Stabilization of histone demethylase PHF8 by USP7 promotes breast carcinogenesis
Qian Wang, … , Zhi Yao, Lei Shi
Qian Wang, … , Zhi Yao, Lei Shi
Published May 16, 2016
Citation Information: J Clin Invest. 2016;126(6):2205-2220. https://doi.org/10.1172/JCI85747.
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Research Article Oncology

Stabilization of histone demethylase PHF8 by USP7 promotes breast carcinogenesis

Abstract

The histone demethylase PHF8 has been implicated in multiple pathological disorders, including X-linked mental retardation and tumorigenesis. However, it is not clear how the abundance and function of PHF8 are regulated. Here, we report that PHF8 physically associates with the deubiquitinase USP7. Specifically, we demonstrated that USP7 promotes deubiquitination and stabilization of PHF8, leading to the upregulation of a group of genes, including cyclin A2, that are critical for cell growth and proliferation. The USP7-encoding gene was also transcriptionally regulated by PHF8, via positive feedback. USP7 was overexpressed in breast carcinomas, and the level of expression positively correlated with expression of PHF8 and cyclin A2 and with the histological grade of breast cancer. We showed that USP7 promotes breast carcinogenesis by stabilizing PHF8 and upregulating cyclin A2 and that the interaction between USP7 and PHF8 is augmented during DNA damage. Moreover, USP7-promoted PHF8 stabilization conferred cellular resistance to genotoxic insults and was required for the recruitment of BLM and KU70, which are both essential for DNA double-strand break repair. Our study mechanistically links USP7 to epigenetic regulation and DNA repair. Moreover, these data support the pursuit of USP7 and PHF8 as potential targets for breast cancer intervention, especially in combination with chemo- or radiotherapies.

Authors

Qian Wang, Shuai Ma, Nan Song, Xin Li, Ling Liu, Shangda Yang, Xiang Ding, Lin Shan, Xing Zhou, Dongxue Su, Yue Wang, Qi Zhang, Xinhua Liu, Na Yu, Kai Zhang, Yongfeng Shang, Zhi Yao, Lei Shi

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Figure 2

USP7 promotes PHF8 stabilization.

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USP7 promotes PHF8 stabilization. (A) MCF-7 cells were transfected with ...
(A) MCF-7 cells were transfected with control siRNA or different sets of USP7 siRNAs. Cellular extracts and total RNA were prepared and analyzed by Western blotting (left panel) and qRT-PCR analysis (right panel), respectively. (B) Experiments analogous to A were performed in U2OS cells. (C) MCF-7 cells were transfected with control siRNA or USP22 siRNA followed by Western blotting analysis. (D) MCF-7 cells were transfected with control siRNA or USP7 siRNA followed by treatment with DMSO or the proteasome inhibitor MG132 (10 μM). Cellular extracts were prepared and analyzed by Western blotting. (E) MCF-7 or U2OS cells transfected with control siRNA or USP7 siRNA were treated with cycloheximide (CHX; 50 μg/ml) and harvested at the indicated time points, followed by Western blotting analysis. Quantitation was done by densitometry with ImageJ software (NIH) with β-actin as a normalizer. (F) U2OS cells synchronized by double-thymidine block were released, and cellular extracts were collected at indicated time points for Western blotting analysis (top panel). Cell cycle profiles of unsynchronized (Unsyn) or synchronized cells were analyzed by FACS (bottom panel). (G) Western blotting analysis of the expression of USP7 and PHF8 in multiple cell lines. (H) Cellular extracts from MCF-7 cells transfected with control siRNA, PHF8 siRNA, or USP7 siRNA were analyzed by Western blotting. Representative images from biological triplicate experiments are shown. In A and B, each bar represents the mean ± SD for biological triplicate experiments. **P < 0.01, 1-way ANOVA.