Ubiquitin-specific protease 7 sustains DNA damage response and promotes cervical carcinogenesis
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
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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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 8

USP7-promoted MDC1 stabilization is required for the growth of cervical cancer cells.

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USP7-promoted MDC1 stabilization is required for the growth of cervical ...
(A) HeLa cells stably expressing the indicated shRNAs and genes were irradiated with different doses of x-ray IR. Colony formation assays were conducted, and representative images from biological triplicate experiments are shown. (B) Experiments analogous to those for A were performed using SiHa cells. Representative images from biological triplicate experiments are shown. (C) HeLa cells stably expressing the indicated shRNAs and GFP vector or GFP-MDC1 were cocultured with equivalent native HeLa cells. The mixed cells were then irradiated with different doses of x-ray IR and analyzed by FACS 10 days later. The percentage of GFP-positive cells relative to GFP-negative cells under IR treatment was normalized to percentages in the untreated control mixture. The ratio reflects cellular fitness. Data represent the mean ± SD from biological triplicate experiments. *P < 0.05 and **P < 0.01, by 1-way ANOVA. (D) Experiments analogous to those for C were performed using SiHa cells. Data represent the mean ± SD from biological triplicate experiments. *P < 0.05 and **P < 0.01, by 1-way ANOVA. (E) Cell viability analysis was performed with SiHa cells stably expressing vector or FLAG-MDC1. Cells were cultured in increasing amounts of GNE-6640 or GNE-6776 followed by x-ray IR (2 Gy). Data represent the mean ± SD from biological triplicate experiments. **P < 0.01, by 2-way ANOVA. (F) SiHa tumors stably expressing shRNAs and the indicated genes were transplanted into athymic mice (n = 12), and half of the mice in each group were randomly chosen and subjected to 10 Gy x-ray IR 1 week after tumor transplantation. Tumor volumes were measured weekly, and tumors were harvested and weighed when mice were sacrificed. Data represent the mean ± SD. *P < 0.05 and **P < 0.01, by 2-way ANOVA for tumor volume analysis and 1-way ANOVA for tumor weight analysis. Images of representative tumors and sacrificed mice are shown.