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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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CHK1 mediates ZEB1 regulation of radiosensitivity(a) Immunoblotting of p-CHK1, CHK1, p-CHK2, CHK2 and GAPDH inSUM159-P2 cells transduced with ZEB1 shRNA, at the indicated time points after 6Gy IR.(b) Immunoblotting of ZEB1, CHK1, γH2AX, H2AX and GAPDH inMCF7 cells transduced with ZEB1, at the indicated time points after 6 Gy IR.(c) Immunoblotting of Zeb1, Chk1 and Gapdh inZeb1+/+,Zeb1+/− andZeb1−/− MEFs.(d) Immunoblotting of ZEB1, CHK1, Cyclin A, p-H3 (S10) and GAPDH inSUM159-P2 cells transfected with ZEB1 siRNA or the scramble control. Cells werearrested overnight with 0.5 μg/ml nocodazole. Mitotic cells were“shaken off” and then released into normal medium. Samples werecollected at the indicated time points and analyzed by western blotting. Cellcycle distribution was gauged by Cyclin A and p-H3 (S10).(e) Clonogenic survival assays of SUM159-P2 cells transfected withCHK1 siRNA. Inset: immunoblotting of CHK1 and GAPDH. n= 3 wells per group.(f) Immunoblotting of CHK1 and GAPDH in ZEB1 shRNA-transducedSUM159-P2 cells with or without ectopic expression of CHK1.(g) Clonogenic survival assays of ZEB1 shRNA-transduced SUM159-P2cells with or without ectopic expression of CHK1. n = 3wells per group.(h) Immunoblotting of ZEB1, CHK1 and GAPDH in SUM159-P0 cellstransfected with ZEB1 alone or in combination with CHK1 siRNA.(i) Clonogenic survival assays of SUM159-P0 cells transfected withZEB1 alone or in combination with CHK1 siRNA. n = 3wells per group.Data in e, g and i are the mean ofbiological replicates from a representative experiment, and error bars indicates.e.m. Statistical significance was determined by a two-tailed, unpairedStudent’s t-test. The experiments were repeated 3times. The source data can be found in Supplementary Table 3. Uncroppedimages of blots are shown in Supplementary Figure 7.
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Figure 4: CHK1 mediates ZEB1 regulation of radiosensitivity(a) Immunoblotting of p-CHK1, CHK1, p-CHK2, CHK2 and GAPDH inSUM159-P2 cells transduced with ZEB1 shRNA, at the indicated time points after 6Gy IR.(b) Immunoblotting of ZEB1, CHK1, γH2AX, H2AX and GAPDH inMCF7 cells transduced with ZEB1, at the indicated time points after 6 Gy IR.(c) Immunoblotting of Zeb1, Chk1 and Gapdh inZeb1+/+,Zeb1+/− andZeb1−/− MEFs.(d) Immunoblotting of ZEB1, CHK1, Cyclin A, p-H3 (S10) and GAPDH inSUM159-P2 cells transfected with ZEB1 siRNA or the scramble control. Cells werearrested overnight with 0.5 μg/ml nocodazole. Mitotic cells were“shaken off” and then released into normal medium. Samples werecollected at the indicated time points and analyzed by western blotting. Cellcycle distribution was gauged by Cyclin A and p-H3 (S10).(e) Clonogenic survival assays of SUM159-P2 cells transfected withCHK1 siRNA. Inset: immunoblotting of CHK1 and GAPDH. n= 3 wells per group.(f) Immunoblotting of CHK1 and GAPDH in ZEB1 shRNA-transducedSUM159-P2 cells with or without ectopic expression of CHK1.(g) Clonogenic survival assays of ZEB1 shRNA-transduced SUM159-P2cells with or without ectopic expression of CHK1. n = 3wells per group.(h) Immunoblotting of ZEB1, CHK1 and GAPDH in SUM159-P0 cellstransfected with ZEB1 alone or in combination with CHK1 siRNA.(i) Clonogenic survival assays of SUM159-P0 cells transfected withZEB1 alone or in combination with CHK1 siRNA. n = 3wells per group.Data in e, g and i are the mean ofbiological replicates from a representative experiment, and error bars indicates.e.m. Statistical significance was determined by a two-tailed, unpairedStudent’s t-test. The experiments were repeated 3times. The source data can be found in Supplementary Table 3. Uncroppedimages of blots are shown in Supplementary Figure 7.

Mentions: We reasoned that ZEB1 regulates radiosensitivity by modulating DDRpathways. CHK1 and CHK2 are two critical effector kinases in DDR and checkpointcontrol28–30, which prompted us to examinetheir status in ZEB1-depleted breast cancer cells. Interestingly, knockdown ofZEB1 in SUM159-P2 cells resulted in a significant reduction in CHK1 proteinlevels in the presence or absence of IR (Fig.4a); in contrast, neither CHK2 total protein level nor itsphosphorylation was affected (Fig. 4a).Moreover, expression of an RNAi-resistant ZEB1 mutant completely reversed theeffect of ZEB1 shRNA on CHK1, γH2AX and clonogenic survival (Supplementary Fig. 3a,b). Conversely, overexpression ZEB1 in MCF7 cells significantlyupregulated CHK1 and promoted the clearance of DNA breaks post-IR (gauged byγH2AX) (Fig. 4b). In addition,Zeb1-deficient mouse embryonic fibroblasts (MEFs) displayeddownregulation of Chk1 (Fig. 4c).


