Androgen-induced Proliferation Research Articles

Artepillin C overcomes apalutamide resistance through blocking androgen signaling in prostate cancer cells

Prostate cancer has a relatively good prognosis, but most cases develop resistance to hormone therapy, leading to castration-resistant prostate cancer (CRPC). Androgen receptor (AR) antagonists and a cytochrome P450 17A1 inhibitor have been used to treat CRPC, but cancer cells readily develop resistance to these drugs. In this study, to improve the therapy of CRPC, we searched for natural compounds which block androgen signaling. Among cinnamic acid derivatives contained in Brazilian green propolis, artepillin C (ArtC) suppressed expressions of androgen-induced prostate-specific antigen and transmembrane protease serine 2 in a dose-dependent manner. Reporter assays revealed that ArtC displayed AR antagonist activity, albeit weaker than an AR antagonist flutamide. In general, aberrant activation of the androgen signaling is involved in the resistance of prostate cancer cells to hormone therapy. Recently, apalutamide, a novel AR antagonist, has been in clinical use, but its drug-resistant cases have been already reported. To search for compounds which overcome the resistance to apalutamide, we established apalutamide-resistant prostate cancer 22Rv1 cells (22Rv1/APA). The 22Rv1/APA cells showed higher AR expression and androgen sensitivity than parental 22Rv1 cells. ArtC inhibited androgen-induced proliferation of 22Rv1/APA cells by suppressing the enhanced androgen signaling through blocking the nuclear translocation of AR. In addition, ArtC potently sensitized the resistant cells to apalutamide by inducing apoptotic cell death due to mitochondrial dysfunction. These results suggest that the intake of Brazilian green propolis containing ArtC improves prostate cancer therapy.

The DNA/RNA helicase DHX9 contributes to the transcriptional program of the androgen receptor in prostate cancer

BackgroundProstate cancer (PC) is the most commonly diagnosed male malignancy and an important cause of mortality. Androgen deprivation therapy is the first line treatment but, unfortunately, a large part of patients evolves to a castration-resistant stage, for which no effective cure is currently available. The DNA/RNA helicase DHX9 is emerging as an important regulator of cellular processes that are often deregulated in cancer.MethodsTo investigate whether DHX9 modulates PC cell transcriptome we performed RNA-sequencing analyses upon DHX9 silencing in the androgen-responsive cell line LNCaP. Bioinformatics and functional analyses were carried out to elucidate the mechanism of gene expression regulation by DHX9. Data from The Cancer Genome Atlas were mined to evaluate the potential role of DHX9 in PC.ResultsWe found that up-regulation of DHX9 correlates with advanced stage and is associated with poor prognosis of PC patients. High-throughput RNA-sequencing analysis revealed that depletion of DHX9 in androgen-sensitive LNCaP cells affects expression of hundreds of genes, which significantly overlap with known targets of the Androgen Receptor (AR). Notably, AR binds to the DHX9 promoter and induces its expression, while Enzalutamide-mediated inhibition of AR activity represses DHX9 expression. Moreover, DHX9 interacts with AR in LNCaP cells and its depletion significantly reduced the recruitment of AR to the promoter region of target genes and the ability of AR to promote their expression in response to 5α-dihydrotestosterone. Consistently, silencing of DXH9 negatively affected androgen-induced PC cell proliferation and migration.ConclusionsCollectively, our data uncover a new role of DHX9 in the control of the AR transcriptional program and establish the existence of an oncogenic DHX9/AR axis, which may represent a new druggable target to counteract PC progression.

