Ferroptosis: A missing puzzle piece in the p53 blueprint?

ABSTRACT

Recent evidence indicates that canonical functions of p53 (i.e., apoptosis and growth arrest) are dispensable for p53-mediated tumor suppression. We have uncovered a novel function of p53 that contributes to tumor suppression through regulation of cystine metabolism, reactive oxygen species responses, and ferroptosis. The p53-mediated ferroptotic response via SLC7A11 denotes an extra layer of defense against tumorigenesis in conjunction with other p53 functions.

Since the discovery of tumor protein p53 (TP53, best known as p53) more than 30 years ago, its role in tumor suppression has generated remarkable interest in the field of cancer biology. Cumulative efforts from the scientific community are constantly expanding the scope of p53 function in mediating tumor suppression, which now covers a diverse array of cellular processes.^1,2 Despite the multiplicity of p53 functions, it has long been thought that p53 thwarts tumorigenesis through its most phenotypically prominent features—apoptosis and cell growth arrest. Recently, however, this “central dogma” of p53-mediated tumor suppressing mechanism has been challenged.

Studies from our group and others have shown that the apoptosis and cell growth arrest functions of p53 may in fact be dispensable for p53-mediated tumor suppression. Knock-in mice that express p53^3KR, an acetylation-deficient p53 variant at specific lysine residues that lacks the ability to transcriptionally activate pro-apoptotic and cell cycle arrest genes (while retaining metabolic regulation), are not tumor prone and exhibit survival curves similar to those of wild-type mice.³ Similarly, studies on a transactivation-compromised mutant variant of p53, p53^25,26, demonstrated that tumor suppression can occur in the absence of a majority of p53 downstream targets.⁴ All this evidence points toward one conclusion: unknown targets of p53 may contribute significantly to its tumor suppressor function. It was this notion that led us to uncover the link between p53 and ferroptosis described in the recent article by Jiang et al.⁵

Embarking on the search for novel p53 targets, we uncovered the SLC7A11 [solute carrier family 7 (anionic amino acid transporter light chain, x_c- system), member 11] gene through microarray screening. SLC7A11 encodes a component of the cystine/glutamate antiporter (system x_c⁻). Inhibition of cystine uptake by system x_c⁻ causes iron-dependent accumulation of lipid reactive oxygen species (ROS) and eventual cell death, a process known as ferroptosis.⁶ Not only is SLC7A11 a novel repression target of p53, but p53^3KR retains the capacity to negatively regulate its expression, and as a result sensitizes cells to undergo ferroptosis, which is triggered independently from other known forms of cell death.⁵ Ferroptosis represents a previously missing puzzle piece in the grand scheme of p53-dependent tumor suppression. Indeed, our work demonstrated that ferroptosis proceeds independently from apoptosis and cell cycle arrest to curb cancer cell growth in p53^3KR-expressing cells and xenografts.⁵ Upon reconstitution of robust SLC7A11 expression in cancer cells ferroptosis is inhibited, presumably by replenishment of intracellular glutathione levels and decreased ROS accumulation.⁷ Ferroptosis also contributes to the embryonic lethality observed in Mdm2^-/- mouse embryos, which could not be rescued by ablating the apoptotic and growth arrest functions of p53 (via p53^3KR knock-in) although the developmental defect was substantially reversed by treatment with a ferroptosis inhibitor.⁵ Overall, p53-mediated ferroptosis could account for the tumor suppressive function of p53^3KR in mice observed in our previous study.

There is a long-standing notion that p53 responds to DNA damage and elicits downstream target effects that either temporarily halt cell cycle progression to repair DNA, or terminate the cell if the damage is irrevocable. DNA damage triggers ATM-CHK2 and ATR-CHK1 signaling pathways, 2 of many pathways that stabilize and activate p53.¹ Although p53 plays a pivotal role in modulating intracellular ROS levels, it is unclear whether p53 responds directly to ROS stress or accumulation. However, our findings revealed that ROS stress can induce ferroptosis in cells that retain p53-dependent repression of SLC7A11, suggesting that at least one downstream effector of p53 constitutes an element in the cellular response to ROS-mediated strain.⁵ The intrinsically lower basal level of SLC7A11 expression in cells with intact p53 function may serve as a preemptive mechanism to guard against ROS-induced cellular damage; ROS accumulation up to a threshold that the cell deems incapacitating (which is dependent on the expression of SLC7A11, and thereby, the capacity of the cell to cope with ROS) will result in elimination to prevent deleterious consequences.

