Introduction: The mode of HPV infection in HNSCC remains poorly understood, and it"s unclear why most patients with HPV-positive HNSCC are non-smokers, whereas HPV-negative HNSCC patients are smokers. Our previous research (n>1,100) suggested an explanation: Smoking induces upregulation of secretory leukocyte protease inhibitor (SLPI), a mucosal protein that competes with HPV for binding to membrane-associated annexin A2 (AnxA2), a key step in HPV cell entry. This study aimed to investigate mechanistic aspects of this hypothesis using transfection assays.

Material and methods: HaCaT and HeLa cell lines were used to examine effects of shRNA-transfection and nicotine exposure on HPV16-PsV uptake. Cells were treated with Lipofectamine™ RNAiMAX with specific shRNA concentrations, while nicotine was added at indicated concentrations. Protein isolation, SLPI and AnxA2 quantification, LDH cytotoxicity assessment, HPV16-PsV uptake measurement, mRNA isolation, cDNA synthesis, and RT-qPCR were conducted.

Results: In vitro, cells transfected with SLPI or AnxA2 shRNA showed that PsVs entered cells significantly better with SLPI downregulation and less with AnxA2 downregulation. Nicotine exposure increased SLPI levels and reduced PsV uptake.

Discussion: Smoking-induced SLPI overexpression hinders HPV cell entry by binding to AnxA2, thus blocking HPV infection. Non-smokers have lower SLPI levels, leaving more unbound AnxA2, which promotes HPV cell entry. These findings suggest a potential paradigm shift in the understanding of HPV-related pathogenesis, particularly in head and neck cancers, and HPV infection overall

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