Abstract
Although the gain of function (GOF) of p53 mutants is well recognized, it remains unclear whether different p53 mutants share the same cofactors to induce GOFs. In a proteomic screen, we identified BACH1 as a cellular factor that recognizes the p53 DNA-binding ___domain depending on its mutation status. BACH1 strongly interacts with p53R175H but fails to effectively bind wild-type p53 or other hotspot mutants in vivo for functional regulation. Notably, p53R175H acts as a repressor for ferroptosis by abrogating BACH1-mediated downregulation of SLC7A11 to enhance tumor growth; conversely, p53R175H promotes BACH1-dependent tumor metastasis by upregulating expression of pro-metastatic targets. Mechanistically, p53R175H-mediated bidirectional regulation of BACH1 function is dependent on its ability to recruit the histone demethylase LSD2 to target promoters and differentially modulate transcription. These data demonstrate that BACH1 acts as a unique partner for p53R175H in executing its specific GOFs and suggest that different p53 mutants induce their GOFs through distinct mechanisms.
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Data availability
RNA-seq data from Cal-33 cells have been deposited to the Gene Expression Omnibus under accession code GSE224730. An analyzed result for this RNA-sequencing is available in Supplementary Table 1. Proteomics data have been deposited in ProteomeXchange via the PRIDE database, with a relevant accession number PXD039886. The human pan-cancer data were derived from the TCGA Research Network: http://cancergenome.nih.gov/ and cBioPortal: https://www.cbioportal.org/. The dataset derived from this resource that supports the findings of this study is available in Source Data Extended Data Fig. 10. Source Data for Figs. 1–8 and Extended Data Figs. 1–10 have been provided as Source Data files. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.
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Acknowledgements
We thank C. Prives and R. Baer for helpful suggestions. We also thank Y. Geno Shi (Harvard Medical School) and K. Igarashi (Tohoku University Graduate School of Medicine) for kind support in this project by providing critical reagents. This work was supported by the National Cancer Institute of the National Institutes of Health under awards R35CA253059, RO1CA258390 and RO1CA254970 to W.G., R35-GM139585 to B.H., R01CA257548 to J.M. and R01CA215607 to K.O. We acknowledge the support from the Herbert Irving Comprehensive Cancer Center (P30 CA13696) and thank staff at the Molecular Pathology and Proteomics of Shared Resources. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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Conception and experimental design was the responsibility of Z.S. and W.G. Methodology and data acquisition was the responsibility of Z.S., N.K., J.Y., Z.L., H.L. and Y.L. Analysis and interpretation of data was carried out by Z.S., N.K., H.Z., W.Z., Q.T., H.K., K.O., S.D., Z. Z., B.H., A.R., J. M. and W.G. Manuscript writing was the responsibility of Z.S. and W.G.
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Extended data
Extended Data Fig. 1 Identification of BACH1 as a specific binding partner of p53R175H.
a, Diagram of the construct of SFB-tagged wild-type p53 DBD + TD domains. DBD, DNA binding ___domain; TD, tetramerization ___domain; SFB tag: S-protein, Flag and streptavidin binding peptide (SBP). b, Silver staining of SDS–PAGE gel loaded with SFB-p53 DBD + TD protein complex purified from H1299 cell line stably overexpressing SFB-p53 DBD + TD protein by double IP (SBP IP + S-protein IP). c, BACH1 peptides sequences identified from Mass-Spec of p53 DBD + TD protein complex. d, Co-IP of SFB-tagged p53 DBD (WT, R175H, R248W, R273H, G245S, R249S and R282W) with Flag-HA (FH)-BACH1. H1299 p53 null cells were co-transfected with SFB-tagged p53 DBD and FH-BACH1 and 24 h later, cells were lysed with NP40 lysis buffer and immunoprecipitated with SBP beads, followed by Western blot analysis of BACH1 and Flag (SFB-p53). e, p53R175H-BACH1 endogenous Co-IP by BACH1 specific antibody in Cal-33 cells. f, Endogenous co-IP of p53 and BACH1 in mutant p53 cell lines MDA-MB-231 (p53R280K). g, Co-IP of SFB-tagged p53R175H truncated domains (100-300-R175H, 1-160 and 98-180-R175H) with Flag-HA (FH)-BACH1 in H1299 cells. h, Co-IP of SFB-tagged BACH1 with p53R175H and its deletion mutant (∆161-180 aa) in H1299 cells. SFB, S-protein-Flag-Streptavidin binding peptide; SBP, streptavidin binding peptide. Gel staining (b) and Western blot experiments (d-h) were repeated three times with similar results and representative images are shown.