FOXO3a-ROS pathway is involved in androgen-induced proliferation of prostate cancer cell

BackgroundAlthough FOXO3a can inhibit the cell proliferation of prostate cancer, its relationship with reactive oxygen species (ROS) in prostate cancer (PCa) has not been reported.MethodsWe analyzed the correlation between the expression of FOXO3a and the antioxidant enzyme catalase in prostate cancer with the TCGA and GEPIA databases. We also constructed a PPI network of FOXO3a via the STRING database. The mRNA and protein expression of FOXO3a and catalase were detected by qRT-PCR or western blotting in LNCaP and 22RV1 cells treated with DHT, R1881, or Enzalutamide. The effects of FOXO3a on catalase expression were tested by over-expressing or knocking down FOXO3a in LNCaP cells. Furthermore, the catalase activity and ROS level were detected in LNCaP cells treated with DHT. Cell proliferation and ROS were also analyzed in LNCaP which was treated with antioxidant.ResultsResults showed that the catalase expression was down-regulated in prostate cancer. A positive correlation between FOXO3a and catalase existed. DHT treatment could significantly reduce FOXO3a and catalase expression at mRNA and protein level in LNCaP cells. Catalase expression partly depended on FOXO3a as over-expression and knockdown of FOXO3a could result in the expresssion change of catalase. DHT treatment was found to inhibit catalase activity and increase ROS level in prostate cancer cell. Our study also demonstrated that antioxidant treatment reduced DHT-induced proliferation and ROS production in prostate cancer cell.ConclusionsWe discovered a novel mechanism by which DHT promotes prostate cancer cell proliferation via suppressing catalase activity and activating ROS signaling via a FOXO3a dependent manner.

Abstract 1235: Selective androgen receptor degraders for the treatment of androgen receptor-positive, triple-negative breast cancer

Abstract Introduction: Triple-negative breast cancer (TNBC) is an aggressive breast cancer with shorter overall survival compared to other breast cancer types. One of the six molecularly-classified TNBC subtypes is the luminal androgen receptor subtype (LAR), which overexpresses androgen receptor (AR) and is dependent on AR for its growth. About 10-20% of TNBCs belong to the LAR subtype. Competitive AR antagonists, enzalutamide and bicalutamide, were effective in preclinical models of LAR TNBC and in clinical trials. This led us to hypothesize that potent selective AR degraders (SARDs), due to their ability to inhibit and degrade the AR, could provide a novel therapeutic approach for the treatment of LAR subtype of TNBC. Description: Western blots, cell line proliferation assays, and gene expression analyses were performed to evaluate novel small molecule SARDs. LAR TNBC cell lines and patient-derived xenografts (PDX) were utilized for in vivo evaluation of the SARDs. Once tumors grew to ~100-300 mm3, mice were randomized and treated orally for four weeks with vehicle, SARD UT-34, SARD UT-105, enzalutamide, or bicalutamide. Tumor volumes were measured twice weekly and tumors were collected at sacrifice for further analyses. Summary: SARDs bind to the N-terminus of the AR and have been characterized in preclinical advanced prostate cancer models. In this study, the SARDs were evaluated in preclinical models of LAR TNBC. Western blot for AR in LAR MDA-MB-453 cells demonstrated degradation of the AR protein by SARDs at low micromolar concentrations. Gene expression studies showed a complete inhibition of androgen-induced AR target gene transcription by the SARDs. Androgen-induced proliferation of MDA-MB-453 cells was inhibited by SARDs. MDA-MB-453 cells implanted subcutaneously in NOD SCID Gamma female mice grew robustly to 100-300 mm3 in 15-20 days. Treatment of tumor-bearing animals with the SARDs completely inhibited or regressed the tumors. Conclusion: These results support the findings that AR is the driver of MDA-MB-453 cell and tumor growth. SARDs with their unique mechanism of action may provide a new therapeutic option to women affected by the LAR subtype of TNBC. Disclosure: This work was partially supported by Oncternal Therapeutics and by an NCI supplement award to R01 (CA229164S1 to author RN). The SARD program has been licensed to Oncternal Therapeutics, Inc. by the University of Tennessee Research Foundation. Author RN is a consultant to Oncternal Therapeutics. Citation Format: Sarah Asemota, Kirsten Young Young, Suriyan Ponnusamy, Thirumagal Thiyagarajan, Dong-Jin Hwang, Yali He, James B. Breitmeyer, Gunnar F. Kaufmann, Duane D. Miller, Ramesh Narayanan. Selective androgen receptor degraders for the treatment of androgen receptor-positive, triple-negative breast cancer [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 1235.

