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. 2014 Sep;16(9):864-75.
doi: 10.1038/ncb3013. Epub 2014 Aug 3.

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

Peijing Zhang  1 , Yongkun Wei  2 , Li Wang  1 , Bisrat G Debeb  3 , Yuan Yuan  4 , Jinsong Zhang  1 , Jingsong Yuan  1 , Min Wang  1 , Dahu Chen  1 , Yutong Sun  2 , Wendy A Woodward  3 , Yongqing Liu  5 , Douglas C Dean  5 , Han Liang  4 , Ye Hu  6 , K Kian Ang  3 , Mien-Chie Hung  7 , Junjie Chen  8 , Li Ma  8
Affiliations

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

Peijing Zhang et al. Nat Cell Biol. 2014 Sep.

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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Conflict of interest statement

COMPETING FINANCIAL INTEREST

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. ZEB1 confers radioresistance on mammary epithelial cells
(a) Immunoblotting of E-cadherin, Vimentin, Snail, Twist, ZEB1 and GAPDH in HMLE cells transduced with Snail, Twist or ZEB1. (b) Clonogenic survival assays of HMLE cells transduced with Snail, Twist or ZEB1. n = 3 wells per group. (c) Immunoblotting of Snail, Twist, ZEB1, E-cadherin, Vimentin and GAPDH in HMLE cells transduced with Snail, Twist or ZEB1 alone or in combination with the siRNA targeting Snail, Twist or ZEB1. (d) Clonogenic survival assays of HMLE cells transduced with Snail, Twist or ZEB1 alone or in combination with the siRNA targeting Snail, Twist or ZEB1. n = 3 wells per group. (e) Immunoblotting of Snail, Twist, ZEB1, E-cadherin, Vimentin and GAPDH in MCF7 cells transduced with Snail, Twist or ZEB1. (f) Clonogenic survival assays of MCF7 cells transduced with Snail, Twist or ZEB1. n = 3 wells per group. Data in b, d and f 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 Student’s 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 Figure 7.
Figure 2
Figure 2. ZEB1 is upregulated in radioresistant cancer cells and promotes tumor radioresistance
(a) Schematic representation of generation of a radioresistant subline (SUM159-P2) from parental SUM159 cells (SUM159-P0). (b) Clonogenic survival assays of SUM159-P0 and SUM159-P2 cells. n = 3 wells per group. (c) Immunoblotting of γH2AX and HSP90 in SUM159-P0 and SUM159-P2 cells treated with 6 Gy IR. (d) Immunoblotting of Snail, Twist, ZEB1 and GAPDH in SUM159-P0 and SUM159-P2 cells. SUM159-P0 cells transfected with Snail or Twist were used as positive controls. (e) Immunoblotting of Snail, Twist, ZEB1 and GAPDH in SUM159-P0 cells transduced with Snail, Twist or ZEB1. (f) Clonogenic survival assays of SUM159-P0 cells transduced with Snail, Twist or ZEB1. n = 3 wells per group. (g) Clonogenic survival assays of SUM159-P2 cells transduced with ZEB1 shRNA (sh-ZEB1). Inset: immunoblotting of ZEB1 and GAPDH. n = 3 wells per group. (h, i) Tumor size of mice bearing control (scramble) or ZEB1 shRNA-transduced SUM159-P2 xenografts. Tumors were locally irradiated with 15 Gy single dose (h) or 2 Gy fractionated dose (XRT) twice per day for 7 consecutive days (i). n = 5 mice per group. General linear model multivariate analysis was performed to determine statistical significance. (j) Immunoblotting of ZEB1 and HSP90 in tumor lysates. Data in b, f, g, h and i are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance in b, f and g was determined by a two-tailed, unpaired Student’s 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 Figure 7.
Figure 3
Figure 3. ZEB1 regulates DNA damage repair
