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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 is upregulated in radioresistant cancer cells and promotes tumorradioresistance(a) Schematic representation of generation of a radioresistantsubline (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 andSUM159-P2 cells treated with 6 Gy IR.(d) Immunoblotting of Snail, Twist, ZEB1 and GAPDH in SUM159-P0 andSUM159-P2 cells. SUM159-P0 cells transfected with Snail or Twist were used aspositive controls.(e) Immunoblotting of Snail, Twist, ZEB1 and GAPDH in SUM159-P0cells transduced with Snail, Twist or ZEB1.(f) Clonogenic survival assays of SUM159-P0 cells transduced withSnail, Twist or ZEB1. n = 3 wells per group.(g) Clonogenic survival assays of SUM159-P2 cells transduced withZEB1 shRNA (sh-ZEB1). Inset: immunoblotting of ZEB1 and GAPDH.n = 3 wells per group.(h, i) Tumor size of mice bearing control (scramble) or ZEB1shRNA-transduced SUM159-P2 xenografts. Tumors were locally irradiated with 15 Gysingle dose (h) or 2 Gy fractionated dose (XRT) twice per day for 7consecutive days (i). n = 5 mice pergroup. General linear model multivariate analysis was performed to determinestatistical significance.(j) Immunoblotting of ZEB1 and HSP90 in tumor lysates.Data in b, f, g, h andi are the mean of biological replicates from a representativeexperiment, and error bars indicate s.e.m. Statistical significance inb, f and g was determined by atwo-tailed, unpaired Student’s t-test. The experimentswere repeated 3 times. The source data can be found in Supplementary Table 3. Uncroppedimages of blots are shown in Supplementary Figure 7.
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Figure 2: ZEB1 is upregulated in radioresistant cancer cells and promotes tumorradioresistance(a) Schematic representation of generation of a radioresistantsubline (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 andSUM159-P2 cells treated with 6 Gy IR.(d) Immunoblotting of Snail, Twist, ZEB1 and GAPDH in SUM159-P0 andSUM159-P2 cells. SUM159-P0 cells transfected with Snail or Twist were used aspositive controls.(e) Immunoblotting of Snail, Twist, ZEB1 and GAPDH in SUM159-P0cells transduced with Snail, Twist or ZEB1.(f) Clonogenic survival assays of SUM159-P0 cells transduced withSnail, Twist or ZEB1. n = 3 wells per group.(g) Clonogenic survival assays of SUM159-P2 cells transduced withZEB1 shRNA (sh-ZEB1). Inset: immunoblotting of ZEB1 and GAPDH.n = 3 wells per group.(h, i) Tumor size of mice bearing control (scramble) or ZEB1shRNA-transduced SUM159-P2 xenografts. Tumors were locally irradiated with 15 Gysingle dose (h) or 2 Gy fractionated dose (XRT) twice per day for 7consecutive days (i). n = 5 mice pergroup. General linear model multivariate analysis was performed to determinestatistical significance.(j) Immunoblotting of ZEB1 and HSP90 in tumor lysates.Data in b, f, g, h andi are the mean of biological replicates from a representativeexperiment, and error bars indicate s.e.m. Statistical significance inb, f and g was determined by atwo-tailed, unpaired Student’s t-test. The experimentswere repeated 3 times. The source data can be found in Supplementary Table 3. Uncroppedimages of blots are shown in Supplementary Figure 7.

Mentions: To determine whether ZEB1 is indeed upregulated in radioresistant tumorcells, we employed γ-ionizing radiation (IR) to select theradioresistant subpopulation from the SUM159 human breast cancer cells whichexpress moderate levels of ZEB1. After a 6 Gray (Gy) dose, survived cells formedcolonies. We pooled the colonies and repeated the dose one more time (Fig. 2a). Cells derived from this selection,named SUM159-P2 cells, displayed increased clonogenic survival upon irradiationcompared with the parental SUM159 cells (SUM159-P0) (Fig. 2b). Irradiation causes DSBs resulting in theformation of γH2AX foci, and the persistence of γH2AX foci marksdelayed repair and correlates with radiosensitivity21–23. At 24 hours after irradiation, γH2AX remainedin SUM159-P0 cells but disappeared in SUM159-P2 cells (Fig. 2c), indicating that this radioresistant sublinehas enhanced clearance of DNA breaks.


