Androgen Receptor-negative Prostate Cancer Cells Research Articles

Dexamethasone inhibits androgen receptor-negative prostate cancer cell proliferation via the GR-FOXO3a-GAS5 axis

BackgroundStudies have shown that glucocorticoid receptor (GR) has inconsistent effects on the proliferation of prostate cancer cells, we found dexamethasone inhibited the proliferation of androgen receptor-negative prostate cancer cells, but the underlying mechanisms remain to be illustrated. MethodsGR expression and its prognosis role were analyzed based on the TCGA dataset. Bioinformatic analysis was performed to identify the candidate of GR downstream, which includes FOXO3a. After overexpressing FOXO3a in PC-3 cells, cell counting kit-8 (CCK-8) and migration assays were performed to evaluate cell proliferation and migration ability. Regulation of FOXO3a on GAS5 was also analyzed by JASPAR and PCR. ResultsGR had low expression in prostate cancer and predicted poor prognosis. FOXO3a was identified as the downstream of GR to inhibit the proliferation of prostate cancer cells. Moreover, FOXO3a directly induces GAS5 expression, forming the GR-FOXO3a-GAS5 signaling pathway. ConclusionOur study showed that GR played a role as a tumor suppressor gene in androgen receptor-negative prostate cancer cells via the GR-FOXO3a-GAS5 axis. Our results suggested patients with prostate cancer should be classified and develop a treatment plan according to the expression of AR and GR.

Anlotinib inhibits the proliferation and induces apoptosis by inactivating the AKT pathway in androgen receptor-negative prostate cancer cells.

Prostate cancer is one of the most frequently diagnosed cancers in men. The medical treatment of metastatic prostate cancer relies heavily on androgen deprivation. The present study aimed to explore the inhibitory effect of anlotinib on androgen receptor (AR)-negative prostate cancer cell lines in vitro and investigate its mechanism of action. Two prostate cancer cell lines, DU145 and PC-3, were treated with different concentrations (0-80 μM) of anlotinib. Cell proliferation was accessed by CCK-8 assay and EdU staining. Cell nuclear morphology was observed after DAPI staining, cell apoptosis level was evaluated by Annexin-V-FITC/PI staining, and western blot was used to detect the proliferation- and apoptosis-related proteins. The potential interaction between anlotinib and AKT was revealed by molecular docking. After treatment with anlotinib, the cell proliferation rate was significantly inhibited in a dose-dependent manner. The DAPI staining showed that anlotinib could induce apoptosis. Further, Annexin V/PI double staining confirmed the occurrence of apoptosis, accompanied by the increase of cleaved caspase-3 and activated PARP. Moreover, anlotinib significantly decreased the phosphorylation of protein kinase B (AKT) and its downstream pathway proteins in prostate cells (p<0.05). Experiments further confirmed that the activation of the AKT pathway reversed the inhibitory effect of anlotinib on DU145 and PC-3 cell proliferation. In addition, molecular docking analysis revealed potential interactions between anlotinib and AKT1 at multiple sites. Overall, the present study suggested that anlotinib could inhibit the proliferation and induce apoptosis in the AR-negative prostate cancer cell lines, possibly via the inactivation of the AKT pathway.

Exploring the Mechanism and Suggesting Combination Therapies for HDAC Inhibitors in Androgen Receptor-Null Prostate Cancer Using Multivariate Statistical Analysis and Data Mining Techniques.

Previously, we showed that novel histone deacetylase (HDAC) inhibitors, N1-hydroxy-N 8-(4-(pyridine-2-carbothioamido)phenyl)octanediamide (Jazz90) and [chlorido(η5-pentamethylcyclopentadienyl)(N1-hydroxy-N8-(4-(pyridine-2-carbothioamido-κ2 N, S)phenyl)octanediamide)rhodium(III)] chloride (Jazz167), have cytostatic and anti-angiogenic effects in androgen receptor-negative prostate cancer cells and are also non-toxic in BALB/c mice. However, only univariate statistical analysis was carried out to determine the role of individual proteins. In this study, multivariate statistical analyses (MVAs) and data mining procedures were carried out with the objective of determining the molecular networks that explain the growth inhibitory potential of Jazz90 and Jazz167 in PC3 cells and to determine potential inhibitors that can be used in combination with these HDAC inhibitors. Lasso regression revealed that angiogenic factors, vascular endothelial growth factor-A (VEGF-A), and vascular endothelial growth factor receptor-2 (VEGFR-2), alongside HDAC inhibition, predicted the reduction in cell number with an adjusted R 2 value of 0.99 following Jazz90 treatment, whereas VEGFR-2, acetylation of histone-3, and HDAC inhibition predicted cell number with an adjusted R 2 value of 0.84 following Jazz167 treatment. These results were further followed up with ridge regression, hierarchical cluster analysis, random forest classification (RFC), and support vector machines. RFC and support vector machines also predicted the treatment groups with a 100% accuracy. MVAs also revealed that Jazz90 should be examined in combination with epithelial to mesenchymal transitioning inhibitors, such as simvastatin and olaparib, whereas Jazz167 should be examined with venetoclax or navitoclax. Future studies should also address the roles of VEGF-A and VEGFR-2 in cellular proliferation, whereas p27 function should be examined for its role in PC3 cell migration.

