DNA damage response as a mechanism for radioresistance in head and neck squamous cell carcinoma

The two major carcinogenic routes for developing Head and neck squamous cell carcinoma (HNSCC) are infection with the human papillomavirus (HPV-positive HNSCC) and the overconsumption of alcohol and/or tobacco (HPV-negative HNSCC). Currently, two-thirds of patients with HNSCC are diagnosed in a locally advanced stage, due to lack of symptoms in earlier stages. The current treatment schedule for many patients with locally advanced disease, independent of HPV status, is the combination of radiotherapy (RT) with chemotherapy, with or without surgery. However, the current treatment options encounter two major obstacles. First, normal tissue toxicity arises with the current treatment options, with one of the most common acute complications being oropharyngeal mucositis (ORM). Hereby the quality of life of patients and treatment adherence are affected, influencing treatment outcome. A promising, non-invasive treatment option for ORM is low-level laser therapy (LLLT), which is able to stimulate wound healing and relief pain, hereby positively affect patient outcome. Second, up to 50% of patients with locally advanced disease develop locoregional recurrences after treatment, with one of the main reasons being the presence of tumor radioresistance. The underlying biological mechanisms are currently not fully understood and treatment options for these patients are still limited. In this thesis, we aimed to focus on these two problems by investigating the biological effects of LLLT in HNSCC cell lines (Chapter III), and investigating the underlying mechanisms of radioresistance in HNSCC cell lines, with a focus on the DNA damage response (DDR) (Chapter IV). Chapter III Biological effects of low-level laser therapy in head and neck squamous cell carcinoma cell lines LLLT is a promising prevention and treatment strategy for the most common RT-induced acute complication, ORM. By stimulating cellular processes, wound healing and pain relief are the main advantages of LLLT. Hereby, LLLT can improve the quality of life of patients and allow treatment adherence. However, by stimulating cellular proliferation, the risk of undesirably stimulating cancer cells is present, since the primary HNSCC tumor site often comprises the LLLT application field. Therefore, we investigated the biological effects of LLLT on HNSCC cell lines and normal epithelial tonsil cells, with the latter being involved in wound healing. The effect of LLLT on proliferation was assessed by exposing HNSCC cell lines and normal epithelial tonsil cells to LLLT with energy densities of 1 and 2 J/cm². HNSCC cell lines showed an increased proliferation rate, whereas no effect in normal epithelial tonsil cells was detected upon LLLT application. Cell cycle distribution, increased levels of mitotic figures and increased percentage of Ki67, a proliferation marker, upon LLLT irradiation, supported the increased proliferative effect seen in HNSCC cells. Mitogenic pathways PI3K and MAPK/ERK were activated in a dose-dependent manner after LLLT in HNSCC cells, which was not found in normal epithelial tonsil cells. In conclusion, we showed that LLLT was able to increase proliferation of HNSCC cells in a dose-dependent manner, by stimulating proliferation pathways. This is an indication that LLLT should be used with extreme caution in a clinical setting for patients with HNSCC undergoing RT in combination with chemotherapy, since tumor cells present in the LLLT application field could be undesirably exposed and stimulated. Chapter IV The role of DDR in radioresistant head and neck squamous cell carcinoma cell lines The presence of radioresistance in a tumor is one of the main reasons for locoregional recurrences after treatment. Several mechanisms influencing RT response have been described, namely hypoxia, cancer stem cells, mitogenic pathways, the DDR and DNA repair capacity, and cell death. However, the exact mechanisms underlying radioresistance are unknown. Since DDR is one of the important mechanisms defining RT outcome, we focused on the role of DDR in radioresistant HNSCC. To mimic patient relapse in vitro, isogenic radioresistant HNSCC cells were generated by exposing parental HNSCC cell lines to a fractionated RT treatment schedule. Radioresistant cells were generated from both HPV-positive and HPV-negative HNSCC, to capture the two major groups of HNSCC. Two radioresistant clones for both HPV-positive parental cell line SCC154 and HPV-negative parental cell line SCC61 were selected. HPV-positive and HPV-negative clones showed increased resistance to RT compared to their parental cell line in vitro and in vivo. In addition, differences in the involvement of the DDR were detected between HPV-positive and HPV-negative radioresistant clones. HPV-positive radioresistant HNSCC showed increased DNA repair capacity and faster cell cycle recovery after irradiation compared to their parental cells. HPV-negative radioresistant HNSCC showed less checkpoint reliance for baseline survival and less genomic instability after RT, indicating increased replicative ability. Possible radiosensitizing strategies for HPV-positive and HPV-negative radioresistant HNSCC were explored by targeting the DDR. Targeted inhibitors for the cell cycle, non-homologous end joining and base excision repair pathway were able to rescue the radioresistant phenotype of HPV-positive and HPV-negative radioresistant clones. Thus, targeting the DDR provided a possible radiosensitizing strategy for both HPV-positive and HPV-negative radioresistant HNSCC. In conclusion, we successfully generated HPV-positive and HPV-negative radioresistant HNSCC with fractionated irradiation. Differences in DDR involvement were detected between radioresistant HPV-positive and HPV-negative HNSCC. In addition, by targeting the DDR with specific inhibitors, possible radiosensitizing strategies for both HPV-positive and HPV-negative radioresistant HNSCC cells were shown.

Publication year:2021

Accessibility:Open