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

Zhang P, Wei Y, Wang L, Debeb BG, Yuan Y, Zhang J, Yuan J, Wang M, Chen D, Sun Y, Woodward WA, Liu Y, Dean DC, Liang H, Hu Y, Ang KK, Hung MC, Chen J, Ma L - Nat. Cell Biol. (2014)

Bottom Line: 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.These findings identify ZEB1 as an ATM substrate linking ATM to CHK1 and the mechanism underlying the association between EMT and radioresistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA.

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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ZEB1 interacts with USP7 which deubiquitinates and stabilizes CHK1(a) SUM159-P2 cells transduced with ZEB1 shRNA were treated with 10μM MG132, irradiated with 6 Gy IR and harvested 6 hr later. Lysates wereimmunoprecipitated with the CHK1 antibody and immunoblotted with antibodiesindicated.(b) A partial list of ZEB1-associated proteins.(c, d) 293T cells were transfected with SFB-ZEB1 (c) orSFB-USP7 (d), followed by pull-down with streptavidin-sepharosebeads (s-s beads) and immunoblotting with antibodies indicated.(e) Top: bacterially purified GST-USP7 was incubated with amyloseresin conjugated with bacterially expressed MBP-GFP or MBP-ZEB1. Proteinsretained on the amylose resin were immunoblotted with the USP7 antibody. Bottom:bacterially purified recombinant proteins were analyzed by SDS-PAGE andCoomassie blue staining. * indicates the predicted position.(f) 293T cells were transfected with SFB-USP7 and treated withcycloheximide (CHX). Cells were harvested at different time points andimmunoblotted with antibodies indicated.(g, h) SUM159-P2 cells were transfected with USP7 siRNA (si-USP7,g) or transduced with ZEB1 shRNA (sh-ZEB1, h), andtreated with cycloheximide. Cells were harvested at different time points andimmunoblotted with antibodies indicated.(i) HA-ubiquitin was co-transfected with SFB-GFP or SFB-USP7 into293T cells. Lysates from cells with or without 6 Gy IR treatment wereimmunoprecipitated with the CHK1 antibody and immunoblotted with the HAantibody. Cells were treated with MG132 (10 μM) for 6 hr beforeharvest.(j) Top: ubiquitinated CHK1 was incubated with SFB-GFP control orSFB-USP7 purified with streptavidin-sepharose beads from 293T cells with orwithout ZEB1 co-transfection. The reaction mixture was then immunoprecipitatedwith 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-P2cells. n = 3 wells per group.Data in k are the mean of biological replicates from arepresentative experiment, and error bars indicate s.e.m. Statisticalsignificance was determined by a two-tailed, unpaired Student’st-test. The experiments were repeated 3 times. The sourcedata can be found in Supplementary Table 3. Uncropped images of blots are shown in Supplementary Figure7.
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Figure 5: ZEB1 interacts with USP7 which deubiquitinates and stabilizes CHK1(a) SUM159-P2 cells transduced with ZEB1 shRNA were treated with 10μM MG132, irradiated with 6 Gy IR and harvested 6 hr later. Lysates wereimmunoprecipitated with the CHK1 antibody and immunoblotted with antibodiesindicated.(b) A partial list of ZEB1-associated proteins.(c, d) 293T cells were transfected with SFB-ZEB1 (c) orSFB-USP7 (d), followed by pull-down with streptavidin-sepharosebeads (s-s beads) and immunoblotting with antibodies indicated.(e) Top: bacterially purified GST-USP7 was incubated with amyloseresin conjugated with bacterially expressed MBP-GFP or MBP-ZEB1. Proteinsretained on the amylose resin were immunoblotted with the USP7 antibody. Bottom:bacterially purified recombinant proteins were analyzed by SDS-PAGE andCoomassie blue staining. * indicates the predicted position.(f) 293T cells were transfected with SFB-USP7 and treated withcycloheximide (CHX). Cells were harvested at different time points andimmunoblotted with antibodies indicated.(g, h) SUM159-P2 cells were transfected with USP7 siRNA (si-USP7,g) or transduced with ZEB1 shRNA (sh-ZEB1, h), andtreated with cycloheximide. Cells were harvested at different time points andimmunoblotted with antibodies indicated.(i) HA-ubiquitin was co-transfected with SFB-GFP or SFB-USP7 into293T cells. Lysates from cells with or without 6 Gy IR treatment wereimmunoprecipitated with the CHK1 antibody and immunoblotted with the HAantibody. Cells were treated with MG132 (10 μM) for 6 hr beforeharvest.(j) Top: ubiquitinated CHK1 was incubated with SFB-GFP control orSFB-USP7 purified with streptavidin-sepharose beads from 293T cells with orwithout ZEB1 co-transfection. The reaction mixture was then immunoprecipitatedwith 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-P2cells. n = 3 wells per group.Data in k are the mean of biological replicates from arepresentative experiment, and error bars indicate s.e.m. Statisticalsignificance was determined by a two-tailed, unpaired Student’st-test. The experiments were repeated 3 times. The sourcedata can be found in Supplementary Table 3. Uncropped images of blots are shown in Supplementary Figure7.

Mentions: Because depletion of ZEB1 downregulated CHK1 protein (Fig. 4a) but not CHK1 mRNA (Supplementary Fig. 4a),and because CHK1 is subject to ubiquitin-dependent degradation followingreplication stress33–35, we reasoned that ZEB1 mayregulate CHK1 protein levels through ubiquitin-dependent mechanisms. Indeed,knockdown of ZEB1 significantly induced the poly-ubiquitination of endogenousCHK1 protein with or without IR (Fig.5a).


