A Selective Inhibitor of Ubiquitin-Specific Protease 4 Suppresses Colorectal Cancer Progression by Regulating beta-Catenin Signaling.
A Selective Inhibitor of Ubiquitin-Specific Protease 4 Suppresses Colorectal Cancer Progression by Regulating beta-Catenin Signaling.
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DOI：
10.33594/000000127
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发表时间：
2019-01-01
期刊：
影响因子：
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通讯作者：
Kim, Kyeong Kyu
中科院分区：
文献类型：
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作者：
Nguyen, Hoa Hong;Kim, Truc;Kim, Kyeong Kyu
背景/目的：调节关键蛋白质稳定性的去泛素化酶(DUBS)的失调与包括癌症在内的许多人类疾病有关。因此，DUBS可以被认为是许多疾病的潜在治疗靶点。其中，由于USP4控制β-连环蛋白的稳定性，USP4已被认为是结肠癌药物的有希望的靶点。β-连环蛋白是参与结直肠癌发生的Wnt信号的关键因子。然而，由于许多DUB具有相似的活性部位结构并表现出广泛的底物特异性，开发潜在的DUB抑制剂一直受到阻碍。方法：通过使用化学文库进行体外脱泛素化活性分析，我们确定了几种潜在的DUB抑制剂。其中，只有中性红(NR)对USP4具有选择性抑制活性。免疫印迹结果表明，在结肠癌细胞中，NR影响β-连环蛋白的蛋白稳定性，实时荧光定量聚合酶链式反应(QRT-PCR)显示其影响β-连环蛋白的靶基因表达。通过克隆形成和细胞迁移分析以及小鼠异种移植模型，进一步评估了NR作为抗癌药物的潜力。结果：我们确定NR是USP4的非竞争性抑制物，并验证了其在结直肠癌中的作用。NR处理的细胞表现出β-连环蛋白稳定性降低和β-连环蛋白靶基因表达减少。此外，用NR处理结肠癌细胞明显减少集落形成和细胞迁移，并将NR注射到小鼠异种移植模型中，减少了肿瘤体积。结论：目前的结果表明，NR可以作为靶向USP4的抗癌药物，并支持开发特异性DUB抑制剂作为治疗药物的可能性。
BACKGROUND/AIMS: Dysregulation of deubiquitinating enzymes (DUBs), which regulate the stability of key proteins, has been implicated in many human diseases, including cancers. Thus, DUBs can be considered as potential therapeutic targets for many diseases. Among them, USP4 has been proposed as a promising target for colon cancer drugs since USP4 controls the stability of beta-catenin, a key factor in the Wnt signaling involved in the tumorigenesis of colorectal cancer. However, developing potential DUB inhibitors has been hindered because many DUBs harbor similar active site structures and show broad substrate specificities.METHODS: By performing in vitro deubiquitinating activity assays using a chemical library, we identified several potential DUB inhibitors. Among them, only neutral red (NR) showed selective inhibitory activity on USP4 in a cell-based assay system. In colon cancer cells, NR affected the protein stability of beta-catenin, as shown by immunoblotting, and it affected the target gene expression of beta-catenin, as shown by quantitative real-time PCR. NR's potential as an anticancer drug was further estimated by colony formation and cell migration assays and by using a mouse xenograft model.RESULTS: We identified NR as an uncompetitive inhibitor of USP4 and validated its effects in colorectal cancer. NR-treated cells showed decreased beta-catenin stability and reduced expression of beta-catenin target genes. Additionally, treating colon cancer cells with NR significantly reduced colony formation and cell migration, and injecting NR into a mouse xenograft model reduced the tumor volume.CONCLUSION: The current results suggest that NR could be developed as an anticancer drug targeting USP4, and they support the possibility of developing specific DUB inhibitors as therapeutic agents.