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 is upregulated in radioresistant cancer cells and promotes tumorradioresistance(a) Schematic representation of generation of a radioresistantsubline (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 andSUM159-P2 cells treated with 6 Gy IR.(d) Immunoblotting of Snail, Twist, ZEB1 and GAPDH in SUM159-P0 andSUM159-P2 cells. SUM159-P0 cells transfected with Snail or Twist were used aspositive controls.(e) Immunoblotting of Snail, Twist, ZEB1 and GAPDH in SUM159-P0cells transduced with Snail, Twist or ZEB1.(f) Clonogenic survival assays of SUM159-P0 cells transduced withSnail, Twist or ZEB1. n = 3 wells per group.(g) Clonogenic survival assays of SUM159-P2 cells transduced withZEB1 shRNA (sh-ZEB1). Inset: immunoblotting of ZEB1 and GAPDH.n = 3 wells per group.(h, i) Tumor size of mice bearing control (scramble) or ZEB1shRNA-transduced SUM159-P2 xenografts. Tumors were locally irradiated with 15 Gysingle dose (h) or 2 Gy fractionated dose (XRT) twice per day for 7consecutive days (i). n = 5 mice pergroup. General linear model multivariate analysis was performed to determinestatistical significance.(j) Immunoblotting of ZEB1 and HSP90 in tumor lysates.Data in b, f, g, h andi are the mean of biological replicates from a representativeexperiment, and error bars indicate s.e.m. Statistical significance inb, f and g was determined by atwo-tailed, unpaired Student’s t-test. The experimentswere repeated 3 times. The source data can be found in Supplementary Table 3. Uncroppedimages of blots are shown in Supplementary Figure 7.
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Related In: Results  -  Collection

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Figure 2: ZEB1 is upregulated in radioresistant cancer cells and promotes tumorradioresistance(a) Schematic representation of generation of a radioresistantsubline (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 andSUM159-P2 cells treated with 6 Gy IR.(d) Immunoblotting of Snail, Twist, ZEB1 and GAPDH in SUM159-P0 andSUM159-P2 cells. SUM159-P0 cells transfected with Snail or Twist were used aspositive controls.(e) Immunoblotting of Snail, Twist, ZEB1 and GAPDH in SUM159-P0cells transduced with Snail, Twist or ZEB1.(f) Clonogenic survival assays of SUM159-P0 cells transduced withSnail, Twist or ZEB1. n = 3 wells per group.(g) Clonogenic survival assays of SUM159-P2 cells transduced withZEB1 shRNA (sh-ZEB1). Inset: immunoblotting of ZEB1 and GAPDH.n = 3 wells per group.(h, i) Tumor size of mice bearing control (scramble) or ZEB1shRNA-transduced SUM159-P2 xenografts. Tumors were locally irradiated with 15 Gysingle dose (h) or 2 Gy fractionated dose (XRT) twice per day for 7consecutive days (i). n = 5 mice pergroup. General linear model multivariate analysis was performed to determinestatistical significance.(j) Immunoblotting of ZEB1 and HSP90 in tumor lysates.Data in b, f, g, h andi are the mean of biological replicates from a representativeexperiment, and error bars indicate s.e.m. Statistical significance inb, f and g was determined by atwo-tailed, unpaired Student’s t-test. The experimentswere repeated 3 times. The source data can be found in Supplementary Table 3. Uncroppedimages of blots are shown in Supplementary Figure 7.
Mentions: To determine whether ZEB1 is indeed upregulated in radioresistant tumorcells, we employed γ-ionizing radiation (IR) to select theradioresistant subpopulation from the SUM159 human breast cancer cells whichexpress moderate levels of ZEB1. After a 6 Gray (Gy) dose, survived cells formedcolonies. We pooled the colonies and repeated the dose one more time (Fig. 2a). Cells derived from this selection,named SUM159-P2 cells, displayed increased clonogenic survival upon irradiationcompared with the parental SUM159 cells (SUM159-P0) (Fig. 2b). Irradiation causes DSBs resulting in theformation of γH2AX foci, and the persistence of γH2AX foci marksdelayed repair and correlates with radiosensitivity21–23. At 24 hours after irradiation, γH2AX remainedin SUM159-P0 cells but disappeared in SUM159-P2 cells (Fig. 2c), indicating that this radioresistant sublinehas enhanced clearance of DNA breaks.

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