Ubiquitin-specific protease 7 sustains DNA damage response and promotes cervical carcinogenesis
Dongxue Su, … , Kai Zhang, Lei Shi
Dongxue Su, … , Kai Zhang, Lei Shi
Published September 4, 2018
Citation Information: J Clin Invest. 2018;128(10):4280-4296. https://doi.org/10.1172/JCI120518.
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Research Article Cell biology Oncology

Ubiquitin-specific protease 7 sustains DNA damage response and promotes cervical carcinogenesis

Abstract

Central to the recognition, signaling, and repair of DNA double-strand breaks (DSBs) are the MRE11-RAD50-NBS1 (MRN) complex and mediator of DNA damage checkpoint protein 1 (MDC1), the interplay of which is essential for initiation and amplification of the DNA damage response (DDR). The intrinsic rule governing the regulation of the function of this molecular machinery remains to be investigated. We report here that the ubiquitin-specific protease USP7 was physically associated with the MRN-MDC1 complex and that the MRN-MDC1 complex acted as a platform for USP7 to efficiently deubiquitinate and stabilize MDC1, thereby sustaining the DDR. Accordingly, depletion of USP7 impaired the engagement of the MRN-MDC1 complex and the consequent recruitment of the downstream factors p53-binding protein 1 (53BP1) and breast cancer protein 1 (BRCA1) at DNA lesions. Significantly, USP7 was overexpressed in cervical cancer, and the level of its expression positively correlated with that of MDC1 and worse survival rates for patients with cervical cancer. We demonstrate that USP7-mediated MDC1 stabilization promoted cervical cancer cell survival and conferred cellular resistance to genotoxic insults. Together, our study reveals a role for USP7 in regulating the function of the MRN-MDC1 complex and activity of the DDR, supporting the pursuit of USP7 as a potential therapeutic target for MDC1-proficient cancers.

Authors

Dongxue Su, Shuai Ma, Lin Shan, Yue Wang, Yuejiao Wang, Cheng Cao, Beibei Liu, Chao Yang, Liyong Wang, Shanshan Tian, Xiang Ding, Xinhua Liu, Na Yu, Nan Song, Ling Liu, Shangda Yang, Qi Zhang, Fuquan Yang, Kai Zhang, Lei Shi

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Figure 3

USP7 promotes MDC1 stabilization.

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USP7 promotes MDC1 stabilization. (A) MCF-7 cells transfected with contr...
(A) MCF-7 cells transfected with control siRNA or different sets of USP7 siRNAs were collected and analyzed by Western blotting and qRT-PCR, respectively. (B) Experiments analogous to the data in A were performed using HeLa cells. (C) Cellular lysates or total RNA from WT and USP7-KO HeLa cells were analyzed by Western blotting and qRT-PCR, respectively. (D) USP7-KO HeLa cells were transfected with control vector, WT USP7 (USP7/WT), or UBL domain–deficient USP7 (USP7/ΔUBL) cells. Cellular extracts were prepared and analyzed by Western blotting. (E) MCF-7 cells were transfected with control siRNA or USP7 siRNA, followed by treatment with CHX (50 μg/ml), and harvested at the indicated time points, followed by Western blotting. (F) MCF-7 cells (left) or U2OS cells (right) transfected with control siRNA or USP7 siRNAs were treated with DMSO or the proteasome inhibitor MG132 (10 μM). Cellular extracts were prepared and analyzed by Western blotting. (G) HeLa cells stably expressing FLAG-MDC1 were transfected with control siRNA or NBS1 siRNAs, and whole-cell lysates were collected for co-IP analysis. (H) HeLa cells transfected with control siRNA or different sets of NBS1 siRNAs were collected and analyzed by Western blotting and qRT-PCR, respectively. (I) USP7-KO HeLa cells were transfected with control vector, USP7/WT, or MATH domain–deficient USP7 (USP7/ΔMATH). Cellular extracts were prepared and analyzed by Western blotting. (J) HeLa cells stably expressing GFP-MDC1 were transfected with FLAG-tagged USP7/WT, USP7/ΔUBL, or USP7/ΔMATH. Whole-cell lysates were collected for co-IP analysis. In A–C and H, data represent the mean ± SD of biological triplicate experiments. **P < 0.01, by 1-way ANOVA (A, B and H) and 2-tailed unpaired Student’s t test (C).