Although p53-mediated cell-cycle arrest, senescence and apoptosis serve as critical barriers to cancer development, emerging evidence suggests that the metabolic activities of p53 are also important. Here we show that p53 inhibits cystine uptake and sensitizes cells to ferroptosis, a non-apoptotic form of cell death, by repressing expression of SLC7A11, a key component of the cystine/glutamate antiporter. Notably, p533KR, an acetylation-defective mutant that fails to induce cell-cycle arrest, senescence and apoptosis, fully retains the ability to regulate SLC7A11 expression and induce ferroptosis upon reactive oxygen species (ROS)-induced stress. Analysis of mutant mice shows that these non-canonical p53 activities contribute to embryonic development and the lethality associated with loss of Mdm2. Moreover, SLC7A11 is highly expressed in human tumours, and its overexpression inhibits ROS-induced ferroptosis and abrogates p533KR-mediated tumour growth suppression in xenograft models. Our findings uncover a new mode of tumour suppression based on p53 regulation of cystine metabolism, ROS responses and ferroptosis.
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Berkers, C. R., Maddocks, O. D., Cheung, E. C., Mor, I. & Vousden, K. H. Metabolic regulation by p53 family members. Cell Metab. 18, 617–633 (2013)
Jackson, J. G. & Lozano, G. The mutant p53 mouse as a pre-clinical model. Oncogene 32, 4325–4330 (2013)
Aylon, Y. & Oren, M. New plays in the p53 theater. Curr. Opin. Genet. Dev. 21, 86–92 (2011)
Junttila, M. R. & Evan, G. I. p53–a Jack of all trades but master of none. Nature Rev. Cancer 9, 821–829 (2009)
Wang, S. J. & Gu, W. To be, or not to be: functional dilemma of p53 metabolic regulation. Curr. Opin. Oncol. 26, 78–85 (2014)
Bieging, K. T. & Attardi, L. D. Deconstructing p53 transcriptional networks in tumor suppression. Trends Cell Biol. 22, 97–106 (2012)
Brady, C. A. et al. Distinct p53 transcriptional programs dictate acute DNA-damage responses and tumor suppression. Cell 145, 571–583 (2011)
Valente, L. J. et al. p53 efficiently suppresses tumor development in the complete absence of its cell-cycle inhibitory and proapoptotic effectors p21, Puma, and Noxa. Cell Rep. 3, 1339–1345 (2013)
Kruse, J. P. & Gu, W. Modes of p53 regulation. Cell 137, 609–622 (2009)
Li, T. et al. Tumor suppression in the absence of p53-mediated cell-cycle arrest, apoptosis, and senescence. Cell 149, 1269–1283 (2012)
Lo, M., Wang, Y. Z. & Gout, P. W. The cystine/glutamate antiporter: a potential target for therapy of cancer and other diseases. J. Cell. Physiol. 215, 593–602 (2008)
Conrad, M. & Sato, H. The oxidative stress-inducible cystine/glutamate antiporter, system : cystine supplier and beyond. Amino Acids 42, 231–246 (2012)
Sato, H., Tamba, M., Kuriyama-Matsumura, K., Okuno, S. & Bannai, S. Molecular cloning and expression of human xCT, the light chain of amino acid transport system . Antioxid. Redox Signal. 2, 665–671 (2000)
Vu, B. T. & Vassilev, L. Small-molecule inhibitors of the p53–MDM2 interaction. Curr. Top. Microbiol. Immunol. 348, 151–172 (2011)
Dixon, S. J. et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149, 1060–1072 (2012)
Huang, Y., Dai, Z., Barbacioru, C. & Sadee, W. Cystine-glutamate transporter SLC7A11 in cancer chemosensitivity and chemoresistance. Cancer Res. 65, 7446–7454 (2005)
Liu, X. X. et al. MicroRNA-26b is underexpressed in human breast cancer and induces cell apoptosis by targeting SLC7A11. FEBS Lett. 585, 1363–1367 (2011)
Guo, W. et al. Disruption of xCT inhibits cell growth via the ROS/autophagy pathway in hepatocellular carcinoma. Cancer Lett. 312, 55–61 (2011)
