Deciphering the mechanism of p53-mediated ferroptosis

Background of p53

It is well known that p53 is a tumor suppressor gene and essential for regulating DNA repair and cell division. Since its discovery in 1979, the p53 gene has been a hot topic in molecular biology and oncology. According to Dolgin, E. et al.’s literature published in?Nature,?#TP53?becomes the most popular human genome in the list of most studied genes in the PubMed database[1]. The TP53 gene provides instructions for making a p53 protein.

According to integrated cancer genomic and epidemiological data analyses, TP53 is the most commonly mutated gene (35%) among mutated driver genes in human cancers[2](Figure 1), it is also the most studied gene in the human genome based on the PubMed database. TP53 is located on the short arm of human chromosome 17 and encodes the p53 tumor suppressor protein.

The p53 protein is often referred to as the “Guardian of the Genome”. The main biological function of the p53 protein is the protection of the DNA integrity of the cell.

In response to intrinsic and extrinsic stress signals, the p53 protein is activated by a variety of post-translational modifications including phosphorylation, acetylation, methylation, ubiquitination, or SUMOylating, etc. These modifications at key sites allow the p53 protein to become stabilized, oligomerize as a tetramer, interact with cofactors, bind to the p53RE, and execute the transcription of the target genes in a tightly controlled and context-dependent manner [Figure 2]. In recent years it was found that p53 plays role in the regulation of ferroptosis. In this article,?the connection between p53 and ferroptosis will be explored

Ferroptosis is a novel form of regulated cell death. The morphological features of ferroptosis include shrunk mitochondria with condensed mitochondrial membrane densities, reduction or vanishing of mitochondria crista, and rupture of outer mitochondrial membrane.The p53 (especially acetylation-defective mutant p53, p533KR) positively regulates ferroptosis by inhibiting expression of SLC7A11 (a specific light-chain subunit of the cysteine/glutamate antiporter).

#SLC7A11 is a subunit of System Xc-, which is responsible for maintaining redox homeostasis by importing cystine. After being transported into cells, cystine is quickly reduced to cysteine, a critical precursor for glutathione, and subsequently reduced glutathione (GSH). GSH biosynthesis is critical to the functional activity of membrane lipid repair enzyme GPX4. Inhibiting System Xc- activity by inhibition of SLC7A11 expression leads to decreased uptake of cystine, eventually resulting in the impaired antioxidant capability of cells and initiation of ferroptosis [Figure 3].?“#Ferroptosis as a p53-mediated activity during tumor suppression”, Jiang et al. reported that p53 inhibits expression of SLC7A11, reduces cystine uptake and induces ferroptosis of cancer cells.

The SLC7A11 gene is a target of p53-mediated transcriptional repression

Jiang, L et al. have demonstrated in the "Ferroptosis as a p53-mediated Activity during Tumour Suppression" published in Nature in 2015 demonstrates that p53 supposes SLC7A11 (a key component of the cystine/glutamate antitransporter) to inhibit cystine uptake and sensitize cells to ferroptosis[7].

By microarray analysis of?Tetracycline-controlled (tet-on) p53-iinduced and non-induced cells,?SLC7A11?was identified as a novel p53 target gene. Western blot showed that p53 activation significantly reduced SLC7A11 protein levels [Figure 4a]. While in U2OS cells under p53-knockdown conditions treated?Nutlin-3?(a p53-MDM2 inhibitor), downregulation of SLC7A11 was abrogated. These data suggest that?the SLC7A11?gene is a target of p53-mediated transcriptional repression.

The acetylation defective mutant p533KR?cells that fails to induce cell cycle arrest, senescence, and apoptosis maintain the ability to regulate SLC7A11 expression and induce ferroptosis. Tet-on p533KR?inducible H1299 cells were resistant to erastin-mediated ferroptosis in the absence of p533KR?induction, while significant cell death was observed upon p533KR?induction together with?Erastin?treatment. But SLC7A11overexpression abrogated p533KR-induced ferroptosis [Figure 4c]. In xenograft tumor models implanted with p53-null H1299 cells, tumor size is markedly reduced upon p533KR?expression induced by tetracycline, while this tumor suppression effects of p533KR?were abrogated by?SLC7A11?overexpression[7]?[Figure 4d-e].

