Nitric oxide (NO) and its oxidative reaction products have been repeatedly shown to block steroid receptor function via nitrosation of zinc finger structures in the DNA-binding domain (DBD). In consequence NO-donors could be of special interest for the treatment of deregulated androgen receptor(AR)-signaling in castration resistant prostate cancer (CRPC).Prostate cancer (PCa) cells were treated with JS-K, a diazeniumdiolate derivate capable of generating large amounts of intracellular NO following activation by glutathione S-transferase. Generation of NO was determined indirectly by the detection of nitrate in tissue culture medium or by immunodetection of nitrotyrosine in the cytoplasm. Effects of JS-K on intracellular AR-levels were determined by western blotting. AR-dimerization was analyzed by mammalian two hybrid assay, nuclear translocation of the AR was visualized in PCa cells transfected with a green fluorescent AR-Eos fusion protein using fluorescence microscopy. Modulation of AR- and WNT-signalling by JS-K was investigated using reporter gene assays. Tumor cell proliferation following JS-K treatment was measured by MTT-Assay.The NO-releasing compound JS-K was shown to inhibit AR-mediated reporter gene activity in 22Rv1 CRPC cells. Inhibition of AR signaling was neither due to an inhibition of nuclear import nor to a reduction in AR-dimerization. In contrast to previously tested NO-donors, JS-K was able to reduce the intracellular concentration of functional AR. This could be attributed to the generation of extremely high intracellular levels of the free radical NO as demonstrated indirectly by high levels of nitrotyrosine in JS-K treated cells. Moreover, JS-K diminished WNT-signaling in AR-positive 22Rv1 cells. In line with these observations, castration resistant 22Rv1 cells were found to be more susceptible to the growth inhibitory effects of JS-K than the androgen dependent LNCaP which do not exhibit an active WNT-signaling pathway.Our results suggest that small molecules able to inhibit WNT- and AR-signaling via NO-release represent a promising platform for the development of new compounds for the treatment of CRPC. Nitric oxide (NO), a free radical gas, is a pleiotropic molecule critical to a number of physiological and pathological processes. NO-releasing drugs are a growing class of promising new therapeutics with applications in a large variety of diseases like cardiovascular and respiratory disorders, osteoporosis, Alzheimer's disease, inflammatory lesions and urinary incontinence -4. Moreoin vitro the development of a castration resistant phenotype is mostly based on the loss of the AR in PCa cells, several clinical studies demonstrated that the AR is rarely lost in CRPC cells in vivo diazen-1-ium-1,2-diolat (DETA/NO) has recently been demonstrated . HoweverStudying the effects of JS-K on AR- and WNT-signalling in the AR-positive PCa cell lines 22Rv1 and LNCaP we were able to demonstrate that only a 100-fold lower concentration of the GST-activated JS-K are necessary to reach comparable biological effects in PCa cells than the commonly used DETA/NO that spontaneously generates NO in physiological fluids. Our data show that NO-prodrugs, capable of generating large intracellular amounts of NO, may be suitable for the treatment of advanced prostate cancer.Renilla reniformis luciferase reporter plasmid pRL-tk-LUC was a product of Promega . Mouse monoclonal antibodies directed against beta actin (ab8224) and nitrotyrosine (ab7048) were products of Abcam . Mouse monoclonal antibody AR441 directed against the N-terminus of the AR was purchased from Dako . The NO-donors O2--1-[(4-ethoxycarbo-nyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K), (Z)-1-[N-(2-Aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO) and the androgen dihydrotestosterone (DHT) were a products of Sigma Aldrich .Probasin promoter driven luciferase-reporter plasmid (pGL3E-Probasin) was kindly provided by Prof. Dr. Z. Culig . The pARt1EosFP expression plasmid coding for a green fluorescent AR-Eos fusion protein (AR-EosFP) was a gift from Prof. F. Oswald . The CMV-promoter driven β-catenin expression plasmid pbCAT, containing human β-catenin cDNA with an activating S33Y mutation was provided by Dr. H. Clevers . TCF-reporter plasmids pTopFlash (TOP) and pFopFlash (FOP), containing three copies of wild-type or mutant TCF-binding sites upstream of a thymidine kinase minimal promoter driving a luciferase gene, were purchased from Upstate Biotechnology . 22Rv1, LNCaP, DU-145 and PC-3 cells were purchased from the American Type Culture Collection . LNCaP-SSR, a castration resistant LNCaP subline ,26, was (1) AR-signalling: Cells were transfected with 200 ng/well of the AR-dependent reporter-plasmid pGL3E-Probasin. The pRL-tk-LUC vector coding for a Renilla luciferase under control of a constitutively active thymidine kinase promoter was co-transfected (80 ng/well) to correct for transfection efficiency. After transfection cells were grown in RPMI 1640 with 5% dextran charcoal treated steroid free, dextran charcoal treated FBS  and treated with 5 nM DHT and different concentrations of JS-K. (2) WNT-signalling: Luciferase reporter plasmids pTopFlash (250 ng/well) and pFopFlash (250 ng/well) were mixed either with pbCAT expression vector (250 ng/well) or insert-free vector (250 ng/well). pRL-tk-luc (80 ng/well) was co-transfected to correct for transfection efficiency. After an incubation period of 12 h in RPMI-1640 with 5% FBSdcc, medium was changed to RPMI-1640 containing 2.5% FBSdcc with increasing concentrations of JS-K.22Rv1 cells were grown on 24-well plates . Transfections were performed using FuGene HD transfection reagent  according to the manufacturer's instructions. AR- and WNT-specific reporter gene assays were performed as recently described . In brieAR- and WNT-reporter activities were analyzed after 24 hours using the Dual Luciferase Reporter Assay System . Experiments were repeated at least three times and performed in triplicates, unless stated otherwise.To investigate the dimerization of the AR we used the CheckMate/FlexiVector Mammalian Two-Hybrid System from Promega . VP16 activation domain (pFN10A-AR) and Gal4 DNA binding domain tagged androgen receptor fusion constructs (pFN11A-AR) were cloned according to the supplier's suggestions by using pSG5-AR as template. DU-145 and PC-3 cells were grown in 24-well plates and co-transfected with 150 ng/well of pFN10A-AR, pFN11A-AR and the Gal4 reporter construct pGL4.31. After transfection, cells were cultured and luciferase activities were determined as recently described .Nuclear translocation of the AR was analyzed in AR-negative DU-145 and PC-3 cells transfected with the green fluorescent AR-EosFP ,29. TherProteins were extracted from cells using RIPA buffer as recently described . ProteinNO was determined indirectly by the photometric measurement of nitrite according to Green et al. For immunocytochemistry 22Rv1 cells were cultured on glass coverslips  washed (2 × 5 min) with PBS and fixed with 3.7% paraformaldehyd for 15 min at room temperature, washed (3 × 10 min) in PBS and permeabilized in 0.2% Triton X-100 with 1% BSA in PBS for 5 min at room temperature. The cells were washed (3 × 10 min) with PBS containing 1% BSA. Cells were then incubated with the monoclonal anti-tyrosine antibody  for 30 min at 37°C. Cells were then washed (3 × 10 min), the nuclei stained with DAPI (2.5 μg/ml in PBS) for 15 min, washed (3 × 10 min) and embedded with glycine and polyvinyle alcohol 4-88 .Cell viability was determined by means of a colorimetric MTT-assay measuring the reduction of the water soluble tetrazolium bromide into its insoluble formazan derivative by functional mitochondria ,33.Data are reported as means ± standard deviation. Analysis was performed with Student's T-test (two tailed for independent samples) with p < 0.05 considered as significant.neg.In a previous paper NO generated by DETA/NO was shown to nitrosate tyrosine residues in proteins to nitrotyrosine . Therefoneg harbouring the inactive JS-K metabolites, showed any nitrotyrosine staining  led to a nitrosation of intracellular proteins as shown by fluorescescence microscopy of nitrotyrosine residues Figure . In contg Figure .Renilla luciferase plasmid under control of a constitutively active thymidine kinase promoter (control of transfection efficiency). Subsequently cells were incubated for 44 hours with 5 nM DHT and increasing concentrations of JS-K or JS-Kneg. JS-K significantly inhibited DHT-induced AR-transactivation in a dose-dependent manner. Inhibition was already significant at a concentration of 2 μM JS-K. In contrast, JS-Kneg was unable to diminish AR transcriptional activity even at higher concentrations  - retinoic X receptor (RXR)-heterodimerization [in vivo, we performed an AR-specific mammalian two hybrid (M2H)-assay in AR-negative PC-3 and DU-145 cells  whereas in presence of DHT the AR was transported to the nucleus  ,37. Subss Figure . As seens Figure .neg had no influence on intracellular AR-levels  that activate TCF/LEF-dependent reporter gene constructs [Several lines of evidence indicate that NO is involved in the regulation of the WNT/β-catenin pathway in various tumors -44. A constructs . As LNCanstructs  we testeneg served as negative control. Cellular proliferation was assessed by means of a colorimetric MTT assay, measuring the reduction of tetrazolium salts to formazan derivatives by functional mitochondria [Based on our observations we tested the antiproliferative effects of JS-K on prostate cancer cell lines. JS-Kchondria ,33. Althchondria  they arechondria ,46,47. Tchondria .A first series of experiments indicated that castration resistant 22Rv1 cells were more susceptible to the growth inhibitory effects of JS-K than the androgen sensitive LNCaP cells Figure . MaximalGiven the highly divergent nature of the AR-positive cell lines 22Rv1 and LNCaP we repeated the proliferation experiments using the androgen sensitive LNCaP and LNCaP-SSR, an AR-positive castration resistant LNCaP-subline. The AR-negative DU-145 served as control (AR off-target effects). Interestingly, the castration resistant LNCaP-SSR cells were more susceptible to the growth inhibitory effects  than the parental LNCaP cells  Figure . In contCurrent treatment for advanced PCa is mainly based on androgen ablation therapies like chemical or surgical castration or application of anti-androgens. Although hormonal therapy is initially very effective, the benefit of androgen withdrawal is only transitory. While still expressing high levels of functional AR, some PCa cells acquire the ability to grow and survive under castrate levels of circulating androgens. The mechanisms involved in an altered AR-signalling of CRPC cells include an over-expression/amplification of the AR (hypersensitive pathway), point mutations broadening ligand-specificity of the receptor (promiscuous pathway) as well as activation of the AR by peptide growth factors or cytokines (outlaw pathway) . A recen4-type zinc fingers, located in the DBD of these receptors [Nitric oxide, a free radical gas, has been repeatedly shown to impair the function of nuclear receptors via targeting their cyseceptors . This iseceptors . As the in vitro using the spontaneous NO-donor Deta/NO [Inhibitory effects of NO on AR-function in PCa cells was first described  Deta/NO . In a re Deta/NO . No card Deta/NO . Althoug Deta/NO ,51 suppoin vivo studies using tumor xenografts in a mouse model, showed that JS-K exhibits a strong anti-tumor