Concomitant Targeting Aggresome Formation and Intracellar Proteolytic Pathways Enhances ER-Stress Mediated Cell Death In Myeloma Cells

ER-stress is caused by an imbalance between the amount of unfolded or misfolded protein in the ER lumen and the capacity of the ER machinery to refold these proteins. This stress induces a coordinated cellular response known as unfolded protein response (UPR). The main functions of UPR are to reduce the amount of protein that enters the ER by suppressing the translational rate and to increase the folding capacity of the ER via translational activation of chaperon proteins. Additionally, if proteins cannot be folded correctly in the ER, they are retrotranslocated to the cytoplasm for degradation via the ubiquitin-proteasome pathway for adaptation. However, if these adaptation strategies fail, apoptosis is triggered with the induction of the pro-apoptotic transcription factor CHOP/GADD153 and with the IRE1 involved in signaling via caspase-12. Autophagy is a highly conserved cellular process in eukaryotes. Intracellular proteins and organelles, including ER, are engulfed in a double-membrane vesicle known as an autophagosome, and are delivered to lysosomes for degradation by lysosomal hydrolases. Autophagy has been regarded as a bulk non-selective degradation system for long-lived proteins and organelles, in contrast to the specific degradation of polyubiquitinated short-lived proteins by proteasome. However, evidence indicates a selective degradation pathway of ubiquitinated protein through autophagy via docking proteins such as p62 and the related protein NBR1. Thus, the two major intracellular protein degradation systems are directly linked. We previously reported that macrolide antibiotics including clarithromycin (CAM) block autophagy flux, and that combined treatment with CAM and proteasome inhibitor bortezomib (BZ) enhances ER-stress-mediated apoptosis in myeloma and breast cancer cells, whereas treatment with CAM alone results in almost no cytotoxicity (Moriya S, et al. Int J Oncol. 2013, Komatsu S, et al. Biochem Biophys Res Commun. 2013). HDAC6, a microtubule-associated deacetylase, is a component of the aggresome and has the capacity to bind both polyubiquitinated misfolded proteins and dynein motors. HDAC6 recruits misfolded protein cargo to dynein motors for transport to aggresomes, which is recognized as a cytoprotective response serving to sequester misfolded proteins and facilitate their clearance by autophagy. Therefore, evidence suggests the existence of an elaborate intracellular network system for processing unfolded proteins. In the present study, we further investigated the combined effect of vorinostat (suberoylanilide hydroxamic acid (SAHA)), which has a potent inhibitory effect for HDAC6 (IC₅₀ 37 nM), with CAM and BZ in myeloma cell lines. SAHA exhibited some cytotoxicity along with an increased acetylation level of α-tubulin, a substrate of HDAC6. Combined treatment of SAHA, CAM, and BZ potently enhanced the apoptosis-inducing effect compared with treatment using each reagent alone or a combination of two of the three. Concomitant treatment with CAM and BZ for blocking autophagy flux and proteasome activity dramatically enhanced aggresome formation in perinuclear region, whereas this aggresome formation was almost completely eliminated in the presence of SAHA. Expression levels of ER-stress-related genes, including the pro-apoptotic transcription factor CHOP, were maximally induced by the simultaneous combination of three reagents. Like myeloma cell lines, a wild-type murine embryonic fibroblast (MEF) cell line exhibited enhanced cytotoxicity and maximally up-regulated Chop after combined treatment with SAHA, CAM, and BZ; however, a Chop KO MEF cell line almost completely canceled this enhanced effect. The specific HDAC6 inhibitor tubacin also exhibited a pronounced cytocidal effect with a combination of CAM plus BZ. These data suggest that simultaneous targeting of intracellular proteolytic pathways and HDAC6 enhances ER-stress-mediated apoptosis in myeloma cells, and also provide novel insight into “ER-stress loading therapy” for myeloma patients.