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 specifically promotes the interaction between USP7 and CHK1(a) 293T cells were transfected with SFB-USP7 alone or incombination with ZEB1, followed by pull-down with streptavidin-sepharose beadsand immunoblotting with antibodies to CHK1, HLTF, p53 and USP7.(b) 293T cells were transfected with ZEB1 siRNA alone or incombination with SFB-USP7, followed by pull-down with streptavidin-sepharosebeads and immunoblotting with antibodies to CHK1, HLTF, p53 and USP7.(c) SUM159-P2 cells were transfected with ZEB1 siRNA, followed byimmunoprecipitation with the USP7 antibody and immunoblotting with antibodies toCHK1 and USP7. Uncropped images of blots are shown in Supplementary Figure 7.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: ZEB1 specifically promotes the interaction between USP7 and CHK1(a) 293T cells were transfected with SFB-USP7 alone or incombination with ZEB1, followed by pull-down with streptavidin-sepharose beadsand immunoblotting with antibodies to CHK1, HLTF, p53 and USP7.(b) 293T cells were transfected with ZEB1 siRNA alone or incombination with SFB-USP7, followed by pull-down with streptavidin-sepharosebeads and immunoblotting with antibodies to CHK1, HLTF, p53 and USP7.(c) SUM159-P2 cells were transfected with ZEB1 siRNA, followed byimmunoprecipitation with the USP7 antibody and immunoblotting with antibodies toCHK1 and USP7. Uncropped images of blots are shown in Supplementary Figure 7.

Mentions: To further understand why ZEB1 regulates the stability of CHK1 but notthe stability of other USP7 substrates (Fig.5h and Supplementary Fig. 4f), we examined the effect of ZEB1 on theinteraction between USP7 and its various substrates. As expected, CHK1, HLTF,p53 and Claspin could be detected in USP7 immunoprecipitates (Fig. 6a and Supplementary Fig. 4g).Interestingly, ectopic expression of ZEB1 markedly enhanced the interaction ofUSP7 with CHK1, but not its association with HLTF, p53 or Claspin (Fig. 6a and Supplementary Fig. 4g). Conversely,knockdown of ZEB1 dramatically decreased the interaction between USP7 (eitheroverexpressed or endogenous) and CHK1, but not HLTF and p53 (Fig. 6b, c). Therefore, ZEB1 specifically promotes theinteraction between USP7 and CHK1.


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 specifically promotes the interaction between USP7 and CHK1(a) 293T cells were transfected with SFB-USP7 alone or incombination with ZEB1, followed by pull-down with streptavidin-sepharose beadsand immunoblotting with antibodies to CHK1, HLTF, p53 and USP7.(b) 293T cells were transfected with ZEB1 siRNA alone or incombination with SFB-USP7, followed by pull-down with streptavidin-sepharosebeads and immunoblotting with antibodies to CHK1, HLTF, p53 and USP7.(c) SUM159-P2 cells were transfected with ZEB1 siRNA, followed byimmunoprecipitation with the USP7 antibody and immunoblotting with antibodies toCHK1 and USP7. Uncropped images 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 6: ZEB1 specifically promotes the interaction between USP7 and CHK1(a) 293T cells were transfected with SFB-USP7 alone or incombination with ZEB1, followed by pull-down with streptavidin-sepharose beadsand immunoblotting with antibodies to CHK1, HLTF, p53 and USP7.(b) 293T cells were transfected with ZEB1 siRNA alone or incombination with SFB-USP7, followed by pull-down with streptavidin-sepharosebeads and immunoblotting with antibodies to CHK1, HLTF, p53 and USP7.(c) SUM159-P2 cells were transfected with ZEB1 siRNA, followed byimmunoprecipitation with the USP7 antibody and immunoblotting with antibodies toCHK1 and USP7. Uncropped images of blots are shown in Supplementary Figure 7.
Mentions: To further understand why ZEB1 regulates the stability of CHK1 but notthe stability of other USP7 substrates (Fig.5h and Supplementary Fig. 4f), we examined the effect of ZEB1 on theinteraction between USP7 and its various substrates. As expected, CHK1, HLTF,p53 and Claspin could be detected in USP7 immunoprecipitates (Fig. 6a and Supplementary Fig. 4g).Interestingly, ectopic expression of ZEB1 markedly enhanced the interaction ofUSP7 with CHK1, but not its association with HLTF, p53 or Claspin (Fig. 6a and Supplementary Fig. 4g). Conversely,knockdown of ZEB1 dramatically decreased the interaction between USP7 (eitheroverexpressed or endogenous) and CHK1, but not HLTF and p53 (Fig. 6b, c). Therefore, ZEB1 specifically promotes theinteraction between USP7 and CHK1.

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