Androgen-induced expression of DRP1 regulates mitochondrial metabolic reprogramming in prostate cancer.

Androgen receptor (AR) signaling plays a central role in metabolic reprogramming for prostate cancer (PCa) growth and progression. Mitochondria are metabolic powerhouses of the cell and support several hallmarks of cancer. However, the molecular links between AR signaling and the mitochondria that support the metabolic demands of PCa cells are poorly understood. Here, we demonstrate increased levels of dynamin-related protein 1 (DRP1), a mitochondrial fission mediator, in androgen-sensitive and castration-resistant AR-driven PCa. AR signaling upregulates DRP1 to form the VDAC-MPC2 complex, increases pyruvate transport into mitochondria, and supports mitochondrial metabolism, including oxidative phosphorylation and lipogenesis. DRP1 inhibition activates the cellular metabolic stress response, which involves AMPK phosphorylation, induction of autophagy, and the ER unfolded protein response, and attenuates androgen-induced proliferation. Additionally, DRP1 expression facilitates PCa cell survival under diverse metabolic stress conditions, including hypoxia and oxidative stress. Moreover, we found that increased DRP1 expression was indicative of poor prognosis in patients with castration-resistant PCa. Collectively, our findings link androgen signaling-mediated mitochondrial dynamics to metabolic reprogramming; moreover, they have important implications for understanding PCa progression.

Pituitary tumor transforming gene binding factor (PBF) is required for androgen-induced prostate cancer proliferation and invasion.

Pituitary tumor transforming gene binding factor (PBF) is a proto-oncogene that plays a role in many cancers; however, its involvement in prostate cancer (PCa) remains unclear. Here, we examined PBF expression in clinical specimens and investigated its regulation and function in human PCa cell lines. Immunohistochemical staining of patient tissues revealed higher PBF expression in PCa than in benign prostatic hyperplasia or adjacent normal prostate specimens. In LNCaP and 22Rv1 cells, PBF expression was upregulated by androgen treatment in a manner partially blocked by the androgen receptor (AR) antagonist bicalutamide. We identified a novel androgen response element in the PBF gene promoter and demonstrated its functional relevance using luciferase reporter assays. Androgen treatment of LNCaP cells induced binding between the endogenous AR and the androgen response element in PBF, as measured by chromatin immunoprecipitation assays. Finally, RNA interference of PBF expression significantly reduced androgen-induced LNCaP cell growth and invasion. Thus, PBF is a novel AR target gene and plays a role in androgen-induced proliferation and metastatic functions in PCa cells.

Functional Studies on Steroid Receptors.

Nuclear receptors play an important role in prostate cancer and the androgen receptor is a key transcription factor in regulation of cellular events. Androgen receptor-associated coregulators may be upregulated or downregulated in prostate cancer. Altered expression of regulators may potentiate androgen-induced proliferation, migration, and invasion. Therapies aimed to modulate the function of coregulators in prostate cancer may be based on the use of small molecule inhibitors. Expression and function of AR-associated proteins could be investigated after overexpression and gene silencing followed by hormonal treatment, real-time RT-PCR and ChIP.

RET-mediated glial cell line-derived neurotrophic factor signaling inhibits mouse prostate development.

In humans and rodents, the prostate gland develops from the embryonic urogenital sinus (UGS). The androgen receptor (AR) is thought to control the expression of morphogenetic genes in inductive UGS mesenchyme, which promotes proliferation and cytodifferentiation of the prostatic epithelium. However, the nature of the AR-regulated morphogenetic genes and the mechanisms whereby AR controls prostate development are not understood. Glial cell line-derived neurotrophic factor (GDNF) binds GDNF family receptor α1 (GFRα1) and signals through activation of RET tyrosine kinase. Gene disruption studies in mice have revealed essential roles for GDNF signaling in development; however, its role in prostate development is unexplored. Here, we establish novel roles of GDNF signaling in mouse prostate development. Using an organ culture system for prostate development and Ret mutant mice, we demonstrate that RET-mediated GDNF signaling in UGS increases proliferation of mesenchyme cells and suppresses androgen-induced proliferation and differentiation of prostate epithelial cells, inhibiting prostate development. We also identify Ar as a GDNF-repressed gene and Gdnf and Gfrα1 as androgen-repressed genes in UGS, thus establishing reciprocal regulatory crosstalk between AR and GDNF signaling in prostate development.

