Limits...
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.

Show MeSH

Related in: MedlinePlus

ZEB1 confers radioresistance on mammary epithelial cells(a) Immunoblotting of E-cadherin, Vimentin, Snail, Twist, ZEB1 andGAPDH 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 andGAPDH in HMLE cells transduced with Snail, Twist or ZEB1 alone or in combinationwith 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 orZEB1. n = 3 wells per group.(e) Immunoblotting of Snail, Twist, ZEB1, E-cadherin, Vimentin andGAPDH 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 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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150825&req=5

Figure 1: ZEB1 confers radioresistance on mammary epithelial cells(a) Immunoblotting of E-cadherin, Vimentin, Snail, Twist, ZEB1 andGAPDH 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 andGAPDH in HMLE cells transduced with Snail, Twist or ZEB1 alone or in combinationwith 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 orZEB1. n = 3 wells per group.(e) Immunoblotting of Snail, Twist, ZEB1, E-cadherin, Vimentin andGAPDH 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 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: To examine the association between EMT and radioresistance, weoverexpressed Snail, Twist or ZEB1 in the experimentally immortalized,non-transformed human mammary epithelial cells18, termed HMLE cells. Each of thesetranscription factors induced EMT – as evidenced by changes inmorphology (Supplementary Fig.1a), downregulation of E-cadherin and upregulation of Vimentin (Fig. 1a), and increased clonogenic survivalupon irradiation (Fig. 1b and Supplementary Fig. 1b).In each case, expression of Snail, Twist and ZEB1 was upregulated; inparticular, overexpression of either Snail or Twist increased ZEB1 expression tothe level as high as that of ZEB1-overexpressing cells (Fig. 1a). Next, we silenced each of the threetranscription factors in HMLE cells overexpressing Snail, Twist or ZEB1, whichdid not cause reversal of EMT (Fig. 1c).Notably, only knockdown of ZEB1 reduced radioresistance (Fig. 1d), suggesting that ZEB1 underlies theassociation between EMT and radioresistance. Consistent with this notion, weobserved upregulation of ZEB1 in the survival fraction of mock-infected HMLEcells (Supplementary Fig.1c); moreover, the survival fraction of ZEB1-depleted HMLE cellsre-expressed ZEB1 (Supplementary Fig. 1c).


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 confers radioresistance on mammary epithelial cells(a) Immunoblotting of E-cadherin, Vimentin, Snail, Twist, ZEB1 andGAPDH 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 andGAPDH in HMLE cells transduced with Snail, Twist or ZEB1 alone or in combinationwith 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 orZEB1. n = 3 wells per group.(e) Immunoblotting of Snail, Twist, ZEB1, E-cadherin, Vimentin andGAPDH 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 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150825&req=5

Figure 1: ZEB1 confers radioresistance on mammary epithelial cells(a) Immunoblotting of E-cadherin, Vimentin, Snail, Twist, ZEB1 andGAPDH 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 andGAPDH in HMLE cells transduced with Snail, Twist or ZEB1 alone or in combinationwith 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 orZEB1. n = 3 wells per group.(e) Immunoblotting of Snail, Twist, ZEB1, E-cadherin, Vimentin andGAPDH 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 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: To examine the association between EMT and radioresistance, weoverexpressed Snail, Twist or ZEB1 in the experimentally immortalized,non-transformed human mammary epithelial cells18, termed HMLE cells. Each of thesetranscription factors induced EMT – as evidenced by changes inmorphology (Supplementary Fig.1a), downregulation of E-cadherin and upregulation of Vimentin (Fig. 1a), and increased clonogenic survivalupon irradiation (Fig. 1b and Supplementary Fig. 1b).In each case, expression of Snail, Twist and ZEB1 was upregulated; inparticular, overexpression of either Snail or Twist increased ZEB1 expression tothe level as high as that of ZEB1-overexpressing cells (Fig. 1a). Next, we silenced each of the threetranscription factors in HMLE cells overexpressing Snail, Twist or ZEB1, whichdid not cause reversal of EMT (Fig. 1c).Notably, only knockdown of ZEB1 reduced radioresistance (Fig. 1d), suggesting that ZEB1 underlies theassociation between EMT and radioresistance. Consistent with this notion, weobserved upregulation of ZEB1 in the survival fraction of mock-infected HMLEcells (Supplementary Fig.1c); moreover, the survival fraction of ZEB1-depleted HMLE cellsre-expressed ZEB1 (Supplementary Fig. 1c).

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