Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2020 Sep 1.
Published in final edited form as: Prostate. 2019 Jan 28;79(6):667–677. doi: 10.1002/pros.23774

Effect of Monoamine oxidase A (MAOA) inhibitors on androgen-sensitive and castration-resistant prostate cancer cells

Shikha Gaur 1, Mitchell E Gross 2, Chun-Peng Liao 2, Bin Qian 1, Jean C Shih 1,3,4
PMCID: PMC7462252  NIHMSID: NIHMS1618625  PMID: 30693539

Abstract

Background:

Monoamine oxidase A (MAOA) is best known for its role in neurotransmitter regulation. Monoamine oxidase inhibitors are used to treat atypical depression. MAOA is highly expressed in high grade prostate cancer and modulates tumorigenesis and progression in prostate cancer. Here, we investigated the potential role of MAOA inhibitors (MAOAIs) in relation to the androgen receptor (AR) pathway and resistance to antiandrogen treatment in prostate cancer.

Methods:

We examined MAOA expression and the effect of MAOI treatment in relation to AR-targeted treatments using the LNCaP, C4–2B, and 22Rv1 human prostate cancer cell lines. MAOA, AR-full length (AR-FL), AR splice variant 7 (AR-V7), and PSA expression was evaluated in the presence of MAOAIs (clorgyline, phenelzine), androgenic ligand (R1881), and antiandrogen (enzalutamide) treatments. An enzalutamide resistance cell line was generated to test the effect of MAOAI treatment in this model.

Results:

We observed that MAOAIs, particularly clorgyline and phenelzine, were effective at decreasing MAOA activity in human prostate cancer cells. MAOAIs significantly decreased growth of LNCaP, C4–2B, and 22Rv1 cells and produced additive growth inhibitory effects when combined with enzalutamide. Clorgyline decreased expression of AR-FL and AR-V7 in 22Rv1 cells and was effective at decreasing growth of an enzalutamide-resistant C4–2B cell line with increased AR-V7 expression.

Conclusions:

MAOAIs decrease growth and proliferation of androgen-sensitive and castration-resistant prostate cancer cells. Clorgyline, in particular, decreases expression of AR-FL and AR-V7 expression and decreases growth of an enzalutamide-resistant cell line. These findings provide preclinical validation of MAOA inhibitors either alone or in combination with antiandrogens for therapeutic intent in patients with advanced forms of prostate cancer.

Keywords: androgen receptor splice variant, clorgyline, enzalutamide, phenelzine, prostate cancer

1 |. INTRODUCTION

Prostate cancer is a major cause of cancer-related morbidity and mortality.1 Androgens, acting through androgen receptor (AR), are required for growth and maintenance of both benign and malignant prostate tissue.2–4 Androgen deprivation therapy (ADT) is the first-line treatment for locally advanced or metastatic prostate cancer. Androgen ablation provides an initial benefit for the majority of patients, but many patients progress within 3–5 years to a state known as castrate-resistant prostate cancer (CRPC).5 AR activity remains critical for tumor growth even in CRPC as demonstrated by the significant improvement in progression-free and overall survival observed with drugs targeting the AR-axis, such as enzalutamide and abiraterone in this population.6 However, resistance to enzalutamide or abiraterone has become a major clinical problem as 20–40% of patients show primary resistance to these agents and acquired secondary resistance develops in the vast majority of the remaining patients.7,8 New approaches are needed to augment the clinical benefit seen by targeting the AR pathways in CRPC.

Prior studies have identified mechanisms of antiandrogen resistance including: expression of dominant active AR splice variants (ARVs),9–11 mutations in AR ligand binding domain12,13 and amplification of full length AR (AR-FL).14,15 Sun and colleagues16 have suggested that constitutively active AR splice variants, induced following castration, play a role in the development of treatment resistance in prostate cancer. The AR-V7 splice variant, also known as AR3, is one of the most abundant and best characterized variants which has been associated with resistance to enzalutamide or abiraterone.17 Thus ARV7 may represent an important predictive biomarker and therapeutic target in treatment-resistant CRPC.

The function of monoamine oxidase (MAO) in the nervous system and other tissues has been extensively studied.18 Two MAO isoenzymes (MAOA and MAOB) function in neurotransmitter regulation. MAO inhibitors (MAOAIs) have been well characterized based on relative selectivity for inhibiting MAOA versus MAOB enzymatic activity.19 MAO inhibitors in clinical use include: moclobemide (reversible MAOA selective); tranylcypromine and phenelzine (both irreversible and non-selective); and selegeline/deprenyl (irreversible, MAOB selective). Clorgyline is a MAOA selective irreversible inhibitor that was previously prescribed as a treatment for depression, but is no longer manufactured for clinical use. Currently, the most widely used MAOA inhibitor in clinical use is phenelzine for atypical/treatment refractory major depression.

