Research ArticleExperimental Studies

Enzalutamide-induced Proteolytic Degradation of the Androgen Receptor in Prostate Cancer Cells Is Mediated Only to a Limited Extent by the Proteasome System

HOLGER H. H. ERB, MARIA A. OSTER, NADINE GELBRICH, CLEMENS CAMMANN, CHRISTIAN THOMAS, ALEXANDER MUSTEA and MATTHIAS B. STOPE
Anticancer Research July 2021, 41 (7) 3271-3279; DOI: https://doi.org/10.21873/anticanres.15113

Abstract

Background/Aim: Androgen receptor (AR) degradation is the primary regulator of androgen receptor activity. This study was designed to investigate the influence of the proteasome on AR protein stability after enzalutamide (Enz) treatment. Materials and Methods: Cell counting after treatment was utilized to assess the effect of Enz on cell proliferation. Changes in mRNA levels were evaluated using reverse transcription-polymerase chain reaction (RT-PCR). Proteasome activity was assessed by measurement of the chymotrypsin-like activity of the beta-5 subunit of the proteasome. Changes in protein levels after treatment with Enz, MG132 (MG), bortezomib (Bor), or their combination were assessed using western blot analysis. Results: Treatment with Enz led to a significant reduction of cell proliferation and AR protein levels. However, AR mRNA levels were unchanged. Inhibition of proteasome activity by MG counteracts the Enz-mediated AR degradation transiently, whereas Bor showed no inhibition of the Enz-mediated AR degradation. Conclusion: Enz-mediated change in AR stability as an early and essential event after treatment was shown. However, investigations of the ubiquitin/proteasome system indicate involvement of several proteases in the Enz-mediated AR degradation process.

Key Words:

The androgen receptor (AR) is a nuclear receptor and is activated by binding androgens, such as testosterone and dihydrotestosterone (1, 2). The AR protein consists of 919 amino acids and has a molecular mass of 110 kDa. The gene is located on the X chromosome at Xq11-12 and has eight exons coding four functional domains, which are homologs to those of the nuclear receptor superfamily (2). These domains include the N-terminal transactivation domain, the DNA-binding domain, the hinge region, and the ligand-binding domain. The AR is involved in various diseases such as androgen insensitivity syndrome, benign prostatic hyperplasia, and prostate cancer (PC).

PC is the most common cancer in the western world, with an estimated record of 1,414,259 new cases diagnosed yearly and 375,304 yearly deaths related to PC worldwide (3). In metastatic PC, one therapy option is the direct inhibition of the AR by the anti-androgen enzalutamide (Enz). It competes with androgens for AR binding and therefore prevents AR activity (4-9).

Enz is a second-generation anti-androgen that has been approved in 2012 to treat metastatic PC (10). It inhibits androgen binding to the AR, AR translocation into the nucleus, AR binding to DNA, and co-regulator recruitment (11). Despite the initial tumor responses to treatment, Enz is only effective for a specified period until the disease progresses and drug resistance occurs (12, 13). The resistance mechanisms to Enz are not fully understood; however, several molecular adaptations such as AR amplification and mutation, changes in co-regulator expression and recruitment, and bypass pathways have been identified (12-18).

Another underlying resistance mechanism appears to be increased AR protein stability (19). In vitro data revealed that cells treated with anti-androgen had reduced AR protein levels. Especially, heat shock proteins (HSP) and the proteasomal machinery have been identified to be involved in this anti-androgen-mediated AR protein degradation (19). Previous studies already described the role of AR protein degradation in the development of castration resistance and PC progression (20). Moreover, in the presence of Enz, HSP reduction was demonstrated, which forms a complex with the AR to control its stability in the cytosol (21). This study investigated the influence of the proteasome on AR protein stability after Enz treatment.

Materials and Methods

Cell culture. The human cell line LNCaP (Cell Lines Service, Eppelheim, Germany) was used as an AR-positive PC model. Propagation was done in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin/streptomycin and 1% pyruvate (PAN Biotech, Aidenbach, Germany) at 37°C and 5% CO2 atmosphere. Cells were passaged two to three times per week.

