Localization Of Androgen Receptor Research Articles

NanoLuc Binary Technology as a methodological approach: an important new tool for studying the localization of androgen receptor and androgen receptor splice variant V7 homo and heterodimers

BackgroundThe androgen/androgen receptor (AR)-signaling axis plays a central role in prostate cancer (PCa). Upon androgen-binding the AR dimerizes with another AR, and translocates into the nucleus where the AR-dimer activates/inactivates androgen-dependent genes. Consequently, treatments for PCa are commonly based on androgen deprivation therapy (ADT). The clinical benefits of ADT are only transitory and most tumors develop mechanisms allowing the AR to bypass its need for physiological levels of circulating androgens. Clinical failure of ADT is often characterized by the synthesis of a constitutively active AR splice variant, termed AR-V7. AR-V7 mRNA expression is considered as a resistance mechanism following ADT. AR-V7 no longer needs androgenic stimuli for nuclear entry and/or dimerization.MethodsOur goal was to mechanistically decipher the interaction between full-length AR (AR-FL) and AR-V7 in AR-null HEK-293 cells using the NanoLuc Binary Technology under androgen stimulation and deprivation conditions.ResultsOur data point toward a hypothesis that AR-FL/AR-FL homodimers form in the cytoplasm, whereas AR-V7/AR-V7 homodimers localize in the nucleus. However, after androgen stimulation, all the AR-FL/AR-FL, AR-FL/AR-V7 and AR-V7/AR-V7 dimers were localized in the nucleus.ConclusionsWe showed that AR-FL and AR-V7 form heterodimers that localize to the nucleus, whereas AR-V7/AR-V7 dimers were found to localize in the absence of androgens in the nucleus.

New approaches concerning the testis of Astyanax lacustris (Characidae): immunohistochemical studies

Astyanax lacustris, locally known as lambari-do-rabo-amarelo, is a study model for Neotropical fish. Testis of A. lacustris shows deep morphophysiological changes throughout the annual reproductive cycle. This work analyzed the distribution of claudin-1, actin, and cytokeratin as elements of the cytoskeleton in germinal epithelium and interstitium; the distribution of type I collagen, fibronectin, and laminin as extracellular matrix compounds; and the localization of androgen receptor in the testis of this species. Claudin-1, cytokeratin, and actin were present in the Sertoli cells and modified Sertoli cells, and actin was also detected in peritubular myoid cells. Type I collagen were in the interstitial tissue, laminin in the basement membrane of germinal epithelium and endothelium, but fibronectin was additionally detected in the germinal epithelium compartment. The labeling of androgen receptor was higher in peritubular myoid cells and undifferentiated spermatogonia, and weaker labeling was detected in type B spermatogonia. Therefore, the present work highlights new aspects of the biology of the testis of A. lacustris, and contribute to amplify the understanding of this organ.

Prevention of seminal vesicle damage by Mucuna pruriens var. pruriens seed extract in chronic unpredictable mild stress mice

Context Thai Mucuna pruriens (L.) DC. var. pruriens (Fabaceae) or T-MP seed extract has been shown to improve sexual performance and sperm quality. Objective This study investigates the preventive effects of T-MP against seminal vesicle damage, apoptotic and Nrf2 protein expression in mice under chronic unpredictable mild stress (CUMS). Materials and methods Forty-eight male ICR mice were divided into four groups: control, CUMS, T-MP300 + CUMS and T-MP600 + CUMS. Mice in control and CUMS groups received distilled water, while those in treated groups were pretreated with T-MP extract (300 or 600 mg/kg BW) for 14 consecutive days. The CMUS and co-treated groups were exposed to one random stressor (of 12 total) each day for 43 days. Components and histopathology of the seminal vesicle were examined, along with localization of androgen receptor (AR) and caspase 3. Expression of seminal AR, tyrosine phosphorylated (TyrPho), heat shock protein 70 (Hsp70), caspases (3 and 9) and nuclear factor erythroid 2-related factor 2 (Nrf2) proteins was investigated. Results T-MP extract at a dose of 600 mg/kg BW improved seminal epithelial damage and secretion of fluid containing essential substances and proteins in CUMS mice. It also increased the expression of AR and TyrPho proteins. Additionally, T-MP increased expression of Nrf2 and inhibited seminal vesicular apoptosis through the suppression of Hsp70 and caspase expression. Conclusion T-MP seeds have an antiapoptotic property in chronic stress seminal vesicle. It is possible to apply this extract for the enhancement of seminal plasma quality.

