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ALOX12 is required for p53-mediated tumour suppression through a distinct ferroptosis pathway

Abstract

It is well established that ferroptosis is primarily controlled by glutathione peroxidase 4 (GPX4). Surprisingly, we observed that p53 activation modulates ferroptotic responses without apparent effects on GPX4 function. Instead, ALOX12 inactivation diminishes p53-mediated ferroptosis induced by reactive oxygen species stress and abrogates p53-dependent inhibition of tumour growth in xenograft models, suggesting that ALOX12 is critical for p53-mediated ferroptosis. The ALOX12 gene resides on human chromosome 17p13.1, a hotspot of monoallelic deletion in human cancers. Loss of one Alox12 allele is sufficient to accelerate tumorigenesis in Eμ-Myc lymphoma models. Moreover, ALOX12 missense mutations from human cancers abrogate its ability to oxygenate polyunsaturated fatty acids and to induce p53-mediated ferroptosis. Notably, ALOX12 is dispensable for ferroptosis induced by erastin or GPX4 inhibitors; conversely, ACSL4 is required for ferroptosis upon GPX4 inhibition but dispensable for p53-mediated ferroptosis. Thus, our study identifies an ALOX12-mediated, ACSL4-independent ferroptosis pathway that is critical for p53-dependent tumour suppression.

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Data availability

The ALOX12 gene expression data for normal versus cancer human tissues were derived from the Oncomine database (https://www.oncomine.org/resource/). The association of ALOX12 with overall survival in patients with pancreatic adenocarcinoma were derived from publicly available clinical information provided by cBioportal for Cancer Genomics databases (http://www.cbioportal.org/). Source data for Figs. 1a,c,f, 2a,b,d,e,g, 3c,d,h, 4b,c,f, 5d–f,h,i, 6b–d,f–h, 7c–e and 8a–g and Supplementary Figs. 1b,d–e, 2b, 3b–d, 5c,d, 6j, 7a–c and 8d have been provided as Supplementary Table 1. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Code availability

No computational code was used in this study.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Acknowledgements

We thank B. Stockwell, R. Baer, X. Jiang, M. Bordyuh and R. Rabadan for critical suggestions in this study. We also appreciate C. D. Funk for critical reagents. This work was supported by the National Cancer Institute of the US National Institutes of Health under Awards 5R01CA216884, 5R01CA190477, 5R01CA085533 and 5RO1CA224272 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.

Author information

B.C. and W.G. conceived the study and experimental design. B.C., N.K., D.C., T. Liu, S.S., T. Li and L.J. conducted the experiments and acquired the data. B.C., O.T. and W.G. analysed and interpreted the data. B.C. and W.G. wrote the manuscript.

Competing interests

The authors declare no competing interests.

Correspondence to Wei Gu.

Integrated supplementary information

Supplementary Figure 1 ALOX12 is critical for p53-mediated ferroptosis.

(a) Schematic diagram of human ALOX family proteins. (b) Q-PCR of ALOX family members from H1299 Tet-on p533KR cells transfected with control siRNA (ctrl) or a pool of ALOX family specific siRNAs as indicated corresponding to Fig. 1a; Error bars are mean ± s.d., n = 3 independent experiments. (c) Western blot analysis of U2OS control or ALOX12 crispr clones #4 and #5. The experiments were repeated twice, independently, with similar results. (d) U2OS control or ALOX12 crispr clone #5, from c, were pre-incubated with Nutlin (10uM) for 12h, then treated with Nutlin (10 uM) and TBH (300 uM) as indicated for 8h. Error bars are mean ± s.d., n = 3 independent experiments. (e) U2OS control or ALOX12 crispr clone #5, from c, were pre-incubated with Nutlin (10 uM) for 12h, then treated with Nutlin (10uM) and TBH (300uM) for indicated times. Error bars are mean ± s.d., n = 3 independent experiments. All P values (b,d,e) were calculated using two-tailed unpaired Student’s t-test. Detailed statistical tests are described in the Methods. Scanned images of unprocessed blots are shown in Supplementary Fig. 9. Raw data are provided in Supplementary Table 1.

Supplementary Figure 2 Regulation of lipid peroxidation levels by p53.