ODM-204: A novel dual inhibitor of CYP17A1 and androgen receptor for the treatment of castration-resistant prostate cancer—Preclinical data.

221 Background: Castration-resistant prostate cancer (CRPC) is characterized by high androgen receptor (AR) expression and persistent activation of AR signaling axis by residual tissue/tumor androgens. Targeting AR and androgen biosynthesis together may be more effective than either alone. ODM-204 is a novel, non-steroidal dual inhibitor of CYP17A1 and AR, which has shown promising results in preclinical studies. Methods: The binding affinity of ODM-204 to wild type AR was determined in rat prostate cytosolic lysates. The potency and functional activity of ODM-204 to human AR were demonstrated in cells stably transfected with the full-length AR and androgen-responsive reporter gene constructs. In addition, assays for AR nuclear translocation and the transactivation of human AR mutants T877A, W741L, and F876L were conducted. The effects of ODM-204 on the growth of androgen-dependent VCaP and LNCaP cells in vitro and subcutaneously grafted VCaP cells in vivo with the oral dose of 50 mg/kg/day were studied. The inhibition of CYP17A1 by ODM-204 was studied in vitro by using human and rat testicular microsomes and a human adrenal cortex cell line, and in vivo in male rats coadministered with luteinizing hormone releasing hormone agonist leuprolide acetate to mimic clinical situation. Results: ODM-204 is a potent inhibitor of both AR and CYP17A1. It binds to AR with a high affinity (Ki=47 nM) and selectivity and has a high potency towards CYP17A1 (IC50=22 nM). In addition, ODM-204 inhibited testosterone-mediated nuclear translocation of AR and the mutant ARs (IC50 values for AR(T877A), AR(W741L), and AR(F876L) were 95, 277, and 6 nM, respectively), and suppressed androgen-induced cell proliferation of LNCaP (IC50=170 nM) and VCaP (IC50=280 nM) cells. In a VCaP xenograft model, ODM-204 showed significant antitumor activity (tumor growth inhibition=66%). In rats, inhibitory effects of leuprolide acetate on testosterone production and androgen-sensitive organ weights were potentiated by ODM-204. Conclusions: ODM-204 is a promising new dual CYP17A1 and AR inhibitor for the treatment of CRPC. Clinical trials in patients with mCRPC will be started in early 2015.

Identification of a Potent Antiandrogen that Targets the BF3 Site of the Androgen Receptor and Inhibits Enzalutamide-Resistant Prostate Cancer

There has been a resurgence of interest in the development of androgen receptor (AR) inhibitors with alternative modes of action to overcome the development of resistance to current therapies. We demonstrated previously that one promising strategy for combatting mutation-driven drug resistance is to target the Binding Function 3 (BF3) pocket of the receptor. Here we report the development of a potent BF3 inhibitor, 3-(2,3-dihydro-1H-indol-2-yl)-1H-indole, which demonstrates excellent antiandrogen potency and anti-PSA activity and abrogates the androgen-induced proliferation of androgen-sensitive (LNCaP) and enzalutamide-resistant (MR49F) PCa cell lines. Moreover, this compound effectively reduces the expression of AR-dependent genes in PCa cells and effectively inhibits tumor growth in vivo in both LNCaP and MR49F xenograft models. These findings provide evidence that targeting the AR BF3 pocket represents a viable therapeutic approach to treat patients with advanced and/or resistant prostate cancer.

