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

Acta Pharmacologica Sinica volume 44, pages 2537–2548 (2023)Cite this article

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.

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Fig. 1: USP14 and UCHL5 may play important roles in the 5-FU resistance of CRC cells.
Fig. 2: b-AP15 effectively inhibits cell viability and cell migration of both 5-FU-sensitive and 5-FU-resistant CRC cell lines.
Fig. 3: b-AP15-induced apoptosis is related to mitochondrial dysfunction and caspase activation.
Fig. 4: b-AP15 suppresses NF-κB activation in both 5-FU-sensitive and 5-FU-resistant CRC cells.
Fig. 5: b-AP15 reverses 5-FU resistance in CRC cells through the induction of ROS.
Fig. 6: b-AP15 restrains the growth of 5-FU-sensitive and -resistant CRC xenografts in nude mice.
Fig. 7: Scheme summarizing the mechanisms of action of b-AP15 on 5-FU-resistant CRC cells.

References

  1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71:209–49.

    Article  PubMed  Google Scholar 

  2. Weitz J, Koch M, Debus J, Hohler T, Galle PR, Buchler MW. Colorectal cancer. Lancet. 2005;365:153–65.

    Article  PubMed  Google Scholar 

  3. Vodenkova S, Buchler T, Cervena K, Veskrnova V, Vodicka P, Vymetalkova V. 5-Fluorouracil and other fluoropyrimidines in colorectal cancer: Past, present and future. Pharmacol Ther. 2020;206:107447.

    Article  CAS  PubMed  Google Scholar 

  4. Longley DB, Harkin DP, Johnston PG. 5-Fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer. 2003;3:330–8.

    Article  CAS  PubMed  Google Scholar 

  5. Douillard JY, Cunningham D, Roth AD, Navarro M, James RD, Karasek P, et al. Irinotecan combined with fluorouracil compared with fluorouracil alone as first-line treatment for metastatic colorectal cancer: a multicentre randomised trial. Lancet. 2000;355:1041–7.

    Article  CAS  PubMed  Google Scholar 

  6. Giacchetti S, Perpoint B, Zidani R, Le Bail N, Faggiuolo R, Focan C, et al. Phase III multicenter randomized trial of oxaliplatin added to chronomodulated fluorouracil-leucovorin as first-line treatment of metastatic colorectal cancer. J Clin Oncol. 2000;18:136–47.

    Article  CAS  PubMed  Google Scholar 

  7. Sethy C, Kundu CN. 5-Fluorouracil (5-FU) resistance and the new strategy to enhance the sensitivity against cancer: Implication of DNA repair inhibition. Biomed Pharmacother. 2021;137:111285.

    Article  CAS  PubMed  Google Scholar 

  8. Blondy S, David V, Verdier M, Mathonnet M, Perraud A, Christou N. 5-Fluorouracil resistance mechanisms in colorectal cancer: From classical pathways to promising processes. Cancer Sci. 2020;111:3142–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Liu J, Leung CT, Liang L, Wang Y, Chen J, Lai KP, et al. Deubiquitinases in cancers: aspects of proliferation, metastasis, and apoptosis. Cancers. 2022;14:3547.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Wang F, Ning S, Yu B, Wang Y. USP14: structure, function, and target inhibition. Front Pharmacol. 2021;12:801328.