Extended Data Fig. 2 p53R175H suppresses BACH1-mediated transcriptional regulation of SLC7A11 and ferroptosis.
a, SLC7A11 promoter sequences containing the BACH1 consensus binding motif (WT or mutated; -118 to -127bp). Wild-type or mutant SLC7A11 promoters were cloned into the pGL3 luciferase reporter vector. b, Dual-luciferase reporter assay for SLC7A11 transcription in H1299 cells co-transfected with control, p53-R175H, BACH1, p53-R175H + BACH1, or WT p53 plasmid, along with SLC7A11 luciferase reporter plasmid (containing mutant motif) and Renilla plasmid. n = 3 technical cell culture replicates. The experiment was repeated three times with similar results. c, Western blot analysis of SLC7A11 expression in HCC1937 cells (p53R306-stop) treated with NC siRNA, p53 siRNA, BACH1 siRNA, or p53 siRNA+BACH1 siRNA for 48 h. The experiment was repeated twice with similar results. d-f, qPCR analysis of SLC7A11 mRNA expression in HuCCT1 cells (d), KLE cells (e) and Cal-33 cells (f) treated with NC siRNA, p53 siRNA, BACH1 siRNA, or p53 siRNA+BACH1 siRNA for 48-72 h. HuCCT1 cells, 48 h; KLE cells, 56 h; Cal-33 cells, 72 h. n = 3 technical replicates. The experiments were repeated twice with similar results. g-j, qPCR analysis of BACH1 targets in Cal-33 cells treated with NC siRNA, p53 siRNA, BACH1 siRNA, or p53 siRNA+BACH1 siRNA for 72 h. HMOX1 (g); GCLM (h); FTH1 (i); FTL (j). n = 3 technical replicates. The experiment was repeated twice with similar results. k, Cell death assay of Cal-33 NC, p53, BACH1, or p53 + BACH1 knockdown cells treated with 1 mM TBH for 8 h in presence or absence of 5 μM ferr-1. n = 3 technical cell culture replicates. The experiment was repeated three times with similar results. l, Western blot analysis of SLC7A11, p53 and BACH1 expression in mouse sarcoma cells. p53R172H/R172H sarcoma cell were from spontaneously-developed sarcoma tumors derived from p53R172H/R172H mice and p53R172H-/- and p53R172H/R172HBACH1-/- cells were made from these p53R172H/R172H cells by CRSIPR method. The experiment was repeated twice with similar results. m, Cell death assay for p53R172H/R172H, p53R172H-/- and p53R172H/R172HBACH1-/- sarcoma cells treated with 0.5 μM erastin for 18 h in the presence or absence of 5 μM ferr-1. n = 3 technical cell culture replicates. The experiment was repeated three times with similar results. Data represent mean of three technical replicates.
Extended Data Fig. 3 Immunohistochemical (IHC) staining and Western blot analysis of Cal-33 xenograft tumors.
a-d, IHC staining for 4-HNE (a), SLC7A11 (b), Ki67 (c) and TUNEL (d). Bar scale=20 μm. e-h, Quantification for 4-HNE (e), SLC7A11 (f), Ki67 (g) and TUNEL (h) staining. n = 5 fields from 5 tumors (each group). P values in e (from left to right): 0.0018; 0.0007. P values in f (from left to right): 0.0003; 0.0001. P values in g (from left to right): 0.5447; 0.5447. P values in h (from left to right): 0.9505; 0.7634. i, Western blot analysis of GPX4 expression. n = 3 tumor samples for each genotype. 4-HNE, SLC7A11 and Ki67 IHC staining was quantified by using the immunoreactive score (IRS) system and TUNEL staining were quantified with percentage of positive cells (see Methods for details). Two-tailed Student’s t-test were used for statistical analysis. ns, not significant, **p < 0.01; ***p < 0.001. Data represent mean + S.E.M.
Extended Data Fig. 4 The role of SLC7A11 in p53R175H-mediated inhibition of ferroptosis and promotion of tumor growth.
