In the present study, PTEN^−/− mice and transgenic adenocarcinoma of mouse prostate (TRAMP) mice were obtained from Jackson Laboratory. All animal protocols were approved by the Animal Care and Use Committee of the Texas A&M Health Science Center at Houston (AUP IACUC 2015-0132-IBT). Normal prostate tissues and prostate tumor tissues were collected from three sets of 11-month-old wild-type, PTEN^−/− and TRAMP mice after mice were sacrificed by performing cervical dislocation by appropriately trained personnel approved on the animal protocol. A total of 150 patients including 112 patients with PCa and 38 with BPH as controls were evaluated in the present study. As previously described (21), these patients had consented to donate prostate tissue samples from surgery or via biopsy and underwent treatment including radiotherapy, prostatectomy, transurethral resection of the prostate (TURP) and hormone therapy between 1999 and 2003 at The Fifth Affiliated Hospital of Guangzhou Medical University, Sun Yat-Sen Memorial Hospital, and The First People's Hospital of Guangzhou and The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China.

All primary samples were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E). Paraffin-embedded, formalin-fixed prostate tissues of patients and mice were immunostained for p62, Keap1 and Nrf2 protein using an immunohistochemical detection kit (GTVision) according to the manufacturer's protocol. Immunostained slides were independently evaluated under a Leica DM3000 microscope by two pathologists in a double-blind manner.

Cell culture, transfection and siRNA. DU145 cells were obtained from the American Type Culture Collection (ATCC); maintained in RPMI-1640 medium (HyClone Laboratories; GE Healthcare Life Sciences) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 units/ml penicillin and 100 µg/ml streptomycin in a humidified incubator at 37°C with 5% CO₂; and sub-cultured for a passage every 3–4 days. Stable DU145 cell strains expressing different levels of p62 were constructed as previously described: C1 carrying the control plasmid vector, OE overexpressing p62, KO with p62 knocked out by CRISPER technology and C2 negative control of KO (22). Validated siRNAs against Keap1 and Nrf2 were purchased from Guangzhou Ribobio Co., Ltd. Cells were treated with 10 mM bafilomycin A1 (BAF) or 10 mM MG-132 for 6 h when necessary.

Whole-cell extracts were prepared with RIPA buffer containing the broad protease inhibitor cocktail (Roche Diagnostics). Nucleoprotein extraction was prepared with NE-PER Nuclear Cytoplasmic Extraction Reagent kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The protein concentration was determined using a BCA kit. Equal amounts of total proteins (25 µg) were separated via 10% SDS-PAGE gel and transferred to PVDF membranes. Then, 5% skimmed milk (Devondale Milk) was used to incubate at room temperature for 2 h. Then the membranes were blotted with primary antibodies against p62 (product code ab56416, dilution 1:2,500), Keap1 (product code ab218815, dilution 1:1,500) and Nrf2 (product code ab194986, dilution 1:1,000; all were from Abcam), Lamin B1 (cat. no. sc-374015, dilution 1:1,000; Santa Cruz Biotechnology, Inc.) or GAPDH (cat. no. AF0911, dilution 1:4,000; Affinity Biosciences), all the primary antibodies were incubated at 4°C overnight. Protein bands were visualized using their respective goat anti-rabbit IgG (H+L) (dilution 1:1,000; incubated at room temperature for an 1 h) or goat anti-mouse IgG (H+L) (dilution 1:1,000; incubated at room temperature for an 1 h) (Wuhan Boster Biological Technology, Ltd.) and ECL reagent (Thermo Fisher Scientific, Inc.). ImageJ^® software (v1.52p; National Institutes of Health) was used for densitometric analysis.

Confocal fluorescent microscopy was performed as previously described (22). Briefly, cells were grown on glass coverslips in 6-well plates for 6 h, washed three times with PBS, fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, and incubated with 2% BSA for 1 h at room temperature. Treated cells were first blotted with an antibody against Keap1 (product code ab139729) or Nrf2 (product code EP1808Y; both from Abcam) for 16 h at 4°C, and subsequently incubated with DYLight649-conjugated goat anti-rabbit IgG (H+L) secondary antibody (cat. no. GAR6492; Multi Sciences) for 1 h at room temperature. After washing with PBS, coverslips were mounted with Fluoromount-G™ (Invitrogen; Thermo Fisher Scientific, Inc.) and visualized under a confocal laser scanning microscope (Zeiss) at an ×20 magnification.

