Abstract 726: HNSCC-associated caspase-8 mutations mediate resistance to apoptosis and up-regulation of immunosuppressive cytokines
Authors: Cui Zhibin , Tal Hadas , Grandis Jennifer R. , Johnson Daniel E. 
Published: August 2019

Abstract
Background: The gene encoding caspase-8 is mutated in 10% of the head and neck squamous cell carcinoma (HNSCC) tumors analyzed by The Cancer Genome Atlas (TCGA). HNSCC patients with caspase-8 mutations tend to have worse prognosis. While the role of wild-type (WT) caspase-8 in mediating extrinsic apoptosis is well known, recent studies indicate that it also acts in a noncatalytic fashion to mediate death ligand induction of immunosuppressive cytokines. To determine the potential impact of HNSCC-associated caspase-8 mutations on anti-tumor immunity and the development of HNSCC, we investigated the functional capacity of caspase-8 mutants to mediate death ligand induction of apoptosis and cytokine production.
Methods: Ininitial studies, we knocked out the endogenous CASP8 gene in HeLa cells using CRISPR-Cas9 technology. The HeLa-CASP8 KO cells were then engineered for doxycycline (DOX)-inducible expression of WT or representative caspase-8 mutants (MT; L105H, D303G, S386* and Q465*). The engineered cells were then stimulated with death ligand (eg. TRAIL) and analyzed for induction of cell death using MTT assays or annexin V staining. Induction of immunosuppressive cytokines was assessed by qPCR and ELISA assays.
Results: HeLa-CASP8 KO cells engineered to express WT caspase-8 underwent rapid apoptosis following TRAIL treatment, as indicated by processing of the caspase-8, PARP cleavage, and cell death assays. By contrast, cells engineered to express the MT caspase-8 proteins failed to undergo apoptosis even when treated for 48 hours with very high concentrations of TRAIL. Treatment of parental HeLa cells, but not HeLa-CASP8 KO cells, with TRAIL led to upregulation of mRNAs for the immunosuppressive cytokines IL-6, IL-8, and CXCL1. Exogenous expression of WT caspase-8 in the HeLa-CASP8 KO cells restored TRAIL induction of the immunosuppressive cytokine mRNAs. Interestingly, exogenous expression of the caspase-8 D303G MT, but not the other MTs (L105H, S386*, Q465*), also restored TRAIL induction of the immunosuppressive cytokines, following treatment for as brief as 6 hours.
Conclusion: Our findings demonstrate that the four representative HNSCC-associated caspase-8 mutations have lost the capacity to mediate TRAIL-induced apoptosis. Hence, HNSCC cells harboring these mutations are likely more resistant to killing by CTLs and NK cells which utilize death receptor-mediated apoptosis to kill target cells. Notably, the D303G caspase-8 MT retained the capacity to mediate TRAIL induction of immunosuppressive cytokines. Expression of the D303G MT, which has been observed in more than one patient, is likely to enhance the immunosuppressive tumor microenvironment, further contributing to HNSCC development.
Citation Format: Zhibin Cui, Hadas Tal, Jennifer R. Grandis, Daniel E. Johnson. HNSCC-associated caspase-8 mutations mediate resistance to apoptosis and up-regulation of immunosuppressive cytokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 726.