Androgen-dependent Prostate Cancer Cells Research Articles

Cytotoxicity of obacunone and obacunone glucoside in human prostate cancer cells involves Akt-mediated programmed cell death.

Obacunone and obacunone glucoside (OG) are naturally occurring triterpenoids commonly found in citrus and other plants of the Rutaceae family. The current study reports the mechanism of cytotoxicity of citrus-derived obacunone and OG on human androgen-dependent prostate cancer LNCaP cells. Both limonoids exhibited time- and dose-dependent inhibition of cell proliferation, with more than 60% inhibition of cell viability at 100 μM, after 24 and 48 h. Analysis of fragmentation of DNA, activity of caspase-3, and cytosolic cytochrome-c in the cells treated with limonoids provided evidence for activation of programmed cell death by limonoids. Treatment of LNCaP cells with obacunone and OG resulted in dose-dependent changes in expression of proteins responsible for the induction of programmed cell death through the intrinsic pathway and down-regulation of Akt, a key molecule in cell signaling pathways. In addition, obacunone and OG also negatively regulated an inflammation-associated transcription factor, androgen receptor, and prostate-specific antigen, and activated proteins related to the cell cycle, confirming the ability of limonoids to induce cytotoxicity through multiple pathways. The results of this study provided, for the first time, an evidence of the cytotoxicity of obacunone and OG in androgen-dependent human prostate cancer cells.

Elevated LIM kinase 1 in nonmetastatic prostate cancer reflects its role in facilitating androgen receptor nuclear translocation.

Prostate cancer affects a large proportion of the male population, and is primarily driven by androgen receptor (AR) activity. First-line treatment typically consists of reducing AR signaling by hormone depletion, but resistance inevitably develops over time. One way to overcome this issue is to block AR function via alternative means, preferably by inhibiting protein targets that are more active in tumors than in normal tissue. By staining prostate cancer tumor sections, elevated LIM kinase 1 (LIMK1) expression and increased phosphorylation of its substrate Cofilin were found to be associated with poor outcome and reduced survival in patients with nonmetastatic prostate cancer. A LIMK-selective small molecule inhibitor (LIMKi) was used to determine whether targeted LIMK inhibition was a potential prostate cancer therapy. LIMKi reduced prostate cancer cell motility, as well as inhibiting proliferation and increasing apoptosis in androgen-dependent prostate cancer cells more effectively than in androgen-independent prostate cancer cells. LIMK inhibition blocked ligand-induced AR nuclear translocation, reduced AR protein stability and transcriptional activity, consistent with its effects on proliferation and survival acting via inhibition of AR activity. Furthermore, inhibition of LIMK activity increased αTubulin acetylation and decreased AR interactions with αTubulin, indicating that the role of LIMK in regulating microtubule dynamics contributes to AR function. These results indicate that LIMK inhibitors could be beneficial for the treatment of prostate cancer both by reducing nuclear AR translocation, leading to reduced proliferation and survival, and by inhibiting prostate cancer cell dissemination.

Arctigenin in combination with quercetin synergistically enhances the antiproliferative effect in prostate cancer cells.

We investigated whether a combination of two promising chemopreventive agents arctigenin (Arc) and quercetin (Q) increases the anticarcinogenic potency at lower concentrations than necessary when used individually in prostate cancer. Androgen-dependent LAPC-4 and LNCaP prostate cancer cells were treated with low doses of Arc and Q alone or in combination for 48 h. The antiproliferative activity of Arc was 10- to 20-fold stronger than Q in both cell lines. Their combination synergistically enhanced the antiproliferative effect, with a stronger effect in androgen receptor (AR) wild-type LAPC-4 cells than in AR mutated LNCaP cells. Arc demonstrated a strong ability to inhibit AR protein expression in LAPC-4 cells. The combination treatment significantly inhibited both AR and PI3K/Akt pathways compared to control. A protein array analysis revealed that the mixture targets multiple pathways particularly in LAPC-4 cells including Stat3 pathway. The mixture significantly inhibited the expression of several oncogenic microRNAs including miR-21, miR-19b, and miR-148a compared to control. The mixture also enhanced the inhibition of cell migration in both cell lines compared to individual compounds tested. The combination of Arc and Q that target similar pathways, at low physiological doses, provides a novel regimen with enhanced chemoprevention in prostate cancer.