Total RNA was isolated using the UNlQ-10 Column TRIzol Total RNA Isolation kit (Sangon Biotech Co., Ltd.) according to the manufacturer's protocol, and reverse-transcribed to cDNA using the PrimeScript™ RT reagent kit (Takara Bio, Inc.). The conditions used were as follows: 30 sec 95°C, followed by 50 cycles of 5 sec 95°C, 34 sec 60°C, then 15 sec 95°C, and 1 min 50°C. The gene-specific primers presented in Table I were obtained from PrimerBank. Differential expression was calculated using the 2^−ΔΔCq method as described (23). Data are presented as the mean ± standard deviation of three independent experiments.

The Annexin V-APC/7AAD Apoptosis Detection system was used to measure rates of apoptosis according to the manufacturer's protocol. Briefly, cells were cultured in RPMI-1640 medium in 10-cm dishes to a confluence of 80–90%, digested with 0.25% trypsin without EDTA, re-suspended in 500 µl of binding buffer supplied with 5 µl APC-Annexin V and 10 µl 7AAD, and incubated at room temperature for 15 min after mixing. The apoptosis rate was detected using BD Accuri C6 flow cytometer and analyzed with the associated software from BD Biosciences.

The rates of cell proliferation were measured using the Real Time Cellular Analysis (RTCA) system. Briefly, cells were harvested at 48 h after infection with siRNA, diluted and seeded into a 16-well strip at a density of 1×10⁴ cells/well. Cell proliferation was monitored in real time with sensor devices placed in the incubator with 5% CO₂.

Cell invasion was continuously monitored every 15 min for 36 h by real-time monitoring on CIM-Plate 16 pre-coated with Matrigel (356234; Corning Inc.) with an RTCA iCELLigence Analyzer (ACEA Biosciences). Briefly, the infected cells were starved for 6 h in serum-starved medium, diluted and seeded into a 16-well strip at a density of 3×10⁵ cells/well in the upper chamber. The lower chamber was filled with medium containing 10% serum.

The detection of ROS was performed using the Cell ROX Orange reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The cell-permeable reagents are non-fluorescent or weakly fluorescent in a reduced state, and emit strong fluorescence signals upon oxidation. DU145 cells in a 6-well plate were incubated in medium containing a final concentration of 5 µM CellROX Orange reagent at 37°C for 30 min and washed three times with pre-warmed PBS after the medium was removed. Levels of ROS of cells were quantified using ImageJ software (v1.52p; National Institutes of Health) based on the fluorescence intensities of cells in captured fields.

Experimental data were presented as the mean ± standard error of at least three independent experiments. Differences in results were analyzed by a two-tailed unpaired t-test using the GraphPad Prism version 7 for Windows (GraphPad Software, Inc.). P<0.05, P<0.01, and P<0.001 were considered to indicate a statistically significant result (*P<0.05, **P<0.01 and ***P<0.001 as indicated in the figures).

It has previously been reported that PCa tissues have higher levels of cytoplasmic p62 than benign prostate hyperplasia (BPH) tissues although p62 whose significance remains unknown was also observed to be distributed in nuclei. In addition, PCa tissues from patients with higher grades or metastasis have significantly higher levels of p62 than those from patients with lower grades or no metastasis (20). The present study further demonstrated that p62 was primarily located in the cytoplasm of epithelial cells of human PCa and BPH tissues (Fig. 1A-C). Keap1 was also primarily located in the cytoplasm of epithelial cells of human PCa and BPH tissues (Fig. 1D and E) while Nrf2 was detected in both the nucleus and cytoplasm (Fig. 1F and G). The levels of p62 and Nrf2 were higher in human PCa than those in BPH (Fig. 1A-C, F and G), while the levels of Keap1 were higher in BPH than those in PCa (Fig. 1D and E). Similarly, higher levels of p62 and Nrf2 and lower levels of Keap1 were detected in PCa tissues collected from PTEN^−/− and TRAMP mice compared with normal prostate tissues collected from normal wild-type mice (Fig. 1H-P). The results indicated that p62 and Nrf2 may act as oncogenes of PCa.

