Conventional screening methods for deubiquitinating enzymes (DUBs) have important limitations. A loss-of-function study based on the knockout of DUB genes in mammalian cells can provide an excellent model for exploring DUB function. Here, we used CRISPR-Cas9 to perform genome-scale knockout of the entire set of genes encoding ubiquitin-specific proteases (USPs), a DUB subfamily, and then systematically screened for DUBs that stabilize the Cdc25A oncoprotein. USP3 was identified as a deubiquitinase of Cdc25A. USP3 depletion reduces the Cdc25A protein level, resulting in a significant delay in cell-cycle progression, and reduces the growth of cervical tumor xenografts in nude mice. Clinically, USP3 expression is positively correlated with Cdc25A protein expression and the poorest survival in breast cancer. We envision that our DUB knockout library kit will facilitate genome-scale screening of functional DUBs for target proteins of interest in a wide range of biomedical fields.
Subscribe to Journal
Get full journal access for 1 year
only $33.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Park MT, Lee SJ. Cell cycle and cancer. J Biochem Mol Biol. 2003;36:60–5.
Vermeulen K, Van Bockstaele DR, Berneman ZN. The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif. 2003;36:131–49.
Sur S, Agrawal DK. Phosphatases and kinases regulating CDC25 activity in the cell cycle: clinical implications of CDC25 overexpression and potential treatment strategies. Mol Cell Biochem. 2016;416:33–46.
Bernadette A, Bernard D. Cell cycle control by the CDC25 phosphatases. Anticancer Agents Med Chem. 2008;8:818–24.
Boutros R, Lobjois V, Ducommun B. CDC25 phosphatases in cancer cells: key players? Good targets? Nat Rev Cancer. 2007;7:495–507.
Xu X, Yamamoto H, Sakon M, Yasui M, Ngan CY, Fukunaga H, et al. Overexpression of CDC25A phosphatase is associated with hypergrowth activity and poor prognosis of human hepatocellular carcinomas. Clin Cancer Res. 2003;9:1764–72.
Donzelli M, Squatrito M, Ganoth D, Hershko A, Pagano M, Draetta GF. Dual mode of degradation of Cdc25 A phosphatase. EMBO J. 2002;21:4875–84.
Pereg Y, Liu BY, O’Rourke KM, Sagolla M, Dey A, Komuves L, et al. Ubiquitin hydrolase Dub3 promotes oncogenic transformation by stabilizing Cdc25A. Nat Cell Biol. 2010;12:400–6.
Ramakrishna S, Kwaku Dad A-B, Beloor J, Gopalappa R, Lee S-K, Kim H. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res. 2014;24:1020–7.
Kim HJ, Lee HJ, Kim H, Cho SW, Kim J-S. Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly. Genome Res. 2009;19:1279–88.
Kim S, Kim D, Cho SW, Kim J, Kim J-S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 2014;24:1012–9.
Ramakrishna S, Cho SW, Kim S, Song M, Gopalappa R, Kim JS, et al. Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nuclease-induced mutations. Nat Commun. 2014;5:3378.
Latifi-Pupovci H, Kuçi Z, Wehner S, Bönig H, Lieberz R, Klingebiel T, et al. In vitro migration and proliferation (“wound healing”) potential of mesenchymal stromal cells generated from human CD271(+) bone marrow mononuclear cells. J Transl Med. 2015;13:315.
Singh SK, Mishra MK, Eltoum I-EA, Bae S, Lillard JW, Singh R. CCR5/CCL5 axis interaction promotes migratory and invasiveness of pancreatic cancer cells. Sci Rep. 2018;8:1323.
Suresh B, Lee J, Hong SH, Kim KS, Ramakrishna S. The role of deubiquitinating enzymes in spermatogenesis. Cell Mol Life Sci. 2015;72:4711–20.
Kee Y, Huang TT. Role of deubiquitinating enzymes in DNA repair. Mol Cell Biol. 2016;36:524–44.
Chandrasekaran AP, Suresh B, Kim HH, Kim KS, Ramakrishna S. Concise review: fate determination of stem cells by deubiquitinating enzymes. Stem Cells. 2017;35:9–16.
Lim KH, Song MH, Baek KH. Decision for cell fate: deubiquitinating enzymes in cell cycle checkpoint. Cell Mol Life Sci. 2016;73:1439–55.
Liu Q, Wu Y, Qin Y, Hu J, Xie W, Qin FX-F, et al. Broad and diverse mechanisms used by deubiquitinase family members in regulating the type I interferon signaling pathway during antiviral responses. Sci Adv. 2018;4:eaar2824.
Biswas K, Philip S, Yadav A, Martin BK, Burkett S, Singh V, et al. BRE/BRCC45 regulates CDC25A stability by recruiting USP7 in response to DNA damage. Nat Commun. 2018;9:537.