activity and is generally well tolerated by the host organism [Commonly used NO-Donors like Deta/NO, spontaneously dissociate in a pH dependent process thereby releasing the free radical NO. Unfortunately NO exhibits only a relatively short half-life in the tissue culture medium or body fluids. Therefore, we tested a potent NO-prodrug, the non-ionic nitroaromatic diazeniumdiolate JS-K. The compound was shown to be activated by glutathione S-transferase (GST), a key phase II detoxification enzyme that is frequently over-expressed in cancer tissue. Among the various isoforms of GSTs which are located in the cytoplasm and the mitochondria, mainly the π isoform is able to form the so called < Meisenheimer complex > which fiorganism ,23,24.In order to test the effects of JS-K on AR signalling, we treated the AR-positive CRPC cell line 22Rv1 grown in presence of DHT with increasing concentrations of JS-K. Generation of intracellular NO was monitored indirectly by detection of nitrotyrosine using fluorescence microscopy. JS-K was able to inhibit the genomic functions of the AR in 22Rv1 cells. The inhibition of AR-signalling was neither due to an inhibition of AR-dimerization nor to an inhibition of nuclear translocation. Interestingly, JS-K was able to diminish intracellular levels of all AR-isoforms in PCa cells. However, in contrast to the dramatic downregulation of AR-V and AR in 22Rv1 cells the intracellular AR levels of LNCaP were only slightly affected.melanogaster ecdysteroid receptor (EcR) when overexpressed in chinese hamster ovary cells, CHO-K1 [So far, studies on intracellular steroid receptor levels following NO-treatment are sparse. In a previous study using Deta/NO as an NO-donor the concentration of the endogenous AR in LNCaP remained unaffected . In cont, CHO-K1 . AlthougThe fact that NO is able to inhibit the canonical WNT-pathway in different tumor cells -44 incluEx3dup) and a C-terminally truncated, constitutively active AR-V [Ex3dup is able to form androgen independent heterodimers with AR-V in 22Rv1 cells, its expression of 3 zinc finger structures makes it probably also more susceptible to the effects of NO. The fact that NO targets the zinc finger structures of wild type AR, mutated AR-forms as well as AR-Vs suggests that NO-donors are promising compounds for the treatment of deregulated AR-activity. Moreover, the observation that CRPC cells like 22Rv1 or LNCaP-SSR, expressing either constitutively active AR-Vs or functionally deregulated full length AR, are more susceptible to the effects of JS-K than LNCaP cells further supports this assumption.In proliferation assays castration resistant 22Rv1 and LNCaP-SSR cells were more susceptible to the growth inhibitory effects of JS-K than LNCaP. Whereas LNCaP cells largely depend on androgenic stimuli, the 22Rv1 and LNCaP-SSR cells are able to grow under androgen deprived conditions. LNCaP cells express a full length AR with a point mutation at position 877 (T877A), enabling the AR to be stimulated by different steroids (promiscuous AR) . In contive AR-V . In the ive AR-V ,48. Althin vitro studies we conclude that GST-activated NO-prodrugs like JS-K show features which might be suited for the treatment of advanced PCa. Therapies targeting the AR and the canonical WNT pathway may lead to a more efficient treatment of CRPC. Therefore, compounds like JS-K may serve as leader compounds for the development of NO-based therapeuticals, targeting key structures in castration resistant prostate cancer cells.From our The authors declare that they have no competing interests.ML, K-D S and MVC conceived the study; ML, WS, AH performed the experiments, MS AJS contributed to data analysis; MVC, K-D S, AJS, MS wrote the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/130/prepubFigure 1S: JS-K does not inhibit AR-dimerization. Complementary data to experiment presented in Figure 3: AR-dimerization was determined in DU-145 cells using the CheckMate Mammalian Two-Hybrid System described in Material and Methods. Results are mean values of three independent experiments performed in quadruplicates. (DOC 20 kb).Click here for fileFigure 2S: NO does not influence hormone-induced nuclear translocation of AR-EosFP in DU-145. Complementary data to experiment presented in Figure Click here for file


The O-type forkhead domain transcription factor (FOXO) is involved in many biological processes such as aging, the oxidative stress response, and growth regulation. FOXO activity is tightly controlled within cells. In particular, growth factor signaling pathways and the oxidative stress response can both stimulate nuclear translocation of this transcription factor. Here, we show that tetrahydrocurcumin (THC), a curcumin metabolite, regulates the oxidative stress response and aging via FOXO. In NIH3T3 cells, THC induced nuclear accumulation of FOXO4, a member of the FOXO family of transcription factors, by inhibiting phosphorylation of protein kinase B (PKB)/Akt. In Drosophila melanogaster, THC attenuated the oxidative stress response, an effect that was blocked in a foxo mutant background. THC also extended the life span of Drosophila under normal conditions, and loss of either foxo or Sir2 activity eliminated this effect. Based on these results, THC may regulate the aging process via an evolutionarily conserved signaling pathway that includes both foxo and Sir2. Drosophila melanogaster, Caenorhabditis elegans, and mammals dCTP and fed to 14-d wild-type (Oregon-R) virgin males or females . The uptake of [32P]dCTP was measured by scintillation counting for 4 min/sample .EtOH (5%) or THC (50 μM in 5% EtOH) was mixed with [


Agonist-mediated endocytic traffic of α1A-AR was assessed by real-time imaging of living, stably transfected human embryonic kidney 293A cells (HEK-293A). α1A-AR was internalized dynamically in cells with agonist stimulation, and actin filaments regulated the initial trafficking of α1A-AR. α1A-AR-induced activation of ERK1/2 but not p38 MAPK was sensitive to disruption of endocytosis, as demonstrated by 4°C chilling, dynamin mutation and treatment with cytochalasin D (actin depolymerizing agent). Activation of protein kinase C (PKC) and C-Raf by α1A-AR was not affected by 4°C chilling or cytochalasin D treatment. U73122 (a phospholipase C [PLC] inhibitor) and Ro 31–8220 (a PKC inhibitor) inhibited α1B-AR- but not α1A-AR-induced ERK1/2 activation. These data suggest that the endocytic pathway is involved in α1A-AR-induced ERK1/2 activation, which is independent of Gq/PLC/PKC signaling.G protein-coupled receptors (GPCRs) activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in α Reconstitution of α1A-AR but not α1B-AR induced ERK1/2 activation and rescued α1ABKO myocytes from cell death induced by norepinephrine, doxorubicin, and H2O21A-AR specifically restored ERK1/2 activation in α1ABKO myocytes suggests that α1A-AR and α1B-AR activate ERK1/2 through differential mechanisms. However, studies to date have not consistently identified major differences in immediate signaling responses initiated by α1A-AR and α1B-AR.α1A-AR is regulated by many mechanisms, including phosphorylation, protein-protein interaction, protein traffic, and transcription 1-ARs activate intracellular effectors, including phospholipase C β (PLCβ), inositol trisphosphate, protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and calcium signals, often through a heterotrimeric G protein-dependent manner 1A-AR variant, which was unable to couple to Gq, could also induce calcium influx when coactivated by β2-AR 1A-AR, even though uncoupled from Gq, may remain competent for induction of signaling events through yet unknown pathways.α2-AR were suppressed in cells transfected with dynamin-mutant K44A (Dyn-K44A), which is defective in GTPase activity 1A-AR endocytic trafficking and activation of MAPKs is still unknown.Increasing evidence has shown the existence of receptor signaling from the endocytic process. For instance, activation of ERK1/2 via epidermal growth factor receptor (EGFR) and β1A-AR. By real-time tracking, we found a time-dependent dynamic pattern of α1A-AR endocytosis with stimulation and the involvement of the cytoskeleton, especially actin-filaments, in this process. This relationship was further examined by colocalization of α1A-AR with reorganized cytoskeletons. We provide evidence for an involvement of endocytosis in α1A-AR-induced activation of ERK1/2, which differs from that of α1B-AR via a Gq/PLC/PKC pathway.We aimed to investigate whether an endocytic process is involved in ERK1/2 activation induced by αCytochalasin D, nocodazole, PE and U73122 were from Sigma . Ro 31–8220, prazosin and phorbol 12-myristate, 13-acetate (PMA) were from Calbiochem . Phospho-p38 MAPK (Thr180/Tyr182), -p42/44 MAPK (Thr202/Tyr204), -PKC (pan) (Ser660), and -C-Raf (Ser338) antibodies were from Cell Signaling Technology . Antibodies against ERK1/2, PKC (pan), C-Raf, p38, FLAG-tag and HA-tag were from Santa Cruz Biotechnology . Horseradish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit antibodies were from Beijing Zhongshan Golden Bridge Biotechnology. Alexa 488-conjugated WGA, Alexa 555 and 633 IgG and Alexa 488-conjugated phalloidin were from Invitrogen. All other chemicals were of analytical grade.1A-AR or α1B-AR were described previously HEK-293A cell lines were obtained from Invitrogen. Receptor constructs and HEK-293A cells stably transfected with α2. Fluorescence was excited at 532-nm by an argon laser . Movies were acquired at a frame rate of 20 Hz by use of MetaMorph software (Molecular Devices). Trajectories from cells observed under the given labeling procedure were plotted and resolved as described previously FLAG-tagged receptors were labeled with anti-FLAG monoclonal antibody (12.5 µg/ml) for 10 min and then Alexa FLour® 555 goat anti-mouse IgG (Invitrogen) (3.75 µg/ml) for 10 min as described previously After drug treatment, cells were fixed for 15 min in 4% paraformaldehyde in PBS and permeabilized with 0.2% Triton X-100. After washes with PBS, cells were incubated for 25 min with TRITC-labeled phalloidin (Sigma). The samples were viewed under a laser scanning confocal microscope  with a Plan-Apo 63×/1.32 oil immersion objective (Leica Microsystems); images were collected by use of Leica TCS SP2 v2.611537. The 488- and 532-nm laser beam was focused by a Leica Apochromat with <200 lW power irradiation. The pinhole size was 1 airy unit.Protein expression was examined by western blot analysis as previously described 1A-AR construct. α1A-AR was detected on the surface of living HEK-293A–α1A-AR cells by use of a monoclonal primary antibody and Alexa-555 IgG  versus time . At the early stage of the activation (first 30 min), the receptor mainly moved at a low velocity at a peak of about 0.3 µm/s. After stimulation for 40 to 60 min, movements became much faster, with high velocity trajectories increased gradually. The main peak of the highest velocity was about 0.8 µm/s. Thus, in general, the active movement of α1A-AR vesicles was slower at the early phase of endocytosis and faster at the later phase.We then resolved the velocities at different time after PE stimulation 1A-AR vesicles largely colocalized with F-actin after 20-min PE stimulation  attenuated α1A-AR- but not α1B-AR-induced ERK1/2 activation 1A-AR and α1B-AR differentially activate downstream effectors, which further underscores the complexity of α1-AR signaling pathways. We found endocytic trafficking involved in α1A-AR induced ERK1/2 activation and that it was independent of