Title: Dissecting the non-canonical functions of p53 through novel target identification and p53 acetylation
Description: It is well established that the p53 tumor suppressor plays a crucial role in controlling cell proliferation and apoptosis upon various types of stress. There is increasing evidence showing that p53 is also critically involved in various non-canonical pathways, including metabolism, autophagy, senescence and aging. Through a ChIP-on-chip screen, we identified a novel p53 metabolic target, pantothenate kinase-1 (PANK1). PanK1 catalyzes the rate-limiting step for CoA synthesis, and therefore, controls intracellular CoA content; Pank1 knockout mice exhibit defect in β-oxidation and gluconeogenesis in the liver after starvation due to insufficient CoA levels. We demonstrated that PANK1 gene is a direct transcriptional target of p53. Although DNA damage-induced p53 upregulates PanK1 expression, depletion of PanK1 expression does not affect p53-dependent growth arrest or apoptosis. Interestingly, upon glucose starvation, PanK1 expression is significantly reduced in HCT116 p53 (-/-) but not in HCT116 p53 (+/+) cells, suggesting that p53 is required to maintain PanK1 expression under metabolic stress conditions.

Moreover, by using p53-mutant mice, we observed that PanK activity and CoA levels are lower in livers of p53-null mice than that of wild-type mice upon starvation. Similar to the case in Pank1 knockout mice, β-oxidation and gluconeogenesis are impaired in p53-null mice. Together, our findings show that p53 is critical in regulating energy homeostasis through transcriptional control of PANK1. Our study on PANK1 led us to the question of how p53 can differentially regulate a diverse array of downstream targets in a context-dependent manner. Studies have shown that p53 acetylation at K120 and K164 lysine residues contribute to p53-mediated apoptosis and growth arrest functions, which was further supported by the 3KR mouse model (K117/161/162R) that mirrors the K120/164R mutations in human p53. These studies also suggest that a potentially large number of p53 targets can still be regulated by p53 in the absence of K120/164 acetylation (K117/161/162R in mouse). To investigate whether additional modifications of p53 can further contribute to promoter-specific transactivation, we conducted a screen using mass spectrometry and identified a novel acetylation site at K101.

Our data demonstrated that K101 in human p53, as well as the homologous K98 lysine residue in mouse p53, can be acetylated by acetyltransferase CBP. Acetylation at this novel site does not contribute to p53 stability or DNA-binding capabilities. Ablation of K98 acetylation in mouse p53 alone does not affect the transcriptional activity of p53. However, simultaneous loss of K98 acetylation with the previously characterized K117/161/162 acetylations (4KR98 p53) significantly abrogates p53-mediated activation of TIGAR and MDM2 genes. The 3KR mouse model, although cannot elicit canonical p53-mediated apoptotic and cell cycle arrest responses, still retains the ability to suppress tumor formation. We, therefore, investigated whether other non-canonical targets of p53 could potentially mediate tumor suppression. By RNA-seq profiling of gene expression in cells expressing 3KR p53, we identified TNFRSF14 (tumor necrosis factor receptor superfamily, member 14) as a novel p53 target. The TNFRSF14 receptor has been shown to be frequently mutated in follicular lymphoma and diffuse large B cell lymphoma, and stimulation by its ligand LIGHT leads to cell death in many cancer cells.

We report that TNFRSF14 is a novel p53 target that can be activated by 3KR p53. Interestingly, transactivation of TNFRSF14 is defective by 4KR98 p53. Furthermore, LIGHT ligand stimulates cell death in TNFRSF14-expressing cells and cells expressing 3KR p53, but not those expressing 4KR98 p53. Altogether, our findings in these studies underscore the extensive scope of p53 functions and provide new insights into the versatility of non-canonical pathways. Not only does p53 mediate tumor suppression through both canonical and non-canonical downstream effectors, p53 can also contribute to cellular homeostasis and energy balance.