Abstract LB-A13: LSD1 activates both PI3K/AKT and AR signaling in prostate cancer cells

Abstract Background: Lysine specific demethylase 1 (LSD1/KDM1A), a specific demethylase of mono- or di-methylated histone lysine 4 (H3K4me1,2, enhancer-associated signature), was first identified as a component of REST repressor complex composed of CoREST and histone deacetylases 1, 2 (HDAC1,2). In hematopoietic malignancies, targeting LSD1 has shown very promising clinical outcomes, potentially through promoting myeloid differentiation. In solid tumors, multiple mechanisms have been proposed for the antitumor effect of inhibiting LSD1. While LSD1 is well known for its transcription repressor activity, it also activates gene transcription through demethylating repressive histone marks, such as H3K9me1,2, and possibly other non-histone proteins. In particular, LSD1 functions as a major androgen receptor (AR) coactivator in prostate cancer (PCa) cells. Indeed, we reported that LSD1 interacts and colocalizes with FOXA1, a pioneer factor of AR, at AR-mediated enhancers, and this interaction may facilitate AR transcription activity. Nonetheless, since AR signaling is critical to PCa development and progression to the lethal stage of castration-resistant PCa (CRPC), studies from us and others highly suggest that targeting LSD1 may be a potential treatment strategy for CRPC, where AR signaling is commonly restored. However, whether LSD1 regulates other major tumor-promoting pathways in PCa cells remains to be determined. Identification of suchcritical LSD1 targets will provide important insights for developing more effective therapeutic strategy for treating PCa. Experiments and Results: To address this question, we conducted RNA-seq analyses of LSD1 inhibitor treated PCa cells. We first confirmed that prolonged LSD1 inhibitor treatment in the presence of androgens broadly impaired AR transcriptional output. Importantly, we found that the LSD1 inhibitor repressed genes were also enriched for PI3K/AKT pathway even in absence of androgen stimulation. We further confirmed that LSD1 inhibition and Knock-out significantly decreased AKT phosphorylation, which interestingly is independent of AR signaling, as targeting LSD1 also decreased AKT phosphorylation in AR negative PCa cells. Through functional annotation analyses and subsequent validations, we identified the regulatory subunit of PI3K, p85α and possibly its isoform p85β, as a critical transcriptional target of LSD1 that mediates its effect on PI3K/AKT pathway activation. Using a combined analysis with ChIP-seq against LSD1 and H3K4me2, we identified a regulatory region on the PIK3R1 gene loci that may be responsible for regulating its gene transcription. We further investigated the effect of LSD1 inhibitor treatment in LuCaP35CR, a patient-derived CRPC xenograft model which is TMPRSS2-ERG positive and PTEN-negative. Using this model, we found that targeting LSD1 impaired the tumor growth and suppressed PI3K/AKT signaling. Conclusion: our study suggests that LSD1 has dual functions in promoting PCa development: 1) it enhances AR signaling through its coactivator function, 2) it activates PI3K/AKT signaling through increasing p85 gene expression. Based on these findings, it is plausible that the effectiveness of LSD1 inhibitor treatment in CRPC may be due to inhibition of both AR signaling and PI3K/AKT signaling pathways. As LSD1 inhibitors are currently being tested in phase II clinical trials of leukemia, our studies can be rapidly translated into clinical trials of CRPC. Citation Format: zifeng wang, Shuai Gao, Changmeng cai. LSD1 activates both PI3K/AKT and AR signaling in prostate cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A13. doi:10.1158/1535-7163.TARG-19-LB-A13

Abstract 853: C1orf116, a gene with unknown function, is a novel driver of epithelial phenotype in epithelial-to-mesenchymal transition in human cancer