a, Identification of SLC7A11-/- Cal-33 cells by Western blot analysis. b, Images of tumors isolated from nude mice implanted with Cal-33 Ctrl and SLC7A11-/- cells. n = 8 tumors. c, Tumor weights for Cal-33 Ctrl and SLC7A11-/- xenografts. n = 8 tumors. P value: <0.0001. d, Representative images of 4-HNE staining of Cal-33 Ctrl and SLC7A11-/- xenograft tumors. Bar scale=20 μm. e, Quantification of 4-HNE staining using the immunoreactive score (IRS) system (n = 6 fields from 6 tumors). P value: 0.0004. f, Identification of p53R175H-/- SLC7A11 overexpressing Cal-33 cells by Western blot analysis. p53R175H-/- SLC7A11 overexpressing Cal-33 cells were made by transducing plenti6-SLC7A11/xCT-V5 lentivirus into p53R175H-/- cells. g, Images of tumors isolated from nude mice implanted with Cal-33 p53R175H-/- and p53R175H-/- +SLC7A11 cells. n = 8 tumors. h, Tumor weights for Cal-33 p53R175H-/- and p53R175H-/- +SLC7A11 xenografts. n = 8 tumors. P value: 0.0008. i, Representative images of 4-HNE staining of Cal-33 p53R175H-/- and p53R175H-/- +SLC7A11 xenograft tumors. Bar scale=20 μm. j, Quantification of 4-HNE staining using the IRS system (n = 6 fields from 6 tumors). P value: <0.0001. k, Representative images of ferroptotic cell death in Cal-33 Ctrl, SLC7A11-/-, p53R175H-/- and p53R175H-/-+ SLC7A11 cells treated with 10 μM erastin for 72 h. Red arrows indicate cells undergoing ferroptotic cell death. Bar scale=50 μm. The images shown are representative of 3 repeats with similar results. l, Cell death assay for Cal-33 Ctrl, SLC7A11-/-, p53R175H-/- and p53R175H-/- + SLC7A11 cells treated with 10 μM erastin for 72 h in presence or absence of 5 μM ferr-1. n = 3 technical cell culture replicates. The experiment was repeated three times with similar results. m, Cell death assay for Cal-33 Ctrl, SLC7A11-/-, p53R175H-/- and p53R175H-/-+SLC7A11 cells treated with 0.85 mM TBH for 7 h in presence or absence of 5 μM ferr-1. n = 3 technical cell culture replicates. The experiment was repeated three times with similar results. Two-tailed Student’s t-test were used for statistical analysis. ***p < 0.001; Data (l, m) represent mean of three technical replicates. Data (c, e, h and j) represent mean + S.E.M.
Extended Data Fig. 5 BACH1/p53R175H–mediated regulation of CEMIP expression is critical for lung metastasis.
a, Diagram of lung metastasis models by tail vein injection in nude mice. Cal-33 cells or other cancer cells were transduced with Tomato fluorescence gene lentivirus (system 1) or luciferase-Tomato lentivirus (system 2) and sorted by FACS and then 0.5~4 million cells (dependent on cell type) were tail vein injected into each mouse. For system 1, 6~8 weeks later, mice were sacrificed and lungs were isolated for imaging using KEYENCE BZ-X800 fluorescence microscope. For system 2, 3~5 weeks later, mice were i.p. injected with luciferase substrate D-luciferin and imaged on IVIS Spectrum Optical Imaging System. b, Representative fluorescence images of liver and kidney isolated from mice tail vein injected with Cal-33 Ctrl cells, related to main Fig. 5a. Scale bar, 500μm. c, Quantitative analysis of total metastatic area (μm2) in lungs, related to main Fig. 5a. n = 7 mice for each group. P values (from left to right): 0.0035; 0.0062; 0.0048; 0.9274; 0.6024. d, H&E staining of lung tissues infiltrated with Cal-33 cells, related to main Fig. 5a. Red arrows indicate the metastatic nodes; panel d is a representative image of 5 lung sections from 5 mice (each group). Bar scale=20 μm. e, Immunohistochemical staining of Ki67 of lung tissues infiltrated with Cal-33 cells, related to main Fig. 5a. Red arrows indicate the Ki67 positive cell clusters; panel e is a representative image of 5 lung sections from 5 mice (each group). Bar scale=20 μm. f, Identification of CEMIP-/- Cal-33 cells made by CRISPR method. g, In vivo luminescence imaging of mice 4 weeks after tail vein injection of 0.5 million of luciferase carrying Cal-33 Ctrl or CEMIP-/- cells. Mice were imaged on IVIS System. n = 5 mice for each group. h, i, Quantitative analysis of total counts of luminescence (h) and average counts of luminescence (i) in lungs, related to panel (g). n = 5 mice for each group. P value (h): 0.0027. P value (i): 0.0028. Two-tailed Student’s t-test were used for statistical analysis. ns, not significant, **p < 0.01; Data represent mean + S.E.M.
