Reciprocal regulation of p53 and malic enzymes modulates metabolism and senescence

Abstract

Cellular senescence both protects multicellular organisms from cancer and contributes to their ageing. The pre-eminent tumour suppressor p53 has an important role in the induction and maintenance of senescence, but how it carries out this function remains poorly understood. In addition, although increasing evidence supports the idea that metabolic changes underlie many cell-fate decisions and p53-mediated tumour suppression, few connections between metabolic enzymes and senescence have been established. Here we describe a new mechanism by which p53 links these functions. We show that p53 represses the expression of the tricarboxylic-acid-cycle-associated malic enzymes ME1 and ME2 in human and mouse cells. Both malic enzymes are important for NADPH production, lipogenesis and glutamine metabolism, but ME2 has a more profound effect. Through the inhibition of malic enzymes, p53 regulates cell metabolism and proliferation. Downregulation of ME1 and ME2 reciprocally activates p53 through distinct MDM2- and AMP-activated protein kinase-mediated mechanisms in a feed-forward manner, bolstering this pathway and enhancing p53 activation. Downregulation of ME1 and ME2 also modulates the outcome of p53 activation, leading to strong induction of senescence, but not apoptosis, whereas enforced expression of either malic enzyme suppresses senescence. Our findings define physiological functions of malic enzymes, demonstrate a positive-feedback mechanism that sustains p53 activation, and reveal a connection between metabolism and senescence mediated by p53.

Figure 1. p53 represses the expression of MEs

a, ME mRNA and protein expression in U2OS cells stably expressing p53 shRNA or control shRNA. Relative ME/actin ratios are given. b, c, mRNA expression (b), total activity, and protein levels (c) of MEs in p53-depleted and control IMR90 cells. Data shown are mean ± SD, n=3. d, ME expression in p53^+/+ and p53^−/− MEFs. e, p53-depleted and control U2OS cells were treated with increasing amounts of ETO and assayed for ME expression. f, p53^+/+ HCT116 cells treated with or without DOX (1 µg/ml) were subjected to ChIP assay with anti-p53 (DO-1), a control mouse IgG, or no antibody (−). *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 2. ME1 and ME2 influence NADPH production, lipid production, and glutaminolysis

a, b, NADPH levels (means ± SD, n=3) in p53-depleted and control IMR90 cells transfected with control, ME1, or ME2 siRNA (a), or in IMR90 cells stably over-expressing wild-type MEs, mutant MEs, or vector control. Protein expression is shown below. c, d, Triglyceride contents (means ± SD, n=3) in p53-depleted and control 3T3-L1 cells transfected with control, ME1, or ME2 siRNA (c), or 3T3-L1 cells stably expressing wild-type MEs, mutant MEs, or vector control (d). mRNA expression is shown below. e, f, Effect of ME1 and ME2 knockdown in p53^−/− HCT116 cells on glucose and glutamine consumption (means ± SD, n=3) (e), and glutaminolytic flux (f).

Figure 3. A role for MEs in suppressing p53-mediated senescence

a, The replicative lifespan (means ± SD, n=3) of IMR90 cells transfected with control, ME1, or ME2 siRNA. b, c, IMR90 cells transfected with ME1, ME2, p53, or control siRNA as indicated. Percentages of SA-β-gal-positive cells (means ± SD, n=3) (b) and numbers of PML NBs (c) are shown. See Supplementary Figs. 9f, g for representative images. d, Percentages of SA-β-gal-positive cells (means ± SD, n=3) (top) and protein expression (bottom) in IMR90 cells at different passages. e, f, Replicative lifespan (mean ± SD, n=3) (e), and protein expression (f) of IMR90 cells with and without overexpression of ME1 or ME2. Arrows indicate the onset of senescence (e).

Figure 4. Mechanisms of p53 activation induced by ME down-regulation and a role of MEs in tumor growth

a, Effect of ME1 and ME2 knockdown on p53 and AMPK activation and Mdm2 expression. ACC: Acetyl-CoA carboxylase. b, p53 and AMPK activation in AMPK^+/+ and AMPK^−/− MEF cells transfected with control or ME2 siRNA. c, ROS levels in IMR90 cells transfected with ME1, ME2, or control siRNA. d, Effect of NAC on AMPK and p53 activation in IMR90 cells transfected with control siRNA or ME2 siRNA. e, f, Average weights of xenograft tumors (mean ± SD, n=6) generated by p53^+/+ and p53^−/− HCT116 cells transfected with ME1, ME2, or control siRNA (e), or p53^+/+ HCT116 cells stably over-expressing wild-type or mutant MEs (f).