P53 pathway inactivation plays an important role in the process of breast cancer tumorigenesis. Post-translational protein modification abnormalities have been confirmed to be an important mechanism underlying inactivation of p53. Numerous deubiquitinating enzymes are aberrantly expressed in breast cancer, and a few deubiquitination enzymes can deubiquitinate and stabilize p53. Here, we report that ovarian tumor (OTU) deubiquitinase 3 (OTUD3) is a deubiquitylase of p53 in breast carcinoma (BC). Correlations between the mRNA expression levels of OTUD3, TP53 and PTEN and the prognosis of BC were assessed with the Kaplan-Meier Plotter tool. OTUD3 protein expression in 80 pairs of specimens in our cohort was examined by immunohistochemistry and western blotting. The relationship among OTUD3, p53, and p21 proteins was analyzed. Half-life analysis and ubiquitylation assay were performed to elucidate the molecular mechanism by which OTUD3 stabilizes p53. The interaction between OTUD3 and p53 in BC cells was verified by a co-immunoprecipitation assay and GST pulldown experiments. MTS assay for proliferation detection, detection of apoptosis induced by cisplatin and colony formation assay were employed to investigate the functional effects of OTUD3 on breast cancer cells. OTUD3 downregulation is correlated with a poor prognosis in BC patients. OTUD3 expression is decreased in breast cancer tissues and not associated with the histological grade. OTUD3 also inhibits cell proliferation and clone formation and increases the sensitivity of BC cells to apoptosis induced by chemotherapy drugs. Reduced OTUD3 expression accompanied by decreased p53 abundance is correlated with human breast cancer progression. Ectopic expression of wild-type OTUD3, but not its catalytically inactive mutant, stabilizes and activates p53. Mechanistically, OTUD3 interacts directly with p53 through the amino-terminal OTU region. Finally, OTUD3 protects p53 from murine double minute 2 (Mdm2)-mediated ubiquitination and degradation, enabling the deubiquitination of p53 in BC cells. In summary, we found that OTUD3 may be a potential therapeutic target for restoring p53 function in breast cancer cells and suggest that the OTUD3-p53 signaling axis may play a critical role in tumor suppression.

中文翻译：

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肿瘤抑制因子OTUD3通过直接去泛化和稳定侵袭性乳腺癌细胞中的p53来诱导生长抑制和细胞凋亡。

P53途径的失活在乳腺癌的肿瘤发生过程中起着重要的作用。翻译后蛋白质修饰异常已被证实是p53失活的重要机制。在乳腺癌中异常表达了许多去泛素化酶，并且一些去泛素化酶可以使p53泛素化。在这里，我们报告卵巢肿瘤（OTU）去泛素化酶3（OTUD3）是乳腺癌（BC）中p53的去泛素化酶。使用Kaplan-Meier绘图仪评估OTUD3，TP53和PTEN的mRNA表达水平与BC预后的相关性。通过免疫组织化学和蛋白质印迹检测了我们队列中80对样本中OTUD3蛋白的表达。分析了OTUD3，p53和p21蛋白之间的关系。进行了半衰期分析和泛素化分析，以阐明OTUD3稳定p53的分子机制。OTUD3和p53在BC细胞中的相互作用通过免疫共沉淀试验和GST下拉实验进行了验证。采用MTS法进行增殖检测，顺铂诱导的凋亡检测和集落形成分析，研究OTUD3对乳腺癌细胞的功能作用。OTUD3的下调与BC患者的不良预后相关。OTUD3表达在乳腺癌组织中降低，并且与组织学分级无关。OTUD3还抑制细胞增殖和克隆形成，并提高BC细胞对化疗药物诱导的凋亡的敏感性。OTUD3表达降低，伴随p53丰度降低与人类乳腺癌的进展相关。野生型OTUD3的异位表达可稳定并激活p53，但不能催化失活。从机理上讲，OTUD3通过氨基末端OTU区直接与p53相互作用。最终，OTUD3保护p53免受鼠类双分2（Mdm2）介导的泛素化和降解，使p53在BC细胞中去泛素化。总而言之，我们发现OTUD3可能是恢复乳腺癌细胞p53功能的潜在治疗靶标，并暗示OTUD3-p53信号轴可能在肿瘤抑制中起关键作用。OTUD3通过氨基末端OTU区直接与p53相互作用。最终，OTUD3保护p53免受鼠类双分2（Mdm2）介导的泛素化和降解，使p53在BC细胞中去泛素化。总而言之，我们发现OTUD3可能是恢复乳腺癌细胞p53功能的潜在治疗靶标，并暗示OTUD3-p53信号轴可能在肿瘤抑制中起关键作用。OTUD3通过氨基末端OTU区直接与p53相互作用。最终，OTUD3保护p53免受鼠类双分2（Mdm2）介导的泛素化和降解，使p53在BC细胞中去泛素化。总而言之，我们发现OTUD3可能是恢复乳腺癌细胞p53功能的潜在治疗靶标，并暗示OTUD3-p53信号轴可能在肿瘤抑制中起关键作用。