Gq/PLC/PKC signaling. By contrast, α1B-AR-induced ERK1/2 activation required Gq/PLC/PKC signaling, as was previously reported 1A-AR uses pathways different from those of α1B-AR to activate ERK1/2.Disruption of endocytosis by 4°C chilling, dynamin mutation and F-actin depolymerizing all indicated that the endocytic process was associated in αq-protein signaling pathway in α1A-AR function. An α1A-AR mutant defective in Gq coupling could also activate calcium influx when coactivated with β2-AR q signaling events in response to α1A-AR stimulation. Activation of calcium influx and PKC may be key events coupling Gq- and Gi-coupled receptor activation to MAPK activation 2+ chelator BAPTA dose-dependently abolished norepinephrine (NE) -stimulated Ca2+ responses but not ERK1/2 activation. The potent PKC inhibitor bisindolylmaleimide I dose dependently inhibited ERK1/2 activation by phorbol ester tumor-promoting agent but not NE. Thus, Ca2+ release and PKC activation are neither necessary nor sufficient for α1A-AR-mediated activation of ERK1/2 in PC12 cells stably transfected with α1A-AR 1A-AR may be independent of traditional Gq-coupled second-messenger pathways. We found that α1A-AR-induced ERK1/2 activation was unaffected by the PLC inhibitor U73122 and PKC inhibitor R0 31–822, which further identifies a non-Gq signaling pathway involved in α1A-AR-mediated ERK1/2 activation.A number of studies have found a non-G2-AR-induced activation of ERK1/2 1A-AR with β-arrestins was much weaker than that of α1B-AR. In addition, α1A-AR did not bind AP50, a subunit of the clathrin adaptor complex AP2. Moreover epinephrine-induced increase of the association of the α1A-AR and β-arrestin 1 or 2 was not statistically significant 1A-AR and α1A-AR-induced ERK1/2 activation is important. PI3K has a potential role in the α1A-AR induced ERK1/2 activation 1A-AR mediated ERK1/2 activation through an endocytosis pathway but remains to be elucidated.Arrestins are critically important for desensitization, endocytosis, and G protein-independent signaling of GPCRs 1A-AR stimulation in cells. Through analysis of receptor tracking in live cells and confocal microscopy, we conclude that the α1A-AR endocytic process is actin-related. Endocytosis and actin reorganization are involved in α1A-AR induced activation of ERK1/2 but not p38, and Gq/PLC/PKC signaling is not required in this process. Thus, we reveal a novel pathway of ERK1/2 activation in α1-AR subtypes. We provide the first biological evidence for a role of endocytosis in the signaling of the α1-AR family, which challenges the classical view that α1A-AR act through Gq/PLC/PKC signaling. Moreover, the mechanism by which α1A-AR induces ERK1/2 activation differs from that of α1B-AR. We provide a possible molecular explanation for the difference between α1A-AR and α1B-AR in activation of ERK1/2 in cardiac myocytes. Because receptor vesicles could link to various intracellular membrane compartments in the endocytic process, the distinct spatiotemporal profile of ERK1/2 activation induced by α1A-AR has profound implications in α1A-AR-mediated survival signaling in cardiac myocytes. In-depth studies of this signaling pathway should add great potential for developing more efficacious and/or safer treatment of heart failure and other clinical conditions.In summary, we identified a putative role of endocytosis in the regulation of the MAPK pathway under αFigure S11A-AR stimulation. HEK-293A–α1A-AR cells were pre-incubated with or without anti-FLAG antibody and Alexa-555 IgG. After phenylephrine (PE) treatment, the phosphorylation level of ERK1/2 did not show significant difference among labeled and unlabeled cells. Prazosin  inhibited the PE-induced ERK1/2 activation in both.Labeling with antibodies did not impair the ERK1/2 signaling induced by α(TIF)Click here for additional data file.


Prostate cancer (PC) is the second most frequently diagnosed cancer and the second leading cause of cancer deaths in man. The treatment of localized PC includes surgery or radiation therapy. In case of relapse after a definitive treatment or in patients with locally advanced or metastatic disease, the standard treatment includes the androgen-deprivation therapy (ADT). By reducing the levels of testosterone and dihydrotestosterone under the castration threshold, the ADT acts on the androgen receptor (AR), even if indirectly. The effects of the ADT are usually temporary and nearly all patients, initially sensitive to the androgen ablation therapy, have a disease progression after an 18–24 months medium term. This is probably due to the selection of the cancer cell clones and to their acquisition of critical somatic genome and epigenomic changes. This review aims to provide an overview about the genetic and epigenetic alterations having a crucial role in the carcinogenesis and in the disease progression toward the castration resistant PC. We focused on the role of the AR, on its signaling cascade and on the clinical implications that the knowledge of these aspects would have on hormonal therapy, on its failure and its toxicity. Figure 1).Prostate cancer (PC) is the second most frequently diagnosed cancer and the second leading cause (the first one is lung cancer) of cancer deaths in man. The estimate of new diagnoses and deaths from PC in 2012, in the United States, amounted to 241,740 and 28,170, respectively . In ItalFigure 2).Data show that mortality from PC is relatively lower compared to the total number of yearly diagnosed cases. Therefore most patients die with this disease rather than from it . The treFigure 3).The androgen deprivation (AD) can be achieved by the use of LHRH analogs  or bilateral orchiectomy . Through the circulating levels of testosterone and dihydrotestosterone (DHT) reduction, the ADT indirectly affects the androgen receptor (AR), which represents the real target of this therapy. The androgen biosynthesis consists in a multi-step process taking place in man’s gonads and adrenals and in which many enzymes are involved. Cytochrome P450c17α, with both 17α-hydroxylase and 17,20-lyase activity, has a key role among these proteins  is a significant event  are up-regulated in PC cells during the CRPC progression. In spite of extremely low levels of circulating androgens, the CRPC progression remains dependent on the androgen-driven activity and PC cells keep the AR pathway active in different ways. An hypothesis standing to explain this observation is that alternative sources of androgenic steroids exist. Pioneer works, confirmed by recent researches in this area, indicated that intraprostatic amount of testosterone and DHT remains moderately high despite their castration serum levels . Locke eAndrogen receptor genetic changes leading to aberrant functioning of the AR pathway may underlie the development of resistance to castration, allowing tumor cells to avoid the normal stimuli to growth. Largely, we are talking about somatic mutations in the AR gene, resulting in an increase of the potential ligands which bind and activate the AR receptor. The AR somatic mutation would include missense mutations like T877A and L701H. The T877A missense mutation has been described for the first time in LNCaP cell lines and affects the LBD of the AR. This alteration allows to ligands different from testosterone and DHT, such as progestins, estrogens, and antiandrogens to activate the AR. This may provide a molecular basis to “withdrawal syndrome”  or repress (co-repressors) the transcriptional activity of the AR. Genes mutation or the aberrant expression of this molecules may participate in PC progression . SeveralThe possibility of a cross-talk between the AR pathway and intracellular signaling cascades activated by IGF-1, KGF, and EGF, leading to transcription of androgen controlled genes, in the absence of the ligand exists . The Erb2 along with a corticosteroid) as standard treatment in the CRPC is based on the survival advantage (approximately 3 months in median OS when compared with mitoxantrone and prednisone) observed in two independent phase III studies . However, most men treated with this drug experienced progression of their disease within 1 year from the start of chemotherapy. Between 2010 and 2011 the cabazitaxel, the sipuleucel-T and the abiraterone acetate were also approved by the FDA for treatment of metastatic CRPC. In addition, a novel new bone-targeting monoclonal antibody, denosumab (Xgeva), and an LHRH antagonist, degarelix (Firmagon), have been introduced into clinical practice. Cabazitaxel is a microtubule inhibitor approved by FDA for the treatment of patients with CRPC progressed after docetaxel. Cabazitaxel, in a phase III multicenter study, in which it was compared with mitoxantrone, showed a statistically significant advantage in terms of median overall survival (15.1 vs. 12.7) and PFS (2.8 vs. 1.4; Currently, chemotherapy represents one of the possible treatment options in the setting of the CRPC, providing modest survival and palliative benefits. However, many other therapeutic options are now available. With the FDA approval of Docetaxel in 2004 prospects on the therapeutic possibilities for this type of cancer has gradually expanded. Docetaxel-based chemotherapy (given every 3 weeks at a dose of 75 mg/mNowadays treatment options available for the CRPC are many and new questions emerge. First, how these new drugs have to be administered in clinical practice. In the era of the personalized cancer therapies, the deep knowledge of underlying mechanisms of the castration resistance of PC has become a topic of primary importance. Research in this field aims to obtain the tools in order to choose the most effective and less toxic therapy for each patient. This is more evident if we think about the impact of the hormone therapy on patients with PC quality life. In this way risks and benefits, ratio of a single therapy, have a greater significance. New biomarkers able to predict which patients may really benefit from a specific therapy without being exposed to an unnecessary toxicity, are required. For the future it should be possible to understand the genetic background of our patients with regard to some critical enzymes which could influence both response and resistance to drugs, before starting a specific therapy. This chance could make it really feasible to personalize the therapy in individual patients.