Androgen receptor (AR) is the primary oncogenic driver of prostate cancer, including aggressive castration-resistant prostate cancer (CRPC). The molecular mechanisms controlling AR activation in general and AR reactivation in CRPC remain elusive. Here we report that monoamine oxidase A (MAOA), a mitochondrial enzyme that degrades monoamine neurotransmitters and dietary amines, reciprocally interacts with AR in prostate cancer. MAOA was induced by androgens through direct AR binding to a novel intronic androgen response element of the MAOA gene, which in turn promoted AR transcriptional activity via upregulation of Shh/Gli-YAP1 signaling to enhance nuclear YAP1–AR interactions. Silencing MAOA suppressed AR-mediated prostate cancer development and growth, including CRPC, in mice. MAOA expression was elevated and positively associated with AR and YAP1 in human CRPC. Finally, genetic or pharmacologic targeting of MAOA enhanced the growth-inhibition efficacy of enzalutamide, darolutamide, and apalutamide in both androgen-dependent and CRPC cells. Collectively, these findings identify and characterize an MAOA–AR reciprocal regulatory circuit with coamplified effects in prostate cancer. Moreover, they suggest that cotargeting this complex may be a viable therapeutic strategy to treat prostate cancer and CRPC.

Significance:

MAOA and AR comprise a positive feedback loop in androgen-dependent and CRPC, providing a mechanistic rationale for combining MAOA inhibition with AR-targeted therapies for prostate cancer treatment.

Prostate cancer is the second commonest cancer and the fifth leading cause of cancer death in men globally (1). Androgen receptor (AR) is considered the primary oncoprotein governing prostate cancer, making AR-targeted therapy currently the principal treatment regimen in prostate cancer. Although initial response rates to androgen deprivation therapy (ADT) exceed 90%, prostate cancer eventually transitions from a hormone-dependent to a castration-resistant disease (CRPC). Most patients develop recurrent tumors 2–3 years after ADT when AR is reactivated despite the low-androgen environment, followed by fatal CRPC (2). There is an unmet clinical need for new molecularly targeted therapies to complement current AR-targeted therapy to improve survival.

Prostate cancer depends exquisitely on AR activity for survival, growth, and progression. In agreement with restored AR activity in CRPC, preclinical studies suggest that AR upregulation alone is sufficient to drive progression to CRPC. A significant portion of CRPCs demonstrate AR upregulation without gene amplification (3). In disease progression, AR is regulated at many levels and cooperates with other genes and signal transduction mechanisms (4). Understanding the mechanisms of AR activation in general and AR reactivation in CRPC specifically will help uncover new druggable molecular targets for rational combination strategies synergizing with AR-targeted therapy. Despite great efforts to identify and characterize genes either regulating AR (AR regulator genes) or affected by AR (AR target genes), molecular targets linking both AR regulators and AR targets for bidirectional cooperative promotion of AR effects are little studied.

Monoamine oxidase A (MAOA), a mitochondrial membrane-bound enzyme, degrades a number of biogenic and dietary monoamines and generates hydrogen peroxide (H2O2), a major source of reactive oxygen species (ROS), as a by-product (5). We and others demonstrated that MAOA is clinically associated with prostate cancer disease progression (6–8). We showed that MAOA induces epithelial–mesenchymal transition and tumor–stromal cell interaction through a ROS–Twist1–Shh/Gli signaling axis to promote prostate cancer metastasis (7, 9). However, the functional and mechanistic link between MAOA and AR in prostate cancer cells, in the AR-driven prostate cancer disease trajectory and treatment response, and in the CRPC setting remains unclear. Filling this knowledge gap will allow precise application of MAOA inhibitors already clinically used as antidepressants as a potential prostate cancer and CRPC therapy synergizing with AR-targeted therapy. This would be especially valuable given prostate cancer's heterogeneous and variable AR status and androgen responsiveness. This study explored previously undiscovered reciprocal cross-talk between MAOA and AR that amplifies the effects of both to promote prostate cancer and CRPC.

Clinical specimens

Hormone-sensitive and CRPC tissue microarrays, including 16 prostate adenocarcinoma specimens from each disease subtype, were provided by the Biobank of Taipei General Veterans Hospital. The study was reviewed and approved by the Institutional Review Board of Taipei General Veterans Hospital, and written informed consent was provided for human samples.

Cell lines

Human prostate cancer LNCaP, VCaP, 22Rv1, and human embryonic kidney 293T cell lines were obtained from American Type Culture Collection. The enzalutamide (Enz)-resistant human prostate cancer C4-2B (C4-2BENZR) cell line was generated as described previously (10). The human prostate cancer C4-2 cell line was provided by Leland W.K. Chung (Cedars-Sinai Medical Center). The human prostate cancer LAPC4 cell line was provided by Michael Freeman (Cedars-Sinai Medical Center). All cell lines were authenticated by short tandem repeat profiling, regularly tested for Mycoplasma by the MycoProbe Mycoplasma Detection Kit (R&D Systems) and used with the number of cell passages below 10.

Plasmids and reagents

A human MAOA lentiviral expression construct was generated by inserting the human MAOA coding region at EcoRI/XbaI sites in pLVX-AcGFP1-N1 vector (Clontech) containing a puromycin-resistant gene. A Dox-inducible MAOA shRNA expression construct was generated by inserting a human MAOA shRNA sequence at NheI/EcoRI sites in EZ-Tet-pLKO-Puro vector (Addgene) containing a puromycin-resistant gene as described previously (11). Primer sequences for constructing MAOA shRNA oligomers are forward 5′-CTAGCCGGATATTCTCTGTCACCAATTACTAGTATTGGTGACAGAGAATATCCGTTTTTG-3′ and reverse 5′-AATTCAAAAACGGATATTCTCTGTCACCAATACTAGTAATTGGTGACAGAGAATATCCGG-3′. An MAOA intron androgen response element (ARE) luciferase reporter construct (MAOA ARE-luc) was generated by inserting the MAOA ARE-centric intronic sequence upstream of a minimal promoter and the Firefly luciferase gene of pGL4.26 vector (Promega). Primer sequences for cloning the MAOA intronic sequence from LNCaP genomic DNA are forward 5′-AAAGGTACCTCTCCAACGTGCCAATCAGG-3′ and reverse 5′- GGGCTCGAGGCAGTTTCTCAATACTAAGCCACT-3′. Human MAOA and nontarget control shRNA lentiviral particles were purchased from Sigma-Aldrich. R1881 was purchased from PerkinElmer or Sigma-Aldrich. Clorgyline, phenelzine, and doxycycline were purchased from Sigma-Aldrich. Verteporfin was purchased from Santa Cruz. Cyclopamine and enzalutamide were purchased from Selleckchem. Darolutamide was purchased from MedKoo Biosciences. Apalutamide was purchased from Toronto Research Chemicals. The Supplementary Materials and Methods provides details on additional plasmids and reagents used in this study.

Biochemical analysis

Total RNA was isolated using the RNeasy Mini Kit (Qiagen) and reverse-transcribed to cDNA by M-MLV reverse transcriptase (Promega) following the manufacturer's instructions. For immunoblots, cells were extracted with RIPA buffer in the presence of a protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific). Blots were performed as described previously (12). The Supplementary Materials and Methods provides details on primary antibodies used for immunoblots. Nuclear and cytoplasmic extracts used for immunoblots were prepared with an NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific). PSA levels in cell culture media or mouse sera were quantified by ELISA (GenWay Biotech or Enzo Life Sciences).

Site-directional mutational analysis of MAOA intron ARE and YAP1 promoter

Site-directed mutagenesis was used to mutate or delete the MAOA ARE cloned in the pGL4.26 vector and mutate the Gli-binding site (GliBS) identified in the 1.6-kb YAP1 promoter, with wild-type (WT) luciferase reporter constructs used as templates. Mutagenesis was carried out by QuickChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) following the manufacturer's instructions. Primer sequences used for mutating or deleting the MAOA ARE were 5′-GCACGGTTCCAGGGAAATTGCGTTCTGCTTG-3′ (Mut 1), 5′-CACGGTTCCAGGGACATTGCATTTTGCTTGACATAAACAATTTC-3′ (Mut 2), and 5′-GCAGAAATTGTTTATGTCAAGGAACCGTGCCCCAAAACA-3′ (Del), with mutated nucleotides underlined. Primer sequence used for mutagenesis of YAP1 promoter was 5′-AGGGATAGCAGGGGTAGGGTGGGAGCTCCTTGAGGATGAAAG-3′ (mutated nucleotides underlined). Mutated or deleted nucleotides were verified by DNA sequencing.

Chromatin immunoprecipitation-qPCR assays

Chromatin immunoprecipitation (ChIP)-qPCR assays were used to determine the association of endogenous AR protein with an MAOA ARE in LNCaP cells grown in phenol red-free medium containing 5% CSS for 72 hours and then treated with R1881 or ethanol for another 24 hours, endogenous AR protein with two known AREs of the PSA and FKBP5 genes in LNCaP cells (shCon and shMAOA), and endogenous Gli1 and Gli2 proteins with a GliBS in YAP1 promoter in LNCaP cells (shCon and shMAOA) by a SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology) following the manufacturer's instructions. Briefly, the chromatin was crosslinked with nuclear proteins, enzymatically digested with micrococcal nuclease followed by sonication, and immunoprecipitated with anti-AR (PG-21, Millipore, RRID: AB_310214; or D6F11, Cell Signaling Technology, RRID: AB_10691711), anti-Gli1 (H-300, Santa Cruz, RRID: AB_2111764), or anti-Gli2 (cat. #ab26056, Abcam, RRID: AB_2111901) antibody. Normal IgG included in the kit was used as a negative control for IP. The immunoprecipitates were pelleted with agarose beads, purified, and subjected to qPCR with primers specifically targeting the ARE-centric MAOA, PSA, and FKBP5 genomic sequences or the GliBS-centric YAP1 promoter region. Details on primers used for qPCR are provided in Supplementary Materials and Methods.