Our work sheds light on an additional layer of defense against cellular injury provided by p53. Here, we propose a model of p53-mediated response to stress that encapsulates various aspects of p53 function (Fig. 1). Both DNA damage and elevation of intracellular ROS as a result of exogenous stress or cellular metabolism can be fatal in excess, but under suitable conditions they could also promote aberrant proliferation and malignant transformation in affected cells. Hence, p53 plays an intricate role in the balance between cell survival and death to maintain cellular integrity. Two divergent p53 functions can ensue upon cellular injury from ROS insult—increased production of the antioxidant glutathione as a result of p53-mediated activation of TP53-inducible glycolysis and apoptosis regulator (TIGAR) and glutaminase 2 (GLS2) to quench excess ROS,^8-10 or ferroptosis, if the ROS are allowed to accrue beyond the state of reversal. If DNA damage were to occur, canonical functions of p53, namely growth arrest/DNA repair and apoptosis, would then decide the fate of the cell depending on the severity of the damage.¹ This model is congruent to the tumor suppressive phenomenon observed in p53^3KR mice, in which the regulation of downstream targets (TIGAR, GLS2, and SLC7A11) that may modulate cell fate according to ROS levels remains intact.

Figure 1.

p53-mediated control of cell survival and death upon cellular injury. Various p53 functions can maintain homeostasis and genomic integrity of cells. Deleterious effects of DNA damage can be eliminated by activation of p53, which may trigger either DNA repair or apoptosis, depending on the severity of the damage. Similarly, p53 can also confer protection against ROS insult. TIGAR and GLS2 can promote antioxidant synthesis (glutathione), whereas p53-mediated repression of SLC7A11 and subsequent ferroptosis can prevent over-accumulation of ROS that may promote tumorigenesis. GSH, glutathione.

Many unanswered questions that have arisen from our work require further investigation. First, it is possible that there are additional components of the ferroptosis pathway that are regulated by p53. Ferroptosis is a fairly novel phenomenon that was just described recently, and certainly there is much yet to be uncovered regarding its mechanism and modulating factors, which may involve p53. Second, evidence to date begs the question of whether p53-mediated ferroptosis requires activation of p53 beyond its basal activity. Our observations in mouse embryonic fibroblast cells led us to believe that the discrepancy in SLC7A11 expression under basal p53 activity (or the lack thereof) can directly influence the extent of the ferroptotic response. However, we also noted that p53 activation induced by Nutlin treatment might sensitize cells to ROS-induced ferroptosis. Given that p53 is a master regulator of an abundance of cellular processes, a better understanding of if and how p53 responds to ROS fluctuation in cells may provide further insight into the upstream signaling of ferroptosis. This leads to the next question: Does p53 directly respond to ROS stress or accumulation? Although nutrient and energy-sensing pathways involving mTOR1/AMPK, which are often associated with redox imbalances, can trigger p53 activation upon metabolic stress, it is still unclear whether there is a direct link between ROS and p53.

In conclusion, uncovering the link between p53 and ferroptosis has opened a new door for p53 biology and brought us one step closer to understanding the mechanism underlying p53-mediated tumor suppression. Our work also sheds light on the potential therapeutic implication of promoting ferroptosis in p53-retaining tumors via ROS-inducing small molecules (i.e., Erastin or tert-butyl hydroperoxide [tBHP]) in the presence of p53 activation (i.e., via Nutlin treatment).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by the National Cancer Institute of the National Institutes of Health under Award 5RO1 CA172023, 5RO1 CA166294, 5RO1CA085533 and 2P01CA080058 to W. G. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. S-J, Wang, and L. Jiang were also supported by NIH cancer biology training grant T32-CA09503.