Oxidative stress-induced ZEB1 acetylation drives a hybrid epithelial-mesenchymal phenotype and promotes lung metastasis in triple-negative breast cancer

Abstract

While epithelial-mesenchymal plasticity (EMP) drives cancer metastasis, its regulation by redox dynamics remains poorly understood. Herein, we identified an oxidative stress-responsive CBP/SIRT1 axis that coordinated ZEB1 acetylation at K1108 to promote lung metastasis in triple-negative breast cancer (TNBC). Mechanistically, the biochemical and functional analyses revealed that the dual-acetyltransferase CBP, through stabilization and autoacetylation by oxidative stress, formed a dynamic partnership with SIRT1 to execute precision lysine modification. This post-translational rheostat triggered the functional metamorphosis of ZEB1. During this process, ZEB1 dissociation from the transcriptional corepressor CtBP, while recruiting CBP, converts ZEB1 into a transcriptional activator of epithelial genes. The resulting hybrid epithelial‒mesenchymal phenotype orchestrated dual metastatic competence-maintaining stromal interaction capacity through partial epithelial‒mesenchymal transition (EMT) while establishing NADPH-driven redox supremacy to circumvent ferroptosis. Importantly, this acetyl switch of ZEB1 revealed a metastasis-specific therapeutic vulnerability in TNBC. Our work thus highlighted ZEB1 acetylation as a redox-interpreted mechanism coupling phenotypic plasticity with stress resistance, proposing targeted disruption of this protein post-translational modification (PTM) circuit as a precision strategy against metastatic progression.

Keywords: Acetylation; Hybrid epithelial-mesenchymal phenotype; Lung metastasis; Oxidative stress; TNBC; ZEB1.

Graphical abstract

Fig. 1

Acetylated ZEB1 is upregulated in lung metastases of TNBC. (A, B) Relative mRNA (A) and protein (B) levels of Zeb1 in primary tumours and lung metastases from PyMT mice (n = 6). (C, D) Relative mRNA (C) and protein (D) levels of Zeb1 in primary tumours and lung metastases from the 4T1 orthotopic metastasis model (n = 6). (E–J) Immunoblotting (IB) and immunoprecipitation (IP) assays with anti-Zeb1 and anti-pan-Kac antibodies in 4T1 (E, F). IgG was used as a negative control. WCL, cytosolic, and nuclear fractions from 4T1 were collected for IB analysis with an anti-pan-Kac antibody (G). MDA-MB-231 cells were under the same treatments (H–J). The data are presented as the means ± SDs. Two-tailed Student's paired t-test was used.

Fig. 2

CBP is the major acetyltransferase for ZEB1 acetylation. (A) IB analysis with an anti-pan-Kac antibody in HEK-293T cells co-transfected with V5-ZEB1 and the indicated HA-tagged acetyltransferases. (B, C) Endogenous Co-IP assays of the interaction between ZEB1 and CBP in MDA-MB-231 cells. (D) IB analysis of CBP and ZEB1 in the WCL, cytosolic, and nuclear fractions of MDA-MB-231 cells. (E) Representative IF images of MDA-MB-231 cells stained with anti-ZEB1 and anti-CBP antibodies. Scale bars, 5 μm. (F) IB analysis with an anti-pan-Kac antibody in HEK-293T cells co-transfected with V5-ZEB1 and different doses of HA-CBP. (G) IB analysis of pan-Kac levels in MDA-MB-231 cells transduced with shRNAs against CBP. (H) IB analysis with an anti-pan-Kac antibody in MDA-MB-231 cells by treatment with different concentrations of A485 for 6 h. (I) IB analysis of HEK-293T cells co-transfected with HA-CBP and V5-tagged ZEB1 truncation mutants. The model diagram of the ZEB1 domain is shown in the top panel. (J) MBP pulldown assays in HEK-293T cells transfected with HA-CBP. Purified MBP-ZEB1-truncated fusion proteins were identified via Coomassie blue staining. (K) IB analysis with an anti-pan-Kac antibody in HEK-293T cells co-transfected with V5-tagged ZEB1 truncation mutants in the presence or absence of HA-CBP.