Peptidomimetic targeting of critical androgen receptor–coregulator interactions in prostate cancer

The growth of advanced prostate cancer depends on androgen receptor signalling, however treatment options are limited. Here we report the disruption of specific protein-protein interactions involving LXXLL motifs in androgen receptor-coregulator proteins such as PELP1 using a novel, small molecule peptidomimetic (D2). D2 is stable, non-toxic and efficiently taken up by prostate cancer cells. Importantly, D2 blocks androgen-induced nuclear uptake and genomic activity of the androgen receptor. Furthermore, D2 abrogates androgen-induced proliferation of prostate cancer cells in vitro with an IC50 of 40 nM, and inhibits tumour growth in a mouse xenograft model. D2 also disrupts androgen receptor-coregulator interactions in ex vivo cultures of primary human prostate tumours. These findings provide evidence that targeting androgen receptor-coregulator interactions using peptidomimetics may be a viable therapeutic approach for patients with advanced prostate cancer.

Overexpression of APRIN inhibits differentiation and proliferation and promotes apoptosis in P19 embryonal carcinoma cells

We have previously demonstrated that androgen-induced proliferation inhibitor (APRIN) expression was upregulated in ventricular septum tissues from patients with ventricular septal defect (VSD). The present study was designed to investigate the effects of APRIN on P19 cell differentiation, proliferation and apoptosis. In this study, we established a stable APRIN-overexpressing P19 embryonal carcinoma cell line that can differentiate into myocardial cells when treated with 1 % dimethyl sulfoxide. Our data showed that mRNA expressions of myocardial cell differentiation-related genes (such as cTnT, α-MHC, GATA4, and MEF2C) in the APRIN-overexpressing P19 cells were downregulated compared to the empty-vector controls. Our findings also indicated that P19 cells overexpressing APRIN had a reduced growth rate and a decreased S phase of the cell cycle. Moreover, the apoptotic rate in P19 cells overexpressing APRIN was significantly higher than that in the controls. In conclusion, our study demonstrates that overexpression of APRIN inhibits differentiation and proliferation and promotes apoptosis in P19 cells, suggesting that APRIN may be involved in the pathogenesis of VSD.

Abstract 351: 2-Methoxyestradiol targets RON kinase and FLIP to inhibit epithelial-mesenchymal transition in prostate cancer

Abstract Prostate Cancer (PCA) is the second leading cause of cancer-related deaths among men in the United States. Despite significant progress in our understanding of prostate biology, early detection and treatment, the five-year survival for hormone refractory metastatic prostate cancer (HRPCA) patients is less than 30%. Current therapies targeting PCA are not effective against the metastatic stage. 2-Methoxyestradiol (2-ME2) is a metabolic product of 17-β-estradiol and is found in trace amounts in the body; it has shown anti-tumorigenic properties in multiple cancer models. Previous studies from our lab have shown that 2-ME2 can induce apoptosis in PCA cells by blocking cell cycle progression, and also inhibits PCA growth and progression in TRAMP mice. However, the precise mechanism of action of 2-ME2 has not yet been elucidated. In order to classify the mechanism behind PCA inhibition by 2-ME2, we tested its effectiveness on an array containing genes associated with epithelial-mesenchymal transition (EMT). We identified Recepteur d'Origine Nantais (RON) as one of the most significantly downregulated genes in response to 2-ME2 treatment. RON has been shown to promote cell invasiveness, migration, survivability, and proliferation in many cancers such as breast, colon, lung, and pancreatic. However, very little is known about RON's signaling mechanism in PCA. We've found RON to be highly increased in advanced stage PCA cell lines. RON knockdown shows a concomitant decrease in the expression of FLICE-like inhibitory protein (FLIP). FLIP is an anti-apoptotic protein which prevents death receptor-induced apoptotic signaling. FLIP has been found to promote EMT as well as androgen-independent PCA growth. Stimulation with the RON ligand HGF yielded elevated expression of RON and FLIP in PCA cell lines while treatment with 2-ME2 caused downregulation. Immunoprecipitation experiments showed RON to be associated with the androgen-induced proliferation inhibitor (APRIN) in PCA cells. Our data suggest that RON/FLIP crosstalk plays a role in prostate cancer. Therefore, targeting RON/FLIP signaling could have potential therapeutic benefit for metastatic PCA. Supported by NIH CA 135451 (APK). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 351. doi:1538-7445.AM2012-351