Several studies point towards a potential role for MAOA in prostate cancer. Previously, investigators have reported a strong correlation between MAOA expression in tissue and the presence of high-grade (Gleason patterns 4 or 5) prostate cancer.20,21 In addition, MAOA expression is associated with higher levels of serum prostate specific antigen (PSA) in patients at diagnosis.22 High MAOA expression in prostate cancer tissues correlated with worse clinical outcomes in patients.21,23 Further we have previously shown that the MAOA gene itself is a target of AR-ligand mediated transcriptional control24 and MAOA gene deletion decreases tumorigenesis and cancer stem cell formation in a PTEN-dependent murine prostate cancer model.25

Clorgyline affects many oncogenic pathways in prostate cancer cells, suggesting clinical value of MAOAIs as a pro-differentiation and anti-oncogenic therapy for high-risk prostate cancer.26,27 The findings collectively define the contribution of MAOA in prostate cancer pathogenesis and suggest the therapeutic potential of MAOAIs in CRPC.

Given the importance of AR and MAOA in prostate cancer, we hypothesized that MAOAIs may potentiate effects of enzalutamide on prostate cancer cells. First, we demonstrate that clorgyline decreases proliferation of prostate cancer cell lines and inhibits expression of AR-dependent genes and exhibits additive effects in combined treatment with enzalutamide, an antiandrogen. Second, we show that clorgyline decreases expression of AR-V7 as a possible mechanism for the pharmacologic interaction between enzalutamide and clorgyline and suggest combined treatment with these agents may have therapeutic potential in patients with advanced prostate cancer.

2 |. MATERIALS AND METHODS

2.1 |. Materials

Human prostate cancer cell lines LNCaP and 22RV1 were purchased from American Type Culture Collection (Manassas, VA). C4–2B28 cells were kindly provided and authenticated by Dr. Leland W. Chung, Cedars-Sinai Medical Center (Los Angeles, CA). Cell culture media and serum were obtained from Invitrogen Life Technologies (Carlsbad, CA). Enzalutamide (MDV-3100) was purchased from Selleckchem (Houston, TX) and R1881, Clorgyline and Phenelzine were obtained from Sigma-Aldrich (St. Louis, MO). Antibodies against different proteins were obtained from Santa Cruz Biotechnologies Inc. (Santa Cruz, CA), Cell signaling technology Inc. (Beverley, MA) or Abcam (Cambridge, MA). HRP Conjugated anti-mouse and anti-rabbit IgG were purchased from Cell signaling technology Inc. and Super signal chemiluminescence substrates were purchased from Thermo Fisher Scientific (Waltham, MA). All other reagents were obtained from VWR (Radnor, PA).

2.2 |. Cell culture

The tumor cell lines were maintained in culture as adherent cells in a monolayer in humidified atmosphere at 37°C and 5% CO2 in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100units/ml penicillin and 0.01 mg/mL streptomycin. For serum starvation experiments the cells were cultured in RPMI-1640 without phenol-red and supplemented with FBS treated with charcoal-dextran (Gene Tex, Irvine, CA). Cells in all cultures were passaged to fresh medium twice a week. The C4–2B enzalutamide resistant (C4–2B-ER), cell line was generated by long-term culture of C4–2B parenteral cells in the presence of 5 μM enzalutamide (fresh medium containing enzalutamide was changed twice/week) for approximately 6 months.

2.3 |. Cell viability assay

The cell viability was measured using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (Promega Corporation, Madison, WI) assay as described earlier.29 IC50 values were calculated from the dose response curve generated from the prostate cancer cell lines in the absence or presence of the drug(s). Briefly, 2000 cells were grown in 96-well plate and after 24 h, a wide range of concentrations of drugs were added to the cells. Next, MTS reagent was added to the wells after 72 h for 2 h according to the manufacturer’s recommendations, and the intensity of the color developed was read at 490 nm using Biotek plate reader (Winooski, VT). Data from cell viability assay (MTS) were expressed as the fraction of cells killed by the individual drugs or the combination of drugs and compared to untreated cells set at 100%. IC50 values were calculated from the dose response curves. For combination experiments the drugs were mixed in 1:1 ratio of IC50 concentration or maximum achievable dosage.

2.4 |. PSA ELISA

Approximately 5 × 104 cells were plated in 6 well plates. The cells were serum starved for 24 h. R1881 and/or clorgyline was added to the cells in fresh medium which was then collected after 24 h. PSA levels were determined using a PSA ELISA kit (American Qualex Antibodies, San Clemente, CA).