Fig. 3

SIRT1 specifically deacetylates ZEB1. (A, B) IB analysis with an anti-pan-Kac antibody in HEK-293T cells co-transfected with V5-ZEB1 and HA-CBP (A) and MDA-MB-231 cells transfected with HA-CBP for 48 h (B), followed by treatment with 2 μM TSA or 5 mM NAM for 12 h. (C) IP analysis with an anti-pan-Kac antibody in HEK-293T cells transfected with the indicated Flag-tagged SIRT family. (D, E) Co-IP analysis of endogenous ZEB1 and SIRT1 in MDA-MB-231 cells. (F) IB analysis of ZEB1 and SIRT1 in the WCL, cytosolic, and nuclear fractions of MDA-MB-231 cells. (G) Representative IF images of MDA-MB-231 cells stained with anti-ZEB1 and anti-SIRT1 antibodies. Scale bars: 5 μm. (H) IB analysis with an anti-pan-Kac antibody in HEK-293T cells co-transfected with V5-ZEB1, HA-CBP, and different doses of Flag-SIRT1. (I) IB analysis with an anti-pan-Kac antibody in MDA-MB-231 cells with SIRT1 knockdown. (J) IB analysis with an anti-pan-Kac antibody in MDA-MB-231 cells by treatment with different concentrations of EX-527 for 8 h. (K) Co-IP analysis in HEK-293T cells co-transfected with V5-tagged ZEB1 truncation mutants and Flag-SIRT1. (L) MBP pulldown assays in HEK-293T cells transfected with Flag-SIRT1. (M) IB analysis with an anti-pan-Kac antibody in HEK-293T cells co-transfected with V5-CZF and HA-CBP in the presence or absence of Flag-SIRT1.

Fig. 4

K1108 is a critical acetylation site for ZEB1. (A) Identification of the acetylation site for ZEB1. HEK-293T cells were transfected with V5-ZEB1 and HA-CBP for 48 h, followed by treatment with 5 mM NAM for 12 h. (B) The indicated acetylated sites in CZF of ZEB1 were identified via MS (top panel). IB analysis was performed with an anti-pan-Kac antibody in HEK-293T cells co-transfected with V5-CZF mutants and HA-CBP (bottom panel). (C) Highly conserved amino acid of ZEB1^K11108 across species. (D) IB analysis with an Ac-ZEB1 antibody in shZEB1/231^WT and shZEB1/231^K1108R cells. (E) IB analysis with Ac-ZEB1 antibody in HEK-293T cells co-transfected with V5-ZEB1^WT in the presence or absence of HA-CBP. (F) IB analysis with an Ac-ZEB1 antibody in MDA-MB-231 cells transfected with HA-CBP. (G) IB analysis with Ac-ZEB1 antibody in HEK-293T cells co-transfected with V5-ZEB1^WT or V5-ZEB1^K1108R in the presence or absence of HA-CBP. (H) IB analysis with Ac-ZEB1 antibody in HEK-293T cells co-transfected with V5-ZEB1^WT in the presence or absence of Flag-SIRT1. (I) IB analysis with an Ac-ZEB1 antibody in MDA-MB-231 cells transfected with Flag-SIRT1. (J) IB analysis with Ac-ZEB1 antibody in HEK-293T cells co-transfected with V5-ZEB1^WT or V5-ZEB1^K1108R in the presence or absence of Flag-SIRT1.

Fig. 5

Acetylation of ZEB1^K1108 switches the transcriptional regulation mode of ZEB1. (A) IB analysis with epithelial- and mesenchymal-related gene antibodies in shCtrl/231, shZEB1/231, shZEB1/231^WT, shZEB1/231^K1108R, and shZEB1/231^K1108Q cells. (B) Dual luciferase assay for the E-cadherin promoter (−178/+92) in the indicated cells. (C) GSEA enrichment analysis of shZEB1/231^K1108Rvs. shZEB1/231^K1108Q cells. NES, normalized enrichment score; FDR, false discovery rate. (D–G) GSEA enrichment analysis of shZEB1/231, shZEB1/231^K1108R, and shZEB1/231^K1108Q cells. (H) Statistical count of upregulated and downregulated DEGs (P < 0.05, FC > 1). (I, J) Venn diagram (I) and heatmap (J) of DEGs upregulated in shZEB1/231^K1108Q cells and downregulated in shZEB1/231^K1108R cells. (K) Relative mRNA levels of ESRP1, F11, and EMB in the indicated cells. (L) Dual luciferase assays for the ESRP1, F11, and EMB promoters in the indicated cells. (M) Co-IP analysis of ZEB1, CBP, and CtBP in shZEB1/231^K1108R and shZEB1/231^K1108Q cells. (N) Co-IP analysis of Zeb1, Cbp, and Ctbp in primary tumours and lung metastases from PyMT and 4T1 orthotopic metastasis models. (O) Sequential ChIP analysis of the co-occupation of E₂-box elements in the E-Cadherin (−23), ESRP1(-100), F11 (−124), and EMB (−190) promoters by association of ZEB1 with CBP or CtBP in shZEB1/231^K1108R and shZEB1/231^K1108Q cells. According to the two-tailed Student's t-test, the error bars represent the means ± SDs, n = 3.

