Abstract 3234: Metformin shifts prostate cancer metabolism towards a less malignant phenotype
Authors: Wirth* Gregory J. , Fendt* Sarah-Maria , Bell Eric L. , Keibler Mark A. , Stephanopoulos Gregory , Olumi Aria F. 
Published: August 2012

Abstract
Introduction Excess nutrient metabolic states such as obesity and hyperinsulinemia are associated with prostate cancer progression. Metformin is the most widely prescribed oral antidiabetic agent and has recently been shown to reduce the risk and mortality of multiple cancer types. Current evidence suggests that on a cellular level, this effect is achieved by an inhibition of mitochondrial Complex I with subsequent activation of AMPK, a central regulator of energy homeostasis. As cancer energetics are adapted to the metabolic requirements of rapidly growing cells, we aimed to assess whether metformin is able to induce a less malignant phenotype in prostate cancer cell lines. Methods Experiments were performed in 3 prostate cancer cell lines (PC3, LNCaP, DU145) treated with 0.5-2.5mM metformin. 13C flux analysis allowed to assess of glucose and glutamine utilization and metabolic pathway activity. Extracellular fluxes were performed to determine O2 consumption, glucose uptake and lactate secretion. Proliferation was assessed with cell counts and protein expression with Western blots. Results Metformin increased glucose uptake and lactate secretion while reducing oxygen consumption in a dose-dependent manner, suggesting a shift form respiratory energy production to fermentation. Accordingly, cell lines that strongly relied on respiration were most sensitive to metformin. Flux analysis revealed that in untreated cells, glucose was directed towards the TCA cycle and constituted the major carbon source for fatty acid synthesis. Metformin inhibited glucose fueling of the TCA cycle, and glutamine was funneled into the TCA cycle and used for fatty acid synthesis instead. Flux analysis showed that this switch was achieved by a partial reversal of the TCA cycle from the usual oxidative metabolism to a reductive flux. In order to further elucidate the effect of metformin on glutamine catabolism, we determined the expression of Sirtuin 4, which in known to downregulate glutamate dehydrogenase, thereby inhibiting the diversion of glutamine into the TCA cycle. Sirtuin 4 expression was decreased under metformin, whereas LNCAP cells in which Sirtuin 4 was knocked down were less sensitive to Metformin and showed a decreased secretion of glutamate. Finally, allosteric inhibition of glutaminase, an enzyme required for mitochondrial glutamine usage, showed a synergistic, anti-proliferative effect with metformin. Conclusion Metformin induced a profound rearrangement of central metabolism in prostate cancer cell lines. Cells shifted ATP synthesis from respiration to less efficient fermentation and resorted to a reductive TCA cycle to uphold biosynthetic processes. Inhibition of this secondary bioenergetic pathway with a glutaminase inhibitor achieved a synergistic anti-tumor effect with metformin. These observed alterations of metabolic fluxes may ultimately provide new targets for cancer therapy.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3234. doi:1538-7445.AM2012-3234