Human papillomavirus positive (HPV+) head and neck squamous cell carcinomas (HNSCC) are characterized by an enhanced radiation sensitivity, which is also evident in cell culture models. This sensitivity is based on a defect in DNA double-strand break repair and especially a defect in homologous recombination (HR) has repeatedly been suggested. Apart from its role in DNA repair, the HR machinery is also critically involved in the protection and restart of stalled replication forks. In the normal viral life cycle the HPV oncoprotein E7 drives infected cells into a replicative state in order to facilitate the replication of viral DNA. Whether HPV-mediated modulations of the replication machinery exist in HPV-driven HNSCC cells and whether these, especially in the light of the proposed HR defect, may translate into altered replication processes of the genomic DNA is currently unknown.
Therefore, we performed comparative studies on DNA-replication using a panel of 6 HPV-positive and 5 HPV-negative HNSCC cell lines using the DNA-fiber-assay to assess replication on the molecular level.
HPV-positive HNSCC cell lines demonstrated a higher variation in replication fork speed with some cell lines showing DNA-elongation rates unmatched in HPV-negative strains. Upon irradiation, the vast majority of HPV-positive cell lines, in contrast to HPV-negative, reduced elongation speed and demonstrated extensive, Mre11-dependent resection of newly replicated DNA. Since a similar resection phenotype has been linked to the activation of the cGAS/STING immune response pathway, we further explored a possible association in our cells. Contrasting previous reports of a generally impaired cGAS/STING pathway in HPV-positive cells, we found that 2 out of the 6 HPV-positive strains demonstrated a generally functionally cGAS/STING response as assessed by nuclear translocation of IRF3 upon transfection of external DNA. Upon radiation, however, only one of the cell lines showed moderate responsiveness.
In summary, HPV+ HNSCC cells demonstrate extensive resection of newly replicated DNA tracks upon DNA damage induction, reminiscent of HR defective cells. While a subfraction of HPV-positive HNSCC cells is in principle able to execute the cGAS/STING pathway, a prominent role for the enhanced radiation sensitivity appears unlikely.