Wu Y, Zhou L, Wang X, Lu J, Zhang R, Liang X, et al. A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A. Cell Discov. 2016;2:16014.
Busino L, Donzelli M, Chiesa M, Guardavaccaro D, Ganoth D, Valerio Dorrello N, et al. Degradation of Cdc25A by β-TrCP during S phase and in response to DNA damage. Nature. 2003;426:87–91.
Mailand N, Falck J, Lukas C, Syljuåsen RG, Welcker M, Bartek J, et al. Rapid destruction of human Cdc25A in response to DNA damage. Science. 2000;288:1425–9.
Bernardi R, Liebermann DA, Hoffman B. Cdc25A stability is controlled by the ubiquitin-proteasome pathway during cell cycle progression and terminal differentiation. Oncogene. 2000;19:2447–54.
Young LM, Pagano M. Cdc25 phosphatases: differential regulation by ubiquitin-mediated proteolysis. Cell Cycle. 2010;9:4613–4.
Jin J, Shirogane T, Xu L, Nalepa G, Qin J, Elledge SJ, et al. SCFbeta-TRCP links Chk1 signaling to degradation of the Cdc25A protein phosphatase. Genes Dev. 2003;17:3062–74.
Emmerich CH, Cohen P. Optimising methods for the preservation, capture and identification of ubiquitin chains and ubiquitylated proteins by immunoblotting. Biochem Biophys Res Commun. 2015;466:1–14.
Hjerpe R, Aillet F, Lopitz-Otsoa F, Lang V, England P, Rodriguez MS. Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. EMBO Rep. 2009;10:1250–8.
Hoffmann I. The role of Cdc25 phosphatases in cell cycle checkpoints. Protoplasma. 2000;211:8–11.
Shen T, Huang S. The role of Cdc25A in the regulation of cell proliferation and apoptosis. Anticancer Agents Med Chem. 2012;12:631–9.
Timofeev O, Cizmecioglu O, Settele F, Kempf T, Hoffmann I. Cdc25 phosphatases are required for timely assembly of CDK1-cyclin B at the G2/M transition. J Biol Chem. 2010;285:16978–90.
López-Contreras AJ, Fernandez-Capetillo O. The ATR barrier to replication-born DNA damage. DNA Repair. 2010;9:1249–55.
Cimprich KA, Cortez D. ATR: an essential regulator of genome integrity. Nat Rev Mol Cell Biol. 2008;9:616–27.
Ruiz S, Mayor-Ruiz C, Lafarga V, Murga M, Vega-Sendino M, Ortega S, et al. A Genome-wide CRISPR screen identifies CDC25A as a determinant of sensitivity to ATR inhibitors. Mol Cell. 2016;62:307–13.
Liu JC, Granieri L, Shrestha M, Wang D-Y, Vorobieva I, Rubie EA, et al. Identification of CDC25 as a common therapeutic target for triple-negative breast cancer. Cell Rep. 2018;23:112–26.
Cangi MG, Cukor B, Soung P, Signoretti S, Moreira G Jr., Ranashinge M, et al. Role of the Cdc25A phosphatase in human breast cancer. J Clin Investig. 2000;106:753–61.
Fang C-L, Lin C-C, Chen H-K, Hseu Y-C, Hung S-T, Sun D-P, et al. Ubiquitin-specific protease 3 overexpression promotes gastric carcinogenesis and is predictive of poor patient prognosis. Cancer Sci. 2018;109:3438–49.
Nicassio F, Corrado N, Vissers JHA, Areces LB, Bergink S, Marteijn JA, et al. Human USP3 is a chromatin modifier required for S phase progression and genome stability. Curr Biol. 2007;17:1972–7.
Lancini C, van den Berk PCM, Vissers JHA, Gargiulo G, Song J-Y, Hulsman D, et al. Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells. J Exp Med. 2014;211:1759–77.
Ray D, Kiyokawa H. CDC25A phosphatase: a rate-limiting oncogene that determines genomic stability. Cancer Res. 2008;68:1251–3.
We are grateful to all members of the SR and KSK labs, especially Naresh Poondla, Janardhan Karapurkar, and Ki-Sang Jo, for their technical support and advice. We sincerely thank Dr Hyun-Hi Kim and Prof. Hansung Jung from Yonsei University for assisting in the IHC experiments. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), which is funded by the Ministry of Education (2018M3A9H3022412, 2017M3A9B3061830, and 2015H1D3A1036065).
Conflict of interest
The authors declare that they have no conflict of interest.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Edited by S. Kumar
About this article
Cite this article
Das, S., Chandrasekaran, A.P., Suresh, B. et al. Genome-scale screening of deubiquitinase subfamily identifies USP3 as a stabilizer of Cdc25A regulating cell cycle in cancer. Cell Death Differ 27, 3004–3020 (2020). https://doi.org/10.1038/s41418-020-0557-5
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research (2020)