Antiandrogenic mechanisms of pesticides in human LNCaP prostate and H295R adrenocortical carcinoma cells.

Several pesticides suspected or known to have endocrine disrupting effects were screened for pro- or antiandrogenic properties by determining their effects on proliferation, prostatic-specific antigen (PSA) secretion and androgen receptor (AR) expression, and AR phosphorylation in androgen-dependent LNCaP human prostate cancer cells, as well as on the expression and catalytic activity of the enzyme CYP17 in H295R human adrenocortical carcinoma cells, an in vitro model of steroidogenesis. Effects on SRD5A gene expression were determined in both cell lines. Benomyl, vinclozolin, and prochloraz, but not atrazine, concentration dependently (1-30 μM) decreased dihydrotestosterone (DHT)-stimulated proliferation of LNCaP cells. All pesticides except atrazine decreased DHT-stimulated PSA secretion, AR nuclear accumulation, and AR phosphorylation on serines 81 and 213 in LNCaP cells. Benomyl and prochloraz, but not vinclozolin or atrazine, decreased levels of CYP17 gene and protein expression, as well as catalytic activity in H295R cells. In the case of prochloraz, some of these effects corresponded with cytotoxicity. H295R cells expressed AR protein and SRD5A1, but not SRD5A2 transcripts. SRD5A1 gene expression in H295R cells was increased by 10 nM DHT, whereas in LNCaP cells significant induction was observed by 0.1 nM DHT. AR protein expression in H295R cells was not increased by DHT. Vinclozolin decreased DHT-induced SRD5A1 gene expression in LNCaP, but not H295R cells, indicating a functional difference of AR between the cell lines. In conclusion, pesticides may exert antiandrogenic effects through several mechanisms that are cell type-specific, including AR antagonism and down-regulation or catalytic inhibition of androgen biosynthetic enzymes, such as CYP17 and SRD5A1.

AZD1152-HQPA induces growth arrest and apoptosis in androgen-dependent prostate cancer cell line (LNCaP) via producing aneugenic micronuclei and polyploidy.

Prostate cancer is the frequent non-cutaneous tumor with high mortality in men. Prostate tumors contain cells with different status of androgen receptor. Androgen receptor plays important roles in progression and treatment of prostate cancer. Aurora B kinase, with oncogenic potential, is involved in chromosome segregation and cytokinesis, and its inhibition is a promising anti-cancer therapy. In the present study, we aimed to investigate the effects of Aurora B inhibitor, AZD1152-HQPA, on survival and proliferation of androgen receptor (AR)-positive prostate cancer cells. LNCaP was used as androgen-dependent prostate cancer cell line. We explored the effects of AZD1152-HQPA on cell viability, DNA content, micronuclei formation, and expression of genes involved in apoptosis and cell cycle. Moreover, the expression of Aurora B and AR were investigated in 23 benign prostatic hyperplasia and 38 prostate cancer specimens. AZD1152-HQPA treatment induced defective cell survival, polyploidy, and cell death in LNCaP cell line. Centromeric labeling with fluorescence in situ hybridization (FISH) showed that the loss of whole chromosomes is the origin of micronuclei, indicating on aneugenic action of AZD1152-HQPA. Treatment of AZD1152-HQPA decreased expression of AR. Moreover, we found weak positive correlations between the expression of Aurora B and AR in both benign prostatic hyperplasia and prostate cancer specimens (r = 0.25, r = 0.41). This is the first time to show that AZD1152-HQPA can be a useful therapeutic strategy for the treatment of androgen-dependent prostate cancer cell line. AZD1152-HQPA induces aneugenic mechanism of micronuclei production. Taken together, this study provides new insight into the direction to overcome the therapeutic impediments against prostate cancer.

LSD1 controls metastasis of androgen-independent prostate cancer cells through PXN and LPAR6.

Lysine-specific demethylase 1 (LSD1) was shown to control gene expression and cell proliferation of androgen-dependent prostate cancer (PCa) cells, whereas the role of LSD1 in androgen-independent metastatic prostate cancer remains elusive. Here, we show that depletion of LSD1 leads to increased migration and invasion of androgen-independent PCa cells. Transcriptome and cistrome analyses reveal that LSD1 regulates expression of lysophosphatidic acid receptor 6 (LPAR6) and cytoskeletal genes including the focal adhesion adaptor protein paxillin (PXN). Enhanced LPAR6 signalling upon LSD1 depletion promotes migration with concomitant phosphorylation of PXN. In mice LPAR6 overexpression enhances, whereas knockdown of LPAR6 abolishes metastasis of androgen-independent PCa cells. Taken together, we uncover a novel mechanism of how LSD1 controls metastasis and identify LPAR6 as a promising therapeutic target to treat metastatic prostate cancer.

