Regulation Of Androgen Receptor Signaling Research Articles

Lemur tyrosine kinase‐2, a novel regulator of androgen receptor signaling in prostate cancer (1095.1)

Androgens and the androgen receptor play essential roles in the development and progression of prostate cancer, which claims roughly around 28,000 lives every year. Contemporary reports have demonstrated that the stability and sensitivity of the androgen receptor can be manipulated through alteration in kinases activity. Recent Genome Wide Analysis Studies argues that a decrease in gene expression of Lemur Tyrosine Kinase 2 (LMTK2) is associated with an increased risk of prostate cancer. We show that manipulation of LMTK2 levels in the prostate adenocarcinoma cell line, LNCaP, markedly affects the expression of androgen dependent genes. Moreover, loss of LMTK2 leads to reduced androgen receptor degradation, enhancing the ability of androgen to activate their target genes. Furthermore, in vivo Proximity Ligation Assays and Co‐immunoprecipiation assay shows that the androgen receptor and LMTK2 are binding partners in LNCaP cells. Using a combination of biochemical, molecular and cellular approaches, our work defines a novel cellular pathway for the androgen receptor regulation in prostate adenocarcinoma cells; pharmacologic manipulation of such regulation will provide innovative and more effective therapies for the treatment of patients with prostate cancer.Grant Funding Source: NIH

MP52-14 SYNERGISTIC EFFECTS OF COMBINATION OF DOCETAXEL AND SAHA ON AR SIGNALING IN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research V1 Apr 2014MP52-14 SYNERGISTIC EFFECTS OF COMBINATION OF DOCETAXEL AND SAHA ON AR SIGNALING IN PROSTATE CANCER Kyung-Sik Han, Taekmin Kwon, In Gab Jeong, Dalsan You, Bumjin Lim, Donghyun Lee, Chunwoo Lee, Sungwoo Hong, Ha-Gyeong Kim, Jung Jin Hwang, Jun Hyuk Hong, Myung-Soo Choo, Tai Young Ahn, and Choung-Soo Kim Kyung-Sik HanKyung-Sik Han More articles by this author , Taekmin KwonTaekmin Kwon More articles by this author , In Gab JeongIn Gab Jeong More articles by this author , Dalsan YouDalsan You More articles by this author , Bumjin LimBumjin Lim More articles by this author , Donghyun LeeDonghyun Lee More articles by this author , Chunwoo LeeChunwoo Lee More articles by this author , Sungwoo HongSungwoo Hong More articles by this author , Ha-Gyeong KimHa-Gyeong Kim More articles by this author , Jung Jin HwangJung Jin Hwang More articles by this author , Jun Hyuk HongJun Hyuk Hong More articles by this author , Myung-Soo ChooMyung-Soo Choo More articles by this author , Tai Young AhnTai Young Ahn More articles by this author , and Choung-Soo KimChoung-Soo Kim More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1624AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail Introduction and Objectives Globally it is the sixth leading cause of cancer-related death in men, and it is now the first in the United Kingdom and second in the United States. At the early stage, androgen is required for cell growth and progression of prostate cancer. Therefore, androgen-deprivation therapy (ADT) is effective to patient with prostate cancer by inhibition of androgen synthesis. With time, however, most patients with prostate cancer eventually lose sensitivity to androgen withdrawal and progress to the development of hormone-refractory prostate cancer (HRPC) or castration-resistant prostate cancer (CRPC). Since the treatment for patient with CRPC is very limited, the novel therapeutic strategies and combination therapy are needed to overcome this resistance. Therefore, we examined whether histone deacetylase inhibitor (HDACI) might enhance the effects of docetaxel in CRPCs. Methods Hormone-independent CWR22Rv1 cells harboring androgen receptor (AR) were treated with docetaxel and/or HDACI, suberoylanilide hydroxamic acid (SAHA). Cell viability was measured with CellTiter-Glo¢ç reagent. AR signaling was analyzed by immunocytochemistry and Western blot of total cell lysates and nuclear fractionated proteins. Results The growth inhibitory effect of sequential treatment with docetaxel and SAHA was synergistic in CWR22Rv1 cells. Combination treatment decreased the levels of androgen receptor (AR), prostate specific antigen (PSA), and anti-apoptotic Bcl-2 proteins (Mcl-1, Bcl-xl) more efficiently, compared with docetaxel or SAHA alone. And combination treatment increased activation of caspase-3 remarkably, while docetaxel or SAHA alone increased it slightly. Moreover, combination treatment induced acetylation and bundling of tubulin, inhibiting nuclear accumulation of AR significantly. These synergistic effects of combination treatment were also observed in hormone-dependent LNCaP cells. Conclusions Docetaxel in combination with SAHA inhibited cell growth efficiently and induced apoptosis by repressing AR expression and translocation to nucleus, resulting in down regulation of AR signaling in hormone-independent CWR22Rv1 cells. These results suggest that combination treatment with docetaxel and SAHA can be a useful therapeutics for the treatment of CRPC patients. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e585 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Kyung-Sik Han More articles by this author Taekmin Kwon More articles by this author In Gab Jeong More articles by this author Dalsan You More articles by this author Bumjin Lim More articles by this author Donghyun Lee More articles by this author Chunwoo Lee More articles by this author Sungwoo Hong More articles by this author Ha-Gyeong Kim More articles by this author Jung Jin Hwang More articles by this author Jun Hyuk Hong More articles by this author Myung-Soo Choo More articles by this author Tai Young Ahn More articles by this author Choung-Soo Kim More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Virtual Screening of PRK1 Inhibitors: Ensemble Docking, Rescoring Using Binding Free Energy Calculation and QSAR Model Development