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Increased expression of histone deacetylases (HDACs) and activation of the PI3K-Akt-mTORC1 pathway are common aberrations in prostate cancer (PCa). For this reason, inhibition of such targets is an exciting avenue for the development of novel therapeutic strategies to treat patients with advanced PCa. Previous reports demonstrated that HDAC inhibitors (HDACi) increases DNA damage and induce greater apoptosis in PCa cell lines that express androgen receptor (AR). In this study we utilized the AR negative PCa cell line and observed that re-expression of AR (PC3-AR) results in greater levels of apoptosis when treated with the pan-DACi, panobinostat (PAN). PAN mediated apoptosis in PC3 and PC3-AR cells was associated with increased levels of double strand DNA breaks, indicated by p-ɣH2AX. Further, PAN treatment in PC3-AR cells resulted in moderate attenuation of the ATM-Akt-ERK DNA damage response pathway. For this reason, we combined PAN with the dual PI3K-mTOR inhibitor, BEZ235. Combination of PAN with BEZ235 resulted in significant attenuation of the DNA damage repair protein ATM and significantly increased anti-tumor activity compared to each single treatment. Overall, superior anti-tumor activity with combination of PAN with BEZ235 was independent of AR status. These findings suggest that this therapeutic strategy should be further developed in clinical trials. Castrate resistant prostate cancer (CRPC) represents an aggressive and incurable phenotype of prostate cancer. Until recently, patients who progressed after receiving androgen deprivation therapy (ADT) had minimal second line therapeutic options available. Within the last 2 years a dramatic shift in the therapeutic landscape for CRPC has emerged, resulting in the FDA approval of 5 novel agents -5. AlthoHistone deactelyases (HDACs) are demonstrated to be over-expressed in multiple cancers, including prostate cancer -8. HDAC Pten [The PI3K-Akt-mTORC1 signaling axis is often constitutively activated in prostate cancer, primarily due to loss of expression/function of the tumor suppressor gene Pten . RecentlPten . Akt actPten . ActivatPten -23.Selective mTORC1 inhibitors including rapamycin and its analogs have been investigated for the treatment of prostate cancer, though with limited success. This failure is thought to be due to the reciprocal feedback loop and activation of rapamycin-insensitive (mTORC2) mTOR complex leading to Akt activation -26 and iBEZ235 (Novartis) is an orally available PI3K/mTOR inhibitor  that demin vivo, combination of panobinostat with BEZ235 resulted in greater anti-tumor activity compared to single treatment against PC3 and PC3-AR tumors. Anti-tumor activity mediated by panobinostat/BEZ235 combination was associated with sustained or increased levels of DNA damage with concurrent reduction of the DDR protein ATM. These data suggest that combination of panobinostat and BEZ235 results in superior therapeutic efficacy that is independent of AR and p53 status. We conclude that combined HDAC and DDR inhibition represents a novel therapeutic strategy for advanced and/or CRPC.The PC3 androgen independent prostate cancer cell line, which is devoid of AR, Pten and p53 expression is shown to be resistant to HDACi mediated apoptosis. In our study, we have utilized a PC3 cell line that expresses AR (PC3-AR) to demonstrate that treatment of PC3-AR cells with the pan-DACi panobinostat induces apoptosis. Panobinostat treatment resulted in comparable reduction of activated Akt and ATM in PC3 cells and PC3-AR cells. However, we observed a dramatic increase of double strand breaks in PC3-AR cells compared to PC3 cells. Further, we determine that 1 population in PC3-AR cells indicating DNA fragmentation, whereas the same treatment resulted in cell cycle arrest of PC3 cells in the G2M phase of the cell cycle  and plasma membrane integrity assessed by uptake of propidium iodide (PI). Initial confirmation of the functional status of AR expressed in PC3-AR cells is shown in It had been previously demonstrated that HDACi possess the ability to induce DNA damage and apoptosis . FurtherCombination of panobinostat and BEZ235 results in increased apoptosis in PC3 and PC3-AR cells. BEZ235 has recently been reported to inhibit the DNA damage response through attenuation of DNA-PKCs and ATM . Investiin vivo by treating male SCID mice bearing PC3 or PC3-AR subcutaneous tumors. Tumor bearing mice were treated with vehicle (N-Methyl-2-pyrrolidone (NMP)/PEG300, 1:9 v/v), PAN (10mg/kg i.p.: Mon-Wed-Fri), BEZ235 (25mg/kg i.p.: Mon-Fri) or combination. Treatment of PC3 or PC3-AR tumor bearing mice did not result in significant toxicity by any treatment , whereas BEZ235 treatment of PC3-AR tumors did not significantly change levels of p-ATM. No significant changes were observed in either PC3 or PC3-AR tumors treated with PAN when compared to vehicle. However, compared to each single treatment, combination treatment in PC3 tumors significantly decreased p-ATM levels  and in PC3-AR tumors  Fig. . CollectAdvances in drug development have resulted in exciting changes to the therapeutic landscape for advanced PCa. For several years, the primary treatment option of CRPC has been limited to docetaxel chemotherapy, which extends survival but for a limited time . While tPten, we proposed that suppression of Akt-Erk1/2 signaling could be a critical axis underlying increased sensitivity to HDACi mediated DSBs and apoptosis. Previously, HDACi have been shown to attenuate activated levels of Akt and Erk1/2 [Apoptosis induced by HDAC inhibition has been demonstrated to be dependent on AR expression . Our stud Erk1/2 , 45. Howin vivo anti-tumor activity via inhibition of Akt and both mTOR signaling complexes. Further, BEZ235 has also been shown to possess inhibitory action towards DNA damage response proteins DNA-PK and ATM in multiple tumor models [in vitro. Therefore, combining PAN with BEZ235 maintained the imbalance between DNA damage and DNA damage repair while simultaneously inhibiting Akt and Erk1/2 signaling, promoting greater accumulation of apoptotic cells.The ability of HDACi to create imbalance between DNA damage and DNA repair mechanisms was exciting, though the modest effect on Akt and Erk1/2 signaling suggested potential escape mechanisms which could be therapeutically exploited to enhance anti-tumor efficacy. Recently, the dual PI3K/mTOR inhibitor BEZ235 has been reported to exhibit potent r models , 44, 46.in vivo showed significant attenuation of tumor growth in PC3 and PC3-AR tumors compared to single agent treatments, which was associated with pronounced loss of tumor proliferation. Combination treatment of PC3 tumors resulted in sustained DNA damage compared with vehicle treated tumors. However this level of DNA damage was less than that observed in PAN single treated tumors. This may be explained by the partial attenuation of DNA damage by BEZ235 single treatment. Moreover, PC3-AR tumors displayed increased DNA damage following combination treatment, comparable to PAN single treatment, and greater than vehicle and BEZ235 treated tumors. Most excitingly, in both tumor models, significant attenuation of the DNA damage response protein ATM occurred with combination therapy compared to single treatments. This is an important finding as it demonstrates the ability of this combination therapy to mediate therapeutic efficacy independent of tumor AR status.The combination of PAN and BEZ235 in vivo dose capable of inducing a biological effect in the PC3 model was relatively low. Further molecular studies will help to dissect the genome and epigenome signature for this susceptibility and will provide the potential predictors of response in patients with PCa.The drug development of HDACi as single agents for the treatment of solid tumors, including prostate cancer, has been disappointing. Rational combination strategies that exploit the transcriptional and prostranslational modifications induced by HDACi should be clinically tested based on convincing preclinical data and robust co-clinical trials. Optimal dosing and schedule of administration should be based on the molecular mechanism targeted by the HDACi. Induction of apoptosis may require relatively high doses given in a pulsed fashion. Interestingly, the required In conclusion, targeting advanced prostate cancer largely involves inhibitors towards androgen synthesis and androgen receptor transactivation. Together, our data provides rationale for the clinical translation of PAN combined with BEZ235 for treatment of advanced prostate cancer, independent of AR status. While targeting the DNA damage/DNA repair axis has been a goal of chemotherapy intervention, toxicity has made this strategy difficult to manage clinically. Both PAN and BEZ235 have returned favorable toxicity profiles in cancer patients and results from our study demonstrate that combination therapy sustains or increases intratumoral DNA damage while attenuating DNA damage repair (decreased ATM) and survival pathway (activated Akt and Erk1/2) signaling independent of AR status Fig. . The opt2. Primary antibodies used were GAPDH (#5174), p-Akt , Akt (#9272), p-Erk1/2 (#4370), Erk1/2 (9102), p-ɣH2AX , p-4EBP1 (#9451), 4EBP1 (9644), p-S6K (#2211), S6K (#2217) all from cell signaling, Ki67  and p-ATM .PC-3 cells were purchased from ATCC and PC3-AR  expressi5) were seeded in a 6-well plate (BD Bioscience) overnight to adhere. Cells were then incubated in the presence of indicated treatments for 24 and 48 hours. Following treatment, adherent and non-adherent cells were collected and washed in 1x PBS and stained by 500ng/ml of propidium iodide (PI) for 5 minutes. FACS Caliber cytometer was used to analyze PI uptake.One million cells (1×105) were seeded in a 6-well plate (BD Bioscience) overnight to adhere and treated as indicated. Following treatments, adherent and non-adherent cells were collected and washed in 1x PBS, and fixed in 50% ethanol at 4°C overnight. Cells were then stained with PI solution containing RNase A (Sigma) for 15 minutes at 37°C. DNA content analysis was performed by a FACS caliber cytometer.One million cells (1×105) were typsinized, washed with 1x PBS, and resuspended in 100ul RIPA buffer (Sigma) supplemented with protease inhibitor cocktail then incubated on ice for 40 minutes. After centrifugation, the protein concentration was quantified using Bradford Protein Assay Kit (Bio-Rad). Fifty micrograms (50μg) of total cell lysates were separated using 4-15% SDS-PAGE gradient gels (Bio-Rad) and then transferred to nitrocellulose membranes. Nitrocellulose membranes (Bio-Rad) were blocked using 5% non-fat milk for 1 hour at room temperature, and then incubated with indicated primary antibodies for overnight. Anti-rabbit horseradish peroxidase-conjugated secondary antibody (Bio-Rad) was used. Immunoblots were developed by enhanced chemiluminescence (PerkinElmer).Cells (1×105) PC-3 and PC3-AR cells were seeded in 6-well plates overnight to adhere. Cells were treated with PAN or BEZ235 for 24 and 48 hours. After treatment, cells were collected, washed with 1x PBS, and incubated with annexin:1x annexin binding buffer (1:100) (BD Bioscience) for 10 minutes at room temperature. Annexin positive cells were detected using a FACS caliber cytometer.One million  was used for detection of all primary antibodies. Staining was visualized using 3,3′-Diaminobenzidine (DAB)  and slides were counterstained with hematoxylin. For quantitation of IHC staining representative images (3-6) were obtained using a Zeiss light microscope (Zeiss). Positive nuclear staining was quantified by Aperio ImageScope (v11.1.2.760).Mice were sacrificed by COad libitum. Five million (5×106) PC3 or PC3-AR cells were subcutaneously injected into intact male SCID mice. Treatment was initiated when tumor size reached ~50mm2, mice were randomized to four treatment groups: (1) Vehicle (N-Methyl-2-pyrrolidone (NMP)/PEG300, 1:9 v/v: Mon-Fri, oral gavage), (2) BEZ235 25mg/kg , (3) PAN 10mg/kg , (4) combination. Mice were weighed weekly to monitor for toxicity and tumor growth was assessed by serial caliper measurements twice weekly.The Institute Animal Care and Use Committee at Roswell Park Cancer Institute approved all mouse protocols used in this study. Mice were housed in an animal facility maintained on a 12-h light/dark cycle, at a constant temperature (22±2°C) and relative humidity (55±15%). Tap water and food were available Data are displayed as the mean ± SE. Differences were determined using one or two-way ANOVA and two-tailed paired t tests where indicated, using GraphPad Prism software. P values less than 0.05 were assigned statistically significant.