Abstract 677: Co-targeting of AR and N-cadherin with Enzalutamide and 2A9 monoclonal antibody to treat castration resistant prostate cancer

Abstract Introduction: Enzalutamide (MDV3100) is an androgen receptor (AR) antagonist that targets various stages of the AR signaling pathway, and has been approved for the treatment of castration resistant prostate cancer (CRPC). Studies show improved survival in men with metastatic CRPC after chemotherapy when treated with Enzalutamide. However, emergence of potential AR-independent mechanisms of castration resistance and resistance to these next generation AR inhibitors has proven to be a challenge. Previously, we reported an association of N-cadherin, a mesenchymal cadherin expressed on the cell surface, with tumor progression and castration resistance. Targeting N-cadherin positive cells with specific monoclonal antibodies affect tumor growth in both AR positive and negative prostate cancers. Here we investigate the effects of co-targeting AR with Enzalutamide and N-cadherin with the N-cadherin specific monoclonal antibody 2A9 in CRPC. Method: N-cadherin was ectopically expressed in androgen dependent prostate cancer cells (LNCaP, MDA-PCa-2b, VCaP) and evaluated in vitro for invasion, growth, and self-renewal in the presence of Enzalutamide and 2A9 monoclonal antibody, raised against extracellular domain 4 of N-cadherin. Enzalutamide-resistant cell lines were generated and characterized. For in vivo studies, castration resistant N-cadherin and AR positive, as well as androgen-dependent tumors were implanted in mice as xenografts. When the tumors reached 100mm^3 in volume, mice were castrated and treated with 5-10mg/kg Enzalutamide and/or 5-10mg/kg 2A9 and monitored for tumor growth, local invasion, and metastasis. The tumors were harvested and analyzed for cell proliferation, apoptosis, and other markers. Results: Ectopic expression of N-cadherin in N-cadherin negative cells showed increase in cell growth and invasion in androgen-deprived conditions. In vitro studies show single or combination treatment of N-cadherin positive and AR positive cells reduce invasion and cell proliferation. Enzalutamide-resistant cell lines show response to 2A9 in androgen-deprived conditions. In vivo studies using CRPC xenograft models expressing N-cadherin and AR show inhibition of tumor growth when the animals are castrated and treated with a combination of Enzalutamide and 2A9 than either treatment alone. Androgen dependent xenograft models in castrated mice also showed significant delay in progression to castration resistance when treated in combination than either treatment alone. Conclusion: Previous studies suggested that therapeutic targeting of N-cadherin with monoclonal antibodies have significant effect in inhibiting tumor progression. The antibody in combination with Enzalutamide showed synergy. Other androgen receptor antagonists and other drugs targeting those potentially involved in downstream pathways may prove to have additional clinical benefit. Citation Format: Evelyn A. Kono, Sean Hyung-Kwon Lee, Tatsuya Shimomura, Shu Lin, Joyce Yamashiro, Robert E. Reiter. Co-targeting of AR and N-cadherin with Enzalutamide and 2A9 monoclonal antibody to treat castration resistant prostate cancer. [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 677. doi:10.1158/1538-7445.AM2014-677

Abstract 616: Transcription-regulating kinases CDK8 and CDK19 as novel therapeutic targets for advanced prostate cancer

