Abstract 4969: Impact of proteasome inhibition of caspase-8 regulation in cancer

Abstract

Abstract Dissection of the extrinsic and intrinsic pathways of apoptosis has lead to the development of new anti-cancer therapeutic strategies. Strategies involve combination of a chemotherapeutic agent with a death inducing ligand, such as TNF-alpha Related Apoptosis Inducing Ligand (TRAIL) to induce apoptosis in cancer cells that are resistant to apoptosis. We recently demonstrated that proteasome inhibition sensitizes TRAIL resistant prostate cancer to TRAIL mediated apoptosis using both in-vitro and in-vivo models, by stabilizing the active p18 subunit of initiator caspase-8. Based on this data indicating a potential relationship between caspase-8 and the proteasome, we hypothesize that caspase-8 ubiquitination and targeting to the 26S proteasome regulates the extrinsic pathway of cancer cell apoptosis. This study pursues the impact of proteasome inhibition on caspase-8 stability, trafficking and activity following death receptor activation. The contribution of caspase-8 polyubiquitination in regulating the extrinsic pathway of apoptosis is determined, using subcellular fractionation, caspase-8 and polyubiquitin immunoprecipitation (IP), and fluorescent microscopy techniques in diverse experimental cell models. The cell lines analyzed include the TRAIL resistant LNCaP prostate cancer cell line and caspase-8 deficient neuroblastoma (NB7) cell line, engineered to re-express and test the wild-type and non-cleavable mutant forms of caspase-8. Our findings indicate that the non-cleavable forms of caspase-8 are capable of inducing apoptosis comparably to wild-type caspase-8 upon treatment with proteasome inhibitor and TRAIL. Moreover we provide evidence that caspase-8 polyubiquitination occurs following caspase-8 processing into its active subunits, processed caspase-8 accumulates at the DISC, and ultimately cycles into the cytosol for proteasomal degradation. This study identifies caspase-8 processing as a significant regulatory mechanism in apoptosis outcomes in cancer cells, but not required for caspase-8 activation. Together these data provide a better understanding of caspase-8 regulation that can be used as a platform for identifying new molecular targets such as E3 ligases responsible for caspase-8 regulation. Further defining caspase-8 regulation can lead to strategies that enhance efficacy of current therapies or lead to development of new more specific caspase-8 driven anti-cancer approaches. Funding provided by DOD GRANT USAMRMC PC073314 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 4969. doi:1538-7445.AM2012-4969