Niphatenones, Glycerol Ethers from the Sponge Niphates digitalis Block Androgen Receptor Transcriptional Activity in Prostate Cancer Cells: Structure Elucidation, Synthesis, and Biological Activity

Extracts of the marine sponge Niphates digitalis collected in Dominica showed strong activity in a cell-based assay designed to detect antagonists of the androgen receptor (AR) that could act as lead compounds for the development of a new class of drugs to treat castration recurrent prostate cancer (CRPC). Assay-guided fractionation showed that niphatenones A (3) and B (4), two new glycerol ether lipids, were the active components of the extracts. The structures of 3 and 4 were elucidated by analysis of NMR and MS data and confimed via total synthesis. Biological evaluation of synthetic analogues of the niphatenones has shown that the enantiomers 7 and 8 are more potent than the natural products in the screening assay and defined preliminary SAR for the new AR antagonist pharmacophore, including the finding that the Michael acceptor enone functionality is not required for activity. Niphatenone B (4) and its enantiomer 8 blocked androgen-induced proliferation of LNCaP prostate cancer cells but had no effect on the proliferation of PC3 prostate cancer cells that do not express functional AR, consistent with activity as AR antagonists. Use of the propargyl ether 44 and Click chemistry showed that niphatenone B binds covalently to the activation function-1 (AF1) region of the AR N-terminus domain (NTD).

Characterization of the Phosphoproteome in Androgen-Repressed Human Prostate Cancer Cells by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Androgen-repressed human prostate cancer, ARCaP, grows and is highly metastatic to bone and soft tissues in castrated mice. The molecular mechanisms underlying the aberrant responses to androgen are not fully understood. Here, we apply state-of-the-art mass spectrometry methods to investigate the phosphoproteome profiles in ARCaP cells. Because protein biological phosphorylation is always substoichiometric and the ionization efficiency of phosphopeptides is low, selective enrichment of phosphorylated proteins/peptides is required for mass spectrometric analysis of phosphorylation from complex biological samples. Therefore, we compare the sensitivity, efficiency, and specificity for three established enrichment strategies: calcium phosphate precipitation (CPP), immobilized metal ion affinity chromatography (IMAC), and TiO(2)-modified metal oxide chromatography. Calcium phosphate precipitation coupled with the TiO(2) approach offers the best strategy to characterize phosphorylation in ARCaP cells. We analyzed phosphopeptides from ARCaP cells by LC-MS/MS with a hybrid LTQ/FT-ICR mass spectrometer. After database search and stringent filtering, we identified 385 phosphoproteins with an average peptide mass error of 0.32 ± 0.6 ppm. Key identified oncogenic pathways include the mammalian target of rapamycin (mTOR) pathway and the E2F signaling pathway. Androgen-induced proliferation inhibitor (APRIN) was detected in its phosphorylated form, implicating a molecular mechanism underlying the ARCaP phenotype.

Abstract 3977: Systematic analysis of microRNAs targeting the androgen receptor in prostate cancer cells

Abstract Androgen receptor (AR) is expressed in all stages of prostate cancer progression and its levels increase in the castration-resistant tumors. However, the mechanisms regulating AR expression remain poorly understood. Here, our aim was to determine the role of miRNAs in regulating AR protein. To this end, we performed a systematic gain-of function screen for 1129 miRNA molecules in prostate cancer cell lines and quantified changes in AR protein content using protein lysate microarrays. We identified 71 unique miRNAs that influenced the level of AR in LNCaP, LAPC-4, 22Rv1, CWR-1R and MDA-PCa-2B cells. Our RNA-sequencing data showed that the 3’UTRs of many genes, including the AR, are much longer than currently used by target prediction programs. Our analyses predicted that the majority of the miRNA regulation of AR would occur through the extended 6 kb 3’UTR. 3’UTR-binding assays validated 13 miRNAs (miR-135b, miR-185, miR-297, miR-299-3p, miR-34a, miR-34c, miR-371-3p, miR-421, miR-449a, miR-449b, miR-634, miR-654-5p, and miR-9) as direct AR regulators. Fifteen AR down-regulating miRNAs decreased androgen-induced proliferation of prostate cancer cells in vitro. Negative correlation of miR-34a and miR-34c expression with AR levels was also found in clinical prostate cancers. To conclude, our results demonstrate that miRNAs interacting with the long 3’UTR of the AR gene are important regulators of AR protein levels. This could be applied for developing novel therapeutic strategies to inhibit AR function and androgen-dependent cell growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3977. doi:10.1158/1538-7445.AM2011-3977