2.5 |. MAOA catalytic activity assay

In a 10-cm dish, 5 × 105 cells were plated in medium supplemented with 10% FBS. After 24 h, cells were cultured in medium with or without serum for 48 h and harvested in PBS (pH 7.4). After centrifugation, the cell pellet was suspended in 800 μL of assay buffer (50 mM sodium phosphate buffer, pH 7.4) and sonicated. Approximately 200 μg of protein lysate from cells was incubated with 100 μM [14C] serotonin in the assay buffer. The reaction products were extracted and radioactivity was measured as previously described.30

2.6 |. Western blot analysis

The cells were collected after treatment with the drugs (24 h) and washed once with PBS and second time with cold PBS containing 0.1 mM orthovanadate. The cells were serum starved for 24 h for R1881 experiments. The whole cell lysates were prepared according to the procedures described previously.29 Protein was measured using Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) and resolved by (25 μg protein per lane) 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The proteins were transferred to PVDF membrane (Amersham, Arlington Heights, IL) and probed with any of the following: AR (1:500), AR-V7 (1:1000), MAOA (1:500) or PSA (1:1000) antibodies. Blots were then incubated with HRP-conjugated secondary antibody (Amersham) followed by ECL western blotting substrate detection from Thermo Fisher Scientific (Waltham, MA). Super signal ELISA Femto substrate was used for detection of ARV7. GAPDH was measured as the protein for loading control. The blots were imaged with Bio-RAD chemiDoc imaging system (Hercules, CA). The ECL bands were scanned, and the optical density for each band was determined using Image lab software (Bio-RAD).

2.7 |. Quantitative real-time PCR

Total cellular RNA was isolated from cells using Direct-Zol RNA mini-prep kit (Zymo Research, Irvine, CA). On-column DNase I digestion was performed during RNA purification to remove Genomic DNA contamination. The RNA concentration and A260:A280 ratio was measured using a Nanodrop spectrophotometer (NanoDrop Technologies Inc., DE). Subsequent cDNA synthesis was carried out with PCR conditions including an initial denaturation step of 3 min at 95°C, followed by 40 cycles of PCR consisting of 30 s at 95°C, 30 s at 60°C, and 40 s at 72°C. All primer sequences used are as follows: AR-V7: F-5′-CAGGGATGACTCTGGGAGAA-3′, R-5′-GCCCTCTAGAGCCCTCATTT-3′, AR-FL: F- 5′-TCTTGTCGTCTTCGGAAATGT-3′, R-5′-AAGCCTCTCCTTCCTCCTGTA-3′, MAOA: F-5′-CTGATCGACTTGCTAAGCTAC-3′, R-5′- ATGCACTGGATGTAAAGCTTC-3′, PSA: F-5′-AGTGCGAGAAGCATTCCCAAC-3′, R-5′-CCAGCAAGATCACGCTTTTGTT-3′, UBE2C: F-5′TGGTCTGCCCTGTATGATGT-3′ R-5′AAAAGCTGTGGGGTTTTTCC-3′, and 18S rRNA-F 5′-CGCCGCTA GAGGTGAAATTC-3′, 18S rRNA-R 5′-CGAACCTCCGACTTTCGTTC-3′. A relative gene-expression quantification method was used to calculate the fold change of mRNA expression according to the comparative Ct method using 18S rRNA as an internal control for normalization.

2.8 |. Statistical analysis

Each data set is presented as mean ± standard error mean of a minimum of three times (independent replicates). Results are compared using two-tailed student’s t-test with Microsoft Excel analysis tools. A P-value < 0.05 was considered statistically significant.

3 |. RESULTS

3.1 |. MAOAI decreases proliferation of prostate cancer cells

We first compared effects of MAO inhibitors in relation to androgen-sensitivity in prostate cancer cell lines. LNCaP cells express androgen receptor (AR) and exhibit androgen-responsive growth. The C4–2B line was derived from LNCaP cells and retains AR expression, but exhibits castratration-resistant/androgen-independent growth.31

As seen in Figure 1, MAOA activity was inhibited in both cell lines. In general, moclobemide, a reversible MAOAI, exhibited the weakest effect on MAOA activity. Deprenyl also exhibited weak effects on MAOA consistent with the known selectivity for MAOB inhibition. Phenelzine, an irreversible MAOA inhibitor, was particularly active in inhibiting MAOA activity with IC50 of ~8 × 10−10 and ~6 × 10−9 M in LNCaP and C4–2B cells, respectively. Similarly, clorgyline was effective at inhibiting MAOA activity with an IC50 of ~5 × 10−12 and ~3 × 10−7 in LNCaP and C4–2B cell, respectively (Figure 1A–C). MAOA activity is presented to compare expression levels at baseline across cell lines (Figure 1D).

FIGURE 1.

FIGURE 1

Open in a new tab

The potency of MAO inhibitors on MAOA activity in prostate cancer cell lines. Effect of various doses of clorgyline, moclobemide, deprenyl, tranylcypromine, and phenelzine on MAOA catalytic activity on LNCaP (A) and C4–2B (B) prostate cancer cell lines was determined. Data is expressed as % MAOA catalytic activity measured in untreated control cells and log (MAOA inhibitor concentration in M). Data is summarized in inset (C). MAOA catalytic activity of LNCaP, C4–2B, and 22Rv1 cells determined by radioassay using serotonin as substrate is summarized (D)

As clorgyline is a highly selective and potent MAOA-specific MAOAI, we studied the effects of clorgyline on proliferation in androgen-sensitive (LNCaP) and castration-resistant (C4–2B and 22Rv1) prostate cancer cell lines. As shown in Figure 2, we observed a dose-dependent decrease in proliferation following 72 h treatment of clorgyline in all three cell lines. Specifically, seventy-two hours of exposure to 80 μM clorgyline led to a ~60% growth reduction of LNCaP (Figure 2A) and C4–2B (Figure 2B) cells and a ~20% reduction in 22Rv1 cell growth (Figure 2C). Thus, clorgyline was more effective in reducing cell growth in these androgen-dependent than castration-resistant cell lines.