Proliferation assay. Cell proliferation was determined using a CASY Cell Counter and Analyzer model TT (Roche Applied Science, Mannheim, Germany). After incubation, adherent cells were resuspended using 0.1% trypsin/0.04% ethylenediaminetetraacetic acid (EDTA; PAN Biotech, Aidenbach, Germany) treatment, and 100 μl of the cell suspension was diluted in 10 ml CASYton solution (Roche Applied Science). To assess the live cell count, 400 μl of this cell suspension was measured in triplicates using a 150 μm capillary. Differentiation into live cells, dead cells, and cell debris was performed by LNCaP-specific gate settings of 12.25 μm (live cells vs. dead cells) and 6.00 μm (dead cells vs. cell debris).

mRNA analysis. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed for quantification of mRNA using the SensiMix SYBR Kit (Bioline, London, UK) and a CFX96 Real-Time PCR Detection System (BioRad, München, Germany). Total RNA was extracted using peqGOLD TriFast (Peqlab, Erlangen, Germany) as described by the manufacturer. Subsequently, 1.0 μg of total RNA was transcribed into complementary DNA using the RevertAid First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany). Detection of AR and the reference gene ribosomal protein large P0 (RPLP0) mRNA was performed using specific oligonucleotides (AR-forward: 5’-TGCCTGATCTGTGGAGATGA-3’, AR-reverse: 5’-CGAAGACGACAAGATGGACA-3’, RPLP0-forward: 5’-CAATGGCAGCATCTACAACC-3’, RPLP0-reverse: 5’-ACTCTTCCTTGGCTTCAACC-3’).

Protein analysis. Adherent cells were treated with lysis buffer (50 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate, 10% glycerol, 0.01% bromophenol blue, 5% 2-mercaptoethanol). 20 μl of protein lysate was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mini-Protean System, BioRad, München, Germany). Subsequently, the protein was transferred to a Protean nitrocellulose membrane (Whatman, Dassel, Germany) using the semi-dry Trans-Blot SD transfer cell (BioRad). After blocking the membranes (Roti block; Carl Roth, Karlsruhe, Germany), proteins were detected with specific antibodies against AR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Cell Signaling Technology (Danvers, MA, USA). Signals were visualized using SuperSignal West Dura chemiluminescent substrate (Thermo Scientific; Waltham, MA, USA) in a ChemiDoc system (BioRad, München, Germany) with Image Lab 5.1 beta software (BioRad).

Proteasome activity assay. Proteasome activity assay determines the chymotrypsin-like activity of the beta5 subunit of the proteasome. After 3 cycles of freeze-thaw in freeze-thaw buffer [10 mM Tris (ph 7.0), 25 mM KCl, 1.1 mM MgCl2, 10% glycerol, 1 mM DTT, complete protease inhibitor cocktail from Roche Applied Science, Mannheim, Germany], protein concentration was determined using Bradford solution (BioRad). Subsequently, 1.0 μg of protein was incubated in assay buffer (30 mM Tris (pH 7.5), 5 mM MgCl2, 10 mM KCl and 10% glycerol) and then transferred to a black 96-well plate. After the addition of 25 μM of the fluorogenic substrate Suc-LLVY-AMC (Bachem, Bubendorf, Switzerland), the preparation was measured in an Infinite M200 plate reader (Tecan, Männedorf, Switzerland) at 37°C (excitation: 360 nm; emission: 460 nm) and compared to control.

Statistical analysis. Statistical analysis of data was performed with Graph Pad Prism 9.1 software (GraphPad Software, La Jolla, CA, USA) using the unpaired Student’s t-test. Data are expressed as mean±standard deviation (SD). p-Values of ≤0.05 were considered significant. All differences highlighted by asterisks were statistically significant as encoded in figures (n.s.: not significant; *p≤0.05; **p≤0.01; ***p≤0.001). All experiments were performed in at least three independent biological replicates.