To examine the association between the levels of p62, Keap1 and Nrf2 proteins, the present study prepared extracts from four different DU145 cell strains expressing different levels of p62. Increasing the expression levels of p62 significantly decreased the levels of Keap1 and enhanced the levels of total Nrf2 and nuclear Nrf2 (Fig. 2A-H), respectively. In contrast, decreasing the expression level of p62 significantly increased levels of Keap1 and reduced levels of total Nrf2 and nuclear Nrf2 (Fig. 2I-P), respectively. In addition, the effects of p62 on the levels of Keap1 and total Nrf2 were further confirmed by immunofluorescence microscopy analyses (Fig. 2Q-T). Therefore, p62 suppressed the levels of Keap1 and enhanced the levels of Nrf2.

In order to determine whether aberrant Nrf2 activity was associated with apoptosis, proliferation and invasion in vitro, DU145 cells overexpressing p62 (OE) were transfected with negative control siRNA (Ctl siRNA) and Nrf2-specific siRNA (Nrf2 siRNA). A knockdown efficiency of 80% was confirmed by qPCR and immunoblotting (Fig. 3A and B). Flow cytometric analyses of cell apoptosis revealed that DU145 cells overexpressing p62 (OE) had lower rates of apoptosis than its control C1 (Fig. 3C and D), while silencing the expression of Nrf2 in OE cells induced prominently higher rates of apoptosis (Fig. 3E and F). In contrast, the overexpression of p62 led to increases in cell proliferation and invasion, while further decreasing the increased levels of Nrf2 expression with siRNA led to decreases in cell proliferation and invasion (Fig. 3G and H). Notably, rates of cell proliferation and invasion between OE and Ctl siRNA cells were different potentially due to the impact of control siRNA (Fig. 3G and H). Therefore, p62 decreased apoptosis and promoted proliferation and invasion of DU145 cells through Nrf2.

In order to investigate the mechanism by which p62 enhances proliferation, invasion and anti-apoptosis, RT-qPCR was used to assess the mRNA levels of Nrf2 target genes such as HO-1, NQO-1, GCLC and glutamate-cysteine ligase modifier subunit (GCLM). It was revealed that p62 increased the mRNA levels of Nrf2 target genes (Fig. 4A-D). Overexpression of p62 led to a decrease in ROS levels, while suppressing the expression of p62 induced an increase in ROS levels (Fig. 4E and F). Thus, p62 increased the expression of antioxidant-responding genes and suppressed ROS.

Numerous studies have demonstrated that Nrf2 protein is degraded through the a proteasomal system (13,24,25). It was revealed in the present study that the levels of Nrf2 in the presence of proteasomal inhibitor MG132 were always increased independent of the levels of p62, while levels of Nrf2 in the presence of autophagy inhibitor BAF were only increased in cells with low levels of p62 (C1 and KO), but remained constant in cells with high levels of p62 (C2 and OE) (Fig. 5). These results indicated that some portions of Nrf2 protein are always degraded through the proteasomal system but some portions of the Nrf2 protein that are modified differentially in the absence of p62 are degraded through the autophagy system.

In order to investigate the association between Keap1 and Nrf2, the expression of Keap1 in DU145 cells with p62 knocked down was suppressed. The levels of Nrf2 were increased (Figs. 2G and H and 6A). The suppression of Keap1 was confirmed in both mRNA (Fig. 6A) and protein levels (Fig. 6B and C). Such suppression caused a significant increase in the levels of Nrf2 protein (Fig. 6B and D) but had no impact on the levels of Nrf2 mRNA (Fig. 6E). Therefore, p62 increased the level of Nrf2 through Keap1 in a post-transcriptional manner.