Caspase-8 mutations associated with head and neck cancer differentially retain functional properties related to TRAIL-induced apoptosis and cytokine induction.

The cysteine protease, caspase-8, undergoes dimerization, processing, and activation following stimulation of cells with death ligands such as TRAIL, and mediates TRAIL induction of the extrinsic apoptosis pathway. In addition, caspase-8 mediates TRAIL-induced activation of NF-κB and upregulation of immunosuppressive chemokines/cytokines, via a mechanism independent of caspase-8 catalytic activity. The gene encoding procaspase-8 is mutated in 10% of human head and neck squamous cell carcinomas (HNSCCs). Despite a paucity of experimental evidence, HNSCC-associated caspase-8 mutations are commonly assumed to be loss of function. To investigate their functional properties and phenotypic effects, 18 HNSCC-associated caspase-8 mutants were expressed in doxycycline-inducible fashion in cell line models wherein the endogenous wild-type caspase-8 was deleted. We observed that 5/8 mutants in the amino-terminal prodomain, but 0/10 mutants in the carboxyl-terminal catalytic region, retained an ability to mediate TRAIL-induced apoptosis. Caspase-8 proteins with mutations in the prodomain were defective in dimerization, whereas all ten of the catalytic region mutants efficiently dimerized, revealing an inverse relationship between dimerization and apoptosis induction for the mutant proteins. Roughly half (3/8) of the prodomain mutants and 9/10 of the catalytic region mutants retained the ability to mediate TRAIL induction of immunosuppressive CXCL1, IL-6, or IL-8. Doxycycline-induced expression of wild-type caspase-8 or a representative mutant led to an increased percentage of T and NKT cells in syngeneic HNSCC xenograft tumors. These findings demonstrate that HNSCC-associated caspase-8 mutants retain properties that may influence TRAIL-mediated apoptosis and cytokine induction, as well as the composition of the tumor microenvironment.

ATR inhibition sensitizes HPV- and HPV+ head and neck squamous cell carcinoma to cisplatin.

OBJECTIVES: Cisplatin is commonly used in the treatment of head and neck squamous cell carcinoma (HNSCC), and the repair of cisplatin-induced DNA damage involves activation of the DNA damage response protein ataxia telangiectasia and Rad3-related (ATR). Resistance to cisplatin therapy exacerbates adverse toxicities and is associated with poor outcomes. Since repair of cisplatin-induced DNA damage contributes to resistance, we hypothesized that inhibition of ATR using AZD6738, a well-tolerated and orally-bioavailable inhibitor, would enhance the sensitivity of HNSCC cells and tumors to cisplatin. MATERIALS AND METHODS: A panel of human papilloma virus-negative (HPV-) and HPV+ HNSCC cell lines were treated with cisplatin in the absence or presence of AZD6738, and effects on cell viability, colony formation, apoptosis signaling, and DNA damage were assessed. The impact of co-treatment with cisplatin plus AZD6738 on the growth of HPV- and HPV+ cell line- and patient-derived xenograft tumors was also examined. RESULTS: Inhibition of ATR with AZD6738 enhanced cisplatin-induced growth inhibition of HNSCC cell lines and tumors, in association with increased apoptosis signaling and DNA damage. Both HPV- and HPV+ models were sensitized to cisplatin by ATR inhibition. CONCLUSION: Inhibition of ATR promotes sensitization to cisplatin in preclinical in vitro and in vivo models of HPV- and HVP+ HNSCC, supporting clinical evaluation of this strategy in this disease.

Signaling by cell surface death receptors: Alterations in head and neck cancer.

Cell surface death receptors are members of the tumor necrosis factor receptor (TNFR) superfamily and mediate signals leading to the induction of apoptosis or necroptosis, as well as NF-κB-mediated cell survival. These biochemical processes play key roles in cell growth, development, tissue homeostasis, and immune responses. The downstream signaling complexes activated by different death receptors can differ significantly and are subject to multiple, distinct regulatory mechanisms. Dysregulation of signaling by the TNFR superfamily contributes to a variety of pathologic conditions, including defective immune responses and cancer. Caspase-8 signaling is important for mediating death receptor signals leading to either apoptosis or NF-κB activation. By contrast, inactivation of caspase-8 or loss of caspase-8 expression shifts death receptor signaling to the necroptosis pathway. Notably, the gene encoding caspase-8 is mutated in roughly ten percent of head and neck cancers. These findings support the hypothesis that alterations in the biochemical pathways mediated by death receptors have important consequences for the development of head and neck, and possibly other, cancers.