Systematic Analysis of MicroRNAs Targeting the Androgen Receptor in Prostate Cancer Cells

Androgen receptor (AR) is expressed in all stages of prostate cancer progression, including in castration-resistant tumors. Eliminating AR function continues to represent a focus of therapeutic investigation, but AR regulatory mechanisms remain poorly understood. To systematically characterize mechanisms involving microRNAs (miRNAs), we conducted a gain-of function screen of 1129 miRNA molecules in a panel of human prostate cancer cell lines and quantified changes in AR protein content using protein lysate microarrays. In this way, we defined 71 unique miRNAs that influenced the level of AR in human prostate cancer cells. RNA sequencing data revealed that the 3'UTR of AR (and other genes) is much longer than currently used in miRNA target prediction programs. Our own analyses predicted that most of the miRNA regulation of AR would target an extended 6 kb 3'UTR. 3'UTR-binding assays validated 13 miRNAs that are able to regulate this long AR 3'UTR (miR-135b, miR-185, miR-297, miR-299-3p, miR-34a, miR-34c, miR-371-3p, miR-421, miR-449a, miR-449b, miR-634, miR-654-5p, and miR-9). Fifteen AR downregulating miRNAs decreased androgen-induced proliferation of prostate cancer cells. In particular, analysis of clinical prostate cancers confirmed a negative correlation of miR-34a and miR-34c expression with AR levels. Our findings establish that miRNAs interacting with the long 3'UTR of the AR gene are important regulators of AR protein levels, with implications for developing new therapeutic strategies to inhibit AR function and androgen-dependent cell growth.

In Silico Analysis To Explore Novel Inhibitors For Human Proto-oncogene Tyrosine Protein Kinase Src

AbstractThe first oncogene and the first non receptor tyrosine kinase, Src, plays a key role in cell morphology, motility, proliferation and survival. Over expression of Src kinase activity disrupts the RAS pathway in signaling pathway, where it loses its ability to hydrolyse GTP and thus, leads to cancer. A wide range of evidences indicated that Src-signaling was important in the oncogenesis of prostate cancer and other tumours. Src-signaling is involved in androgen-induced proliferation of prostate cancer in cancer tissue of patients having castration-refractory prostate cancer. Once prostate cancer becomes castration-resistant, bone metastases become significant problem for which treatment options are limited. As Src is involved in multiple signaling pathways, central to prostate cancer development, progression, and metastasis, in addition to normal and pathologic osteoclast activities, Src inhibition becomes a valid therapeutic strategy for investigation. Existing Src kinase inhibitors are less efficient towards prostate cancer and bone metastasis. Hence an in silico work was carried out to identify novel potent inhibitors. Three published inhibitors of human Src kinase those are currently under clinical trial such as AZDO530, bosutinib and dasatinib were subjected to high-throughput screening from more than million entries of Ligand.Info Meta-Database, based on the assumption that small molecules with similar structure have similar pharmacological properties. The ligand dataset of 1152 generated through this approach were prepared using LigPrep to generate possible conformations of each ligand molecule, and at the same time duplicate conformers, conformations with reactive functional group and ADME violaters were rejected. The human Src kinase co-crystal structure with AZDO530 was analyzed to find the inhibitor binding site. The crystal structure was optimized and energy was minimized applying OPLS force field in Maestro v9.0. Glide 5.5 docking was performed to predict the binding orientation of prepared ligand molecule into a grid of 20 x 20 x 20 Å created around the centroid of optimized human Src kinase. Ten lead molecules with good binding affinity with human Src were identified. In silico pharmacokinetics study for these ten lead molecules had shown no ADME violation. Analysis of Lead ‘1’ - human Src docking complex had revealed a XP Gscore of -11.56 kcal/mol with highly stabilized hydrogen bond network with Ala390, Asn391, Lys295, W543 and W640 and good Van der Waals interactions. The docking complex coincides well with the native co-crystallized human Sac and inhibitor AZDO530 complex. Thus, Fisetin, identified as Lead ‘1’ in the present study would be highly useful for developing potential drug molecules for treatment of advanced prostate cancer.