Hippocampal synaptic dysfunction and spatial memory impairment in omeprazole-treated rats.

Although the association of prolonged use of proton pump inhibitors, such as omeprazole, with memory impairment has been reported more than two decades ago, its underlying molecular mechanism is yet to be determined. Thus, in this study, we aimed to determine the mechanisms underlying the effect of prolonged omeprazole treatment on hippocampal synaptic function and spatial memory in male rats. Adult rats were subcutaneously administered with omeprazole for 12 or 24 weeks. Spatial memory was assessed using the Morris water maze (MWM) test. We examined the hippocampal protein expression of synaptic plasticity proteins, including the AMPA receptor subunit GluA1, postsynaptic density-95 (PSD-95), and activity-regulated cytoskeleton-associated protein (Arc), and the hippocampal expression and localization of androgen receptor (AR). In the MWM test, the escape latency was found to be significantly higher, and the number of platform crossings and the time spent in the target quadrant were significantly lower in the rats treated with omeprazole compared to the control rats. Hypomagnesemia and lower bone and brain Mg2+ content were also detected in the omeprazole-treated groups compared with the control group. The expression of GluA1, PSD-95, and Arc in the hippocampus and the expression of AR in the dentate gyrus and CA1 of the hippocampus were significantly lower in the omeprazole-treated groups than in the control group. These results suggest that prolonged omeprazole treatment might lead to memory deficit by impairing glutamate receptor trafficking or synaptic anchoring. Hypomagnesemia and brain Mg2+ deficiency may be, at least in part, involved in omeprazole-induced memory impairment.

The effect of PARP inhibition on androgen receptor localization and activity in castration resistant prostate cancer.

e17037 Background: The poly(ADP-ribose) polymerase inhibitors (PARPi) olaparib and rucaparib have been approved for the treatment of metastatic castration resistant prostate cancer (mCRPC) in the setting of homologous recombination deficiency (HRD). Additionally, PARPi have been shown to modulate androgen receptor (AR) signaling, with a recent report demonstrating mono-ADP-ribosylation of cysteine residues in AR by Parp7. Here, we further evaluated the effect of PARPi on AR activity and localization. Methods: The effect of PARPi on cell growth and survival of CRPC cell lines was evaluated in vitro and in vivo. AR ribosylation was assessed in CRPC cell lines by immunoprecipitation (IP) assays and proximity ligation assays (PLA). The subcellular localization of AR was determined by quantitative immunofluorescence microscopy in CRPC cell lines and xenograft models. Changes in AR activity with PARPi treatment were evaluated by a luciferase reporter assay and AR target gene expression in a PDX model. Finally, PARPi mediated alteration in the AR protein interactome was evaluated by liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics. Results: The PARPi olaparib and talazoparib, and to a lesser extent veliparib, inhibited CRPC cell growth. Evidence of AR ribosylation was seen by IP and PLA. PARPi treatment of multiple in vitro and in vivo prostate cancer models resulted in a shift of AR subcellular localization, from predominantly nuclear to cytoplasmic compartments. In luciferase reporter assays, AR transactivation activity was decreased after PARPi treatment in a dose dependent manner. In vivo, in prostate cancer xenograft models, decreased AR target gene expression was seen upon PARPi treatment. LC-MS/MS proteomic studies revealed that PARP inhibition resulted in significant changes in the composition of AR interaction partners, in particular of proteins related to intracellular trafficking and nuclear transport. This suggests a potential link between altered AR complex assembly and the observed changes in AR subcellular localization in the context of PARPi treatment. Conclusions: We describe a novel sequela of PARPi therapy to alter AR localization and activity in CRPC. Single agent PARP inhibition can alter prostate cancer cell growth in vitro and in vivo. With PARPi treatment, AR localization is shifted to the cytoplasm, the AR interactome is altered, and AR transcriptional activity decreased. These findings implicate a collateral mechanism of PARPi in preventing prostate cancer cell growth/survival that may augment previously described mechanisms related to HDR and synthetic lethality. It is essential to understand the role of PARPi beyond synthetic lethality in the context of HRD in order to better define the spectrum of response to PARPi across patients, and for development of biology-informed combination therapies.