Protein kinase C Related Kinase 1 (PRK1) has been shown to be involved in the regulation of androgen receptor signaling and has been identified as a novel potential drug target for prostate cancer therapy. Since there is no PRK1 crystal structure available to date, multiple PRK1 homology models were generated in order to address the protein flexibility. An in-house library of compounds tested on PRK1 was docked into the ATP binding site of the generated models. In most cases a correct pose of the inhibitors could be identified by ensemble docking, while there is still a challenge of finding a reasonable scoring function that is able to rank compounds according to their biological activity. We estimated the binding free energy for our data set of structurally diverse PRK1 inhibitors using the MM-PB(GB)SA and QM/MM-GBSA methods. The obtained results demonstrate that a correlation between calculated binding free energies and experimental IC50 values was found to be usually higher than using docking scores. Furthermore, the developed approach was tested on a set of diverse PRK1 inhibitors taken from literature, which resulted in a significant correlation. The developed method is computationally inexpensive and can be applied as a postdocking filter in virtual screening as well as for optimization of PRK1 inhibitors in order to prioritize compounds for further biological characterization.

Abstract 3577: The role of Checkpoint Kinase 2 in the regulation of androgen receptor signaling and prostate cancer cell growth during progression to castration resistance.

Abstract As the second leading cause of cancer deaths in men, prostate cancer is a persistent, significant challenge for clinicians and researchers. Almost all patients with disseminated prostate cancer initially respond to androgen deprivation therapy. However, virtually every patient will relapse due to the growth of castration-resistant cancer cells and develop metastatic and lethal disease. Even with the recent development of new androgen ablation treatments such as Abiraterone and Enzalutamide, castration-resistant prostate cancer is still incurable. We hypothesize that compensatory signaling mechanisms that limit the effectiveness of androgen ablation can be overcome with therapeutic strategies targeting kinase cascades. In order to identify signaling pathways that regulate AR activity and prostate cancer cell growth we screened a panel of shRNAs targeting the human kinome in LNCaP prostate cancer cells grown in the presence and absence of androgen. We discovered that knockdown of Checkpoint Kinase 2 (CHK2) dramatically increased LNCaP prostate cancer cell proliferation. This observation is clinically relevant since CHK2 inactivating mutations arise in over 10% of prostate cancer patients and CHK2 expression decreases as prostate cancer progresses to a castration-resistant disease. Consistent with these clinical observations, CHK2 knockdown did not affect cell growth in the castration resistant C4-2 cell line, suggesting that the selection of the castration resistant C4-2 line from LNCaP involved the same effector as observed in clinical specimens. Consistent with this, CHK2 expression is lower in C4-2 cells compared to LNCaP. We determined that CHK2 knockdown increases androgen receptor (AR) transcriptional activity on both androgen activated and androgen repressed genes, providing evidence that CHK2 affects prostate cancer cell proliferation, at least in part, through the AR. These data suggest that CHK2 is a negative regulator of androgen sensitivity and prostate cancer cell growth and that CHK2 is lost during the progression to castration resistance. We are examining the role of CHK2 in regulating androgen-AR signaling, growth, and survival of prostate cancer cells; specifically we are identifying the crucial intracellular proteins that mediate CHK2 signaling and growth regulation of prostate cancer cells. Since CHK2 signaling is activated by DNA damage, such as that triggered by radiation therapy and brachytherapy, understanding how CHK2 signaling regulates the AR will provide critical insights into how we can combine current therapies with androgen blockade for greater clinical effectiveness. Citation Format: Huy Q. Ta, Melissa L. Ivey, Daniel Gioeli. The role of Checkpoint Kinase 2 in the regulation of androgen receptor signaling and prostate cancer cell growth during progression to castration resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3577. doi:10.1158/1538-7445.AM2013-3577

