Expression Of Androgen-responsive Genes Research Articles

Abstract B026: Identification of small RNAs that function as multi-target therapeutics and as tools for novel target discovery in prostate cancer

Abstract Introduction: A major contributor to prostate cancer (PCa) mortality is the development of resistance to single agents targeting androgen receptor (AR) signaling. We have developed an unbiased shRNA-based negative selection screen to identify small RNAs (shRNAs and siRNAs) that inhibit PCa cell growth and survival (toxic sRNAs). Computational analyses of the most toxic sRNAs identified revealed that each can simultaneously target multiple known AR-coregulatory mRNAs. AR-coregulators modulate AR transcriptional response and are essential to AR signaling. We have proposed that the identified toxic sRNAs inhibit androgen signaling through seed-mediated targeting of androgen regulatory gene networks, resulting in inhibition of cell growth and viability. The goal of the current studies was to begin to analyze the broad therapeutic potential of these toxic sRNAs for PCa. We focused on analyzing i) their potential to thwart cell growth and/or viability when delivered to preclinical models of PCa, and ii) the targetome of the toxic sRNAs as a mean to uncover novel AR-coregulators that represent a targetable axis within androgen signaling. Methods: i) cationic lipid nanoparticles (LNPs), that efficiently deliver the toxic siRNAs to PCa cell lines, were used to deliver toxic siRNAs to organoids grown from PCa patient derived xenografts (PDX) models. 3D image analysis using the Ominer® software package was used to determine organoid count and size, the numbers of nuclei per organoid, and the fraction of dead cells to discriminate between cytotoxic and cytostatic effects. ii) to identify novel AR-coregulators, toxic siRNA direct targets were identified by sequencing of transcripts captured by biotinylated toxic siRNAs. Directly bound targets, were subjected to pathway enrichment and network analysis followed by identification of hub genes (Cytoscape). The effect of individually silencing potential AR-coregulators on the expression of androgen responsive genes was analyzed using Nanostring. Results and Conclusions: i) The effect of the toxic siRNAs on growth and viability of the PCa PDX-derived organoids will be presented and discussed as compelling preliminary data for therapeutic testing in more complex pre-clinical models (PCa PDXs). ii) Based on bioinformatic and downstream analysis of the direct targets of two toxic siRNAs, we selected 12 potentially novel AR- coregulators, with varied known functions including chromatin modifiers, transcriptional regulators, etc. Silencing of several of these potential AR-coregulators significantly affect expression of numerous AR responsive genes. Future experiments will focus on analyzing their interaction and functional association with the AR (i.e. effect of AR binding to chromatin). In conclusion, we have developed an assay that allowed the identification of toxic sRNAs for PCa cells and demonstrated their potential as therapeutics and as a tool for discovery of potential novel targets for PCa. Citation Format: Joshua M Corbin, Hamilton Young, Stefan Wilhelm, Adam S Asch, Chao Xu, Maria J Ruiz Echevarria. Identification of small RNAs that function as multi-target therapeutics and as tools for novel target discovery in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B026.

Calcium Activation of the Androgen Receptor in Prostate Cells.

Although prostate cancer patients initially respond to androgen deprivation therapy, most patients progress to a resistant phenotype. Castration resistance is due, in part, to intratumoral and/or adrenal synthesis of androgens, overexpression or mutation of the androgen receptor (AR), stabilization of AR by chaperones, and ligand-independent activation of AR. Increasing evidence also links disruption of calcium homeostasis to progression of prostate cancer. Our previous study shows that heavy metal cadmium activates the AR through a ligand-independent mechanism. Cadmium mimics calcium in biological systems due to their similar ionic charge and radius. This study determines whether calcium activates AR and whether first- and second-generation antiandrogens block the ability of calcium to activate the receptor. The expression of androgen-responsive genes and calcium channels was measured in prostate cells using a quantitative real-time polymerase chain reaction assay. Cell growth was measured. To ask whether calcium activates AR, prostate cells were treated with calcium in the absence and presence of the first-generation antiandrogens hydroxyflutamide and bicalutamide and the second-generation antiandrogen enzalutamide, and the expression of androgen-responsive genes and cell growth was measured. In the normal PWR-1E cells and HEK293T cells transiently expressing AR, treatment with calcium increased the expression of androgen-responsive genes by approximately 3-fold. The increase was blocked by enzalutamide but was not consistently blocked by the first-generation antiandrogens. In LNCaP cells which contain a mutant AR, treatment with calcium also increased the expression of androgen-responsive genes by approximately 3-fold, and the increase was more effectively blocked by enzalutamide than by hydroxyflutamide or bicalutamide. Treatment with calcium also increased cell growth that was blocked by enzalutamide. To ask whether dysregulation of calcium channels is associated with castration resistance, calcium channels were measured in the normal PWR-1E prostate cells, the hormone-responsive LNCaP cells, and the castration-resistant VCaP and 22RV1 cells. Compared to normal prostate cells, the hormone-responsive and hormone-resistant cells overexpressed several calcium channels. The results of this study show that calcium activates AR and increases cell growth and that calcium channels are overexpressed in hormone-responsive and hormone-resistant prostate cancer cells. Taken together, the results suggest a novel role of calcium in the castration-resistant phenotype.