Abstract Most of castration-refractory prostate cancers (CRPC) continue to express androgen receptor (AR) that maintains its activity at low levels or even in the absence of androgen, driving tumor cell proliferation. Androgen-depleting drugs and androgen antagonists have little or no effect against AR+ cancers that are fully androgen-independent (AI). On the other hand, experimental compounds that cause AR degradation prevent AR-mediated repression of tumor-promoting genes, a tumor-suppressive effect of AR. We report a novel druggable target for CRPC, which mediates the induction but not the repression of gene expression by AR and inhibits the growth of AI prostate cancer (PCa) cells. This target is two closely related transcription-regulating kinases CDK8/CDK19 that unlike some other members of the CDK family are not required for cell cycle progression but play critical roles in several transcriptional signaling pathways. CDK8 and CDK19 are highly expressed in AR+ PCa cells and elevated under the conditions of androgen deprivation; CDK19 is overexpressed in AR+ PCa cell lines and in metastatic clinical PCa. A novel selective CDK8/19 kinase inhibitor (Senexin B) blocks ligand-induced AR-mediated transcriptional activation in androgen-dependent (AD) PCa cells but does not affect the AR-mediated gene repression. The CDK8/19 inhibitor also inhibits ligand-independent AR activation and AI cell growth in AI-PCa cells resistant to the anti-androgenic drug enzalutamide, both in vitro and in a mouse xenograft model. We have also discovered that CDK8/19 inhibition decreases NFκB mediated transcriptional activation of a group of tumor-promoting chemokines, such as CXCL1, CXCL2 and IL8, suggesting that CDK8/19 may promote prostate cancer chemoresistance and metastasis through the NFκB pathway. The above results suggest that CDK8/19 is an especially attractive therapeutic target for advanced PCa, which controls both AR-driven cell proliferation and other oncogenic pathways. Citation Format: Mengqian Chen, Vimala Kaza, Jiaxin Liang, Martina McDermott, Igor Roninson. Transcription-regulating kinases CDK8 and CDK19 as novel therapeutic targets for advanced prostate cancer. [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 616. doi:10.1158/1538-7445.AM2014-616

Abstract 1387: Convergent CREB1/FoxA1 transcriptional activity defines castration-resistant prostate cancer gene expression profile

Abstract To identify novel pathways and therapeutic targets in lethal, castration-resistant prostate cancer (CRPC), we extended our previous findings that the overexpression of several G1/S and G2/M phase cell cycle genes in CRPC vs. androgen-dependent prostate cancer (ADPC) cells relies on the cooperative activity of the transcription factors (TFs) FoxA1 and CREB1, and sought to comprehensively characterize their role in supporting CRPC growth. We have taken an integrated genomics approach to reveal the CREB1/FoxA1-coregulated gene expression profile of ADPC (LNCaP) and CRPC (LNCaP-abl) cell line models. Chromatin-immunoprecipitation combined with high-throughput sequencing (ChIP-seq) was performed using antibodies against CREB1 and FoxA1 to reveal genome-wide TF binding sites. Our results indicate that these factors adopt divergent binding patterns in each cell line and that the CREB1 and FoxA1 cistromes exhibit a higher degree of overlap in CRPC vs. ADPC. RNA-seq was also performed in each cell line following transfection with Control-, FoxA1-, or CREB1-targeting small interfering RNA (siRNA) molecules. We identified putative direct CREB1/FoxA1-coregulated genes by noting instances of TF binding near cooperatively coregulated genes (i.e. those up- or down-regulated by both CREB1 and FoxA1 knockdown). Unlike previous analyses, which found that cooperative TF activity occurs primarily in the event of TF co-occupancy of gene regulatory elements, we found that non-overlapping as well as overlapping CREB1/FoxA1 binding events were significantly enriched within cooperatively coregulated target gene loci compared to genes that were unresponsive to TF knockdown. Gene Ontology (GO) analysis of LNCaP-abl-coregulated genes found that CREB1/FoxA1 enhance the expression of critical CRPC pathways including “DNA Repair” and “Aurora Kinase,” while they repress pathways such as “Apoptosis” and, consistent with previous findings, “Androgen Receptor Signaling.” Importantly, CREB1/FoxA1-up-regulated and down-regulated pathway elements exhibit overexpression or under-expression, respectively, in LNCaP-abl vs. LNCaP as well as in public gene expression datasets of metastatic vs. primary prostate cancer. Finally, we performed several proof-of-concept analyses to demonstrate that the CREB1/FoxA1-coregulatory pathway is sensitive to compounds that target CREB1 phosphorylation, a critical event in the transactivation of CREB1 mediated in large part by the related AGC kinase family members PKA and Akt/PKB. Together these results reveal that CREB1 and FoxA1 assume overlapping roles in determining the expression of genes/networks relevant to experimental models and clinical cases of advanced prostate cancer, and though targeting transcription factors has proven challenging, this coregulatory pathway may in fact represent a therapeutic target owing to its kinase-dependent activation. Citation Format: Benjamin D. Sunkel, Dayong Wu, Xiangtao Liu, Zhenqing Ye, Victor Jin, Qianben Wang. Convergent CREB1/FoxA1 transcriptional activity defines castration-resistant prostate cancer gene expression profile. [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 1387. doi:10.1158/1538-7445.AM2014-1387