Proximity ligation assay

Cells were seeded on chamber slides and fixed with 4% formaldehyde for 10 minutes at room temperature, washed twice with PBS containing 0.02% Tween 20, and permeabilized with 0.5% Triton X-100/PBS solution (blocking solution) for 30 minutes at room temperature. Primary antibodies against AR (N-20, rabbit IgG, Santa Cruz, RRID: AB_1563391) or YAP1 (63.7, mouse IgG, Santa Cruz, RRID: AB_1131430) were incubated in blocking solution at 4°C overnight. Assay was then performed with the Duolink In Situ Red Starter Kit Mouse/Rabbit (Duolink, Sigma-Aldrich) according to the manufacturer's instructions using anti-mouse MINUS and anti-rabbit PLUS proximity ligation assay (PLA) probes (Duolink). Images were acquired by a Nikon Ti-E inverted microscope or a Zeiss Axio Imager M2 upright microscope using a ×40 objective and analyzed for fluorescence per nucleus with inForm (PerkinElmer) or HALO (Indica Labs) software.

Animal studies

All animal studies received prior approval from the Washington State University IACUC and complied with IACUC recommendations. Male 4- to 6-week-old SCID, SCID/beige, and NSG mice were purchased from Envigo or Jackson Laboratory and housed in the animal research facility at Washington State University. To determine MAOA's effect on AR-dictated prostate cancer development and growth, 4 × 106 LNCaP cells expressing Dox-inducible MAOA shRNA were mixed 1:1 with Matrigel (BD Biosciences) for bilateral subcutaneous injection into SCID/beige mice. One week after tumor inoculation, mice were randomized into two groups (20 mice/group) and fed a diet with 625 mg/kg Dox (Dox+, Envigo) or a normal diet (Dox−) ad libitum. Two weeks later, tumor-bearing mice of both groups were randomly separated into two subgroups to undergo surgical castration or not, followed by tumor size measurement by caliper 3 times a week. To determine MAOA's effect on CRPC development and growth, 4 × 106 C4-2BENZR or 22Rv1 cells, both expressing Dox-inducible MAOA shRNA, were mixed 1:1 with Matrigel and bilaterally injected subcutaneously into NSG or SCID mice, respectively. Mice implanted with C4-2BENZR cells were orally administered with Enz (10 mg/kg) every other day after tumor inoculation, whereas mice implanted with 22Rv1 cells received castration 2 weeks prior to tumor inoculation. One week after tumor inoculation, mice were randomly divided into two groups (12 mice/group or 7 mice/group for C4-2BENZR or 22Rv1 cells, respectively) and fed a Dox+ or a Dox− diet. Tumor size was measured every other day by caliper after the formation of palpable tumors. Tumor volume was calculated by as length × width2 × 0.52. At the endpoints, tumors were dissected and weighed. Tumor samples and mouse sera were collected for subsequent biochemical and IHC analyses.

IHC and quantum dot labeling analysis

IHC analysis of xenograft and clinical tumor samples was performed using antibodies against MAOA (H-70, Santa Cruz, RRID: AB_2137260), YAP1 (63.7, Santa Cruz), AR (N-20, Santa Cruz), Ki-67 (D2H10, Cell Signaling Technology, RRID: AB_2636984), or cleaved caspase-3 (Asp175, Cell Signaling Technology, RRID: AB_2341188) following a published protocol (7). Cell-based IHC staining intensity and percentage of positive expression for individual proteins were analyzed by HALO software. MAOA IHC staining in human tissue microarrays was scored by a semiquantitative method taking into account both staining intensity (I) and quantity based on the proportion of tumor cells stained (q) to obtain a final score defined as the product of I × q in the range of 0–12 as described previously (7). All scoring was performed by a pathologist. The IHC staining protocol was modified for double quantum dot (QD) labeling as described previously (13). The human CRPC tissue microarray was stained with antibodies against MAOA (H-70, Santa Cruz) and YAP1 (63.7, Santa Cruz) sequentially by single QD labeling. Cell-based averages of QD intensity counts for MAOA and nuclear YAP1 expression in each sample were analyzed by inForm software after areas of interest were defined using manual tissue segmentation by a pathologist.

Bioinformatics analysis

The human prostate cancer data sets used for coexpression correlation studies were downloaded from the Oncomine database by licensed access or the cBioPortal for Cancer Genomics database. For analysis of ChIP-seq data sets GSE43720, GSE55062, and GSE65478 available in the Gene-Expression Omnibus database, Bowtie was used to map the human hg19 genome, and unique mapped reads were used for peak calling, using MACS2 to perform the peak calling and ChIPseeker for peak annotation.

Statistical analysis

Data are presented as the mean ± SEM as indicated in figure legends. Comparisons were analyzed by unpaired two-tailed Student t test. Correlations were determined by Pearson correlation. A P value less than 0.05 was considered statistically significant.

MAOA reciprocally interacts with AR in prostate cancer cells

To seek initial evidence of MAOA-AR cross-talk in prostate cancer, we analyzed multiple clinical data sets for a MAOA association with AR. We found that MAOA mRNA expression was positively correlated with prediagnosis and pretreatment serum PSA levels in the Taylor 3 data set (Fig. 1A; ref. 14). MAOA was also positively coexpressed with three bona fide AR target genes, including PSA, TMPRSS2, and FKBP5, at the transcript level in the same data set (Fig. 1B), as corroborated by a similar mRNA coexpression pattern from additional publicly available data sets (Supplementary Table S1).

Figure 1.

MAOA reciprocally interacts with AR in prostate cancer cells. A and B, Pearson correlation analysis of MAOA with prediagnosis/pretreatment serum PSA levels (A) or PSA, TMPRSS2, and FKBP5 (B) in the Taylor 3 data set. C, Western blot of MAOA and PSA upon R1881 stimulation (10 nmol/L) at indicated times in LNCaP and VCaP cells. D, qPCR of MAOA by R1881 (10 nmol/L, 24 hours) in LNCaP cells (n = 3). E, Genomic browser representation of DNase I, H3K27ac, and AR binding in ARE-centric MAOA intron 3, with the nucleotides identical to the canonical ARE underlined, in GSE43720, GSE55062, and GSE65478. F, ChIP-qPCR of AR occupancy at the MAOA and PSA AREs and an irrelevant sequence (NC) by R1881 (10 nmol/L, 24 hours) in LNCaP cells. Fold enrichment of AR was normalized to IgG in each group (n = 3). G, Determination of MAOA ARE-luc activity in WT, mutated (Mut1 and Mut2), or deleted (Del) forms by R1881 (10 nmol/L, 24 hours) in LNCaP cells. H, Western blot of MAOA and AR in control and MAOA-OE LAPC4 cells. I, qPCR of MAOA, AR, and AR target genes in control and MAOA-OE LAPC4 cells (n = 3). J, ELISA of PSA secretion in culture media from control and MAOA-OE LAPC4 cells (n = 3). K, Representative nuclear AR staining and quantification of per-nucleus intensity in control (n = 33) and MAOA-OE (n = 91) LAPC4 cells upon R1881 stimulation (10 nmol/L, 6 hours). Scale bars, 20 μm. Data, mean ± SEM. **, P < 0.01; ns, not significant.

Figure 1.

MAOA reciprocally interacts with AR in prostate cancer cells. A and B, Pearson correlation analysis of MAOA with prediagnosis/pretreatment serum PSA levels (A) or PSA, TMPRSS2, and FKBP5 (B) in the Taylor 3 data set. C, Western blot of MAOA and PSA upon R1881 stimulation (10 nmol/L) at indicated times in LNCaP and VCaP cells. D, qPCR of MAOA by R1881 (10 nmol/L, 24 hours) in LNCaP cells (n = 3). E, Genomic browser representation of DNase I, H3K27ac, and AR binding in ARE-centric MAOA intron 3, with the nucleotides identical to the canonical ARE underlined, in GSE43720, GSE55062, and GSE65478. F, ChIP-qPCR of AR occupancy at the MAOA and PSA AREs and an irrelevant sequence (NC) by R1881 (10 nmol/L, 24 hours) in LNCaP cells. Fold enrichment of AR was normalized to IgG in each group (n = 3). G, Determination of MAOA ARE-luc activity in WT, mutated (Mut1 and Mut2), or deleted (Del) forms by R1881 (10 nmol/L, 24 hours) in LNCaP cells. H, Western blot of MAOA and AR in control and MAOA-OE LAPC4 cells. I, qPCR of MAOA, AR, and AR target genes in control and MAOA-OE LAPC4 cells (n = 3). J, ELISA of PSA secretion in culture media from control and MAOA-OE LAPC4 cells (n = 3). K, Representative nuclear AR staining and quantification of per-nucleus intensity in control (n = 33) and MAOA-OE (n = 91) LAPC4 cells upon R1881 stimulation (10 nmol/L, 6 hours). Scale bars, 20 μm. Data, mean ± SEM. **, P < 0.01; ns, not significant.

