Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, 04763, South Korea.
Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, South Korea.
Cancer Lett. 2022 Jan 28;525:146-157. doi: 10.1016/j.canlet.2021.10.032. Epub 2021 Nov 3.
The NADPH oxidase (Nox) family of enzymes is solely dedicated in the generation of reactive oxygen species (ROS). ROS generated by Nox are involved in multiple signaling cascades and a myriad of pathophysiological conditions including cancer. As such, ROS seem to have both detrimental and beneficial roles in a number of cellular functions, including cell signaling, growth, apoptosis and proliferation. Regulatory mechanisms are required to control the activity of Nox enzymes in order to maintain ROS balance within the cell. Here, we performed genome-wide screening for deubiquitinating enzymes (DUBs) regulating Nox organizer 1 (NoxO1) protein expression using a CRISPR/Cas9-mediated DUB-knockout library. We identified cylindromatosis (CYLD) as a binding partner regulating NoxO1 protein expression. We demonstrated that the overexpression of CYLD promotes ubiquitination of NoxO1 protein and reduces the NoxO1 protein half-life. The destabilization of NoxO1 protein by CYLD suppressed excessive ROS generation. Additionally, CRISPR/Cas9-mediated knockout of CYLD in PC-3 cells promoted cell proliferation, migration, colony formation and invasion in vitro. In xenografted mice, injection of CYLD-depleted cells consistently led to tumor development with increased weight and volume. Taken together, these results indicate that CYLD acts as a destabilizer of NoxO1 protein and could be a potential tumor suppressor target for cancer therapeutics.
NADPH 氧化酶（Nox）家族的酶专门用于生成活性氧物种（ROS）。Nox 产生的 ROS 参与多种信号级联和无数病理生理状况，包括癌症。因此，ROS 在许多细胞功能中似乎具有有害和有益的作用，包括细胞信号转导、生长、凋亡和增殖。需要调节机制来控制 Nox 酶的活性，以维持细胞内 ROS 的平衡。在这里，我们使用 CRISPR/Cas9 介导的 DUB 敲除文库进行了全基因组筛选，以寻找调节 Nox 组织者 1（NoxO1）蛋白表达的去泛素化酶（DUB）。我们确定了圆柱瘤（CYLD）作为调节 NoxO1 蛋白表达的结合伴侣。我们证明，CYLD 的过表达促进 NoxO1 蛋白的泛素化，并降低 NoxO1 蛋白的半衰期。CYLD 对 NoxO1 蛋白的不稳定抑制了过量的 ROS 生成。此外，PC-3 细胞中 CRISPR/Cas9 介导的 CYLD 敲除促进了体外细胞增殖、迁移、集落形成和侵袭。在异种移植小鼠中，注射耗尽 CYLD 的细胞一致导致肿瘤发展，体重和体积增加。总之，这些结果表明 CYLD 作为 NoxO1 蛋白的稳定剂，可能是癌症治疗的潜在肿瘤抑制靶标。