Abstract Metastatic prostate cancer is an incurable lethal disease and is the cause of death for 28,000 men in the US annually. In order for a cancer cell to metastasize from the primary tumor, it must move. In a critical early event of metastasis, a rare population of cells in the primary tumor undergoes a phenotype switch from a proliferating epithelial cell to gaining the migratory capacity of a mesenchymal cell, a process, termed epithelial-to-mesenchymal transition (EMT). To identify previously overlooked regulators of EMT, we undertook a multi-study gene expression array analysis (15 published gene expression studies; 95 total samples; 6 cancer types) and identified C1orf116 as a candidate driver of the epithelial phenotype in EMT across multiple cancer types. Of note, C1orf116 is an unnamed gene of unknown function that is largely uncharacterized in any setting, including in cancer biology or in EMT. To substantiate its potential role in EMT, we queried the NCI-60 cell line collection gene expression dataset and found that C1orf116 shared a similar expression pattern with epithelial marker genes (ESRP1, ESRP2, OVOL1, OVOL2) and a converse expression pattern to mesenchymal marker genes (CDH2, SNAI2, VIM). Moreover, we found that C1orf116 expression is decreased in metastatic prostate cancer tumors compared to localized disease (Oncomine), suggesting the potential for clinical translation. To specifically interrogate the role of C1orf116 in EMT, we utilized the PC3 cell line, an in vitro model of prostate cancer EMT. We found that C1orf 116 expression was higher in an epithelial PC3 cell line (PC3-epi) compared to PC3 cells that had undergone a stable EMT (PC3-emt). Moreover, shRNA-mediated knockdown of C1orf116 expression in PC3-epi cells resulted in decreased gene expression of canonical epithelial markers (OVOL1, ESRP1, CDH1) and elevated expression of standard mesenchymal markers (CDH2, ZEB1, VIM), suggesting a role as a driver of the epithelial phenotype. Interestingly, the sole publication specifically studying C1orf116 describes it as Specifically Androgen Regulated Gene (SARG). The authors demonstrate the presence of an androgen response element upstream of the C1orf116 start site the in androgen-responsive prostate cancer cell line LNCaP. Our data indicate a role for C1orf116 in the androgen receptor-negative prostate cancer cell line PC3, suggesting a non-androgen-mediated role for C1orf116 in prostate cancer. In addition to elucidating its role in EMT, work is underway to characterize the RNA and protein products of C1orf116. Alternative splicing results in two transcripts of varying length that likely give rise to at least two protein product sizes. RNA sequencing and mass spectrometry will reveal transcript and protein identity and provide insight into potential isoform switching regulating EMT and other processes. Citation Format: Sarah R. Amend, James Hernandez, Princy Parsana, Kenneth J. Pienta. C1orf116, a gene with unknown function, is a novel driver of epithelial phenotype in epithelial-to-mesenchymal transition in human 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 853. doi:10.1158/1538-7445.AM2017-853

Cyproterone acetate enhances TRAIL-induced androgen-independent prostate cancer cell apoptosis via up-regulation of death receptor 5

BackgroundVirtually all prostate cancer deaths occur due to obtaining the castration-resistant phenotype after prostate cancer cells escaped from apoptosis and/or growth suppression initially induced by androgen receptor blockade. TNF-related apoptosis-inducing ligand (TRAIL) was an attractive cancer therapeutic agent due to its minimal toxicity to normal cells and remarkable apoptotic activity in tumor cells. However, most localized cancers including prostate cancer are resistant to TRAIL-induced apoptosis, thereby creating a therapeutic challenge of inducing TRAIL sensitivity in cancer cells. Herein the effects of cyproterone acetate, an antiandrogen steroid, on the TRAIL-induced apoptosis of androgen receptor-negative prostate cancer cells are reported.MethodsCell apoptosis was assessed by both annexin V/propidium iodide labeling and poly (ADP-ribose) polymerase cleavage assays. Gene and protein expression changes were determined by quantitative real-time PCR and western blot assays. The effect of cyproterone acetate on gene promoter activity was determined by luciferase reporter assay.ResultsCyproterone acetate but not AR antagonist bicalutamide dramatically increased the susceptibility of androgen receptor-negative human prostate cancer PC-3 and DU145 cells to TRAIL-induced apoptosis but no effects on immortalized human prostate stromal PS30 cells and human embryonic kidney HEK293 cells. Further investigation of the TRAIL-induced apoptosis pathway revealed that cyproterone acetate exerted its effect by selectively increasing death receptor 5 (DR5) mRNA and protein expression. Cyproterone acetate treatment also increased DR5 gene promoter activity, which could be abolished by mutation of a consensus binding domain of transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP) in the DR5 gene promoter. Cyproterone acetate increases CHOP expression in a concentration and time-dependent manner and endoplasmic reticulum stress reducer 4-phenylbutyrate could block cyproterone acetate-induced CHOP and DR5 up-regulation. More importantly, siRNA silencing of CHOP significantly reduced cyproterone acetate-induced DR5 up-regulation and TRAIL sensitivity in prostate cancer cells.ConclusionsOur study shows a novel effect of cyproterone acetate on apoptosis pathways in prostate cancer cells and raises the possibility that a combination of TRAIL with cyproterone acetate could be a promising strategy for treating castration-resistant prostate cancer.

PD32-07 GALETERONE SHOWS ANTI-TUMOR ACTIVITY IN MULTIPLE PRE-CLINICAL MODELS THAT EXPRESS ANDROGEN RECEPTOR SPLICE VARIANTS, SUPPORTING CORRELATIVE PATIENT DATA SEEN IN ARMOR2

