Abstract
Abstract TNF-related apoptosis-inducing ligand (TRAIL)-mediated cell death is currently being utilized in newly developed cancer therapies. The molecular basis of normal cell resistance to TRAIL-mediated apoptosis, and thus therapeutic index, remains largely unexplored. We investigated TRAIL sensitivity in normal fibroblasts (HFF, WI-38, MRC-5) and found that normal fibroblasts have decreased protein expression of initiator caspase-8 that is required for complete TRAIL signaling. TRAIL treatment caused caspase-8 cleavage in TRAIL-sensitive cancer cells. Yet, no caspase-8 cleavage was observed in normal fibroblasts after TRAIL treatment. TRAIL treated fibroblasts had modest increase in caspase-8 activity compared to TRAIL-susceptible cancer cells. Previous studies have found caspase-8 polyubiquitination necessary for caspase-8 aggregation and complete activation. Additionally, reports show deubiquitination of caspase-8 limits caspase-8 activity. Therefore, we examined caspase-8 ubiquitination in normal fibroblasts. Caspase-8 ubiquitination, analyzed by Western blotting, in normal fibroblasts was found to be decreased compared to TRAIL-sensitive cancer cells. TRAIL sensitivity in normal fibroblasts after treatment with deubiquitinase inhibitor PR-619 was also assessed. Normal fibroblasts had markedly decreased cell viability after co-treatment with TRAIL and PR-619 compared to TRAIL-only treated fibroblasts. Cancer cell viability after co-treatment with TRAIL and PR-619 was similar to cancer cells merely treated with TRAIL. Normal fibroblasts co-treated with TRAIL and PR-619 had increased caspase-8 cleavage, PARP cleavage, and caspase-8 activity. The increased cell death seen in normal fibroblasts with TRAIL and PR-619 co-treatment was greatly inhibited when caspase inhibitor ZVAD was added, demonstrating the cell death with TRAIL and PR-619 was caspase-mediated. Our data suggest that caspase-8 deubiquitination may be a contributing factor in normal fibroblast resistance to TRAIL-mediated apoptosis. Our current studies are focused on investigating the role of deubiquitinase A20, previously found to deubiquitinate caspase-8, in normal fibroblast caspase-8 deubiquitination. Citation Format: Roslyn N. Crowder, David T. Dicker, Wafik S. El-Deiry. Altered caspase-8 protein expression and activity in TRAIL-resistant normal human fibroblasts. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2938. doi:10.1158/1538-7445.AM2013-2938
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