CDC6 is an oncogenic protein whose expression level fluctuates during the cell cycle. Although several E3 ubiquitin ligases responsible for the ubiquitin-mediated proteolysis of CDC6 have been identified, the deubiquitination pathway for CDC6 has not been investigated. The proteome-wide deubiquitinase (DUB) screening was used to identify the potential regulator of CDC6. Immunofluorescence, protein half-life and deubiquitination assays were performed to determine the protein stability of CDC6. Gain- and loss-of-function experiments were implemented to analyse the impacts of OUTD6A-CDC6 axis on tumour growth and chemosensitivity in vitro. N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced conditional Otud6a knockout (CKO) mouse model and tumour xenograft model were performed to analyse the role of OTUD6A-CDC6 axis in vivo. Tissue specimens were used to determine the association between OTUD6A and CDC6. OTUD6A interacts with, depolyubiquitinates and stabilizes CDC6 by removing K6-, K33-, and K48-linked polyubiquitination. Moreover, OTUD6A promotes cell proliferation and decreases sensitivity to chemotherapy by upregulating CDC6. CKO mice are less prone to BCa tumorigenesis induced by BBN, and knockdown of OTUD6A inhibits tumour progression in vivo. Furthermore, OTUD6A protein level has a positive correlation with CDC6 protein level, and high protein levels of OTUD6A and CDC6 are associated with poor prognosis in patients with bladder cancer. We reveal an important yet missing piece of novel DUB governing CDC6 stability. In addition, our findings propose a model for the OTUD6A-CDC6 axis that provides novel insights into cell cycle and chemosensitivity regulation, which may become a potential biomarker and promising drug target for cancer treatment.

中文翻译：

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OTUD6A 对 CDC6 的去泛素化促进肿瘤进展和化疗耐药

CDC6 是一种致癌蛋白，其表达水平在细胞周期中波动。尽管已经鉴定了几种负责泛素介导的 CDC6 蛋白水解的 E3 泛素连接酶，但 CDC6 的去泛素化途径尚未得到研究。全蛋白质组去泛素酶 (DUB) 筛选用于鉴定 CDC6 的潜在调节因子。进行免疫荧光、蛋白质半衰期和去泛素化测定以确定 CDC6 的蛋白质稳定性。进行功能获得和丧失实验来分析 OUTD6A-CDC6 轴对体外肿瘤生长和化疗敏感性的影响。采用N-丁基-N-(4-羟丁基)亚硝胺(BBN)诱导的条件性Otud6a敲除(CKO)小鼠模型和肿瘤异种移植模型来分析OTUD6A-CDC6轴在体内的作用。使用组织标本来确定 OTUD6A 和 CDC6 之间的关联。 OTUD6A 通过去除 K6、K33 和 K48 连接的多泛素化来与 CDC6 相互作用、去多泛素化并稳定 CDC6。此外，OTUD6A 通过上调 CDC6 促进细胞增殖并降低对化疗的敏感性。 CKO 小鼠不太容易发生 BBN 诱导的 BCa 肿瘤发生，并且 OTUD6A 的敲低可抑制体内肿瘤的进展。此外，OTUD6A蛋白水平与CDC6蛋白水平呈正相关，OTUD6A和CDC6蛋白水平高与膀胱癌患者预后不良相关。我们揭示了控制 CDC6 稳定性的新颖 DUB 的一个重要但缺失的部分。此外，我们的研究结果提出了 OTUD6A-CDC6 轴的模型，该模型为细胞周期和化​​学敏感性调节提供了新的见解，这可能成为潜在的生物标志物和有前途的癌症治疗药物靶点。