Investigating PAD-mediated citrullination in HPV-driven head and neck squamous cell carcinoma

Citrullination is a post-translational modification catalyzed by peptidyl-arginine deiminases (PADs), which convert peptidyl-arginine into peptidyl-citrulline. The PAD family in humans com- prises five isozymes (PADs 1-4 and 6), which are implicated in various diseases, including cancer. Our previous findings revealed a novel mechanism by which high-risk human papillomaviruses (HPVs) manipulate host regulatory pathways involved in the cell cycle and survival to enhance viral fitness, implying that PADs may be promising targets for developing new host-targeting antivirals to prevent cervical cancer progression. In this study, we further explore the impact of PAD-mediated protein citrullination on HPV infection, in the context of the head and neck squa- mous cell carcinoma (HNSCC). Here we demonstrate using RT-qPCR that PAD1, PAD2, and PAD4 are expressed and modulated in HNSCC biopsies compared to normal mucosa, though their regulation is not influenced by HPV status as tested by nested-PCR. Additionally, we conducted an immunohistochemical (IHC) analysis focusing on PAD1 and PAD4 in a cohort of formalin-fixed paraffin-embedded HNSCC samples. According to preliminary results, PAD1 expression appears to be associated with tu- mor differentiation; specifically, we observed intense IHC positivity in poor differentiated HNSCC. Additional investigation is necessary for PAD4; however, its expression seems to overlap with PAD1 tumor expression. To further corroborate these data, we took ad- vantage of an in vitro cellular model of immortalized keratinocytes (NOKs) expressing the major HPV oncoproteins E6 and E7. Preliminary results revealed that both PAD ex- pression and overall citrullination are not significantly modulated by HPV oncoproteins. In conclusion, in the context of HNSCC, the expression of PAD1 and PAD4 is modulated accor- ding to tumor differentiation, although HPV infection does not appear to significantly influence this regulation.