Abstract | BACKGROUND: METHODS: We employed a CRISPR/Cas9 system to perform a genome-wide knockout of ubiquitin-specific proteases (USPs) and used western blot analysis to screen for DUBs that regulate REST protein abundance. The interaction between USP3 and REST was confirmed by immunoprecipitation and Duolink in situ proximity assays. The deubiquitinating effect of USP3 on REST protein degradation, half-life, and neuronal differentiation was validated by immunoprecipitation, in vitro deubiquitination, protein-turnover, and immunostaining assays. The correlation between USP3 and REST expression was assessed using patient neuroblastoma datasets. The USP3 gene knockout in neuroblastoma cells was performed using CRISPR/Cas9, and the clinical relevance of USP3 regulating REST-mediated neuroblastoma tumorigenesis was confirmed by in vitro and in vivo oncogenic experiments. RESULTS: We identified a deubiquitinase USP3 that interacts with, stabilizes, and increases the half-life of REST protein by counteracting its ubiquitination in neuroblastoma. An in silico analysis showed a correlation between USP3 and REST in multiple neuroblastoma cell lines and identified USP3 as a prognostic marker for overall survival in neuroblastoma patients. Silencing of USP3 led to a decreased self-renewal capacity and promoted retinoic acid-induced differentiation in neuroblastoma. A loss of USP3 led to attenuation of REST-mediated neuroblastoma tumorigenesis in a mouse xenograft model. CONCLUSION: The findings of this study indicate that USP3 is a critical factor that blocks neuronal differentiation, which can lead to neuroblastoma. We envision that targeting USP3 in neuroblastoma tumors might provide an effective therapeutic differentiation strategy for improved survival rates of neuroblastoma patients.
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Authors | Janardhan Keshav Karapurkar, Min-Seong Kim, Jencia Carminha Colaco, Bharathi Suresh, Neha Sarodaya, Dong-Ho Kim, Chang-Hwan Park, Seok-Ho Hong, Kye-Seong Kim, Suresh Ramakrishna |
Journal | Journal of experimental & clinical cancer research : CR
(J Exp Clin Cancer Res)
Vol. 42
Issue 1
Pg. 121
(May 12 2023)
ISSN: 1756-9966 [Electronic] England |
PMID | 37170124
(Publication Type: Journal Article)
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Copyright | © 2023. The Author(s). |
Chemical References |
- Transcription Factors
- Ubiquitin-Protein Ligases
- Ubiquitin-Specific Proteases
- USP3 protein, human
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Topics |
- Animals
- Humans
- Mice
- Cell Differentiation
(genetics)
- Cell Transformation, Neoplastic
(genetics)
- CRISPR-Cas Systems
- Neuroblastoma
(genetics)
- Neurons
(physiology)
- Transcription Factors
(metabolism)
- Ubiquitin-Protein Ligases
(metabolism)
- Ubiquitin-Specific Proteases
(metabolism)
- Ubiquitination
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