Abstract LB-74: The NLR-related protein NWD1 is associated with prostate cancer and modulates androgen receptor signaling

Abstract Prostate cancer (PCa) is among the leading causes of cancer-related death in men. Androgen receptor (AR) signaling plays a seminal role in prostate development and homeostasis, and dysregulation of this pathway is intimately linked to prostate cancer pathogenesis and progression. Here, we identify the cytosolic NLR-related protein NWD1 as a novel modulator of AR signaling. We determined that expression of NWD1 becomes elevated during prostate cancer progression, based on analysis of primary tumor specimens. Experiments with cultured cells showed that NWD1 expression is up-regulated by the sex-determining region Y (SRY) proteins. Gene silencing by short hairpin RNA (shRNA), in conjunction with transcriptional profiling, showed that NWD1 is required for expression of PDEF (prostate-derived Ets factor), which is known to bind and co-regulate AR. Of note, NWD1 modulates AR protein levels. Silencing NWD1 in PCa cell lines reduces the levels of androgen-stimulated accumulation of nuclear AR and suppresses activity of androgen-driven reporter genes. NWD1 knockdown potently suppressed growth of androgen-dependent LNCaP prostate cancer cells, thus showing its functional importance in an AR-dependent tumor cell model. Proteomics analysis suggested that NWD1 associates with various molecular chaperones commonly related to AR complexes. Altogether, our data suggest a role for tumor-associated over-expression of NWD1 in the dysregulation of AR signaling in PCa. Citation Format: Ricardo G. Correa, Maryla Krajewska, Carl F. Ware, Motti Gerlic, John C. Reed. The NLR-related protein NWD1 is associated with prostate cancer and modulates androgen receptor signaling. [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 LB-74. doi:10.1158/1538-7445.AM2014-LB-74

Abstract 3312: The antibiotic salinomycin cotargets two pivotal pathways in prostate cancer

Abstract Background: The androgen receptor (AR) and PI3K/AKT/mTOR signaling axis are two key pathways that are activated in prostate cancer (PC). Although interference with the AR axis is the common first-line treatment for controlling metastatic recurrent PC, fast-growing PC progresses rapidly on hormone therapy and chemotherapy to a terminal condition within 18 to 24 months. Inhibitors of the PI3K/AKT/mTOR axis, such as rapamycin and the dual inhibitor BEZ-235 (Novartis), are ineffective in treating metastatic PC in part due to feedback regulations that activate the AR axis. Reciprocally, inhibition of AR activity reduces the expression of an AKT phosphatase, which is an AR target gene, with consequent increase of the AKT kinase activity. The antibiotic salinomycin, a product of the bacterium Streptomyces albus, has known anti-neoplastic activity, and in a screen of 16,000 small molecules, salinomycin was found to be the most potent in causing apoptosis of breast cancer stem cells. We previously reported that salinomycin treatment elevated intracellular oxidative stress and induced growth arrest of PC cells due to apoptosis. Design and Results: Here we report further elaboration of the underlying mechanism for growth arrest of androgen-dependent (LNCaP) and castration-resistant (C4-2B) human prostate cancer cells by salinomycin at sub-micromolar concentrations. We report that salinomycin can significantly reduce mTOR activity in both LNCaP and C4-2B cells without altering the mTOR activity of non-malignant RWPE-1 prostate epithelial cells. The specificity of the salinomycin effect is evidenced by the result that other anti-cancer agents such as docetaxel, the plant lignan deoxypodophyllotoxin and vitamin D did not alter mTOR activity. Preliminary results suggest that salinomycin may also induce autophagy in PC cells. Importantly, AR expression was markedly reduced in salinomycin-treated prostate cancer cells, suggesting dual targeting of the AR pathway and PI3K/AKT/mTOR axis by this antibiotic. Conclusion: We conclude that salinomycin targets two pivotal pathways that promote prostate cancer progression. Unlike other PI3K/AKT/mTOR inhibitors, the AR-activating feedback cross regulation did not compromise the therapeutic potential of salinomycin. Salinomycin-targeted interventions of the survival signals for castration-resistant PC will further avoid chemotherapy-induced toxicity. Preclinical validation of our results will pave the way for testing the efficacy of salinomycin against prostate cancer in a clinical setting. Supported by a Merit-Review grant from the Department of Veterans Affairs. Citation Format: Nooshin Mirkheshti, Chung Song, Bandana Chatterjee. The antibiotic salinomycin cotargets two pivotal pathways in prostate cancer. [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 3312. doi:10.1158/1538-7445.AM2014-3312

Identification of an antibody fragment specific for androgen-dependent prostate cancer cells.