Fig. 6

Acetylated ZEB1^K1108 allows tumour cells to adapt to oxidative stress. (A) IB analysis with Ac-ZEB1 antibody in MDA-MB-231 cells by treatment with 400 μM H₂O₂ for 3 h. (B) IB analysis with Ac-ZEB1 antibody in MDA-MB-231/DAAO cells by treatment with 10 mM D-Ala for 6 h. (C) IB analysis with Ac-ZEB1 antibody in MDA-MB-231 cells by treatment with 100 μM sodium arsenite for 3 h. (D) IB analysis with Ac-ZEB1 antibody in MDA-MB-231 cells treated with 400 μM H₂O₂ for 3 h, followed by treatment with catalase for 1.5 h. (E) Representative phase contrast images of MDA-MB-231 cells treated with 400 μM H₂O₂ for 3 h. (F, G) Analysis of colony formation (F) and cell viability (G) in shZEB1/231^WT and shZEB1/231^K1108R cells by treatment with 150 μM H₂O_2.(H) GSEA enrichment analysis of transcriptome data from shZEB1/231^K1108Q cells vs. shZEB1/231^K1108R cells. (I) Relative mRNA levels of ME1, ME2, and G6PDH in shZEB1/231^K1108R and shZEB1/231^K1108Q cells. (J) Relative NADPH/NADP⁺ ratio in shZEB1/231^WT, shZEB1/231^K1108R, and shZEB1/231^K1108Q cells by treatment with 400 μM H₂O₂ for 3 h. (K) Relative GSH/GSSG ratio in shZEB1/231^WT, shZEB1/231^K1108R, and shZEB1/231^K1108Q cells by treatment with 400 μM H₂O₂ for 3 h. (L) IB analysis with Ac-ZEB1, ACSL4, and GPX4 in shZEB1/231^WT and shZEB1/231^K1108R cells by treatment with 400 μM H₂O₂ for 3 h. (M, N) Representative BODIPY™ 581/591 C11 staining images of shZEB1/231^WT and shZEB1/231^K1108R cells by treatment with 400 μM H₂O₂ for 3 h. (O) Representative transmission electron microscopy images of mitochondrial ferroptosis in shZEB1/231^WT and shZEB1/231^K1108R cells by treatment with 400 μM H₂O₂ for 3 h. The red arrow indicates mitochondria. According to the two-tailed Student's t-test, the error bars represent the means ± SDs, n = 3.

Fig. 7

ZEB1^K1108 acetylation promotes breast cancer lung metastasis in vivo. (A) Schematic of the 4T1 orthotopic metastasis model (top panel) with shZeb1/4T1^WT and shZeb1/4T1^K1094R cells. The lower panel shows tumours derived from shZeb1/4T1^WT and shZeb1/4T1^K1094R-bearing mice. (B) The approximate volume of tumours from the indicated tumour-bearing mice. (C) Representative images and HE-stained sections of metastatic lung nodules from the indicated tumour-bearing mice. (D–F) The approximate number of metastatic foci (D), the metastatic foci area occupying the total lung area (E), and the lung weight (F) in the indicated tumour-bearing mice. (G) Representative images of IHC staining using Ac-Zeb1 and Zeb1 antibodies in primary tumours and lung metastases. (H) Representative IF images of E-cadherin and Vimentin in primary tumours and lung metastases. Two-tailed Student's t-test was used. The error bars represent the means ± SDs, n = 6.