Evaluation Of Anti-Tumor Effects Of Ver-155008, A Functional Inhibitor Of Hsp70, Through Macroautophagy In Mesothelioma Cells

Abstract Pleural mesothelioma is a poor prognostic malignancy because the effect of therapies remains limited; and thus, it is urgent to elucidate the pathophysiology leading to newer treatment. Our previous investigation showed that growth of mesothelioma cells was inhibited by suppression of dishevelled-3 on Wnt signaling pathway, accompanied with reduction of heat shock protein 70 (HSP70), which works as a cell-protective molecular chaperon against heat stress, oxidative stress, heavy metals and others, by refolding proteins. Furthermore, HSP70 has been showed to affect cell signaling such as Akt/mTOR pathway, which negatively regulates macroautophagy. This study evaluated effects of VER-155008, an adenosine-derived functional inhibitor of HSP70, on cell growth and macroautophagy of mesothelioma cell lines. Mesothelioma cell lines including MSTO-211H (211H), NCI-H2452 (H2452) and NCI-H28 (H28) were cultured with 0.5-40 µM VER-155008 for cell viability analysis by water-soluble tetrazolium salt-8, colony formation assay, cell cycle analysis and the following analyses. Western blotting confirmed the expression of HSP70, Akt, phosphorylated (p-) Akt, JNK, p-JNK, light chain 3A (LC3A) and lysosome-associated membrane protein 2A (LAMP2A). For evaluation of macroautophagy, a plasmid, pMRX-IP-GFP-LC3-RFP-LC3ΔG, was transfected to mesothelioma cells. The transfected cells generate GFP-LC3-RFP-LC3ΔG, and it was divided into GFP-LC3 and RFP-LC3ΔG by inherent ATG4. GFP-LC3 decreases by macroautophagy activation, while RFP-LC3ΔG serves as an internal control. VER-155008 suppressed cell viability dose-dependently, and colony formation of 211H, H2452 and H28. In 211H and H28 cells, the proportions of cell count in G1 phase significantly increased, accompanied with inhibition of cell growth. In western blot analysis, VER-155008 reduced expression of p-Akt, while the expression of Akt, p-JNK, JNK, LC3A, LAMP2A and HSP70 were not altered. In macroautophagy analysis by the use of mesothelioma cells, which were transfected with the plasmid and stably expressed GFP-LC3-RFP-LC3ΔG, 20 µM VER-155008, 5.0 µM gefitinib and deprivation of fetal bovine serum (FBS) induced macroautophagy. In addition, VER-155008 enhanced FBS-deprivation-induced macroautophagy. From these results, functional inhibition of HSP70 by VER-155008 suppressed cell growth in pleural mesothelioma cells, accompanied with enhancement of macroautophagy. Citation Format: Kosuke Sakai, Maya Inoue, Hiroaki Nishimura, Shintaro Mikami, Yoshiki Kuwabara, Akitoshi Kojima, Maiko Toda, Yumiko Ogawa, Satoshi Kikuchi, Yusuke Hirata, Yuriko Mikami, Hiroyuki Kyoyama, Gaku Moriyama, Akihiko Gemma, Kazutsugu Uematsu. Evaluation of anti-tumor effects of VER-155008, a functional inhibitor of HSP70, through macroautophagy in mesothelioma cells [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 1830.