The Deubiquitinating Enzyme USP1 is Auto-Ubiquitinated and Destabilized by ML323 in Colorectal Cancer Cells

Xu, Xin; MEI, Xiao; Han, Kunkun; Yang, Yili; Ruili, Li; Wu, Guanting

doi:10.14744/ejmo.2023.46574

The Deubiquitinating Enzyme USP1 is Auto-Ubiquitinated and Destabilized by ML323 in Colorectal Cancer Cells

Xin XU, (Center for Self-Propelled Nanotechnologies, College of Biotechnology, Suzhou Industrial Park Institute of Services Outsourcing Suzhou, Jiangsu, P. R. China)

Xiao MEI, (Center for Self-Propelled Nanotechnologies, College of Biotechnology, Suzhou Industrial Park Institute of Services Outsourcing Suzhou, Jiangsu, P. R. China)

Kunkun HAN, (Center for Self-Propelled Nanotechnologies, College of Biotechnology, Suzhou Industrial Park Institute of Services Outsourcing Suzhou, Jiangsu, P. R. China)

Guanting WU, (Department of General Surgery, The Affiliated Jiangsu Shengze Hospital of Nanjing Medical University, Suzhou, Jiangsu, P. R. China)

Li Ruili, (Center for Self-Propelled Nanotechnologies, College of Biotechnology, Suzhou Industrial Park Institute of Services Outsourcing Suzhou, Jiangsu, P. R. China)

Yili YANG (Center for Self-Propelled Nanotechnologies, College of Biotechnology, Suzhou Industrial Park Institute of Services Outsourcing Suzhou, Jiangsu, P. R. China)

Eurasian Journal of Medicine and Oncology

5 1

Yıl: 2023 Cilt: 7 Sayı: 2 Sayfa Aralığı: 174 - 179 Metin Dili: İngilizce DOI: 10.14744/ejmo.2023.46574 İndeks Tarihi: 05-07-2023

The Deubiquitinating Enzyme USP1 is Auto-Ubiquitinated and Destabilized by ML323 in Colorectal Cancer Cells

Öz:

Objectives: Our previous study indicated that USP1 inhibitor ML323 downregulated USP1 in colorectal cancer (CRC) cells, but the specific mechanism was still unknown. Methods: CRC cells were lysed for immunoblotting to detect protein expressions. Quantitative real-time PCR was per- formed to examine mRNA levels. Cycloheximide chase assays were carried out to evaluate the half-life of USP1. Co- immunoprecipitation was used to analyze the polyubiquitination of USP1. Results: USP1 protein stability was enhanced by the proteasome inhibitor MG132 in CRC cells. The wild-type USP1 was upregulated by MG132, but not its catalytic mutant. Additionally, the polyubiquitination of USP1 was enhanced by MG132 as well, which indicated USP1 was degraded through the ubiquitin-proteasome pathway. Meanwhile, we confirmed ML323 downregulated USP1 expression in CRC cells, and cycloheximide chase assay also revealed ML323 reduced USP1 protein stability. Further results showed ML323-induced USP1 downregulation and destabilization were abolished by MG132. Moreover, USP1 protein destabilization was not reversed by the caspase inhibitor Z-VAD, which further suggested ML323-induced USP1 downregulation was not dependent on the effects of cell death in CRC cells. Conclusion: Our results showed USP1 was auto-ubiquitinated, and ML323 destabilized USP1 through the ubiquitin- proteasome pathway in CRC cells, providing a theoretical basis for anti-CRC drugs’ development targeting USP1.