BackgroundProstate cancer is the most-diagnosed non-skin cancer among males in the US, and the second leading cause of cancer-related death. Current methods of treatment and diagnosis are not specific for the disease. This work identified an antibody fragment that binds selectively to a molecule on the surface of androgen-dependent prostate cancer cells but not benign prostatic cells.ResultsAntibody fragment identification was achieved using a library screening and enrichment strategy. A library of 109 yeast-displayed human non-immune antibody fragments was enriched for those that bind to androgen-dependent prostate cancer cells, but not to benign prostatic cells or purified prostate-specific membrane antigen (PSMA). Seven rounds of panning and fluorescence-activated cell sorting (FACS) screening yielded one antibody fragment identified from the enriched library. This molecule, termed HiR7.8, has a low-nanomolar equilibrium dissociation constant (Kd) and high specificity for androgen-dependent prostate cancer cells.ConclusionsAntibody fragment screening from a yeast-displayed library has yielded one molecule with high affinity and specificity. With further pre-clinical development, it is hoped that the antibody fragment identified using this screening strategy will be useful in the specific detection of prostate cancer and in targeted delivery of therapeutic agents for increased efficacy and reduced side effects.

Phorbol ester stimulates ethanolamine release from the metastatic basal prostate cancer cell line PC3 but not from prostate epithelial cell lines LNCaP and P4E6.

Background:Malignancy alters cellular complex lipid metabolism and membrane lipid composition and turnover. Here, we investigated whether tumorigenesis in cancer-derived prostate epithelial cell lines influences protein kinase C-linked turnover of ethanolamine phosphoglycerides (EtnPGs) and alters the pattern of ethanolamine (Etn) metabolites released to the medium.Methods:Prostate epithelial cell lines P4E6, LNCaP and PC3 were models of prostate cancer (PCa). PNT2C2 and PNT1A were models of benign prostate epithelia. Cellular EtnPGs were labelled with [1-3H]-Etn hydrochloride. PKC was activated with phorbol ester (TPA) and inhibited with Ro31-8220 and GF109203X. D609 was used to inhibit PLD (phospholipase D). [3H]-labelled Etn metabolites were resolved by ion-exchange chromatography. Sodium oleate and mastoparan were tested as activators of PLD2. Phospholipase D activity was measured by a transphosphatidylation reaction. Cells were treated with ionomycin to raise intracellular Ca2+ levels.Results:Unstimulated cell lines release mainly Etn and glycerylphosphorylEtn (GPEtn) to the medium. Phorbol ester treatment over 3h increased Etn metabolite release from the metastatic PC3 cell line and the benign cell lines PNT2C2 and PNT1A but not from the tumour-derived cell lines P4E6 and LNCaP; this effect was blocked by Ro31-8220 and GF109203X as well as by D609, which inhibited PLD in a transphosphatidylation reaction. Only metastatic PC3 cells specifically upregulated Etn release in response to TPA treatment. Oleate and mastoparan increased GPEtn release from all cell lines at the expense of Etn. Ionomycin stimulated GPEtn release from benign PNT2C2 cells but not from cancer-derived cell lines P4E6 or PC3. Ethanolamine did not stimulate the proliferation of LNCaP or PC3 cell lines but decreased the uptake of choline (Cho).Conclusions:Only the metastatic basal PC3 cell line specifically increased the release of Etn on TPA treatment most probably by PKC activation of PLD1 and increased turnover of EtnPGs. The phosphatidic acid formed will maintain a cancer phenotype through the regulation of mTOR. Ethanolamine released from cells may reduce Cho uptake, regulating the membrane PtdEtn:PtdCho ratio and influencing the action of PtdEtn-binding proteins such as RKIP and the anti-apoptotic hPEBP4. The work highlights a difference between LNCaP cells used as a model of androgen-dependent early stage PCa and androgen-independent PC3 cells used to model later refractory stage disease.