Yes‐associated protein, YAP, functions as a positive regulator of androgen receptor signaling in prostate cancer cells

Androgen receptor (AR) plays a central role in prostate tumor biology. Here, we studied the impact of yes‐associated protein (YAP) oncogene, a nuclear effector of the hippo tumor suppressor network, on AR signaling. We first showed that native YAP formed protein complexes with endogenous AR, and the complex formation that mainly occurred in cell nuclei was enhanced by androgen, as revealed by immunoprecipitation (IP) and western blots. Our promoter reporter analysis demonstrated that enforced YAP expression increased the activation of AR‐dependent promoter reporter gene, and that the carboxyl‐terminal site of YAP, which consists of WW and coiled‐coiled domains, appeared to be responsible for AR transactivation. Chromatin‐IP experiment demonstrated that YAP interacted with the DNA regions where AR also binds, and that androgen enhanced the YAP and chromatin DNA complexes. We also provide evidence that YAP induction promoted an androgen‐dependent and ‐independent cell growth, and that YAP knockdown had the opposite effects. Our data indicate that YAP is a binding partner and physiologic positive regulator of AR and suggest that altered Hippo‐YAP signaling could play a critical role in prostate tumor progression in humans.

Epigenetic Repression of miR-31 Disrupts Androgen Receptor Homeostasis and Contributes to Prostate Cancer Progression

Androgen receptor signaling plays a critical role in prostate cancer pathogenesis. Yet, the regulation of androgen receptor signaling remains elusive. Even with stringent androgen deprivation therapy, androgen receptor signaling persists. Here, our data suggest that there is a complex interaction between the expression of the tumor suppressor miRNA, miR-31, and androgen receptor signaling. We examined primary and metastatic prostate cancer and found that miR-31 expression was reduced as a result of promoter hypermethylation, and importantly, the levels of miR-31 expression were inversely correlated with the aggressiveness of the disease. As the expression of androgen receptor and miR-31 was inversely correlated in the cell lines, our study further suggested that miR-31 and androgen receptor could mutually repress each other. Upregulation of miR-31 effectively suppressed androgen receptor expression through multiple mechanisms and inhibited prostate cancer growth in vivo. Notably, we found that miR-31 targeted androgen receptor directly at a site located in the coding region, which was commonly mutated in prostate cancer. In addition, miR-31 suppressed cell-cycle regulators including E2F1, E2F2, EXO1, FOXM1, and MCM2. Together, our findings suggest a novel androgen receptor regulatory mechanism mediated through miR-31 expression. The downregulation of miR-31 may disrupt cellular homeostasis and contribute to the evolution and progression of prostate cancer. We provide implications for epigenetic treatment and support clinical development of detecting miR-31 promoter methylation as a novel biomarker.

Targeting the regulation of androgen receptor signaling by the heat shock protein 90 cochaperone FKBP52 in prostate cancer cells

Drugs that target novel surfaces on the androgen receptor (AR) and/or novel AR regulatory mechanisms are promising alternatives for the treatment of castrate-resistant prostate cancer. The 52 kDa FK506 binding protein (FKBP52) is an important positive regulator of AR in cellular and whole animal models and represents an attractive target for the treatment of prostate cancer. We used a modified receptor-mediated reporter assay in yeast to screen a diversified natural compound library for inhibitors of FKBP52-enhanced AR function. The lead compound, termed MJC13, inhibits AR function by preventing hormone-dependent dissociation of the Hsp90-FKBP52-AR complex, which results in less hormone-bound receptor in the nucleus. Assays in early and late stage human prostate cancer cells demonstrated that MJC13 inhibits AR-dependent gene expression and androgen-stimulated prostate cancer cell proliferation.