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1. Dekker E, Tanis PJ, Vleugels JLA, Kasi PM, Wallace MB. Colorec- tal cancer. Lancet 2019;394:1467–80.
2. Li J, Ma X, Chakravarti D, Shalapour S, DePinho RA. Genetic and biological hallmarks of colorectal cancer. Genes Dev 2021;35:787–820.
3. Mahmoud NN. Colorectal cancer: Preoperative evaluation and staging. Surg Oncol Clin N Am 2022;31:127–41.
4. Piawah S, Venook AP. Targeted therapy for colorectal cancer metastases: A review of current methods of molecularly tar- geted therapy and the use of tumor biomarkers in the treat- ment of metastatic colorectal cancer. Cancer 2019;125:4139– 47.
5. Harrigan JA, Jacq X, Martin NM, Jackson SP. Deubiquitylating enzymes and drug discovery: emerging opportunities. Nat Rev Drug Discov 2018;17:57–78.
6. Varshavsky A. The ubiquitin system, autophagy, and regulat- ed protein degradation. Annu Rev Biochem 2017;86:123–8.
7. Carroll EC, Marqusee S. Site-specific ubiquitination: De- constructing the degradation tag. Curr Opin Struct Biol 2022;73:102345.
8. Komander D, Clague MJ, Urbé S. Breaking the chains: struc- ture and function of the deubiquitinases. Nat Rev Mol Cell Biol 2009;10:550–63.
9. Han S, Wang R, Zhang Y, Li X, Gan Y, Gao F, et al. The role of ubiquitination and deubiquitination in tumor invasion and metastasis. Int J Biol Sci 2022;18:2292–303.
10. Lange SM, Armstrong LA, Kulathu Y. Deubiquitinases: From mechanisms to their inhibition by small molecules. Mol Cell 2022;82:15–29.
11. García-Santisteban I, Peters GJ, Giovannetti E, Rodríguez JA. USP1 deubiquitinase: cellular functions, regulatory mecha- nisms and emerging potential as target in cancer therapy. Mol Cancer 2013;12:91.
12. Xu X, Li S, Cui X, Han K, Wang J, Hou X, et al. Inhibition of ubiq- uitin specific protease 1 sensitizes colorectal cancer cells to DNA-damaging chemotherapeutics. Front Oncol 2019;9:1406.
13. Xu X, Huang A, Cui X, Han K, Hou X, Wang Q, et al. Ubiquitin specific peptidase 5 regulates colorectal cancer cell growth by stabilizing Tu translation elongation factor. Theranostics 2019;9:4208–20.
14. Sun Y, Sha B, Huang W, Li M, Zhao S, Zhang Y, et al. ML323, a USP1 inhibitor triggers cell cycle arrest, apoptosis and au- tophagy in esophageal squamous cell carcinoma cells. Apop- tosis 2022;27:545–60.
15. Xu X, Wang J, Han K, Li S, Xu F, Yang Y. Antimalarial drug me - floquine inhibits nuclear factor kappa B signaling and induces apoptosis in colorectal cancer cells. Cancer Sci 2018;109:1220– 9.
16. Hou X, Xia J, Feng Y, Cui L, Yang Y, Yang P, et al. USP47-me - diated deubiquitination and stabilization of TCEA3 attenu- ates pyroptosis and apoptosis of colorectal cancer cells in- duced by chemotherapeutic doxorubicin. Front Pharmacol 2021;12:713322.
17. Dexheimer TS, Rosenthal AS, Liang Q, Chen J, Villamil MA, Kerns EH, et al. Discovery of ML323 as a novel inhibitor of the USP1/UAF1 deubiquitinase complex. Probe Reports from the NIH Molecular Libraries Program. Available at: https://www. ncbi.nlm.nih.gov/books/NBK259186/. Accessed Jun 2, 2023.
18. Liang Q, Dexheimer TS, Zhang P, Rosenthal AS, Villamil MA, You C, et al. A selective USP1-UAF1 inhibitor links deubiquitination to DNA damage responses. Nat Chem Biol 2014;10:298–304.
19. Hodul M, Ganji R, Dahlberg CL, Raman M, Juo P. The WD40- repeat protein WDR-48 promotes the stability of the deubiq- uitinating enzyme USP-46 by inhibiting its ubiquitination and degradation. J Biol Chem 2020;295:11776–88.
20. Paiva SL, Crews CM. Targeted protein degradation: elements of PROTAC design. Curr Opin Chem Biol 2019;50:111–9.
21. Qi SM, Dong J, Xu ZY, Cheng XD, Zhang WD, Qin JJ. PROTAC: An effective targeted protein degradation strategy for cancer therapy. Front Pharmacol 2021;12:692574.
22. Zhao L, Zhao J, Zhong K, Tong A, Jia D. Targeted protein degra- dation: mechanisms, strategies and application. Signal Trans- duct Target Ther 2022;7:113.

