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Genome-scale screening of deubiquitinase subfamily identifies USP3 as a stabilizer of Cdc25A regulating cell cycle in cancer

Abstract

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.

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Fig. 1: DUBKO library kit design and application for genome-scale screening for functional DUBs.
Fig. 2: Screening for functional DUBs that regulate Cdc25A stability using the DUBKO library kit.
Fig. 3: USP3 interacts with Cdc25A and regulates Cdc25A stability.
Fig. 4: USP3 deubiquitinates Cdc25A and extends the half-life of Cdc25A protein.
Fig. 5: Loss of USP3 destabilizes Cdc25A, leading to cell-cycle arrest.
Fig. 6: The role of USP3 in the pathogenesis of cancer.
Fig. 7: USP3 overexpression in human breast cancer correlates with increased Cdc25A expression and poor prognosis.

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Acknowledgements

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).

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Correspondence to Hyongbum Henry Kim or Kye-Seong Kim or Suresh Ramakrishna.

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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

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