    Article  CAS  PubMed  Google Scholar 

  11. Ma YS, Wang XF, Zhang YJ, Luo P, Long HD, Li L, et al. Inhibition of USP14 deubiquitinating activity as a potential therapy for tumors with p53 deficiency. Mol Ther Oncolytics. 2020;16:147–57.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Han KH, Kwak M, Lee TH, Park MS, Jeong IH, Kim MJ, et al. USP14 inhibition regulates tumorigenesis by inducing autophagy in lung cancer in vitro. Int J Mol Sci. 2019;20:5300.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Liao Y, Xia X, Liu N, Cai J, Guo Z, Li Y, et al. Growth arrest and apoptosis induction in androgen receptor-positive human breast cancer cells by inhibition of USP14-mediated androgen receptor deubiquitination. Oncogene. 2018;37:1896–910.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Zhang J, Xu H, Yang X, Zhao Y, Xu X, Zhang L, et al. Deubiquitinase UCHL5 is elevated and associated with a poor clinical outcome in lung adenocarcinoma (LUAD). J Cancer. 2020;11:6675–85.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Liu D, Song Z, Wang X, Ouyang L. Ubiquitin C-terminal hydrolase L5 (UCHL5) accelerates the growth of endometrial cancer via activating the Wnt/beta-Catenin signaling pathway. Front Oncol. 2020;10:865.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Arpalahti L, Laitinen A, Hagstrom J, Mustonen H, Kokkola A, Bockelman C, et al. Positive cytoplasmic UCHL5 tumor expression in gastric cancer is linked to improved prognosis. PLoS One. 2018;13:e0193125.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Arpalahti L, Hagstrom J, Mustonen H, Lundin M, Haglund C, Holmberg CI. UCHL5 expression associates with improved survival in lymph-node-positive rectal cancer. Tumour Biol. 2017;39:1010428317716078.

    Article  PubMed  Google Scholar 

  18. D’Arcy P, Brnjic S, Olofsson MH, Fryknas M, Lindsten K, De Cesare M, et al. Inhibition of proteasome deubiquitinating activity as a new cancer therapy. Nat Med. 2011;17:1636–40.

    Article  PubMed  Google Scholar 

  19. Zhang X, Pellegrini P, Saei AA, Hillert EK, Mazurkiewicz M, Olofsson MH, et al. The deubiquitinase inhibitor b-AP15 induces strong proteotoxic stress and mitochondrial damage. Biochem Pharmacol. 2018;156:291–301.

    Article  CAS  PubMed  Google Scholar 

  20. Jiang L, Sun Y, Wang J, He Q, Chen X, Lan X, et al. Proteasomal cysteine deubiquitinase inhibitor b-AP15 suppresses migration and induces apoptosis in diffuse large B cell lymphoma. J Exp Clin Cancer Res. 2019;38:453.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Morgan EL, Toni T, Viswanathan R, Robbins Y, Yang X, Cheng H, et al. Inhibition of USP14 promotes TNFalpha-induced cell death in head and neck squamous cell carcinoma (HNSCC). Cell Death Differ. 2023;30:1382–96.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Yang Y, Cao L, Guo Z, Gu H, Zhang K, Qiu Z. Deubiquitinase UCHL5 stabilizes ELK3 to potentiate cancer stemness and tumor progression in pancreatic adenocarcinoma (PAAD). Exp Cell Res. 2022;421:113402.

    Article  CAS  PubMed  Google Scholar 

  23. Chow PM, Dong JR, Chang YW, Kuo KL, Lin WC, Liu SH, et al. The UCHL5 inhibitor b-AP15 overcomes cisplatin resistance via suppression of cancer stemness in urothelial carcinoma. Mol Ther Oncolytics. 2022;26:387–98.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Jiang L, He Q, Chen X, Liu A, Ding W, Zhang H, et al. Inhibition of proteasomal deubiquitinases USP14 and UCHL5 overcomes tyrosine kinase inhibitor resistance in chronic myeloid leukaemia. Clin Transl Med. 2022;12:e1038.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Wen C, Chen J, Zhang D, Wang H, Che J, Qin Q, et al. Pseudolaric acid B induces mitotic arrest and apoptosis in both 5-fluorouracil-sensitive and -resistant colorectal cancer cells. Cancer Lett. 2016;383:295–308.

    Article  CAS  PubMed  Google Scholar 

  26. Wen C, Huang L, Chen J, Lin M, Li W, Lu B, et al. Gambogic acid inhibits growth, induces apoptosis, and overcomes drug resistance in human colorectal cancer cells. Int J Oncol. 2015;47:1663–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Chen X, Huang J, Yu C, Liu J, Gao W, Li J, et al. A noncanonical function of EIF4E limits ALDH1B1 activity and increases susceptibility to ferroptosis. Nat Commun. 2022;13:6318.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Koulich E, Li X, DeMartino GN. Relative structural and functional roles of multiple deubiquitylating proteins associated with mammalian 26S proteasome. Mol Biol Cell. 2008;19:1072–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Tian Z, D’Arcy P, Wang X, Ray A, Tai YT, Hu Y, et al. A novel small molecule inhibitor of deubiquitylating enzyme USP14 and UCHL5 induces apoptosis in multiple myeloma and overcomes bortezomib resistance. Blood. 2014;123:706–16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Huxford T, Huang DB, Malek S, Ghosh G. The crystal structure of the IkappaBalpha/NF-kappaB complex reveals mechanisms of NF-kappaB inactivation. Cell. 1998;95:759–70.

