extracellular flux analyzer protocol: oligomycin at 18 minutes, FCCP at 36 minutes, and antimycin A+rotenone at 54 minutes. Basal respiration (rate prior to oligomycin injection minus nonmitochondrial respiration) and ATP production (basal respiration minus proton leak) were higher in U87-BevS/EV than U87-BevS/GLUT3. P < 0.0001, Wilcoxon-Mann-Whitney test, n = 46/group. For ECAR and OCR curves in A and B, error bars represent SDs. (C) Glycogen synthase kinase-3β inhibitor GSK-3 IX (10 μM) caused more cell death in bevacizumab-resistant (U87-BevR) cells than U87-BevS cells after 24 (P = 0.04), 48 (P = 0.005), and 72 (P = 0.006) hours. Student’s t test, n = 6/group. 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, **P < 0.01. (D) Subcutaneous xenografts derived from U87-BevS/GLUT3 and U87-BevS/EV cells were treated with bevacizumab or control IgG antibody (n = 6/group). After 20 days, GLUT3 overexpression promoted tumor growth in vivo (P = 0.006) while bevacizumab reduced tumor growth (P < 0.0001), with no interaction between these variables (P = 0.1, ANOVA). For tumor volumes, values and error bars represent mean ± SEM.