Ubiquitin Carboxyl-Terminal Hydrolase-L1 (UCH-L1) is a deubiquitinating enzyme that is highly expressed throughout the central and peripheral nervous system and in cells of the diffuse neuroendocrine system. Aberrant function of UCH-L1 has been associated with neurological disorders such as Parkinson's disease and Alzheimer's disease. Moreover, UCH-L1 exhibits a variable expression pattern in cancer, acting either as a tumour suppressor or promoter, depending on the type of cancer. In non-small cell lung carcinoma primary tumour samples, UCH-L1 is highly expressed and is associated with an advanced tumour stage. This suggests UCH-L1 may be involved in oncogenic transformation and tumour invasion in NSCLC. However, the functional significance of UCH-L1 in the progression of NSCLC is unclear. The aim of this study was to investigate the role of UCH-L1 using NSCLC cell line models and to determine if it is clinically relevant as a prognostic marker for advanced stage disease.UCH-L1 expression in NSCLC cell lines H838 and H157 was modulated by siRNA-knockdown, and the phenotypic changes were assessed by flow cytometry, haematoxylin & eosin (H&E) staining and poly (ADP-ribose) polymerase (PARP) cleavage. Metastatic potential was measured by the presence of phosphorylated myosin light chain (MLC2). Tumour microarrays were examined immunohistochemically for UCH-L1 expression. Kaplan-Meier curves were generated using UCH-L1 expression levels and patient survival data extracted from Gene Expression Omnibus data files.Expression of UCH-L1 was decreased by siRNA in both cell lines, resulting in increased cell death in H838 adenocarcinoma cells but not in the H157 squamous cell line. However, metastatic potential was reduced in H157 cells. Immunohistochemical staining of UCH-L1 in patient tumours confirmed it was preferentially expressed in squamous cell carcinoma rather than adenocarcinoma. However the Kaplan-Meier curves generated showed no correlation between UCH-L1 expression levels and patient outcome.Although UCH-L1 appears to be involved in carcinogenic processes in NSCLC cell lines, the absence of correlation with patient survival indicates that caution is required in the use of UCH-L1 as a potential prognostic marker for advanced stage and metastasis in lung carcinoma. Ubiquitination is a highly diverse and complex post-translational modification responsible for controlling protein expression and activity in a vast array of cellular processes such as proteasomal degradation, cell cycle regulation, protein trafficking, inflammation and DNA repair ,2. RemovUCH-L1 have been detected in cases of familial PD. In particular the I93 M amino-acid substitution has been linked to a rare inherited form of PD known as PARK5 [UCHL-1 is highly expressed in the central and peripheral nervous system, reproductive tissue and neuroendocrine (NE) cells, although it is expressed in most adult tissues ,10. In bas PARK5 , whereasas PARK5 .UCH-L1 promoter region [In cancer, UCH-L1 exhibits highly variable expression patterns seemingly in a tumor-specific manner. UCH-L1 can act as a tumor-suppressor and is silenced in ovarian , hepatocr region ,20-22. Or region , lung car region , myelomar region , prostatr region , osteosar region  and pancr region . Severalr region ,29-31.In non-small cell lung carcinoma (NSCLC) UCH-L1 is consistently highly expressed in both cell lines and primary tumour samples when compared to normal lung tissue where the expression of UCH-L1 is confined solely to cells of the neuroendocrine (NE) system. The presence of high levels of UCH-L1 has also been associated with an advanced tumor stage suggesting a possible role of UCH-L1 in oncogenic transformation and tumor invasion in NSCLC ,33. A coThe distinction between different types of NSCLC was until quite recently, clinically unimportant. It was necessary only to decide if a patient had NSCLC or small cell carcinoma, a determination which can be made robustly on morphology. With the development of drugs such as Pemetrexed (Alimta™), which shows more activity against non-squamous NSCLC and Bevacizumab (Avastin™), which is contraindicated for use in squamous cell carcinoma, the further classification of NSCLC type is now the clinical standard. The distinction is made on the basis of morphology, histochemistry (mucin staining with Alcian blue/Periodic acid Schiff) and immunohistochemistry for thyroid transcription factor 1 (TTF-1), cytokeratins (CK) 5/6 and p63 amongst other possible combinations. Squamous differentiation is indicated by positivity with CK5/6 and p63 whilst TTF-1 is negative . TherefoTo establish whether UCH-L1 plays an important role in the pathogenesis of lung carcinoma we used two NSCLC cell lines of different subtypes to investigate the phenotypic effects observed following silencing of UCH-L1. We found that UCH-L1 expression increases apoptotic resistance in the adenocarcinoma cell line (H838) and promotes cell migration in the H157 squamous cell carcinoma cell line. Also, in NSCLC tumor samples we showed that UCH-L1 is preferentially expressed in squamous cell carcinoma. To examine the importance of UCH-L1 in patient samples we analyzed NSCLC patient survival data but despite the oncogenic role found in the NSCLC cell lines, no correlation between UCH-L1 expression and survival was evident.® F12 (Ham) Nutrient Mixture (Invitrogen), supplemented with 10% FBS, 1% Penicillin/Streptomycin, 1% L-glutamine and 1% Non-Essential Amino Acids. The cells were grown in a humidified incubator  at 37°C with 5% CO2.All cell lines were maintained in RPMI 1640 medium containing 10% fetal bovine serum , 100 U/ml penicillin and 100 μg/ml streptomycin , except BEAS-2B, MPP-89 and REN cells which were maintained in GIBCOUCH-L1 mRNA expression in parental and UCH-L1 siRNA-treated H157 and H838 cells was measured by quantitative-PCR (q-PCR). Primers and probes for UCH-L1 (assay ID: Hs00188233_m1) and 18S RNA internal control (assay ID: Hs99999901_s1) were obtained from Applied Biosystems . Reactions were carried out on the ABI Prism 7500 system equipped with a 96-well thermal cycler as previously described [escribed . BrieflyDNA was extracted from each cell line using the DNeasy Blood and Tissue Kit . PCR-directed sequencing was performed using standard protocols (primers available on request). The DNA sequencing data was viewed and analysed using Chromas Lite software  and SeqMan™ II software .Western blot analysis was used to detect the expression level of proteins as previously described . Primary5/ml. For each transfection reaction, 5 μl of siPORT NeoFX reagent was applied to 95 μl of Opti-MEM medium (Invitrogen), incubated at room temperature for 10 min, then mixed with an equal volume of UCH-L1 siRNA solution . After incubation at room temperature for 10 min, the siRNA transfection complexes were dispersed into 6-well plates and overlaid by cell suspensions, gently mixed and incubated for 48 to 72 hr at 37°C, 5% CO2. Transfection efficiency was assessed by q-PCR and Western blot.UCH-L1 siRNA  was transiently transfected into H838 and H157 cells in 6-well plates using siPORT NeoFX transfection agent according to the manufacturer's recommendations . Briefly, prior to the transfection, cells were trypsinised then resuspended in media without antibiotics at a cell density of 1 × 10Phase-contrast microscopy with a Zeiss Axiovert 200 phase-contrast microscope  equipped with an Orca camera  was used to observe the morphological changes in H838 cells 48 hr post-transfection of UCH-L1 siRNA.Transiently transfected H838 cells were grown on coverslips. At 48 hr after transfection, the cells were fixed in 90% ethanol, stained with haematoxylin & eosin (H&E) and viewed under light microscope for signs of apoptosis. The cells with abnormal nuclear features such as a fragmented nucleus or breakdown of the nuclear membrane were classified as apoptotic. For each slide, the numbers of apoptotic cells in 20 different fields at 250× magnification were counted.At 72 hr post-transfection cells were harvested by trypsinisation and fixed by ice-cold 70% ethanol for 1 hr. The fixed cells were washed twice with PBS and stained with 0.5 ml of 40 μg/ml propidium iodide (PI) at 37°C for 30 min protected from light. The PI-stained samples were analyzed by the BD™ LSR II FACS instrument and the BD™ FACS Flow Supply System  and a total of 10,000 events were analyzed. The hypodiploid sub-population in sub-G1/G0 phase was regarded as apoptotic cells and the percentages of these cells were calculated using the BD™ FACS Diva software v.6.1.2.Formalin-fixed paraffin wax-embedded cell blocks of H157, H838 and BEAS-2B cells and paraffin wax embedded sections from 140 samples of NSCLC were stained for UCHL-1 expression. Briefly, sections were pre-treated in a 750 W microwave oven  for 22 minutes, cooled rapidly, washed in Tris-buffered Saline and were incubated in mouse anti-UCHL-1  overnight at 4°C. Localisation was achieved using Envision peroxidise kit as recommended by the manufacturer . All sections were counterstained in Meyer's haematoxylin. Immunoreactivity was assessed by two observers and percentage positive agreed. A cut-off value of 10% was used for UCH-L1 results. Selected sections were incubated with mouse immunoglobulin as negative controls. All tissues were used under regional ethical permission  and sourced from the Belfast Health & Social Care Trust, ISU Abxis Co  and US Biomax Inc .Three relevant publicaly available lung cancer datasets  which contained whole-genome profiles and associated patient outcome data were identified in the Gene Expression Omnibus (GEO) database repository. GSE13213 consisted of whole-genome expression profiles of 117 adenocarcinoma samples with the associated outcome data of "days survival". GSE3141 consisted of 111 primary lung tumour samples with associated survival data stated in "months survival" and GSE8894 contained gene expression profiles from primary tumours from 138 lung cancer patients with associated "recurrence free survival (months)" outcome data. Expression profiles for GSE13212 were generated using the Agilent-014850 Whole Human Genome Microarray 4 × 44 K G4112F platform which contains 1 probe for the UCH-L1 gene (A_23P132956). For both GSE3141 and GSE8894 datasets, gene expression profiles were generated using