Abstract 215: Metabolic and mitochondrial consequences of p53 upregulation and their relation to cell fate outcomes

Background: p53 regulates a plethora of biological processes including cellular metabolism and mitochondrial function. Using the small molecule MDM2 agonist MI-63, we previously demonstrated differential p53 upregulation and induction of global metabolomics profiles in malignant MCF-7 and non-malignant MCF-10A mammary epithelial cells undergoing different cell fates (apoptosis vs. cytostasis). In particular, p53 upregulation suppressed glycolytic metabolite pools in MCF-10A, while increasing their levels in MCF-7 cells. We postulated that this opposing metabolic response may be linked to their different cell fate outcomes. In this study, we evaluated the association between p53 induced effects on live cell metabolic flux and cell fate using different p53wt cell lines subjected to comparable p53 upregulation. Methods: We employed four p53wt cell lines: MCF-7, ZR-75-1, SJSA-1 and MCF-10A. Cell fate outcomes upon p53 upregulation (24hr, 10μM MI63) were assessed by Western blotting for cytostasis (p21) and apoptosis markers (cleaved PARP, PUMA). Expression changes in enzymes involved in proline metabolism were similarly measured. Real-time measurements of oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) under control and MI-63 treatments were assessed by the Seahorse Extracellular Flux Analyzer, and specific acidification contributions from respiration and glycolysis to total ECAR were computed. Results: p53 upregulation induced apoptosis in the malignant MCF-7, ZR-75-1 and SJSA-1 cells, but not in non-malignant MCF-10A cells. MI-63 treatment decreased basal OCR in MCF-7 cells (t-test p = 0.046), but not the other cell lines. No significant differences in maximum OCR were observed between control and MI-63 treatments in any of the cell lines tested. As well, p53 upregulation did not produce any significant change in total ECAR. However, in MCF-7 cells, glycolytic ECAR was significantly increased (MI-63/Control: 4.36, Tukey test p = 0.001), consistent with our earlier metabolomics conclusions. In contrast, no significant changes in glycolytic ECAR were produced in MCF-10A, ZR-75-1 or SJSA-1 cells (MI-63/Control: 1.27, 1.32 and 1.33 respectively). Interestingly, p53 upregulation induced expression of proline dehydrogenase (PRODH) in all three malignant cell lines, but not in MCF-10A cells; and coordinated changes in other proline catabolizing enzymes (P5C dehydrogenase, P5C reductase) were seen in MCF-7 cells. Conclusion: These findings indicate that p53 induced effects on oxygen consumption and glycolysis are cell context dependent but are not likely responsible for the different cell fate outcomes observed following p53 upregulation. Rather, our evidence suggests a potential mechanistic link between p53 induced effects on proline metabolism and cell fate outcomes, indicating that this stress-activated mitochondrial pathway deserves further study. Citation Format: Yau Christina, Katya Frazier, Daniel Rothschild, Shona Mookerjee, Martin Brand, Gary Scott, Christopher C. Benz. Metabolic and mitochondrial consequences of p53 upregulation and their relation to cell fate outcomes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 215.