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Targeting proteasomal deubiquitinases USP14 and UCHL5 with b-AP15 reduces 5-fluorouracil resistance in colorectal cancer cells

Wa Ding1,2, Jin-xiang Wang3, Jun-zheng Wu1, Ao-chu Liu1, Li-ling Jiang1, Hai-chuan Zhang1, Yi Meng1, Bing-yuan Liu1, Guan-jie Peng1, En-zhe Lou1, Qiong Mao1, Huan Zhou1, Dao-lin Tang4, Xin Chen1, Jin-bao Liu1, Xian-ping Shi1
1 The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People’s Hospital, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, Sino-French Hoffmann Institute, School of Basic Medical Sciences, Guangzhou Medical University, Qingyuan 511500, China
2 Guangdong Provincial Education Department Key Laboratory of Nano-Immunoregulation Tumour Microenvironment, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou 510260, China
3 Guangdong Provincial Key Laboratory of Digestive Cancer Research, Digestive Diseases Center, Precision Medicine Center, Department of Biobank, The Seventh Affiliated Hospital, Sun Yat-Sen University, Shenzhen 518107, China
4 Department of Surgery, UT Southwestern Medical Center, Dallas, TX 75390, USA
Correspondence to: Xin Chen: chenxin@gzhmu.edu.cn, Jin-bao Liu: jliu@gzhmu.edu.cn, Xian-ping Shi: xianping.shi@gzhmu.edu.cn,
DOI: 10.1038/s41401-023-01136-0
Received: 3 January 2023
Accepted: 9 July 2023
Advance online: 1 August 2023

Abstract

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.
Keywords: USP14; UCHL5; deubiquitinase; 5-FU-resistant colorectal cancer; proteasome deubiquitinase inhibitors; b-AP15

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