Affymetrix Human Genome U133 Plus 2.0 Array which contains 2 probesets for the UCH-L1 gene . The Series Matrix files were downloaded from GEO for all 3 datasets. Normalized expression data and associated outcome data were imported into the Partek Genomics Suite . Patients were separated into quartiles based on expression levels of the UCH-L1 gene for each dataset. The survival times for each quartile were compared using Kaplan-Meier survival analysis and the log-rank test.n = 3. Intergroup comparisons were made by Student's t test with P < 0.05 considered statistically significant.All experiments were carried out with a minimum of UCH-L1 mRNA expression  were screened for UCH-L1 positivity by immunohistochemistry Figure  and 7B. To investigate if the potential oncogenic role of UCH-L1 observed in the cell line model is reflected in patients, Kaplan-Meier plots were generated for NSCLC patients based on UCH-L1 expression. To do this three microarray-based gene expression studies with associated patient outcome data  were identified that were available from the NCBI's Gene Expression Ombnibus (GEO). Normalized microarray data and phenotype data were downloaded and samples were separated into quartiles according to UCH-L1 expression levels. Kaplan-Meier survival analysis, including the log-rank test, was performed on each of the quartiles. No significant difference in survival was observed between the quartiles for all three datasets  stain positive for UCH-L1 by immunohistochemistry. This confirms previous reports that UCH-L1 is highly expressed in NSCLC cell lines and primary tumours. UCH-L1 staining also correlates with histology as squamous cell carcinomas express the protein more frequently than adenocarcinomas. Although Sasaki et al  found noThe functional role of UCH-L1 in lung tumourigenesis however remains elusive, therefore following confirmation of high UCH-L1 expression we examined the phenotypic effects in NSCLC cell lines. The expression of UCH-L1 was reduced using siRNA in both squamous cell carcinoma H157) and adenocarcinoma H838) cell lines. Knockdown of UCH-L1 in H838 cells shows morphological differences indicative of apoptosis and cell death was confirmed by H&E staining, cell cycle analysis and the presence of PARP cleavage. Although other studies have not examined the effect of UCH-L1 specifically in H838 cells, UCH-L1 has been associated with apoptosis in several cases. In neuronal cells and testicular germ cells UCH-L1 is viewed as an apoptosis-promoting protein due to its role in balancing the levels of pro-apoptotic and anti-apoptotic proteins ,11,12. I7 and ade cell linIn contrast to H838 cells, our study reveals UCH-L1 knockdown causes no difference in morphology, apoptosis or proliferation in H157 cells but does reduce the capacity for cell migration. MLC2, a protein responsible for cell movement, is phosphorylated during cell invasion . In thisTaken together our results suggest that in addition to the correlation of UCH-L1 expression with histological type, the functional effects of UCH-L1 on NSCLC cells may also be subtype-dependent. Analysis of UCH-L1 in the large cell carcinoma cell line H1299 presents yet another different role for this protein in NSCLC since UCH-L1 was found to be antiproliferative in this case and the authors concluded that it is expressed as a response to tumour growth .In vivo studies of UCH-L1 expression in the lung have also demonstrated a role for UCH-L1 in lung carcinogenesis in two separate reports. When BALB/C nude mice were injected with UCH-L1-expressing metastatic melanoma cells, black melanoma colonies were generated in the lungs but when melanoma cells treated with UCH-L1 siRNA were introduced there was a significant decrease in the number of metastatic lung colonies [Our cell line studies suggest that UCH-L1 expression may be important in the pathogenesis of lung cancer. colonies . Additiocolonies  demonstrTo assess the relevance of UCH-L1 in patient samples we looked at whether high or low UCH-L1 expression resulted in any difference in survival status of NSCLC patients. Despite the evidence supporting a role for UCH-L1 in lung carcinogenesis in the cell line study, UCH-L1 status was not significantly associated with patient outcome. This was particularly surprising considering high UCH-L1 expression in NSCLC was previously correlated with an advanced tumour stage. However, Sasaki et al  also faiIn conclusion, this study shows the expression of UCH-L1 in NSCLC is variable and dependent on histological type. In cell line models UCH-L1 appears to have an oncogenic role in NSCLC leading to increased apoptotic resistance in H838 adenocarcinoma cells and a greater capacity for migration in the squamous cell carcinoma cell line (H157).Despite the promising observations in the NSCLC cell lines following UCH-L1 knockdown, translation to the clinical setting did not indicate any correlation with patient survival. Thus caution is required when using UCH-L1 as a prognostic marker in isolation for advanced stage and metastasis in lung carcinoma as other factors may be involved. Clearly further investigation would be required to establish whether UCH-L1 is part of a pathway that influences prognosis in lung cancer.The authors declare that they have no competing interests.KSO performed siRNA knockdown, apoptosis and metastatic potential assays, and prepared the manuscript. ZS conceived the study and designed the siRNA knockdown and apoptosis assays. WMB generated Kaplan-Meier curves, analyzed patient survival data, and prepared the manuscript. KOH generated Kaplan-Meier curves, analyzed patient survival data, reviewed all statistics and reviewed the manuscript. TRL conceived the study, participated in the design and prepared the manuscript. PM performed immunohistochemical analysis of tumours microarrays and prepared the manuscript. MJP conceived the study, participated in the design and prepared the manuscript. All authors read and approved the final manuscript.Loss of UCH-L1 expression did not affect cell proliferation of H838 cells. CyQUANT® assays were performed at two different time points of 24 and 48 hr post-transfection with UCH-L1 siRNA in H838 cells. The results from 3 experiments are shown graphically. Statistical analysis showed no significant difference between UCH-L1 siRNA-treated and controls.Click here for fileKaplan-Meier analysis in the GSE13213 dataset based on UCH-L1 expression. A. Kaplan-Meier analysis for patients separated into above and below the median of UCH-L1 expression in the GSE13213 dataset. B Kaplan-Meier analysis for patients separated into quartiles based on UCH-L1 expression. The first and fourth quartiles are included in the graph. The UCH-L1 gene is represented by a single probe (A-23P132956).Click here for fileKaplan-Meier analysis in the GSE3141 dataset based on UCH-L1 expression represented by probesets 1555834_at and 201387_s_at. A. Kaplan-Meier analysis for patients separated into above and below the median expression of UCH-L1 based on probeset 1555834_at signal intensities. B. Kaplan-Meier analysis for patients separated into quartiles based on UCH-L1 expression represented by probeset 1555834_at. The first and fourth quartiles are included in the graph. C. Kaplan-Meier analysis for patients separated into above and below the median expression of UCH-L1 based on probeset 201387_s_at signal intensities. D. Kaplan-Meier analysis for patients separated into quartiles based on UCH-L1 expression represented by 201387_s_at. The first and fourth quartiles are included in the graph.Click here for fileKaplan-Meier analysis in the GSE8894 dataset based on UCHL-1 expression represented by 2 probesets 1555834_at and 201387_s_at. A. Kaplan-Meier analysis for patients separated into above and below the median expression of UCH-L1 based on probeset 1555834_at signal intensities. B. Kaplan-Meier analysis for patients separated into quartiles based on UCH-L1 expression represented by probeset 1555834_at. The first and fourth quartiles are included in the graph. C. Kaplan-Meier analysis for patients separated into above and below the median expression of UCH-L1 based on probeset 201387_s_at signal intensities. D. Kaplan-Meier analysis for patients separated into quartiles based on UCH-L1 expression represented by 201387_s_at. The first and fourth quartiles are included in the graph.Click here for file


We used concentration addition (CA) and independent action (IA) to formulate additivity expectations, and Schild plot analyses to investigate competitive AR antagonism.Methods: We used the MDA-kb2 assay to test a combination of eight AR antagonists that did not also possess AR agonist properties (“pure” antagonists; 8 mix: fludioxonil, fenhexamid, Results: A good agreement between the effects of the mixture of eight “pure” AR antagonists and the responses predicted by CA was observed. Schild plot analysis revealed that the 8 mix acted by competitive AR antagonism. However, the observed responses of the 5 mix and the 13 mix fell within the “prediction window” boundaries defined by the predicted regression curves of CA and IA. Schild plot analysis with these mixtures yielded anomalous responses incompatible with competitive receptor antagonism.Conclusions: A mixture of widely used pesticides can, in a predictable manner, produce combined AR antagonist effects that exceed the responses elicited by the most potent component alone. Inasmuch as large populations are regularly exposed to mixtures of antiandrogenic pesticides, our results underline the need for considering combination effects for these substances in regulatory practice. These ptor (AR) , which ctor (AR) , 2009. Din vitro  concentration addition , and b) independent action . For CA, it is assumed that all compounds have a similar mechanism of action , whereas for IA, it is assumed that all mixture components affect the same end point via different sites or modes of action . Both additivity models assume no interaction between the compounds, neither on a physicochemical level nor in their toxicokinetics and toxicodynamics.At present, there are 1,252 registered active ingredients in pesticide formulations in the United States . There are 411 registered entities in Europe, with another 72 pending registration . With suin vivo  and to four antiandrogenic contaminants