Regression of Castrate-Recurrent Prostate Cancer by a Small-Molecule Inhibitor of the Amino-Terminus Domain of the Androgen Receptor

Castration-recurrent prostate cancer (CRPC) is suspected to depend on androgen receptor (AR). The AF-1 region in the amino-terminal domain (NTD) of AR contains most, if not all, of the transcriptional activity. Here we identify EPI-001, a small molecule that blocked transactivation of the NTD and was specific for inhibition of AR without attenuating transcriptional activities of related steroid receptors. EPI-001 interacted with the AF-1 region, inhibited protein-protein interactions with AR, and reduced AR interaction with androgen-response elements on target genes. Importantly, EPI-001 blocked androgen-induced proliferation and caused cytoreduction of CRPC in xenografts dependent on AR for growth and survival without causing toxicity.

Abstract 2083: microRNA-616 induces androgen-independent growth of prostate cancer cells through suppression of TFPI-2 expression

Abstract Prostate cancer is the most frequently diagnosed malignant tumor and the second leading cause of cancer deaths in North America. One of the most troubling aspects of prostate cancer is that, after androgen deprivation therapy, androgen-dependent (AD) prostate cancer inescapably progresses to an androgen-independent (AI) state, for which no effective treatment has been developed. Although several molecular pathways have been suggested to explain the pathogenesis of this disease, to date the mechanisms for the development and progression of prostate cancer remain largely unknown. A better understanding of the molecular events by which AD prostate cancer cells acquire the ability to resist androgen ablation may aid in the development of more effective therapies against this disease. microRNAs (miRNAs) are a class of small, non-coding RNAs that regulate gene expression through the specific targeting of the 3’-untranslated regions (UTRs) of their target mRNAs, thereby inducing degradation of the mRNA itself or impairing its translation. The aim of this project is to examine the differential miRNA profiles in AD and AI prostate cancer. AD LNCaP-FGC and AI LNCaP-LNO, two sublines derived from the same parental prostate cancer cell line LNCaP but bearing different androgen-responsiveness, were analyzed for expression of 419 selected miRNAs. A significant overexpression of miR-616 was identified in AI LNCaP-LNO as well as in malignant prostate tissues than opposed to AD LNCaP-FGC and benign prostate tissues, respectively. Consistently, expression studies of miR-616 in cell lines and xeongrafts representing AD and AI prostate cancer stages, revealed that miR-616 is overexpressed in the more aggressive AI cells (PC3, DU145, C4-2B) when compared with the less aggressive androgen-responsive (AR) cells (22rv1), AD cells (LNCaP, LAPC-4, LAPC-9) or the immortalized, normal prostate cells (HPr-1, NPTx). Stable miR-616 overexpression and knockdown by lentiviral-based approach stimulated and diminished androgen-induced prostate cancer cell proliferation in vitro, respectively. More importantly, inhibition of miR-616 repressed prostate tumor growth in vivo and was sufficient to impart castration resistance as evident by its reduced ability to grow in an in vivo animal model where castration was performed. Subsequent integrated mRNA expression profiling findings (LNCaP-FGC with stable miR-616 overexpression or empty vector) with in silico predictions identified tumor suppressor tissue factor pathway inhibitor-2 (TFPI-2) as a candidate downstream mRNA target of miR-616. Taken together, our results suggest that miR-616 acts as an oncogene, contributing to the pathogenesis of androgen-independent prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2083.