FIGURE 2.

FIGURE 2

Open in a new tab

Clorgyline decreases proliferation of androgen-dependent and androgen-independent prostate cancer cells determined by MTS assay. A total of 2–5 × 103 LNCaP (A), C4–2B (B), and 22Rv1 (C) prostate cancer cells were seeded into 96-well plates. The cells were serum starved for 24 h before the addition of indicated concentrations of R1881 and clorgyline for 72 h. The viability for each cell line was assessed with the MTS assay. Cell number in untreated control was set at 100%. Each data set represents the mean ± SD of three independent populations (n = 3). *P < 0.05

3.2 |. Clorgyline inhibits ligand-dependent AR-signaling

Next, we investigated the effects of clorgyline on AR-signaling. Prostate specific antigen (PSA, KLK3) is an AR-transcriptional target gene which encodes a secreted protein widely used as a diagnostic and prognostic biomarker in prostate cancer patients. The AR gene is itself regulated by androgenic activity, therefore we examined effects of clorgyline on endogenous PSA and AR expression levels. Clorgyline decreased PSA and AR expression by ~60% and 30%, respectively, at the mRNA (Figure 3A) and ~90% and 60%, respectively, at the protein level (Figure 3B). Next we investigated if clorgyline is able to decrease ligand-induced increases in PSA and AR expression. As expected, R1881 (methyltrienolone, a synthetic AR-ligand) increased the AR levels by close to 1.5 fold and PSA levels by more than twofold over baseline. Clorgyline significantly decreased ligand-induced PSA and AR expression. Finally, we examined the dose dependent effect of clorgyline on PSA secretion from LNCaP cells with an ELISA assay. R1881 increased PSA secretion by more than 20 fold over baseline while clorgyline decreased the amount of PSA in a dose dependent manner both in the presence or absence of R1881 (Figure 3C). Taken together, these results indicate that clorgyline can suppress the AR-signaling pathway in prostate cancer cells.

FIGURE 3.

FIGURE 3

Open in a new tab

Clorgyline blocks ligand-induced androgen signaling in prostate cancer cells. LNCaP cells were treated with vehicle (Ctr), 10nM R1881 (R1881), 10μM clorgyline (Clg) or both (R1881 + Clg) for 24 h in fresh medium after serum starvation for 24 h. The cells were collected and processed for (A) RT-PCR or (B) Western blot analysis. Each experiment was repeated at least three times and shows a representative blot. (B) The bar diagram (mean ± SEM, n = 3)) showing the fold change in the intensity of the band with respect to GAPDH and relative to untreated group (Ctr), (C) 20 000 cells were plated into each well of 6-well plate. The cells were starved for 24 h before the addition of indicated concentrations of clorgyline and absence or presence of 1nM R1881. The medium was collected after 24 h and change in PSA secretions were determined in cell free medium using prostatic-specific antigen ELISA kit. Each data set represents the mean ± SD of three independent populations (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001

3.3 |. MAOAIs add to antiproliferative effects of enzalutamide

Antiandrogens and androgen deprivation therapy (ADT) are a mainstay of therapy for prostate cancer as antiandrogens compete with androgens for binding to the androgen receptor.32 As our prior results suggested an interaction between MAOA and androgen pathways,24 we investigated the interaction between MAOAIs and enzalutamide (a non-steroidal antiandrogen) on cell proliferation in prostate cancer cell lines. Dose response curves for LNCaP and 22Rv1 cells treated with varying doses of clorgyline with and without 2 μM enzalutamide are shown (Figure 4A,B). Enzalutamide at 2 μM concentration inhibited cell proliferation by 35.3 ± 2.7% and 11.6 ± 1.1% (P < 0.05) in LNCaP and 22Rv1 cells, respectively. Clorgyline, when combined with enzalutamide, decreased cell proliferation by more than 20% as compared to clorgyline alone in both cell lines. Similar effects were observed with phenelzine combined with enzalutamide (Figure 4C,D). Phenelzine also decreased cell viability in a dose dependent manner in LNCaP and 22Rv1 cells which was further decreased by co-treatment with 2 μM enzalutamide. These data are summarized in Table 1. We conclude that MAOAIs exhibit an additive effect combined with enzalutamide in androgen-sensitive and androgen-insensitive prostate cancer cell lines.

FIGURE 4.