    Article  CAS  PubMed  Google Scholar 

  31. Danesh Pouya F, Rasmi Y, Nemati M. Signaling pathways involved in 5-FU drug resistance in cancer. Cancer Invest. 2022;40:516–43.

    Article  CAS  PubMed  Google Scholar 

  32. Okazaki T, Sakon S, Sasazuki T, Sakurai H, Doi T, Yagita H, et al. Phosphorylation of serine 276 is essential for p65 NF-kappaB subunit-dependent cellular responses. Biochem Biophys Res Commun. 2003;300:807–12.

    Article  CAS  PubMed  Google Scholar 

  33. Karin M, Yamamoto Y, Wang QM. The IKK NF-kappa B system: a treasure trove for drug development. Nat Rev Drug Discov. 2004;3:17–26.

    Article  CAS  PubMed  Google Scholar 

  34. Grumont RJ, Strasser A, Gerondakis S. B cell growth is controlled by phosphatidylinosotol 3-kinase-dependent induction of Rel/NF-kappaB regulated c-myc transcription. Mol Cell. 2002;10:1283–94.

    Article  CAS  PubMed  Google Scholar 

  35. Kawakami H, Tomita M, Matsuda T, Ohta T, Tanaka Y, Fujii M, et al. Transcriptional activation of survivin through the NF-kappaB pathway by human T-cell leukemia virus type I tax. Int J Cancer. 2005;115:967–74.

    Article  CAS  PubMed  Google Scholar 

  36. Hinz M, Krappmann D, Eichten A, Heder A, Scheidereit C, Strauss M. NF-kappaB function in growth control: regulation of cyclin D1 expression and G0/G1-to-S-phase transition. Mol Cell Biol. 1999;19:2690–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Paniagua Soriano G, De Bruin G, Overkleeft HS, Florea BI. Toward understanding induction of oxidative stress and apoptosis by proteasome inhibitors. Antioxid Redox Signal. 2014;21:2419–43.

    Article  CAS  PubMed  Google Scholar 

  38. Fricker LD. Proteasome inhibitor drugs. Annu Rev Pharmacol Toxicol. 2020;60:457–76.

    Article  CAS  PubMed  Google Scholar 

  39. Paulus A, Akhtar S, Caulfield TR, Samuel K, Yousaf H, Bashir Y, et al. Coinhibition of the deubiquitinating enzymes, USP14 and UCHL5, with VLX1570 is lethal to ibrutinib- or bortezomib-resistant Waldenstrom macroglobulinemia tumor cells. Blood Cancer J. 2016;6:e492.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Wu T, Li C, Zhou C, Niu X, Li G, Zhou Y, et al. Inhibition of USP14 enhances anti-tumor effect in vemurafenib-resistant melanoma by regulation of Skp2. Cell Biol Toxicol. 2022 Jun 1. https://doi.org/10.1007/s10565-022-09729-x.

  41. Fu Y, Ma G, Liu G, Li B, Li H, Hao X, et al. USP14 as a novel prognostic marker promotes cisplatin resistance via Akt/ERK signaling pathways in gastric cancer. Cancer Med. 2018;7:5577–88.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Chen X, Shi X, Zhao C, Li X, Lan X, Liu S, et al. Anti-rheumatic agent auranofin induced apoptosis in chronic myeloid leukemia cells resistant to imatinib through both Bcr/Abl-dependent and -independent mechanisms. Oncotarget. 2014;5:9118–32.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Chen X, Yang Q, Xiao L, Tang D, Dou QP, Liu J. Metal-based proteasomal deubiquitinase inhibitors as potential anticancer agents. Cancer Metastasis Rev. 2017;36:655–68.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Ma YS, Wang XF, Yu F, Wu TM, Liu JB, Zhang YJ, et al. Inhibition of USP14 and UCH37 deubiquitinating activity by b-AP15 as a potential therapy for tumors with p53 deficiency. Signal Transduct Target Ther. 2020;5:30.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Rowinsky EK, Paner A, Berdeja JG, Paba-Prada C, Venugopal P, Porkka K, et al. Phase 1 study of the protein deubiquitinase inhibitor VLX1570 in patients with relapsed and/or refractory multiple myeloma. Invest New Drugs. 2020;38:1448–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Longley DB, Johnston PG. Molecular mechanisms of drug resistance. J Pathol. 2005;205:275–92.

