br Protein degradation in the cell is mainly
Protein degradation in the cell is mainly performed by the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway. In the UPS, proteins labeled by a chain of ubiquitin are recognized and then degraded by the 26 S proteasome consisting of a 19S regulatory particle
Corresponding authors at: Key Laboratory of Protein Modification and Degradation, State Key Laboratory of Respiratory Disease, School of Basic Medical Sciences; Aﬃliated Cancer Hospital and institute of Guangzhou Medical University, Guangzhou, Guangdong 511436, China.
E-mail addresses: [email protected] (J. Liu), [email protected] (H. Huang).
1 Equal contribution.
and a 20S proteasome. Nevertheless, deubiquitinating enzymes (DUBs) can reverse target protein degradation by removing ubiquitin/ubi-quitin-like chains. DUBs function to regulate multiple cellular pro-cesses, including DNA damage responses, Olaparib control, and many signaling pathways (Aressy et al., 2010; Song and Rape, 2008). Ap-proximately 100 putative DUBs are encoded in human genome and classified into six families based on their catalytic and structural fea-tures (Hussain et al., 2009). Among these enzymes, three diﬀerent DUBs (i.e., USP14, UCHL5 and Rpn11) are present in the 19S regulatory particle in mammalian cells. Rpn11 is a metalloprotease and an ob-ligatory subunit of the 19 S proteasome complexes (Brnjic et al., 2014; D'Arcy and Linder, 2012; Wang et al., 2015; Weissman et al., 2011). USP14 and UCHL5 are cysteine proteases involved in deubiquitination at the proteasome, and belonging to the USP and the UCH families, respectively. USP14 and UCHL5 are overexpressed in various carci-noma cells, providing a potential therapeutic target (Mialki et al., 2013). Several small molecule inhibitors have been developed to target USP14 and UCHL5; some of them have been used in preclinical tests (D'Arcy and Linder, 2014).
Auranofin (Aur), otherwise known as a thioredoxin reductase in-hibitor (Fiskus et al., 2014; Rigobello et al., 2004), has also been re-ported as a USP14/UCHL5 selective inhibitor (Huang et al., 2016; Liu et al., 2014). Aur exerts potent anti-tumor eﬀects through regulating proteolytic activities of the 19 S. In our previous report, we have de-monstrated that Aur's thioredoxin reductase inhibition is not necessary for its proteasome inhibition and that proteasome inhibition is required for cytotoxicity mediated by Aur (Liu et al., 2014). Our prior studies also have shown that USP14 stabilizes androgen receptor by deubi-quitination in breast cancer and PCa (Liao et al., 2017, 2018), sug-gesting that Aur might be beneficial to PCa treatment. Since this pre-mise has not yet been examined, the aim of the present study was to fill this gap. By examining the eﬀect of Aur on PCa and exploring the un-derlying mechanisms, the present study provides further compelling support for a promising anti-cancer strategy in which inhibition of USP14 and UCHL5 with Aur suppresses the growth and progression of androgen receptor-positive PCa both in vitro and in vivo.
2. Material and methods
Auranofin (C20H35AuO9PS) was obtained from Enzo Life Sciences International, Inc. (Plymouth Meeting, PA) and suspended in DMSO at 10 mmol/L concentration, aliquoted and stored at −80 °C. MG132 was purchased from Selleckchem (Houston, TX, USA). Antibodies were obtained from following sources: anti- androgen receptor (Abcam, USA); anti-Bcl-2 (15071s), anti-caspase cleave 3(9661 s), anti-PARP (9542s), anti-CDK4 (1279s), anti-cyclinD1 (29269), anti-p21 (2947P), anti-CHOP (2895 s), anti-eIF2α (53249D7D3), phospho-eIF2α (3398Sd9g8), anti-ubiquitin (3936s), anti-K48-linkage Specific Polyubiquitin (8081s) (Cell Signaling Technology, MA, USA); anti-GAPDH (Bioworld Technology, St. Louis Park, MN, USA). MTS assay (CellTiter 96 Aqueous One Solution reagent) was gained from Promega Corporation. Apoptosis Detection Kit was obtained from Keygen Company in Nanjing, China. Dynabeads antibody coupling kit was obtained from Life technologies.