Close modal

To understand the regulatory relationship between MAOA and AR, we first investigated whether AR controls MAOA in prostate cancer cells. The androgen-dependent LNCaP and castration-resistant VCaP human prostate cancer cell lines, which both express AR and respond to androgens, showed time dependently increased MAOA and PSA protein expression under R1881 synthetic androgen treatment (Fig. 1C), and a 1.5-fold increase of MAOA mRNA expression by R1881 in LNCaP cells (Fig. 1D), suggesting androgenic regulation of MAOA at the transcriptional level. To determine whether AR directly binds to the MAOA gene locus for transcriptional activation, we analyzed three ChIP-seq data sets with subjects assembled from cultured human prostate cancer cells and clinical prostate tumors. We found enriched AR occupancy at a site downstream from the transcription start site (TSS) of MAOA across most samples. Examining the AR-bound sequence, we further identified a consensus ARE, GGGACAttgCGTTCT (+53,107 to +53,121 with the MAOA TSS set as +1) in MAOA intron 3 with high homology (10 out of 12 bp) with the canonical ARE GGT/AACAnnnTGTTCT for AR binding (Fig. 1E; ref. 15). ChIP-qPCR assays validated direct AR interaction with this sequence, which showed significant AR association with the intronic ARE of MAOA gene as well as a known ARE in the distal enhancer of the PSA gene in LNCaP cells upon R1881 stimulation, paralleled by minimal AR binding at both AREs in the absence of R1881. An irrelevant genomic sequence used as a negative control demonstrated no AR occupancy regardless of R1881 induction (Fig. 1F). To determine whether the MAOA ARE is functional, we inserted the corresponding MAOA ARE-centric intron sequence upstream of the minimal promoter-driven luciferase gene to construct an MAOA ARE-luc reporter. Compared with the WT MAOA ARE-luc notably inductive to R1881, mutation of select nucleotides in the ARE (Mut 1 and Mut 2) or deletion of the ARE (Del) made the reporter no longer responsive to R1881 (Fig. 1G).

Next, we examined whether MAOA influences AR in prostate cancer cells. Stably enforced expression of MAOA in AR-positive androgen-responsive human prostate cancer LAPC4 cells with low levels of MAOA resulted in elevated expression of MAOA at both the protein and mRNA levels (Fig. 1H and I). Forced MAOA expression led to mRNA activation of several AR target genes (Fig. 1I) and a more than 4-fold increase in PSA protein secretion (Fig. 1J). Interestingly, MAOA overexpression (OE) caused no changes in AR expression at the protein and mRNA levels (Fig. 1H and I), or the extent of AR nuclear translocation upon R1881 stimulation, a regulatory mechanism necessary for AR activation, as examined by quantitating nuclear AR staining levels (Fig. 1K).

Conversely, we stably silenced MAOA expression with a shRNA in both LNCaP and castration-resistant, enzalutamide (Enz)-resistant C4-2B (C4-2BENZR) human prostate cancer cells. The C4-2BENZR cell line was established by chronic exposure of LNCaP-derived human CRPC C4-2B cells to Enz, a second-generation antiandrogen drug for CRPC, at gradually increasing doses to develop resistance (10). Consistent with observations in MAOA-OE LAPC4 cells, MAOA knockdown (KD) did not affect AR protein expression or the extent of AR nuclear translocation by R1881 in either LNCaP and C4-2BENZR cells (Fig. 2A and B) or 2 additional AR-positive human CRPC cell lines, C4-2 and 22Rv1 (Supplementary Fig. S1A). In contrast, MAOA KD prevalently decreased the mRNA levels of a panel of AR target genes, including PSA, TMPRSS2, and PLZF, in LNCaP and C4-2BENZR cells (Fig. 2C). In the absence or presence of various doses of R1881, MAOA KD reduced the activity of an AR-dependent luciferase reporter (PSA-luc), an androgen-responsive PSA enhancer–promoter fused sequence placed upstream of the luciferase gene, in LNCaP and C4-2BENZR cells (Fig. 2D). Moreover, MAOA KD attenuated the R1881 responsiveness of PSA and TMPRSS2 at the protein secretion and/or mRNA levels within 48 hours in LNCaP and C4-2BENZR cells (Fig. 2E and F). Similarly, MAOA silencing repressed induction of PSA protein secretion and PSA/TMPRSS2 mRNA by R1881 in C4-2 cells as well as the response of KLK2 mRNA to R1881 in 22Rv1 cells where KLK2 was reportedly more androgen responsive than PSA in 22Rv1 cells (Supplementary Fig. S1B–S1D; ref. 16). ChIP-qPCR assays then demonstrated lower AR occupancy at individual AREs of PSA and FKBP5 gene loci in MAOA-KD LNCaP cells compared with controls (Fig. 2G). Collectively, these findings imply reciprocal cross-talk between MAOA and AR in both androgen-dependent and CRPC cells.

Figure 2.

MAOA silencing suppressed AR transcriptional activity in prostate cancer cells. A, Western blot of MAOA and AR in control (shCon) and MAOA-KD (shMAOA) LNCaP and C4-2BENZR cells. B, Representative nuclear AR staining and quantification of per-nucleus intensity in the indicated control (LNCaP, n = 160; C4-2BENZR, n = 663) and MAOA-KD (LNCaP, n = 154; C4-2BENZR, n = 760) cells upon R1881 stimulation (10 nmol/L, 6 hours). Scale bars, 20 μm. C, qPCR of AR target genes in the indicated control and MAOA-KD cells (n = 3). D, Determination of PSA-luc activity by R1881 at indicated concentrations for 24 hours in the indicated control and MAOA-KD cells (n = 3). E, ELISA of time-dependent fold induction of PSA by R1881 (10 nmol/L) in the indicated control and MAOA-KD cells (n = 3). F, qPCR of time-dependent fold induction of PSA and TMPRSS2 by R1881 (10 nmol/L) in the indicated control and MAOA-KD cells (n = 3). G, ChIP-qPCR of AR occupancy at PSA and FKBP5 AREs in control and MAOA-KD LNCaP cells. Fold enrichment of AR was normalized to IgG in each group (n = 3). Data, mean ± SEM. *, P < 0.05; **, P < 0.01; ns, not significant.

Figure 2.

MAOA silencing suppressed AR transcriptional activity in prostate cancer cells. A, Western blot of MAOA and AR in control (shCon) and MAOA-KD (shMAOA) LNCaP and C4-2BENZR cells. B, Representative nuclear AR staining and quantification of per-nucleus intensity in the indicated control (LNCaP, n = 160; C4-2BENZR, n = 663) and MAOA-KD (LNCaP, n = 154; C4-2BENZR, n = 760) cells upon R1881 stimulation (10 nmol/L, 6 hours). Scale bars, 20 μm. C, qPCR of AR target genes in the indicated control and MAOA-KD cells (n = 3). D, Determination of PSA-luc activity by R1881 at indicated concentrations for 24 hours in the indicated control and MAOA-KD cells (n = 3). E, ELISA of time-dependent fold induction of PSA by R1881 (10 nmol/L) in the indicated control and MAOA-KD cells (n = 3). F, qPCR of time-dependent fold induction of PSA and TMPRSS2 by R1881 (10 nmol/L) in the indicated control and MAOA-KD cells (n = 3). G, ChIP-qPCR of AR occupancy at PSA and FKBP5 AREs in control and MAOA-KD LNCaP cells. Fold enrichment of AR was normalized to IgG in each group (n = 3). Data, mean ± SEM. *, P < 0.05; **, P < 0.01; ns, not significant.

Close modal

MAOA activates AR-interacting YAP1 in a Gli1/2-dependent manner

Because MAOA activates AR transcriptional activity without affecting AR mRNA/protein expression and nuclear translocation, we speculated that MAOA may regulate AR through alternative mechanisms. We first investigated whether MAOA induces the expression levels of AR-interacting transcription factors and cofactors to modulate AR effects. We conducted qPCR-based screening of a group of transcription factors and cofactors known as nuclear AR interactors controlling AR transcriptional activity in MAOA-manipulated cells (17). We identified YAP1 as the top candidate because of its relatively larger differences in expression levels between control and MAOA-manipulated cells in each cell pair as well as its consistent pattern of expression-level changes in response to MAOA across all cell pairs compared with the other genes (Fig. 3A). YAP1, a transcriptional coactivator and Hippo pathway effector, was recently reported to physically interact with AR to promote prostate cancer and CRPC growth and invasion (18–20). Using both MAOA-KD and -OE cell lines, we showed that MAOA increased YAP1 protein expression while decreasing phospho-YAP1 (Ser127) protein expression in whole-cell lysates, congruent with the observations that MAOA caused higher YAP1 and lower phospho-YAP1 protein levels in nuclear and cytoplasmic fractions, respectively (Fig. 3B; Supplementary Fig. S2A). Immunofluorescence assays also revealed less YAP1 protein accumulation in the nucleus in response to MAOA inactivation in C4-2BENZR cells (Supplementary Fig. S2B). We found reduced mRNA expression of multiple YAP1 target genes (CTGF, IGFBP3, and AMOTL2) in MAOA-KD LNCaP and C4-2BENZR cells compared with controls, which corresponded to elevated mRNA expression of several YAP1 target genes (CTGF, Cyr61, and AMOTL2) in MAOA-OE LAPC4 cells relative to controls (Fig. 3C). Using the 8xGTIIC luciferase reporter in which luciferase expression is driven by a YAP1-responsive synthetic promoter, we demonstrated a decrease of reporter activity when MAOA was silenced in both LNCaP and C4-2BENZR cells. This decrease weakened until abolished dose dependently when MAOA-KD cells were treated with verteporfin, a small-molecule inhibitor of YAP1 (21), suggesting the specificity of this reporter to YAP1 (Fig. 3D). Bioinformatics analysis further indicated downregulation of androgen-responsive/AR-dependent and YAP1-directed gene signatures enriched in MAOA-KD LNCaP and C4-2BENZR cells compared with controls using RNA-seq coupled with gene set enrichment analysis (Fig. 3E). Importantly, MAOA and YAP1 demonstrated a significant positive coexpression correlation in multiple prostate cancer clinical data sets (Supplementary Fig. S2C).

Figure 3.