(a) GPX4 activity is measured by glutathione peroxidase assay kit in U2OS cells with or without Nutlin (10uM) treatment. Error bars are mean ± s.d., n = 3 independent experiments. (b) Lipid peroxidation levels in U2OS cells treated with Nutlin (10uM) and TBH (300uM) were assessed by flow cytometry using C11-BODIPY dye (c) Quantification of lipid peroxidation levels (b) is shown. Error bars are mean ± s.d., n = 3 independent experiments. (d) Representative phase-contrast images of H1299 Tet-on p533KR GPX4 crispr cells transfected with GPX4 vectors. Ferr-1 (1uM) was then withdrew from the cells (Scale bars, 100µm). The experiments were repeated three times, independently, with similar results. (e) Lipid peroxidation levels in H1299 Tet-on p533KR GPX4 crispr cells transfected with GPX4 were assessed by flow cytometry using C11-BODIPY dye. The experiments were repeated three times, independently, with similar results. All P values (a,c) were calculated using two-tailed unpaired Student’s t-test. Raw data are provided in Supplementary Table 1.

Supplementary Figure 3 ALOX12 is critical for p53-mediated ferroptosis in H1299 Tet-on p533KR cells.

(a) Western blot analysis of H1299 Tet-on p533KR control or ALOX12 crispr clones #3 and #4. The experiments were repeated twice, independently, with similar results. (b) H1299 Tet-on p533KR control or ALOX12 crispr clones #4, from a, were pre-incubated with doxycycline (0.5ug/ml) for 12h, then treated with doxycycline (0.5 ug/ml), and TBH (60uM) as indicated for 8h. Quantification of cell death is shown. Error bars are mean ± s.d., n = 3 independent experiments. (c) H1299 Tet-on p533KR control or ALOX12 crispr clones #4, from a, were pre-incubated with doxycycline (0.5ug/ml) for 12h, then treated with doxycycline (0.5 ug/ml), and TBH (60uM) for indicated times. Quantification of cell death is shown. Error bars are mean ± s.d., n = 3 independent experiments. (d)Tumor weight (mg) (each group has n = 3 independent samples) was determined shown in Fig. 2f. Error bars are mean ± s.d. (e) Western blot analysis of xenograft tumors from H1299 Tet-on p533KR control and ALOX12 crispr shown in Fig. 2f. The experiments were repeated twice, independently, with similar results. All P values were calculated using two-tailed unpaired Student’s t-test. Scanned images of unprocessed blots are shown in Supplementary Fig. 9. Raw data are provided in Supplementary Table 1.

Supplementary Figure 4 Characterization of Eµ-myc; ALOX12+/- tumors.

(a) Semi-quantitative PCR of full-length p53 of tumor samples from Eµ-myc; ALOX12+/- and Eµ-myc; p53+/- mice, corresponding to Fig. 3d–g; WT, wild-type; LOH, loss of heterozygosity. The experiments were repeated twice, independently, with similar results. (b) PCR from genomic DNA to genotype ALOX12 of tumor samples from Eµ-myc; ALOX12+/− and control samples, corresponding to Fig. 3d–g; WT, wild-type allele; NULL, null allele. The experiments were repeated twice, independently, with similar results. (c) Western blot analysis of the spleen tissues from WT and ALOX12-/- mice with or without irradiation treatment. The experiments were repeated twice, independently, with similar results. (d) Representative immunohistochemistry staining of caspase-3 on spleen from WT and ALOX12-/- mice. Scale bars, 50 µm. The experiments were repeated three times, independently, with similar results. (e) The effects on apoptosis by loss of ALOX12. Representative immunohistochemistry staining of TUNEL on tumors from Eµ-Myc mice vs. Eµ-Myc/ALOX12+/− mice. Scale bars, 20 µm. The experiments were repeated three times, independently, with similar results. Scanned images of unprocessed blots and gels are shown in Supplementary Fig. 9.

Supplementary Figure 5 ALOX12 is downregulated in human cancers.

