5-氟尿嘧啶（5-FU）是结直肠癌（CRC）患者的一线治疗方法，但对5-FU耐药性的产生仍然是一个巨大的挑战。脱泛素酶在蛋白质降解通路中扮演着关键角色，这与癌症发展和化疗耐药性密切相关。在本研究中，我们调查了针对蛋白酶体脱泛素酶USP14和UCHL5的靶向抑制对CRC发展和5-FU耐药性的影响。通过分析GEO数据集，我们发现CRC组织中USP14和UCHL5的mRNA表达水平明显增加，并与CRC患者的生存率呈负相关。USP14和UCHL5的敲除导致5-FU耐药性的CRC细胞系（RKO-R和HCT-15R）对5-FU的敏感性增加，而在5-FU敏感的CRC细胞中过表达USP14和UCHL5则降低了5-FU的敏感性。USP14和UCHL5的特异性抑制剂B-AP15（1-5μM）剂量依赖性地抑制了RKO、RKO-R、HCT-15和HCT-15R细胞的存活。此外，B-AP15处理减少了CRC细胞的恶性表型，包括细胞增殖和迁移，并通过损害蛋白体功能和增加活性氧（ROS）产生诱导5-FU敏感和5-FU耐药的CRC细胞死亡。此外，B-AP15抑制了NF-κB通路的激活，抑制了细胞增殖。在5-FU敏感和5-FU耐药的CRC裸鼠异种移植瘤模型中，B-AP15（8 mg·kg-1·d-1，腹腔注射）有效抑制了两种类型瘤的生长。这些结果表明，USP14和UCHL5在CRC的发展和5-FU耐药性中发挥了重要作用。用B-AP15靶向USP14和UCHL5可能成为治疗CRC的有希望的治疗策略。© 2023年。该作者，独家授权给中国科学院上海药物研究所和中国药理学会。

5-Fluorouracil (5-FU) is the first-line treatment for colorectal cancer (CRC) patients, but the development of acquired resistance to 5-FU remains a big challenge. Deubiquitinases play a key role in the protein degradation pathway, which is involved in cancer development and chemotherapy resistance. In this study, we investigated the effects of targeted inhibition of the proteasomal deubiquitinases USP14 and UCHL5 on the development of CRC and resistance to 5-FU. By analyzing GEO datasets, we found that the mRNA expression levels of USP14 and UCHL5 in CRC tissues were significantly increased, and negatively correlated with the survival of CRC patients. Knockdown of both USP14 and UCHL5 led to increased 5-FU sensitivity in 5-FU-resistant CRC cell lines (RKO-R and HCT-15R), whereas overexpression of USP14 and UCHL5 in 5-FU-sensitive CRC cells decreased 5-FU sensitivity. B-AP15, a specific inhibitor of USP14 and UCHL5, (1-5 μM) dose-dependently inhibited the viability of RKO, RKO-R, HCT-15, and HCT-15R cells. Furthermore, treatment with b-AP15 reduced the malignant phenotype of CRC cells including cell proliferation and migration, and induced cell death in both 5-FU-sensitive and 5-FU-resistant CRC cells by impairing proteasome function and increasing reactive oxygen species (ROS) production. In addition, b-AP15 inhibited the activation of NF-κB pathway, suppressing cell proliferation. In 5-FU-sensitive and 5-FU-resistant CRC xenografts nude mice, administration of b-AP15 (8 mg·kg-1·d-1, intraperitoneal injection) effectively suppressed the growth of both types of tumors. These results demonstrate that USP14 and UCHL5 play an important role in the development of CRC and resistance to 5-FU. Targeting USP14 and UCHL5 with b-AP15 may represent a promising therapeutic strategy for the treatment of CRC.© 2023. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.