Macrolide Antibiotics Block Autophagy Flux and Sensitize to Bortezomib Via Endoplasmic Reticulum-Stress-Mediated CHOP Induction in Myeloma Cells
Authors: Moriya Shota , Che Xiao-Fang , Komatsu Seiichiro , Abe Akihisa , Kawaguchi Tomohiro , Gotoh Akihiko , Inazu Masato , Tomoda Akio , Miyazawa Keisuke 
Published: October 2019

Abstract
Abstract 4992
Macroautophagy (hereafter, “autophagy”) is a highly conserved cellular process of self-degradation in eukaryotes. Intracellular proteins and organelles including the endoplasmic reticulum (ER) are engulfed in a double-membrane vesicle called 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, recent reports revealed the selective degradation pathway of ubiquitinated protein through autophagy via docking proteins such as p62 and the related protein NBR1, having both a microtubule-associated protein 1 light chain 3 (LC3)-interacting region and a ubiquitin-associated domain. LC3 is essential for autophagy and is associated with autophagosome membranes after processing. By binding ubiquitin via their C-terminal ubiquitin-associated domains, p62-mediated degradation of ubiquitinated cargo occurs by selective autophagy. Thus the two major intracellular degradation systems are directly linked. We have reported on the inhibition of autophagy using the autophagy inhibitor bafilomycin A1enhanced bortezomib (BZ)-induced apoptosis by burdening ER stress in multiple myeloma (MM) cell lines. It was also reported that clarithromycin (CAM) attenuated or blocked autophagy flux, probably mediated through inhibiting the lysosomal function. We therefore investigated whether simultaneous inhibition of protein degradation systems such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome system by a macrolide antibiotic enhances the loading of ER-stress and ER–stress-mediated CHOP (CADD153) induction, followed by transcriptional activation for proapoptotic genes.
BZ potently induces autophagy, ER–stress, and apoptosis in MM cell lines (e. g. U266, IM-9, and RPMI8226). The macrolide antibiotics including CAM, concanamycin A, erythromycin (EM), and azithromycin (AZM) all blocked autophagy flux, as assessed by intracellular accumulation of LC3B-II and p62. Combined treatment of BZ and CAM or AZM enhanced cytotoxicity in MM cell lines, although treatment with either CAM or AZM alone exhibited almost no cytotoxicity. This combination also substantially enhanced aggresome formation, intracellular ubiquitinated proteins, and induced the proapoptotic transcription factor CHOP. Expression levels of the proapoptotic genes transcriptionally regulated by CHOP (e. g. BIM, BAX, DR5, and TRB3) were all enhanced by combined treatment with BZ plus CAM, compared with treatment with each reagent alone. Like the MM cell lines, the CHOP+/+ murine embryonic fibroblast (MEF) cell line exhibited enhanced cytotoxicity and up-regulation of CHOP and its transcriptional targets with a combination of BZ and one of the macrolides. In contrast, CHOP−/− MEF cells exhibited resistance against BZ and almost completely canceled enhanced cytotoxicity with a combination of BZ and a macrolide.
These data suggest that ER-stress mediated CHOP induction is involved in pronounced cytotoxicity. Simultaneously targeting two major intracellular protein degradation systems such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome system by a macrolide antibiotic enhances ER-stress-mediated apoptosis in MM cells. This result suggests the therapeutic possibility of using a macrolide antibiotic with a proteasome inhibitor for MM therapy.

Disclosures:
No relevant conflicts of interest to declare.