Impact of add-back FSH on human and mouse prostate following gonadotropin ablation by GnRH antagonist treatment

Objective During androgen ablation in prostate cancer by the standard gonadotropin-releasing hormone (GnRH) agonist treatment, only luteinizing hormone (LH) is permanently suppressed while circulating follicle-stimulating hormone (FSH) rebounds. We explored direct prostatic effects of add-back FSH, after androgen ablation with GnRH antagonist, permanently suppressing both gonadotropins.Methods The effects of recombinant human (rFSH) were examined in mice treated with vehicle (controls), GnRH antagonist degarelix (dgx), dgx + rFSH, dgx + flutamide, or dgx + rFSH + flutamide for 4 weeks. Prostates and testes size and expression of prostate-specific and/or androgen-responsive genes were measured. Additionally, 33 young men underwent dgx-treatment. Seventeen were supplemented with rFSH (weeks 1–5), and all with testosterone (weeks 4–5). Testosterone, gondotropins, prostate-specific antigen (PSA), and inhibin B were measured.Results In dgx and dgx + flutamide treated mice, prostate weight/body weight was 91% lower than in controls, but 41 and 11%, respectively, was regained by rFSH treatment (P = 0.02). The levels of seminal vesicle secretion 6, Pbsn, Nkx3.1, beta-microseminoprotein, and inhibin b were elevated in dgx + rFSH-treated animals compared with only dgx treated (all P < 0.05). In men, serum inhibin B rose after dgx treatment but was subsequently suppressed by testosterone. rFSH add-back had no effect on PSA levels.Conclusions These data provide novel evidence for the direct effects of FSH on prostate size and gene expression in chemically castrated mice. However, in chemically castrated men, FSH had no effect on PSA production. Whether FSH effects on the prostate in humans also require suppression of the residual adrenal-derived androgens and/or a longer period of rFSH stimulation, remains to be explored.

Abstract 1008: Comparative proteomics and transcriptomics analysis of the impact of androgen stimulation and darolutamide inhibition in a prostate cancer model

Abstract Androgen receptor (AR) signaling is the major driver in prostate cancer. Darolutamide is a high-affinity AR inhibitor recently approved for non-metastatic castration-resistant prostate cancer. The impact of the AR on gene transcription has been extensively examined but the downstream effects on protein levels are still poorly understood. Here we generated for the first time comprehensive quantitative proteomic and transcriptomic data from the androgen-sensitive, prostate cancer cell line VCaP treated with the androgen R1881 and darolutamide. For the proteomic analysis, a comprehensive and quantitative label-free protein expression profiling was performed using nanoscale liquid chromatography coupled to tandem mass spectrometry. For the transcriptomic analysis, sequencing was performed via single-end, 50 base-pair reads with an average depth of 21 million reads per sample. FASTQ reads were mapped with STAR aligner to the human genome GRCh38 and quantified with RSEM. Further analysis was performed in R statistical software. We investigated the expression levels of 12900 protein-coding genes and 8721 translated proteins. Androgen treatment resulted in increased expression of known androgen response genes, as well as of genes from the mTORC1 pathway, cell cycle and other pathways previously shown to be affected by androgens. This was paralleled by upregulation of the corresponding protein levels in the majority of cases. Importantly, a detailed comparative analysis of transcriptomic and proteomic data revealed a few notable differences in regulation. In androgen-treated prostate cancer cells, there was a strong increase of FOLH1, AMACR and FKBP5 transcript levels (10 to 40-fold) but only a moderate increase of the corresponding protein levels (2 to 6-fold). Additional darolutamide treatment led to strong down-regulation of androgen-induced gene expression and of the corresponding protein levels. Here also there were a few instances where protein and transcript levels were differentially regulated. The discrepancy in transcript and protein levels seen after androgen and anti-androgen treatment suggests differential post-transcriptional regulation of protein abundance to occur in some instances. In conclusion, we found that darolutamide strongly reversed androgen-induced activation of gene expression programs related to cell proliferation and survival, both at the transcript and protein levels. We furthermore identified for the first time androgen-activated genes whose function is additionally regulated at the post-transcription level. Citation Format: Ekaterina Nevedomskaya, Simon J. Baumgart, Tatsuo Sugawara, Ralf Lesche, Dominik Mumberg, Bernard Haendler. Comparative proteomics and transcriptomics analysis of the impact of androgen stimulation and darolutamide inhibition in a prostate cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1008.

A prospective clinical and transcriptomic feasibility study of oral-only hormonal therapy with radiation for unfavorable prostate cancer in men 70 years of age and older or with comorbidity.

Androgen deprivation therapy (ADT) improves outcomes in unfavorable-risk prostate cancer (PCa) treated with radiation therapy (RT). It was hypothesized that replacing luteinizing hormone-releasing hormone (LHRH) agonists with a 5-α-reductase inhibitor (5-ARI) would improve hormonal health-related quality of life (HRQOL) without differentially suppressing androgen-responsive (AR) gene expression. Patients with localized unfavorable-risk PCa, aged ≥70 years or Charlson Comorbidity Index score ≥2 were treated with oral ADT (oADT), consisting of 4 months of bicalutamide, a 5-ARI, and RT at 78 Gy. The primary end point was Expanded Prostate Cancer Index Composite HRQOL at 6 months ≤30%, and improvement compared with a synchronous standard of care (SOC) cohort receiving 4 months of bicalutamide and long-term LHRH agonist with RT. RNA sequencing was performed from matched pre-/post-ADT prostate tumor biopsies in a subset of men. Differential gene and pathway expressional changes were examined using gene set enrichment. Between 2011 and 2018, 40 and 30 men were enrolled in the oADT and SOC cohorts, respectively. Median follow-up was 40 months. Those with ≤30% decline in hormonal HRQOL at 6 months was 97% (oADT) and 93% (SOC). The average 6-month hormonal decline was 1% (oADT) versus 12% (SOC; P = .04). The 4-year freedom from biochemical failure was 88% (oADT) versus 81% (SOC; P = .48). RNA sequencing (n = 9) showed similar numbers of downregulated and upregulated genes between the treatment groups (fold-change = 2; false-discovery rate-adjusted P ≤ .05). Both treatments comparably decreased the expression of 20 genes in canonical androgen receptor signaling. For men with PCa undergoing RT, oral versus standard ADT may improve 6-month QOL and appears to have a similar impact on androgen-responsive gene expression.

The brominated flame retardants TBECH and DPTE alter prostate growth, histology and gene expression patterns in the mouse

The brominated flame retardants (BFRs), 1,2-dibromo-4-(1,2 dibromoethyl)cyclohexane (TBECH) and 2,3-dibromopropyl-2,4,6-tribromophenyl ether (DPTE) bind to the androgen receptor (AR). in vitro bioassays have shown that TBECH is a potent androgen agonist while DPTE is a potent AR antagonist. Both TBECH and DPTE alter gene expression associated with AR regulation. However, it remains to be determined if TBECH and DPTE can affect the prostate. For this reason, we exposed CD1 mice to a 1:1 mixture of TBECH diastereomers α and β, a 1:1 mixture of γ and δ, and to DPTE, and tested their effects on prostate growth, histology and gene expression profiles. Castrated mice were used to study the androgenic effects of TBECHαβ and TBECHγδ while the antagonistic effects of DPTE were studied in non-castrated mice. We observed that testosterone and TBECHγδ increased body and prostate weights while TBECHαβ affected neither of them; and that DPTE had no effect on body weight but reduced prostate weight drastically. Histomorphometric analysis of the prostate revealed epithelial and glandular alterations in the TBECHγδ group comparable to those in testosterone group while alterations in the TBECHαβ group were less pronounced. DPTE displayed androgen antagonist activity reminiscent of castration. The transcription profile of the prostate was altered by castration and exposure to testosterone and to TBECHγδ reversed several of these changes. Testosterone and TBECHγδ also regulated the expression of several androgen responsive genes implicated in prostate growth and cancer. While DPTE resulted in a drastic reduction in prostate weight, it only affected a small number of genes. The results indicate that TBECHγδ and DPTE are of high human health concern as they may contribute to changes in prostate growth, histology and function.

Sea buckthorn (Hippophae rhamnoides L.) inhibits cellular proliferation, wound healing and decreases expression of prostate specific antigen in prostate cancer cells in vitro

Abstract Androgen receptor (AR) is a ligand-dependent transcription factor that governs the expression of androgen-responsive genes. If somehow AR is retained in the cytoplasm and its shuttling into the nucleus is inhibited it cannot activate the target androgen responsive genes and prevent prostate cancer progression. The primary goal of our study was to evaluate plant based functional food extract from sea buckthorn (Hippophae rhamnoides L.) that can target AR nuclear localization in prostate cancer cells and inhibit prostate cancer growth in vitro. Differential extraction using various solvents based on polarity revealed that the end phase aqueous cocktail extracted from leaves of Hippophae rhamnoides L. (SKICDDL-3) can effectively target AR and downregulate androgen responsive genes, PSA, ELL2, EAF2 and CALR significantly in vitro. Colony formation Unit assay and Wound healing assay further shows that SKICDDL-3 can effectively inhibit proliferation and migration of castration resistant C4-2 prostate cancer cells in vitro.

Androgen modulation of XBP1 is functionally driving part of the AR transcriptional program.

Prostate cancer development and progression is largely dependent on androgen receptor (AR) signaling. AR is a hormone-dependent transcription factor, which binds to thousands of sites throughout the human genome to regulate expression of directly responsive genes, including pro-survival genes that enable tumor cells to cope with increased cellular stress. ERN1 and XBP1 - two key players of the unfolded protein response (UPR) - are among such stress-associated genes. Here, we show that XBP1 levels in primary prostate cancer are associated with biochemical recurrence in five independent cohorts. Patients who received AR-targeted therapies had significantly lower XBP1 expression, whereas expression of the active form of XBP1 (XBP1s) was elevated. In vitro results show that AR-induced ERN1 expression led to increased XBP1s mRNA and protein levels. Furthermore, ChIP-seq analysis revealed that XBP1s binds enhancers upon stress stimuli regulating genes involved in UPR processes, eIF2 signaling and protein ubiquitination. We further demonstrate genomic overlap of AR- and XBP1s-binding sites, suggesting genomic conversion of the two signaling cascades. Transcriptomic effects of XBP1 were further studied by knockdown experiments, which lead to decreased expression of androgen-responsive genes and UPR genes. These results suggest a two-step mechanism of gene regulation, which involves androgen-induced expression of ERN1, thereby enhancing XBP1 splicing and transcriptional activity. This signaling cascade may prepare the cells for the increased protein folding, mRNA decay and translation that accompanies AR-regulated tumor cell proliferation.

The Homeodomain Transcription Factor NKX3.1 Modulates Bladder Outlet Obstruction Induced Fibrosis in Mice.

Fibrosis is an irreversible remodeling process characterized by the deposition of collagen in the extracellular matrix of various organs through a variety of pathologies in children, leading to the stiffening of healthy tissues and organ dysfunction. Despite the prevalence of fibrotic disease in children, large gaps exist in our understanding of the mechanisms that lead to fibrosis, and there are currently no therapies to treat or reverse it. We previously observed that castration significantly reduces fibrosis in the bladders of male mice that have been partially obstructed. Here, we investigated if the expression of androgen response genes were altered in mouse bladders after partial bladder outlet obstruction (PO). Using a QPCR microarray and QRTPCR we found that PO was sufficient to increase expression of the androgen response gene Nkx3.1. Consistent with this was an increase in the expression of NKX3.1 protein. Immunofluorescent antibody localization demonstrated nuclear NKX3.1 in most bladder cells after PO. We tested if genetic deletion of Nkx3.1 alters remodeling of the bladder wall after PO. After PO, Nkx3.1KO/KO bladders underwent remodeling, demonstrating smaller bladder area, thickness, and bladder: body weight ratios than obstructed, wild type controls. Remarkably, Nkx3.1KO/KO specifically affected histological parameters of fibrosis, including reduced collagen to muscle ratio. Loss of Nkx3.1 altered collagen and smooth muscle cytoskeletal gene expression following PO which supported our histologic findings. Together these findings indicated that after PO, Nkx3.1 expression is induced in the bladder and that it mediates important pathways that lead to tissue fibrosis. As Nkx3.1 is an androgen response gene, our data suggest a possible mechanism by which fibrosis is mediated in male mice and opens the possibility of a molecular pathway mediated by NKX3.1 that could explain sexual dimorphism in bladder fibrosis.

The long non-coding RNA PCA3: an update of its functions and clinical applications as a biomarker in prostate cancer.

Prostate cancer antigen 3 (PCA3) is an overexpressed prostate long non-coding RNA (lncRNA), transcribed from an intronic region at the long arm of human chromosome 9q21–22. It has been described that PCA3 modulates prostate cancer (PCa) cell survival through modulating androgen receptor (AR) signaling, besides controlling the expression of several androgen responsive and cancer-related genes, including epithelial–mesenchymal transition (EMT) markers and those regulating gene expression and cell signaling. Also, PCA3 urine levels have been successfully used as a PCa diagnostic biomarker. In this review, we have highlighted recent findings regarding PCA3, addressing its gene structure, putative applications as a biomarker, a proposed origin of this lncRNA, roles in PCa biology and expression patterns. We also updated data regarding PCA3 interactions with cancer-related miRNAs and expression in other tissues and diseases beyond the prostate. Altogether, literature data indicate aberrant expression and dysregulated activity of PCA3, suggesting PCA3 as a promising relevant target that should be even further evaluated on its applicability for PCa detection and management.

Abstract 2629: Constitutive AhR signaling enhances androgen receptor signaling and growth in prostate cancer cells

Abstract The aryl hydrocarbon receptor (AhR) has been reported to interact with multiple signaling pathways during prostate cancer progression to castration resistant prostate cancer (CRPC) including the androgen receptor. Previously published data from our lab shows that AhR protein is overexpressed and constitutively active in castration resistant (DU145, PC3 and PC3M) prostate cancer cells. Pharmacological inhibition of AhR in these advanced prostate cancer cell lines reduced the growth rate under androgen depleted conditions. Furthermore, shRNA mediated depletion of AhR expression in castration resistant C4-2 cells, which were derived from hormone sensitive LNCaP cells, resulted in decreased expression of androgen responsive genes as well as a decreased growth rate which was comparable to LNCaP cell growth. In the present study, we evaluate cloneA (moderate increase in AhR) and CloneB (high increase in AhR) for alterations in AhR and AR signaling. AhR was overexpressed in LNCaP using PLNCX2 retrovirus vector containing AhR cDNA to determine if ectopic overexpression alone induces a castrate resistant phenotype. The highly overexpressed AhR clone (CloneB) illustrated further increase in transcriptional and promotor activity for AhR and AR compared to the moderately overexpressed AhR clone (Clone A) and control. Western blot analysis showed more AhR, AR, cSrc, and pSrc protein expression in clones compared to empty vector control. AhR overexpression was found to induce several biological properties such as migration, invasion, proliferation, and promotion of G1 to S phase during the cell cycle. Bicalutamide treatment had no effect on AR transcriptional activity in either clone, proving resistance to anti-androgen therapy. Our results confirm that overexpression of AhR induces constitutive AhR activity and stimulates androgen receptor signaling. This suggests a role for AhR in the development of CRPC. Citation Format: Maryam Ghotbaddini, Sakura McLaughlin, Kailen Turner, Devyn Pirtle, Joann B. Powell. Constitutive AhR signaling enhances androgen receptor signaling and growth in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2629.

Alteration of androgen receptor expression, apoptosis and cell proliferation in cryptorchid suckling, nursery and growing-finishing pigs

Cryptorchidism, a condition of one or two undescended testicles, is a common congenital disease in pigs, causing loss in the pig industry. One of the major factors affecting testicular descent is the androgen receptor (AR), which binds to androgen and then regulates the expression of androgen-responsive genes in the inguinoscrotal phase of testicular descent. AR expression has been reported to regulate apoptosis in testicular stem cells. The present study aimed to immunohistochemically examine AR and Ki-67 protein expression and apoptosis detection in unilateral undescended testicles (UDT) and descended testicles in cryptorchid pigs (DT) of suckling (aged 1–2 weeks), nursery (aged 6 weeks) and growing-finishing pigs (aged 12, 15 and 20 weeks) and in normal testicles (NT) at 1–2 and 12 weeks of age. At 1–2 weeks, decreased expression of AR was observed in UDT and DT compared with NT and was lower than that at 6–20 weeks. The expression of Ki-67, a marker of cell proliferation, in UDT and DT at 12 weeks was lower than that in NT at the same age. In addition, Ki-67 expression in UDT at 6 and 12 weeks was lower than that in UDT at 1–2 and 15–20 weeks. More testicular apoptosis was revealed in UDT at 1–2 weeks than in DT and NT at the same age. At 15–20 weeks, more apoptosis was detected in UDT than in DT. Positive correlation of AR expression in DT at 6 and 12 weeks was also noted, in addition to the association of the expression of AR and Ki-67 in NT at 12 weeks. Taken together, this study unveiled the low expression of AR and high apoptosis detection in UDT, whereas low expression of AR and low apoptosis detection were noted in DT in suckling piglets. Diminished cell proliferation was shown in UDT at 6–12 weeks, whereas high apoptosis was observed in UDT at 15–20 weeks. High expression of AR was shown only in nursery pigs. Distinct expression of AR in DT and NT at 1–2 and 12 weeks indicated that both conditions were not interchangeable.

Anti-androgenic therapy with finasteride improves cardiac function, attenuates remodeling and reverts pathologic gene-expression after myocardial infarction in mice

Maladaptive cardiac remodeling after myocardial infarction (MI) is increasingly contributing to the prevalence of chronic heart failure. Women show less severe remodeling, a reduced mortality and a better systolic function after MI compared to men. Although sex hormones are being made responsible for these differences, it remains currently unknown how this could be translated into therapeutic strategies. Because we had recently demonstrated that inhibition of the conversion of testosterone to its highly active metabolite dihydrotestosterone (DHT) by finasteride effectively reduces cardiac hypertrophy and improves heart function during pressure overload, we asked here whether this strategy could be applied to post-MI remodeling. We found increased abundance of DHT and increased expression of androgen responsive genes in the mouse myocardium after experimental MI. Treatment of mice with finasteride for 21 days (starting 7 days after surgery), reduced myocardial DHT levels and markedly attenuated cardiac dysfunction as well as hypertrophic remodeling after MI. Histological and molecular analyses showed reduced MI triggered interstitial fibrosis, reduced cardiomyocyte hypertrophy and increased capillary density in the myocardium of finasteride treated mice. Mechanistically, this was associated with decreased activation of myocardial growth-signaling pathways, a comprehensive normalization of pathological myocardial gene-expression as revealed by RNA deep-sequencing and with direct effects of finasteride on cardiac fibroblasts and endothelial cells. In conclusion, we demonstrated a beneficial role of anti-androgenic treatment with finasteride in post-MI remodeling of mice. As finasteride is already approved for the treatment of benign prostate disease, it could potentially be evaluated as therapeutic strategy for heart failure after MI.

KDM8/JMJD5 as a dual coactivator of AR and PKM2 integrates AR/EZH2 network and tumor metabolism in CRPC

During the evolution into castration or therapy resistance, prostate cancer cells reprogram the androgen responses to cope with the diminishing level of androgens, and undergo metabolic adaption to the nutritionally deprived and hypoxia conditions. AR (androgen receptor) and PKM2 (pyruvate kinase M2) have key roles in these processes. We report in this study, KDM8/JMJD5, a histone lysine demethylase/dioxygnase, exhibits a novel property as a dual coactivator of AR and PKM2 and as such, it is a potent inducer of castration and therapy resistance. Previously, we showed that KDM8 is involved in the regulation of cell cycle and tumor metabolism in breast cancer cells. Its role in prostate cancer has not been explored. Here, we show that KDM8’s oncogenic properties in prostate cancer come from its direct interaction (1) with AR to affect androgen response and (2) with PKM2 to regulate tumor metabolism. The interaction with AR leads to the elevated expression of androgen response genes in androgen-deprived conditions. They include ANCCA/ATAD2 and EZH2, which are directly targeted by KDM8 and involved in sustaining the survival of the cells under hormone-deprived conditions. Notably, in enzalutamide-resistant cells, the expressions of both KDM8 and EZH2 are further elevated, so are neuroendocrine markers. Consequently, EZH2 inhibitors or KDM8 knockdown both resensitize the cells toward enzalutamide. In the cytosol, KDM8 associates with PKM2, the gatekeeper of pyruvate flux and translocates PKM2 into the nucleus, where the KDM8/PKM2 complex serves as a coactivator of HIF-1α to upregulate glycolytic genes. Using shRNA knockdown, we validate KDM8’s functions as a regulator for both androgen-responsive and metabolic genes. KDM8 thus presents itself as an ideal therapeutic target for metabolic adaptation and castration-resistance of prostate cancer cells.

Androgen Signaling in Esophageal Adenocarcinoma Cell Lines In Vitro

BackgroundWe showed previously that nuclear localization of the androgen receptor (AR) and expression of the androgen-responsive gene FK506-binding protein 5 (FKBP5) in esophageal adenocarcinoma (EAC) tissues were associated with decreased patient survival, suggesting a role for androgens in this cancer.AimTo investigate the effect of the AR ligand 5α-dihydrotestosterone (DHT) on AR-expressing EAC cell lines in vitro.Methods and ResultsIn tissue resection specimens from EAC patients, FKBP5 expression was positively associated with proliferation as measured by Ki-67 expression. We stably transduced AR into three AR-negative EAC cell lines, OE33, JH-EsoAd1, and OE19, to investigate androgen signaling in vitro. In the AR-expressing cell lines, 10 nM DHT, the concentration typically used to study AR signaling, induced changes in the expression of androgen-responsive genes and inhibited proliferation by inducing cell cycle arrest and senescence. At lower DHT concentrations near the half maximal inhibitory concentration (IC50), the AR-expressing cell lines proliferated and there were changes in the expression of androgen-responsive genes. In direct co-culture with cancer-associated fibroblast-like PShTert myofibroblasts, 10 nM DHT induced changes in the expression of androgen-responsive genes but did not inhibit proliferation.ConclusionsThis is the first study to show that EAC cell lines respond to androgen in vitro. Proliferation together with the expression of androgen-responsive genes was dependent on the concentration of DHT, or the presence of a permissive microenvironment, consistent with observations in the tissues. These findings are consistent with a role for androgen signaling in EAC.

Inhibition of Androgen Receptor Nuclear Localization and Castration-Resistant Prostate Tumor Growth by Pyrroloimidazole-based Small Molecules.

The androgen receptor (AR) is a ligand-dependent transcription factor that controls the expression of androgen-responsive genes. A key step in androgen action, which is amplified in castration-resistant prostate cancer (CRPC), is AR nuclear translocation. Small molecules capable of inhibiting AR nuclear localization could be developed as novel therapeutics for CRPC. We developed a high-throughput screen and identified two structurally-related pyrroloimidazoles that could block AR nuclear localization in CRPC cells. We show that these two small molecules, 3-(4-ethoxyphenyl)-6,7-dihydro-5H-pyrrolo[1,2-a]imidazole (EPPI) and 3-(4-chlorophenyl)-6,7-dihydro-5H-pyrrolo[1,2-a]imidazole (CPPI) can inhibit the nuclear localization and transcriptional activity of AR and reduce the proliferation of AR-positive but not AR-negative prostate cancer cell lines. EPPI and CPPI did not inhibit nuclear localization of the glucocorticoid receptor or the estrogen receptor, suggesting they selectively target AR. In LNCaP tumor xenografts, CPPI inhibited the proliferation of relapsed LNCaP tumors. These findings suggest that EPPI and CPPI could serve as lead structures for the development of therapeutic agents for CRPC. Mol Cancer Ther; 16(10); 2120-9. ©2017 AACR.

Abstract 1584: Wnt/beta-catenin and Foxa2 axis activates AR signaling in castration resistant prostate cancer

Abstract Background: Prostate cancer (PCa) is the leading cancer among men in the world. Androgen deprivation therapy is a common treatment to cease prostate growth. However, androgen deprivation eventually fails and PCa relapses, evolving into castration resistant prostate cancer (CRPCa). It has been established that androgen receptor (AR) continues to be the central player in mediating CRPCa. However, the mechanism by which the AR signaling remains active is unclear. We have reported that activation of Wnt/beta-catenin signaling pathway promotes castrate-resistant progression. Our study has also shown that activation of the Wnt/beta-catenin pathway induces the expression of Foxa2, a fork-head transcription factor that is expressed in embryonic prostate &amp; advanced stage PCa. Here, we study the Wnt pathway and the role of Foxa2 in retaining active AR signaling in advanced PCa in the absence of androgens. TRAMP mice, SV40 T-antigen transgenic mouse models for PCa, were used. It has been recognized that in this model, T-antigen, which is driven by androgen-responsive probasin promoter, remains expressed following androgen ablation. In this study, we identified a mechanism that sustains an active AR signaling in CRPCa. Methods: To study whether the Wnt/beta-catenin pathway is active in advanced stage PCa, we crossed the TRAMP mice with GFP-Wnt reporter mice. Some of the TRAMP/Wnt-reporter mice were castrated to study how androgen deprivation affects Wnt/beta-Catenin signaling and promotes PCa progression. Prostates were analyzed using IHC and IF staining. We also established a prostate epithelial cell line that stably expresses Foxa2. Cell proliferation was examined with IncuCyte Zoom software. The expression of AR target genes was assessed by using quantitative RT-PCR and Western blotting. ChIP was conducted to assess the recruitment of transcriptional machinery to the promoters of AR-regulated genes. Results: Utilizing the TRAMP/Wnt mouse model, we show that Wnt/beta-Catenin signaling is active in advanced stage PCa. Additionally, our data showed that Foxa2, the downstream target of Wnt/beta-Catenin signaling, drives the expression of androgen-responsive genes, regardless of androgen deprivation. Furthermore, cell proliferation assays revealed that the expression of Foxa2 enabled an androgen-independent growth of prostatic cells. Using this model system, we further explored the mechanism that activates the transcription of AR-responsive genes in the absence of androgens. Our data indicated that Foxa2 is capable of recruiting H3k27Ac to AR-responsive promoters. Conclusion: Together, these data indicate that Wnt signaling is activated in advanced stage PCa, driving Foxa2 expression, and promoting AR signaling to persist. This represents a novel mechanism that enables advanced PCa to retain AR signaling pathway following androgen ablation. Targeting both Wnt signaling and Foxa2 may pose as novel therapeutics for CRPCa. Citation Format: Zachary M. Connelly, Shu Yang, Jiahe Li, Robert Matusik, Xiuping Yu. Wnt/beta-catenin and Foxa2 axis activates AR signaling in castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1584. doi:10.1158/1538-7445.AM2017-1584

Cranberry intervention in patients with prostate cancer prior to radical prostatectomy. Clinical, pathological and laboratory findings.

Recently, we described an inverse association between cranberry supplementation and serum prostate specific antigen (PSA) in patients with negative biopsy for prostate cancer (PCa) and chronic nonbacterial prostatitis. This double blind placebo controlled study evaluates the effects of cranberry consumption on PSA values and other markers in men with PCa before radical prostatectomy. Prior to surgery, 64 patients with prostate cancer were randomized to a cranberry or placebo group. The cranberry group (n=32) received a mean 30 days of 1500 mg cranberry fruit powder. The control group (n=32) took a similar amount of placebo. Selected blood/urine markers as well as free and total phenolics in urine were measured at baseline and on the day of surgery in both groups. Prostate tissue markers were evaluated after surgery. The serum PSA significantly decreased by 22.5% in the cranberry arm (n=31, P<0.05). A trend to down-regulation of urinary beta-microseminoprotein (MSMB) and serum gamma-glutamyltranspeptidase, as well as upregulation of IGF-1 was found after cranberry supplementation. There were no changes in prostate tissue markers or, composition and concentration of phenolics in urine. Daily consumption of a powdered cranberry fruit lowered serum PSA in patients with prostate cancer. The whole fruit contains constituents that may regulate the expression of androgen-responsive genes.

