Efavirenz causes epithelial-to-mesenchymal transition in human oral keratinocytes harboring HPV

Objectives: Although highly active antiretroviral therapy (HAART) drastically reduced the morbidity and mortality associated with acquired immunodeficiency syndrome (AIDS), recent studies show emergence of non-AIDS defining malignancies (NADM), such as skin, head/neck, and lung cancers. Presumed risk factors of NADM involve infection with oncogenic viruses and chronic exposure to HAART, including reverse transcriptase inhibitors such as efavirenz (EFV). The current study evaluated phenotypic effects of EFV on human oral keratinocytes harboring human papillomavirus (HPV). Methods: HOK-16B cells immortalized with “high risk” HPV type 16 were exposed chronically to sublethal and clinical dose of EFV. Phenotypic changes were compared between cells with and without EFV exposure. Effect of EFV on cell behavior and tissue architecture was determined in organotypic raft cultures. Expression levels of proteins involved in cell cycle regulation and migration were determined by Western blotting. mRNA levels of keratinocyte differentiation marker genes were detected by quantitative reverse transcription PCR (RT-qPCR). Results: HOK-16B cells chronically exposed to EFV exhibited features resembling epithelial-to-mesenchymal transition (EMT), including spindle-shaped morphology, ability to replicate in serum-containing media, enhanced migration, and acquisition of invasive properties. EFV treatment also led to reduced expression of keratinocyte differentiation markers, such as involucrin, cytokeratin (types 1, 10, and 14), loricrin, and fillagrin. Consistent with EMT phenotype, EFV treatment notably reduced the expression of E-cadherin, which is epithelial cell marker. HOK-16B cells developed stratified epithelium with cornified layer in organotypic raft cultures. However, EFV-treated cells failed to undergo terminal differentiation in raft cultures and exhibited invasion into the submucosal layer. Conclusion: Our data suggest that chronic exposure to EFV causes EMT phenotype in human oral keratinocytes. Since EMT is associated with malignant phenotype in cancer, this effect may contribute to development of NADM in HIV+ patients. This study was supported in part by grants, R01DE18295 and K02DE18959, from NIDCR/NIH.