A prognostic model for human papillomavirus‑negative head and neck squamous cell carcinoma based on a novel ferroptosis‑related gene signature: Development, validation and elucidation of its relationship with the immune microenvironment

Head and neck squamous cell carcinoma (HNSCC) can be categorized as human papillomavirus (HPV)-positive or -negative. The present study explored the potential prognostic value of ferroptosis-related genes (FRGs) and their impact on the tumor immune microenvironment in HPV-negative HNSCC compared with HPV-positive HNSCC, based on data from The Cancer Genome Atlas online database. The bioinformatics analysis results were then verified through cell culture and histological analyses. The prognostic model was developed by evaluating the correlations between prognostic value and the expression of FRGs in HPV-negative HNSCC. This was based on differential analysis of data from The Cancer Genome Atlas between the HPV-negative and -positive patient subgroups. The relationships of the identified hub FRGs with immune cell infiltration were determined by CIBERSORT R scripts for the HPV-negative subgroup. Tribbles pseudokinase 3 (TRIB3) expression in both HPV-negative and -positive HNSCC cells was verified by reverse transcription-quantitative polymerase chain reaction, western blotting and immunohistochemistry. The role of TRIB3 in mediating ferroptosis in both HPV-positive and -negative cells was validated by transmission electron microscopy and intracellular Fe²⁺, reactive oxygen species (ROS), glutathione (GSH) and malondialdehyde (MDA) assessments. As a result, it was verified that the prognostic model for HPV-negative patients had a good performance. TRIB3 expression was higher in HPV-negative samples than in HPV-positive samples. Additionally, following cell transfection, <i>TRIB3</i> knockdown increased intracellular Fe²⁺, MDA and ROS levels, decreased GSH levels and diminished or eliminated mitochondrial ridges in HPV-negative cells. By contrast, <i>TRIB3</i> overexpression decreased intracellular Fe²⁺, MDA and ROS, while increasing GSH in HPV-positive cells. In conclusion, the constructed model may indicate the prognosis of patients with HPV-negative HNSCC. Moreover, <i>TRIB3</i>, via its involvement in ferroptosis, may serve as a potential therapeutic target for both HPV-positive and HPV-negative HNSCC. Thus, the present study has provided a starting point for further investigation into the therapeutic potential of targeting <i>TRIB3</i>.