(a) γH2AX and DAPI staining of SUM159-P2 cells transduced with ZEB1 shRNA, 24 hr after 6 Gy IR. Scale bar: 10 μm. (b) Immunoblotting of ZEB1, γH2AX, H2AX and GAPDH in SUM159-P2 cells transduced with ZEB1 shRNA, at the indicated time points after 6 Gy IR. (c, d) Images (c) and data quantification (d) of comet assays of SUM159-P2 cells transduced with ZEB1 shRNA, at the indicated time points after 6 Gy IR. n = 62 cells per group. Scale bar in (c): 50 μm. (e) Immunoblotting of ZEB1 and GAPDH in U2OS_DR-GFP cells transfected with ZEB1 siRNA alone or in combination with ZEB1. (f) HR repair assays of U2OS_DR-GFP cells transfected with ZEB1 siRNA alone or in combination with ZEB1. n = 3 wells per group. Data in d and f 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 Student’s 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 Figure 7.
Figure 4
Figure 4. CHK1 mediates ZEB1 regulation of radiosensitivity
(a) Immunoblotting of p-CHK1, CHK1, p-CHK2, CHK2 and GAPDH in SUM159-P2 cells transduced with ZEB1 shRNA, at the indicated time points after 6 Gy IR. (b) Immunoblotting of ZEB1, CHK1, γH2AX, H2AX and GAPDH in MCF7 cells transduced with ZEB1, at the indicated time points after 6 Gy IR. (c) Immunoblotting of Zeb1, Chk1 and Gapdh in Zeb1+/+, Zeb1+/− and Zeb1−/− MEFs. (d) Immunoblotting of ZEB1, CHK1, Cyclin A, p-H3 (S10) and GAPDH in SUM159-P2 cells transfected with ZEB1 siRNA or the scramble control. Cells were arrested overnight with 0.5 μg/ml nocodazole. Mitotic cells were “shaken off” and then released into normal medium. Samples were collected at the indicated time points and analyzed by western blotting. Cell cycle distribution was gauged by Cyclin A and p-H3 (S10). (e) Clonogenic survival assays of SUM159-P2 cells transfected with CHK1 siRNA. Inset: immunoblotting of CHK1 and GAPDH. n = 3 wells per group. (f) Immunoblotting of CHK1 and GAPDH in ZEB1 shRNA-transduced SUM159-P2 cells with or without ectopic expression of CHK1. (g) Clonogenic survival assays of ZEB1 shRNA-transduced SUM159-P2 cells with or without ectopic expression of CHK1. n = 3 wells per group. (h) Immunoblotting of ZEB1, CHK1 and GAPDH in SUM159-P0 cells transfected with ZEB1 alone or in combination with CHK1 siRNA. (i) Clonogenic survival assays of SUM159-P0 cells transfected with ZEB1 alone or in combination with CHK1 siRNA. n = 3 wells per group. Data in e, g and i 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 Student’s 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 Figure 7.
Figure 5
Figure 5. ZEB1 interacts with USP7 which deubiquitinates and stabilizes CHK1
(a) SUM159-P2 cells transduced with ZEB1 shRNA were treated with 10 μM MG132, irradiated with 6 Gy IR and harvested 6 hr later. Lysates were immunoprecipitated with the CHK1 antibody and immunoblotted with antibodies indicated. (b) A partial list of ZEB1-associated proteins. (c, d) 293T cells were transfected with SFB-ZEB1 (c) or SFB-USP7 (d), followed by pull-down with streptavidin-sepharose beads (s-s beads) and immunoblotting with antibodies indicated. (e) Top: bacterially purified GST-USP7 was incubated with amylose resin conjugated with bacterially expressed MBP-GFP or MBP-ZEB1. Proteins retained on the amylose resin were immunoblotted with the USP7 antibody. Bottom: bacterially purified recombinant proteins were analyzed by SDS-PAGE and Coomassie blue staining. * indicates the predicted position. (f) 293T cells were transfected with SFB-USP7 and treated with cycloheximide (CHX). Cells were harvested at different time points and immunoblotted with antibodies indicated. (g, h) SUM159-P2 cells were transfected with USP7 siRNA (si-USP7, g) or transduced with ZEB1 shRNA (sh-ZEB1, h), and treated with cycloheximide. Cells were harvested at different time points and immunoblotted with antibodies indicated. (i) HA-ubiquitin was co-transfected with SFB-GFP or SFB-USP7 into 293T cells. Lysates from cells with or without 6 Gy IR treatment were immunoprecipitated with the CHK1 antibody and immunoblotted with the HA antibody. Cells were treated with MG132 (10 μM) for 6 hr before harvest. (j) Top: ubiquitinated CHK1 was incubated with SFB-GFP control or SFB-USP7 purified with streptavidin-sepharose beads from 293T cells with or without ZEB1 co-transfection. The reaction mixture was then immunoprecipitated with the FLAG antibody and immunoblotted with the CHK1 antibody. Bottom: purified SFB-USP7 was immunoblotted with antibodies to ZEB1 and USP7. (k) Clonogenic survival assays of USP7 siRNA-transfected SUM159-P2 cells. n = 3 wells per group. Data in k 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 Student’s 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 Figure 7.