(a) Analysis of ALOX12 mRNA underexpression (blue) in different cancer types derived from Oncomine. (b) Box plots derived from gene expression data in Oncoming comparing the expression of ALOX12 mRNA in different cancer types. For Cervical normal tissues n = 10 independent samples, tumor tissues n = 21 independent samples; Head and Neck normal tissues n = 13 independent samples, tumor tissues n = 41 independent samples; Esophageal normal tissues n = 17 independent samples, tumor tissues n = 17 independent samples; Acute Myeloid Leukemia, normal tissues n = 8 independent samples, tumor tissues n = 285 independent samples. Box plots with centre line at median, box limits at 25th/75th centiles. (c) Kaplan-Meier survival curves were generated for overall survival (months) by stratifying patient samples with Pancreatic Adenocarcinoma (TCGA, Provisional) from cBioPortal (n = 186) based on ALOX12 expression levels. The patients were quartiled for ALOX12 expression [ALOX12 low (n = 45; black) and ALOX12 high (n = 45; red)], Log-rank Mantel-Cox test was used (p = 0.016). (d) Sequence alignment of Alox12 proteins from different species downloaded from HomoloGene and aligned using Clustal Omega Multiple Sequence Alignment. Cancer-associated residues (aa 372, 381, and 562) bolded and shown in red. (e) Representative phase-contrast images of H1299 Tet-on p533KR cells treated with doxycycline (0.5 ug/ml), TBH (60 uM), Ferr-1 (2 uM), and ML-355 (4 uM) as indicated for 8h (Scale bars, 100 µm). The experiments were repeated three times, independently, with similar results. (f) H1299 Tet-on p533KR cells were pre-incubated with doxycycline (0.5 ug/ml) for 12h, then treated with doxycycline (0.5 ug/ml), TBH (60 uM), Ferr-1 (2 uM), and ML-355 (4 uM) as indicated for 8 h. Quantification of cell death is shown. Error bars are mean ± s.d., n = 3 independent experiments. (g) U2OS ALOX12 crispr cells transfected with control, ALOX12, or mutant ALOX12 G381R vectors were pre-incubated with Nutlin (10 uM) for 12 h, then treated with TBH (300uM) and Nutlin (10uM) as indicated for 8h. Quantification of cell death is shown. Error bars are mean ± s.d., n = 3 independent experiments. All P values were calculated using two-tailed unpaired Student’s t-test. Raw data are provided in Supplementary Table 1.

Supplementary Figure 6 Interaction of SLC7A11 with ALOX family proteins.

(a, b, c, d) Western blot analysis of the interaction between SLC7A11 and ALOX family proteins (a, b, c, d). 293T cells were co-transfected with indicated constructs, and the extract was analysed by Co-IP assays. The western blot experiments were repeated twice, independently, with similar results. (e, f) Western blot analysis of the protein levels of ALOX12, ALOX15 and SLC7A11 (e, f). H1299 cells were transfected with indicated constructs. The western blot experiments were repeated twice, independently, with similar results. (g) Schematic diagram of WT and mutant SLC7A11. (h) Western blot analysis for the interactions of ALOX12 with WT, SLC7A11∆N, SLC7A11∆C. The western blot experiments were repeated twice, independently, with similar results. (i) Western blot analysis of the interaction of ALOX12 with SLC7A11 ∆91-140. The western blot experiments were repeated twice, independently, with similar results. (j) The effects of ALOX12 activity by SLC7A11-wt and SLC7A11∆91-140. Quantification of cell death is shown. Error bars are mean ± s.d., n = 3 independent experiments. P values were calculated using two-tailed unpaired Student’s t-test. Scanned images of unprocessed blots are shown in Supplementary Fig. 9. Raw data are provided in Supplementary Table 1.

Supplementary Figure 7 Loss of ALOX12 has no effect on Erastin- or RSL-3 -induced ferroptosis.

(a) WT, ALOX12+/−, ALOX12−/− MEFs were treated with different concentrations of Erastin (1, 2.5, 5 and 10 uM) for 12 h. Quantification of cell death is shown. Error bars are mean ± s.d., n = 3 independent experiments. (b) U2OS control or ALOX12 crispr cells, were treated with Erastin (20 uM) and Ferr-1 (2 uM) as indicated for 12 h. Quantification of cell death is shown. Error bars are mean ± s.d., n = 3 independent experiments. (c) Representative phase-contrast images of U2OS control or ALOX12 crispr cells treated with RSL-3 (4 uM) and Ferr-1 (2 uM) as indicated for 12h (Scale bars, 100 µm). The experiments were repeated three times, independently, with similar results. (d) U2OS control or ALOX12 crispr cells treated with RSL-3 (4 uM) and Ferr-1 (2 uM as indicated for 12 h. Quantification of cell death is shown. Error bars are mean ± s.d., n = 3 independent experiments. All P values (a,b,d) were calculated using two-tailed unpaired Student’s t-test. Raw data are provided in Supplementary Table 1

Supplementary Figure 8 Further characterization of ALOX12-mediated ferroptosis.

