Abstract 4330: Everolimus-induced hyperglycemia: Dual efficacy of metformin
Authors: Ariaans Gerke , Jong Steven de , Vries Elisabeth G.E. de , Jalving Mathilde 
Published: May 2015

Abstract
The anti-cancer drug and mTOR inhibitor everolimus often causes hyperglycemia in patients. This side-effect potentially reduces its efficacy and may cause complications in the long term. The diabetes drug metformin, a mitochondrial complex 1 inhibitor, may counteract hyperglycemia while enhancing the anti-tumor efficacy of everolimus. Here, we investigated the growth inhibitory properties of both drugs and their combination in vitro with an emphasis on their effects on glucose metabolism.
The breast cancer cell lines MCF-7, T47D and MDA-MB-231 were cultured in media containing 11 mM (high), 5.5 mM or 2.75 mM (low) glucose and treated with 1-10 mM metformin or 1-10 nM everolimus for 2 or 4 days. Proliferation, colony formation and apoptosis were measured. Intracellular effects were analyzed using glucose transporter 4 (GLUT4) staining. The Seahorse XF analyzer was used to measure mitochondrial respiration and glycolysis.
Metformin (5 mM) added to high glucose medium reduced proliferation by 40-60%, depending on the cell line. Under low glucose conditions metformin reduced proliferation by 70-80%. The efficacy of everolimus in high glucose conditions was comparable to its efficacy in low glucose conditions. Combining metformin and everolimus resulted in at least 40% growth inhibition of the three cell lines under all glucose conditions. After 48h of treatment with 5 mM metformin basal mitochondrial respiration of all cell lines decreased to 20-40% of control. Simultaneously, glycolysis rate increased to 150-270% of control. Everolimus treatment (10 nM) for 48 h decreased mitochondrial respiration to the same extent, but did not affect glycolysis rate. Decreased GLUT4 membrane expression by at least 40% compared to the untreated control may explain this observation. The effects of metformin and everolimus on glucose metabolism were independent of glucose concentration.
Since metformin strongly increased glycolysis rate, the effect of glucose depletion and replenishment on metformin induced cell death and proliferation inhibition was determined. 5 mM metformin treatment in high glucose medium for 4 days did not induce cell death in any cell line. However, the same treatment in low glucose media resulted in up to 50% apoptotic cells. Daily renewal of low glucose media reversed this effect. Metformin-induced growth inhibition in high or low glucose concentrations was not affected by medium replenishment in short term survival assays and clonogenic assays.
Our study provides evidence that everolimus anti-tumor efficacy is not affected by high glucose concentrations. Metformin, however, was more effective at low glucose concentrations due to its glycolysis inducing capacity. Addition of metformin to everolimus treatment may be beneficial, especially under low glucose conditions as found within tumours. Moreover, this combination is appealing because of their complementary effects on cellular metabolism.
Citation Format: Gerke Ariaans, Steven de Jong, Elisabeth G.E. de Vries, Mathilde Jalving. Everolimus-induced hyperglycemia: Dual efficacy of metformin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4330. doi:10.1158/1538-7445.AM2014-4330