Seed extract of Thai Mucuna pruriens reduced male reproductive damage in rats induced by chronic stress

Context Thai Mucuna pruriens (L.) DC. var. pruriens (Fabaceae) (TMP) is known to enrich reproduction but preventive effects on stress related adverse reproductive parameters are not documented. Objective This study investigates the protective property of TMP seed extract on reproductive damage under chronic stress (CS). Materials and methods Male Sprague-Dawley rats were divided into four groups. The control and CS groups received distilled water, whereas the pre-treated rats received the aqueous TMP seed extract at doses of 150 and 300 mg/kg BW for 20 days before co-treatments with CS induction (immobilization and forced swimming) for 81 days. Serum was used to determine the cortisol and testosterone levels. Histology of testis and epididymis was observed with localization of androgen receptor (AR). Sperm parameters and the expression of steroidogenic acute regulatory (StAR), cytochrome P450 family 11 subfamily a member 1 (CYP11A1), AR, HSP70, caspases (3 and 9) and tyrosine phosphorylation (TyrPho) proteins were investigated. Results TMP extract improved cortisol level (0.84 ± 0.02 µg/dL) and protected against the damage of reproductive tissues and sperm parameters (count [49.78 ± 3.74 million sperm/mL], viability [90.01 ± 1.17%] and precocious acrosome reaction [1.38 ± 0.48%]). Expression of testicular StAR, CYP11A1, AR and HSP70 proteins was improved. Caspase expression was decreased in treated rats. TMP increased AR expression in CS sperm. Moreover, TyrPho protein expression was corrected after TMP administration. Conclusions TMP seed protected against adverse reproductive parameters in CS via improvements of functionally testicular markers and reductions of apoptotic proteins. It is possible to develop the TMP beans as an alternative medicine in treating of male subfertility caused by CS.

Receptor-related aspects of reduced lubrication in women of reproductive age with hypoandrogenism

Aim — to investigate receptivity of the vaginal epithelium in women with the female sexual dysfunction and hypoandrogenism.Materials and methods. The immunohistochemical investigation has been performed to define the density of androgen receptors (AR) and estrogen receptors (ER) in the distal part of the vaginal tract of women of reproductive age with hypoandrogenic provision of their hormonal background. For immunohistological examination, a biopsy of the lower third of the vaginal mucosa was taken using a dermopunch with a diameter of 3 mm in 20 women with female sexual dysfunction (FSD) and hypo­andro­genism. The control group consisted of 20 women of reproductive age without FSDmanifestations and disturbances in their hormonal levels. The survey was carried out at the stage of pre-conceptional preparation. Location: Ukrainian Scientific and Prac­tical Center for Endocrine Surgery, Transplantation of Endocrine Organs and Tissues of the Ministry of Health of Ukraine.Results. Immunoreactivity of ERα in the epithelium in the main and control groupswas 47.2 ± 22.2 and 86.1 ± 8.0 (p < 0.001), in the stroma — respectively 13.9 ± 16.8 and 86.1 ± 8.3 (p < 0.001), that is, immunoreactivity in the epithelium was significantly higher thanin the stroma (p < 0,01). The ERα locationin the vaginal epithelium was the samein both groups: cellsof the basal-intermediate layer along the basement membrane and the intermediate layer. No cells, expressing ERα, were detected in the surface layer. In stromal cells, ERαcontained in fibroblasts and smooth muscle cells. ERβ was found in cellsof thebasal — parabasal layer, the intermediate layer, occasionally —on the surface layerof vaginal epithelium, as well as in fibroblasts, vascular endothelium, where both nuclear andcytoplasmic staining was revealed. According to the results of immunohistochemical study of AR invagina, the proportion of AR-positive cells in healthy women was negligible (3.8 ± 2.2), that is considerably less than in patients with androgen deficiency (18.8 ± 8.3) and compared withother receptors. AR were localized mainly in the basal layer alongbasement membrane. Single AR-positive fibroblasts occurred in the stroma.Conclusions. Estrogen receptors α (ERα) are involved in the regulation of the processes of proliferation and differentiation of the vaginal epithelium under the influence of estrogen, as evidenced by a 1.8-fold increase in the density of stained ERα in women of the control group. The absence of correlations between ЕRα and ЕRβ receptors (r = 0.24; p < 0.05) may indicate the ability to change in the same tissue depending on the strength of estrogen effects on the body of a woman. Topical localization of androgen receptors in the basal layer testifies in favour of the safe use of local formsof androgensto overcome the reduced lubrication.

