Abstract 1227: Identification of the molecular target of azithromycin as an autophagy inhibitor and its potential application in cancer therapeutics
Authors: Takano Naoharu , Yamada Yumiko , Tokuhisa Mayumi , Hino Hirotsugu , Hiramoto Masaki , Miyazawa Keisuke 
Published: September 2020

Abstract
Autophagy is a self-digestive cellular mechanism that enables survival under nutrient depleted conditions. In normal cells, autophagy has been known to prevent tumorigenesis by removal of genotoxicity-causing mitochondria, as well as transformed cells. In contrast, once cancer has been established, autophagy appears to play a cytoprotective role to adapt to the insufficiently vascularized hypoxic and low nutrient environment; and also works for metastasis and therapeutic resistance. Therefore, autophagic inhibitors could be suitable candidates for cancer therapeutics. However, the only two autophagic inhibitors clinically available at present are chloroquine (CQ) and hydroxychloroquine (HCQ). We previously reported that azithromycin (AZM), a macrolide antibiotic, has an inhibitory effect on autophagy and that combined administration of AZM with various anti-cancer drugs, such as a tyrosine kinase inhibitor and a proteasomal inhibitor, appears to enhance the anti-cancer effect. With an aim to explore the clinical application of AZM for cancer therapy, we attempted to identify the molecular target of AZM for autophagy inhibition. High throughput affinity purification using AZM-conjugated magnetic nano-beads was used to identify several candidates for the AZM-binding protein involved in autophagy inhibition from human NSCLC-derived A549 cell lysates. Knockdown of one of the candidate genes resulted in a prominent accumulation of autophagosomes and lysosomes, leading to autophagy inhibition. Fluorescence microscopic observation of A549 cells expressing GFP-LC3 and LAMP1-mCherry markers suggested that AZM suppressed the fusion between autophagosomes and lysosomes. Comparison of autophagy inhibitory capacity using the GFP-LC3/RFP-LC3ΔG fluorescent reporter system revealed an almost equivalent autophagic inhibition between AZM and HCQ but less cytotoxicity by itself at the same concentrations. Suppression of lysosomal acidification was not observed upon AZM administration as same as in the case of HCQ administration. However, unlike HCQ, AZM did not increase the levels of HIF-1α and phospho-ERK1/2. This indicates the existence of different autophagy inhibitory mechanisms for AZM and HCQ. In the A549 murine xenograft model, daily oral administration of AZM (100 μg/g/day) significantly suppressed tumor cell growth. These results from the present study encourage the use of AZM as an autophagy inhibitor for cancer therapy.
Citation Format: Naoharu Takano, Yumiko Yamada, Mayumi Tokuhisa, Hirotsugu Hino, Masaki Hiramoto, Keisuke Miyazawa. Identification of the molecular target of azithromycin as an autophagy inhibitor and its potential application in cancer therapeutics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1227.