MAOA activates AR-interacting YAP1 in a Gli1/2-dependent manner. A, qPCR-based heat map depicting differential expressions of AR-interacting transcription factors and cofactors in the indicated cell pairs. Log2 scale was used to indicate relative gene expression from an average of three replicates, with gene expression in controls set as 1. B, Western blot of YAP1 and phospho-YAP1 (S127) in total cell lysates along with nuclear and cytoplasmic fractions of the indicated cells. C, qPCR of YAP1 target genes in the indicated control and MAOA-KD cells (n = 3). D, Determination of YAP1-responsive 8xGTIIC-luc activity by verteporfin (24 hours) in the indicated control and MAOA-KD cells (n = 3). E, Gene set enrichment analysis of the indicated gene sets for the comparisons of MAOA-KD versus control LNCaP and C4-2BENZR cells. F, Determination of YAP1 promoter activity by cyclopamine (20 μmol/L, 48 hours) in control and MAOA-KD LNCaP cells (n = 3). G, Sequences of the canonical GliBS (top), a putative GliBS in YAP1 promoter (middle), and introduced point mutations (bottom, italic and red) to inactivate the GliBS, with the YAP1 TSS set as +1. H, Determination of WT and mutant (Mut) YAP1 promoter activity by cyclopamine (20 μmol/L, 48 hours) in 293T cells (n = 3). I, ChIP-qPCR of Gli1 and Gli2 occupancy at the GliBS-centric YAP1 promoter in control and MAOA-KD LNCaP cells (n = 3). J, Pearson correlation analysis of YAP1-Gli1/Gli2 in the TCGA (n = 498) and Beltran (n = 49) data sets. Data, mean ± SEM. *, P < 0.05; **, P < 0.01; ns, not significant.

Figure 3.

MAOA activates AR-interacting YAP1 in a Gli1/2-dependent manner. A, qPCR-based heat map depicting differential expressions of AR-interacting transcription factors and cofactors in the indicated cell pairs. Log2 scale was used to indicate relative gene expression from an average of three replicates, with gene expression in controls set as 1. B, Western blot of YAP1 and phospho-YAP1 (S127) in total cell lysates along with nuclear and cytoplasmic fractions of the indicated cells. C, qPCR of YAP1 target genes in the indicated control and MAOA-KD cells (n = 3). D, Determination of YAP1-responsive 8xGTIIC-luc activity by verteporfin (24 hours) in the indicated control and MAOA-KD cells (n = 3). E, Gene set enrichment analysis of the indicated gene sets for the comparisons of MAOA-KD versus control LNCaP and C4-2BENZR cells. F, Determination of YAP1 promoter activity by cyclopamine (20 μmol/L, 48 hours) in control and MAOA-KD LNCaP cells (n = 3). G, Sequences of the canonical GliBS (top), a putative GliBS in YAP1 promoter (middle), and introduced point mutations (bottom, italic and red) to inactivate the GliBS, with the YAP1 TSS set as +1. H, Determination of WT and mutant (Mut) YAP1 promoter activity by cyclopamine (20 μmol/L, 48 hours) in 293T cells (n = 3). I, ChIP-qPCR of Gli1 and Gli2 occupancy at the GliBS-centric YAP1 promoter in control and MAOA-KD LNCaP cells (n = 3). J, Pearson correlation analysis of YAP1-Gli1/Gli2 in the TCGA (n = 498) and Beltran (n = 49) data sets. Data, mean ± SEM. *, P < 0.05; **, P < 0.01; ns, not significant.

Close modal

In addition to the qPCR array approach, we also examined whether MAOA regulates the assembly and stability of AR complexes, especially through the AR-interacting proteins affected by MAOA, likely more for the degree of AR binding than their expression levels. We carried out proteomic analysis of AR-bound proteins with quantitation of their AR-binding affinity in control and MAOA-KD LNCaP cells. Mass spectrometry following AR pulldown revealed 491 AR-bound proteins expressed in the nucleus and enriched in LNCaP cells, which were narrowed down to 33 high-confidence hits with differential degrees of AR binding between control and MAOA-KD cells by statistical analysis. Literature mining further filtered out a prostate cancer growth-activating protein (LMNA) with lower binding affinity to AR and five prostate cancer growth-repressed proteins (PDCD4, ENDOD1, DHRS7, FUS, and HDAC1) with higher binding affinity to AR in MAOA-KD cells compared with controls (Supplementary Fig. S3A; Supplementary Data Set S1; refs. 22–27). To determine whether these proteins mediated MAOA's effect on AR, we first showed that their corresponding gene-expression levels were barely changed in MAOA-KD LNCaP cells compared with controls (Supplementary Fig. S3B). Surprisingly, we found that after individual KD, none of these genes affected several canonical AR target gene expressions or PSA protein secretion in LNCaP cells (Supplementary Fig. S3C–S3E), implying that MAOA is not likely to regulate AR activity by modulating the interactions of these proteins with AR. In addition, we demonstrated the negligible effect of MAOA on AR protein stability by similar AR protein levels in response to cycloheximide to inhibit protein synthesis or MG132 to inhibit protein degradation in control and MAOA-KD LNCaP cells (Supplementary Fig. S4A–S4C). Based on these findings, we decided to focus on YAP1 as a possible mediator of MAOA's action on AR.

Next, we sought to find out how MAOA activates YAP1 in prostate cancer cells. Given the induction of YAP1 mRNA by MAOA, we surmised that MAOA might regulate YAP1 at the transcriptional level and investigated the underlying mechanism. We previously demonstrated, using AR-negative PC-3 cells as the principal model, that MAOA elicits a ROS-Twist1-Shh/Gli signaling cascade to promote prostate cancer metastasis (7, 9). Mechanistically, MAOA generates ROS via oxidative deamination to stabilize HIF1α protein and subsequently induce the VEGF-A–mediated AKT/FOXO1 pathway, resulting in the nuclear export of transcription repressor FOXO1 to activate Twist1 transcription and gene expression (7). In turn, Twist1 upregulates the transcription of Shh through direct interaction with an E-box on the Shh promoter (9). Subsequent to binding to transmembrane protein Ptch1 and relieving the downstream depression of SMO, Shh activates Gli1 and Gli2 transcription factors, facilitating Gli translocation to the nucleus and occupancy at target gene promoters for transactivation (28). These findings led us to speculate that Shh/Gli signaling might be a candidate mechanism for MAOA's transcriptional activation of YAP1, which is coincidentally supported by a recent study revealing that hedgehog pathway enhances YAP1 at both the mRNA and protein levels during liver regeneration (29).

To prove this idea, we first examined whether MAOA could induce the ROS–Twist1–Shh/Gli signaling axis in AR-positive androgen-dependent or CRPC cells as MAOA did in AR-negative PC-3 cells. We demonstrated MAOA's ability to promote intracellular ROS production, Twist1 and Twist1′s upstream regulators (HIF1α, AKT, and FOXO1) in AR-expressing cells (Supplementary Fig. S5A–S5C). Moreover, we observed that the antioxidant N-acetylcysteine (NAC) decreased MAOA-induced Twist1 expression in LAPC4 cells whereas addition of H2O2, the by-product released from MAOA-mediated enzymatic reactions, diminished Twist1 repression caused by MAOA silencing in LNCaP and C4-2BENZR cells, suggesting ROS-dependent MAOA activation of Twist1 in AR-positive cells (Supplementary Fig. S5D). Then we assessed whether MAOA could further induce Shh/Gli signaling via ROS and Twist1 in AR-positive cells. To this end, we modulated the intracellular ROS and Twist1 expression levels in MAOA-manipulated AR-expressing cells, and examined Shh and Gli1 (a direct transcriptional target of Gli1 itself) expression as well as Gli-luc reporter activity, in which 8 copies of the GliBS upstream of the luciferase gene drive luciferase expression to indicate Gli transcriptional activity. We showed that NAC treatment or siRNA-mediated Twist1 KD reduced MAOA-induced Shh/Gli1 expression and Gli-luc activity in LAPC4 cells, whereas H2O2 treatment or forced expression of Twist1 restored Shh/Gli1 levels and Gli-luc activity in MAOA-KD LNCaP and C4-2BENZR cells (Supplementary Fig. S6A–S6C). We also found that Twist1 is positively coexpressed with Shh, Gli1, and Gli2 at the transcript level in clinical samples from the Taylor 3 data set (Supplementary Fig. S6D). Collectively, these data as in line with our prior findings reveal that MAOA upregulates Shh/Gli signaling through ROS and Twist1 in AR-positive androgen-dependent or CRPC cells.

Because MAOA induced Shh mRNA expression and Gli-luc activity in multiple AR-positive prostate cancer cell lines (Supplementary Fig. S7A and S7B), we then determined whether Shh/Gli signaling mediates MAOA's control of YAP1 transcription by directly regulating YAP1 promoter. We used a human 1.6-kb YAP1 promoter Gaussia luciferase reporter with concurrent expression of secreted alkaline phosphatase as internal control. We observed a 48% decrease of normalized YAP1 promoter activity in MAOA-KD LNCaP cells compared with controls, which was diminished upon cyclopamine treatment, an inhibitor of SMO for blockade of Shh/Gli signaling (Fig. 3F). After testing a series of truncated YAP1 promoter reporter constructs, we found the middle ∼500 bp of the 1.6-kb promoter most responsive to MAOA silencing. Examining the ∼500-bp promoter sequence, we identified a consensus GliBS with the nucleotides of the half-site identical to the canonical ones (Fig. 3G; ref. 30). We generated a YAP1 promoter reporter construct with a mutant GliBS and found that the mutated (Mut) reporter was no longer suppressed by cyclopamine, unlike its WT counterpart, which showed a 47% decrease by cyclopamine (Fig. 3H). ChIP-qPCR assays then demonstrated 33% and 44% lower association of endogenous Gli1 and Gli2 proteins with the GliBS, respectively, after MAOA KD in LNCaP cells compared with controls (Fig. 3I). There was a significant positive correlation between YAP1 and Gli1/Gli2 mRNA expression in both the TCGA primary prostate cancer and the Beltran CRPC data sets (31), as clinical evidence supporting Gli-dependent MAOA activation of YAP1 expression (Fig. 3J; Supplementary Fig. S7C). These data in aggregate indicate MAOA's ability to induce YAP1 through downstream ROS/Twist1-dependent activation of Shh/Gli signaling for direct Gli1/2 interaction with a GliBS in YAP1 promoter.