A Deep Insight of Spermatogenesis and Hormone Levels of Aqua-Cultured Turbot (Scophthalmus maximus)

Turbot (Scophthalmus maximus) is an important marine fish both in Europe and North China. Although there are plenty of studies on the reproduction of turbot, the complete cytological process of spermatogenesis remains unclear. In this study, we investigated the submicroscopic structure of total 23 types of male germ cells throughout the breeding season, with a relatively complete process of the primary spermatocytes. We found that the spermatid tail formed early at Spermatid II, and there were at least 16 spherical mitochondria in the spermatozoa. The hepatosomatic index (HSI) and gonadosomatic index (GSI) both peaked during the breeding season. Preliminary analysis showed that the vitality of mature sperm was negatively correlated with the proportion of sperm deformity. The serum 3,5,3′-triiodothyronine (T3), 5-hydroxytryptamine (5-HT), testosterone (T), 17α,20β-Dihydroxy-4-pregnen-3-one (17α,20β-DHP), and 17β-estradiol (E2) all increased during the maturity period, with the change of T content most noticeable. Whereas in the testis, an overall high level of 11-ketotestosterone (11-KT) was more remarkable. The expression and localization of androgen receptor (AR) mRNA showed that the AR was highly expressed at the stages of II (15 – 70 g), with a slight rebound at the mature stages [IV(2200 g) to V(2500 g)], whose change was ahead to the changes of T and 11-KT. Fluorescence in situ hybridization (FISH) analysis showed that the AR mainly distributed in but not limited to Sertoli cells. This study represents the most complete overview of the reproductive cycle and spermatogenesis of turbot, which provides an important reference for the reproduction research and the guidance of flatfish breeding.

Erratum Notice for Immunohistochemical Localization of Androgen Receptor in Rat Caput Epididymis during Postnatal Development

This article is an erratum notice for the following article: Gur FM, Timurkaan S. Immunohistochemical localization of androgen receptor in rat caput epididymis during postnatal development. J Clin Exp Invest. 2011;2(3):260-6. https://doi.org/10.5799/ahinjs.01.2011.03.0051

Mitochondrial localization, import, and mitochondrial function of the androgen receptor

Nuclear localization of androgen receptor (AR) directs transcriptional regulation of a host of genes, referred to as genomic signaling. Additionally, nonnuclear or nongenomic activities of the AR have long been described, but understanding of these activities remains elusive. Here, we report that AR is imported into and localizes to mitochondria and has a novel role in regulating multiple mitochondrial processes. Employing complementary experimental approaches of AR knockdown in AR-expressing cells and ectopic AR expression in AR-deficient cells, we demonstrate an inverse relationship between AR expression and mitochondrial DNA (mtDNA) content and transcription factor A, mitochondrial (TFAM), a regulator of mtDNA content. We show that AR localizes to mitochondria in prostate tissues and cell lines and is imported into mitochondria in vitro We also found that AR contains a 36-amino-acid-long mitochondrial localization sequence (MLS) capable of targeting a passenger protein (GFP) to the mitochondria and that deletion of the MLS abolishes the import of AR into the mitochondria. Ectopic AR expression reduced the expression of oxidative phosphorylation (OXPHOS) subunits. Interestingly, AR also controlled translation of mtDNA-encoded genes by regulating expression of multiple nuclear DNA-encoded mitochondrial ribosomal proteins. Consistent with these observations, OXPHOS supercomplexes were destabilized, and OXPHOS enzymatic activities were reduced in AR-expressing cells and restored upon AR knockdown. Moreover, mitochondrial impairment induced AR expression and increased its translocation into mitochondria. We conclude that AR localizes to mitochondria, where it controls multiple mitochondrial functions and mitonuclear communication. Our studies also suggest that mitochondria are novel players in nongenomic activities of AR.