Abstract 4383: Nrf1 and Nrf2 mediated regulation of androgen receptor signaling in androgen-independent prostate cancer cells

Abstract Androgen deprivation is often used for the treatment of prostate cancer (PCa) patients. However, the development of androgen independent (AI) PCa remains a significant barrier to successful therapy. Interestingly, the androgen receptor (AR) is functionally expressed in all stages of PCa, although AI-PCa cells often overexpress AR and employ alternative signaling cascades to enhance AR signaling. Elevated reactive oxygen species (ROS) levels are associated with increased aggressiveness in PCa. Delineation of how these pathways are utilized by AI-PCa cells will implicate novel targets for abrogation of aggressive tumor cell growth in the later stages of PCa. We have used two sets of syngeneic PCa cell lines to emulate the progression of PCa in vitro from a non-tumorigenic stage (RWPE-1 and RWPE2 cells) to a tumorigenic and androgen-dependent (LNCaP cells) and androgen-independent (C4-2B cells) state. PSA luciferase assays showed that C4-2B cells have 7-8 fold higher activation of the AR regulated gene (PSA) than LNCaP cells after 1nM and 10nM DHT treatment for 24hrs. We have also observed that LNCaP and C4-2B cells have the ability to produce higher levels of (ROS), most likely due to an increased expression of the ROS producing enzymes, the NADPH oxidases, NOX4 and NOX5. Maintenance of higher ROS levels in C4-2B cells may allow red-ox pathways to be used for mitogenic purposes. These cells also overexpress the antioxidant proteins, Thioredoxin-1 (Trx-1) and Peroxiredoxin-1 (Prx-1), which may prevent the detrimental effects of long term exposure to ROS produced by NOX4 and NOX5. Red-ox pathways have been shown to modify hormone receptor signaling in both breast and prostate cancer. Trx-1 can differentially modulate estrogen receptor (ER) signaling in breast cancer and Prx-1 is known to bind directly to AR and enhance AR transactivation in PCa cells. The overexpression of these antioxidant proteins in AI-PCa suggests that these cells can use antioxidants to enhance AR mediated signaling, even under androgen deprived conditions. Hence, suppression of AR dependent PCa cell growth may be achieved by suppressing the induction of Prx-1 and Trx-1. Induction of these antioxidant proteins are dictated by two redox sensitive transcription factors, Nrf1 and Nrf2. C4-2B cells showed the highest expression of Nrf1 and the lowest expression of Nrf2 at the mRNA and protein levels. Indeed, decreased Nrf2 expression has also been associated with PCa aggressiveness. It is likely that a balance between Nrf1 and Nrf2 signaling regulates the expression of Prx-1 and Trx-1, which may ultimately regulate AR activity in C4-2B cells. A clearer understanding of how the Nrf1/Nrf2 balance mediates antioxidant protein expression and AR mediated gene regulation may lead to new avenues for treatment of hormone independent PCa. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4383.

FKBP51 Promotes Assembly of the Hsp90 Chaperone Complex and Regulates Androgen Receptor Signaling in Prostate Cancer Cells

Prostate cancer progression to the androgen-independent (AI) state involves acquisition of pathways that allow tumor growth under low-androgen conditions. We hypothesized that expression of molecular chaperones that modulate androgen binding to AR might be altered in prostate cancer and contribute to progression to the AI state. Here, we report that the Hsp90 cochaperone FKBP51 is upregulated in LAPC-4 AI tumors grown in castrated mice and describe a molecular mechanism by which FKBP51 regulates AR activity. Using recombinant proteins, we show that FKBP51 stimulates recruitment of the cochaperone p23 to the ATP-bound form of Hsp90, forming an FKBP51-Hsp90-p23 superchaperone complex. In cells, FKBP51 expression promotes superchaperone complex association with AR and increases the number of AR molecules that undergo androgen binding. FKBP51 stimulates androgen-dependent transcription and cell growth, and FKBP51 is part of a positive feedback loop that is regulated by AR and androgen. Finally, depleting FKBP51 levels by short hairpin RNA reduces the transcript levels of genes regulated by AR and androgen. Because the superchaperone complex plays a critical role in determining the ligand-binding competence and transcription function of AR, it provides an attractive target for inhibiting AR activity in prostate cancer cells.