FIGURE 4

Open in a new tab

Clorgyline and phenelzine enhances anti-proliferative effects of enzalutamide in LNCaP and 22Rv1 cells. 5000 cells from each prostate cancer cell line were seeded into each well of 96-well plates. Indicated concentrations of clorgyline (Clg) in the presence or absence of 2 μM enzalutamide (Enz) were added for 72 h and cell proliferation rate was assessed using MTS assay in (A) LNCaP and (B) 22Rv1 cells. The same experimental setup was used to study effects of phenelzine (Phen) at the indicated concentrations in companion with 2 μM enzalutamide (C and D). Each data point is calculated as % of untreated control and represents mean ± SEM of three independent experiments (n = 3). *P < 0.05 for clorgyline vs clorgyline with enzalutamide

TABLE 1.

IC50 of clorgyline or phenelzine with or without enzalutamide in prostate cancer cell lines

LNCaP (μM) C4–2B (μM)
Enzalutamide 27.25 ± 2.5 46.5 ± 3.2
Clorgyhne 63.7 ± 8.2* 73.2 ± 3.0
Clorgyhne + Enzalutamide (2 μM) 12.3 ± 6.0* 40.0 ± 5.2
Phenelzme 310 ± 49 >400
Phenelzme + Enzalutamide (2 μM) 180 ± 20 306 ± 52
Open in a new tab

Half maximal Inhibitory concentration (IC50) of Clorgyline, Phenelzine, and Enzalutamide in Prostate Cancer Cell Lines. IC50 were calculated with MTS assay from cells treated in the presence of clorgyline or phenelzine and/or enzalutamide (2 μM). (n = 3).

*

P < 0.05 for LNCaP cells treated with clorgyline + enzalutamide versus clorgyline alone.

3.4 |. MAOAIs decrease full length and AR-variant androgen receptor expression

While enzalutamide is a standard treatment for CRPC, nearly all patients will eventually develop clinical resistance and progressive disease.7,32,33 Recently, expression of a constitutively active, AR-V7 produced by alternative splicing of the AR-FL transcript has been associated with resistance to antiandrogen therapy17 Therefore, we explored effects of clorgyline on AR-FL and AR-V7 expression.

As 22Rv1 cells express both AR-FL and AR-V7 (Figure 5A), we observed enzalutamide produced a significant increase in AR-FL and AR-V7 expression at both protein and mRNA levels which were decreased by co-treatment with 2 μM clorgyline (Figure 5A,B, upper panels).

FIGURE 5.

FIGURE 5

Open in a new tab

Clorgyline decreases androgen receptor-full length (AR-FL) and AR-variant 7 (AR-V7) expression in 22Rv1 cells. 22Rv1 cells were treated with vehicle (Ctr), 2 μM enzalutamide (Enz), 10 μM clorgyline (Clg), or enzalutamide and clorgyline (Enz + Clg) for 24 h in fresh medium for 24 h. The cells were collected and processed for protein (A) and mRNA (B) expression analysis. Each experiment was repeated at least three times and average results are plotted. (A) Representative Western blot (upper panel) and semi-quantiative expression of AR-FL and AR-V7 (lower panel) in 22Rv1 cells following indicated treatments is shown with GAPDH present as a loading control. Bar diagram (mean ± SEM, n = 3) showing the fold change in the intensity of the band with respect to GAPDH and relative to untreated group (Ctr), (B) 22Rv1 cells were treated with vehicle (Ctr), 10 μM clorgyline, (Clg) 2 μM enzalutamide (Enz) or enzalutamide and clorgyline (Enz + Clg) for24 h. RNA was isolated and mRNA expressions for AR-FL, AR-V7, PSA and UBE2C were measured and normalized to 18S housekeeping gene. The histogram showing the mean ± SEM of three independent experiments, n = 3. *P < 0.05

As AR-V7 splice variant is known to be induced by antiandrogen treatment, we next examined effects of co-treatment of 22Rv1 cells with enzalutamide and clorgyline. Our results show that enzalutamide treatment in 22Rv1 cells showed upregulated expression of AR-V7 by approximately 30%, which was inhibited by co-treatment with clorgyline (Figure 5A,B, upper panels).

AR-V7 regulates a subset of genes that are unique from AR-FL. UBE2C has been shown to be induced by AR-V7.4 Therefore, we examined mRNA expression of PSA and UBE2C along with AR-FL and AR-V7 in relation to clorgyline and enzalutamide treatment in the 22Rv1 cell line (Figure 5B, lower panels). Enzalutamide produced no change in the expression of UBE2C in 22Rv1 cell. However, clorgyline alone, or combined with enzalutamide, significantly decreased the UBE2C expression by 0.7 ± 0.1 and 0.4 ± 0.1 fold, respectively (Figure 5B, lower panels). Together these results show a preclinical validation of clorgyline as promising inhibitor of AR-FL, AR-V7 and their regulatory genes either alone or in combination with current anti-androgen therapy, indicating the possibility of using MAOAIs in suppression of CRPC in clinic.

