Splicing of HPV16 E6 promotes aggressive invasion in oropharyngeal cancer via endocytosis of E-cadherin

Human papillomavirus-positive oropharyngeal squamous cell carcinoma (HPV+ OPSCC) is now the leading HPV+ cancer in the United States and United Kingdom. Despite high cure rates, a significant subset of patients have aggressive HPV+ OPSCC that recurs; deciphering the underlying mechanisms will identify biomarkers that delineate patient subgroups for personalized treatment. In a comprehensive investigation using complementary clinically-relevant models of HPV+ OPSCC, we demonstrated that elevated expression of a HPV16 E6 spliced isoform (E6*I) compared to full-length E6 (E6FL), is critical for aggressive invasion. Introduction of splice switching oligonucleotides (SSOs) to inhibit E6FL splicing, effectively mitigated invasion. Mechanistic studies revealed that aggressive invasion occurs via E6*I-induced endocytosis of E-cadherin (ECAD) from the cell membrane, consistent with partial epithelial-to-mesenchymal transition (p-EMT). The clinical relevance of this mechanism was validated in patient cohorts where a reduced ratio of E6FL to all E6 splice variants was associated with high p-EMT signature, lower membrane:cell ECAD, and worse recurrence-free survival. Together, our findings show that low membrane:cell ECAD ratio in pre-treatment biopsies of HPV+ OPSCC, could stratify patients according to risk; patients with a low ratio may not be candidates for treatment de-intensification trials. Importantly, since ECAD detection is by immunohistochemistry, which is widely used even in diagnostic pathology laboratories with limited resources, membrane:cell ECAD ratio could be a clinically scalable biomarker. Targeting E6FL splicing with SSOs should be explored further as a logical strategy for therapeutic intervention in patients with HPV+ OPSCC.