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) III1 Apr 2016PD32-07 GALETERONE SHOWS ANTI-TUMOR ACTIVITY IN MULTIPLE PRE-CLINICAL MODELS THAT EXPRESS ANDROGEN RECEPTOR SPLICE VARIANTS, SUPPORTING CORRELATIVE PATIENT DATA SEEN IN ARMOR2 Vincent Njar, Amina Zoubeidi, Eva Corey, Elahe Mostaghel, Andrew Kwegyir-Afful, Senthilmurugan Ramalingam, and Douglas Jacoby Vincent NjarVincent Njar More articles by this author , Amina ZoubeidiAmina Zoubeidi More articles by this author , Eva CoreyEva Corey More articles by this author , Elahe MostaghelElahe Mostaghel More articles by this author , Andrew Kwegyir-AffulAndrew Kwegyir-Afful More articles by this author , Senthilmurugan RamalingamSenthilmurugan Ramalingam More articles by this author , and Douglas JacobyDouglas Jacoby More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.683AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Galeterone is a selective, multitargeted, small molecule that disrupts androgen signaling at multiple points in the pathway by enhancing androgen receptor (AR) degradation, inhibiting androgen biosynthesis, and antagonizing androgen binding to the AR. AR splice variant-7 (AR-V7) is a truncated, constitutively active splice variant of the AR that lacks the ligand binding domain (LBD), and has been implicated in castration resistant prostate cancer (CRPC) progression. AR-V7 expression is detected in approximately 12-26% of men with metastatic CRPC (mCRPC) prior to second-generation anti-androgens or chemotherapy, and AR-V7 expression has been clinically associated with resistance to enzalutamide (Xtandi) and abiraterone (Zytiga). In this study, multiple experimental approaches examined whether, and by what mechanism, galeterone inhibits the growth of prostate cancer cells or tumors expressing AR splice variants, such as AR-V7. METHODS To measure effects of galeterone on prostate tumor cell signaling and growth, we monitored reductions in AR protein levels, and have used in vitro measures of AR-dependent gene expression and tumor cell viability. In addition, we assessed in vivo efficacy of galeterone in mouse xenograft models and clinically by evaluating PSA responses in patients whose CTCs express truncated AR. RESULTS Our findings show that galeterone attenuates tumor cell proliferation, AR signaling and xenograft tumor growth using cells and tumors that express AR splice variants. Galeterone treatment also causes dose-dependent reductions in AR protein. Reductions in AR splice variant protein were observed in cells that co-express wild-type AR, and in AR negative prostate cancer cells that transiently express AR splice variant protein. These findings support the PSA responses seen clinically with galeterone treatment in men with mCRPC whose tumors contain truncated AR. CONCLUSIONS Galeterone's unique mechanism of AR protein downregulation occurs in both splice variant and full-length AR, supporting that galeterone-induced AR downregulation is independent of the AR LBD. Because preclinical and clinical data support an opportunity for treatment in this setting, Tokai is evaluating galeterone in an ongoing global pivotal trial designed to determine whether treatment with galeterone results in a statistically significant increase in radiographic progression-free survival as compared to enzalutamide in approximately 148 treatment-naïve, AR-V7+ mCRPC patients. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e763 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Vincent Njar More articles by this author Amina Zoubeidi More articles by this author Eva Corey More articles by this author Elahe Mostaghel More articles by this author Andrew Kwegyir-Afful More articles by this author Senthilmurugan Ramalingam More articles by this author Douglas Jacoby More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract 5065: Interleukin-1β secreted into the bone metastatic niche by androgen receptor-negative prostate cancer cells enables skeletal metastasis

Abstract Advanced prostate cancer (PCa) frequently leads to bone metastasis, a major cause of morbidity and mortality. Limited knowledge of the molecular interactions underpinning the behavior of cancer cells in the bone microenvironment has stymied the development of curative therapies. Signaling between cancer cells and stroma likely plays a crucial role in dictating the fate of disseminated tumor cells (DTCs), and its disruption could interfere with the supportive role of the metastatic niche and thus hamper tumor growth. We previously identified interleukin-1β (IL-1β) as a major mediator of the bone-metastatic behavior of human prostate cancer cells inoculated into the left cardiac ventricle of SCID mice. Here, we show not only that pharmacologic disruption of IL-1β signaling using the IL-1 receptor (IL-1R) antagonist anakinra diminishes the metastatic potential of PCa cells in vivo, but also that IL-1β secreted by PCa cells can prompt bone stromal cells to acquire a carcinoma-associated fibroblast (CAF) phenotype. Emphasizing the role of the stroma, we demonstrate that SCID mice null for IL-1R display diminished formation of bone metastases relative to wild-type when inoculated with highly metastatic IL-1β-expressing PCa cells. In this same model we find that IL-1β supports early survival of DTCs in the bone. Interestingly, while the aggressively-metastatic androgen receptor (AR)-negative PC3-ML cell line expresss high levels of IL-1β, the numerous AR(+) PCa cell lines we examined each lack IL-1β expression. This was recapitulated in human tissue specimens of PCa bone metastases examined by a combination of Laser Capture Microdissection and qPCR. In these lesions, a relevant fraction of PCa cells lack AR transcript and express IL-1β, whereas AR(+) cells were uniformly negative for this cytokine. As AR(+) PCa cells are consistently non-metastatic in our pre-clinical model, we speculated that IL-1β-mediated induction of CAFs in the bone may enable growth of AR(+) PCa cells that would otherwise be unable to initiate a metastatic niche. To test this hypothesis, we co-inoculated PC3-ML and AR(+) PCa cells and evaluated their metastatic abilities using two-color labeling in both multispectral microscopy and bioluminescence imaging. We found that the presence of AR(-), IL-1β-expressing cells allowed AR(+) PCa cells both to colonize the bone and persist in macroscopic mixed tumors in the long term. Our study thus conclusively confirms the existence of AR(-) PCa cells in skeletal metastases, describes a negative correlation between AR and IL-1β expression, and implicates this cytokine in a form of metastatic cooperation between phenotypically distinct cancer cells. These discoveries may be exploited therapeutically to alter the microenvironment of the metastatic niche, with the aim to impair establishment and progression of bone metastatic disease in PCa patients. Citation Format: Kristina S. Shahriari, Fei Shen, QingXin Liu, Danielle L. Jernigan, Ramanpreet Kaur, Alessandro Fatatis. Interleukin-1β secreted into the bone metastatic niche by androgen receptor-negative prostate cancer cells enables skeletal metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5065. doi:10.1158/1538-7445.AM2015-5065