Montes de Oca Luna, R., Wagner, D. S. & Lozano, G. Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378, 203–206 (1995)
Jones, S. N., Roe, A. E., Donehower, L. A. & Bradley, A. Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 378, 206–208 (1995)
Gannon, H. S. & Jones, S. N. Using mouse models to explore MDM–p53 signaling in development, cell growth, and tumorigenesis. Genes Cancer 3, 209–218 (2012)
Mendrysa, S. M. et al. Mdm2 is critical for inhibition of p53 during lymphopoiesis and the response to ionizing irradiation. Mol. Cell. Biol. 23, 462–472 (2003)
Marine, J. C. & Lozano, G. Mdm2-mediated ubiquitylation: p53 and beyond. Cell Death Differ. 17, 93–102 (2010)
Chavez-Reyes, A. et al. Switching mechanisms of cell death in mdm2- and mdm4-null mice by deletion of p53 downstream targets. Cancer Res. 63, 8664–8669 (2003)
Yang, W. S. et al. Regulation of ferroptotic cancer cell death by GPX4. Cell 156, 317–331 (2014)
Hughes, R. H., Silva, V. A., Ahmed, I., Shreiber, D. I. & Morrison, B., III Neuroprotection by genipin against reactive oxygen and reactive nitrogen species-mediated injury in organotypic hippocampal slice cultures. Brain Res. 1543, 308–314 (2014)
Wang, Z., Jiang, H., Chen, S., Du, F. & Wang, X. The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways. Cell 148, 228–243 (2012)
Lu, M. et al. Restoring p53 function in human melanoma cells by inhibiting MDM2 and cyclin B1/CDK1-phosphorylated nuclear iASPP. Cancer Cell 23, 618–633 (2013)
Wade, M. & Wahl, G. M. Targeting Mdm2 and Mdmx in cancer therapy: better living through medicinal chemistry? Mol. Cancer Res. 7, 1–11 (2009)
Wang, P. Y. et al. Increased oxidative metabolism in the Li-Fraumeni syndrome. N. Engl. J. Med. 368, 1027–1032 (2013)
Liang, Y., Liu, J. & Feng, Z. The regulation of cellular metabolism by tumor suppressor p53. Cell Biosci. 3, 9 (2013)
Bensaad, K. et al. TIGAR, a p53-inducible regulator of glycolysis and apoptosis. Cell 126, 107–120 (2006)
Cairns, R. A., Harris, I. S. & Mak, T. W. Regulation of cancer cell metabolism. Nature Rev. Cancer 11, 85–95 (2011)
Cheung, E. C., Ludwig, R. L. & Vousden, K. H. Mitochondrial localization of TIGAR under hypoxia stimulates HK2 and lowers ROS and cell death. Proc. Natl Acad. Sci. USA 109, 20491–20496 (2012)
Cheung, E. C. et al. TIGAR is required for efficient intestinal regeneration and tumorigenesis. Dev. Cell 25, 463–477 (2013)
Hu, W. et al. Glutaminase 2, a novel p53 target gene regulating energy metabolism and antioxidant function. Proc. Natl Acad. Sci. USA 107, 7455–7460 (2010)
Suzuki, S. et al. Phosphate-activated glutaminase (GLS2), a p53-inducible regulator of glutamine metabolism and reactive oxygen species. Proc. Natl Acad. Sci. USA 107, 7461–7466 (2010)
Schmidt, D. et al. ChIP-seq: using high-throughput sequencing to discover protein-DNA interactions. Methods 48, 240–248 (2009)
Zheng, H. et al. A posttranslational modification cascade involving p38, Tip60, and PRAK mediates oncogene-induced senescence. Mol. Cell 50, 699–710 (2013)
Kon, N. et al. Inactivation of HAUSP in vivo modulates p53 function. Oncogene 29, 1270–1279 (2010)
This work was supported by the National Cancer Institute of the National Institutes of Health under awards 5R01CA172023, 5RO1CA166294, 5RO1CA169246, 5RO1CA085533 and 2P01CA080058 to W.G. It was also supported by the National Cancer Institute under award 2P01CA097403 to R.B. and 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. L.J. and S.-J.W. were supported by NIH cancer biology training grant T32-CA09503. We thank S. Mendrysa for Mdm2 mutant mice.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 SLC7A11 expression is downregulated by p53 and identification of p53 binding sites for mouse Slc7a11 gene.