MAOA promotes AR transcriptional activity by enhancing nuclear YAP1–AR interaction

Investigating whether YAP1 mediates MAOA's effect on AR transactivation, we showed 41% and 59% decreases of R1881-induced PSA-luc activity in LNCaP and C4-2BENZR cells, respectively, where MAOA expression was silenced compared with controls. These decreases were abolished by YAP1 inhibition through verteporfin (Fig. 4A). A similar pattern of PSA and PLZF mRNA expression in the absence or presence of verteporfin was also observed in MAOA-KD LNCaP and C4-2BENZR cells compared with controls (Fig. 4B). Next, we examined the direct YAP1–AR interaction, reportedly the mechanism by which YAP1 modulates AR activity (19). In situ PLA visualized endogenous YAP1–AR protein complexes in both the nucleus and cytoplasm of cells, in agreement with the fact that YAP1 and AR can reside in the nucleus as well as the cytoplasm, making their interaction possible in both compartments. Quantitative analysis of the fluorescence restricted to the nucleus revealed a 3.8-fold increase in nuclear YAP1–AR interaction in MAOA-OE LAPC4 cells and a 59% and 44% reduction in MAOA-KD LNCaP and C4-2BENZR cells, respectively, compared with controls. Parallel incubation of an AR antibody only as a negative control in the assay demonstrated undetectable fluorescence in all cell lines (Fig. 4C and D). A coimmunoprecipitation assay also revealed less YAP1 bound with AR protein in MAOA-KD LNCaP whole-cell lysates compared with controls (Supplementary Fig. S8).

Figure 4.

MAOA promotes nuclear YAP1–AR interaction for enhanced AR transcriptional activity. A, Determination of PSA-luc activity by R1881 (10 nmol/L, 24 hours) ± verteporfin (5 μmol/L, 24 hours) in the indicated control and MAOA-KD cells (n = 3). B, qPCR of PSA and PLZF by verteporfin (5 μmol/L, 24 hours) in the indicated control and MAOA-KD cells (n = 3). C, Representative PLA staining of YAP1–AR interaction in the indicated cell pairs. AR antibody incubation alone served as negative control. Scale bars, 20 μm. D, Quantitation of nuclear YAP1–AR interaction by per-nucleus fluorescence intensity in the indicated cell pairs. Data, mean ± SEM. *, P < 0.05; **, P < 0.01; ns, not significant.

Figure 4.

MAOA promotes nuclear YAP1–AR interaction for enhanced AR transcriptional activity. A, Determination of PSA-luc activity by R1881 (10 nmol/L, 24 hours) ± verteporfin (5 μmol/L, 24 hours) in the indicated control and MAOA-KD cells (n = 3). B, qPCR of PSA and PLZF by verteporfin (5 μmol/L, 24 hours) in the indicated control and MAOA-KD cells (n = 3). C, Representative PLA staining of YAP1–AR interaction in the indicated cell pairs. AR antibody incubation alone served as negative control. Scale bars, 20 μm. D, Quantitation of nuclear YAP1–AR interaction by per-nucleus fluorescence intensity in the indicated cell pairs. Data, mean ± SEM. *, P < 0.05; **, P < 0.01; ns, not significant.

Close modal

Silencing MAOA suppressed AR-dictated prostate tumor development and growth

We demonstrated the necessity of MAOA for maintaining AR activity in prostate cancer cultures, which sheds light on MAOA's supportive role in AR-dictated prostate tumor behavior in vivo, including castration-resistant tumors where functional reactivated AR provides survival and growth signals (32). To test this idea, we established multiple prostate cancer xenograft mouse models to assess MAOA's function in controlling AR-governed prostate cancer and CRPC development and growth. Our findings of MAOA's effect on AR signaling suggested that suppressing MAOA might reduce the viability of AR-dependent xenografts preinjection, rendering baseline growth inequivalent at the starting point. This is especially likely based on previous studies, including ours, indicating MAOA's ability to promote AR-dependent prostate cancer cell growth (7, 8, 33). To address this concern, we generated a Dox-dependent inducible MAOA shRNA expression construct and stably expressed it in LNCaP, C4-2BENZR, and 22Rv1 cells. We observed a notable reduction of MAOA protein expression as well as cell growth upon Dox stimulation consistently across all cell lines, validating the efficacy of induced MAOA KD (Supplementary Fig. S9A and S9B).

To examine the effect of MAOA on AR-directed prostate tumor development and growth, we subcutaneously implanted LNCaP cells stably expressing Dox-inducible MAOA shRNA into mice. One week after inoculation, mice were randomly separated into two groups and given either a Dox-containing (Dox+) diet or a normal (Dox−) diet. Mice fed a Dox+ diet leading to induced tumor MAOA KD formed fewer tumors compared with control mice over a 2-week observation period after Dox administration (Fig. 5A). Two weeks after treatment, tumor-bearing mice from both groups were further randomized into two subgroups to receive surgical castration (Cx+) or not (Cx−). Castration stopped tumor growth for 2–3 weeks followed by regrowth of castration-resistant tumors (Fig. 5B). Dox-induced tumor MAOA KD markedly slowed tumor growth, evidenced by smaller tumor volumes and lower tumor weights and serum PSA levels at the endpoint, in both castrated and intact mice compared with controls (Fig. 5B–E). Strikingly, silencing tumor MAOA expression suppressed tumor growth more dramatically than castration, implying the superiority of MAOA ablation over castration for limiting AR signaling. Combining castration and tumor MAOA inactivation retarded tumor growth the most among all groups and nearly halted the growth of relapsed LNCaP tumors after castration in mice (Fig. 5B). Even more appealing, further quantitative analysis revealed that MAOA silencing resulted in a significantly greater fold reduction in average endpoint tumor volume, tumor weight, and serum PSA level in castrated mice compared with intact mice (Supplementary Fig. S10A–S10C), suggesting MAOA's particular importance in regulating CRPC tumor growth. Similar to in vitro findings, silencing MAOA inhibited AR activity by reduced expression of several canonical AR target genes in both hormone-naïve and castration-resistant LNCaP xenograft samples (Fig. 5F). By characterizing tumor samples, we demonstrated decreased MAOA protein expression in Dox-treated tumors in both castrated and intact mice, indicating effective and sustainable Dox-induced MAOA KD under in vivo conditions. Ki-67 staining of tumor samples revealed an average 76% and 60% drop in Ki-67+ cells in MAOA-KD tumors compared with controls in castrated and intact mice,, respectively. Higher cleaved caspase-3 staining correlating to increased apoptosis was also shown in MAOA-KD tumors compared with controls regardless of castration status. Consistent with in vitro findings, we found minimal changes in the percentage of nuclear AR+ cells and nuclear AR expression, parallel with reduced nuclear YAP1 levels, in MAOA-KD tumors in both castrated and intact mice compared with controls (Fig. 5G and H; Supplementary Fig. S11A).

Figure 5.

MAOA is essential for driving AR-dictated prostate cancer development and growth in mice. A, Kaplan–Meier tumor-free curves of mice inoculated with LNCaP cells expressing a Dox-inducible MAOA shRNA and fed a Dox− or a Dox+ diet (n = 40). B, Tumor growth curves of mice fed a Dox− or a Dox+ diet in combination with the state of castration (Cx+) or not (Cx−; n = 5–10, which applies to C and D). C and D, Tumor weights (C) and anatomic tumor images (D) at the endpoint. E, ELISA of endpoint serum PSA levels (n = 4–7). F, qPCR of indicated genes in tumor samples (n = 3). G, Representative MAOA, Ki-67, cleaved caspase-3 (c-Cas3), AR, and YAP1 IHC staining in tumor samples. Scale bars, 20 μm. H, Quantification of the percentage of Ki-67+ and c-Cas3+ cells in tumor samples (n = 3). Data, mean ± SEM. *, P < 0.05; **, P < 0.01.

Figure 5.

MAOA is essential for driving AR-dictated prostate cancer development and growth in mice. A, Kaplan–Meier tumor-free curves of mice inoculated with LNCaP cells expressing a Dox-inducible MAOA shRNA and fed a Dox− or a Dox+ diet (n = 40). B, Tumor growth curves of mice fed a Dox− or a Dox+ diet in combination with the state of castration (Cx+) or not (Cx−; n = 5–10, which applies to C and D). C and D, Tumor weights (C) and anatomic tumor images (D) at the endpoint. E, ELISA of endpoint serum PSA levels (n = 4–7). F, qPCR of indicated genes in tumor samples (n = 3). G, Representative MAOA, Ki-67, cleaved caspase-3 (c-Cas3), AR, and YAP1 IHC staining in tumor samples. Scale bars, 20 μm. H, Quantification of the percentage of Ki-67+ and c-Cas3+ cells in tumor samples (n = 3). Data, mean ± SEM. *, P < 0.05; **, P < 0.01.

