NSD2 upregulation is driven by high-risk HPV E6/E7 and disrupts epithelial differentiation in HPV-associated head and neck cancer

Abstract Background Head and Neck Squamous Cell Carcinoma (HNSCC) are classified in two main subtypes: HPV-positive (HPV+), driven by human papillomavirus (HPV) infections, and HPV-negative (HPV-), associated with environmental risk factors. Despite molecular and clinicopathological differences, neither subtype has effective tailored therapies. Since high-risk HPV oncoproteins E6/E7 affect several epigenetic regulators, characterizing the epigenetic landscape of HPV+ and HPV- HNSCC may uncover novel subtype-specific biomarkers and therapeutic targets. Methods Histone post-translational modifications were profiled in HPV+ and HPV- HNSCC tissues and cell lines using super-SILAC mass spectrometry. The same analysis was performed and combined with RNA-sequencing on E6/E7-transduced human primary keratinocytes (HKs) to identify relevant histone modifiers affected by HPV oncoproteins. Candidate gene was validated via E6/E7-mediated-siRNA knockdown in HPV + cell lines. Western Blot, RT-qPCR and Immunohistochemistry assessed gene expression. NSD2 expression was examined in patients’ tissue samples, TCGA data and 14 HNSCC cell lines. shRNA-mediated NSD2 knockdown followed by RNA-seq, cell proliferation and migration assays evaluated its oncogenic role in HNSCC. CaCl2 treatments were used to investigate NSD2’s role in epithelial differentiation, while ALDH-positive cells were quantified by flow-cytometry. NSD2 overexpression was used to confirm results. Results HPV+ HNSCC exhibited elevated H3K36me2 levels, compared to HPV-. This alteration is driven by E6/E7-induced NSD2 upregulation. NSD2, a histone methyltransferase specific for H3K36 di-methylation is overexpressed in HPV+ relative to HPV- HNSCC and in both subtypes compared to normal tissue, suggesting crucial implications in HNSCC. Functional assays revealed that NSD2 promotes cell proliferation and migration in both the subtypes. Notably, we identified a novel role for NSD2 in inhibiting epithelial cell differentiation, particularly in HPV+ HNSCC, where its upregulation mediates the E6/E7-induced differentiation blockade. Conclusions We identified a novel HPV-driven epigenetic signature in HNSCC marked by increased H3K36me2 and its writer, NSD2. Our study highlights H3K36me2 as a potential biomarker for patient stratification and positions NSD2 as a promising therapeutic target across HNSCC subtypes, modulating both common and subtype-specific oncogenic pathways. Specifically, NSD2 inhibition in HPV+ tumors restores epithelial differentiation, offering a potential strategy to arrest tumor progression.