APA	Xu X, MEI X, Han K, Wu G, Ruili L, Yang Y (2023). The Deubiquitinating Enzyme USP1 is Auto-Ubiquitinated and Destabilized by ML323 in Colorectal Cancer Cells. , 174 - 179. 10.14744/ejmo.2023.46574
Chicago	Xu Xin,MEI Xiao,Han Kunkun,Wu Guanting,Ruili Li,Yang Yili The Deubiquitinating Enzyme USP1 is Auto-Ubiquitinated and Destabilized by ML323 in Colorectal Cancer Cells. (2023): 174 - 179. 10.14744/ejmo.2023.46574
MLA	Xu Xin,MEI Xiao,Han Kunkun,Wu Guanting,Ruili Li,Yang Yili The Deubiquitinating Enzyme USP1 is Auto-Ubiquitinated and Destabilized by ML323 in Colorectal Cancer Cells. , 2023, ss.174 - 179. 10.14744/ejmo.2023.46574
AMA	Xu X,MEI X,Han K,Wu G,Ruili L,Yang Y The Deubiquitinating Enzyme USP1 is Auto-Ubiquitinated and Destabilized by ML323 in Colorectal Cancer Cells. . 2023; 174 - 179. 10.14744/ejmo.2023.46574
Vancouver	Xu X,MEI X,Han K,Wu G,Ruili L,Yang Y The Deubiquitinating Enzyme USP1 is Auto-Ubiquitinated and Destabilized by ML323 in Colorectal Cancer Cells. . 2023; 174 - 179. 10.14744/ejmo.2023.46574
IEEE	Xu X,MEI X,Han K,Wu G,Ruili L,Yang Y "The Deubiquitinating Enzyme USP1 is Auto-Ubiquitinated and Destabilized by ML323 in Colorectal Cancer Cells." , ss.174 - 179, 2023. 10.14744/ejmo.2023.46574
ISNAD	Xu, Xin vd. "The Deubiquitinating Enzyme USP1 is Auto-Ubiquitinated and Destabilized by ML323 in Colorectal Cancer Cells". (2023), 174-179. https://doi.org/10.14744/ejmo.2023.46574

APA	Xu X, MEI X, Han K, Wu G, Ruili L, Yang Y (2023). The Deubiquitinating Enzyme USP1 is Auto-Ubiquitinated and Destabilized by ML323 in Colorectal Cancer Cells. Eurasian Journal of Medicine and Oncology, 7(2), 174 - 179. 10.14744/ejmo.2023.46574
Chicago	Xu Xin,MEI Xiao,Han Kunkun,Wu Guanting,Ruili Li,Yang Yili The Deubiquitinating Enzyme USP1 is Auto-Ubiquitinated and Destabilized by ML323 in Colorectal Cancer Cells. Eurasian Journal of Medicine and Oncology 7, no.2 (2023): 174 - 179. 10.14744/ejmo.2023.46574
MLA	Xu Xin,MEI Xiao,Han Kunkun,Wu Guanting,Ruili Li,Yang Yili The Deubiquitinating Enzyme USP1 is Auto-Ubiquitinated and Destabilized by ML323 in Colorectal Cancer Cells. Eurasian Journal of Medicine and Oncology, vol.7, no.2, 2023, ss.174 - 179. 10.14744/ejmo.2023.46574
AMA	Xu X,MEI X,Han K,Wu G,Ruili L,Yang Y The Deubiquitinating Enzyme USP1 is Auto-Ubiquitinated and Destabilized by ML323 in Colorectal Cancer Cells. Eurasian Journal of Medicine and Oncology. 2023; 7(2): 174 - 179. 10.14744/ejmo.2023.46574
Vancouver	Xu X,MEI X,Han K,Wu G,Ruili L,Yang Y The Deubiquitinating Enzyme USP1 is Auto-Ubiquitinated and Destabilized by ML323 in Colorectal Cancer Cells. Eurasian Journal of Medicine and Oncology. 2023; 7(2): 174 - 179. 10.14744/ejmo.2023.46574
IEEE	Xu X,MEI X,Han K,Wu G,Ruili L,Yang Y "The Deubiquitinating Enzyme USP1 is Auto-Ubiquitinated and Destabilized by ML323 in Colorectal Cancer Cells." Eurasian Journal of Medicine and Oncology, 7, ss.174 - 179, 2023. 10.14744/ejmo.2023.46574
ISNAD	Xu, Xin vd. "The Deubiquitinating Enzyme USP1 is Auto-Ubiquitinated and Destabilized by ML323 in Colorectal Cancer Cells". Eurasian Journal of Medicine and Oncology 7/2 (2023), 174-179. https://doi.org/10.14744/ejmo.2023.46574