Figure 6
Figure 6. ZEB1 specifically promotes the interaction between USP7 and CHK1
(a) 293T cells were transfected with SFB-USP7 alone or in combination with ZEB1, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to CHK1, HLTF, p53 and USP7. (b) 293T cells were transfected with ZEB1 siRNA alone or in combination with SFB-USP7, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to CHK1, HLTF, p53 and USP7. (c) SUM159-P2 cells were transfected with ZEB1 siRNA, followed by immunoprecipitation with the USP7 antibody and immunoblotting with antibodies to CHK1 and USP7. Uncropped images of blots are shown in Supplementary Figure 7.
Figure 7
Figure 7. ATM phosphorylates and stabilizes ZEB1
(a) 293T cells were transfected with SFB-ZEB1 and treated with IR, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to ATM and FLAG. (b) SUM159-P2 cells were transduced with ATM shRNA and treated with IR. Lysates were immunoblotted with antibodies to p-ATM, ATM, ZEB1, CHK1 and GAPDH. (c) SUM159-P2 cells with or without Ku55933 pretreatment (10 μM, 1 hr) were treated with IR (6 Gy) and CHX (50 μg/ml), harvested at different time points, immunoprecipitated with the ZEB1 antibody and immunoblotted with antibodies to p-S/TQ and ZEB1. (d) 293T cells were transfected with SFB-ZEB1 and treated with IR, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to p-S/TQ and ZEB1. (e) Endogenous ZEB1 was immunoprecipitated from SUM159-P0 and SUM159-P2 cells and immunoblotted with antibodies to p-S/TQ and ZEB1. (f) Consensus ATM phosphorylation site on human ZEB1 (S585) and alignment with the conserved site on mouse, rat and Xenopus Zeb1. (g) 293T cells were transfected with wild-type, the S585A or S585D mutant of SFB-ZEB1 and treated with IR, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to p-S/TQ and ZEB1. (h) Immunopurified wild-type ZEB1 or the S585A mutant was incubated with immunopurified wild-type ATM or the kinase-dead (KD) mutant in kinase buffer containing 32P-ATP. After reaction, proteins were resolved by SDS-PAGE and subjected to autoradiography and immunoblotting with antibodies to ZEB1 and p-ATM. Purified GST-p53 was used as a positive control for ATM kinase activity. (i) HeLa cells were co-transfected with SFB-GFP and wild-type, the S585A or S585D mutant of SFB-ZEB1, treated with CHX with or without IR, harvested at different time points and immunoblotted with antibodies to FLAG. SFB-GFP serves as the control for transfection. (j) Clonogenic survival assays of SUM159-P0 cells transfected with wild-type ZEB1 or the mutant. n = 3 wells per group. Data in j 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 Student’s 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 Figure 7.
Figure 8
Figure 8. ZEB1 correlates with CHK1 protein levels and poor clinical outcome in human breast cancer
(a) Immunohistochemical staining of ZEB1 and CHK1 in representative carcinoma specimens on the NCI breast cancer progression tissue microarrays. Brown staining indicates positive immunoreactivity. Scale bar: 50 μm. (b) Correlation between ZEB1 and CHK1 protein levels in human breast tumors. Statistical significance was determined by a χ2 test. R is the correlation coefficient. (c) Kaplan-Meier curves showing the distant relapse-free survival of patients with high or low expression of ZEB1 in their breast tumors. Statistical significance was determined by a log-rank test. (d) The working model of regulation of radiosensitivity and DDR by ZEB1.
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