Sagar N Pawar1, Rohit Waghole1, Sushil S Pawar2, Prasad Sulkshane1,3,4, Priyanka Rajput1, Abhay Uthale1,3, Swapnil Oak1,3, Prajakta Kalkar1, Harshada Wani1, Rahul Patil2, Sudhir Nair3,5, Pallavi Rane6, Tanuja Teni7,8. 1. Teni Lab, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre (TMC), Kharghar, Navi Mumbai, Maharashtra, India. 2. KBH Dental College and Hospital, Panchwati, Nashik, Maharashtra, India. 3. Homi Bhabha National Institute (HBNI), Mumbai, Maharashtra, India. 4. Glickman Lab, Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel. 5. Department of Surgical Oncology, Tata Memorial Centre, Mumbai, Maharashtra, India. 6. Clinical Research Secretariat, ACTREC, TMC, Kharghar, Navi Mumbai, Maharashtra, India. 7. Teni Lab, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre (TMC), Kharghar, Navi Mumbai, Maharashtra, India. tteni@actrec.gov.in. 8. Homi Bhabha National Institute (HBNI), Mumbai, Maharashtra, India. tteni@actrec.gov.in.
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.
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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