(a) Schematic diagram of the strategy to generate ACSL4 conditional knockout mice. (b) Validation of ACSL4 knockout by southern blot. The experiments were repeated twice, independently, with similar results. (c) Validation of ACSL4 knockout by genotyping. The experiments were repeated twice, independently, with similar results. (d) U2OS cells were pre-incubated with Nutlin (10 uM) for 12 h, then treated with Nutlin (10 uM), Paraquat (1 mM), Ferr-1 (2 uM), and ML-355 (4 uM) as indicated for 8 h. Quantification of cell death is shown. Error bars are mean ± s.d., n = 3 independent experiments . P values were calculated using two-tailed unpaired Student’s t-test. (e) Schematic diagram of human chromosome 17p13.1 (upper panel) and mouse chromosome 11 (lower panel). ALOX12 and p53 locations indicated with distance between the two genes; MB, megabase. Scanned images of unprocessed blots are shown in Supplementary Fig. 9. Raw data are provided in Supplementary Table 1.

Supplementary Figure 9 Unprocessed images of all gels and blots.

Unprocessed images for Fig. 1b,d,g and 2c. Unprocessed images for Fig. 3a,g and 4c-d. Unprocessed images for Fig. 5a-c,f-g and 6a,e. Unprocessed images for Fig. 7a-b,d-e. Unprocessed images for Supplementary Fig. 1c, 3a,e and 4a-b. Unprocessed images for Supplementary Fig. 4c. Unprocessed images for Supplementary Fig. 6a-f. Unprocessed images for Supplementary Fig. 6h-i and 8b-c.

Supplementary information

Supplementary Information

Supplementary Figures 1–9 and Supplementary Table titles and legends.

Reporting Summary

Supplementary Table 1

Statistics source data.

Supplementary Table 2

Somatic Mutations in Cancer (COSMIC) cancer database identified somatic mutations in individuals with cancer mapping to the lipoxygenase domain of ALOX12.

Supplementary Table 3

The information of primary and secondary antibodies.

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Fig. 1: ALOX12 is essential for p53-mediated ferroptosis upon ROS stress.
Fig. 2: ALOX12 is critical for p53-mediated tumour growth suppression.
Fig. 3: Loss of one Alox12 allele is sufficient to accelerate tumorigenesis in Eμ-Myc lymphoma models.
Fig. 4: ALOX12 missense mutations from human cancers abrogate ALOX12 enzymatic activity and p53-mediated ferroptosis.
Fig. 5: Mechanistic insight into p53-mediated activation of ALOX12.
Fig. 6: p53-mediated ferroptosis is ALOX12 dependent but ACSL4 independent.
Fig. 7: Mechanistic insight into p53-mediated ferroptosis.
Fig. 8: ALOX12 in regulating p53-mediated ferroptosis in human cancer lines.
Supplementary Figure 1: ALOX12 is critical for p53-mediated ferroptosis.
Supplementary Figure 2: Regulation of lipid peroxidation levels by p53.
Supplementary Figure 3: ALOX12 is critical for p53-mediated ferroptosis in H1299 Tet-on p533KR cells.
Supplementary Figure 4: Characterization of Eµ-myc; ALOX12+/- tumors.
Supplementary Figure 5: ALOX12 is downregulated in human cancers.
Supplementary Figure 6: Interaction of SLC7A11 with ALOX family proteins.
Supplementary Figure 7: Loss of ALOX12 has no effect on Erastin- or RSL-3 -induced ferroptosis.
Supplementary Figure 8: Further characterization of ALOX12-mediated ferroptosis.
Supplementary Figure 9: Unprocessed images of all gels and blots.