Chromatographic lipophilicity as a predictor of antiproliferative activity of 17-picolyl and 17-picolinylidene androstane derivatives toward prostate cancer

The present study describes the quantitative retention - (anticancer) activity relationship analysis of a series of 17-picolyl and 17-picolinylidene androstane derivatives based on the chromatographic data obtained by reversed-phase high performance liquid chromatography (C18-RP-HPLC) in a mobile phase consisting of 90% of methanol and 10% of water. The obtained retention factor or chromatographic lipophilicity factor (logk0.90) was correlated with the antiproliferative activity of studied compounds (IC50) toward androgen-receptor negative prostate cancer cell line (AR-neg. PC-3). Correlation analysis was carried out by applying non-linear artificial neural networks (ANNs) method. Statistical validation showed that the obtained ANNs can successfully predict the IC50 values of structurally similar androstane derivatives.

Analysis of tumor necrosis factor α-induced and nuclear factor κB-silenced LNCaP prostate cancer cells by RT-qPCR.

Prostate cancer is the second leading cause of morbidity and mortality in males in the Western world. In the present study, LNCaP, which is an androgen receptor-positive and androgen-responsive prostate cancer cell line derived from lymph node metastasis, and DU145, which is an androgen receptor-negative prostate cancer cell line derived from brain metastasis, were investigated. TNFα treatment decreased p105 and p50 expression and R1881 treatment slightly decreased p105 expression but increased p50 expression with or without TNFα induction. As an aggressive prostate cancer cell line, DU145 transfected with six transmembrane protein of prostate (STAMP)1 or STAMP2 was also exposed to TNFα. Western blotting indicated that transfection with either STAMP gene caused a significant increase in NFκB expression following TNFα induction. In addition, following the treatment of LNCaP cells with TNFα, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed with a panel of apoptosis-related gene primers. The apoptosis-related genes p53, p73, caspase 7 and caspase 9 showed statistically significant increases in expression levels while the expression levels of MDM2 and STAMP1 decreased following TNFα induction. Furthermore, LNCaP cells were transfected with a small interfering NFκB (siNFκB) construct for 1 and 4 days and induced with TNFα for the final 24 h. RT-qPCR amplifications were performed with apoptosis-related gene primers, including p53, caspases and STAMPs. However, no changes in the level of STAMP2 were observed between cells in the presence or absence of TNFα induction or between those transfected or not transfected with siNFκB; however, the level of STAMP1 was significantly decreased by TNFα induction, and significantly increased with siNFκB transfection. Silencing of the survival gene NFκB caused anti-apoptotic STAMP1 expression to increase, which repressed p53, together with MDM2. NFκB silencing had varying effects on a panel of cancer regulatory genes. Therefore, the effective inhibition of NFκB may be critical in providing a targeted pathway for prostate cancer prevention.

Abstract 4950: Hypercholesterolemia promotes prostate cancer PC-3 metastases in orthotopic xenograft mice

Abstract Hypercholesterolemia has been proposed as a potential risk factor for advanced prostate cancer, and use of cholesterol-lowering drugs, statins, inversely correlates with advanced prostate cancer risk. Hypercholesterolemia increases growth of androgen-sensitive LNCaP xenograft in vivo, potentially via androgen signaling. The aim of this study was to determine if hypercholesterolemia affects castration-resistant prostate tumor progression using an androgen receptor-negative prostate cancer cell line PC-3. Compared to control media, cholesterol-deficient media reduced PC-3 proliferation, migration and anchorage-independent growth in vitro. While adding cholesterol did not significantly increase proliferation or anchorage-independent growth, cholesterol replacement in cholesterol-deficient media significantly increased PC-3 transmigration. In order to determine the in vivo effect, mice were randomly assigned to normal or hypercholesterolemic, isocaloric diet groups (N=14 and 15, respectively). After two weeks, hypercholesterolemic diet significantly increased circulating cholesterol but did not increase body weight. PC-3 cells stably expressing luciferase were orthotopically injected into the dorsolateral prostate. Tumor progression and metastases were monitored by in vivo and ex vivo optical bioluminescence imaging for 6 weeks. Strikingly, the results show that diet-induced hypercholesterolemia accelerated tumor metastases to lymph nodes, lung, proximal and distant bones without significantly affecting primary tumor growth. The metastases were confirmed histopathologcally. Hypercholesterolemia was not associated with elevated weight or circulating testosterone. This is the first study to directly demonstrate a causal relationship between hypercholesterolemia and prostate tumor metastases mediated through androgen-independent mechanisms, highlighting the potential clinical benefit of cholesterol lowering therapy such as statins in advanced, castration-resistant, prostate cancer patients. Citation Format: Hyeongsun Moon, Laura Sharpe, Eunju Choi, Helle Bielefeldt-Ohmann, Zeyad Nassar, Marie-Odile Parat, Mathias Francois, C Soon Lee, Andrew Brown, Pamela Russell, Kerry Inder, Michelle Hill. Hypercholesterolemia promotes prostate cancer PC-3 metastases in orthotopic xenograft mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4950. doi:10.1158/1538-7445.AM2014-4950

Hypermethylation-mediated transcriptional repression of TMPRSS2 in androgen receptor-negative prostate cancer cells.

