BACKGROUND The 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. METHODS Cell 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. RESULTS Pharmacological 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. CONCLUSION This 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.

中文翻译：

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USP7是一种新型的去泛素酶，可维持PLK1蛋白的稳定性并调节有丝分裂中的染色体排列。

背景技术去泛素化酶USP7已被鉴定为致癌基因，其在一系列癌症类型的肿瘤发生和治疗抗性中具有关键作用。最近，已经开发出了小分子抑制剂来靶向USP7。但是，USP7抑制剂的抗癌机制仍然难以捉摸。方法采用紫水晶法检测细胞活力或克隆形成能力。通过流式细胞术分析细胞凋亡或细胞周期，并通过荧光显微镜观察染色体错位。通过串联亲和纯化和高通量蛋白质组学检测到PLK1和USP7的蛋白质相互作用，并通过共免疫沉淀，GST下拉和蛋白质共定位进一步证实。评估了肿瘤组织的USP7水平与紫杉烷抗性之间的相关性。结果P5091抑制USP7的药理作用可延缓细胞增殖并诱导细胞凋亡。进一步的研究表明，P5091诱导细胞周期停滞在G2 / M期，尤其是诱导染色体错位，表明USP7在有丝分裂中的关键作用。在与PLK1相互作用的蛋白复合物中检测到USP7蛋白。USP7通过其PBD结构域通过催化活性与PLK1蛋白相互作用。USP7作为一种去泛素化酶，可通过C223位点维持PLK1蛋白质的稳定性，相反，USP7被P5091抑制可通过泛素化-蛋白酶体途径促进PLK1的蛋白质降解。通过过表达PLK1，已经被RNAi耗尽的USP7停止在有丝分裂中诱导染色体错位，并再次支持细胞增殖和细胞存活。USP7和PLK1均在耐紫杉烷的癌细胞中过表达，并且与肿瘤组织中的MP得分呈负相关。RNAi引起的USP7或PLK1敲低使紫杉烷抗性细胞对紫杉烷细胞的杀伤作用非常敏感。结论这是第一份报道PLK1是USP7去泛素酶的新型底物，并且USP7维持了PLK1的蛋白质稳定性。USP7抑制可诱导细胞凋亡和细胞周期G2 / M停滞，并通过诱导PLK1的蛋白质降解来克服紫杉烷抗性，从而导致有丝分裂中的染色体错位。并且USP7维持了PLK1的蛋白质稳定性。USP7抑制可诱导细胞凋亡和细胞周期G2 / M停滞，并通过诱导PLK1的蛋白质降解来克服紫杉烷抗性，从而导致有丝分裂中的染色体错位。并且USP7维持了PLK1的蛋白质稳定性。USP7抑制可诱导细胞凋亡和细胞周期G2 / M停滞，并通过诱导PLK1的蛋白质降解来克服紫杉烷抗性，从而导致有丝分裂中的染色体错位。