Arrestin2 modulates androgen receptor activation.

Androgen receptor (AR) has a pivotal role in the growth and survival of prostate cancer (PCa). Arrestin2 (Arr2) is a ubiquitous scaffolding/adaptor protein first characterized as a regulator of G protein-coupled receptor signaling. In this study, we report that Arr2 additionally functions as a positive regulator of AR expression and function in PCa cells. Expression level of Arr2 correlates with that of AR, and knockdown of Arr2 inhibits the expression of AR and its effectors prostate-specific antigen, transmembrane protease serine 2, FK506-binding protein 51 and fatty acid synthase. Mechanistically, the knockdown of Arr2 attenuates the binding of AR to androgen response elements and consequently decreases transcription of AR-regulated genes. The inhibition of AR by Arr2 knockdown occurs in both androgen-dependent and castration-resistant PCa (CRPC) cells, although the effect is more prominent in CRPC. Arr2 knockdown inhibits the in vitro CRPC cell proliferation, prostasphere growth and invasion, as well as the in vivo prostate tumor formation, local invasion and distant metastasis. These results illustrate a new role for Arr2 in the expression and activation of AR and its potential relevance as a target for therapeutic intervention and monitoring of disease progression.

Transcriptome analysis reveals a dynamic and differential transcriptional response to sulforaphane in normal and prostate cancer cells and suggests a role for Sp1 in chemoprevention.

Epidemiological studies provide evidence that consumption of cruciferous vegetables, like broccoli, can reduce the risk of cancer development. Sulforaphane (SFN) is a phytochemical derived from cruciferous vegetables that induces anti-proliferative and pro-apoptotic responses in prostate cancer cells, but not in normal prostate cells. The mechanisms responsible for this cancer-specific cytotoxicity remain unclear. We utilized RNA sequencing and determined the transcriptomes of normal prostate epithelial cells, androgen-dependent prostate cancer cells, and androgen-independent prostate cancer cells treated with SFN. SFN treatment dynamically altered gene expression and resulted in distinct transcriptome profiles depending on prostate cell line. SFN also down-regulated the expression of genes that were up-regulated in prostate cancer cells. Network analysis of genes altered by SFN treatment revealed that the transcription factor Specificity protein 1 (Sp1) was present in an average of 90.5% of networks. Sp1 protein was significantly decreased by SFN treatment in prostate cancer cells and Sp1 may be an important mediator of SFN-induced changes in expression. Overall, the data show that SFN alters gene expression differentially in normal and cancer cells with key targets in chemopreventive processes, making it a promising dietary anti-cancer agent.

Targeted BikDD expression kills androgen-dependent and castration-resistant prostate cancer cells.

Targeted gene therapy is a promising approach for treating prostate cancer after the discovery of prostate cancer-specific promoters such as prostate-specific antigen, rat probasin, and human glandular kallikrein. However, these promoters are androgen dependent, and after castration or androgen ablation therapy, they become much less active or sometimes inactive. Importantly, the disease will inevitably progress from androgen-dependent (ADPC) to castration-resistant prostate cancer (CRPC), at which treatments fail and high mortality ensues. Therefore, it is critical to develop a targeted gene therapy strategy that is effective in both ADPC and CRPC to eradicate recurrent prostate tumors. The human telomerase reverse transcriptase-VP16-Gal4-WPRE integrated systemic amplifier composite (T-VISA) vector we previously developed, which targets transgene expression in ovarian and breast cancer, is also active in prostate cancer. To further improve its effectiveness based on androgen response in ADPC progression, the ARR2 element (two copies of androgen response region from rat probasin promoter) was incorporated into T-VISA to produce AT-VISA. Under androgen analog (R1881) stimulation, the activity of AT-VISA was increased to a level greater than or comparable to the cytomegalovirus promoter in ADPC and CRPC cells, respectively. Importantly, AT-VISA demonstrated little or no expression in normal cells. Systemic administration of AT-VISA-BikDD encapsulated in liposomes repressed prostate tumor growth and prolonged mouse survival in orthotopic animal models as well as in the transgenic adenocarcinoma mouse prostate model, indicating that AT-VISA-BikDD has therapeutic potential to treat ADPC and CRPC safely and effectively in preclinical setting.