3.5 |. Clorgyline can restore enzalutamide sensitivity to an enzalutamide resistant cell line

Our results suggest MAOAIs may re-sensitize enzalutamide resistant cells to androgen-directed therapies. Therefore, we tested this approach in vitro by generating an enzalutamide-resistant cell line. Specifically, the C4–2B-ER subline was generated by continuous maintenance of the C4–2B cell line in the culture medium containing 5 μM enzalutamide for more than 5 months. C4–2B-ER cells showed no decrease in cell proliferation even at enzalutamide concentration as high as 20 μM (parental C4–2B decreased significantly by >20% cell proliferation at this concentration, Figure 6A). Interestingly, the MAOA protein expression and enzymatic activity were increased in C4–2B-ER cells compared with parental cells (Figure 6B,C).

FIGURE 6.

FIGURE 6

Open in a new tab

C4–2B cells chronically treated with enzalutamide (C4–2B-ER) express AR-V7, increase expression of MAOA and are sensitive to clorgyline: (A) C4–2B and C4–2B-ER cells (5000) were plated in 96-well plate and treated with increasing concentration of enzalutamide for 72 h followed by MTS assay. Dose response curves were generated. Cell number in untreated control (Ctr) was set at 100%. N = 3, (B) Expression of AR-FL, AR-V7, PSA, and MAOA by western blot analysis in C4–2B and C4–2B-ER cells. (C) MAOA catalytic activity measured in C4–2B and C4–2B-ER cells. (D) Histogram showing mRNA expression of AR-FL, AR-V7, and MAOA measure by RT-PCR and (E) comparison of dose response curves for C4–2B and C4–2B-ER cells treated with either increasing concentrations of clorgyline or enzalutamide for three independent populations, *P < 0.05

Next, we examined the mRNA expression of AR-FL, AR-V7, and MAOA in these cell lines. C4–2B-ER cells showed significant increase in expression of AR-FL, PSA, and MAOA (Figure 6D). Consistent with an earlier report by Liu et al,34 the C4–2B-ER cells also expressed AR-V7 (Figure 6B). Most importantly, we observed that clorgyline inhibited the growth of C4–2B-ER cells at levels comparable to the parental cells (Figure 6E). These data suggest that MAOA may participate in the progression of enzalutamide resistance through AR-V7 in prostate cancer and that MAOA inhibition may restore enzalutamide-sensitivity to enzalutamide resistant prostate cancer in CRPC patients.

4 |. DISCUSSION

In this study, we evaluated the growth inhibitory effects of MAOAIs and enzalutamide in cell culture models of prostate cancer. Our goal was to elucidate possible molecular mechanisms of action for the compounds contributing to the antiproliferative capacity of human prostate cancer cells. We found that clorgyline effectively inhibited the growth of prostate cancer cell lines regardless of their sensitivity to androgen or androgen antagonist. Moreover, MAOAIs exhibit additive effects when combined with antiandrogens as treatment for androgen-responsive prostate cancer cell lines. Of greater interest, we identified clorgyline as a potent inhibitor of AR-V7 expression in a cell line model for androgen independent prostate cancer. Thus, MAOA inhibitors may be part of a strategy to overcome antiandrogen resistance by downregulation of AR-V7 in enzalutamide-resistant patients.

Previous studies have shown strong correlation between MAOA expression and high Gleason grade prostate cancer.20,21 MAOA promotor and catalytic activity is increased by androgen receptor agonist R1881.24 Androgen receptor remains a driving force in the development and progression of prostate cancer and it is maintained throughout prostate cancer progression. Consistent with previous reports, our results show that R1881 induced cell proliferation, MAOA catalytic activity, and expression of AR-FL and its target gene PSA in androgen sensitive cells. Clorgyline, a selective MAOA inhibitor, decreased R1881 induced cell growth, MAOA catalytic activity, and AR expression in androgen-sensitive cells (Figure 3). Clorgyline also inhibited the growth of androgen-independent prostate cancer cells (Figure 2). Together, these results suggest that clorgyline inhibits mitogenic pathways downstream from the androgen receptor.

Androgen deprivation therapy (ADT) is the first line treatment for locally advanced or metastatic prostate cancer and provides initial benefits to most patients. However, a majority patients will eventually develop therapeutic resistance. Our results demonstrate that combined treatment with clorgyline and enzalutamide showed a greater decrease in cell growth compared to enzalutamide alone (Figures 5 and 6). 22Rv1 and C4–2B cells are androgen independent and exhibited minimal decrease in cell proliferation with enzalutamide. However, clorgyline enhanced the effect of enzalutamide in these cells. These results suggest that concomitant treatment of prostate cancer with a MAOA inhibitor and an antiandrogen may be an effective strategy to treat antiandrogen-resistant prostate cancer.

