Suppression of oxidative phosphorylation confers resistance against bevacizumab in experimental glioma.

Although bevacizumab initially shows high response rates in gliomas and other tumours, therapy resistance usually develops later. Because anti-angiogenic agents are supposed to induce hypoxia, we asked whether rendering glioma cells independent of oxidative phosphorylation modulates their sensitivity against hypoxia and bevacizumab. LNT-229 glioma cells without functional mitochondria (rho0 ) and control (rho+ ) cells were generated. LNT-229 rho0 -cells displayed reduced expression of oxidative phosphorylation-related genes and diminished oxygen consumption. Conversely, glycolysis was up-regulated in these cells, as shown by increased lactate production and stronger expression of glucose transporter-1 and lactate dehydrogenase-A. However, hypoxia-induced cell death in vitro was nearly completely abolished in the LNT-229 rho0 -cells, these cells were more sensitive towards glucose restriction and the treatment with the glycolysis inhibitor 2-deoxy-D-glucose. In an orthotopic mouse xenograft experiment, bevacizumab induced hypoxia as reflected by elevated Hypoxia-inducible factor 1-alpha staining in both, rho+ - and rho0 -tumours. However, it prolonged survival only in the mice bearing rho+ -tumours (74 days vs. 105 days, p = 0.024 log-rank test) and had no effect on survival in mice carrying LNT-229 rho0 -tumours (75 days vs. 70 days, p = 0.52 log-rank test). Interestingly, inhibition of glycolysis in vivo with 2-deoxy-D-glucose re-established sensitivity of rho0 -tumours against bevacizumab (98 days vs. 80 days, p = 0.0001). In summary, ablation of oxidative phosphorylation in glioma cells leads to a more glycolytic and hypoxia-resistant phenotype and is sufficient to induce bevacizumab-refractory tumours. These results add to increasing evidence that a switch towards glycolysis is one mechanism how tumour cells may evade anti-angiogenic treatments and suggest anti-glycolytic strategies as promising approaches to overcome bevacizumab resistance.

Activation of AMP-activated kinase modulates sensitivity of glioma cells against epidermal growth factor receptor inhibition.

The epidermal growth factor (EGFR) pathway is frequently activated in glioblastoma but the clinical efficacy of EGFR inhibitors in malignant glioma has been disappointing. The reasons for the failure of the mechanisms of resistance of these inhibitors are unclear, but may involve factors of the tumor microenvironment such as limited glucose availability and hypoxia. It was therefore examined whether glucose and oxygen influenced the response of glioma cells to EGFR inhibition. Decreased levels of glucose and oxygen led to resistance against the EGFR inhibitor PD153035, whereas high glucose amounts and normoxia sensitised glioma cells towards the inhibitor. Low levels of glucose and oxygen stimulated AMP-activated kinase (AMPK) in glioma cells. 2DG, an inhibitor of glycolysis, and the AMPK activator A769662 reduced glucose consumption, induced phosphorylation of AMPK and mimicked the effects of low glucose availability on the toxicity of PD153035. Similarly, 2DG reduced toxicity of imatinib in K562 leukemia cells. In contrast, inhibition of AMPK by compound C or by short-hairpin (sh)-mediated gene suppression increased cell death induced by the EGFR inhibitor and reverted the protective effects of 2DG and A769662. In conclusion, cytotoxicity of EGFR inhibition can be diminished by AMPK activation in glioma cells. These results may provide one explanation for the low activity of EGFR inhibitors in clinical trials and suggest antagonism of AMPK or of AMPK-regulated metabolic alterations as a promising approach to enhance their therapeutic efficacy.