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 6

Decreased oxidative phosphorylation in a xenograft model of antiangiogenic therapy resistance.

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Decreased oxidative phosphorylation in a xenograft model of antiangiogen...
(A) Flow cytometry of bevacizumab-resistant glioma cell line–derived xenograft (U87-BevR) and bevacizumab-sensitive (U87-BevS) cells treated with MitoTracker revealed no difference in number of mitochondria per cell between the cells (P = 0.07, Student’s t test). (B) Incubating cultured U87-BevS and U87-BevR cells with JC-1 fluorochrome revealed the red-to-green ratio, an indicator of mitochondrial membrane potential, to be lower in U87-BevR than U87-BevS cells in normoxia (P = 0.03) and hypoxia (P = 0.03). Furthermore, normoxic U87-BevR cells had a remarkably reduced membrane potential, on par with hypoxic U87-BevS cells (P = 0.2), indicating the level of mitochondrial impairment in cells from bevacizumab-resistant xenografts. Wilcoxon-Mann-Whitney test, n = 4/group. (C) Oxygen consumption rate (OCR) analysis over time when treating cells 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. U87-BevS had greater basal respiration before adding oligomycin (P = 0.03) and ATP production (basal respiration minus the proton leak between the post-oligomycin curve and the post-antimycin A+rotenone curve; P = 0.008) than U87-BevR, without a change in spare respiratory capacity (maximal respiration after adding FCCP minus basal respiration; P = 0.8) or proton leak (P = 0.3). Wilcoxon-Mann-Whitney test. For OCR curves, error bars represent SDs. (D) ATP production was higher in mitochondria isolated from U87-BevS cells and grown in 10 mM galactose and normoxia (20% oxygen), conditions selecting for oxidative phosphorylation, than mitochondria from U87-BevR cells (P = 0.03), a difference that was eliminated in hypoxia (P = 0.1). Wilcoxon-Mann-Whitney test, n = 4/group. (E) Western blot revealed decreased HSP60 expression in U87-BevR xenografts compared with U87-BevS xenografts (n = 5 tumors/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.