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)

CHK1 mediates ZEB1 regulation of radiosensitivity(a) Immunoblotting of p-CHK1, CHK1, p-CHK2, CHK2 and GAPDH inSUM159-P2 cells transduced with ZEB1 shRNA, at the indicated time points after 6Gy IR.(b) Immunoblotting of ZEB1, CHK1, γH2AX, H2AX and GAPDH inMCF7 cells transduced with ZEB1, at the indicated time points after 6 Gy IR.(c) Immunoblotting of Zeb1, Chk1 and Gapdh inZeb1+/+,Zeb1+/− andZeb1−/− MEFs.(d) Immunoblotting of ZEB1, CHK1, Cyclin A, p-H3 (S10) and GAPDH inSUM159-P2 cells transfected with ZEB1 siRNA or the scramble control. Cells werearrested overnight with 0.5 μg/ml nocodazole. Mitotic cells were“shaken off” and then released into normal medium. Samples werecollected at the indicated time points and analyzed by western blotting. Cellcycle distribution was gauged by Cyclin A and p-H3 (S10).(e) Clonogenic survival assays of SUM159-P2 cells transfected withCHK1 siRNA. Inset: immunoblotting of CHK1 and GAPDH. n= 3 wells per group.(f) Immunoblotting of CHK1 and GAPDH in ZEB1 shRNA-transducedSUM159-P2 cells with or without ectopic expression of CHK1.(g) Clonogenic survival assays of ZEB1 shRNA-transduced SUM159-P2cells with or without ectopic expression of CHK1. n = 3wells per group.(h) Immunoblotting of ZEB1, CHK1 and GAPDH in SUM159-P0 cellstransfected with ZEB1 alone or in combination with CHK1 siRNA.(i) Clonogenic survival assays of SUM159-P0 cells transfected withZEB1 alone or in combination with CHK1 siRNA. n = 3wells per group.Data in e, g and i are the mean ofbiological replicates from a representative experiment, and error bars indicates.e.m. Statistical significance was determined by a two-tailed, unpairedStudent’s t-test. The experiments were repeated 3times. The source data can be found in Supplementary Table 3. Uncroppedimages of blots are shown in Supplementary Figure 7.
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Figure 4: CHK1 mediates ZEB1 regulation of radiosensitivity(a) Immunoblotting of p-CHK1, CHK1, p-CHK2, CHK2 and GAPDH inSUM159-P2 cells transduced with ZEB1 shRNA, at the indicated time points after 6Gy IR.(b) Immunoblotting of ZEB1, CHK1, γH2AX, H2AX and GAPDH inMCF7 cells transduced with ZEB1, at the indicated time points after 6 Gy IR.(c) Immunoblotting of Zeb1, Chk1 and Gapdh inZeb1+/+,Zeb1+/− andZeb1−/− MEFs.(d) Immunoblotting of ZEB1, CHK1, Cyclin A, p-H3 (S10) and GAPDH inSUM159-P2 cells transfected with ZEB1 siRNA or the scramble control. Cells werearrested overnight with 0.5 μg/ml nocodazole. Mitotic cells were“shaken off” and then released into normal medium. Samples werecollected at the indicated time points and analyzed by western blotting. Cellcycle distribution was gauged by Cyclin A and p-H3 (S10).(e) Clonogenic survival assays of SUM159-P2 cells transfected withCHK1 siRNA. Inset: immunoblotting of CHK1 and GAPDH. n= 3 wells per group.(f) Immunoblotting of CHK1 and GAPDH in ZEB1 shRNA-transducedSUM159-P2 cells with or without ectopic expression of CHK1.(g) Clonogenic survival assays of ZEB1 shRNA-transduced SUM159-P2cells with or without ectopic expression of CHK1. n = 3wells per group.(h) Immunoblotting of ZEB1, CHK1 and GAPDH in SUM159-P0 cellstransfected with ZEB1 alone or in combination with CHK1 siRNA.(i) Clonogenic survival assays of SUM159-P0 cells transfected withZEB1 alone or in combination with CHK1 siRNA. n = 3wells per group.Data in e, g and i are the mean ofbiological replicates from a representative experiment, and error bars indicates.e.m. Statistical significance was determined by a two-tailed, unpairedStudent’s t-test. The experiments were repeated 3times. The source data can be found in Supplementary Table 3. Uncroppedimages of blots are shown in Supplementary Figure 7.
Mentions: We reasoned that ZEB1 regulates radiosensitivity by modulating DDRpathways. CHK1 and CHK2 are two critical effector kinases in DDR and checkpointcontrol28–30, which prompted us to examinetheir status in ZEB1-depleted breast cancer cells. Interestingly, knockdown ofZEB1 in SUM159-P2 cells resulted in a significant reduction in CHK1 proteinlevels in the presence or absence of IR (Fig.4a); in contrast, neither CHK2 total protein level nor itsphosphorylation was affected (Fig. 4a).Moreover, expression of an RNAi-resistant ZEB1 mutant completely reversed theeffect of ZEB1 shRNA on CHK1, γH2AX and clonogenic survival (Supplementary Fig. 3a,b). Conversely, overexpression ZEB1 in MCF7 cells significantlyupregulated CHK1 and promoted the clearance of DNA breaks post-IR (gauged byγH2AX) (Fig. 4b). In addition,Zeb1-deficient mouse embryonic fibroblasts (MEFs) displayeddownregulation of Chk1 (Fig. 4c).

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