Intrafollicular level of steroid hormones and the expression of androgen receptor in the equine ovary at puberty

Steroidogenic activity in the equine ovary from birth to puberty has been poorly investigated. This study aimed to examine the capability of the ovarian follicles of prepubertal and pubertal fillies to produce steroid hormones and to evaluate the expression and cellular localization of androgen receptor (AR) in their ovaries. The ovaries of 6–18 month-old fillies were divided into two groups: prepubertal (PrP) – without preovulatory follicle (pF) and corpus luteum (CL), and ovulating/postpubertal (Ov/pB) – with pF and/or CL in at least one of the gonads. Adult mares (Me) were used as a control. The concentration of progesterone (P4), testosterone (T) and estradiol (E2) in follicular fluid (FF) was measured by radioimmunoassay. AR distribution was assessed by immunohistochemistry, while AR protein expression was examined by Western blot analysis. In the female groups, E2 concentration in FF of small follicles (<10 mm) was low and increased with the diameter of the follicle reaching the greatest value in pF (Ov/pB and Me group). In follicles (11–30 mm) of PrP fillies, the concentration of E2 was similar to that from Ov/pB fillies, but less than half (P < 0.05) than in Me follicles. In FF from all classes of follicles of Ov/pB fillies, the concentration of all steroids was similar to that in Me. AR immunolocalization, predominantly nuclear, was observed in all types of follicular cells (granulosa and theca cells) as well as in stroma and luteal cells. The pattern of staining was dependent on the follicle size and the group of females. In smaller antral follicles and in pF, the nuclear AR staining in granulosa cells was stronger than that found in follicles of 21–25 mm. In theca interna cells of pF, both nuclear and faint cytoplasmic reactions were seen. In luteal cells, AR labeling was noted in the nuclei and the cytoplasm: the strongest one in the early CL and almost negative in the late CL. AR protein expression in filly and mare ovarian tissues was confirmed by Western blot analysis and detected as a single band at approximately 110 kDa. In summary, the ovaries of fillies aged at least 6 months are capable of active steroidogenesis. ARs are present either in the cell nuclei or cytoplasm of all compartments of the equine ovary. AR expression in follicular and stroma cells may indicate the sensitivity of the filly ovarian tissue to androgens, the impact of androgens on folliculogenesis and the development of the equine ovary via a receptor-mediated pathway.

External Genital Development, Urethra Formation, and Hypospadias Induction in Guinea Pig: A Double Zipper Model for Human Urethral Development

To determine whether the guinea pig phallus would be an appropriate model of human penile development, we characterized the embryology and sexual differentiation of guinea pig external genitalia and attended to induce hypospadias in males and tubular urethra formation in females pharmacologically. The external genitalia of guinea pig were collected from genital swelling initiation to newborn stages, and scanning electronic microscopy and histology were performed to visualize the morphology and structure. Immunohistochemistry was used to determine the androgen receptor localization. Bicalutamide and methyltestosterone were given to pregnant dams to reveal the role and timing of androgen in guinea pig penile masculinization. Canalization and dorsal-to-ventral movement of the urethral canal develops the urethral groove in both sexes, and then the males perform distal-opening-proximal-closing to form tubular urethra. More nuclear-localized androgen receptor is found in proximal genital tubercles of males than in females at (E) 29. Antiandrogen treatment at E26-E30 can cause hypospadias, and methyltestosterone administration at E27-E31 can induce tubular urethra formation in females. Fetal development of the guinea pig phallus is homologous to that of humans. Although guinea pig has structures similar to mouse, the urethral groove and the tubular urethra formation are more similar to humans. Antiandrogen treatment causes hypospadias in males and additional androgen induces tubular urethra formation in females. Thus, guinea pig is an appropriate model for further study of cellular and molecular mechanisms involved in distal-opening-proximal-closing in tubular urethra formation and the evaluation of the pathophysiological processes of hypospadias.

