Title: Dissecting the role of p53-mediated metabolic regulation in tumor suppression
Description: The p53 tumor suppressor protein has been well-characterized for its role in inducing growth arrest, senescence, and apoptosis upon various types of stresses. Recently, however, roles of p53 have expanded beyond the canonical functions, and now include cellular processes such as metabolism, oxidative balance, and ferroptosis. Through RNA-seq screening, we first identified phosphoglycerate dehydrogenase (PHGDH), a rate-limiting enzyme in the serine biosynthesis pathway, as a novel metabolic target of p53. p53 suppresses PHGDH expression and inhibits de novo serine biosynthesis. Notably, upon serine starvation, p53-mediated cell death is significantly enhanced in response to Nutlin-3 treatment. Moreover, PHGDH has been demonstrated to be frequently amplified in human melanomas. We found that PHGDH overexpression significantly suppresses the apoptotic response, whereas RNAi-mediated knock-down of endogenous PHGDH promotes apoptosis under the same treatment. Together, our findings demonstrate an important role of p53 in regulating serine biosynthesis through suppressing PHGDH expression, and reveal serine deprivation as a novel approach to sensitize p53-mediated apoptotic responses in human melanoma cells.

In addition, we also identified spermidine/spermine N1-acetyltransferase 1 (SAT1) as a novel metabolic 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 CRISPR-cas9-mediated knockout of SAT1 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. Together, these data indicate a novel regulatory role of p53 in polyamine metabolism and provide insight into the regulation of p53-mediated ferroptotic responses.

Our studies on PHGDH and SAT1 led us to the question of whether these unconventional functions of p53 contribute to its role as a tumor suppressor. In fact, previous view regarding the mechanism of p53-mediated tumor suppression, which was long thought to be growth arrest, apoptosis, and senescence, has recently been challenged by several knockout and knock-in mouse studies. Previously, we established mice (p533KR/3KR) in which p53 acetylation at lysine residues K117, K161, and K162 were abolished by replacing lysine with arginine. p533KR/3KR mice completely lost p53-mediated cell cycle arrest, apoptosis, and senescence functions in response to stresses. However, unlike p53-null mice which rapidly develop spontaneous thymic lymphomas, all of the p533KR/3KR mice remain tumor-free, indicating that other aspects of p53 functions are sufficient to prevent tumor formation. Notably, p533KR retains the ability to regulate metabolic targets including TIGAR and SAT1, as well as ferroptosis regulator SLC7A11.

In this study, we have identified two novel acetylation sites- K98 and K136, in the mouse p53 DNA-binding domain. Whereas loss of K98 or K136 acetylation (p53K98R, p53K136R) alone has modest effect on p53 transcriptional activity, simultaneous mutations at all of these acetylation sites (p534KR98: K98R+3KR, p534KR136: K136R+3KR, p535KR: K98R+K136R+3KR) completely abolish the ability of p53 to regulate TIGAR, SAT1, and SLC7A11. In addition, p534KR98, p534KR136, and p535KR are defective in Erastin-induced ferroptosis. Notably, p534KR98/4KR98, p534KR136/4KR136, and p535KR/5KR knock-in mice lost intact tumor suppression and developed spontaneous tumors. This suggests that p53-mediated ferroptosis may function as a critical barrier to prevent tumor formation independently from growth arrest, apoptosis, and senescence. Interestingly, both p534KR98/4KR98 and p534KR136/4KR136 mice displayed significantly delayed tumorigenesis comparing with p53-null and p535KR/5KR mice. We found that unlike p535KR, p534KR98 retains the capacity to inhibit mammalian target of rapamycin (mTOR) signaling pathway through activating the expression of two mTOR negative regulators, Sestrin2 and DDIT4.

Altogether, our findings underscore the extensive scope of p53 functions in metabolic regulation, oxidative stress response, and ferroptosis, and provide novel insights into the tumor suppression mechanism of p53.