Repression of <scp>ROBO1</scp> Enhances Cisplatin Sensitivity in Hypermethylated Subtype of <scp>HPV</scp> ‐Positive Head and Neck Carcinoma

Although human papillomavirus-positive (HPV(+)) head and neck squamous cell carcinoma (HNSCC), particularly oropharyngeal squamous cell carcinoma (OPSCC), is generally associated with favorable outcomes, a subset of HPV(+) OPSCC remains resistant to chemoradiotherapy. We previously stratified clinical OPSCC cases using DNA methylome profiling and identified a significant correlation between elevated DNA methylation levels and favorable treatment response. To elucidate the molecular basis of this correlation, we performed integrated methylome analyses of 64 HPV(+) HNSCC cases in The Cancer Genome Atlas (TCGA) and 29 HPV(+) OPSCC cases in our Chiba cohort. We identified 595 genes showing significantly higher DNA methylation in good responders in each cohort. Additionally, using HNSCC cell lines, we extracted genes that exhibited both high DNA methylation and decreased expression in cisplatin (CDDP) sensitive cells. Twelve genes were consistently hypermethylated across both clinical cohorts, among which two genes, ROBO1 and SULT4A1, showed statistical significance and effect size. Functional validation demonstrated that ROBO1 knockdown in unmethylated HNSCC cells significantly suppressed cellular growth (p < 0.05), and additional exposure to CDDP further synergistically reduced cellular growth (p < 0.05). Conversely, ROBO1 overexpression conferred resistance to CDDP. CDDP resistance in unmethylated HNSCC cells led to upregulation of ROBO1 expression, suggesting an inverse relationship between ROBO1 silencing and drug resistance. Mechanistically, ROBO1 suppression increased DNA damage and apoptosis following CDDP treatment. These indicate that DNA hypermethylation of ROBO1 causally correlates with better treatment response of HPV(+) HNSCC, and its chemosensitivity might be enhanced by silencing of methylation target genes.