title: Effect of radiotherapy on DNA signalling cascades of head and neck cancer cells
Creators: name: Dashi, Tobias
Publisher: name: Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)
Year: 2025

description: Head and neck squamous cell carcinomas (HNSCC) are the most common type of cancer in the head and neck region. Recent discoveries have shown that besides risk factors such as tobacco and alcohol consumption, infection with the oncogenic human papillomavirus (HPV) does play a major role in developing this malignancy (Johnson et al., 2020). Regarding therapy, immune checkpoint inhibitors such as pembrolizumab have made their way into clinics in the last decade, enhancing particularly the effects of radiotherapy (RT) as they strengthen the host’s immune system by blocking immune suppressive molecules that get also up-regulated after standard treatments. However, they sometimes lead to severe side effects resulting in having to cancel the therapy (Ramos-Casals et al., 2020). Latest research has shown that DNA damage response is connected to expression of immune modulatory molecules and can influence the immunogenicity of tumour cells, especially after being radiated. By this, DNA is shifted into the cytoplasm and DNA sensing pathways are activated. This axis may give an additional possibility of interfering with immunogenic molecules. We therefore aimed to analyse the expression of DNA sensing molecules in HNSCC tumour cells following RT and how this impacts on activation of immune modulators such as interferon (IFN)-β. As model system we used four human cell lines, two being HPV-negative (Cal33, HSC4), and two being HPV-positive (UM-Scc-47 and UD-Scc-2), irradiated them with a hypo-fractioned RT schedule (5x3 Gy) or with a single high-dose of RT (19.3 Gy) and examined their IFN-β release 24 hours after the last radiation. Furthermore, we analysed the expression of DNA signalling molecules such as cGAS/STING and RIG-I as well as IFNs and interferon-stimulated genes (ISGs). Additionally, HSC4 tumour cells were treated with inhibitors of the RIG-I pathway and the effect on cytokine release and immune checkpoint expression was analysed. Hypo-fractioned radiotherapy led to increased apoptosis and necrosis in three of the four HNSCC cell lines (Cal33, UD-Scc-2 and UM-Scc-47) compared to the single high-dose. Both radiation schemes led to an increase of IFN-β protein in the supernatant or mRNA expression for the HSC4 cell line and upregulation of RIG-I mRNA, whereas no difference in cGAS/STING expression was found. Also, the ISGs IFIT1 and IFIT3 showed a higher expression after radiation. No changes were detected for the other three examined HNSCC tumour cell lines. Trex1 expression, being an DNA-degrading enzyme, was unaffected by radiation for all four cell lines. Inhibition of the RIG-I pathway for the HSC4 cell line led to both a missing increase of ISGs expression and a missing upregulation of the immunosuppressive immune checkpoint molecules PD-L1 and PD-L2 compared to the irradiated control group. We conclude that for the HSC4 cell line, the RIG-I and not the cGAS/STING pathway might be the driving factor for the induction of ISGs. Additionally, immune checkpoint expression of PD- L1 and PD-L2 is altered by DNA sensing pathway inhibition of RIG-I, whereas IFN-β secretion remains unaffected. As this was observed in one out of four HNSCC tumour cell lines, the RT- modulated DNA sensing response seems to be a rather individual event, also not being dependent on the HPV status of the tumour cells.