in vivo  , pirimiphos-methyl , tebuconazole , chlorpropham , methiocarb , cyprodinil , and pyrimethanil . In addition, vinclozolin was included because of its high ERR (79.8), its known in vivo potency  was purchased from Steraloids Ltd. ; dimethomorph and methiocarb ; and all other pesticides (> 97% purity) were purchased from Sigma-Aldrich . All test compounds were dissolved in > 99.7%-purity ethanol to make stock solutions to be used in the assays.MDA-kb2 assay. MDA-kb2 cells  are human breast cancer cells stably transfected with a firefly luciferase reporter gene that is driven by an androgen–response element-containing promoter  containing 10% charcoal-stripped fetal calf serum (Invitrogen Ltd.) in white luminometer plates. After 28 hr, luciferase activity was determined with SteadyGlo assay reagent  and measured in a plate reader . For regression analysis, cells were exposed to eight serial dilutions of selected pesticides with or without DHT (0.25 nM). Subsequent to the initial testing range of 1.17 nM–150 µM, the mixtures’ concentrations were modified to reflect the potency and toxicity of each individual mixture. For Schild plot analysis, cells were coexposed with eight serial dilutions of DHT (0.009–20 nM) and fixed concentrations of pesticide mixtures (150–6.25 µM), which varied according to the individual activity/toxicity of each mixture. For all testing scenarios, the following controls were run on each plate: medium, ethanol (0.25%), DHT coexposure (0.25 nM), DHT serial dilutions (0.009–20 nM) with ethanol (0.25%), and procymidone (0.005–3.2 µM) with DHT (0.25 nM). All concentrations were tested in duplicate over two plates; each mixture stock was measured at least twice in separate experiments, and mixtures were independently tested at least three times  by two experimenters. For comparative purposes, luminescence was normalized to DHT alone at the coexposure concentration  and solvent-only (ethanol) controls .promoter . Detailspromoter . BrieflyCytotoxicity as a confounding factor. The MDA-kb2 assay measures decreases in luminescence of the DHT agonist that occur as a result of receptor antagonism. Because the luminescence signal can also be driven down by cytotoxicity, it is important to distinguish antagonism from interfering cytotoxicity, and we adopted well-established procedures  gene] in order to eliminate the possible interfering effects of cell proliferation. Briefly, 4 µg DNA was incubated with 6 µL TurboFect  in 400 µL of serum-free Leibowitz L-15 medium (Invitrogen Ltd.) for 20 min. MDA-kb2 cells were transfected with the Renilla construct for 48 hr prior to following the normal procedure for the MDA-kb2 assay. In order to read both the luciferase and Renilla signals, after 28 hr of incubation, luciferase activity was determined with Dual-Glo Reporter assay reagent (Promega UK Ltd.), which employs the sequential addition of two reconstituted reagents with luminescence measurement after each reagent addition . The first reagent provides the necessary substrate for firefly luciferase, and the second reagent quenches this activity while at the same time activating Renilla luciferase. Cells transfected with the Renilla construct were exposed to the 5 mix IC10 only; for regression analysis, 5 mix  was coexposed with a fixed concentration of DHT (0.25 nM), and for Schild plot analysis, serial concentrations of DHT (0.009–20 nM) with various fixed concentrations of 5 mix (110–13.75 µM).Renilla Test mixtures. All mixtures were designed as fixed-ratio equipotent mixtures. We tested three distinct pesticide mixtures: an 8 mix, a 5 mix, and a 13 mix. The 8 mix comprised eight “pure” AR antagonists ; the 5 mix comprised five antagonists with additional agonist properties ; and the 13 mix comprised the eight “pure” antagonists together with the five “mixed” antagonists. Fixed-mixture ratios were calculated in proportion to the concentrations of the individual mixture components that led to a suppression of DHT effects by 1%, 10%, 20%, or 50% . The 13 mix was tested at four fixed mixture ratios , and the 8 mix and 5 mix were tested at two fixed mixture ratios  . The mathematical and statistical procedures used for calculating mixture effects according to CA and IA are described by Schild plot calculations. To confirm applicability of the MDA-kb2 assay to Schild plot analysis, we first determined concentration–effect curves for the agonist DHT in the presence of various concentrations of flutamide. From the concentration–effect curves, we estimated a series of concentration ratios  for a given effect. This was determined for several concentrations of the antagonists. To get the most accurate results, we used a 50% inhibition—the concentration ratio calculated as the IC50 in the presence of antagonist divided by the IC50 in the absence of antagonist. The Schild plot analysis was then based on the linear regression:50DHT + A/IC50DHT – 1) = –log(DK) + θ × log(Ac). [1]log(ICDK is the (unknown) dissociation constant of the antagonist, Ac the concentration of the antagonist “A” held fixed in the experiments, and θ the slope parameter. The unknown parameters, θ and log(DK), were estimated by ordinary least squares. If the regression is linear with a slope of 1, the antagonism is competitive and, by definition, the agonist and antagonist act at the same sites  and the solvent controls (n = 8), which were placed on the same plate. Luminescence readings from pesticides tested in the absence of DHT were divided by the mean of the solvent controls from the same plate and analyzed for negative and positive trends . All data from the same test compound were pooled and statistical concentration–response regression analyses were conducted by using the best-fit approach to derive ICs for androgenicity  and in all other cases were never outside the range predicted by CA and IA. Cytotoxicity was only observed at high mixture concentrations  . The overlap with antiandrogenic responses was negligible and did not interfere with the detection of AR antagonistic responses ]. When tested on their own, none of the individual pesticides in the mixtures showed AR agonistic effects at their concentration in the mixture. The androgenicity of 5 mix and 13 mix therefore appears to be a genuine combination effect. Indications for toxicity were detected only at high tested concentrations and are unlikely to interfere with the mixture assessment.There was good agreement of the 8 mix responses with those predicted by CA over the entire concentration range and for both tested mixture ratios , Table 1http://dx.doi.org/10.1289/ehp.1205391)], indicating that agonist and antagonist acted in a competitive manner at the same receptor site .By performing Schild plot analysis with the pure antiandrogen flutamide and DHT, we were able to confirm competitive receptor antagonism. Increasing concentrations of this antagonist shifted the dose–response curve of the agonist DHT to the left [see Supplemental Material, Figure S1A,B  . These sRenilla luciferase construct in our assay. This construct produces luminescence in proportion to cell number, independent of AR activation. There was no dose–response relationship between rising concentrations of the 5 mix and Renilla luminescence of the MDA-kb2 cells and no differences in luminescence between ethanol only  and the DHT background concentration only . The same applied to DHT , the positive control procymidone , and to any concentration of the 5 mix with DHT  and the 5 mix on its own  (data not shown). This indicates that cell proliferation was not the cause of the increased luminescence observed with this mixture and DHT, and that the supramaximal responses were the consequence of phenomena at the receptor.To investigate whether stimulation of the maximal response with the 5 mix and DHT was the result of cell proliferation, we used a This is the first time that a mixture of widely used pesticides has been shown to produce combined AR antagonist effects that exceed the responses elicited by the most potent component alone. Furthermore, these mixture effects occurred in a quite predictable manner. The responses of the 8 mix composed of “pure” AR antagonists agreed very well with the combined effects predicted by CA. However, the combined effects seen with the two mixtures containing AR antagonists that also showed AR agonist properties (5 mix and 13 mix) were somewhat lower than anticipated by CA and fell between the “prediction window” boundaries defined by the predicted regression curves of CA and IA. These deviations are highly unlikely to be due to experimental artefacts because the mixtures were tested independently by different experimenters who prepared several independent mixture stock solutions.By conducting Schild plot analysis, we were able to pinpoint competitive AR antagonism as a key factor that influenced agreement of the experimentally observed responses with the CA predictions. The combination of 8 “pure” AR antagonists (8 mix), well predicted by CA, produced Schild plots typical of competitive receptor antagonism. In contrast, anomalous supramaximal effects were observed with the 5 mix and 13 mix in experiments where increasing concentrations of these two mixtures were combined with DHT. These anomalies suggest that the 5 mix and 13 mix displaced DHT from the AR in ways not compatible with competitive antagonism. This allows us to infer that the lack of competitive AR antagonism is the likely cause of the observed deviations from CA. Chemicals that display mixed androgenic/antiandrogenic activity interact in ways with the amino acid residues in the AR binding domain that are distinct from those of “pure” AR antagonists . Chemicain vivo. The applicability of CA for mixtures composed of AR antagonists was tested in a rat developmental toxicity model with flutamide, procymidone (both “pure” antagonists) and vinclozolin (which liberates metabolites that possess mixed AR antagonistic and agonistic properties)  . In thesperties) . Althoug than IA . Further than IA . Therefoin vivo data for pesticides tested in this mixture  nature of CA. It is well known that people are exposed to mixtures of pesticides, and therefore the additive nature of these pollutants is a cause for concern.Widely used pesticides act additively (1.1 MB) PDFClick here for additional data file.


