Abstract
BackgroundThe deubiquitinase USP7 has been identified as an oncogene with key roles in tumorigenesis and therapeutic resistance for a series of cancer types. Recently small molecular inhibitors have been developed to target USP7. However, the anticancer mechanism of USP7 inhibitors is still elusive.MethodsCell viability or clonogenicity was tested by violet crystal assay. Cell apoptosis or cell cycle was analyzed by flow cytometry, and chromosome misalignment was observed by a fluorescent microscopy. The protein interaction of PLK1 and USP7 was detected by tandem affinity purification and high throughput proteomics, and further confirmed by co-immunoprecipitation, GST pull-down and protein co-localization. The correlation between USP7 level of tumor tissues and taxane-resistance was evaluated.ResultsPharmacological USP7 inhibition by P5091 retarded cell proliferation and induced cell apoptosis. Further studies showed that P5091 induced cell cycle arrest at G2/M phase, and particularly induced chromosome misalignment, indicating the key roles of USP7 in mitosis. USP7 protein was detected in the PLK1-interacted protein complex. USP7 interacts with PLK1 protein through its PBD domain by catalytic activity. USP7 as a deubiquitinase sustained PLK1 protein stability via the C223 site, and inversely, USP7 inhibition by P5091 promoted the protein degradation of PLK1 through the ubiquitination-proteasome pathway. By overexpressing PLK1, USP7 that had been depleted by RNAi ceased to induce chromosome misalignment in mitosis and again supported cell proliferation and cell survival. Both USP7 and PLK1 were overexpressed in taxane-resistant cancer cells, and negatively correlated with the MP scores in tumor tissues. Either USP7 or PLK1 knockdown by RNAi significantly sensitized taxane-resistant cells to taxane cell killing.ConclusionThis is the first report that PLK1 is a novel substrate of USP7 deubiquitinase, and that USP7 sustained the protein stability of PLK1. USP7 inhibition induces cell apoptosis and cell cycle G2/M arrest, and overcomes taxane resistance by inducing the protein degradation of PLK1, resulting in chromosome misalignment in mitosis.
Highlights
The deubiquitinase Ubiquitin specific protease 7 (USP7) has been identified as an oncogene with key roles in tumorigenesis and therapeutic resistance for a series of cancer types
USP7 depletion impaired cell colony formation and retarded cell proliferation by inducing G2/M cell cycle arrest and chromosome misalignment in mitosis USP7 has been identified as an oncogene, and plays a key role in tumorigenesis in several cancer types, including prostate cancer
To further explore the mechanisms by which USP7 depletion impaired cell colony formation and retarded cell proliferation, we tested changes of cell cycle distribution when USP7 was knockdown with siRNA
Summary
The deubiquitinase USP7 has been identified as an oncogene with key roles in tumorigenesis and therapeutic resistance for a series of cancer types. Small molecular inhibitors have been developed to target USP7. Protein stability is critical for normal cellular homeostasis. In addition to the autophagy-lysosome system, the ubiquitin-proteasome system (UPS) takes up approximately 80 to 90% of intracellular protein degradation [1]. In UPS-induced protein degradation, ubiquitin binds to target proteins and catalyzes them by a hierarchical cascade comprising E1, E2 and E3 ubiquitin ligases [2]. The ubiquitination is removed from the labeled proteins or from polyubiquitin chains by deubiquitinating enzymes (or deubiquitinases, DUBs). DUBs are critical in cellular growth, survival and homeostasis, and are responsible for the turnover, localization and activity of their substrate proteins. Aberrant DUB activity results in a series of diseases, including cancer [3, 4]
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