br Aur downregulates the protein level
3.5. Aur downregulates the protein level of androgen receptor
Our previous studies have demonstrated that USP14 could regulate the stability of androgen receptor proteins in PCa and breast carci-nomas (Liao et al., 2017, 2018). Inhibition of USP14 significantly in-creased the degradation of androgen receptor protein. To verify whe-ther Aur can aﬀect the stability of androgen receptors via inhibiting UCHL5 and USP14, the androgen receptor protein levels were assessed using western blot. We found that Aur caused decreases in androgen receptor protein expression in androgen receptor-positive PCa cells in a dose-dependent manner (Fig. 5A and B). The androgen receptor sig-naling pathway mediates survival and growth of PCa via multiple me-chanisms. Recent studies have shown that a few DUBs can modulate the localization or function of target proteins. USP10 deubiquitinates an-drogen receptor in the cytoplasm and increases its nuclear import and transcriptional activity (Faus et al., 2005).
To better understand the expression of androgen receptors in the nucleus and in the cytoplasm and explore whether Aur could co-translocate with androgen receptors, we examined the androgen re-ceptor protein distribution and expression in LNcap and 22RV1 cells exposed to Aur (1, 2 μM) using confocal microscopy. As shown in
Fig. 3. Aur induced proteasome inhibition and ER stress in PCa cells. (A) Cell lysates were extracted from 22RV1 cells treated with or without Aur for 24 h and the lysates were incubated with the HA-UbVS probe for 1 h. The protein levels of native USP14 and UCHL5 as well as their UbVS-bound counterparts were im-munodetected using the primary cck-8 against USP14 and UCHL5, respectively. Quantitative data were shown. GAPDH was used as an internal control. *P < 0.05, #P < 0.01 vs the control treatment group. (B) Cell viability analysis was performed on prostate cancer cells post either USP14 siRNA, UCHL5 siRNA or the combination treatment for 48 h.(C) 22RV1 and LNcap cells were exposed to the indicated concentrations of Aur for 24 h. Total proteins were extracted from the treated cells. Western blot assay was used to assess the total ubiquitinated proteins (Ub-prs.), K48-linked ubiquitin chain conjugated proteins (K48-), eIF2α, phosphorylated eIF2α (P-eIF2α) and CHOP expression. Quantitative data were shown. GAPDH was used as a loading control. *P < 0.05, #P < 0.01 vs the control treatment group.
Fig. 5C, androgen receptor was located in both the nucleus and cyto-plasm, albeit mainly in nucleus; Aur decreased the abundance of an-drogen receptor in the nucleus and cytoplasm but did not seem to alter androgen receptor nucleus to cytoplasm ratio. These results demon-strate that Aur downregulates the abundance of the total androgen receptor in androgen receptor-positive PCa cells.
3.6. Aur promotes androgen receptor protein degradation and inhibits androgen receptor transcription
In addition, we also tested whether Aur would enhance the de-gradation of androgen receptor proteins using the cycloheximide chase assay. The assay showed that the half-life of androgen receptor was approximately 6 h in LNcap and 22RV1 cells but more importantly Aur
Fig. 4. Aur suppressed cell cycle progression in PCa cells. (A) LNcap and 22RV1 cells were treated with Aur at the indicated dose for 24 h. Cells were harvested and fixed in 70% ethanol. Flow cytometry was used to record the distribution of cell cycle in each group. Representative images are shown and (B) the percentage of cells in each phase were calculated from three independent replicates. Mean ± S.D. (n = 3). (C) Protein lysates were extracted from PCa cells treated with Aur for 24 h. CDK4, cyclin D1 and p21 proteins were analyzed using western blot. Quantitative data were shown. GAPDH was used as a loading control. *P < 0.05, #P < 0.01 vs the control treatment group.
treatment induced rapid decreases in androgen receptor half-life in both PCa cell lines (Fig. 6A and B). To further confirm that the UPS is in-volved in androgen receptor degradation in Aur-treated PCacells, we detected the expression of ubiquitinated androgen receptors using co-IP and found that the level of ubiquitinated androgen receptors was dra-matically increased in Aur-treated group (Fig. 6C). Lastly, we tested whether Aur also would regulate the mRNA level of androgen re-ceptors. RT-qPCR results revealed that Aur decreased the mRNA ex-pression of the androgen receptor and the PSA gene (a target gene of androgen receptor) (Fig. 6D). These results suggest that Aur also reg-ulate androgen receptor expression at both protein and mRNA levels.
PCa is the most prevalent malignancy among men and a serious threat to men's health worldwide. Over recent years, ADT and anti-androgen therapy are used as the standard method to suppress the progression of PCa. However, these treatments may eventually lose their eﬀectiveness due to the development of the CRPC in many patients (Ingersoll et al., 2015). Additionally, there is an increased risk of myocardial infarction and stroke in patients treated with ADT (Bosco et al., 2015; Zhao et al., 2014). When ADT fails, alternative treatment approaches targeting androgen receptors need to be employed.