Inhibition of deubiquitination by PR-619 induces apoptosis and autophagy via ubi-protein aggregation-activated ER stress in oesophageal squamous cell carcinoma

Objectives: Targeting the deubiquitinases (DUBs) has become a promising avenue for anti-cancer drug development. However, the effect and mechanism of pan-DUB inhibitor, PR-619, on oesophageal squamous cell carcinoma (ESCC) cells remain to be investigated.

Materials and methods: The effect of PR-619 on ESCC cell growth and cell cycle was evaluated by CCK-8 and PI staining. Annexin V-FITC/PI double staining was performed to detect Apoptosis. LC3 immunofluorescence and acridine orange staining were applied to examine Autophagy. Intercellular CA²⁺ concentration was monitored by Fluo-3AM fluorescence. The accumulation of ubi-proteins and the expression of the endoplasmic reticulum (ER) stress-related protein and CaMKKβ-AMPK signalling were determined by immunoblotting.

Results: PR-619 could inhibit ESCC cell growth and induce G2/M cell cycle arrest by downregulating cyclin B1 and upregulating p21. Meanwhile, PR-619 led to the accumulation of ubiquitylated proteins, induced ER stress and triggered Apoptosis by the ATF4-Noxa axis. Moreover, the ER stress increased cytoplasmic CA²⁺ and then stimulated Autophagy through CA²⁺ -CaMKKβ-AMPK signalling pathway. Ubiquitin E1 inhibitor, PYR-41, could reduce the accumulation of ubi-proteins and alleviate ER stress, G2/M cell cycle arrest, Apoptosis and Autophagy in PR-619-treated ESCC cells. Furthermore, blocking Autophagy by chloroquine or bafilomycin A1 enhanced the cell growth inhibition effect and Apoptosis induced by PR-619.

Conclusions: Our findings reveal an unrecognized mechanism for the cytotoxic effects of general DUBs inhibitor (PR-619) and imply that targeting DUBs may be a potential anti-ESCC strategy.