Although the potential molecular mechanisms underlying the development of androgen resistance are under intensive investigation, preclinical studies have shown that AR-V7 contributes to escape from and resistance to anti-androgen therapy.35 The AR-V7 splice variant lacks the androgen-binding site, but retains the DNA-binding and amino-terminal activation domains producing a constitutively active AR. The presence of AR-V7 positive circulating tumor cells has been proposed to select patients for cytotoxic chemotherapy over androgen receptor signaling inhibitors (enzalutamide or abiraterone).17,36 Our results confirmed increased expression of AR-V7 in 22Rv1 cells, but not in LNCaP and C4–2B cells. Consistent with previous results, enzalutamide did not alter the expression of AR-V7 in these cells. Clorgyline, on the other hand, inhibited the expression of AR-V7 (Figures 3 and 5). In addition, prolonged treatment of C4–2B cells with enzalutamide produced an enzalutamide-resistant subline which exhibited significantly higher expression of AR-FL, AR-V7, PSA, and MAOA as well as increased MAOA catalytic activity as compared to parental controls. Most importantly, MAOAI treatment decreased the growth of this enzalutamide-resistant cell line. These results suggest significant effects of MAOA treatment on the androgen pathway in both androgen-sensitive and androgen-insensitive prostate cancer cells.

In summary, our results show MAOAIs decrease androgen receptor activation and add to anti-proliferative effects of antiandrogen-treatment in both androgen-sensitive and androgen-insensitive prostate cancer cells. Further, our results show that MAOAIs can decrease AR-V7 splice variant expression and growth of an enzalutamide-resistant cell line. Our findings highlight the importance of MAOA in prostate cancer and suggest a role for MAOAIs in treating patients with advanced prostate cancer. A phase 2 trial examining potential anti-cancer effects of phenelzine in non-metastatic recurrent prostate cancer is currently ongoing (NCT02217709).

ACKNOWLEDGMENTS

This work was supported by the Tsai Family Fund and Boyd and Elsie Welin Professorship to J.C. Shih. We thank Abbey Kardy for performing the PSA ELISA assays. This work was supported by the Tsai Family Fund and Boyd and Elsie Welin Professorship to J.C. Shih.

Funding information

Tsai Family Fund

Footnotes

CONFLICTS OF INTEREST

All authors have no conflicts of interest to disclose.