Prostate cancer is the most common type of cancer for men in the developed world. Androgen receptor (AR) is very important in prostate cancer progression. TMPRSS2 is an AR signaling downstream gene and closely related to prostate carcinogenesis. DNA methylation is a key mechanism to influence gene expression. Though previous reports have shown that AR signaling plays a critical role in the regulation of TMPRSS2 in prostate cancer, hardly any studies have examined whether the DNA methylation has been involved in the regulation of TMPRSS2. In the present study, we demonstrated that AR-negative prostate cancer (PCa) cells showed low expression levels and hypermethylation of TMPRSS2. In contrast, AR-positive PCa cells displayed high levels and hypomethylation of TMPRSS2. Treatment with the DNA methylation inhibitor 5-Aza-2'-deoxycytidine reversed the low expression levels of TMPRSS2 in the AR-negative PCa cells. Additionally, we found that the level of DNA methyltransferases 1 (DNMT1) was high in AR-negative PCa cells, in which hypermethylation of TMPRSS2 and low expression level of TMPRSS2 were observed. Collectively, these data suggest that the high level of DNMT1 might be the mechanism for the hypermethylation-mediated transcriptional repression of TMPRSS2 in AR-negative PCa cells.

Androgen receptor is negatively correlated with the methylation-mediated transcriptional repression of miR-375 in human prostate cancer cells

Androgen receptor (AR) plays a critical role during the development and progression of prostate cancer in which microRNA miR-375 is overexpressed and correlated with tumor progression. Although DNA methylation is a key mechanism for the repression of gene expression, the relationship between AR and the expression or the hypermethylation of miR-375 is unknown. In this study, we found that AR-positive prostate cancer (PCa) cells showed high expression levels and hypomethylation of the miR-375. In contrast, AR-negative PCa cells displayed low levels and hypermethylation of the miR-375. Addition of 5-Aza-2′-deoxycytidine, a specific inhibitor of DNA methylation, into the culture medium reversed the low expression levels of miR-375 in the AR negative PCa cells. In addition, the total activity levels of DNA methyltransferases (DNMTs) were high in AR-negative PCa cells, in which hypermethylation of miR-375 promoter and low expression levels of miR-375 were observed. Taken together, these findings indicate that the negative correlation between AR and total DNMT activity is one of mechanisms to influence the methylation status of miR-375 promoter, which in turn regulates the expression of miR-375.

Essential Role of NADPH Oxidase-Dependent Reactive Oxygen Species Generation in Regulating MicroRNA-21 Expression and Function in Prostate Cancer

Oncogenic microRNAs (miRs) promote tumor growth and invasiveness. One of these, miR-21, contributes to carcinogenesis in prostate and other cancers. In the present study, we tested the hypothesis that NADPH oxidase-dependent reactive oxygen species (ROS) regulate the expression and function of miR-21 and its target proteins, maspin and programmed cell death 4 (PDCD4), in prostate cancer cells. The highly aggressive androgen receptor negative PC-3M-MM2 prostate cancer cells demonstrated high expression of miR-21 and p47(phox) (an essential subunit of NADPH oxidase). Using loss-of-function strategy, we showed that transfection of PC-3M-MM2 cells with anti-miR-21- and p47(phox) siRNA (si-p47(phox)) led to reduced expression of miR-21 with concurrent increase in maspin and PDCD4, and decreased the invasiveness of the cells. Tail-vein injections of anti-miR-21- and si-p47(phox)-transfected PC-3M-MM2 cells in severe combined immunodeficient mice reduced lung metastases. Clinical samples from patients with advanced prostate cancer expressed high levels of miR-21 and p47(phox), and low expression of maspin and PDCD4. Finally, ROS activated Akt in these cells, the inhibition of which reduced miR-21 expression. The levels of NADPH oxidase-derived ROS are high in prostate cancer cells, which have been shown to be involved in their growth and migration. This study demonstrates that ROS produced by this pathway is essential for the expression and function of an onco-miR, miR-21, in androgen receptor-negative prostate cancer cells. These data demonstrate that miR-21 is an important target of ROS, which contributes to the highly invasive and metastatic phenotype of prostate cancer cells.