Immunohistochemical localization of androgen receptors in female mole rat (Spalax leucodon) tissues

Androgens exert their effects through androgen receptors (AR) in tissues. We investigated the distribution of AR in female mole rat tissues. Tissues were excised, fixed with 10% formalin and embedded in paraffin. Sections were stained after microwave antigen retrieval for immunohistochemistry. Immunostaining of AR immunostaining was detected in the nucleus or cytoplasm of the cells in the cerebral cortex, cerebellum, anterior pituitary, lung, liver, uterus and skin. Granulosa and some thecal cells in the ovary, cardiac muscle cells and adipose cells exhibited a nuclear reaction for AR. In the kidney, labeling of AR was restricted to the cytoplasm of tubule cells. We found that AR could be detected using immunohistochemistry in the nucleus or cytoplasm or both in the presence of androgens.

Impact of adjustable cryogel properties on the performance of prostate cancer cells in 3D.

BackgroundBiochemical and physical characteristics of extracellular environment play a key role in assisting cell behavior over different molecular pathways. In this study, we investigated how the presence of chemical binding sites, the pore network and the stiffness of designed scaffolds affected prostate cancer cells.MethodsA blend of poly hydroxyethyl methacrylate–alginate–gelatin scaffold was synthesized by cryogelation process using polyethyleneglycol diacrylate (PEGda) and glutaraldehyde as cross linkers. The chemical and mechanical scaffold properties were varied by concentration of gelatin and PEGda, respectively. The pore network was modified by applying different ‘freezing time’. Growth, spheroid formation and localization of androgen receptor (AR) were measured to evaluate cell response within various cryogel types.ResultsInsufficient porosity in combination with a brittle nature affects cell growth negatively. Spheroid size was reduced by porosity, elasticity as well as by the absence of the cell adhesive motif composed of arginine, glycine und aspartic acid (RGD). Localization of AR indicates its activity and should be under normal culture conditions in the nucleus. But in this study, we could investigate for the first time that AR remains in the cytoplasm when AR positive prostate cancer cells are cultured in scaffolds without RGD as well as in case of an insufficient pore network (total porosity under 10 %) and a too less stiffness of around 10 kPa.ConclusionsThe results indicate that for getting a reliable preclinical drug screening a three-dimensional prostate model system with appropriate biochemical and physical surrounding is needed.

TM4SF3 and AR: A Nuclear Complex that Stabilizes Both Proteins

Transmembrane 4 superfamily 3 (TM4SF3) was identified as a novel androgen-regulated gene in prostate cancer (PCa) cells. Our data demonstrate that TM4SF3 exhibits androgen-induced repression of the mRNA but up-regulation of the protein. The androgen positive effect on the TM4SF3 protein is of significant interest in view of the procancer functions of both androgens and tetraspanin proteins. Androgen positively regulates TM4SF3 protein stability by inhibiting its proteasome-dependent degradation. This androgen stabilization of TM4SF3 is involved in promoting PCa cell invasion and migration of both androgen-dependent and androgen-independent PCa cells. Although confirming androgen up-regulation of the TM4SF3 protein, we observed that TM4SF3 is localized not only to the membrane, but also, surprisingly, the nuclei of PCa cells. This novel nuclear localization of TM4SF3 depends on androgen-induced nuclear localization of androgen receptor (AR) in both androgen-dependent and androgen-independent PCa cell lines. TM4SF3 interacts with AR both in PCa cell types and in vitro, strongly suggesting a direct interaction. This direct interaction is required for the stabilization of not only TM4SF3, but also remarkably AR, because down-regulation of TM4SF3 resulted in reduced AR protein levels. As expected of an important AR regulator, TM4SF3 regulates androgen-dependent gene expression in and proliferation of PCa cells. Importantly, a direct correlation between AR and TM4SF3 protein levels and nuclear colocalization were also observed in prostate tumors, strongly suggesting that the mutual stabilization resulting from the AR-TM4SF3 interaction is found in tumors and that this interaction is important in PCa biology.

