Abstract 252: Altered caspase-8 localization and decreased caspase-8 sumoylation in TRAIL-resistant normal human fibroblasts
Authors: Roslyn N. Crowder, David T. Dicker, Wafik S. El-Deiry
Journal: Cancer Research (2012)

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 previously 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. We examined caspase-8 stability in cycloheximide treated normal fibroblasts and SW480 human colon cancer cells. Normal fibroblast caspase-8 stability was found to be similar to SW480 cells. Additionally, we investigated caspase-8 modulation in normal fibroblasts to uncover any drug treatments or modified growth conditions that result in increased caspase-8 protein expression and enhanced susceptibility to TRAIL-mediated apoptosis. Normal fibroblasts treated for 24, 48, and 72 hours with non-lethal doses of histone deacetylase inhibitor Trichostatin A or DNA methylation inhibitor 5-azacytidine had no change in caspase-8 protein expression. Our results suggest epigenetic silencing of caspase-8 is not responsible for TRAIL resistance in normal fibroblasts. However, normal fibroblasts serum starved with media containing 0.5% fetal calf serum for 48 hours, resulted in increased caspase-8 protein expression but not effector caspase-3 expression. Serum starved normal fibroblasts treated with TRAIL had increased caspase-3 activity and an increase in the percentage of sub-G1 positive cells compared to TRAIL treated cells in normal growth media. No increase in TRAIL-mediated apoptosis was observed in serum starved SW480 cells. Since previous studies have detected caspase-8 targets in both the cytoplasm and nucleus, we examined caspase-8 localization in normal fibroblasts. Little caspase-8 was found in nuclear extracts isolated from normal fibroblasts. Alternatively, SW480 caspase-8 expression was observed in both cytoplasmic and nuclear fractions. Sumoylated caspase-8 (p75), previously found to be associated with caspase-8 nuclear localization, was noted in nuclear fractions in both normal fibroblasts and SW480 cells. Yet, like total caspase-8 expression, sumoylated caspase-8 expression was found to be decreased in normal fibroblasts compared to SW480 colon cancer cells. Thus, decreased nuclear caspase-8 may be a contributing factor in normal fibroblast resistance to TRAIL-mediated apoptosis. Our current studies are focused on further analysis of caspase-8 sumoylation/desumoylation as well as caspase-8 post-translational modifications that may suppress caspase activity and nuclear translocation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 252. doi:1538-7445.AM2012-252