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

Zhang P, Wei Y, Wang L, Debeb BG, Yuan Y, Zhang J, Yuan J, Wang M, Chen D, Sun Y, Woodward WA, Liu Y, Dean DC, Liang H, Hu Y, Ang KK, Hung MC, Chen J, Ma L - Nat. Cell Biol. (2014)

ZEB1 interacts with USP7 which deubiquitinates and stabilizes CHK1(a) SUM159-P2 cells transduced with ZEB1 shRNA were treated with 10μM MG132, irradiated with 6 Gy IR and harvested 6 hr later. Lysates wereimmunoprecipitated with the CHK1 antibody and immunoblotted with antibodiesindicated.(b) A partial list of ZEB1-associated proteins.(c, d) 293T cells were transfected with SFB-ZEB1 (c) orSFB-USP7 (d), followed by pull-down with streptavidin-sepharosebeads (s-s beads) and immunoblotting with antibodies indicated.(e) Top: bacterially purified GST-USP7 was incubated with amyloseresin conjugated with bacterially expressed MBP-GFP or MBP-ZEB1. Proteinsretained on the amylose resin were immunoblotted with the USP7 antibody. Bottom:bacterially purified recombinant proteins were analyzed by SDS-PAGE andCoomassie blue staining. * indicates the predicted position.(f) 293T cells were transfected with SFB-USP7 and treated withcycloheximide (CHX). Cells were harvested at different time points andimmunoblotted with antibodies indicated.(g, h) SUM159-P2 cells were transfected with USP7 siRNA (si-USP7,g) or transduced with ZEB1 shRNA (sh-ZEB1, h), andtreated with cycloheximide. Cells were harvested at different time points andimmunoblotted with antibodies indicated.(i) HA-ubiquitin was co-transfected with SFB-GFP or SFB-USP7 into293T cells. Lysates from cells with or without 6 Gy IR treatment wereimmunoprecipitated with the CHK1 antibody and immunoblotted with the HAantibody. Cells were treated with MG132 (10 μM) for 6 hr beforeharvest.(j) Top: ubiquitinated CHK1 was incubated with SFB-GFP control orSFB-USP7 purified with streptavidin-sepharose beads from 293T cells with orwithout ZEB1 co-transfection. The reaction mixture was then immunoprecipitatedwith 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-P2cells. n = 3 wells per group.Data in k are the mean of biological replicates from arepresentative experiment, and error bars indicate s.e.m. Statisticalsignificance was determined by a two-tailed, unpaired Student’st-test. The experiments were repeated 3 times. The sourcedata can be found in Supplementary Table 3. Uncropped images of blots are shown in Supplementary Figure7.
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Figure 5: ZEB1 interacts with USP7 which deubiquitinates and stabilizes CHK1(a) SUM159-P2 cells transduced with ZEB1 shRNA were treated with 10μM MG132, irradiated with 6 Gy IR and harvested 6 hr later. Lysates wereimmunoprecipitated with the CHK1 antibody and immunoblotted with antibodiesindicated.(b) A partial list of ZEB1-associated proteins.(c, d) 293T cells were transfected with SFB-ZEB1 (c) orSFB-USP7 (d), followed by pull-down with streptavidin-sepharosebeads (s-s beads) and immunoblotting with antibodies indicated.(e) Top: bacterially purified GST-USP7 was incubated with amyloseresin conjugated with bacterially expressed MBP-GFP or MBP-ZEB1. Proteinsretained on the amylose resin were immunoblotted with the USP7 antibody. Bottom:bacterially purified recombinant proteins were analyzed by SDS-PAGE andCoomassie blue staining. * indicates the predicted position.(f) 293T cells were transfected with SFB-USP7 and treated withcycloheximide (CHX). Cells were harvested at different time points andimmunoblotted with antibodies indicated.(g, h) SUM159-P2 cells were transfected with USP7 siRNA (si-USP7,g) or transduced with ZEB1 shRNA (sh-ZEB1, h), andtreated with cycloheximide. Cells were harvested at different time points andimmunoblotted with antibodies indicated.(i) HA-ubiquitin was co-transfected with SFB-GFP or SFB-USP7 into293T cells. Lysates from cells with or without 6 Gy IR treatment wereimmunoprecipitated with the CHK1 antibody and immunoblotted with the HAantibody. Cells were treated with MG132 (10 μM) for 6 hr beforeharvest.(j) Top: ubiquitinated CHK1 was incubated with SFB-GFP control orSFB-USP7 purified with streptavidin-sepharose beads from 293T cells with orwithout ZEB1 co-transfection. The reaction mixture was then immunoprecipitatedwith 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-P2cells. n = 3 wells per group.Data in k are the mean of biological replicates from arepresentative experiment, and error bars indicate s.e.m. Statisticalsignificance was determined by a two-tailed, unpaired Student’st-test. The experiments were repeated 3 times. The sourcedata can be found in Supplementary Table 3. Uncropped images of blots are shown in Supplementary Figure7.
Mentions: Because depletion of ZEB1 downregulated CHK1 protein (Fig. 4a) but not CHK1 mRNA (Supplementary Fig. 4a),and because CHK1 is subject to ubiquitin-dependent degradation followingreplication stress33–35, we reasoned that ZEB1 mayregulate CHK1 protein levels through ubiquitin-dependent mechanisms. Indeed,knockdown of ZEB1 significantly induced the poly-ubiquitination of endogenousCHK1 protein with or without IR (Fig.5a).

Bottom Line: 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.These findings identify ZEB1 as an ATM substrate linking ATM to CHK1 and the mechanism underlying the association between EMT and radioresistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA.

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.

Show MeSH
Related in: MedlinePlus