Although p53–mediated cell–cycle arrest, senescence and apoptosis serve as critical barriers to cancer development, emerging evidence suggests that the metabolic activities of p53 are also important. Here we show that p53 inhibits cystine uptake and sensitizes cells to ferroptosis, a non–apoptotic form of cell death, by repressing expression of SLC7A11, a key component of the cystine/glutamate antiporter. Notably, p533KR, an acetylation–defective mutant that fails to induce cell–cycle arrest, senescence and apoptosis, fully retains the ability to regulate SLC7A11 expression and induce ferroptosis upon reactive oxygen species (ROS)–induced stress. Analysis of mutant mice shows that these non–canonical p53 activities contribute to embryonic development and the lethality associated with loss of Mdm2. Moreover, SLC7A11 is highly expressed in human tumours, and its overexpression inhibits ROS–induced ferroptosis and abrogates p533KR–mediated tumour growth suppression in xenograft models. Our findings uncover a new mode of tumour suppression based on p53 regulation of cystine metabolism, ROS responses and ferroptosis.

The NAD-dependent histone deacetylation of Sir2 connects cellular metabolism with gene silencing as well as aging in yeast. Here, we show that mammalian Sir2alpha physically interacts with p53 and attenuates p53-mediated functions. Nicotinamide (Vitamin B3) inhibits an NAD-dependent p53 deacetylation induced by Sir2alpha, and also enhances the p53 acetylation levels in vivo. Furthermore, Sir2alpha represses p53-dependent apoptosis in response to DNA damage and oxidative stress, whereas expression of a Sir2alpha point mutant increases the sensitivity of cells in the stress response. Thus, our findings implicate a p53 regulatory pathway mediated by mammalian Sir2alpha. These results have significant implications regarding an important role for Sir2alpha in modulating the sensitivity of cells in p53-dependent apoptotic response and the possible effect in cancer therapy.

Summary Cancer immunotherapy restores and/or enhances effector function of CD8+ T cells in the tumor microenvironment1,2. CD8+ T cells activated by cancer immunotherapy execute tumor clearance mainly by inducing cell death through perforin-granzyme- and Fas/Fas ligand-pathways3,4. Ferroptosis is a form of cell death that differs from apoptosis and results from iron-dependent lipid peroxide accumulation5,6. Although it was mechanistically illuminated in vitro7,8, emerging evidence has shown that ferroptosis may be implicated in a variety of pathological scenarios9,10. However, the involvement of ferroptosis in T cell immunity and cancer immunotherapy is unknown. Here, we find that immunotherapy-activated CD8+ T cells enhance ferroptosis-specific lipid peroxidation in tumor cells, and in turn, increased ferroptosis contributes to the anti-tumor efficacy of immunotherapy. Mechanistically, interferon gamma (IFNγ) released from CD8+ T cells downregulates expression of SLC3A2 and SLC7A11, two subunits of glutamate-cystine antiporter system xc-, restrains tumor cell cystine uptake, and as a consequence, promotes tumor cell lipid peroxidation and ferroptosis. In preclinical models, depletion of cyst(e)ine by cyst(e)inase in combination with checkpoint blockade synergistically enhances T cell-mediated anti-tumor immunity and induces tumor cell ferroptosis. Expression of system xc- is negatively associated with CD8+ T cell signature, IFNγ expression, and cancer patient outcome. Transcriptome analyses before and during nivolumab therapy reveal that clinical benefits correlate with reduced expression of SLC3A2 and increased IFNγ and CD8. Thus, T cell-promoted tumor ferroptosis is a novel anti-tumor mechanism. Targeting tumor ferroptosis pathway constitutes a therapeutic approach in combination with checkpoint blockade.

The traditional view of p53 activation includes three steps—p53 stabilization, DNA binding, and transcriptional activation. However, recent studies indicate that each step of p53 activation is more complex than originally anticipated. Moreover, both genetic studies in mice and in vitro studies with purified components suggest that the classical model may not be sufficient to explain all aspects of p53 activation in vivo. To reconcile these differences, we propose that antirepression, the release of p53 from repression by factors such as Mdm2 and MdmX, is a key step in the physiological activation of p53.