REFERENCES

  • 1.Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65:87–108. [DOI] [PubMed] [Google Scholar]
  • 2.Roy AK, Tyagi RK, Song CS, et al. Androgen receptor: structural domains and functional dynamics after ligand-receptor interaction. Ann NY Acad Sci. 2001;949:44–57. [DOI] [PubMed] [Google Scholar]
  • 3.Zhou Y, Bolton EC, Jones JO. Androgens and androgen receptor signaling in prostate tumorigenesis. J Mol Endocrinol. 2015;54:R15–R29. [DOI] [PubMed] [Google Scholar]
  • 4.Hu R, Lu C, Mostaghel EA, et al. Distinct transcriptional programs mediated by the ligand-dependent full-length androgen receptor and its splice variants in castration-resistant prostate cancer. Cancer Res. 2012;72:3457–3462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Harris WP, Mostaghel EA, Nelson PS, Montgomery B. Androgen deprivation therapy: progress in understanding mechanisms of resistance and optimizing androgen depletion. Nat Clinical Pract. 2009;6:76–85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Schweizer MT, Yu EY. Persistent androgen receptor addiction in castration-resistant prostate cancer. J Hematol Oncol. 2015;8:128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Beer TM, Armstrong AJ, Rathkopf DE, et al. Enzalutamide in metastatic prostate cancer before chemotherapy. N Engl J Med. 2014;371: 424–433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Ryan CJ, Smith MR, de Bono JS, et al. Abiraterone in metastatic prostate cancer without previous chemotherapy. N Eng J Med. 2013;368:138–148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Hu R, Dunn TA, Wei S, et al. Ligand-independent androgen receptor variants derived from splicing of cryptic exons signify hormone-refractory prostate cancer. Cancer Res. 2009;69:16–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Watson PA, Arora VK, Sawyers CL. Emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer. Nat Rev Cancer. 2015;15:701–711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Lu J, Van der Steen T, Tindall DJ. Are androgen receptor variants a substitute for the full-length receptor? Nat Rev Urol. 2015;12:137–144. [DOI] [PubMed] [Google Scholar]
  • 12.Eisermann K, Wang D, Jing Y, et al. Androgen receptor gene mutation, rearrangement, polymorphism. Transl Androl Urol. 2013;2:137–147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Taplin ME, Rajeshkumar B, Halabi S, et al. Androgen receptor mutations in androgen-independent prostate cancer: cancer and leukemia group B study 9663. J Clin Oncol. 2003;21:2673–2678. [DOI] [PubMed] [Google Scholar]
  • 14.Linja MJ, Savinainen KJ, Saramaki OR, Tammela TL, Vessella RL, Visakorpi T. Amplification and overexpression of androgen receptor gene in hormone-refractory prostate cancer. Cancer Res. 2001;61:3550–3555. [PubMed] [Google Scholar]
  • 15.Merson S, Yang ZH, Brewer D, et al. Focal amplification of the androgen receptor gene in hormone-naive human prostate cancer. Br J Cancer. 2014;110:1655–1662. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Sun S, Sprenger CC, Vessella RL, et al. Castration resistance in human prostate cancer is conferred by a frequently occurring androgen receptor splice variant. J Clin Invest. 2010;120:2715–2730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Antonarakis ES, Lu C, Wang H, et al. AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer. N Engl J Med. 2014;371:1028–1038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Tipton KF, Boyce S, O’Sullivan J, Davey GP, Healy J. Monoamine oxidases: certainties and uncertainties. Curr Med Chem. 2004;11: 1965–1982. [DOI] [PubMed] [Google Scholar]
  • 19.Wimbiscus M, Kostenko O, Malone D. MAO inhibitors: risks, benefits, and lore. Cleve Clin J Med. 2010;77:859–882. [DOI] [PubMed] [Google Scholar]
  • 20.True L, Coleman I, Hawley S, et al. A molecular correlate to the Gleason grading system for prostate adenocarcinoma. Proc Natl Acad Sci USA. 2006;103:10991–10996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Wu JB, Shao C, Li X, et al. Monoamine oxidase A mediates prostate tumorigenesis and cancer metastasis. J Clin Invest. 2014;124: 2891–2908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Gordon RR, Wu M, Huang CY, et al. Chemotherapy-induced monoamine oxidase expression in prostate carcinoma functions as a cytoprotective resistance enzyme and associates with clinical outcomes. PLoS ONE. 2014;9:e104271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Flamand V, Zhao H, Peehl DM. Targeting monoamine oxidase A in advanced prostate cancer. J Cancer Res Clin Oncol. 2010;136:1761–1771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Ou XM, Chen K, Shih JC. Glucocorticoid and androgen activation of monoamine oxidase A is regulated differently by R1 and Sp1. J Biol Chem. 2006;281:21512–21525. [DOI] [PubMed] [Google Scholar]
  • 25.Liao CP, Lin TP, Li PC, et al. Loss of MAOA in epithelia inhibits adenocarcinoma development, cell proliferation and cancer stem cells in prostate. Oncogene. 2018;37:5175–5190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Zhao H, Flamand V, Peehl DM. Anti-oncogenic and pro-differentiation effects of clorgyline, a monoamine oxidase A inhibitor, on high grade prostate cancer cells. BMC Med Genomics. 2009;2:55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Peehl DM, Coram M, Khine H, Reese S, Nolley R, Zhao H. The significance of monoamine oxidase-A expression in high grade prostate cancer. J Urol. 2008;180:2206–2211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Thalmann GN, Anezinis PE, Chang SM, et al. Androgen-independent cancer progression and bone metastasis in the LNCaP model of human prostate cancer. Cancer Res. 1994;54:2577–2581. [PubMed] [Google Scholar]
  • 29.Gaur S, Chen L, Yang L, Wu X, Un F, Yen Y. Inhibitors of mTOR overcome drug resistance from topoisomerase II inhibitors in solid tumors. Cancer Lett. 2011;311:20–28. [DOI] [PubMed] [Google Scholar]
  • 30.Geha RM, Rebrin I, Chen K. Shih JC. Substrate and inhibitor specificities for human monoamine oxidase A and B are influenced by a single amino acid. J Biol Chem. 2001;276:9877–9882. [DOI] [PubMed] [Google Scholar]
  • 31.Wu HC, Hsieh JT, Gleave ME, Brown NM, Pathak S, Chung LW. Derivation of androgen-independent human LNCaP prostatic cancer cell sublines: role of bone stromal cells. Int J Cancer. 1994;57:406–412. [DOI] [PubMed] [Google Scholar]
  • 32.Saad F Evidence for the efficacy of enzalutamide in postchemotherapy metastatic castrate-resistant prostate cancer. Ther Adv Urol. 2013;5:201–210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Hussain M, Fizazi K, Saad F, et al. PROSPER: A phase 3, randomized, double-blind, placebo (PBO)-controlled study of enzalutamide (ENZA) in men with nonmetastatic castration-resistant prostate cancer (M0 CRPC). J Clin Oncol. 2018;36:3. [Google Scholar]
  • 34.Liu C, Lou W, Armstrong C, Zhu Y, Evans CP, Gao AC. Niclosamide suppresses cell migration and invasion in enzalutamide resistant prostate cancer cells via Stat3-AR axis inhibition. Prostate. 2015;75: 1341–1353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Luo J, Attard G, Balk SP, et al. Role of androgen receptor variants in prostate cancer: report from the 2017 mission androgen receptor variants meeting. Eur Urol. 2018;73:715–723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Scher HI, Graf RP, Schreiber NA, et al. Assessment of the validity of nuclear-localized androgen receptor splice variant 7 in circulating tumor cells as a predictive biomarker for castration-resistant prostate cancer. JAMA Oncol. 2018;4:1179–1186. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES