Prostate Androgen Receptor Research Articles

Effects of Lupeol on Estrogen and Androgen Receptor-Positive Breast and Prostate Cancer Cells

Background: Different reports have shown that prostate and breast cancers are the most common cancers worldwide. Lupeol, a dietary triterpene, provides various beneficial effects including anti-cancer properties. The current study aims to investigate the anti-proliferative and antioxidant effects of lupeol, in line with the effects of lupeol on the expression of estrogen and androgen receptors in breast (MCF-7) and prostate (LNCaP) cancer cell lines. Methods: MCF-7 and LNCaP cells were incubated with increasing concentrations of the lupeol (1, 10, and 100 µM) for 24h. The cytotoxicity of the lupeol was assessed by MTT and Neutral Red assays. Moreover, TAC (total antioxidant capacity), and gene expression of androgen and estrogen receptors were measured by spectrophotometric and qPCR methods, respectively. Overall, 17 beta-estradiol (E2) (9 nM) and dehydroepiandrosterone (DHEA) (5 µM) were selected as positive controls. Results: The highest concentration of the lupeol induced cytotoxic effects on MCF-7 and LNCaP cells. Various levels of lupeol at specified time intervals increased TAC levels in comparison with the control group. Moreover, the expression levels of estrogen receptors (α and β) and androgen receptors were negatively affected by lupeol. Conclusion: The findings of our study indicate that lupeol could serve as a promising, and accessible multi-functional anti-tumor agent against hormone-positive breast and prostate cancers.

Androgen receptor and estrogen receptor variants in prostate and breast cancers

The androgen receptor (AR) and estrogen receptor alpha (ERα) are steroid receptor transcription factors with critical roles in the development and progression of prostate and breast cancers. Advances in the understanding of mechanisms underlying the ligand-dependent activation of these transcription factors have contributed to the development of small molecule inhibitors that block AR and ERα actions. These inhibitors include competitive antagonists and degraders that directly bind the ligand binding domains of these receptors, luteinizing hormone releasing hormone (LHRH) analogs that suppress gonadal synthesis of testosterone or estrogen, and drugs that block specific enzymes required for biosynthesis of testosterone or estrogen. However, resistance to these therapies is frequent, and is often driven by selection for tumor cells with alterations in the AR or ESR1 genes and/or alternatively spliced AR or ESR1 mRNAs that encode variant forms AR or ERα. While most investigations involving AR have been within the context of prostate cancer, and the majority of investigations involving ERα have been within the context of breast cancer, important roles for AR have been elucidated in breast cancer, and important roles for ERα have been elucidated in prostate cancer. Here, we will discuss the roles of AR and ERα in breast and prostate cancers, outline the effects of gene- and mRNA-level alterations in AR and ESR1 on progression of these diseases, and identify strategies that are being developed to target these alterations therapeutically.

The Combination of Curcumae Radix and Syzygium Aromaticum Extracts Mitigates Benign Prostatic Hyperplasia through Anti-Proliferative and Anti-Inflammatory Effects.

Benign prostatic hyperplasia (BPH) is a urinary tract disorder that primarily affects geriatric males. Natural materials have been developed to treat and prevent symptoms of BPH. However, a few natural functional foods has been conclusively proven to cure or prevent symptoms of BPH. The study aimed to investigate the anti-proliferative mechanism of Curcumae Radix (CR) and Syzygium aromaticum (SA) extracts using RWPE-1 cells and testosterone propionate (TP)-induced BPH rats. In vitro experiments were performed to assess the synergistic anti-proliferative effects of an equal mixture of CR and SA extracts on TP-treated RWPE-1 cells. In animal experiments, TP-induced BPH rats were administrated with saline, CR and SA extracts at 50, 100, and 200 mg/kg or finasteride at 1 mg/kg daily for 6 weeks. Body weight, prostate weight, dihydrotestosterone (DHT), androgen receptor (AR), and prostate-specific antigen (PSA) levels were measured. Additionally, extracellular signal-regulated kinase and NF-κB levels, oxidative stress, and inflammatory stress responses in the prostate were also analyzed. In this study, the combination of CR and SA extracts synergistically inhibited cell proliferation compared with the effect of each extract in TP-treated RWPE-1 cells. CR and SA extracts inhibited increasing of prostate weight, thickness of prostate epithelium, the level of PSA and DHT in serum. The expression of protein and gene of PSA and AR in prostate of TP-induced BPH rats were also suppressed by the administration of CR and SA extracts. Furthermore, reactive oxygen species and inflammation axis, NOX4-iNOS-COX2 and its associated representative inflammatory cytokine, interleukin-8 were also reduced in the CR and SA extracts-administered BPH rats. The results suggest that the combination of CR and SA extracts improves BPH through its anti-inflammatory and anti-oxidative effects, demonstrating great potential as an anti-BPH medicine.

18F]-FDHT PET for the Imaging of Androgen Receptor in Prostate and Breast Cancer: A Systematic Review.

The aim of this systematic review is to provide a comprehensive overview of the role of fluoro-5α-dihydrotestosterone ([18F]-FDHT) for the in vivo imaging of androgen receptors (AR) through positron emission tomography (PET) in metastatic breast (mBC) and metastatic castration-resistant prostate cancer (mCRPC). Relevant studies published from 2013 up to May 2023 were selected by searching Scopus, PubMed and Web of Science. The selected imaging studies were analyzed using a modified version of the critical Appraisal Skills Programme (CASP). Eleven studies encompassing 321 patients were selected. Seven of the eleven selected papers included 266 subjects (82.2%) affected by mCRPC, while four encompassed 55 (17.2%) patients affected by mBC. [18F]-FDHT PET showed a satisfying test/retest reproducibility, and when compared to a histochemical analysis, it provided encouraging results for in vivo AR quantification both in mCRPC and mBC. [18F]-FDHT PET had a prognostic relevance in mCRPC patients submitted to AR-targeted therapy, while a clear association between [18F]-FDHT uptake and the bicalutamide response was not observed in women affected by AR-positive mBC. Further studies are needed to better define the role of [18F]-FDHT PET, alone or in combination with other tracers (i.e., [18F]-FDG/[18F]-FES), for patients' selection and monitoring during AR-targeted therapy, especially in the case of mBC.

DeepAR: a novel deep learning-based hybrid framework for the interpretable prediction of androgen receptor antagonists

Drug resistance represents a major obstacle to therapeutic innovations and is a prevalent feature in prostate cancer (PCa). Androgen receptors (ARs) are the hallmark therapeutic target for prostate cancer modulation and AR antagonists have achieved great success. However, rapid emergence of resistance contributing to PCa progression is the ultimate burden of their long-term usage. Hence, the discovery and development of AR antagonists with capability to combat the resistance, remains an avenue for further exploration. Therefore, this study proposes a novel deep learning (DL)-based hybrid framework, named DeepAR, to accurately and rapidly identify AR antagonists by using only the SMILES notation. Specifically, DeepAR is capable of extracting and learning the key information embedded in AR antagonists. Firstly, we established a benchmark dataset by collecting active and inactive compounds against AR from the ChEMBL database. Based on this dataset, we developed and optimized a collection of baseline models by using a comprehensive set of well-known molecular descriptors and machine learning algorithms. Then, these baseline models were utilized for creating probabilistic features. Finally, these probabilistic features were combined and used for the construction of a meta-model based on a one-dimensional convolutional neural network. Experimental results indicated that DeepAR is a more accurate and stable approach for identifying AR antagonists in terms of the independent test dataset, by achieving an accuracy of 0.911 and MCC of 0.823. In addition, our proposed framework is able to provide feature importance information by leveraging a popular computational approach, named SHapley Additive exPlanations (SHAP). In the meanwhile, the characterization and analysis of potential AR antagonist candidates were achieved through the SHAP waterfall plot and molecular docking. The analysis inferred that N-heterocyclic moieties, halogenated substituents, and a cyano functional group were significant determinants of potential AR antagonists. Lastly, we implemented an online web server by using DeepAR (at http://pmlabstack.pythonanywhere.com/DeepAR). We anticipate that DeepAR could be a useful computational tool for community-wide facilitation of AR candidates from a large number of uncharacterized compounds.

Cell-by-cell quantification of the androgen receptor in benign and malignant prostate leads to a better understanding of changes linked to cancer initiation and progression.

The androgen receptor (AR) plays a crucial role in the development and homeostasis of the prostate and is a key therapeutic target in prostate cancer (PCa). The gold standard therapy for advanced PCa is androgen deprivation therapy (ADT), which targets androgen production and AR signaling. However, resistance to ADT develops via AR-dependent and AR-independent mechanisms. As reports on AR expression patterns in PCa have been conflicting, we performed cell-by-cell AR quantification by immunohistochemistry in the benign and malignant prostate to monitor changes with disease development, progression, and hormonal treatment. Prostates from radical prostatectomy (RP) cases, both hormone-naïve and hormone-treated, prostate tissues from patients on palliative ADT, and bone metastases were included. In the normal prostate, AR is expressed in >99% of luminal cells, 51% of basal cells, and 61% of fibroblasts. An increase in the percentage of AR negative (%AR-) cancer cells along with a gradual loss of fibroblastic AR were observed with increasing Gleason grade and hormonal treatment. This was accompanied by a parallel increase in staining intensity of AR positive (AR+) cells under ADT. Staining AR with N- and C-terminal antibodies yielded similar results. The combination of %AR- cancer cells, %AR- fibroblasts, and AR intensity score led to the definition of an AR index, which was predictive of biochemical recurrence in the RP cohort and further stratified patients of intermediate risk. Lastly, androgen receptor variant 7 (ARV7)+ cells and AR- cells expressing neuroendocrine and stem markers were interspersed among a majority of AR+ cells in ADT cases. Altogether, the comprehensive quantification of AR expression in the prostate reveals concomitant changes in tumor cell subtypes and fibroblasts, emphasizing the significance of AR- cells with disease progression and palliative ADT.

Design, synthesis, and characterization of PROTACs targeting the androgen receptor in prostate and lung cancer models.

Although the androgen receptor (AR) is a validated target for the treatment of prostate cancer, resistance to antiandrogens necessitates the development of new therapeutic modalities. Exploiting the ubiquitin-proteasome system with proteolysis-targeting chimeras (PROTACs) has become a practical approach to degrade specific proteins and thus to extend the portfolio of small molecules used for the treatment of a broader spectrum of diseases. Herein, we present three subgroups of enzalutamide-based PROTACs in which only the exit vector was modified. By recruiting cereblon, we were able to demonstrate the potent degradation of AR in lung cancer cells. Furthermore, the initial evaluation enabled the design of an optimized PROTAC with a rigid linker that degraded AR with a DC50 value in the nanomolar range. These results provide novel AR-directed PROTACs and a clear rationale for further investigating AR involvement in lung cancer models.

Abstract 2365: Intracellular interaction of downstream molecular mediators of miR-1207-3p in prostate cancer cells

Abstract Prostate cancer (PCa) is the second most common cancer diagnosed among men in the United States. Human chromosome 8q24 is the most important PCa susceptibility locus. This region contains the MYC oncogene, which is involved in early PCa initiation. Downstream to MYC is the PVT1 gene, which is often amplified in PCa. PVT1 is a non-protein coding gene that encodes six annotated microRNAs (miRNAs), including miR-1207-3p. Our laboratory has previously demonstrated miR1207-3p to be a significant modulator in PCa and revealed that miR-1207-3p is significantly underexpressed in PCa cells and histologically confirmed PCa tissues compared to normal prostatic cells and tissues. We have also shown that miR-1207-3p has tumor suppressive activity in vivo. Moreover, we discovered that miR-1207-3p directly targets fibronectin type III domain containing 1 (FNDC1), which was found to be overexpressed in PCa cells and there is a concomitant overexpression of (FN1)/androgen receptor (AR)/c-MYC. In an effort to better understand the role of FNDC1/FN1/AR/c-MYC in PCa progression we examined the interaction between FNDC1/FN1/AR/c-MYC and found direct physical interaction through coimmunoprecipitation. We also examined the spatial localization of the FNDC1/FN1/AR/c-MYC pathway by performing immunofluorescence staining in PCa cells. Single staining analysis revealed that FNDC1 and c-MYC localize to the nucleus and cytoplasm while AR localizes to the nucleus and FN1 localizes to the cytoplasm in PCa cells. However, in normal prostate epithelial cells FNDC1, AR and c-MYC localize to the nucleus whereas FN1 localize to the cytoplasm. These results suggest that the underexpression of miR1207-3p may be affecting the localization of FNDC1 to cytoplasm and thus promoting interaction between FNDC1/FN1/AR/c-MYC. Future studies include examining the effect of miR-1207 on the spatial organization and interactions of the FNDC1/FN1/AR/c-MYC pathway through immunofluorescence and co-immunoprecipitation assays. Thus, understanding this molecular interaction could reveal interesting insight into the biology of microRNA-1207-3p regulation in PCa. Citation Format: Priyanka Ghosh, Oloronseun Ogunwobi. Intracellular interaction of downstream molecular mediators of miR-1207-3p in prostate cancer cells [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 2365.

CBP/p300: Critical Co-Activators for Nuclear Steroid Hormone Receptors and Emerging Therapeutic Targets in Prostate and Breast Cancers.

Simple SummaryThe CREB-binding protein (CBP) and p300 are paralogous lysine acetyltransferases that serve as critical co-activators for transcription factors involved in diverse signaling pathways in cancer. Work in the last two decades has firmly established CBP and p300 as important regulators of nuclear hormone signaling mediated by nuclear receptors, such as the androgen receptor (AR) and estrogen receptor (ER). The AR and ER promote tumor growth in hormone-dependent prostate and breast cancer, respectively. Inhibitors of androgen and estrogen signaling are the standard-of-care therapeutics for treating these cancers. However, resistance to current therapies remains a significant clinical problem. Inhibition of CBP and p300 as a means to block the transactivation activity of the AR and ER is an emerging therapeutic strategy for prostate and breast cancers. This review describes how CBP and p300 regulate androgen and estrogen signaling and discusses therapeutic potential of newly discovered potent CBP/p300 inhibitors for treating prostate and breast cancer.The CREB-binding protein (CBP) and p300 are two paralogous lysine acetyltransferases (KATs) that were discovered in the 1980s–1990s. Since their discovery, CBP/p300 have emerged as important regulatory proteins due to their ability to acetylate histone and non-histone proteins to modulate transcription. Work in the last 20 years has firmly established CBP/p300 as critical regulators for nuclear hormone signaling pathways, which drive tumor growth in several cancer types. Indeed, CBP/p300 are critical co-activators for the androgen receptor (AR) and estrogen receptor (ER) signaling in prostate and breast cancer, respectively. The AR and ER are stimulated by sex hormones and function as transcription factors to regulate genes involved in cell cycle progression, metabolism, and other cellular functions that contribute to oncogenesis. Recent structural studies of the AR/p300 and ER/p300 complexes have provided critical insights into the mechanism by which p300 interacts with and activates AR- and ER-mediated transcription. Breast and prostate cancer rank the first and forth respectively in cancer diagnoses worldwide and effective treatments are urgently needed. Recent efforts have identified specific and potent CBP/p300 inhibitors that target the acetyltransferase activity and the acetytllysine-binding bromodomain (BD) of CBP/p300. These compounds inhibit AR signaling and tumor growth in prostate cancer. CBP/p300 inhibitors may also be applicable for treating breast and other hormone-dependent cancers. Here we provide an in-depth account of the critical roles of CBP/p300 in regulating the AR and ER signaling pathways and discuss the potential of CBP/p300 inhibitors for treating prostate and breast cancer.

The role of the androgen receptor in prostate cancer‐induced platelet aggregation and platelet‐induced invasion

BackgroundMetastatic prostate cancer progresses from a hormone sensitive androgen receptor expressing phenotype to a hormone insensitive androgen receptor‐independent subtype with low overall survival. Human platelets contribute to metastasis via tumor cell‐induced platelet aggregation, which in part enhances cancer cell invasion. Given the more aggressive nature of hormone insensitive prostate cancer, we hypothesized that androgen receptor‐negative prostate cancer cells exhibit higher platelet aggregation potency and invasive response compared to cells with androgen receptor. ObjectiveTo characterize the role of androgen receptors in prostate cancer‐induced platelet aggregation and platelet‐induced invasion. MethodsTumor cell‐induced platelet aggregation experiments were performed with platelets from healthy human donors and benign prostate (RWPE‐1) and prostate cancer cell lines positive (LNCaP) and negative for androgen receptor (DU145 and PC3). Immunoblot measured prostate cancer prothrombin. Modified Boyden chamber invasion assays and zymography were performed to assess the effects of platelets on prostate cancer cell invasion and matrix metalloproteinase (MMP) expression, respectively. ResultsAndrogen receptor‐positive prostate cancer cell lines failed to induce platelet aggregation. However, androgen receptor‐inhibited and ‐negative cell lines all induced platelet aggregation, which was abolished by dabigatran. Androgen receptor‐inhibited and ‐negative cell lines demonstrated greater expression of prothrombin than androgen receptor‐positive cells. Platelets enhanced invasion and MMP‐2 and ‐9 expression by androgen receptor‐inhibited and negative prostate cancer cells, but not that of the androgen receptor‐positive cells. ConclusionsAndrogen receptor loss within prostate cancer results in increased thrombogenicity due to upregulation of prothrombin expression. Reciprocally, platelets enhance invasion of androgen receptor‐negative prostate cancer cells via increased MMP expression.

Efficacy and safety of apalutamide in Japanese patients with nonmetastatic castration-resistant prostate cancer: a subgroup analysis of a randomized, double-blind, placebo-controlled, Phase-3 study

BackgroundIn the global Phase-3 Selective Prostate Androgen Receptor Targeting with ARN-509 study, apalutamide plus ongoing androgen deprivation therapy (ADT) significantly increased metastasis-free survival (MFS) and improved other clinical outcomes in men with nonmetastatic castration-resistant prostate cancer (nm-CRPC) who were at high risk of developing metastases. In this subpopulation analysis of Selective Prostate Androgen Receptor Targeting with ARN-509 study, the efficacy and safety of apalutamide plus ADT were evaluated in Japanese patients with nm-CRPC.MethodsThe primary efficacy end point was MFS. Secondary efficacy end points were time to metastasis, progression-free survival, symptomatic progression, initiation of cytotoxic chemotherapy, and overall survival. Safety and pharmacokinetic parameters were also assessed.ResultsFifty-five Japanese patients with ongoing ADT were randomized (apalutamide: n = 34, placebo: n = 21). Median treatment duration was 5.7 months in the apalutamide group and 11.0 months in the placebo group. Median MFS was not reached in the apalutamide group (95% confidence interval: 10.97, not estimable) and was 18.23 months (95% confidence interval: 11.04, 18.50) in the placebo group. Secondary end points were improved in the apalutamide group. The safety profile of apalutamide with ADT was comparable with the global population, and no new safety signals were identified in this Japanese subpopulation. Although, apalutamide exposure tended to be higher in the Japanese subpopulation compared with the non-Japanese population, this was likely to be explained by body weight and considered not clinically meaningful.ConclusionIn the Japanese subpopulation, treatment with apalutamide with ADT resulted in favorable efficacy outcomes with comparable benefit-risk profile to the global population with nm-CRPC who are at high-risk of developing metastases.

Neonatal exposure to aluminum chloride disrupts branching morphogenesis and hormonal signaling of the ventral male prostate and female prostate of gerbils

BackgroungExposure to environmental pollutants in critical developmental windows may predispose the prostate to permanent changes in its homeostasis. Thus, it is essential to know the effects that environmental toxics, such as aluminum, can cause during the development of this gland. The aim of this study was to evaluate the effects of neonatal aluminum exposure on the ventral male prostate and the female prostate of 15 days old gerbils. MethodsMale and female gerbils were exposed orally to 10 mg/kg/day of aluminum chloride from the 1st to the 14th postnatal day life. At 15 days of life, gerbils were euthanized and their prostates were collected for biometric, morphological, morphometric, immunohistochemical and three-dimensional reconstruction analyzes. ResultsAl exposure caused a reduction in body weight in males and a significant increase in serum testosterone levels in females. Prostate branching morphogenesis was intensified in males, who had greater length, number and area of prostatic epithelial buds. Additionally, Al altered the prostate hormonal regulation of males and females, causing up regulation of the androgen receptor and estrogen receptor alpha in the female prostate, and increased immunostaining of the androgen receptor in the ventral male prostate. These changes were associated with an increased rate of epithelial and stromal cell proliferation in both sexes. ConclusionTogether, these results indicate that Al altered the neonatal development of the prostate and that this metal acted as an endocrine disruptor in this gland.

Abstract 5080: Decursinol intercepts LNCaP human prostate cancer xenograft growth bypassing androgen receptor-prostate specific antigen axis

Abstract The slow-growing nature of most prostate cancers (PCa) provides multiple windows of opportunity to block or delay disease progression to reduce patient suffering and mortality. This is especially true following the failure of radical prostatectomy (RP) and radiation therapy (RT) of localized PCa with biochemical recurrence detectable by a rise in plasma/serum prostate specific antigen (PSA) and prior to androgen deprivation therapy (ADT). Costly aside, ADT is not curative and causes many adverse effects which negatively affect the quality of life of the patients. Currently there is no standard of care regimen for post-RP/RT patients with rising PSA to stump the trajectory. Many patients seek food or supplement-based interventions with a hope to delay ADT. The root of Korean Angelica gigas Nakai (AGN) contains decursin (D) and its isomer decursinol angelate (DA) as the major pyranocoumarins. We have previously shown that daily gavage of AGN and D/DA suppressed the growth of androgen receptor (AR)+ and AR- xenograft tumors and TRAMP neuroendocrine carcinomas. We also showed that D/DA rapidly converted to decursinol (DOH) in rodents and humans, and DOH inhibited LNCaP-AR xenograft growth, therefore the likely in vivo active compound. To assess merit of DOH to intercept recurrent PCa, we tested in NSG SCID mice bearing s.c. inoculated human LNCaP PCa xenograft (a) its efficacy to inhibit tumor growth and b) serum PSA as an efficacy metric. A day prior to start of daily DOH gavage (120 mg/kg body weight) for the tumor bearing mice, blood was drawn through tail vein for baseline PSA. After 4 weeks, the mice were bled for post-PSA and their tumors/prostate were dissected and weighed. Gavage delivered DOH did not affect body weight, but significantly reduced the tumor volume and weight without decreasing the weight of the prostate, distinct from ADT drugs. The post-PSA showed a significant and tight linear correlation with the tumor weight for mice in the vehicle group (r2=0.94, n=6). Five of 7 DOH-treated mice showed post-PSA/tumor weight plot on the same regression line but two non-responders displayed exaggerated post-PSA/weight ratio. The much slower PSA rise in the DOH-responders (2.9 fold, n=5) than vehicle group (7.8 fold, n=6, p=0.031) directly reflected a reduction of tumor burden by DOH treatment. Histological (H&E) analysis showed that DOH responder tumors had greater necrosis and less hemorrhage than non-responders and control tumors. Immunohistochemistry staining for AR and PSA did not detect any reduction of either protein in the responder tumors which contained reduced mitotic marker phospho-histone 3. Our preclinical modeling therefore suggests serum PSA as a reliable efficacy metric for DOH-mediated PCa interception in post-PR/RT patients, involving mitosis inhibition and necrosis without affecting the cancer AR/PSA axis. Citation Format: Sangyub Kim, Chongtao Qin, Deepkamal N. Karelia, Arati Sharma, Cheng Jiang, Junxuan Lu. Decursinol intercepts LNCaP human prostate cancer xenograft growth bypassing androgen receptor-prostate specific antigen axis [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 5080.

Membrane androgen receptors (OXER1, GPRC6A AND ZIP9) in prostate and breast cancer: A comparative study of their expression

Accumulating evidence during the last decades revealed that androgens exert membrane-initiated actions leading to the modulation of significant cellular processes, important for cancer cell growth and metastasis (including prostate and breast), that involve signaling via specific kinases. Collectively, many nonclassical, cell surface-initiated androgen actions are mediated by novel membrane androgen receptors (mARs), unrelated to nuclear androgen receptors. Recently, our group identified the G protein coupled oxo-eicosanoid receptor 1 (OXER1) (a receptor of the arachidonic acid metabolite, 5-oxoeicosatetraenoic acid, 5-oxoETE) as a novel mAR involved in the rapid effects of androgens. However, two other membrane proteins, G protein-coupled receptor family C group 6 member A (GPRC6A) and zinc transporter member 9 (ZIP9) have also been portrayed as mARs, related to the extranuclear action of androgens. In the present work, we present a comparative study of in silico pharmacology, gene expression and immunocytochemical data of the three receptors in various prostate and breast cancer cell lines. Furthermore, we analyzed the immunohistochemical expression of these receptors in human tumor and non-tumoral specimens and provide a pattern of expression and intracellular distribution.

Abstract A066: Rb and ZBTB7A cooperatively mediate the transcriptional repression function of androgen receptor

Abstract Prostate cancer (PCa) is one of the most common cancers in men. Primary PCa depends on androgen for growth and survival by binding to the C-terminal ligand-binding domain of androgen receptor (AR), a transcriptional factor that regulates multiple gene expression. The standard treatment of PCa is surgical or medical castration (known as androgen deprivation therapy, ADT) to reduce circulating androgens. However, patients invariably relapse into a more aggressive castration-resistant prostate cancer (CRPC) and AR remains important in this stage of disease. Although AR is well known for its transcriptional activator function, it can also suppress the expression of a subset of genes, including AR itself, multiple androgen synthetic genes, and a group of DNA replication genes. Mechanistically, we have shown that AR globally recruits hypophosphorylated retinoblastoma protein (Rb) to the promoters/enhancers of DNA replication gene loci and strengthens the activity of Rb-E2F suppressor complex. This novel activity of AR provides one molecular basis for treating CRPC patients with high level androgens. Importantly, we have also shown in vitro that this tumor-suppressor function of AR can be enhanced by CDK4/6 inhibitors, which block Rb phosphorylation. Although most CRPC are Rb-positive, recent sequencing studies indicate that ~10-15% of CRPC showed Rb loss in their chromosome. Therefore, we have generated tetracycline-inducible Rb-silencing CRPC cell lines to further examine the impact of Rb-loss on the transcriptional repression activity of AR. Consistently, we have found that Rb-silencing globally impaired the AR repression on DNA replication genes. Furthermore, we have also examined the AR-independent function of Rb and found that Rb-loss led to the activation of neuroendocrine genes. To further understand the mechanisms of how AR performs its transcriptional repression activity, we analyzed AR ChIP-seq data and androgen-regulated gene profile. This analysis resulted in the identification of ZBTB7A binding motif as the most enriched motif within AR repression sites. ZBTB7A, also known as LRF/POKEMON, is a zinc finger and BTB domain containing transcription repressor consisting of a protein-protein interacting BTB domain at N-terminus and DNA binding zinc fingers at C-terminus. Even though ZBTB7A has been identified as a proto-oncogene, a recent study using transgenic mouse models shows that it functions as a tumor suppressor in driving PCa progression with PTEN-null background. Therefore, we hypothesize that ZBTB7A might play a key role in regulating the Rb-dependent transcriptional repressor activity of AR. To globally determine this function of ZBTB7A, we carried out ZBTB7A ChIP-seq to identify high confident binding peaks. Motif analysis on these peaks indicated the marked enrichment of binding motif of ZBTB family members, suggesting that these binding sites are specific for ZBTB7A binding. Significantly, ~30% of these ZBTB7A binding sites overlapped with AR binding sites, particularly at promoter region. Using the unbiased Binding and Expression Target Analysis, we further found that these ZBTB7A and AR cobinding sites were significantly associated with the expression of AR-repressed genes. More importantly, the binding of ZBTB7A at these sites highly associated with Rb binding and silencing ZBTB7A impaired the AR repression activity on the DNA replication genes, suggesting that ZBTB7A may cooperate with Rb in regulating AR repressor activity. By coimmunoprecipitation assays, we found that ZBTB7A physically interacts with AR and Rb and these interactions are enhanced by androgen stimulation, suggesting that ZBTB7A is an additional component in the AR repressor complex. Furthermore, by additional ChIP-seq analyses we have also found that ZBTB7A binding at those AR repression sites was rapidly increased upon androgen stimulation, further indicating that AR may recruit ZBTB7A to the chromatin and ZBTB7A and Rb may cooperatively mediate the transcriptional activity of AR. This study provides novel insights in the molecular basis of transcriptional repression function of AR and may have significant therapeutic impact on enhancing current androgen therapy to treat CRPC patients by enhancing the expression or activity of ZBTB7A. Citation Format: Wanting Han, Dong Han, Sujun Chen, Shuai Gao, Housheng He, Changmeng Cai. Rb and ZBTB7A cooperatively mediate the transcriptional repression function of androgen receptor [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A066.

Myofibroblast androgen receptor expression determines cell survival in co-cultures of myofibroblasts and prostate cancer cells in vitro.

Fibroblasts express androgen receptor (AR) in the normal prostate and during prostate cancer development. We have reported that loss of AR expression in prostate cancer-associated fibroblasts is a poor prognostic indicator. Here we report outcomes of direct and indirect co-cultures of immortalised AR-positive (PShTert-AR) or AR-negative (PShTert) myofibroblasts with prostate cancer cells.In the initial co-cultures the AR-negative PC3 cell line was used so AR expression and signalling were restricted to the myofibroblasts. In both direct and indirect co-culture with PShTert-AR myofibroblasts, paracrine signalling to the PC3 cells slowed proliferation and induced apoptosis. In contrast, PC3 cells proliferated with PShTert myofibroblasts irrespective of the co-culture method. In direct co-culture PC3 cells induced apoptosis in and destroyed PShTerts by direct signalling. Similar results were seen in direct co-cultures with AR-negative DU145 and AR-positive LNCaP and C4-2B prostate cancer cell lines. The AR ligand 5α-dihydrotestosterone (DHT) inhibited the proliferation of the PShTert-AR myofibroblasts, thereby reducing the extent of their inhibitory effect on cancer cell growth.These results suggest loss of stromal AR would favour prostate cancer cell growth in vivo, providing an explanation for the clinical observation that reduced stromal AR is associated with a poorer outcome.

Combined oral contraceptives promote androgen receptor and oestrogen receptor alpha upregulation in the female prostate (Skene's paraurethral glands) of adult gerbils (Meriones unguiculatus).

The aim of this study was to evaluate the effects of cyproterone acetate (CPA) and ethinyloestradiol (EE) alone or in combination on the female prostate of adult gerbils. Adult females were exposed for 21 days to daily oral doses of CPA (1mgkg-1), EE (10µgkg-1) or a combination of CPA and EE. Female prostatic complexes were removed, weighed and subjected to morphological, stereological, immunohistochemical and ultrastructural analyses. CPA treatment caused epithelial atrophy and decreased prostate secretory activity. The EE treatment group showed glandular hyperplasia, a high cell-proliferation index and an increase in androgen and oestrogen receptor α (AR and ERα) immunoreactivity. Combined treatment (CPA+EE) caused adverse effects, such as an increase in cell proliferation, higher AR and ERα immunoreactivity, prostatic intraepithelial neoplasia, cell degeneration and aging. In conclusion, the CPA-only treatment promoted antiandrogenic effects on the female gerbil prostate, whereas EE-only had a potent oestrogenic activity. However, when combined, EE overlapped the effects of CPA, changing the pattern of glandular hormonal regulation and stimulating the development of prostatic lesions in female gerbils.

Serotonin regulates prostate growth through androgen receptor modulation

Aging and testosterone almost inexorably cause benign prostatic hyperplasia (BPH) in Human males. However, etiology of BPH is largely unknown. Serotonin (5-HT) is produced by neuroendocrine prostatic cells and presents in high concentration in normal prostatic transition zone, but its function in prostate physiology is unknown. Previous evidence demonstrated that neuroendocrine cells and 5-HT are decreased in BPH compared to normal prostate. Here, we show that 5-HT is a strong negative regulator of prostate growth. In vitro, 5-HT inhibits rat prostate branching through down-regulation of androgen receptor (AR). This 5-HT’s inhibitory mechanism is also present in human cells of normal prostate and BPH, namely in cell lines expressing AR when treated with testosterone. In both models, 5-HT’s inhibitory mechanism was replicated by specific agonists of 5-Htr1a and 5-Htr1b. Since peripheral 5-HT production is specifically regulated by tryptophan hydroxylase 1(Tph1), we showed that Tph1 knockout mice present higher prostate mass and up-regulation of AR when compared to wild-type, whereas 5-HT treatment restored the prostate weight and AR levels. As 5-HT is decreased in BPH, we present here evidence that links 5-HT depletion to BPH etiology through modulation of AR. Serotoninergic prostate pathway should be explored as a new therapeutic target for BPH.