Inhibitory effects of Rhenium-188-labeled Herceptin on prostate cancer cell growth: A possible radioimmunotherapy to prostate carcinoma

Purpose: Herceptin is widely used in treating Her2-overexpressing breast cancer. However, the application of Herceptin in prostate cancer is still controversial. Our previous results have indicated the relevance of Her2 in the transition of the androgen requirement in prostate cancer cells. In this study, the effects of radioimmunotherapy against Her2 in prostate cancer were investigated.Materials and methods: DU145, an androgen receptor-negative prostate cancer cell line, was used in vitro and in vivo to evaluate the effects of Herceptin labeled with a beta emitter, Rhenium-188 (Re-188). Its effects on cell growth, extent of apoptosis, the bio-distribution of Re-188 labeled Herceptin (Re-H), and protein levels were determined.Results: Treatments with Re-188 and Re-H reduced the proliferation of DU145 cells in dose- and time-dependent manners compared to the Herceptin-treated group. Growth inhibition and apoptosis were induced after Re-H treatment; growth inhibition was more distinct in cells with high Her2/p-Her2 levels. Our in vivo xenograft studies revealed that Re-H treatment significantly retarded tumor growth and altered the levels of apoptosis-related proteins. The bio-distribution of Re-H in mice demonstrated a tissue-specific pattern. Importantly, the levels of p35 protein, which is related to cancer cell survival and invasion, dramatically decreased after Re-H treatment.Conclusions: Our data demonstrate that Re-188-labeled Herceptin effectively inhibited the growth of DU145 cells compared to the Herceptin- and Re-188-treated cohorts. This implies that targeting Her2 by both radio- and immuno- therapy might be a potential strategy for treating patients with androgen-independent prostate cancer.

Activation of β-catenin signaling in androgen receptor-negative prostate cancer cells.

To study Wnt/β-catenin in castrate-resistant prostate cancer (CRPC) and understand its function independently of the β-catenin-androgen receptor (AR) interaction. We carried out β-catenin immunocytochemical analysis, evaluated TOP-flash reporter activity (a reporter of β-catenin-mediated transcription), and sequenced the β-catenin gene in MDA prostate cancer 118a, MDA prostate cancer 118b, MDA prostate cancer 2b, and PC-3 prostate cancer cells. We knocked down β-catenin in AR-negative MDA prostate cancer 118b cells and carried out comparative gene-array analysis. We also immunohistochemically analyzed β-catenin and AR in 27 bone metastases of human CRPCs. β-Catenin nuclear accumulation and TOP-flash reporter activity were high in MDA prostate cancer 118b but not in MDA prostate cancer 2b or PC-3 cells. MDA prostate cancer 118a and MDA prostate cancer 118b cells carry a mutated β-catenin at codon 32 (D32G). Ten genes were expressed differently (false discovery rate, 0.05) in MDA prostate cancer 118b cells with downregulated β-catenin. One such gene, hyaluronan synthase 2 (HAS2), synthesizes hyaluronan, a core component of the extracellular matrix. We confirmed HAS2 upregulation in PC-3 cells transfected with D32G-mutant β-catenin. Finally, we found nuclear localization of β-catenin in 10 of 27 human tissue specimens; this localization was inversely associated with AR expression (P = 0.056, Fisher's exact test), suggesting that reduced AR expression enables Wnt/β-catenin signaling. We identified a previously unknown downstream target of β-catenin, HAS2, in prostate cancer, and found that high β-catenin nuclear localization and low or no AR expression may define a subpopulation of men with bone metastatic prostate cancer. These findings may guide physicians in managing these patients.

A Single Therapeutic miRNA Rescues the Oncolysis of Androgen Activated Prostate Specific Virus in Androgen Independent Cell Model

Prostate specific conditionally replicating adenoviruses (CRAd) are made by placing a tissue specific promoter upstream of one or more of the viral genes required for replication (e.g., E1A, E1B). However, one major problem associated with these vectors is their dependency for androgen receptor (AR), to transactivate the immediate early gene of the virus. This absolute necessity of androgens for prostate specific promoters renders them less useful in patients that have undergone or are currently on total androgen ablation therapy. Therefore, an alternative approach is needed for the existing vectors to be useful in clinical settings. To overcome this problem, we have generated a prostate specific CRAd by introducing a single therapeutic miRNA (miR-34c) that was identified in our library screen to synergize with viral oncolysis. Overexpression of miR-34c from the backbone of virus not only helped in suppressing the proliferation of AR positive cells but also rescued the oncolysis of androgen activated prostate specific virus in androgen independent cell model. To our knowledge this is the first report describing the utility of a single therapeutic miRNA construct to rescue viral oncolysis in advanced androgen resistant AR negative prostate cancer cell line model.

Abstract CN08-05: A combinatorial approach to targeting androgen receptor signaling in prostate cancer.