a, Messenger RNA levels of SLC7A11 in tet-on wild-type p53 stable line and parental H1299 cells treated with doxycycline (0.1 µg ml−1). b, U2OS cells were treated with doxorubicin (0.2 µg ml−1) and mRNA was quantified. c, Osteosarcoma cell lines, U2OS (p53 wild type) and SAOS-2 (p53 null) cells, were treated with doxorubicin (0.2 µg ml−1) and mRNA levels were determined. d, Lung cancer cell lines, H1299 (p53 null) and H460 (p53 wild type) cells, were treated with doxorubicin (0.2 µg ml−1) and RT–PCR was used to determine mRNA expression. e, The breast cancer cell line MCF7 was treated with doxorubicin (0.2 µg ml−1) for indicated duration and RT–qPCR was used to measure mRNA expression. f, RT–qPCR were used to determine the mRNA level of Slc7a11 in MEFs with indicated genotype. g, Schematic diagram representing potential p53 binding locations and sequences on the mouse Slc7a11 gene. TSS, transcription start site; light blue box, 5′-UTR. h, ChIP–qPCR was performed on MEFs that were treated with nutlin (10 µM) for 6 h. All qPCR was performed in two technical replicates and mean ± s.d. are shown. All experiments were repeated independently three times.
a, Quantification of cell death as shown in Fig. 3a. Error bars are s.d. from two technical replicates. b, Kinetics of cell death induced by erastin (1 µM) over a 24-h period in MEFs with indicated genotypes. Technical replicates were performed and mean ± s.d. are shown (n = 2). c, Transmission electron microscopy image of wild-type MEFs that were treated with TNFα (20 ng ml−1) and CHX (5 µg ml−1) for 16 h with arrows pointing to fragmented nuclei. d, Wild-type MEFs were treated with mouse TNFα (20 ng ml−1) and CHX (5 µg ml−1) or erastin (1 µM) for 8 h followed by western blots. e, TUNEL assay was carried out using wild-type MEFs treated as in d. f, Quantification of TUNEL signals for e. Mean ± s.d. from ten random microscope views are shown (magnification, ×20). g, MEFs with indicated p53 status were treated with erastin (4 µM) and specific cell death inhibitors for 8 h before images were taken (magnification, ×10). 3-MA, 3-methylademine. All experiments were repeated at least three times and representative data are shown.
a, Wild-type MEF cells were starved in DMEM medium deprived of glucose, sodium pyruvate or l-glutamine for 2 h with or without 3-methylademine (2 mM) followed by western blots. b, Wild-type MEFs were treated for 48 h with TNFα (20 ng ml−1), SMAC mimetic (100 nM) and Z-VAD-FMK (10 µg ml−1) to induced necroptosis with or without the presence of necrostatin-1 (10 µg ml−1) (magnification, ×10). c, Quantification of cell death as shown in b. PI, propidium iodide. Mean ± s.d. from two technical replicates are shown. d, Wild-type MEFs were treated for 48 h with TNFα (20 ng ml−1), SMAC mimetic (100 nM) and necrostatin-1 (10 µg ml−1) to induce apoptosis with or without the presence of Z-VAD-FMK (10 µg ml−1) (magnification, ×10). e, Quantification of cell death as shown in d. Mean ± s.d. from two technical replicates are shown. f, p533KR/3KR MEFs were treated with erastin (4 µM) and various chemicals that block ferroptosis for 24 h before the percentage of cell death was determined; error bars, s.d. from two technical replicates. DMSO, dimethyl sulfoxide; DFO, deferoxamine; U0126, 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene; β-ME, β-mercaptoethanol; NAC, N-acetyl-l-cysteine. All experiments were independently repeated three times.
a–c, Quantitative RT–PCR was used to determine the expression levels of SLC7A11 in paired normal and cancer tissues from colon (a), kidney (b) and liver (c); average expression levels from normal tissues were normalized to 1 in each type of cancer. Mean ± s.d. from two technical replicates are shown. d, Representative heamotoxylin and eosin (H&E) and immunofluorescence staining of SLC7A11 on frozen sections of paired patient cancer and adjacent normal tissues. Magnifcation, ×20. N, normal tissue; C, cancer tissue. Blue, DAPI; green, anti-ATP1A1; red, anti-SLC7A11. e, DNA sequencing was performed on colon cancer samples and specific mutations were identified. Independent experiments were repeated three times and representative data are shown.
