Abstract 1736: The anticancer activity of the DUB inhibitor b-AP15 is associated with accumulation of proteasome bound ubiquitin and oxidative stress

Abstract

Abstract Background: The ubiquitin proteasome system (UPS) is the major cellular degradation system for damaged or short-lived proteins. Tumor cells are dependent on a functioning UPS, making it an ideal target for the development of anti-cancer therapies. Bortezomib, an inhibitor of the proteolytic activities of the proteasome core particle (20SCP), is approved for treatment of multiple myeloma and mantle cell lymphoma; however several problems have emerged including dose-limiting toxicity and tumor relapse. We previously identified b-AP15 as a novel inhibitor of the proteasome associated deubiquitinases (DUBs) USP14 and UCH37/UCHL5. Treatment with b-AP15 induced cell death in several in vitro and in vivo cancer models. The aim of this study was to investigate how the different modes of UPS inhibition affect the structure of the 26S proteasome and cellular response as well as the potential for proteasome DUB inhibitors as a therapeutic option for cancer. Methods: HCT116 colon carcinoma cells were cultured in McCoy's 5A modified medium/10% fetal calf serum. For protein expression analysis cell extracts were resolved by Tris-Acetate gel and transferred to nitrocellulose membrane for western blotting. Proteasome integrity and cellular distribution of polyubiquitin chains were assessed in cell extracts subjected to density gradient centrifugation, conducted in 4-24% linear glycerol gradients, followed by western blot analysis. Apoptotic response was assessed with M30-CytoDeath ELISA, measuring level of caspase-cleavage of the endogenous cellular substrate keratin-18. Results: We found that inhibitors of proteasome function do not alter proteasome assembly or structure; however treatment induced the accumulation of polyubiquitinated proteins associated with the 26S proteasome. Comparison of 20S CP and 19S DUB inhibition showed that b-AP15 induced more rapid and sustained apoptotic response in comparison to bortezomib. Proteasome inhibition has been previously associated with endoplasmic reticulum and oxidative stress, however our findings show that reactive oxygen species (ROS) are main mediators of b-AP15 induced apoptosis. b-AP15 strongly induces several genes associated with oxidative stress response; hemoxygenase-1 (HMOX1), p38-MAPK, c-JUN and Jun N-terminal kinase (JNK). Additionally, treatment with a pharmacological inhibitor of JNK and several ROS scavengers decreased the levels of apoptosis in response to b-AP15 treatment. Conclusions: Cellular response to b-AP15 mediated proteasome inhibition is distinct from clinically used bortezomib; therefore b-AP15 has a potential clinical use, in particular for treatment of malignancies resistant to other proteasome inhibitors. Citation Format: Magdalena Mazurkiewicz, Slavica Brnjic, Mårten Fryknäs, Chao Sun, Xiaonan Zhang, Rolf Larsson, Pádraig D'Arcy, Stig Linder. The anticancer activity of the DUB inhibitor b-AP15 is associated with accumulation of proteasome bound ubiquitin and oxidative stress. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1736. doi:10.1158/1538-7445.AM2015-1736