Results

Enzalutamide treatment induces AR degradation in LNCaP cells. Previous studies indicated that Enz does not achieve its growth inhibitory effect by antagonizing AR signaling alone and suppresses cellular protein expression, including the AR protein itself (21). In the AR-positive cell line LNCaP, 10 μM Enz significantly suppresses cell growth within 72 h (Figure 1A). On mRNA level, Enz did not alter the expression of AR mRNA levels (Figure 1B). However, the growth reduction was accompanied by a rapid decrease in intracellular AR concentration. Within just 4 h, AR protein concentration was already significantly lower compared to control cells (Figure 1C and D).

Figure 1.
Figure 1.

Enzalutamide treatment induces androgen receptor degradation in LNCaP cells. (A) Tumor cell growth of LNCaP cells after 24, 48, and 72 h incubation with 10 μM Enzalutamide (Enz). Data were assessed by cell counting, normalized to the 0 h time point, and shown as mean±SD of four independent experiments. (B) AR mRNA levels after Enz-exposure of LNCaP cells for 24, 48, and 72 h. mRNA levels were determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and normalized to ribosomal protein large P0 (RPLP0). (C) Representative western blot for androgen receptor (AR) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein after Enz-exposure of LNCaP cells. (D) Densitometry of AR protein expression levels relative to GAPDH after Enz-exposure. Data are normalized to the corresponding untreated control (dashed line) and shown as box and whisker (min to max) of three independent western blot experiments.

Inhibition of proteasome activity by MG132 counteracts the Enz-mediated AR degradation transiently. Proteasomes are protein complexes degrading unneeded or damaged proteins by proteolysis (22, 23). Therefore, it was evaluated if proteasomes mediate the observed effects of Enz on the AR protein levels. The proteasome inhibitor MG132 (MG; 5 μM) led to a significant reduction in total proteasome activity up to 4 h in LNCaP cells; however, after 6 h of incubation, inhibition of the proteasome activity was not consistent anymore (Figure 2A). In line with the proteasome activity, the cells exhibited a significant increase in AR protein concentration up to 4 h (Figure 2B and C).

Figure 2.
Figure 2.

Inhibition of proteasome activity by MG132 counteracts the enzalutamide-mediated androgen receptor degradation transiently. (A) Proteasome activity after 2, 4, and 6 h MG132 (MG) treatment. Chymotrypsin-like activity of the proteasome 20S subunit was used to determine proteasome activity. Data were normalized to the untreated condition (dashed line) and shown as box and whisker (min to max) of four independent experiments. (B) Representative western blot for androgen receptor (AR) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein after MG treatment of LNCaP cells. (C) Densitometry of AR protein expression levels relative to GAPDH after MG treatment of LNCaP cells. Data are normalized to the corresponding untreated control (dashed line) and shown as box and whisker (min to max) of three independent western blot experiments. (D) Schematic representation of methodology for AR protein stability experiments with MG. (E) Representative western blot for AR and GAPDH protein after 2 h of enzalutamide (Enz), MG, and Enz plus MG treatment of LNCaP cells. (F) Densitometry of AR protein expression levels relative to GAPDH of LNCaP cells after 2 h treatment of Enz, MG, and Enz plus MG. Data are normalized to the corresponding untreated control (dashed line) and shown as box and whisker (min to max) of three independent western blot experiments. (G) Representative western blot for AR and GAPDH protein after 4 h of Enz, MG, and Enz plus MG treatment of LNCaP cells. (H) Densitometry of AR protein expression levels relative to GAPDH of LNCaP cells after 4 h treatment of Enz, MG, and Enz plus MG. Data are normalized to the corresponding untreated control (dashed line) and shown as box and whisker (min to max) of three independent western blot experiments.