Close modal

Next, we used two CRPC cell lines, C4-2BENZR and 22Rv1, both stably expressing Dox-inducible MAOA shRNA, to establish subcutaneous xenografts in mice to assess MAOA's effect on CRPC tumor development and growth. To mimic the real CRPC tumor environment, mice implanted with C4-2BENZR cells were administered Enz continuously after inoculation to create an AR-repressed host environment analogous to a castrate environment as well as to maintain Enz resistance as the tumors developed, whereas mice implanted with 22Rv1 cells received prior surgical castration. Mice were randomized into two groups to receive either a Dox+ or a Dox− diet to induce tumor MAOA KD or not, one week after inoculation in both the C4-2BENZR and 22Rv1 xenograft models. We demonstrated a substantial reduction in C4-2BENZR tumor formation frequency and tumor growth, including smaller tumor sizes and lower tumor weights and serum PSA levels at the endpoint, in Dox-fed mice compared with controls (Fig. 6A–E). In 22Rv1 xenografts, Dox treatment remarkably delayed the onset of tumor formation and repressed tumor growth compared with controls (Fig. 6F). Tumor samples showed downregulated expression of several canonical AR target genes, indicating decreased AR transactivation, in both MAOA-silenced C4–2BENZR and 22Rv1 tumors (Fig. 6K). We also confirmed continued Dox-induced MAOA KD in both C4-2BENZR and 22Rv1 tumors by IHC analysis. Ki-67 staining of tumor samples revealed an average 51% and 91% drop of Ki-67+ cells in MAOA-KD C4-2BENZR and 22Rv1 tumors, respectively, compared with controls. A roughly 2- and 4-fold increase of cleaved caspase-3 staining indicative of enhanced tumor cell apoptosis was shown in MAOA-silenced C4-2BENZR and 22Rv1 tumors compared with controls. In line with the in vitro observations, we detected no changes in the percentage of nuclear AR positivity and nuclear AR staining levels, but reduced nuclear YAP1 expression, in both MAOA-KD C4–2BENZR and 22Rv1 tumors compared with controls (Fig. 6L and M; Supplementary Fig. S11B and S11C). Based on these findings, we firmly concluded that MAOA is essential for AR-dictated prostate cancer and CRPC development and growth in mice.

Figure 6.

MAOA silencing suppressed CRPC development and growth in mice. A, Kaplan–Meier tumor-free curves of mice inoculated with C4-2BENZR cells expressing a Dox-inducible MAOA shRNA and fed a Dox− or a Dox+ diet (n = 24). B, C4-2BENZR tumor growth curves of mice fed a Dox− or a Dox+ diet (Dox−, n = 18; Dox+, n = 8 tumors/group, which applies to C and D). C and D, C4-2BENZR tumor weights (C) and anatomic tumor images (D) at the endpoint. E, ELISA of endpoint serum PSA levels from C4-2BENZR tumor–bearing mice (n = 6). F, Kaplan–Meier tumor-free curves of mice inoculated with 22Rv1 cells expressing a Dox-inducible MAOA shRNA and fed a Dox− or a Dox+ diet (n = 14). G, 22Rv1 tumor growth curves of mice fed a Dox− or a Dox+ diet (Dox−, n = 14; Dox+, n = 13, which applies to H and I). H and I, 22Rv1 tumor weights (H) and anatomic tumor images (I) at the endpoint. J, ELISA of endpoint serum PSA levels from 22Rv1 tumor–bearing mice (n = 7). K, qPCR of indicated genes in tumor samples (n = 6). L, Representative MAOA, Ki-67, c-Cas3, AR, and YAP1 IHC staining in tumor samples. Scale bars, 20 μm. M, Quantification of the percentage of Ki-67+ and c-Cas3+ cells in tumor samples (n = 3). Data, mean ± SEM. *, P < 0.05; **, P < 0.01.

Figure 6.

MAOA silencing suppressed CRPC development and growth in mice. A, Kaplan–Meier tumor-free curves of mice inoculated with C4-2BENZR cells expressing a Dox-inducible MAOA shRNA and fed a Dox− or a Dox+ diet (n = 24). B, C4-2BENZR tumor growth curves of mice fed a Dox− or a Dox+ diet (Dox−, n = 18; Dox+, n = 8 tumors/group, which applies to C and D). C and D, C4-2BENZR tumor weights (C) and anatomic tumor images (D) at the endpoint. E, ELISA of endpoint serum PSA levels from C4-2BENZR tumor–bearing mice (n = 6). F, Kaplan–Meier tumor-free curves of mice inoculated with 22Rv1 cells expressing a Dox-inducible MAOA shRNA and fed a Dox− or a Dox+ diet (n = 14). G, 22Rv1 tumor growth curves of mice fed a Dox− or a Dox+ diet (Dox−, n = 14; Dox+, n = 13, which applies to H and I). H and I, 22Rv1 tumor weights (H) and anatomic tumor images (I) at the endpoint. J, ELISA of endpoint serum PSA levels from 22Rv1 tumor–bearing mice (n = 7). K, qPCR of indicated genes in tumor samples (n = 6). L, Representative MAOA, Ki-67, c-Cas3, AR, and YAP1 IHC staining in tumor samples. Scale bars, 20 μm. M, Quantification of the percentage of Ki-67+ and c-Cas3+ cells in tumor samples (n = 3). Data, mean ± SEM. *, P < 0.05; **, P < 0.01.

Close modal

MAOA expression is elevated and associated with AR and YAP1 in human CRPC

CRPC is a fatal prostate cancer disease stage with the remarkable feature of near-universal AR reactivation (34, 35). This prompted us to examine MAOA expression and its clinical association with AR alongside the CRPC characteristics developed during disease progression. We first performed histologic analysis of a tissue panel of hormone-sensitive prostate cancer (HSPC) and CRPC and found elevated MAOA protein expression in CRPC relative to HSPC (Fig. 7A and B). Two CRPC data sets, Beltran and Abida (36), showed a positive association of MAOA mRNA expression with AR score defined by overall assessment of a group of AR target genes (Fig. 7C and D). We also demonstrated a positive mRNA coexpression correlation between MAOA and two AR target genes, TMPRSS2 and NKX3-1, in the Beltran data set (Fig. 7E). Then we analyzed our CRPC cohort to address whether YAP1 is associated with MAOA in the clinical CRPC setting. Using double QD labeling analysis, we found that MAOA and active YAP1, which reside in the cytoplasm and nucleus, respectively, were positively coexpressed on a single-cell basis in CRPC (Fig. 7F and G). This was corroborated by a positive correlation between MAOA and YAP1 mRNA expression in the Beltran data set (Fig. 7H). Further, higher mRNA expression of both MAOA and YAP1 was associated with disease recurrence in the Glinsky data set (Fig. 7I; ref. 37). These results strongly support the clinical significance of MAOA-AR cross-talk in CRPC.

Figure 7.

MAOA expression associated with AR and YAP1 is elevated in human CRPC. A and B, Representative MAOA IHC staining (A) and quantification (B) in a prostate cancer cohort containing HSPC (n = 16) and CRPC (n = 16). Scale bars, 20 μm. Data, mean ± SEM. *, P < 0.05. C and D, Pearson correlation analysis of MAOA with AR score in the Beltran (C) and Abida (D) data sets. E, Pearson correlation analysis of MAOA with TMPRSS2 and NKX3-1 in the Beltran data set. F and G, Representative MAOA and YAP1 double QD staining (F) and corresponding Pearson correlation analysis (G) in a CRPC cohort. Scale bars, 20 μm. H, Pearson correlation analysis of MAOA and YAP1 in the Beltran data set. I, Kaplan–Meier recurrence-free curves of prostate cancer patients categorized by MAOA/YAP1 mRNA levels from the Glinsky data set.

Figure 7.

MAOA expression associated with AR and YAP1 is elevated in human CRPC. A and B, Representative MAOA IHC staining (A) and quantification (B) in a prostate cancer cohort containing HSPC (n = 16) and CRPC (n = 16). Scale bars, 20 μm. Data, mean ± SEM. *, P < 0.05. C and D, Pearson correlation analysis of MAOA with AR score in the Beltran (C) and Abida (D) data sets. E, Pearson correlation analysis of MAOA with TMPRSS2 and NKX3-1 in the Beltran data set. F and G, Representative MAOA and YAP1 double QD staining (F) and corresponding Pearson correlation analysis (G) in a CRPC cohort. Scale bars, 20 μm. H, Pearson correlation analysis of MAOA and YAP1 in the Beltran data set. I, Kaplan–Meier recurrence-free curves of prostate cancer patients categorized by MAOA/YAP1 mRNA levels from the Glinsky data set.

Close modal

MAOA inhibition enhances antiandrogen drug efficacy

Given the current clinical use of MAOA inhibitors (38), we evaluated the therapeutic potential of synergizing MAOA inhibition with antiandrogen drugs to suppress prostate cancer cell growth. We tested the combinational effects of MAOA inactivation by either shRNA or small-molecule inhibitors with three antiandrogen drugs, Enz, darolutamide (Daro), and apalutamide (Apa), all second-generation antiandrogen drugs currently used clinically to treat CRPC (39), in both LNCaP and its lineage-related castration-resistant C4-2 prostate cancer cell lines. We first showed that shRNA-mediated MAOA KD enhanced Enz, Daro, and Apa growth inhibition in both LNCaP and C4-2 cells with up to 4- and 2-fold decreases of IC50 values, respectively (Fig. 8A and B). Next, we evaluated the effectiveness of two conventional MAOA inhibitors, clorgyline and phenelzine, in promoting antiandrogen drug efficacy. Phenelzine is clinically prescribed as an antidepressant in the United States (40). We found that Enz, Daro, and Apa suppressed LNCaP and C4-2 cell survival further by up to 50% and 53%, respectively, in the presence of clorgyline or phenelzine compared with antiandrogen drug treatment alone (Fig. 8C–F). These findings suggest that MAOA targeting has significant potential for combinational use with AR-targeted therapy to treat AR-driven prostate cancer and CRPC.

Figure 8.

Genetically and pharmacologically inhibiting MAOA enhanced antiandrogen drug efficacy. A and B, MTS cell proliferation assays of control and MAOA-KD LNCaP (A) and C4-2 (B) cells by Enz, Daro, or Apa at various doses for 5 days (n = 3). C–F, Cell counting assays of LNCaP (C and D) and C4-2 (E and F) cells by Enz, Daro, or Apa (5 μmol/L for each) together with clorgyline (1 μmol/L) or phenelzine (2 μmol/L) for 5 days (n = 3). Average cell numbers in control group with no treatment were set as 100%. G, Schematic summarizing MAOA–AR reciprocal interaction in prostate cancer. Data, mean ± SEM. *, P < 0.05; **, P < 0.01.