Abstract When androgen deprivation therapy (ADT) for prostate cancer fails and the disease progresses to a castrate-resistant state (castrate-resistant prostate cancer; CRPC), therapeutic options for patients are limited. It is now recognized that the majority of CRPC still express the androgen receptor (AR) and remain dependent on AR signaling for growth and survival. To limit growth or evolution of AR-dependent disease and thereby improve patient survival, we propose that a treatment strategy that simultaneously targets multiple features of the AR signaling axis will more effectively eliminate AR-dependent prostate cancer cells than single agent treatment strategies. We have shown that pharmacologically low doses of an AR antagonist (bicalutamide) in combination with either a heat shock protein 90 inhibitor (Hsp90i; 17-AAG) or a histone deacetylase inhibitor (vorinostat), results in synergistic growth suppression and induction of caspase-dependent death in AR-dependent prostate cancer cells. These drug combinations did not alter steady state protein levels of the AR, but suppressed AR activity, as evidenced by reduced prostate specific antigen levels. As expected, the drug combinations were not effective in androgen-independent, AR negative prostate cancer cells. To elucidate the mechanisms of drug synergy, we performed microarray studies to identify genetic programs modulated by each combination therapy or bicalutamide alone. Resultant data suggest that the combination treatments did not markedly enhance abrogation of androgen signaling compared with bicalutamide alone. Rather, the combination treatments regulated the expression of unique gene sets that were enriched for cell cycle, apoptosis, MAPK and insulin signaling compared to individual agents. To assess the potential efficacy of the combinatorial approach in human disease, we have developed a unique approach to study human prostate cancer where tumor specimens collected from men undergoing radical prostatectomy are cultured as explants for up to 7 days with maintenance of tissue integrity, cell proliferation and androgen signaling. Using this ex vivo strategy, we demonstrated that human prostate tumor tissue responds to the novel synthetic Hsp90 inhibitors (HSP990 and AUY922) with a marked reduction in proliferation and AR levels. Importantly, these studies have revealed a dissociation between client protein modulation (i.e. the established clinical biomarker of Hsp90 inhibition, HSP70) and proliferative response to Hsp90 inhibition, which was not seen in prostate cancer cell lines in vitro. Collectively, these findings suggest that the ex vivo methodology may better predict individual patient response to Hsp90 inhibitors and could be useful as a pre-clinical model of drug development, including identification of robust biomarkers of response. Currently we are using human tumor explants to identify optimal combinations of AR-targeting agents and potential markers of response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr CN08-05.

Androgen receptor mediates the expression of UDP‐glucuronosyltransferase 2 B15 and B17 genes

Enhanced androgen receptor (AR) activity by increased testosterone availability may play important roles in prostate cancer progressing to castration resistant state. Comparison of expression profiles in androgen dependent and independent prostate tumors demonstrated a marked increase of the expression of UDP-glucuronosyltransferase 2B15 (UGT2B15), an androgen catabolic enzyme. We investigated mechanisms controlling the differential expression of UGT2B15 and B17 in response to androgen treatments. Gene expression was determined by RT-PCR. The association of AR with UGT2B15/B17 genes was determined by Chromatin immuno-precipitation (CHIP). RNA interference was used to knock-down gene expression. UGT2B15 and B17 genes were not expressed in AR negative prostate cancer cell lines, PC3 and DU145, while they were expressed in AR positive cell lines, LNCaP, LNCaP-abl (an androgen independent LNCaP sub-line), and VCaP. The expression levels of UGT2B15/B17 were up-regulated in LNCaP-abl comparing to those in LNCaP. These results suggest the requirement of AR for the expression of UGT2B15/B17. Treatment with DHT down-regulated the expression of UGT2B15/B17 in LNCaP in a time and dose dependent manner and this down-regulation was competitively antagonized by flutamide and bicalutimide, suggesting a pathway mediated by AR. Further CHIP experiments demonstrated the direct interaction of AR with the promoter regions of UGT2B15/B17 genes. Knocking down AR expression in LNCaP significantly reduced the expression of UGT2B15/B17 and completely inhibited the DHT-induced down-regulation of UGT2B15/B17 genes. We demonstrated that UGT2B15 and B17 are primary androgen-regulated genes and AR is required for both their basal expression and their androgen-regulated expression.

Nitric oxide-mediated inhibition of androgen receptor activity: possible implications for prostate cancer progression

Chronic inflammation increases the risk of cancer and many cancers, including prostate cancer, arise at sites of chronic inflammation. Inducible nitric oxide synthase (iNOS) is an enzyme dominantly expressed during inflammatory reactions. Although synthesis of high amounts of nitric oxide (NO) by iNOS has been demonstrated in pathophysiological processes, such as acute or chronic inflammation, autoimmune diseases or tumorigenesis, the role of iNOS activity in most of these diseases is poorly understood. Analysing prostate cancer biopsies by immunohistochemistry we found iNOS protein expression in tumor cells strongly paralleled by nitrotyrosine suggesting that iNOS is fully active. In vitro, NO inhibits androgen receptor-dependent promoter activity and prostate specific antigen production as well as DNA-binding activity of the androgen receptor (AR) in a concentration-dependent manner. Inhibition of the activity of androgen receptor-dependent reporter constructs is neither owing to diminished AR protein levels nor owing to an inhibition of its nuclear import. In addition, NO inhibits the proliferation of androgen receptor-positive prostate cancer cells significantly more efficiently than proliferation of androgen receptor-negative prostate cancer cells. In summary, our findings suggest that intratumoral iNOS activity favors development of prostate cancer cells that are able to proliferate androgen receptor-independently, thereby promoting prostate tumor progression.