VIMENTIN, SNAIL and ZEB1 was observed in the presence of constitutively active variants. In conclusion, our findings highlight novel consequences of constitutively active AR variants on the regulation of mesenchymal markers in prostate cancer.Androgen receptor (AR) signaling pathway remains the foremost target of novel therapeutics for castration-resistant prostate cancer (CRPC). However, the expression of constitutively active AR variants lacking the carboxy-terminal region in CRPC may lead to therapy inefficacy. These AR variants are supposed to support PCa cell growth in an androgen-depleted environment, but their mode of action still remains unresolved. Moreover, recent studies indicate that constitutively active AR variants are expressed in primary prostate tumors and may contribute to tumor progression. The aim of this study was to investigate the impact of constitutively active AR variants on the expression of tumor progression markers. N-cadherin expression was analyzed in LNCaP cells overexpressing the wild type AR or a constitutively active AR variant by qRT-PCR, Western blot and immunofluorescence. We showed here for the first time that N-cadherin expression was increased in the presence of constitutively active AR variants. These results were confirmed in C4-2B cells overexpressing these AR variants. Although N-cadherin expression is often associated with a downregulation of E-cadherin, this phenomenon was not observed in our model. Nevertheless, in addition to the increased expression of N-cadherin, an upregulation of other mesenchymal markers expression such as  Prostate cancer (PCa) is the most common cancer in men over 50 years of age and the second cause of male mortality due to cancer in Europe. Androgens signaling plays a key role in PCa cells proliferation or survival Genetic and splicing events affecting the androgen receptor (AR) gene have been linked to CRPC. Constitutively active AR variants, lacking the carboxy-terminal region that encompasses the ligand binding domain and the activation function 2, might contribute to the progression of PCa into castration resistance. These constitutively active AR variants result from premature stop codons due to nonsense mutations as reported for the ARQ640X in vitro and in vivo in the absence of androgen The role of constitutively active AR variants in CRPC has been shown in many studies Recent studies suggest that constitutively active AR variants could play a role in tumor progression. Indeed, although these constitutively active AR variants are already expressed in primary prostate tumors, their expression is all the more expressed in bone metastasis N-cadherin, which belongs to cadherin superfamily, is located at adherens junctions in nervous, endothelial or mesenchymal cells and is involved in tumor progression More recently, studies have shown that castration-resistant PCa is associated with an upregulation of N-cadherin expression in cellular models as well as PCa xenografts and clinical samples of CRPC N-CADHERIN as well as VIMENTIN, SNAIL and ZEB1 are upregulated in the presence of constitutively active AR variants in PCa.The aim of this work was to show a possible link between the presence of constitutively active AR variants and the expression of tumor progression markers. More particularly, we focused on the impact of constitutively active AR variants on the expression of N-cadherin and other mesenchymal markers. In the present study, we have shown that The human prostate carcinoma LNCaP cell line, clone FGC and the 22Rv1 cell line  was maintained in RPMI-1640 complete medium containing 10% fetal calf serum (FCS), 10 mM HEPES, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin  and 1mM sodium pyruvate .C4-2B cell line  was maintained in DMEM medium supplemented with 20% Ham's F12, 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin, 5 µg/mL insulin, 13.65 pg/mL triiodo-thyronine, 4.4 µg/mL apo-transferrin human, 0.244 µg/mL d-biotin and 12.5 µg/mL adenine .For immunofluorescence experiments, the wild type androgen receptor (AR) (AR-WT) and the constitutively active AR Q640X and AR Q670X TM transfection reagent  was used according to the manufacturer's protocol. LNCaP cells were seeded in 10 cm dishes at 1×106 cells/dish or in 6-wells plate at 2×105/well. Three days later, the medium was changed and cells were transfected with 10 µg of the indicated plasmid using 20 µl of JetPEI transfection reagent for 10 cm dishes or with 3 µg of plasmid using 6 µl of JetPEI for 6-wells plate. Medium was changed 48 h after and cells were incubated up to 9 days according to the experiments. The medium was changed every two days and for incubations beyond 4 post-transfection days, cells were incubated in the presence of 400 µg/mL geneticin .For transfections, the JetPEILNCaP cells were seeded in 6-wells plate in complete medium and transfected as previously described. Twenty four hours later, medium was changed to phenol red free RPMI-1640 supplemented with 5% dextran-coated charcoal-stripped FCS (DCC-FCS) and with the indicated concentration of dihydrotestosterone (DHT)  or vehicle (ethanol).For experiment with MDV3100, transfected LNCaP cells were incubated in RPMI-1640 supplemented with 5% DCC-FCS containing the indicated DHT dose and 100 nM MDV3100  or vehicle . To confirm the effects of androgens on N-cadherin expression, 22Rv1 cells were grown in RPMI-1640 with 100 nM or 1 µM MDV3100, or DMSO.6 cells/dish and were transfected with pEGFP-ARWT or pEGFP-ARQ640X. Four days after transfection, cells were trypsinized and sorted thanks to the green fluorescence (EGFP) with a BD FACSAria-II cell sorter . Total RNA was extracted from EGFP negative (non-transfected) and EGFP positive (transfected) cells and was used to analyze gene expression by qRT-PCR.LNCaP cells were seeded in 10cm dishes at 1×10N-CADHERIN, E-CADHERIN, VIMENTIN, SNAIL, TWIST1, and ZEB1 mRNA levels were quantified using real-time PCR with LightCycler 480 . For PCR reactions, 5 µL LightCycler® 480 SYBR Green I Master  and 1 µL specific primers (β-ACTIN or PBGD (Porphobilinogen deaminase) . Amplification specificity was verified by analyzing melting curve and by electrophoresis migration. All experiments were realized in triplicate and repeated 3 times. Relative quantification was used to determinate fold change in expression level by the ΔΔCt method. Each value is expressed as the mean ΔΔCt ± SEM. Results were analyzed with Student t test and p-value <0.05 was considered significant.Total cellular RNA was extracted from cell lines using NucleoSpin® RNA II assay  according to the manufacturer's procedure. RNA concentrations and purity were quantified measuring the absorbance at 260 nm and 280 nm . The reverse transcription was performed from 400 ng or 1 µg RNA using RT Omniscript assay . RNA were diluted into 13 µL and denatured at 65°C during 5 minutes. A 7 µL reaction mix containing 1×RT template, 0.5 mM of each dNTP, 1 µM oligo dT, 10U RNase inhibitor and 4U Omniscript Reverse Transcriptase was added and the reaction was incubated 1 h at 37°C. The reaction was stopped by heating to 93°C for 5 minutes.  primers  .Cells were lysed in buffer containing 10 mM Tris-HCl pH7, 140 mM NaCl, 3 mM MgCl2, 0.5×Igepal, 5 mM DTT, 1× phosphatase inhibitor, and 1× protease inhibitor. Protein concentration for each sample was quantified using BCA Protein Assay  according to the manufacturer's procedure. A quantity of 15 µg to 100 µg of total proteins was loaded on 7,5% SDS-PAGE. After migration and transfer to nitrocellulose membrane, membranes were saturated with PBS/0.1%Tween/2%ECL and incubated at 4°C overnight with 0.1 µg/mL mouse monoclonal anti N-cadherin  or 1 µg/mL mouse monoclonal anti AR  antibody. β-actin (0.2 µg/mL)  was used as internal control. After washes, immunocomplexes were detected with 0.2 µg/mL HRP-conjugated goat anti mouse , or 0.5 µg/mL rat anti mouse IgG2a secondary antibodies , and finally revealed by chemiluminescence /PBS for 30 min at room temperature. Cultures were incubated with 2.5 µg/mL anti N-cadherin mouse monoclonal antibody  or isotypic antibody  at 4°C overnight. After washing in PBS, LNCaP cells were incubated with 2 µg/mL Alexa Fluor 568-conjugated goat anti mouse  for 1 h and nuclei were stained with 0.1 µg/mL DAPI solution for 20 min at 30°C. Images were captured with the Leica LAS AF6000 fluorescence microscope using LAS AF software (Leica).Lab-Tek II chamber slides (2 wells) were coated with LNCaP medium for two hours and 1×10N-CADHERIN mRNA level was determined by qRT-PCR in LNCaP cells overexpressing the constitutively active AR Q640X or AR-V7, or the AR-WT as control  or EGFP positive (transfected cells) fractions by qRT-PCR. Consistent with above results, N-CADHERIN expression was undetectable in both EGFP- negative and positive fractions following LNCaP transfection with pEGFP-ARWT. However, upon transfection with pEGFP-ARQ640X, N-CADHERIN expression was increased in EGFP positive cells overexpressing the constitutively active AR, but not in the EGFP negative fraction  to activate endogenous target genes. To explore the effect of the endogenous AR-FL present in LNCaP cells on the ability of constitutively active AR variants to induce N-cadherin expression, LNCaP cells overexpressing AR-WT or a constitutively androgen variant were incubated in the presence of 100 nM DHT or vehicle, and N-cadherin expression was analyzed by qRT-PCR. In accordance with our previous results, no N-cadherin expression was observed in cells overexpressing AR-WT. Interestingly, a 1.4-fold decrease in N-cadherin expression level was observed when cells overexpressing AR Q640X or AR-V7 were cultured in the presence of 100 nM DHT compared to vehicle . In addiAll together, these data suggest that DHT-activated AR-FL could compete with constitutively active androgen receptor for regulating N-cadherin expression.E-CADHERIN mRNA levels in LNCaP cells upon transfection with ARQ640X or AR-V7 expression plasmid did not show any significant difference compared with controls  repeats in intron 1 These findings again highlight the link between AR signaling pathway and N-cadherin expression. Recent studies suggest that AR negatively regulates N-cadherin expression N-CADHERIN expression. For example, in prostate cancer, N-CADHERIN expression was associated with a nuclear translocation of Twist1 TWIST1 mRNA levels, constitutively active AR variants might enhance nuclear translocation of Twist1, which could in turn induce N-CADHERIN expression after binding to the E-box within the first intron of N-CADHERIN.However, constitutively active AR variants could also indirectly control N-CADHERIN expression.These hypotheses deserve to be studied in further studies to understand how constitutively active AR variants regulate VIMENTIN, SNAIL and ZEB1. These results are consistent with a recent study, which showed an increase of mesenchymal markers in tumors from patients treated with androgen deprivation therapy In this study, we have also shown that constitutively active AR variants were associated with an increased expression of mesenchymal markers as in vitro cell culture The expression of mesenchymal markers reported here in the presence of constitutively active AR variants was not associated with a downregulation of E-cadherin in our model. The inverse correlation between N-cadherin upregulation and E-cadherin downregulation is still debated. Indeed, McKeithen and colleagues, and more recently Tiwari and colleagues report a co-expression of both E- and N-cadherins in tumor cells Further studies are warranted to understand functional consequences of N-cadherin and other mesenchymal markers upregulation in the presence of constitutively active AR variants. N-cadherin expression is widely associated with tumor progression notably owing to its role in migration and invasion. Indeed, N-cadherin favors the migration of cancer cells via cytoskeleton reorganization and lamellipodia formation There is presently great interest in the mode of action of constitutively active AR variants in CRPC. In this study, we have shown for the first time that constitutively active AR variants induce N-cadherin expression and other mesenchymal markers in PCa. These findings support the hypothesis that these constitutively active AR variants could contribute to systemic dissemination of PCa cells, and reinforce the importance to target these AR variants in PCa.Figure S1N-cadherin expression was upregulated in C4-2B cells in the presence of constitutively active AR variants. N-cadherin expression was assessed by qRT-PCR in C4-2B cells overexpressing AR Q640X variant or transfected with empty plasmid (C3) 4 days after transfection. Parental C4-2B cells were used as control. N-CADHERIN expression was normalized to β-ACTIN and calculated using the ΔΔCt method. Results are presented as the mean of ΔΔCt ± SEM from three independent experiments. NS: Not Significant * P<0.05, **P<0.01 and ***P<0.001.(TIF)Click here for additional data file.Figure S2DHT activated AR-FL repressed N-cadherin expression induced by constitutively active AR variants. 22Rv1 cells were cultured in complete medium supplemented with 100 nM and 1 µM of MDV3100 or DMSO. N-cadherin expression was analyzed by qRT-PCR four days after and was normalized to PBGD. The fold change was expressed as relative values to parental cell line 22Rv1 under normal condition. NS: Not Significant * P<0.05, **P<0.01 and ***P<0.001.(TIF)Click here for additional data file.