Effects of CASP8 mutations on radiotherapy and inflammatory responses in cancer cells

Abstract

CASP8 mutations occur most commonly in head and neck squamous cell carcinoma (HNSCC), with a mutation frequency of 10,7 %. Alterations in this gene have been shown to impair the effects of radiotherapy in HNSCC patients and are associated with poor overall survival. Thus, it is of great importance to identify the means cancer cells use to escape cell death and develop resistance to cancer therapies in order to find predictive markers. Caspase-8 is a cysteine protease, encoded by the CASP8 gene that plays a crucial role in both extrinsic apoptosis and inflammatory signaling, while acting as an inhibitor of necroptosis. In apoptotic signaling, the catalytic activation of Caspase-8 by cleavage and dimerization is required, while in inflammatory signaling, procaspase-8 acts as a scaffold for FADDosome formation independently from its catalytic activity. CASP8 mutations have a complex role in HNSCC; mutations have been shown to impair and inhibit death receptor-mediated cell death as well as to promote activation of NF-κB-dependent inflammatory signaling. Previous studies have shown that HNSCC-associated CASP8 mutations could inhibit the activation of apoptotic signaling and cell death following death receptor stimulation. Moreover, mutations have been shown to both impair and enhance the NF-κB- dependent inflammatory signaling, depending on the mutation site. These findings indicate the diverse functional properties of HNSCC-associated CASP8 mutations, rather than only being loss- of-function mutations. This thesis aimed to investigate the contributions of nine HNSCC-associated CASP8 mutations (L7V, L62P, R71T, S99F, L105H, D303G, S375*, T441I and Q465*) to radiotherapy response in cancer cells. In addition, the effects of CASP8 mutations on inflammatory signaling mediated by death receptor stimulation in cancer cells were studied. CASP8 mutations were generated by site-directed mutagenesis and cloned using Gateway cloning. HeLa CASP8-/- cells were then virally transduced with mutation plasmids. Created stable CASP8 mutation cell lines were irradiated, after which cell viabilities were measured using the MTT assay. The effects of CASP8 mutations on the cytokine and chemokine expression following irradiation and TRAIL treatment were analysed by qPCR. Based on the findings from this study, mutations T441I and Q465* exhibited higher cell viability following irradiation compared to WT CASP8, suggesting that these mutations could potentially impair the activation of cell death signaling after irradiation. Moreover, seven out of nine CASP8 mutants were more sensitive to irradiation than WT CASP8. This study also showed that most of the CASP8 mutants retained their ability to activate NF-κB-dependent inflammatory signaling following irradiation or death receptor stimulation by TRAIL. However, further studies are required to determine whether mutations T441I and Q465* have potential to act as predictive markers in HNSCC.