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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 regulates DNA damage repair(a) γH2AX and DAPI staining of SUM159-P2 cells transducedwith ZEB1 shRNA, 24 hr after 6 Gy IR. Scale bar: 10 μm.(b) Immunoblotting of ZEB1, γH2AX, H2AX and GAPDH inSUM159-P2 cells transduced with ZEB1 shRNA, at the indicated time points after 6Gy IR.(c, d) Images (c) and data quantification(d) of comet assays of SUM159-P2 cells transduced with ZEB1shRNA, 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 cellstransfected with ZEB1 siRNA alone or in combination with ZEB1.(f) HR repair assays of U2OS_DR-GFP cells transfected with ZEB1siRNA alone or in combination with ZEB1. n = 3 wellsper group.Data in d and f are the mean of biological replicatesfrom a representative 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 3: ZEB1 regulates DNA damage repair(a) γH2AX and DAPI staining of SUM159-P2 cells transducedwith ZEB1 shRNA, 24 hr after 6 Gy IR. Scale bar: 10 μm.(b) Immunoblotting of ZEB1, γH2AX, H2AX and GAPDH inSUM159-P2 cells transduced with ZEB1 shRNA, at the indicated time points after 6Gy IR.(c, d) Images (c) and data quantification(d) of comet assays of SUM159-P2 cells transduced with ZEB1shRNA, 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 cellstransfected with ZEB1 siRNA alone or in combination with ZEB1.(f) HR repair assays of U2OS_DR-GFP cells transfected with ZEB1siRNA alone or in combination with ZEB1. n = 3 wellsper group.Data in d and f are the mean of biological replicatesfrom a representative 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: After IR treatment, γH2AX foci persist longer in radiosensitivecell lines than in radioresistant lines23. In ZEB1 shRNA-expressing SUM159-P2 cells but not cellsinfected with a scrambled control, we observed persistence of γH2AX foci24 hours after IR (Fig. 3a, b), indicatingthat ZEB1-depleted cells were less able to repair DNA lesions. To directly gaugedamaged DNA, we performed a comet assay to detect both single- and double-strandDNA breaks. 24 hours after IR, ZEB1-depleted SUM159-P2 cells exhibited a4.5-fold increase in the comet ‘tail moment’ (=percentage of the DNA in the tail × length of the tail in μm)– a previously described measure of DNA damage24, compared with the control cells (Fig. 3c, d).


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 regulates DNA damage repair(a) γH2AX and DAPI staining of SUM159-P2 cells transducedwith ZEB1 shRNA, 24 hr after 6 Gy IR. Scale bar: 10 μm.(b) Immunoblotting of ZEB1, γH2AX, H2AX and GAPDH inSUM159-P2 cells transduced with ZEB1 shRNA, at the indicated time points after 6Gy IR.(c, d) Images (c) and data quantification(d) of comet assays of SUM159-P2 cells transduced with ZEB1shRNA, 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 cellstransfected with ZEB1 siRNA alone or in combination with ZEB1.(f) HR repair assays of U2OS_DR-GFP cells transfected with ZEB1siRNA alone or in combination with ZEB1. n = 3 wellsper group.Data in d and f are the mean of biological replicatesfrom a representative 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 3: ZEB1 regulates DNA damage repair(a) γH2AX and DAPI staining of SUM159-P2 cells transducedwith ZEB1 shRNA, 24 hr after 6 Gy IR. Scale bar: 10 μm.(b) Immunoblotting of ZEB1, γH2AX, H2AX and GAPDH inSUM159-P2 cells transduced with ZEB1 shRNA, at the indicated time points after 6Gy IR.(c, d) Images (c) and data quantification(d) of comet assays of SUM159-P2 cells transduced with ZEB1shRNA, 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 cellstransfected with ZEB1 siRNA alone or in combination with ZEB1.(f) HR repair assays of U2OS_DR-GFP cells transfected with ZEB1siRNA alone or in combination with ZEB1. n = 3 wellsper group.Data in d and f are the mean of biological replicatesfrom a representative 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: After IR treatment, γH2AX foci persist longer in radiosensitivecell lines than in radioresistant lines23. In ZEB1 shRNA-expressing SUM159-P2 cells but not cellsinfected with a scrambled control, we observed persistence of γH2AX foci24 hours after IR (Fig. 3a, b), indicatingthat ZEB1-depleted cells were less able to repair DNA lesions. To directly gaugedamaged DNA, we performed a comet assay to detect both single- and double-strandDNA breaks. 24 hours after IR, ZEB1-depleted SUM159-P2 cells exhibited a4.5-fold increase in the comet ‘tail moment’ (=percentage of the DNA in the tail × length of the tail in μm)– a previously described measure of DNA damage24, compared with the control cells (Fig. 3c, d).

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