Anti-tumor effects of everolimus and metformin are complementary and glucose-dependent in breast cancer cells

Background: Clinical efficacy of the mTOR inhibitor everolimus is limited in breast cancer and regularly leads to side-effects including hyperglycemia. The AMPK inhibitor and anti-diabetic drug metformin may counteract everolimus-induced hyperglycemia, as well as enhancing anti-cancer efficacy. We investigated the glucosedependent growth-inhibitory properties of everolimus, metformin and the combination in breast cancer cell lines. Methods: The breast cancer cell lines MCF-7, MDA-MB-231 and T47D were cultured in media containing 11 mM or 2.75 mM glucose with 21% or 1% oxygen. Everolimus and metformin treated cells were subjected to cytotoxicity and clonogenic assays, western blotting, FACS and metabolic measurements. Results: Everolimus was less effective in MCF7 cells under low glucose conditions compared to high glucose conditions (IC50 of > 50 nM vs 29.1 +/- 1.4 nM) in a short-term survival assay, while sensitivity of MDA-MB-231 and T47D cells to everolimus was lost under low glucose conditions. In contrast, metformin was more effective in low than in high glucose conditions in MCF7 (IC50 of 1.8 +/- 1.2 mM vs > 5 mM) and MDA-MB231 cells (1.5 +/- 1.3 mM vs 2.6 +/- 1.2 mM). Metformin sensitivity of T47D cells was independent of glucose concentrations. Everolimus combined with metformin additively inhibited cell survival, clonogenicity, mTOR signaling activity and mitochondrial respiration. These effects were not the result of enhanced autophagy or apoptosis induction. Similar results were observed under hypoxic conditions. Conclusion: Metformin-induced effects are additive to the anti-proliferative and colony inhibitory properties of everolimus through inhibition of mitochondrial respiration and mTOR signaling. These results warrant further in vivo investigation of everolimus combined with metformin as a putative anti-cancer therapy.