3087 - Modulating Non-Canonical Neddylation of TGFßRII to Enhance Radiotherapy Response in Oral Squamous Cell Carcinoma
Presenter(s)
H. L. Lee1,2, P. H. Wu3, J. F. Chiou4, C. H. Lin3, H. W. Lu5, C. C. Kuo6, Y. F. Lin7, and I. C. Lai8; 1TMU Proton Center, Taipei City, Taiwan, 2Taipei Medical University, Taipei City, Taiwan, 3Taipei Medical University Hospital, Taipei City, Taipei City, Taiwan, 4Department of Radiology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan, 5Taipei Medical University Shuang-Ho Hospital, New Taipei City, Taiwan, 6Radiation Oncology, Taipei Medical University Hospital, Taipei, Taiwan, 7Taipei Medical University, Taipei City, Taipei City, Taiwan, 8Department of Heavy Particles & Radiation Oncology, Taipei Veterans General Hospital, Taipei, Taiwan
Purpose/Objective(s): Radiotherapy is a primary treatment for oral squamous cell carcinoma (OSCC), but radioresistance often leads to treatment failure. Neddylation, a ubiquitin-like modification, plays a role in cancer progression, yet its impact on OSCC radioresistance remains unclear. This study investigates the role of neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8) in OSCC radioresistance and evaluates the therapeutic potential of targeting non-canonical TGFß receptor type II (TGFßRII) neddylation to enhance radiosensitivity in refractory OSCC.
Materials/Methods: NEDD8 expression was analyzed in OSCC patient samples and cell lines using quantitative RT-PCR, immunohistochemistry, and Western blotting, with correlations to radiotherapy outcomes. Functional assays, including clonogenic survival and cell viability tests, assessed the effects of NEDD8 knockdown and overexpression on radiosensitivity. MLN4924, a selective NEDD8-activating enzyme (NAE) inhibitor, was used to block neddylation, and its effects on OSCC radiosensitivity were evaluated. Gene Set Enrichment Analysis (GSEA) and Western blotting examined downstream signaling alterations. To assess the role of non-canonical TGFßRII neddylation, CRISPR/Cas9 knockout of c-CBL, an E3 ligase regulating TGFßRII neddylation, was performed, followed by analyses of TGFßRII protein stability and radiation-induced cytotoxicity.
Results: NEDD8 was significantly upregulated in OSCC tumors compared to adjacent normal tissues, with higher levels correlating with reduced radiosensitivity and poorer patient outcomes. NEDD8 knockdown increased radiosensitivity, while overexpression induced resistance. MLN4924 treatment reversed radioresistance in a dose-dependent manner, restoring irradiation response in refractory OSCC cells. Mechanistic studies showed MLN4924 suppressed TGFß signaling, a pathway implicated in OSCC radioresistance. Inhibiting TGFß signaling restored radiosensitivity in NEDD8-overexpressing cells. Notably, CRISPR/Cas9-mediated c-CBL knockout destabilized and degraded TGFßRII, further enhancing radiation-induced cytotoxicity.
Conclusion:
This study identifies non-canonical TGFßRII neddylation as a key mechanism in OSCC radioresistance and demonstrates that targeting c-CBL-mediated neddylation enhances radiosensitivity. These findings suggest a promising therapeutic strategy to overcome radioresistance and improve OSCC radiotherapy outcomes.