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 October 1, 2018; First 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 1

USP7 is physically associated with the MRN-MDC1 complex.

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USP7 is physically associated with the MRN-MDC1 complex. (A) Immunopurif...
(A) Immunopurification and mass spectrometric analysis of MRE11-, NBS1-, or MDC1-containing protein complexes. Cellular extracts from HeLa cells stably expressing FLAG-MRE11, FLAG-NBS1, or FLAG-MDC1 were immunopurified with anti-FLAG affinity beads and eluted with FLAG peptide. The eluates were resolved on SDS/PAGE and silver stained, followed by mass spectrometric analysis. The percentage of peptide coverage of the indicated proteins is shown. (B) Co-IP analysis of the association between USP7 and the MRN-MDC1 complex. Whole-cell lysates from HeLa and MCF-7 cells were immunoprecipitated and then immunoblotted with antibodies against the indicated proteins. α, anti-. (C) FPLC analysis of the native protein complex. Nuclear extracts from HeLa cells were fractionated on Superose 6 size-exclusion columns with high-salt buffer. Chromatographic elution profiles and Western blot analysis of the chromatographic fractions with antibodies against the indicated proteins are shown. Equal volumes from each fraction were analyzed, and the elution positions of the calibration proteins with known molecular masses (kDa) are indicated. The boxed area indicates fractions in which endogenous USP7 was coeluted with the MRN-MDC1 complex. (D) Experiments analogous to those for C were performed with a USP7-containing protein complex purified from FLAG-USP7–expressing HeLa cells. The boxed area indicates fractions in which FLAG-USP7 was coeluted with the MRN-MDC1 complex. (E) Confocal microscopic analysis of USP7 and MRN-MDC1 complex subcellular localization. HeLa cells were fixed and immunostained with antibodies against the indicated proteins. Representative images from biological triplicate experiments are shown. Scale bars: 10 μm.