ATM-mediated stabilization of ZEB1 promotes DNA damage response and radioresistance through CHK1

Abstract

Epithelial–mesenchymal transition (EMT) is associated with characteristics of breast cancer stem cells, including chemoresistance and radioresistance. However, it is unclear whether EMT itself or specific EMT regulators play causal roles in these properties. Here we identify an EMT-inducing transcription factor, zinc finger E-box binding homeobox 1 (ZEB1), as a regulator of radiosensitivity and DNA damage response. Radioresistant subpopulations of breast cancer cells derived from ionizing radiation exhibit hyperactivation of the kinase ATM and upregulation of ZEB1, and the latter promotes tumour cell radioresistance in vitro and in vivo. Mechanistically, ATM phosphorylates and stabilizes ZEB1 in response to DNA damage, ZEB1 in turn directly interacts with USP7 and enhances its ability to deubiquitylate and stabilize CHK1, thereby promoting homologous recombination-dependent DNA repair and resistance to radiation. These findings identify ZEB1 as an ATM substrate linking ATM to CHK1 and the mechanism underlying the association between EMT and radioresistance.

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Figure 1: ZEB1 confers radioresistance on mammary epithelial cells.
Figure 2: ZEB1 is upregulated in radioresistant cancer cells and promotes tumour radioresistance.
Figure 3: ZEB1 regulates DNA damage repair.
Figure 4: CHK1 mediates ZEB1 regulation of radiosensitivity.
Figure 5: ZEB1 interacts with USP7, which deubiquitylates and stabilizes CHK1.
Figure 6: ZEB1 specifically promotes the interaction between USP7 and CHK1.
Figure 7: ATM phosphorylates and stabilizes ZEB1.
Figure 8: ZEB1 correlates with CHK1 protein levels and poor clinical outcome in human breast cancer.

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Acknowledgements

We thank the shRNA and ORFeome Core at MD Anderson Cancer Center and Z. Gong, A. Lin, J. Wang, W. Wang, G. Wan and X. Lu for reagents and technical assistance. We thank A. Postigo, H-L. Piao and J. Kim for discussion. This work is supported by the NIH grants R00CA138572, R01CA166051 and R01CA181029 (to L.M.) and a CPRIT Scholar Award R1004 (to L.M.). L.M. is an R. Lee Clark Fellow of The University of Texas MD Anderson Cancer Center. B.G.D. and W.A.W. are supported by a Komen Foundation Grant KG101478. Y.H. is supported in part by NIH U54CA151668. We wish to dedicate this work to the memory of K. Kian Ang.

Author information

P.Z. and L.M. conceived and designed the project. P.Z. performed and analysed most of the experiments. Y.W. and M-C.H. performed studies on tissue microarrays of human patient samples. L.W. and K.K.A. performed tumour radiosensitivity studies. B.G.D. and W.A.W. established the radioresistant subline. Y.Y. and H.L. performed computational data analysis. J.Z. and D.C. made some constructs. J.Y. and J.C. provided DR-GFP-expressing U2OS cells and performed tandem-affinity purification and mass spectrometry analysis. M.W. maintained mice. Y.S. maintained shRNA and ORF clones. Y.L. and D.C.D. provided Zeb1-deficient MEFs. Y.H. contributed to discussion and revision of the manuscript. P.Z. and L.M. wrote the manuscript with input from all other authors.

Correspondence to Li Ma.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 3 Induction of EMT by Snail, Twist or ZEB1.

(a) Phase contrast images of HMLE cells transduced with Snail, Twist or ZEB1. Scale bar, 50 μm. (b) Images of clonogenic assays of HMLE cells transduced with Snail, Twist or ZEB1. (c) Immunoblotting of ZEB1 and GAPDH in mock-infected HMLE cells or HMLE cells transduced with ZEB1 alone or in combination with transfection of ZEB1 siRNA. C: control (non-irradiated); SF: survival fraction collected 3 weeks after 6-Gy irradiation.

Supplementary Figure 4 ZEB1, SNAI1 and TWIST1 mRNA levels are not substantially increased in SUM159-P2 cells.