Kinase modulation of androgen receptor signaling: implications for prostate cancer.

Androgens and androgen receptors play essential roles in the development and progression of prostate cancer, a disease that claims roughly 28,000 lives annually. In addition to androgen biding, androgen receptor activity can be regulated via several post-translational modifications such as ubiquitination, acetylation, phosphorylation, methylation & SUMO-ylation. Off these modifications, phosphorylation has been the most extensively studied. Modification by phosphorylation can alter androgen receptor localization, protein stability and transcriptional activity, ultimately leading to changes in the biology of cancer cells and cancer progression. Understanding, role of phosphorylated androgen receptor species holds the key to identifying a potential therapeutic drug target for patients with prostate cancer and castrate resistant prostate cancer. Here, we present a brief review of recently discovered protein kinases phosphorylating AR, focusing on the functional role of phosphorylated androgen receptor species in prostate cancer and castrate resistant prostate cancer.

MiRNAs dysregulated in association with Gleason grade regulate extracellular matrix, cytoskeleton and androgen receptor pathways

The Gleason grading system is an important determinant of treatment decisions and prognosis in prostate cancer. It has a number of limitations, including significant inter-observer variability, creating a need for biological parameters to accurately assess the Gleason grade. The objective of this study was to determine the molecular correlates of the different Gleason grades. Global miRNA expression was analysed in pure regions of each Gleason grade. Bioinformatics analysis was performed to predict miRNA-mediated signalling. We experimentally validated the effect of miRNAs on target gene expression and cellular functions using cell line models. We also examined the correlation of miRNAs with biochemical failure, metastasis and prognosis. We identified miRNAs that are differentially expressed between grades 3 and 5, and the top biological processes associated with Gleason grade transition were extracellular matrix (ECM)-mediated signalling, focal adhesion kinase- and mitogen-activated kinase pathways. Transfection with miR-29c, miR-34a and miR-141 repressed genes involved in ECM-mediated pathways, such as SRC, PRKCA, COL1A1, ITGB1 and MAPK13, and decreased cell proliferation and migration. Furthermore, miR-29c and miR-34a influenced downstream pathways that affect actin cytoskeleton organization and androgen receptor localization. Finally, miR-29c, miR-34a, miR-141 and miR-148a showed inverse correlations with biochemical recurrence, but were independent of other clinical parameters. Our results demonstrate the potential role of miRNAs as independent prognostic markers and pave the road for a biological-based reclassification of the Gleason grading system.

Effects of androgen on immunohistochemical localization of androgen receptor and Connexin 43 in mouse ovary

Androgens have essential roles in the regulation of follicular development and female fertility. Androgen excess is the leading defect in polycystic ovary syndrome (PCOS) patients and involved in the ovarian dysfunction. The aim of this study was to elucidate the regarding regulatory role of androgen in the follicular development of female mouse. Immunohistochemical staining and Western blot analyses were performed to detect androgen receptor (AR) and Connexin 43 (Cx43) expression in ovaries from both control and testosterone-treated group mice. In this study, localizations of AR and Cx43 were dramatically altered in testosterone-treated mouse ovaries. In addition, AR expression was significantly increased, whereas Cx43 expression was markedly decreased after testosterone treatment. Alterations of AR and Cx43 expression by testosterone with concomitant reduction of MII oocytes. Overall, these results suggest the involvement of androgen in the regulation of AR and Cx43 localizations in mouse ovary. Alterations of AR and Cx43 expression by testosterone may affect normal folliculogenesis. Together these findings will enable us to begin understanding the important roles of AR and Cx43 actions in the regulation of follicular development, as well as providing insights into the role of AR and Cx43 actions in the androgen-associated reproductive diseases such as PCOS.

