CCN6是一种细胞外矩阵蛋白，对乳腺癌的肿瘤发生和进展起到关键调节作用。虽然CCN6的抗肿瘤功能已经广泛研究，但其蛋白水平的调控机制仍不清楚。本研究旨在调查泛素化和去泛素酶（DUBs）对CCN6的调控。通过筛选分析找到了OTUB1作为CCN6的DUB。运用多种生化方法阐明OTUB1在CCN6调控中的分子机制。通过细胞实验和移植模型研究了OTUB1-CCN6互作在乳腺癌中的作用。通过免疫组化和Western blot确定了OTUB1和CCN6在人乳腺癌中的相关性。我们发现CCN6的蛋白水平受泛素蛋白酶体系统控制。OTUB1通过直接与CCN6的连接域相互作用来抑制CCN6的K48泛素化和降解。此外，OTUB1以一种非经典方式抑制CCN6的泛素化。OTUB1的缺失导致CCN6减少，并增加乳腺癌细胞的迁移、增殖和存活能力。CCN6的补充可以消除OTUB1缺失对乳腺癌的影响。重要的是，OTUB1在人乳腺癌中表达下调，并与CCN6水平呈正相关。本研究确定了OTUB1作为乳腺癌中CCN6的新调控因子。© 2023 The Authors. Shanghai Institute of Clinical Bioinformatics 代表 John Wiley & Sons Australia, Ltd 发表的《临床与转化医学》出版物

CCN6 is a matricellular protein that critically regulates the tumourigenesis and progression of breast cancer. Although the tumour-suppressive function of CCN6 has been extensively studied, molecular mechanisms regulating protein levels of CCN6 remain largely unclear. This study aims to investigate the regulation of CCN6 by ubiquitination and deubiquitinating enzymes (DUBs) in breast cancer.A screening assay was performed to identify OTUB1 as the DUB for CCN6. Various biochemical methods were applied to elucidate the molecular mechanism of OTUB1 in the regulation of CCN6. The role of OTUB1-CCN6 interaction in breast cancer was studied with cell experiments and the allograft model. The correlation of OTUB1 and CCN6 in human breast cancer was determined by immunohistochemistry and Western blot.We found that CCN6 protein levels were controlled by the ubiquitin-proteasome system. The K48 ubiquitination and degradation of CCN6 was inhibited by OTUB1, which directly interacted with CCN6 through its linker domain. Furthermore, OTUB1 inhibited the ubiquitination of CCN6 in a non-canonical manner. Deletion of OTUB1, concomitant with reduced CCN6 abundance, increased the migration, proliferation and viability of breast cancer cells. Supplementation of CCN6 abolished the effect of OTUB1 deletion on breast cancer. Importantly, OTUB1 expression was downregulated in human breast cancer and positively correlated with CCN6 levels.This study identified OTUB1 as a novel regulator of CCN6 in breast cancer.© 2023 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.