Figure 8.

Genetically and pharmacologically inhibiting MAOA enhanced antiandrogen drug efficacy. A and B, MTS cell proliferation assays of control and MAOA-KD LNCaP (A) and C4-2 (B) cells by Enz, Daro, or Apa at various doses for 5 days (n = 3). C–F, Cell counting assays of LNCaP (C and D) and C4-2 (E and F) cells by Enz, Daro, or Apa (5 μmol/L for each) together with clorgyline (1 μmol/L) or phenelzine (2 μmol/L) for 5 days (n = 3). Average cell numbers in control group with no treatment were set as 100%. G, Schematic summarizing MAOA–AR reciprocal interaction in prostate cancer. Data, mean ± SEM. *, P < 0.05; **, P < 0.01.

Close modal

In summary, our data show that AR promotes MAOA through direct binding to an intronic ARE of MAOA, and in a reciprocal manner MAOA induces Shh/Gli signaling via ROS-dependent Twist1, which activates YAP1 to enhance nuclear YAP1–AR interaction, thereby upregulating AR transcriptional activity in AR-dominant prostate cancer cells (Fig. 8G).

This study showed that MAOA synergizes with AR through reciprocal cross-talk to amplify AR-directed prostate cancer disease progression, including the aggressive castration-resistant variant. Our previous study revealed that MAOA upregulation could be under the concerted control of aberrant oncogenic signaling, including activation of c-Myc and loss of PTEN and p53, at different disease stages (7). This study provides an additional regulatory mechanism for elevated MAOA expression in prostate cancer through androgenic signaling. MAOA activation by androgens in neuroblastoma cells through direct AR interaction with an ARE in the proximal promoter of MAOA has been reported (41). However, we identified a previously undescribed functional intronic ARE in the MAOA gene locus, demonstrated in both prostate cancer cultured cells and clinical data sets, suggesting that AR may regulate MAOA in a cell context–dependent manner.

CRPC is a significant clinical challenge mainly due to AR reactivation after escape from ADT. We found increased MAOA expression and MAOA association with AR activity in human CRPC. In line with clinical reexpression of most if not all of the genes known to be under AR regulation in castration-resistant tumors (34, 35), our observations support the role of MAOA as an AR target gene in CRPC. Compelling evidence indicates that reactivated AR is functional and fuels castration-resistant tumor regrowth after a period of regression, rendering AR a viable therapeutic target even after castration resistance develops (3). Our discovery of a new mechanism where MAOA is regulated by AR and in turn controls AR transcriptional activity in prostate cancer cells may reveal a positive feed-forward loop augmenting AR signaling in CRPC. This provides a rationale for targeting MAOA to untangle the cross-talk between MAOA and AR as a potential CRPC therapy. Indeed, we demonstrated in preclinical xenograft mouse models that MAOA inactivation significantly impeded CRPC development and growth. Pharmacologic inhibition of MAOA also enhanced the efficacy of three second-generation antiandrogen drugs in CRPC cells. These findings call for further evaluation of MAOA inhibitors for clinical application in CRPC to complement current therapies targeting the AR axis.

Our data indicate that MAOA promotes AR transactivation through upregulation of YAP1 and enhanced nuclear YAP1–AR interaction. YAP1, a transcriptional coactivator that regulates diverse cellular processes, was recently reported to act as a physiologic binding partner and positive regulator of AR in prostate cancer through both androgen-dependent and -independent mechanisms in different disease states. YAP1–AR interaction also contributes to the switch from androgen-dependent to castration-resistant growth in prostate cancer (19). These mechanistic details support YAP1-dependent MAOA upregulation of AR in both androgen-dependent and CRPC cells. YAP1 utilizes the WW/SH3 domain to interact specifically with the N-terminal transactivation domain (NTD) of AR (19). This protein–protein interaction mode makes YAP1 interaction with AR variants possible, especially those lacking a C-terminal ligand-binding domain (LBD) while maintaining NTD (e.g., AR-V7) as observed in prostate cancer following ADT (42, 43). Thus, it seems likely that MAOA might have an activating effect on AR variants through the same YAP1-dependent mechanism. This provides a rationale for antagonizing MAOA in CRPC, which is prone to develop resistance to the more highly potent antiandrogen drugs like Enz owing to the emergence of LBD-deficient AR variants for constitutive activation of AR signaling.

One of the salient mechanistic findings of our study is that MAOA activates YAP1 through downstream ROS/Twist1-mediated Shh/Gli signaling, wherein Gli1/2 directly binds to a GliBS on the YAP1 promoter to activate YAP1 transcription. YAP1 is amplified and upregulated in hedgehog-associated medulloblastomas and was also recently found to have functional interplay with hedgehog signaling in different development and disease states (44, 45). Despite these mechanistic advances, this study provides an alternative mechanism to YAP1 regulation by hedgehog signaling. In addition, numerous studies have demonstrated cross-talk between hedgehog signaling and androgen signaling under certain conditions depending on the tumor microenvironment (46–48). In this study, we demonstrated that MAOA-dependent autocrine Shh/Gli signaling activates YAP1 to enhance AR transactivation, distinct from the paracrine signaling we previously found supporting MAOA-elicited tumor–stromal cell interaction to promote metastasis (9), suggesting that MAOA/Shh signaling might be context dependent in regulating different aspects of prostate tumor behavior. Hedgehog signaling has also been shown to support androgen signaling and the growth of androgen-deprived and -independent prostate cancer cells (46), which might sustain YAP1-mediated MAOA upregulation of AR and the resulting AR-driven phenotype in CRPC.

In conclusion, this study uncovered MAOA's reciprocal cross-talk with AR, amplifying the effects of both to promote prostate cancer development and growth dictated by AR signaling. This provides new insights into the mechanistic basis of AR regulation and functions in prostate cancer. We also provided strong preclinical evidence for targeting MAOA, alone or in combination with AR-targeted therapy, to disengage the MAOA/AR complex as a potential therapy for prostate cancer and CRPC.

No disclosures were reported.

J. Wei: Data curation, formal analysis, investigation, and methodology. L. Yin: Data curation and formal analysis. J. Li: Data curation and formal analysis. J. Wang: Formal analysis. T. Pu: Data curation and formal analysis. P. Duan: Data curation and formal analysis. T. Lin: Provision of experimental material. A.C. Gao: Provision of experimental material. B. Wu: Conceptualization, data curation, formal analysis, supervision, funding acquisition, investigation, methodology, writing–original draft, and project administration.