Immunoelectron microscope localization of androgen receptors and proliferating cell nuclear antigen in the epithelial cells of albino rat ventral prostate

Androgen receptor (AR) and proliferating cell nuclear antigen (PCNA) play a crucial role in development and progression of various prostatic diseases including prostatic carcinoma that is a leading cause of death in males. Previous studies have evaluated the expression pattern of AR and PCNA in prostate epithelial cells using immunohistochemistry (IHC). However, this technique has limited ability to identify their precise subcellular localization. Therefore, the aim of this study was to localize, subcellularly, AR and PCNA in the secretory epithelial cells of rat ventral prostate using post embedding immunogold-electron microscopy. The ventral lobes were dissected from six adult male albino rats after being perfused with paraformaldehyde. Some specimens were immuno-labeled with AR or PCNA and others were processed for immunoelectron microscope of AR and PCNA using 15-nm gold conjugated secondary antibodies. The results showed that, by immunoperoxidase reaction, AR and PCNA were localized diffusely throughout the nuclei of the epithelial cells of prostatic acini without visible cytoplasmic expression. However, the higher resolution immuno-electron microscopy was able to detect AR and PCNA in the nucleus and some cytoplasmic organelles. In conclusion, this study emphasizes the importance of immuoelectron microscopy in precise localization of AR and PCNA at the subcelullar levels in the secretory epithelial cells of the rat prostatic acini. These findings will help to further understand the mechanism of action of these receptors under normal and pathological conditions that could have future clinical application after careful human investigation.

Abstract 923: Analysis of microtubule perturbations and androgen receptor localization in circulating tumor cells from castration resistant prostate cancer patients as predictive biomarkers of clinical response to docetaxel chemotherapy

Abstract Although the recent availability of novel treatment for castration resistant prostate cancer (CRPC) has shown improvements in overall survival, CRPC is incurable and lethal disease. It has been recognized that tumor progression despite castrate levels of androgens is still associated with active signaling from the androgen receptor (AR) which affects gene transcription following its nuclear accumulation. Taxanes bind to and stabilize cellular microtubules (MTs) perturbing the fine and intricate organization of the microtubule network thus, impairing MT-based cell processes like cell division and intracellular trafficking. Our recent work showed that AR translocation from the cytoplasm to the nucleus is MT-dependent and that taxane treatment sequesters AR into the cytoplasm inhibiting the receptors transcriptional activity. While this novel mechanism of taxane activity may explain the clinical efficacy of taxanes in CRPC, clinically we still fail to understand the molecular basis of patient response or resistance to taxanes. We hypothesized that engagement of the MT-AR pathway by the taxanes can be used as read-out of effective drug-target engagement in cancer cells and can be used as biomarker predictive of taxane activity in CRPC patients. To assess the predictive role of these biomarkers, we are currently conducting a prospective clinical study in which we isolate circulating tumor cells (CTCs) from the peripheral blood of CRPC patient undergoing docetaxel treatment. We have enrolled 50 of the 80 total patients, from which we isolated CTCs before (at baseline) and during docetaxel treatment (at day 8 of first cycle of docetaxel and upon relapse) using two different approaches: the EpCAM-based immunomagnetic enrichment (CellSearch) and the ficolling technique to isolate unenriched PBMCs inclusive of CTCs. To avoid issues with leucocyte contamination we subject the EpCAM captured cells are to staining for DAPI , PSMA, CD-45, AR and Tubulin and image them by confocal microscopy. CTCs are defined as nucleated, PSMA+, CD-45- cells. The unenriched PBMC population is also subjected to the same multiplex confocal microscopy protocol. Microtubule network morphology and AR subcellular localization in then assessed in CTCs and each biomarker alone and in combination is correlated with clinical response to docetaxel treatment defined by PCWG2 recommendation. So far we have collected and analyzed baseline and cycle 1-Day 8 samples from all 50 patients and relapse samples from 31 patients. Determination of effective drug-target engagement on C1D8 may allow early detection of molecular response to treatment, or lack of molecular response, which will ultimately allow for chemotherapy customization for the individual patient. Citation Format: Shinsuke Tasaki, Matthew Sung, Alexandre Matov, Giuseppe Galletti, Elan Diamond, Neil Bander, Kathy Zhou, Scott Tagawa, David Nanus, Paraskevi Giannakakou. Analysis of microtubule perturbations and androgen receptor localization in circulating tumor cells from castration resistant prostate cancer patients as predictive biomarkers of clinical response to docetaxel chemotherapy. [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 923. doi:10.1158/1538-7445.AM2014-923