The NAD-dependent deacetylase SIR2 and the forkhead transcription factor DAF-16 regulate lifespan in model organisms, such as yeast and C. elegans. Here we show that the mammalian SIR2 ortholog SIRT1 deacetylates and represses the activity of the forkhead transcription factor Foxo3a and other mammalian forkhead factors. This regulation appears to be in the opposite direction from the genetic interaction of SIR2 with forkhead in C. elegans. By restraining mammalian forkhead proteins, SIRT1 also reduces forkhead-dependent apoptosis. The inhibition of forkhead activity by SIRT1 parallels the effect of this deacetylase on the tumor suppressor p53. We speculate how down-regulating these two classes of damage-responsive mammalian factors may favor long lifespan under certain environmental conditions, such as calorie restriction.

The p53 tumour suppressor is a short-lived protein that is maintained at low levels in normal cells by Mdm2-mediated ubiquitination and subsequent proteolysis. Stabilization of p53 is crucial for its tumour suppressor function. However, the precise mechanism by which ubiquitinated p53 levels are regulated in vivo is not completely understood. By mass spectrometry of affinity-purified p53-associated factors, we have identified herpesvirus-associated ubiquitin-specific protease (HAUSP) as a novel p53-interacting protein. HAUSP strongly stabilizes p53 even in the presence of excess Mdm2, and also induces p53-dependent cell growth repression and apoptosis. Significantly, HAUSP has an intrinsic enzymatic activity that specifically deubiquitinates p53 both in vitro and in vivo. In contrast, expression of a catalytically inactive point mutant of HAUSP in cells increases the levels of p53 ubiquitination and destabilizes p53. These findings reveal an important mechanism by which p53 can be stabilized by direct deubiquitination and also imply that HAUSP might function as a tumour suppressor in vivo through the stabilization of p53.

Upon DNA damage and other types of stress, p53 induces either cell-cycle arrest or apoptosis depending on the cellular context. However, the molecular mechanisms that govern the choice between cell-cycle arrest and apoptosis are not well understood. Here, we show that Tip60 is required for both cell growth arrest and apoptosis mediated by p53 and also induces its acetylation specifically at lysine 120 (K120) within the DNA-binding domain. Interestingly, this modification is crucial for p53-dependent apoptosis but is dispensable for its mediated growth arrest. K120 is a recurrent site for p53 mutation in human cancer, and the corresponding acetylation-defective tumor mutant (K120R) abrogates p53-mediated apoptosis, but not growth arrest. Thus, our study demonstrates that Tip60-dependent acetylation of p53 at K120 modulates the decision between cell-cycle arrest and apoptosis, and it reveals that the DNA-binding core domain is an important target for p53 regulation by posttranslational modifications.

Summary Cell-cycle arrest, apoptosis, and senescence are widely accepted as major mechanisms by which p53 inhibits tumor formation. Nevertheless, it remains unclear whether they are rate-limiting steps in tumor suppression. Here, we have generated mice bearing lysine to arginine mutations at one (p53K117R) or three (p533KR; K117R+K161R+K162R) of the critical p53 acetylation sites. While p53K117R/K117R cells are competent for p53-mediated cell-cycle arrest and senescence, but not apoptosis, all three of these processes are ablated in p533KR/3KR cells. Surprisingly, unlike p53-null mice, which rapidly succumb to spontaneous thymic lymphomas, early-onset tumor formation does not occur in either p53K117R/K117R or p533KR/3KR animals. Notably, p533KR retains the ability to modulate energy metabolism and reactive oxygen species (ROS) production by regulating metabolic p53 target genes. These findings underscore the crucial role of acetylation in differentially modulating p53 responses and suggest that unconventional activities of p53, such as metabolic regulation and antioxidant function, are critical for suppression of early-onset spontaneous tumorigenesis.