    Article  CAS  PubMed  Google Scholar 

  47. Rosini E, Pollegioni L. Reactive oxygen species as a double-edged sword: The role of oxidative enzymes in antitumor therapy. Biofactors. 2022;48:384–99.

    Article  CAS  PubMed  Google Scholar 

  48. Kurozumi N, Tsujioka T, Ouchida M, Sakakibara K, Nakahara T, Suemori SI, et al. VLX1570 induces apoptosis through the generation of ROS and induction of ER stress on leukemia cell lines. Cancer Sci. 2021;112:3302–13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Jarvius M, Fryknas M, D’Arcy P, Sun C, Rickardson L, Gullbo J, et al. Piperlongumine induces inhibition of the ubiquitin-proteasome system in cancer cells. Biochem Biophys Res Commun. 2013;431:117–23.

    Article  CAS  PubMed  Google Scholar 

  50. Kang KA, Ryu YS, Piao MJ, Shilnikova K, Kang HK, Yi JM, et al. DUOX2-mediated production of reactive oxygen species induces epithelial mesenchymal transition in 5-fluorouracil resistant human colon cancer cells. Redox Biol. 2018;17:224–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Bechtel W, Bauer G. Catalase protects tumor cells from apoptosis induction by intercellular ROS signaling. Anticancer Res. 2009;29:4541–57.

    CAS  PubMed  Google Scholar 

  52. Nobrega-Pereira S, Fernandez-Marcos PJ, Brioche T, Gomez-Cabrera MC, Salvador-Pascual A, Flores JM, et al. G6PD protects from oxidative damage and improves healthspan in mice. Nat Commun. 2016;7:10894.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (82170177/H0809 and 81670154/H0812), the Foundation of Innovation Projects of General Colleges and Universities in Guangdong Province (2020KTSCX10), the Natural Science Foundation of Guangdong Province (2021A1515011334 and 2023A1515011976), Key Discipline of Guangzhou Education Bureau (Basic Medicine) (201851839), the open research funds from the Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People’s Hospital (202011-203), the Innovation Team of General Universities in Guangdong Province (2022KCXTD021), and the Foundation of Guangzhou Science and Technology Innovation Committee (202201010811) to XPS.

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Author notes

  1. These authors contributed equally: Wa Ding, Jin-xiang Wang, Jun-zheng Wu, Ao-chu Liu

Authors and Affiliations

  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

    Wa Ding, Jun-zheng Wu, Ao-chu Liu, Li-ling Jiang, Hai-chuan Zhang, Yi Meng, Bing-yuan Liu, Guan-jie Peng, En-zhe Lou, Qiong Mao, Huan Zhou, Xin Chen, Jin-bao Liu & Xian-ping Shi

  2. Guangdong Provincial Education Department Key Laboratory of Nano-Immunoregulation Tumour Microenvironment, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, 510260, China

    Wa Ding

  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

    Jin-xiang Wang

  4. Department of Surgery, UT Southwestern Medical Center, Dallas, TX, 75390, USA

    Dao-lin Tang

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  1. Wa Ding
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Contributions

XPS, JBL, and XC designed the study. WD, JXW, JZW, and ACL conducted the experiments and analyzed the data. LLJ, HCZ, YM, BYL, GJP, EZL, QM, and HZ participated in the experiments. WD and XC wrote the manuscript. DLT edited the manuscript.

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Correspondence to Xin Chen, Jin-bao Liu or Xian-ping Shi.

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Ding, W., Wang, Jx., Wu, Jz. et al. Targeting proteasomal deubiquitinases USP14 and UCHL5 with b-AP15 reduces 5-fluorouracil resistance in colorectal cancer cells. Acta Pharmacol Sin 44, 2537–2548 (2023). https://doi.org/10.1038/s41401-023-01136-0

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