(a) qPCR of ZEB1, SNAI1 and TWIST1 in SUM159-P0 and SUM159-P2 cells. n = 3 samples per group. (b) Clonogenic survival assays of U2OS cells transfected with ZEB1 siRNA. n = 3 wells per group. Inset: immunoblotting of ZEB1 and GAPDH. Data in a and b are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance was determined by a two-tailed, unpaired Students t-test. The experiments were repeated 3 times. The source data can be found in Supplementary Table 3. Uncropped images of blots are shown in Supplementary Fig. 7.

Supplementary Figure 5 Effect of ZEB1 on CHK1, radiosensitivity and the G2 checkpoint.

(a) Immunoblotting of ZEB1, γH2AX, H2AX and GAPDH in ZEB1 shRNA-transduced SUM159-P2 cells with or without ectopic expression of an RNAi-resistant ZEB1 mutant (ZEB1-RE), at the indicated time points after 6 Gy IR. (b) Clonogenic survival assays of ZEB1 shRNA-transduced SUM159-P2 cells with or without ectopic expression of an RNAi-resistant mutant (ZEB1-RE). n = 3 wells per group. (c) Percentage of the G2/M population. SUM159-P2 cells were transduced with ZEB1 shRNA, treated with 6-Gy IR and analysed by flow cytometry. n = 3 wells per group. Data in b and c are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance was determined by a two-tailed, unpaired Students t-test. The experiments were repeated 3 times. The source data can be found in Supplementary Table 3. Uncropped images of blots are shown in Supplementary Fig. 7.

Supplementary Figure 6 ZEB1 specifically regulates the protein stability of the USP7 target CHK1.

(a) qPCR of CHK1 in SUM159-P2 cells transduced with ZEB1 shRNA. n = 3 samples per group. (b) Quantification of CHK1 protein levels (normalized to GAPDH) in Fig. 5f. (c) Quantification of CHK1 protein levels (normalized to GAPDH) in Fig. 5g. (d) SUM159-P2 cells were transfected with the scramble control or USP7 siRNA (si-USP7) and then treated with 50 μg ml−1 cycloheximide (CHX). Cells were harvested at different time points as indicated and then immunoblotted with antibodies to ZEB1, USP7 and GAPDH. (e) Quantification of CHK1 protein levels (normalized to GAPDH) in Fig. 5h. (f) SUM159-P2 cells were treated with 50 μg ml−1 cycloheximide (CHX), harvested at different time points as indicated and then immunoblotted with antibodies to Claspin, ZEB1 and GAPDH. (g) 293T cells were transfected with SFB–USP7 alone or in combination with ZEB1, followed by pull-down with streptavidin-sepharose beads (s-s beads) and immunoblotting with the antibody to Claspin. Data in a are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance was determined by a two-tailed, unpaired Students t-test. The experiments were repeated 3 times. The source data can be found in Supplementary Table 3. Uncropped images of blots are shown in Supplementary Fig. 7.

Supplementary Figure 7 ATR does not regulate ZEB1.

(a) 293T cells were transfected with SFB–ZEB1, followed by pull-down with streptavidin-sepharose beads (s-s beads) and immunoblotting with antibodies to ATR and ATM. (b) SUM159-P2 cells were transfected with ATR siRNA and treated with IR. Lysates were immunoblotted with antibodies to p-ATR, ATR, ZEB1 and GAPDH. (c) SUM159-P2 cells were pretreated with ETP-46464 and treated with IR. Lysates were immunoblotted with antibodies to ZEB1, p-CHK1, CHK1, p-CHK2, CHK2 and GAPDH.

Supplementary Figure 8 ATM-dependent phosphorylation of ZEB1 at S585 is critical for radiation-induced stabilization of ZEB1 but not the interaction between ZEB1 and USP7.

(a) 293T cells were transfected with SFB–ZEB1 (wild-type, S585A or S585D), followed by pull-down with streptavidin-sepharose beads (s-s beads) and immunoblotting with the USP7 antibody. (b,c) Quantification of ZEB1 proteins levels (normalized to co-transfected GFP) in Fig. 7i. Uncropped images of blots are shown in Supplementary Fig. 7.

Supplementary Table 1 List of ZEB1-interacting proteins identified by TAP-MS analysis.
Supplementary Table 2 Vectors used in this study.

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Zhang, P., Wei, Y., Wang, L. et al. ATM-mediated stabilization of ZEB1 promotes DNA damage response and radioresistance through CHK1. Nat Cell Biol 16, 864–875 (2014) doi:10.1038/ncb3013

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