br Androgen receptor is a nuclear receptor transcriptional
Androgen receptor is a nuclear receptor/transcriptional factor with transcriptional activity that depends on the transactivation domains: AF-1 and AF-2 domains, as well as LBD domains. The basic transcrip-tional machinery consists of the following processes: first, androgen receptor signaling is activated by the binding of androgen to androgen receptor in the cytosol. Upon androgen binding, androgen receptor conformation is changed and the nuclear localization signal is un-masked, resulting in translocation of the ligand-activated androgen receptor to the nucleus where androgen receptor binds the androgen-responsive elements of target genes. Recent studies have indicated that
androgen receptor dominates the development and progression of PCa (Ferraldeschi et al., 2015), suggesting that androgen receptor signaling is the critical target for the treatment of PCa. In addition, most CRPC also depends on the transcriptional activity of androgen receptors for cell survival and growth. The crosstalk between androgen receptor and epidermal growth factor receptor drives the progession of PCa (Hsieh et al., 2013; Izumi et al., 2012; Munoz-Moreno et al., 2014; Sugita et al., 2004). EGF increased transcriptional activity of androgen receptor by activating MAPK pathways in cck-8 cancer cells (Kue et al., 2002). Recent studies have explored the post-translation modification of an-drogen receptors, including phosphorylation, acetylation, ubiquitina-tion and methylation, in order to change androgen receptor transcrip-tional activity. Yet, many of them have suggested that ubiquitination has a central role in androgen receptor activity regulation (Liao et al., 2017; McClurg et al., 2018). Many E3 ubiquitin ligases participate in the ubiquitination of androgen receptors and thereby regulate the an-drogen receptor stability. For example, MDM2 is involved in the an-drogen receptor ubiquitylation and degradation by AKT and has been established as an androgen receptor E3 ubiquitin ligase (Gaughan et al., 2005; Lin et al., 2002).
A few DUBs are over-expressed in cancer cells and some of them are emerging as novel targets for anticancer strategies. Indeed, several DUBs have shown to play a regulatory role in androgen receptor ex-pression or transcriptional activation. For example, USP7 modulates androgen receptor transcriptional output by restraining the binging of androgen receptor to chromatin (Chen et al., 2015). Androgen alters the position of the androgen receptor LBD domain, resulting in USP7-mediated deubiquitination of androgen receptors. Inhibition of USP7 can weaken androgen-regulated androgen receptor activation in PCa cells. Moreover, USP10 increases androgen receptor activation and transcriptional activity by binding and deubiquitinating androgen re-ceptor (Draker et al., 2011; Faus et al., 2005). Deubiquitinases on the 19S proteasome have been reported as a class of original biomarkers in
Fig. 5. Aur aﬀected androgen receptor expression but not its localization. (A-B) Total protein extracts from 22RV1 or LNcap cells that were exposed to the in-dicated concentration of Aur for 24 h were subject to western blot analyses for androgen receptor and GAPDH. Quantitative data were shown. *P < 0.05, #P < 0.01 vs the control treatment group.(C)LNcap and 22RV1 cells treated with Aur (1 μM) for 12 h were fixed and subject to immunofluorescence staining for androgen receptor. Nuclei were stained with DAPI. Three independent ex-periments were performed. Representative fluorescence micrographs are shown. Scale bar = 10 µm.
tumor tissues. UCHL5 and USP14 are essential for the degradation of proteins. The total K48-linked poly-ubiquitin conjugated proteins are accumulated by inhibition of UCHL5 and USP14, thus implying that UCHL5 and USP14 are involved in proteasomal degradation of most proteins. However, the mechanism through which USP14 and UCHL5 regulate proteasomal degradation of a specific protein remains largely unknown. In our previous study, we have reported that anti-rheumatoid agent Aur could inhibit UCHL5 and USP14, in addition to its well-known inhibition of thioredoxin reductases (Fiskus et al., 2014; Liu et al., 2014; Rigobello et al., 2004). Aur is clinically used to treat rheumatoid arthritis and US Food and Drug Administration has ap-proved its use for phase II clinical trial for cancer therapy. Hence, a better understanding of the molecular mechanism underlying the an-ticancer eﬀect of Aur is warranted.
Our data show that Aur inhibits cell proliferation in androgen European Journal of Pharmacology 846 (2019) 1–11
receptor-positive prostate cancer cells, suppresses the growth of sub-cutaneous xenografts of PCa in nude mice, and notably decreases tumor weight, without aﬀecting body weight, thus indicating that Aur may be a safe agent to treat PCa. In the present study, Aur induced higher levels of cell apoptosis, following caspase-3 activation, PARP cleavage and accumulation of ubiquitin proteins. Moreover, Aur arrested cell cycle at the G0/G1 phase in LNcap and 22RV1 cells. It decreased the levels of CDK4 and cyclin D1 and upregulated p21 in androgen receptor-positive PCa cells. Additionally, Aur downregulated total androgen receptor protein expression although it did not alter androgen receptor nuclear localization in LNcap and 22RV1 cells. Consistent with the inhibitory role of Aur on USP14 and UCHL5, we found that ubiquitinated an-drogen receptor was increased and androgen receptor half-life was significantly shortened following the Aur treatment. Indeed, we have previously shown that USP14 functions as a DUB for androgen re-ceptors and thereby slows down the proteasomal degradation of an-drogen receptors (Liao et al., 2017, 2018). It is known that Aur induces intracellular reactive oxygen species generation and inhibits thior-edoxin reductase activity (Fiskus et al., 2014; Rigobello et al., 2004). In our previous report (Liu et al., 2014), we have shown that Aur can inhibit 19 S proteasome associated deubiquitinases USP14 and UCHL5, and cell apoptosis induced by Aur occurs after proteasome inhibition, but thioredoxin reductase inhibition by Aur is not required for pro-teasome inhibition. Accordingly, we concluded that downregulating androgen receptor by promoting its degradation through Aur is de-pendent on USP14 inhibition, but not on thioredoxin reductase in-hibition. In addition, we found that Aur decreased the mRNA level of androgen receptors, thus suggesting that transcriptional activity of androgen receptors was inhibited by Aur. These data demonstrate that Aur induces antiproliferation of PCa cells by modulating not only the post-translational modification but also the transcriptional activity of androgen receptors (Fig. 6E). Froscio et al. has reported that Aur's thioredoxin reductase inhibition induced decreased activity of PKC which mediates androgen receptor signaling (Baek et al., 2017; Froscio et al., 1989; Peterziel et al., 1999). However, tertiary butylhy-droquinone which decrease reactive oxygen species generation, did not block Aur-induced proteasome inhibition (Liu et al., 2014). Hence, we speculated that Aur inhibited androgen receptor signaling by PKC pathway and promoted the degradation of androgen receptor result from USP14 and UCHL5 inhibition.