Extended Data Figure 5 Cell death induced by p533KR and erastin is ferroptosis and effect of SLC7A11 overexpression on colony formation.
a, Representative phase-contrast images of cell cultures as treated in Fig. 4b (magnification, ×10). b, p533KR tet-on stable line cells were treated as indicated and the percentage of cell death was quantified (DFO, deferoxamine; U0126, 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene; β-ME, beta-mercaptoethanol; NAC, N-acetyl-l-cysteine). Mean ± s.d. from two technical replicates are shown. c, H1299 cells were transfected with indicated plasmids followed by western blot 24 h later. HA, haemagglutinin d, Representative images of colony formation assay in 10-cm plates as transfected in c. e, Quantification of colony formation assay as shown in d. Numbers of colonies formed in control plates were normalized to 100 and mean ± s.d. from two technical replicates are shown. All experiments were repeated three times with representative data shown.
a, Representative gel images for genotyping of Mdm2 status in p533KR/3KR background mice. b, Summary of numbers of live embryos and pups recovered from p533KR/3KRMdm2+/− intercross breeding. c, Haematoxylin and eosin (H&E) and immunohistochemistry staining of p53 and TUNEL assay on E7.5 embryos of indicated genotype (magnification, ×20). d, Percentage of cells with positive TUNEL or BrdU were determined by counting 100 cells in each section from three different embryos. Error bars, s.d.; N.S., not significant. e, Whole-embryo extracts from E9.5 embryos were used for western blot. As positive controls, thymus protein lysate from irradiated (IR) wild-type mouse (8 Gy) was used. f, Messenger RNA expression levels of Puma were determined by RT–qPCR using E9.5 embryos with indicated genotype (n = 3 for p533KR/3KRMdm2+/+ and n = 5 for p533KR/3KRMdm2−/−; error bars, s.d.; N.S, not significant). g, Representative images of whole-mount senescence-associated β-galactosidase staining using E9.5 mouse embryos with indicated genotype (magnification, ×2). h, Same protocol as in g was used to stain control wild-type embryos and embryos of HAUSP heterozygous knockouts, which express β-galactosidase40 (magnification, ×2). i, Late passage senescent wild-type MEFs were stained for senescence-associated β-galactosidase activity using the same protocol as in g (magnification, ×10).
a, Representative morphologies of E11.5 mouse embryos of indicated genotype (magnification, ×3). b, Representative embryos recovered from p533KR/3KRMdm2+/− intercross with or without ferr-1 injection. The dashed line marked by an asterisk highlights the body of a dead p533KR/3KRMdm2−/− embryo, which disintegrated upon further dissection (magnification, ×1.5). c, Head-to-tail lengths of p533KR/3KRMdm2−/− embryos were measured and compared to p533KR/3KRMdm2+/+ controls (n = 4 for each group of p533KR/3KRMdm2−/− embryos with or without ferr-1 treatment; error bars, s.d.). d, Representative haematoxylin and eosin (H&E) staining of eye structures of p533KR/3KRMdm2+/+ and p533KR/3KRMdm2−/− mouse embryos (magnification, ×20 and ×40 as indicated).
a, Percentage of cell death as shown in Fig. 6a was quantified. Mean ± s.d. from two technical replicates are shown. b, U2OS cells with stable knockdown of p53 were treated by nultin (10 µM) for 24 h followed by addition of ROS (tert-butyl hydroperoxide, 350 µM) for 4 h. Western blots were performed. c, Quantification of cell death as shown in Fig. 6c. Mean ± s.d. from two technical replicates are shown. d, U2OS cells were treated with nutlin (10 µM) for 24 h first, followed by ROS (tert-butyl hydroperoxide, 350 µM) along with indicated cell death inhibitors; cell death were quantified 24 h later. Error bars, s.d. from two technical replicates. e, U2OS cells were treated with DNA-damaging agents (etoposide, 20 µM; doxorubicin, 0.2 µg ml−1) for 48 h with or without the presence of ferr-1 (2 µM) (magnification, ×10); cell death was quantified in f with mean ± s.d. shown (n = 2 technical replicates). All data were repeated three times independently.
a, Schematic diagram showing the procedure for generation of Slc7a11-BAC transgenic mice. b, Snap shot of BACs surrounding mouse Slc7a11 genes. BAC (RP24-242E11) that contains only the Slc7a11 gene was selected for injection. c, PCR at both ends of the BAC construct identified founders (no. 21 and no. 22) as positive BAC transgenic mice. d, Germline transmission was confirmed from both founders identified in c. NC, no template control. e, Thymus and brain tissues from 3-week-old litter mates of control and Slc7a11-BAC transgenic mice were lysed and examined by western blots. f, MEF cells with indicated genotypes were treated as in Fig. 6e for 2 h and mRNA levels were determined by RT–qPCR. Mean ± s.d. from two technical replicates are shown. g, Representative images of cells treated as in Fig. 6e (magnification, ×10).
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Jiang, L., Kon, N., Li, T. et al. Ferroptosis as a p53-mediated activity during tumour suppression. Nature 520, 57–62 (2015). https://doi.org/10.1038/nature14344
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