To analyze whether inhibition of proteasomal protein degradation diminishes the Enz-induced suppression of AR expression, LNCaP cells were treated with Enz combined with the proteasome inhibitor MG. For this purpose, LNCaP cells were treated for 24 h with Enz, followed by Enz and MG (Figure 2D). Based on previous experiments (Figure 2A), Enz and MG were incubated for 2 h (Figure 2E and F) and 4 h (Figure 2G and H).

Single incubation with Enz and MG resulted in the expected suppression or restoration of AR levels (Figure 2E-H). However, the combination of Enz plus MG resulted in an initial restoration of AR protein levels after 2 h incubation (Figure 2E and F) followed by a re-suppression of the AR protein levels after 4 h (Figure 2G and H).

Inhibition of proteasome activity by Bor does not counteract Enz-mediated AR degradation in LNCaP cells. To validate the results obtained with MG, the proteasome inhibitor Bortezomib (Bor) was used. Bor (20 nM) led to a significant reduction in total proteasome activity in LNCaP cells throughout the entire incubation period (Figure 3A). Concomitantly, the cells exhibited a significant increase in AR protein concentration (Figure 3B and C).

Figure 3.
Figure 3.

Inhibition of proteasome activity by bortezomib does not counteract enzalutamide-mediated androgen receptor degradation in LNCaP cells. (A) Proteasome activity after 2, 4, and 6 h Bortezomib (Bor) treatment. Chymotrypsin-like activity of the proteasome 20S subunit was used to determine proteasome activity. Data were normalized to the untreated condition (dashed line) and shown as box and whisker (min to max) of four independent experiments. (B) Representative western blot for androgen receptor (AR) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein after Bor treatment of LNCaP cells. (C) Densitometry of AR protein expression levels relative to GAPDH after Bor treatment of LNCaP cells. Data are normalized to the corresponding untreated control (dashed line) and shown as box and whisker (min to max) of three independent western blot experiments. (D) Schematic representation of methodology for AR protein stability experiments with Bor. (E) Representative western blot for AR and GAPDH protein after 2 h of Enzalutamide (Enz), Bor, and Enz plus Bor treatment of LNCaP cells. (F) Densitometry of AR protein expression levels relative to GAPDH of LNCaP cells after 2 h treatment of Enz, Bor, and Enz plus Bor. Data are normalized to the corresponding untreated control (dashed line) and shown as box and whisker (min to max) of three independent western blot experiments. (G) Representative western blot for AR and GAPDH protein after 4 h of Enz, Bor, and Enz plus Bor treatment of LNCaP cells. (H) Densitometry of AR protein expression levels relative to GAPDH of LNCaP cells after 4 h treatment of Enz, Bor, and Enz plus Bor. Data are normalized to the corresponding untreated control (dashed line) and shown as box and whisker (min to max) of three independent western blot experiments.

To analyze whether Bor also inhibits Enz-induced AR degradation, LNCaP cells were treated with Enz combined with Bor. In line with the MG experiments, LNCaP cells were treated 24 h with Enz, followed by Enz plus Bor (Figure 3D). Single incubation with Enz and MG resulted in the expected suppression or restoration of AR levels (Figure 3E-H). However, in contrast to the results with MG, incubation of Enz and Bor did not restore the AR protein levels at the tested time points (Figure 3E-H).

Discussion

The nonsteroidal second-generation anti-androgen Enz is a promising agent for patients with metastatic PC, demonstrating improved survival in castration-resistant as well as in hormone-sensitive state; however, relapse and development of therapy resistance remain the main reasons for poor survival (9, 12, 24, 25). Several molecular mechanisms in anti-androgen resistance have been described, including concepts of target modification, bypass signaling, histologic transformation, cancer stem cells, and crosstalk between androgen-sensitive and androgen-insensitive cells (12, 18, 26, 27). AR protein regulation has also been described as a regulator of AR activity and a possible mechanism in therapy resistance (19). Puhr et al. revealed that PIAS1 is a determinant act as a co-activator of AR signaling through enhanced AR stabilization. Subsequently, the efficiency of Abiraterone or Enz treatment is decreased in the presence of PIAS1 and, therefore, may favor therapy resistance (28). STAT5 has been described as a regulator of AR stability and involved in castration- and anti-androgen resistance (17, 20, 29). HSPs have also been linked to the regulation of AR stability and therefore influence AR activity (21). In Enz-resistant PC, the HSP70 protein protects the AR of ubiquitination and degradation and affects the AR inhibitory effects of the anti-androgen (30). This study aimed to shed light on the mechanism behind the influence of Enz on AR protein stability. The present study was also able to confirm that treatment with Enz led to a decrease in cell proliferation accompanied by a significant reduction in AR protein levels after 4 h (17, 20, 21, 28-30).