In addition, it was found that high levels of reactive oxygen species (ROS) can trigger p53-mediated ferroptosis. As shown in Figure 5a, no significant cell death was observed in p533KR?induction alone or in ROS activator treatment; whereas the p533KR?and ROS group induced substantial cell death, which was rescued by overexpression of SLC7A11 (Figure 5b)[7].

ALOX12 is essential for the p53-mediated ferroptosis pathway

In a follow-up study, Chu B. et al. reported that #ALOX12-mediated, ACSL4-independent ferroptosis pathway is critical for p53-dependent tumor suppression[8]. The?ALOX12?gene is located on human chromosome 17p13.1, a labile site for monoallelic deletions in human cancers.

ALOX12 was found to be essential for p53-mediated ferroptosis under ROS stress. ALOX12 depletion had no apparent effect on p53 levels or the expression of its transcriptional targets such as SLC7A11, Mdm2, and p21, but it rescued p53-mediated ferroptosis (Figure 6a-b)[8].

Next, it was found that p53-mediated ferroptosis under ROS stress was regulated independently of GPX4. As shown in Figure 6c, high levels of endogenous lipid peroxidation could be detected in GPX4-knockout cells, whereas lipid peroxidation levels were significantly reduced after ectopic expression of GPX4. At the same time, RSL-3 (GPX4 inhibitor) could abrogate the reducing effect of GPX4 on lipid peroxidation, while the activation of p53 had no effect on it[8].

Then, the effect of ALOX12 on p53-mediated tumor growth inhibition was investigated. Tetracycline-induced p533KR?expression significantly reduced tumor cell growth. Moreover, ALOX12 knockdown ablated the tumor suppressive effect of p533KR?[Figure 6c-d]. These data suggested that ALOX12 is critical for the tumor cell growth inhibitory activity of p53[8].

iPLA2β is a key regulator of the p53-mediated ferroptosis pathway

In June 2022, Chen Du et al. reported a mechanism in an article?"iPLA2β-mediated lipid detoxification controls p53-driven ferroptosis independent of GPX4"?in?Nature CommunicationsIn?this proposed mechanism iPLA2β is a key regulator of p53 activation-induced ferroptosis under high ROS stress conditions and p53 induces ferroptosis in a GPX4-independent manner[9][Figure 7].

The p53 levels were not affected by ACSL4 and GPX4, in the ACSL4/GPX4 double knockout (ACSL4-/-/GPX4-/-) human osteosarcoma cell line U2OS. At the same time, p53-mediated transcriptional activation of p21 or repression of SLC7A11 remained unchanged (Fig. 8a). However, when ACSL4-/-/GPX4-/-?cells were exposed to TBH and Nutlin, ferroptosis cell death apparently occurred, which was specifically blocked by ferroptosis inhibitors (Fig. 8b).

Conclusion:

The tumor suppressor gene?TP53?is the most studied gene in cancer research. Ferroptosis is a novel form of regulated cell death that has sparked a research frenzy since its discovery. The discovery of the connection between p53 and ferroptosis paves a new way for the development of p53-related drugs for cancer treatment.

References

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[3].Nature. 2017 Nov 23;551(7681):427-431.

[4].Oncogene. 2005 Apr 18;24(17):2899-908.

[5].Ou M, et al.Mol Metab. 2022 Jul;61:101502.

[6].Cell Death Differ. 2016 Mar;23(3):369-79.

[7].Nature. 2015 Apr 2;520(7545):57-62.

[8].Nat Cell Biol. 2019 May;21(5):579-591.

[9].Nat Commun. 2021 Jun 15;12(1):3644.