Abstract 5160: Global metabolomic profiles distinguish malignant from non-malignant human mammary epithelial cell responses to endogenous p53 upregulation by MDM2 antagonism

Abstract

Abstract Wildtype (wt) p53 can alter metabolic pathways and mitochondrial function but it is unclear whether these responses are similar in normal and malignant cells, where it is also hypothesized that p53wt can modulate the Warburg effect. Therefore, we used a small molecule inhibitor of MDM2, MI-63 (Ding et al., J. Med. Chem. 2006; Ascenta/Sanofi), to induce ∼5-fold intracellular increases in p53wt protein levels within 24 h of treating malignant (MCF-7) or non-malignant (MCF10A) human mammary epithelial cells, whereupon global biochemical changes were assayed in replicate (x6) extracts using various mass spectrometry platforms (GC/MS, LC/MS/MS; Metabolon, Durham, NC). Extracts from similarly treated p53mut malignant SKBr3 cells were also assessed. Normalized (to total protein) and imputed data from 310 known and detected biochemicals were compared for treatment effects (Wilcoxon rank sum test, p<0.05). Treatment significantly altered levels of 93 biochemicals (58 up, 35 down) in the malignant MCF-7 cells, 62 (39 up, 23 down) in the non-malignant MCF10A cells, and 24 (20 up, 4 down) in the malignant p53mut SKBr3 cells. Common to both MCF-7 and MCF10A, p53wt upregulation increased NAD+ and NADP+ levels, responses not seen in SKBr3 cells. Upregulation of p53wt selectively suppressed polyamines (spermidine, putrescine) in MCF10A, whose response to MI-63 is cytostatic unlike the apoptotic response of MCF-7 or treatment unaffected growth of SKBr3 cells. Relative to MCF10A, MCF-7 cells showed opposing energy-related biochemical responses in glucose, glucose-6-phophosphate, 3-phosphoglycerate and pyruvate pools, along with increases in the glutaminolysis product succinate, suggesting that p53wt upregulation enhances glycolytic and mitochondrial (TCA) production of pyruvate in malignant MCF-7 cells yet decreases glycolysis and pyruvate production in non-malignant MCF10A cells. This conclusion that p53wt upregulation increases glycolysis, pyruvate production and mitochondrial ATP synthesis in MCF-7 was confirmed using live cell extracellular flux analysis of oxygen consumption and acid production (Seahorse XF), which showed opposing treatment responses by MCF10A and no treatment effects in SKBr3. Altogether, these findings point to opposing metabolic and bioenergetic responses induced by p53wt upregulation in malignant and non-malignant mammary epithelial cells, consistent with p53wt induced reversal of the Warburg effect, and offering new insights into pathways associated with different therapeutic and cell fate responses induced by novel small molecule MDM2 antagonists like MI-63. 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 5160. doi:1538-7445.AM2012-5160