HPV16 E7-Associated SERPINB3 Suppression and MYC-Related Epithelial Plasticity in Head and Neck Squamous Cell Carcinoma

Background: Human papillomavirus (HPV) infection defines a distinct subtype of head and neck squamous cell carcinoma (HNSCC). Although HPV-positive (HPV+) HNSCC generally shows better overall survival than HPV-negative (HPV−) disease, it is frequently associated with cervical lymph node metastasis. However, the epithelial cell states and viral gene-associated mechanisms underlying HPV−related metastatic progression remain incompletely understood. This study aimed to identify metastasis-associated epithelial subpopulations in HPV+ HNSCC and explore the potential role of HPV16 E7 in regulating metastatic programs. Methods: Public single-cell RNA-sequencing datasets from paired primary and metastatic HNSCC samples were integrated and analyzed to characterize malignant epithelial subpopulations. Copy number variation (CNV) inference, clustering, pathway enrichment, stemness scoring, and trajectory analysis were performed to define metastasis-associated epithelial states. TCGA transcriptomic data and tissue-based validation were used to support candidate gene screening. In vitro functional assays were performed using SERPINB3-knockdown and HPV16 early gene-overexpressing CAL27 cell models. Results: Single-cell analysis identified stem-like metastatic epithelial subpopulations in HPV+ and HPV− HNSCC. In HPV+ metastatic lesions, an ALDH2+/LAMB3+ epithelial subpopulation showed elevated epithelial–mesenchymal transition activity and stem-like features. SERPINB3 displayed a dynamic expression pattern during HPV+ metastatic progression. Functional assays showed that SERPINB3 knockdown enhanced CAL27 cell migration and invasion and was associated with activation of MYC- and epithelial–mesenchymal transition-related transcriptional programs. Among HPV16 early genes, E5, E6, E6*, and E7 showed different degrees of SERPINB3 suppression, while E7-expressing cells exhibited distinct transcriptional alterations associated with epithelial plasticity and metastatic-related programs. Conclusions: This study identifies a stem-like metastatic epithelial state in HPV-associated HNSCC and suggests a potential HPV16 early gene-SERPINB3-MYC-related regulatory mechanism involved in metastatic epithelial plasticity.