Extended Data Fig. 6 The role of SLC7A11 and ACSL4 in lung metastasis of Cal-33 cells.
a, In vivo luminescence imaging of mice 4 weeks after tail vein injection of 0.5 million of luciferase carrying Cal-33 cells. SLC7A11-/- Cal-33 cells were made by CRISPR method and SLC7A11 overexpressing cells were made by transduction of SLC7A11 lentivirus into Cal-33 p53R175H-/- cells. Mice were i.p. injected with luciferase substrate D-luciferin and imaged on IVIS Spectrum Optical Imaging System. b, Western blot analysis of SLC7A11 and p53 expression in the above cell lines. b was repeated three times with similar results and a representative result is shown. c, Quantitative analysis of total counts of luminescence in lungs for each group, related to panel (a). n = 7 mice for each group. P values (from left to right): 0.9398; 0.00097; 0.5446. d, In vivo luminescence imaging of mice 3 weeks after tail vein injection of 0.5 million of luciferase carrying control or ACSL4-/- Cal-33 cells. ACSL4-/- Cal-33 cells were made by CRISPR method. Mice were i.p. injected with luciferase substrate D-luciferin and imaged on IVIS Spectrum Optical Imaging System. e, Western blot analysis of ACSL4 expression in the above cell lines. e was repeated three times with similar results and a representative result is shown. f, Quantitative analysis of total counts of luminescence in lungs for each group, related to panel (d). n = 6 mice for each group. P value: 0.9049. Two-tailed Student’s t-test were used for statistical analysis. ns, not significant, ***p < 0.001; Data represent mean + S.E.M.
Extended Data Fig. 7 p53R175H regulates BACH1 targets through recruiting LSD2.
a, ChIP analysis of the recruitment of p53R175H to SLC7A11 promoter (-128 to -114) in A549 p53-/- cells transfected with p53R175H alone or p53R175H + BACH1 plasmids. n = 3 technical replicates. The experiment was repeated twice with similar results. b, Coomassie blue staining of SDS–PAGE gel loaded with SFB-p53R175H protein complex purified from H1299 cells stably expressing SFB-p53R175H by double IP (SBP IP + S-protein IP). The experiment was repeated three times with similar results. c, LSD2/KDM1B peptides sequences identified from Mass-Spec of SFB-p53R175H complex. d, Co-IP of SFB-tagged p53R175H with Flag-HA (FH)-tagged BACH1 or its deletion mutant ∆466-515 in H1299 cells. The experiment was repeated three times with similar results. e, ChIP analysis with LSD2 antibody in Cal-33 p53R175H-/-BACH1-/- cells. Cal-33 p53R175H-/-BACH1-/- cells were transfected with LSD2, LSD2 + BACH1, LSD2 + BACH1∆466-515, LSD2 + BACH1 + p53R175H, or LSD2 + BACH1∆466-515 + p53R175H plasmids and then cells lysates were ChIPed with LSD2 antibody, followed by qPCR analysis of the recruitment of LSD2 to SLC7A11 promoter. n = 3 technical replicates. The experiment was repeated twice with similar results. f, Western blot analysis of SLC7A11 expression in TOV-112D cells transfected with NC, p53, or LSD2 siRNAs for 48 h. The experiment was repeated twice with similar results. g, ChIP analysis of the recruitment of LSD2 to CEMIP promoter (-2036 to -2022) in Cal-33 native cells treated with NC, BACH1, or p53 siRNA for 48 h. n = 3 technical replicates. The experiment was repeated twice with similar results. h, ChIP analysis with LSD2 antibody in Cal-33 p53R175H-/-BACH1-/- cells. Cal-33 p53R175H-/-BACH1-/- cells were transfected with LSD2, LSD2 + BACH1, LSD2 + BACH1∆466-515, LSD2 + BACH1 + p53R175H, or LSD2 + BACH1∆466-515 + p53R175H plasmids and then cells lysates were ChIPed with LSD2 antibody, followed by qPCR analysis of the recruitment of LSD2 to CEMIP promoter. n = 3 technical replicates. The experiment was repeated twice with similar results. Data represent mean of three technical replicates.