The authors thank Xiangyan Li (Cedars–Sinai Medical Center) for technical help, Jen-Ming Huang (Cedars–Sinai Medical Center) for insightful discussion, Leland W.K. Chung (Cedars–Sinai Medical Center) for comprehensive support of this study, and Gary Mawyer for editorial assistance. This work was supported by NIH/NCI grant R37CA233658, DOD Prostate Cancer Research Program grant W81XWH-19-1-0279, and WSU start-up funding (to B.J. Wu).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Bray
F
,
Ferlay
J
,
Soerjomataram
I
,
Siegel
RL
,
Torre
LA
,
Jemal
A
. 
Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries
.
CA Cancer J Clin
2018
;
68
:
394
424
.
2.
Saad
F
,
Fizazi
K
. 
Androgen deprivation therapy and secondary hormone therapy in the management of hormone-sensitive and castration-resistant prostate cancer
.
Urology
2015
;
86
:
852
61
.
3.
Graham
L
,
Schweizer
MT
. 
Targeting persistent androgen receptor signaling in castration-resistant prostate cancer
.
Med Oncol
2016
;
33
:
44
.
4.
Culig
Z
,
Santer
FR
. 
Androgen receptor signaling in prostate cancer
.
Cancer Metastasis Rev
2014
;
33
:
413
27
.
5.
Shih
JC
,
Chen
K
,
Ridd
MJ
. 
Monoamine oxidase: from genes to behavior
.
Annu Rev Neurosci
1999
;
22
:
197
217
.
6.
True
L
,
Coleman
I
,
Hawley
S
,
Huang
CY
,
Gifford
D
,
Coleman
R
, et al
A molecular correlate to the Gleason grading system for prostate adenocarcinoma
.
Proc Natl Acad Sci U S A
2006
;
103
:
10991
6
.
7.
Wu
JB
,
Shao
C
,
Li
X
,
Li
Q
,
Hu
P
,
Shi
C
, et al
Monoamine oxidase A mediates prostate tumorigenesis and cancer metastasis
.
J Clin Invest
2014
;
124
:
2891
908
.
8.
Flamand
V
,
Zhao
H
,
Peehl
DM
. 
Targeting monoamine oxidase A in advanced prostate cancer
.
J Cancer Res Clin Oncol
2010
;
136
:
1761
71
.
9.
Wu
JB
,
Yin
L
,
Shi
C
,
Li
Q
,
Duan
P
,
Huang
JM
, et al
MAOA-dependent activation of Shh-IL6-RANKL signaling network promotes prostate cancer metastasis by engaging tumor-stromal cell interactions
.
Cancer Cell
2017
;
31
:
368
82
.
10.
Liu
C
,
Lou
W
,
Zhu
Y
,
Yang
JC
,
Nadiminty
N
,
Gaikwad
NW
, et al
Intracrine androgens and AKR1C3 activation confer resistance to enzalutamide in prostate cancer
.
Cancer Res
2015
;
75
:
1413
22
.
11.
Frank
SB
,
Schulz
VV
,
Miranti
CK
. 
A streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector
.
BMC Biotechnol
2017
;
17
:
24
.
12.
Wu
JB
,
Chen
K
,
Ou
XM
,
Shih
JC
. 
Retinoic acid activates monoamine oxidase B promoter in human neuronal cells
.
J Biol Chem
2009
;
284
:
16723
35
.
13.
Hu
P
,
Chu
GC
,
Zhu
G
,
Yang
H
,
Luthringer
D
,
Prins
G
, et al
Multiplexed quantum dot labeling of activated c-Met signaling in castration-resistant human prostate cancer
.
PLoS One
2011
;
6
:
e28670
.
14.
Taylor
BS
,
Schultz
N
,
Hieronymus
H
,
Gopalan
A
,
Xiao
Y
,
Carver
BS
, et al
Integrative genomic profiling of human prostate cancer
.
Cancer Cell
2010
;
18
:
11
22
.
15.
Roche
PJ
,
Hoare
SA
,
Parker
MG
. 
A consensus DNA-binding site for the androgen receptor
.
Mol Endocrinol
1992
;
6
:
2229
35
.
16.
Denmeade
SR
,
Sokoll
LJ
,
Dalrymple
S
,
Rosen
DM
,
Gady
AM
,
Bruzek
D
, et al
Dissociation between androgen responsiveness for malignant growth vs. expression of prostate specific differentiation markers PSA, hK2, and PSMA in human prostate cancer models
.
Prostate
2003
;
54
:
249
57
.
17.
Heemers
HV
,
Tindall
DJ
. 
Androgen receptor (AR) coregulators: a diversity of functions converging on and regulating the AR transcriptional complex
.
Endocr Rev
2007
;
28
:
778
808
.
18.
Jiang
N
,
Hjorth-Jensen
K
,
Hekmat
O
,
Iglesias-Gato
D
,
Kruse
T
,
Wang
C
, et al
In vivo quantitative phosphoproteomic profiling identifies novel regulators of castration-resistant prostate cancer growth
.
Oncogene
2015
;
34
:
2764
76
.
19.
Kuser-Abali
G
,
Alptekin
A
,
Lewis
M
,
Garraway
IP
,
Cinar
B
. 
YAP1 and AR interactions contribute to the switch from androgen-dependent to castration-resistant growth in prostate cancer
.
Nat Commun
2015
;
6
:
8126
.
20.
Zhang
L
,
Yang
S
,
Chen
X
,
Stauffer
S
,
Yu
F
,
Lele
SM
, et al
The hippo pathway effector YAP regulates motility, invasion, and castration-resistant growth of prostate cancer cells
.
Mol Cell Biol
2015
;
35
:
1350
62
.
21.
Wang
C
,
Zhu
X
,
Feng
W
,
Yu
Y
,
Jeong
K
,
Guo
W
, et al
Verteporfin inhibits YAP function through up-regulating 14-3-3sigma sequestering YAP in the cytoplasm
.
Am J Cancer Res
2016
;
6
:
27
37
.
22.
Araya
S
,
Kratschmar
DV
,
Tsachaki
M
,
Stucheli
S
,
Beck
KR
,
Odermatt
A
. 
DHRS7 (SDR34C1): a new player in the regulation of androgen receptor function by inactivation of 5α-dihydrotestosterone?
J Steroid Biochem Mol Biol
2017
;
171
:
288
95
.
23.
Brooke
GN
,
Culley
RL
,
Dart
DA
,
Mann
DJ
,
Gaughan
L
,
McCracken
SR
, et al
FUS/TLS is a novel mediator of androgen-dependent cell-cycle progression and prostate cancer growth
.
Cancer Res
2011
;
71
:
914
24
.
24.
Gaughan
L
,
Logan
IR
,
Cook
S
,
Neal
DE
,
Robson
CN
. 
Tip60 and histone deacetylase 1 regulate androgen receptor activity through changes to the acetylation status of the receptor
.
J Biol Chem
2002
;
277
:
25904
13
.
25.
Zennami
K
,
Choi
SM
,
Liao
R
,
Li
Y
,
Dinalankara
W
,
Marchionni
L
, et al
PDCD4 is an androgen-repressed tumor suppressor that regulates prostate cancer growth and castration resistance
.
Mol Cancer Res
2019
;
17
:
618
27
.
26.
Qiu
J
,
Peng
S
,
Si-Tu
J
,
Hu
C
,
Huang
W
,
Mao
Y
, et al
Identification of endonuclease domain-containing 1 as a novel tumor suppressor in prostate cancer
.
BMC Cancer
2017
;
17
:
360
.
27.
Kong
L
,
Schafer
G
,
Bu
H
,
Zhang
Y
,
Zhang
Y
,
Klocker
H
. 
Lamin A/C protein is overexpressed in tissue-invading prostate cancer and promotes prostate cancer cell growth, migration and invasion through the PI3K/AKT/PTEN pathway
.
Carcinogenesis
2012
;
33
:
751
9
.
28.
McMillan
R
,
Matsui
W
. 
Molecular pathways: the hedgehog signaling pathway in cancer
.
Clin Cancer Res
2012
;
18
:
4883
8
.
29.
Swiderska-Syn
M
,
Xie
G
,
Michelotti
GA
,
Jewell
ML
,
Premont
RT
,
Syn
WK
, et al
Hedgehog regulates yes-associated protein 1 in regenerating mouse liver
.
Hepatology
2016
;
64
:
232
44
.
30.
Kinzler
KW
,
Vogelstein
B
. 
The GLI gene encodes a nuclear protein which binds specific sequences in the human genome
.
Mol Cell Biol
1990
;
10
:
634
42
.
31.
Beltran
H
,
Prandi
D
,
Mosquera
JM
,
Benelli
M
,
Puca
L
,
Cyrta
J
, et al
Divergent clonal evolution of castration-resistant neuroendocrine prostate cancer
.
Nat Med
2016
;
22
:
298
305
.
32.
Schweizer
MT
,
Yu
EY
. 
Persistent androgen receptor addiction in castration-resistant prostate cancer
.
J Hematol Oncol
2015
;
8
:
128
.
33.
Gordon
RR
,
Wu
M
,
Huang
CY
,
Harris
WP
,
Sim
HG
,
Lucas
JM
, 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
.
34.
Chandrasekar
T
,
Yang
JC
,
Gao
AC
,
Evans
CP
. 
Mechanisms of resistance in castration-resistant prostate cancer (CRPC)
.
Transl Androl Urol
2015
;
4
:
365
80
.
35.
Katzenwadel
A
,
Wolf
P
. 
Androgen deprivation of prostate cancer: Leading to a therapeutic dead end
.
Cancer Lett
2015
;
367
:
12
7
.
36.
Abida
W
,
Cyrta
J
,
Heller
G
,
Prandi
D
,
Armenia
J
,
Coleman
I
, et al
Genomic correlates of clinical outcome in advanced prostate cancer
.
PNAS
2019
;
116
:
11428
36
.
37.
Glinsky
GV
,
Berezovska
O
,
Glinskii
AB
. 
Microarray analysis identifies a death-from-cancer signature predicting therapy failure in patients with multiple types of cancer
.
J Clin Invest
2005
;
115
:
1503
21
.
38.
Bortolato
M
,
Chen
K
,
Shih
JC
. 
Monoamine oxidase inactivation: from pathophysiology to therapeutics
.
Adv Drug Deliv Rev
2008
;
60
:
1527
33
.
39.
Rice
MA
,
Malhotra
SV
,
Stoyanova
T
. 
Second-generation antiandrogens: from discovery to standard of care in castration resistant prostate cancer
.
Front Oncol
2019
;
9
:
801
.
40.
Shulman
KI
,
Herrmann
N
,
Walker
SE
. 
Current place of monoamine oxidase inhibitors in the treatment of depression
.
CNS Drugs
2013
;
27
:
789
97
.
41.
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
25
.
42.
Haile
S
,
Sadar
MD
. 
Androgen receptor and its splice variants in prostate cancer
.
Cell Mol Life Sci
2011
;
68
:
3971
81
.
43.
Antonarakis
ES
,
Lu
C
,
Wang
H
,
Luber
B
,
Nakazawa
M
,
Roeser
JC
, et al
AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer
.
N Engl J Med
2014
;
371
:
1028
38
.
44.
Du
K
,
Hyun
J
,
Premont
RT
,
Choi
SS
,
Michelotti
GA
,
Swiderska-Syn
M
, et al
Hedgehog-YAP signaling pathway regulates glutaminolysis to control activation of hepatic stellate cells
.
Gastroenterology
2018
;
154
:
1465
79
.
45.
Fernandez
LA
,
Northcott
PA
,
Dalton
J
,
Fraga
C
,
Ellison
D
,
Angers
S
, et al
YAP1 is amplified and up-regulated in hedgehog-associated medulloblastomas and mediates Sonic hedgehog-driven neural precursor proliferation
.
Genes Dev
2009
;
23
:
2729
41
.
46.
Chen
M
,
Feuerstein
MA
,
Levina
E
,
Baghel
PS
,
Carkner
RD
,
Tanner
MJ
, et al
Hedgehog/Gli supports androgen signaling in androgen deprived and androgen independent prostate cancer cells
.
Mol Cancer
2010
;
9
:
89
.
47.
Peng
YC
,
Levine
CM
,
Zahid
S
,
Wilson
EL
,
Joyner
AL
. 
Sonic hedgehog signals to multiple prostate stromal stem cells that replenish distinct stromal subtypes during regeneration
.
Proc Natl Acad Sci U S A
2013
;
110
:
20611
6
.
48.
Yamamichi
F
,
Shigemura
K
,
Behnsawy
HM
,
Meligy
FY
,
Huang
WC
,
Li
X
, et al
Sonic hedgehog and androgen signaling in tumor and stromal compartments drives epithelial-mesenchymal transition in prostate cancer
.
Scand J Urol
2014
;
48
:
523
32
.