Abstract 4643: The role of subcellular localization of interleukin-1alpha in head and neck cancer

Abstract Interleukin-1α (IL-1α) is a pro-inflammatory cytokine upregulated in multiple solid malignancies, including head and neck squamous cell carcinomas (HNSCCs) and has been associated with tumor growth, metastasis, immunosuppression, and therapy resistance. Unfortunately, targeting circulating IL-1α in clinical trials has shown disappointing results for cancer patients. Our previous research revealed that high nuclear IL-1α expression is associated with perineural invasion and poor progression free survival. However, little is known about the importance of subcellular localization of IL-1α. This study aims to explore the differences in nuclear and cytoplasmic IL-1α expression on tumor aggressiveness. Cal-27 HNSCC cells were transfected with four plasmid constructs of different forms of IL-1α: 1 - precursor/full length (FL) IL-1α (1-271 AA) which localizes in both the nucleus and cytoplasm; 2 - N-terminal/propiece (N-tern) IL-1α (1-112 AA) which localizes in the nucleus; 3 - C-terminal/matured (C-term) IL-1α (113-271 AA) which localizes in the cytoplasm; and 4 - pGenLenti control (CON) construct. Intracellular IL-1α expression was validated using PCR and western blot, subcellular localization was validated using subcellular fractionation kits prior to western blot, and secreted IL-1α and IL-1α-signaling endpoints (e.g. IL-6, IL-8) were analyzed by ELISA. Differences in cell growth and epithelial-mesenchymal transition (EMT) markers were assessed among the IL-1α-expressing cell lines. IL-1α gene expression was significantly increased in all IL-1α construct-expressing cell lines (FL, C-term, N-term) compared to CON. Whole cell lysates showed significantly increased precursor and matured IL-1α protein expression in the FL cells; and matured IL-1α in C-term expressing cells compared to CON. Subcellular fractionation showed increased precursor IL-1α in FL and matured IL-1α in C-term expressing cells in the cytoplasmic fraction; while higher precursor IL-1α was found in nuclear fraction of FL expressing cells. IL-1α secretion was significantly increased from both FL and C-term expressing cells while the IL-1 signaling endpoints IL-6 and IL-8 were increased in the FL expressing cells only. C-term expressing cells grew significantly more rapidly compared to the other cell lines and demonstrated “mesenchymal-like” morphology. To confirm, western blot revealed an increase in the expression of the mesenchymal markers N-cadherin and vimentin, and a complete loss of epithelial markers E-cadherin and claudin. Overall, these findings suggest that the subcellular localization of IL-1α may play a complex role in tumor growth and EMT. Future work is needed to understand the significance of IL-1α subcellular localization to improve more efficient targeting of the IL-1 pathway for cancer therapy. Citation Format: Ishrat Nourin Khan, M M Hasibuzzaman, Andrean L. Simons-Burnett. The role of subcellular localization of interleukin-1alpha in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4643.