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Molecular Diagnostics

Elevated USP9X drives early-to-late-stage oral tumorigenesis via stabilisation of anti-apoptotic MCL-1 protein and impacts outcome in oral cancers

Abstract

Background

Overexpression of anti-apoptotic MCL-1 protein in oral squamous cell carcinoma (OSCC) is linked to disease progression, therapy resistance and poor outcome. Despite its characteristic short half-life owing to ubiquitin–proteasome-dependent degradation, oral tumours frequently show elevated MCL-1 protein expression. Hence, we investigated the role of deubiquitinase USP9X in stabilising MCL-1 protein and its contribution to oral tumorigenesis.

Methods

Expression of MCL-1 and USP9X was assessed by immunoblotting and immunohistochemistry in oral cancer cell lines and tissues. The association between MCL-1 and USP9X was confirmed by coimmunoprecipitation and immunofluorescence. Cell death assessment was performed by MTT, flow cytometry and clonogenic assays.

Results

Both USP9X and MCL-1 are significantly elevated in oral premalignant lesions and oral tumours versus normal mucosa. USP9X interacts with and deubiquitinates MCL-1, thereby stabilising it. Pharmacological inhibition of USP9X potently induced cell death in OSCC cells in vitro and in vivo. The elevated expression of USP9X and MCL-1 correlated with poor prognosis in OSCC patients.

Conclusion

We demonstrate the oncogenic role of USP9X in driving early-to-late stages of oral tumorigenesis via stabilisation of MCL-1, suggesting its potential as a prognostic biomarker and therapeutic target in oral cancers.

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Fig. 1: MCL-1 protein expression correlates with USP9X expression in oral cell lines and tissues.
Fig. 2: USP9X interacts with MCL-1 protein in OSCC cells and stabilises it.
Fig. 3: WP1130 potently induces cell death in OSCC cells.
Fig. 4: High MCL-1 and USP9X expression correlates with disease relapse and aggressive tumours in oral cancer patients.
Fig. 5: High co-expression of MCL-1 and USP9X is associated with dismal outcome in OSCC patients.

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Acknowledgements

We thank Dr S. Gollins, the University of Pittsburg for kindly providing the SCC029B cell line. P.S. was supported by Senior Research Fellowship from the Department of Biotechnology, Government of India. We sincerely thank members of the light microscopy, electron microscopy, flow cytometry, histology and animal house facility at ACTREC. We extend our thanks to all the members of the Teni laboratory for their technical help, suggestions and critical comments while preparing the paper.

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Contributions

Conceptualisation: P.S., S.S.P. and T.T.; data curation: P.S., S.N.P., R.W., S.S.P., A.U., S.O., P. Rajput and T.T.; formal analysis: P.S., R.W., S.S.P., P. Rane, R.P. and S.N.; methodology: P.S., S.N.P., R.W., S.S.P., A.U., S.O., P. Rajput, P.K. and H.W.; funding acquisition: P.S., S.N.P., R.P., S.N. and T.T.; writing original draft: P.S.; review and editing: P.S., R.P., S.N., P. Rane and T.T.; resources: S.N.P., R.W., S.S.P., R.P., S.N. and T.T.; supervision: S.N.P., R.P., S.N. and T.T.

Corresponding author

Correspondence to Tanuja Teni.

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This work was approved by the Institutional Ethics Committee (IEC-III) of TMC-ACTREC (Project No. 900211) and was performed in accordance with the Declaration of Helsinki. All patients provided written informed consent. The animal studies were reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of TMC-ACTREC constituted under the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.

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All the data generated or analysed in this study are included in this published article and supplementary information file. If required, the clinical outcomes/follow-up and biomarker data are readily available. Supplementary information is available at the British Journal of Cancer’s website.

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The authors declare no competing interests.

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This project was supported by an Intramural grant from Tata Memorial Centre.

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Sulkshane, P., Pawar, S.N., Waghole, R. et al. Elevated USP9X drives early-to-late-stage oral tumorigenesis via stabilisation of anti-apoptotic MCL-1 protein and impacts outcome in oral cancers. Br J Cancer (2021). https://doi.org/10.1038/s41416-021-01421-x

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