Abstract 4571: Semaphorin/plexin signalling promotes nucleocytoplasmic trafficking of nuclear receptors

Abstract Androgen receptor (AR) signalling is critical to prostate cancer progression and androgen deprivation therapy (ADT) is the first line of treatment for metastatic prostate cancer. Acquired resistance to ADT results from androgen-independent reactivation of the AR pathway primarily. Prostate tumours also bypass androgen blockade by the upregulation of the glucocorticoid receptor (GR). Both AR and GR are nuclear receptors that bind their respective ligands in the cytoplasm and then translocate to the nucleus where they act as transcription factors. Plexins are cell surface receptors for semaphorins. PlexinB1 is overexpressed and mutated in several cancers including prostate cancer and deletion of the plexinB1 gene reduces metastasis in mouse models, indicating an oncogenic role for plexinB1 in prostate cancer. We have found that activation of plexinB1 by semaphorin4D (Sema4D) results in AR phosphorylation at serine 81, activation of AR-transcriptional activity and increased expression of androgen-responsive genes, effects reversed by plexinB1 knockdown. PlexinB1 activation increases the translocation of both AR-GFP constructs and endogenous AR to the nucleus in prostate cancer cells. Furthermore knockdown of AR in the prostate cancer cell line LNCaP reduces Sema4D-induced anchorage-independent growth of these cells. To determine if the Sema4D/plexinB1 signalling pathway also affects translocation of other nuclear receptors to the nucleus, we have also studied the effect of Sema4D treatment on nuclear trafficking of the glucocorticoid receptor (GR). Prostate cancer cells were transfected with a GR-GFP construct and the subcellular localisation of GR-GFP was monitored upon Sema4D treatment by immunocytochemistry and confocal microscopy. The nuclear translocation of endogenous GR was also assessed following Sema4D stimulation by immunocytochemistry and subcellular fractionation studies. We found that activation of plexinB1 by Sema4D results in a significant increase in the percentage of cells with nuclear GR-GFP and in the nuclear localisation of endogenous GR in prostate cancer cells. These findings show that plexinB1 activation has a role in the trafficking and activation of nuclear receptors and so may have a role in resistance to ADT in late stage prostate cancer. Citation Format: Magali Williamson, John R. Masters. Semaphorin/plexin signalling promotes nucleocytoplasmic trafficking of nuclear receptors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4571.

MP66-06 THE ROLE OF THE PKD1-β-CATENIN-ANDROGEN RECEPTOR AXIS IN PROSTATE CANCER PROGRESSION

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II1 Apr 2016MP66-06 THE ROLE OF THE PKD1-β-CATENIN-ANDROGEN RECEPTOR AXIS IN PROSTATE CANCER PROGRESSION Bita Nick Kholgh, Michael B. Rothberg, Sittadjody Sivanandane, Xiaolan Fang, and K.C. Balaji Bita Nick KholghBita Nick Kholgh More articles by this author , Michael B. RothbergMichael B. Rothberg More articles by this author , Sittadjody SivanandaneSittadjody Sivanandane More articles by this author , Xiaolan FangXiaolan Fang More articles by this author , and K.C. BalajiK.C. Balaji More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1280AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prior studies have described androgen receptor (AR) involvement in the nuclear translocation of β-catenin; in turn, β-catenin acts as coactivator of AR and enhances AR-mediated transcription. It has been shown that phosphorylation of β-catenin at the threonine 120 (T120) residue decreases active β-catenin (ABC) expression. Currently the only known upstream kinase for T120-β-catenin phosphorylation is protein kinase D1 (PKD1), a novel tumor and metastasis suppressor in prostate cancer (PCa). In progressive PCa, down regulation of PKD1 increases the proliferation and invasion of prostatic cells as well as induces epithelial to mesenchymal transition. PKD1 also influences AR transcriptional activity. METHODS In order to study the role of the PKD1-β-catenin-AR axis in progressive PCa, we transfected androgen sensitive LNCaP cells with one of three constructs: wild type β-catenin, T120 phosphorylated-β-catenin, or an empty vector. Chromatin Immunoprecipitation (CHIP) assay demonstrated a β-catenin and PKD1 transcription site association, which was subsequently abolished following mutation of T120-β-catenin to its unphosphorylated form. RESULTS Western blot experiments on all groups of transfected cells showed the nuclear translocation of T120 phosphorylated-β-catenin mutant transfected cells were higher compared to cells transfected with wild type-β-catenin or an empty vector, confirming increased levels of ABC in the nucleus. Interestingly, the expression of AR in cells transfected with T120 phosphorylated-β-catenin mutant was significantly lower compared to controls. Despite a decrease in AR activity, the expression of androgen response genes in the presence of androgen was significantly increased in the mutant transfected cells compared to controls, which suggests that ABC could activate androgen response genes independent from AR. CONCLUSIONS In conclusion, our study has identified several novel findings: 1) ABC is associated with the PKD1 transcription site suggesting a possible role for β-catenin in transcriptional regulation of PKD1, 2) the T120-β-catenin mutant down regulates AR expression, suggesting that phosphorylation of T120 by PKD1 is necessary for co-activation of AR expression by β-catenin and, 3) there exists a novel complex autoregulatory mechanism of PKD1, AR, and β-catenin expression, transport, and function. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e875 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Bita Nick Kholgh More articles by this author Michael B. Rothberg More articles by this author Sittadjody Sivanandane More articles by this author Xiaolan Fang More articles by this author K.C. Balaji More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...