Extended Data Fig. 8 Sequential ChIP analysis of p53R175H-BACH1-LSD2 complex and diagram for differential regulation of repression targets and activation targets of BACH1 by p53R175H and LSD2.
a, Diagram for sequential ChIP analysis. Sequential ChIP analysis was followed the established protocol by Beischlag et al 2018 (Ref. 52). Co-enrichment of BACH1/p53R175H/LSD2 in gene promoter was analyzed by qPCR. b-e, Single ChIP assay (by BACH1 specific antibody) and sequential ChIP assay (firstly ChIP-ed by BACH1 antibody, secondly by p53 DO-1 antibody and thirdly by LSD2 antibody) for SLC7A11 promoter (b), CEMIP promoter (c), GCLM promoter (d) and FTH1 promoter (e) using Cal-33 cells. n = 3 technical replicates. The experiment was repeated twice with similar results. f. Diagram for differential regulation of repression targets (for example, SLC7A11) and activation targets (for example, CEMIP) of BACH1 by p53R175H and LSD2. BACH1 has opposite functions in transcription on two different types of target genes: on one hand, it acts as a transcriptional repressor to downregulate a number of targets such as SLC7A11 critically involved in ferroptosis; on the other hand, BACH1 can also function as a transcriptional activator to induce pro-metastatic targets such as CEMIP to promote cancer metastasis. The interaction between LSD2 and BACH1 is very weak and unstable when p53R175H is absent, but this interaction is significantly enhanced in the presence of p53R175H expression since p53R175H can strongly interact with both BACH1 and LSD2. p53R175H is able to abrogate BACH1-mediated repression of SLC7A11 through the recruitment of LSD2 demethylase. This formation of p53R175H-BACH1-LSD2 complex modifies the histone methylation status at the promoter of SLC7A11 and subsequently abrogates its transcriptional repression mediated by BACH1. Conversely, the recruitment of LSD2 demethylase by p53R175H to the promoter of CEMIP results in enhanced transcriptional activation of CEMIP by BACH1. Data represent mean of three technical replicates.
Extended Data Fig. 9 BACH1 suppresses tumor growth in p53 wild-type cell line HCT116-derived xenograft tumors.
HCT116 BACH1 inducible cells were made from HCT116 BACH1-/- cells by transfecting BACH1 inducible plasmid. HCT116 inducible cells were pre-incubated with or without doxycycline (Dox) for 48 h in vitro and subcutaneously injected into BALB/c nude mice and mice were fed with or without doxycycline diet. a, Western blot analysis of SLC7A11, BACH1 and p53 expression in HCT116 BACH1 inducible cells in the presence or absence of doxycycline. The experiment was repeated three times with similar results. b, Tumors isolated from nude mice implanted with HCT116 BACH1 inducible cells fed with or without doxycycline diet. Mice were sacrificed at day 20. n = 10 tumors. c, Tumor weights. n = 10 tumors. P value: <0.0001. d, Representative images of 4-HNE staining of HCT116 xenograft tumors. Bar scale=20μm. e, Quantification of 4-HNE staining using the immunoreactive score (IRS) system. n = 6 fields from 6 tumors. P value: <0.0001. P values were calculated by two-tailed Student’s t-test. ***p < 0.001; Data represent mean + S.E.M.
Extended Data Fig. 10 Physiological relevance of p53 R175H mutation in cancer patients.
a, b. Violin plots of SLC7A11/ CEMIP expression in cancer patients with distinct p53 mutation. Analyses are based on data from cBioPortal Pan-cancer studies and specific cancer types studies (271 pan-cancer and specific cancer studies were included). a, SLC7A11; b, CEMIP. P values were calculated by two-tailed Student’s t-test. n numbers are shown on the panels. P values in a (from left to right): 0.000039; 0.8038; 0.6746; 0.9791. P values in b (from left to right): 0.0092; 0.7587; 0.916; 0.7466. c-i, Correlation between BACH1 expression level and patients’ overall survival in different p53 mutation status. c, p53R175H; d, p53R248Q/W; e, p53R273H; f, p53G245S; g, p53R249S; h, p53R282W; i, p53 wild-type. Distinct p53 mutation patients’ BACH1 expression and survival data were obtained from TCGA pan-cancer database (xenabrower.net). P values of survival curves were calculated by Log-rank (Mantel-Cox) test. n numbers are shown on the panels. P values: 0.0233 (c); 0.4013 (d); 0.9842 (e); 0.6665 (f); 0.6148 (g); 0.8513 (h); 0.0195 (i).
Supplementary information
Supplementary Information
Supplementary Fig.1
Supplementary Table 1
Table 1. Cal-33 knockdown cells RNA-seq TPM values. Table 2. Antibodies used in this study. Table 3. Oligonucleotides used in this study.
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Statistical Source Data for Figs. 1 and 3–8 and Extended Data Figs. 2–10.
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Su, Z., Kon, N., Yi, J. et al. Specific regulation of BACH1 by the hotspot mutant p53R175H reveals a distinct gain-of-function mechanism. Nat Cancer 4, 564–581 (2023). https://doi.org/10.1038/s43018-023-00532-z
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DOI: https://doi.org/10.1038/s43018-023-00532-z
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