A rare castration-resistant progenitor cell population is highly enriched in Pten-null prostate tumours.

Castration-resistant prostate cancer is a lethal disease. The cell type(s) that survive androgen deprivation remain poorly described, despite global efforts to understand the various mechanisms of therapy resistance. We recently identified in wild-type (WT) mouse prostates a rare population of luminal progenitor cells that we called LSCmed according to their FACS profile (Lin- /Sca-1+ /CD49fmed ). Here, we investigated the prevalence and castration resistance of LSCmed in various mouse models of prostate tumourigenesis (Pb-PRL, Ptenpc-/- , and Hi-Myc mice). LSCmed prevalence is low (∼8%, similar to WT) in Hi-Myc mice, where prostatic androgen receptor signalling is unaltered, but is significantly higher in the two other models, where androgen receptor signalling is decreased, rising up to more than 80% in Ptenpc-/- prostates. LSCmed tolerate androgen deprivation and persist or are enriched 2-3 weeks after castration. The tumour-initiating properties of LSCmed from Ptenpc-/- mice were demonstrated by regeneration of tumours in vivo. Transcriptomic analysis revealed that LSCmed represent a unique cell entity as their gene expression profile is different from luminal and basal/stem cells, but shares markers of each. Their intrinsic androgen signalling is markedly decreased, explaining why LSCmed tolerate androgen deprivation. This also illuminates why Ptenpc-/- tumours are castration-resistant since LSCmed represent the most prevalent cell type in this model. We validated CK4 as a specific marker for LSCmed on sorted cells and prostate tissues by immunostaining, allowing for the detection of LSCmed in various mouse prostate specimens. In castrated Ptenpc-/- prostates, there was significant proliferation of CK4+ cells, further demonstrating their key role in castration-resistant prostate cancer progression. Taken together, this study identifies LSCmed as a probable source of prostate cancer relapse after androgen deprivation and as a new therapeutic target for the prevention of castrate-resistant prostate cancer. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract 5795: Functional and mechanistic interrogation of BET Bromodomain degraders for the treatment of metastatic castration resistant prostate cancer

Abstract Metastatic castration-resistant prostate cancer (mCRPC), a lethal disease primarily driven by androgen receptor (AR) signaling, results in approximately 30,000 annual deaths in the U.S. While the AR is necessary for the function, survival, and differentiation of normal prostatic tissue, AR function and signaling shift from tumor suppressive to tumor promoting during prostate carcinogenesis. Surgical or chemical castration targeting the AR signaling axis has been the mainstay of prostate cancer (PCa) treatment. Unfortunately, despite castration, PCa will inevitably progress into castration resistant prostate cancer (CRPC) in which AR remains an important oncogene. Current CRPC therapeutics, such as second-generation AR antagonists, elicit only temporary responses and patients eventually succumb to the disease. Therefore, new therapeutics against the AR axis in CRPC are urgently needed. One such novel strategy for targeting the AR-pathway and inhibiting the growth of CRPC has been the use of bromodomain and extraterminal (BET) protein inhibitors such a JQ1, which disrupt AR and oncogenic c-MYC transcriptional activity. While BET inhibitors may seem to be attractive candidate drugs for clinical translation, their off-target effects, such as binding to other proteins, toxicity, and the rapid development of treatment resistance, limit their translation. However, a new class of molecules that target BET proteins through proteasomal degradation can improve efficacy and specificity over standard inhibitors. Based on our preliminary findings, we have hypothesized that pharmacologic BET degradation represents an important advance in CRPC treatment and may provide a novel therapeutic strategy that can enhance efficacy and disrupt resistance to AR-targeted therapy. Utilizing prostate cancer cell lines, organoids, and patient derived xenografts, we performed RNA-seq, ChIP-seq, and proteomic studies to assay the effects of BET inhibitors and degrader compounds in preclinical models. These inhibitors affected AR-positive prostate cancer cells preferentially over AR-negative cells, and proteomic and genomic mechanistic studies confirmed the disruption of oncogenic AR and MYC signaling both in vivo and in vitro. In conclusion, we are developing highly potent, small molecules that lead to the proteasomal-degradation of BET proteins. With optimized in vivo properties, BET degraders promise to be a novel potential therapeutic strategy for patients with mCRPC. Citation Format: Steven Kregel, Rohit Malik, Irfan A. Asangani, Ester Fernandez-Salas, Kari Wilder-Romans, Xia Jiang, Thekkelnaycke Rajendiran, Xuhong Cao, Corey Speers, Shaomeng Wang, Arul M. Chinnaiyan. Functional and mechanistic interrogation of BET Bromodomain degraders for the treatment of metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5795.