Hydrogen Sulfide Represses Androgen Receptor Transactivation by Targeting at the Second Zinc Finger Module

Androgen receptor (AR) signaling is indispensable for the development of prostate cancer from the initial androgen-dependent state to a later aggressive androgen-resistant state. This study examined the role of hydrogen sulfide (H(2)S), a novel gasotransmitter, in the regulation of AR signaling as well as its mediation in androgen-independent cell growth in prostate cancer cells. Here we found that H(2)S inhibits cell proliferation of both androgen-dependent (LNCaP) and antiandrogen-resistant prostate cancer cells (LNCaP-B), with more significance on the latter, which was established by long term treatment of parental LNCaP cells with bicalutamide. The expression of cystathionine γ-lyase (CSE), a major H(2)S producing enzyme in prostate tissue, was reduced in both human prostate cancer tissues and LNCaP-B cells. LNCaP-B cells were resistant to bicalutamide-induced cell growth inhibition, and CSE overexpression could rebuild the sensitivity of LNCaP-B cells to bicalutamide. H(2)S significantly repressed the expression of prostate-specific antigen (PSA) and TMPRSS2, two AR-targeted genes. In addition, H(2)S inhibited AR binding with PSA promoter and androgen-responsive element (ARE) luciferase activity. We further found that AR is post-translationally modified by H(2)S through S-sulfhydration. Mutation of cysteine 611 and cysteine 614 in the second zinc finger module of AR-DNA binding domain diminished the effects of H(2)S on AR S-sulfhydration and AR dimerization. These data suggest that reduced CSE/H2S signaling contributes to antiandrogen-resistant status, and sufficient level of H(2)S is able to inhibit AR transactivation and treat castration-resistant prostate cancer.

Zeb1 promotes androgen independence of prostate cancer via induction of stem cell-like properties.

Current androgen deprivation therapy often leads to androgen independence. However, mechanism of the therapeutic failure is still not well understood. Here, we demonstrate elevated expression of Zeb1 in androgen-independent prostate cancer cells and prostate tumors of castrated PTEN conditional knockout mice. While Zeb1 shRNA resulted in a sensitization of androgen-independent prostate cancer cells, forced Zeb1 expression caused androgen-dependent prostate cancer cells to be more resistant to androgen deprivation. Moreover, such effects appeared to be mediated by induction of pluripotent genes or stem cell-like properties. Collectively, these findings suggest that inhibition of Zeb1 might be a potential therapeutic strategy for treatment of androgen-independent prostate cancer.

Overexpression of α (1,6) fucosyltransferase associated with aggressive prostate cancer

Aberrant protein glycosylation is known to be associated with the development of cancers. The aberrant glycans are produced by the combined actions of changed glycosylation enzymes, substrates and transporters in glycosylation synthesis pathways in cancer cells. To identify glycosylation enzymes associated with aggressive prostate cancer (PCa), we analyzed the difference in the expression of glycosyltransferase genes between aggressive and non-aggressive PCa. Three candidate genes encoding glycosyltransferases that were elevated in aggressive PCa were subsequently selected. The expression of the three candidates was then further evaluated in androgen-dependent (LNCaP) and androgen-independent (PC3) PCa cell lines. We found that the protein expression of one of the glycosyltransferases, α (1,6) fucosyltransferase (FUT8), was only detected in PC3 cells, but not in LNCaP cells. We further showed that FUT8 protein expression was elevated in metastatic PCa tissues compared to normal prostate tissues. In addition, using tissue microarrays, we found that FUT8 overexpression was statistically associated with PCa with a high Gleason score. Using PC3 and LNCaP cells as models, we found that FUT8 overexpression in LNCaP cells increased PCa cell migration, while loss of FUT8 in PC3 cells decreased cell motility. Our results suggest that FUT8 may be associated with aggressive PCa and thus is potentially useful for its prognosis.

Effect of enzalutamide (EZ) exposure time on androgen receptor (AR) splice variant (SV) expression and gene transcription correlates in androgen-dependent (AD) prostate cancer (PC) cells.

e22066 Background: The emergence of castration-resistant (CR) PC limits the benefits of androgen deprivation therapy (ADT) in controlling recurrent AD PC. ARSVs lacking the ligand-binding domain ar...