Although p53-mediated cell-cycle arrest, senescence, and apoptosis remain critical barriers to cancer development, the emerging role of p53 in cell metabolism, oxidative responses, and ferroptotic cell death has been a topic of great interest. Nevertheless, it is unclear how p53 orchestrates its activities in multiple metabolic pathways into tumor suppressive effects. Here, we identified the SAT1 (spermidine/spermine N 1 -acetyltransferase 1) gene as a transcription target of p53. SAT1 is a rate-limiting enzyme in polyamine catabolism critically involved in the conversion of spermidine and spermine back to putrescine. Surprisingly, we found that activation of SAT1 expression induces lipid peroxidation and sensitizes cells to undergo ferroptosis upon reactive oxygen species (ROS)-induced stress, which also leads to suppression of tumor growth in xenograft tumor models. Notably, SAT1 expression is down-regulated in human tumors, and CRISPRcas9-mediated knockout of SAT1 expression partially abrogates p53-mediated ferroptosis. Moreover, SAT1 induction is correlated with the expression levels of arachidonate 15-lipoxygenase (ALOX15), and SAT1-induced ferroptosis is significantly abrogated in the presence of PD146176, a specific inhibitor of ALOX15. Thus, our findings uncover a metabolic target of p53 involved in ferroptotic cell death and provide insight into the regulation of polyamine metabolism and ferroptosis-mediated tumor suppression.SAT1 | p53 | polyamine metabolism | ferroptosis | tumor suppression

Summary Although previous studies indicate that loss of p53-mediated cell cycle arrest, apoptosis, and senescence, does not completely abrogate its tumor suppression function, it is unclear how the remaining activities of p53 are regulated. Here we identified an acetylation site at lysine K98 in mouse p53 (or K101 for human p53). While the loss of K98 acetylation (p53K98R) alone has very modest effects on p53-mediated transactivation, simultaneous mutations at all four acetylation sites (p534KR: K98R+3KR(K117R+K161R+K162R)) completely abolish its ability to regulate metabolic targets such as TIGAR and SLC7A11. Notably, in contrast to p533KR, p534KR is severely defective in suppressing tumor growth in mouse xenograft models. Moreover, p534KR is still capable of inducing the p53-Mdm2 feedback loop but p53-dependent ferroptotic responses are markedly abrogated. Together, these data indicate the critical role of p53 acetylation in ferroptotic responses and its remaining tumor suppression activity.

Mammalian ectoapyrase (CD39) is an integral membrane protein with two transmembrane domains and a large extracellular region. The enzymatic activity of ectoapyrase is inhibited by most detergents used for membrane protein solubilization. In contrast, the enzymatic activities of soluble E-type ATPases, including potato tuber (Solanum tuberosum) apyrase and parasite ectoATPase, are not affected by detergents. Here we show that ectoapyrase is a tetramer and that detergents that reduce the activity of the enzyme promote dissociation of the tetramer to monomers. We expressed a secreted form of the ectoapyrase in COS-7 cells by fusing the signal peptide of murine CD4 with the extracellular domain of the ectoapyrase. The soluble ectoapyrase is catalytically active and its activity is not affected by detergents. Mutants of the ectoapyrase with only the NH 2 -or the COOH-terminal transmembrane domain are membrane-bound, and their activity is no longer affected by detergents. The enzymatic activity of all of the mutant proteins is less than that of the native enzyme. These results suggest that the proper contacts between the transmembrane domains of the monomers in the tetramer are necessary for full enzymatic activity.Ectoapyrases, or ATP diphosphohydrolases, hydrolyze extracellular nucleotide tri-and diphosphates in the presence of Ca 2ϩ or Mg 2ϩ (1). The rate of hydrolysis of nucleotide diphosphates is ϳ50% of that of the triphosphates. Mammalian ectoapyrases play important roles in many biological processes, including the modulation of neural cell activities (for a review, see Ref.2), prevention of intravascular thrombosis (3, 4), and regulation of immune responses (5). The enzymatic activity of ectoapyrases suggests that their functions might be to regulate purinergic signaling systems (6).The amino acid sequence of potato apyrase (7) revealed regions of similarity between the potato apyrase and several other proteins: garden pea nucleotide triphosphatase (8), Toxoplasma gondii nucleotide triphosphatase (9), yeast guanosine diphosphatase (10), and the lymphocyte cell activating antigen CD39 (11). We showed that CD39 has ectoapyrase activity (5) and that it is likely that the gene for CD39 is the only ectoapyrase gene in the mammalian genome (12). Subsequently, it was shown that CD39 also has ecto-ADPase activity in endothelial cells and is responsible for inhibition of ADP-induced platelet aggregation (3, 4). The ecto-ATPases in chicken gizzard (13), humans (CD39L1 (14)), and rats (15) are also encoded by genes similar to that for CD39. These latter enzymes hydrolyze nucleoside diphosphates at less than 10% of the rate of ATP hydrolysis (15).Mammalian ectoapyrases (CD39) are integral membrane proteins with two transmembrane domains (one at each end of the protein), small cytoplasmic NH 2 -and COOH-terminal segments, and a large extracellular domain (11) with enzymatic activity (5). This arrangement is unusual for ecto-enzymes, since these are usually attached to the membrane by a single protein or lipid link (16, 17)...