In contrast, AR mRNA did not change after Enz treatment indicating a post-transcriptional regulation of the AR by Enz. This conclusion is strengthened by chromatin immunoprecipitation experiments showing no androgen response elements in the AR gene promotor and also no regulation of the AR gene transcription by the AR (31, 32). The posttranscriptional regulation is also verified by Kemppainen et al., who demonstrated intracellular AR degradation after androgen deprivation with a half-time of approximately 1 h (33).

As the data presented here reveal that Enz does not influence AR mRNA levels, the AR protein stability must be controlled by direct regulation mechanisms such as phosphorylation and the ubiquitin/proteasome system (34). Therefore, using the proteasome inhibitor MG, an attempt was made to reverse the Enz-induced protein degradation of the AR. The inhibitor MG is a peptide aldehyde, which effectively blocks the proteolytic activity of the 26S proteasome complex (35). The results revealed a transient inhibition of the Enz-induced AR degradation for 2 h, diminished after 4 h. This transient rescue of the AR protein indicates a potential role of the ubiquitin/proteasome system in the mechanism of action of Enz. MG has a low selectivity specific for all three catalytic subunits of the proteasome (beta1, beta2, beta5) and also inhibits other proteases such as cathepsins and calpain (36).

As Cathepsin D and calpain have been reported to be involved in AR degradation, the distinctly more specific proteasome inhibitor Bor, acting primarily on the beta5 subunit, has been used to validate the involvement of the proteasome in the Enz-mediated degradation of the AR (37, 38).

The experiments performed here demonstrate that Bor suppresses protease activity for the entire experimental period but cannot prevent Enz-mediated degradation of the AR. This result reveals a complex and proteasome-independent mechanism responsible for the degradation of the AR after treatment with Enz. Indeed, protease inhibition studies have shown that the AR turnorver is also controlled by a panel of cytosolic proteases independent of gene expression and proteasome system (39, 40).

Our study identified the change in AR stability as an early and essential event after treatment with Enz. However, investigations of the ubiquitin/proteasome system failed to reveal the exact mechanism behind the Enz-mediated AR degradation. Therefore, more molecular analyses are mandatory to decipher the role of AR degradation in response to Enz.

Footnotes

  • ↵* These Authors contributed equally to the study.

  • This article is freely accessible online.

  • Authors’ Contributions

    Conceptualization: HHHE, MBS. Methodology: HHHE, AO, NG, CC, US. Formal analysis: AO, NG, MBS. Investigation: HHHE, AO, MBS. Writing-Original Draft Preparation: HHHE, MBS. Writing-Review & Editing: HHHE, CT, A.M., MBS. Supervision: CT, A.M., MBS.

  • Conflicts of Interest

    The Authors declare that they have no competing interests.

  • Received May 19, 2021.
  • Revision received May 28, 2021.
  • Accepted June 15, 2021.

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Enzalutamide-induced Proteolytic Degradation of the Androgen Receptor in Prostate Cancer Cells Is Mediated Only to a Limited Extent by the Proteasome System
HOLGER H. H. ERB, MARIA A. OSTER, NADINE GELBRICH, CLEMENS CAMMANN, CHRISTIAN THOMAS, ALEXANDER MUSTEA, MATTHIAS B. STOPE
Anticancer Research Jul 2021, 41 (7) 3271-3279; DOI: 10.21873/anticanres.15113
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