Nemo‐like kinase induces apoptosis and inhibits androgen receptor signaling in prostate cancer cells

The mitogen-activated protein kinases (MAPKs) regulate cell growth, differentiation, and stress responses, and many critical signaling pathways are subject to cross-regulation by MAPK signaling. Previous studies have yielded evidence of cross-talk between the MAPK pathways and androgen receptor (AR) signaling, which plays a critical role in growth control of both normal prostate and prostate cancer (PCa). Objective of this study was to evaluate the expression of MAPK-like protein nemo-like kinase (NLK) in PCa and its effects on AR-mediated transcription. Real-time PCR and IHC were used to evaluate levels of NLK in prostatic samples. Effects of over-expression of NLK on apoptosis and proliferation were determined using Western blot and flow cytometry. Effects on AR signaling were evaluated using over-expression and knockdown of NLK in PCa cells in combination with PCR, Western blotting and reporter assays. Our results show that the expression of NLK is decreased in PCa metastases in comparison to normal prostate epithelium and primary PCa. Our results also show that over-expression of NLK resulted in induction of apoptosis, which was more pronounced in AR-expressing LNCaP versus AR-negative PC-3 cells. Higher levels of NLK decreased levels of AR mRNA and protein as well as inhibited AR-mediated transcription. NLK expression is altered during PCa progression and it is involved in regulation of AR signaling in these cells. A deeper understanding of the roles of NLK in regulation of AR-mediated transcription and control of PCa progression may point the way to new modes of therapeutic intervention in this disease.

Pias3-Dependent SUMOylation Directs Rod Photoreceptor Development

Specification of retinal rod photoreceptors is determined by several different transcription factors that activate expression of rod-specific genes and repress expression of cone photoreceptor-specific genes. The mechanism by which this dual regulation occurs is unclear. We have found that Pias3, a transcriptional coregulator and E3 SUMO ligase that is selectively expressed in developing photoreceptors, promotes the differentiation of rod photoreceptors while preventing rods from adopting cone photoreceptor-like characteristics. Pias3 binds the photoreceptor-specific transcription factors Crx and Nr2e3 and is specifically targeted to the promoters of photoreceptor-specific genes. Pias3 SUMOylates Nr2e3, converting it into a potent repressor of cone-specific gene expression. Rod- and cone-specific promoters are bound by hyperSUMOylated proteins in rod photoreceptors, and blocking SUMOylation in photoreceptors results in cells with morphological and molecular features of cones and an absence of rod-specific markers. Our data thus identify Pias3-mediated SUMOylation of photoreceptor-specific transcription factors as a key mechanism of rod specification.

FOXP1 is an androgen-responsive transcription factor that negatively regulates androgen receptor signaling in prostate cancer cells

Androgen and androgen receptor (AR) play important roles in the formation and the progression of prostate cancer. AR activates its target genes by recruiting various coregulators and transcriptional factors. Here we show that the FOXP1 forkhead transcription factor is a novel androgen-regulated gene. By sequencing DNA fragments obtained from chromatin immunoprecipitation (ChIP), a bona-fide AR binding site (ARBS) is identified in an intron region of FOXP1 gene. FOXP1 can be induced by androgen in hormone-sensitive prostate cancer LNCaP cells at both mRNA and protein levels. In particular, a smaller FOXP1 variant, FOXP1D, is upregulated in response to androgen. Notably, we demonstrate that FOXP1 directly interacts with AR and negatively regulates AR signaling ligand-dependently, as exemplified by the transcriptional repression of PSA gene regulated by androgen-dependent FOXP1 recruitment on its enhancer region. We show that several other forkhead transcription factors are also androgen-responsive in LNCaP cells. Our study provides a new insight to the function of forkhead transcription factors that modulates AR signaling as an androgen-regulated transcriptional factor, which would contribute to the tumorigenesis of prostate cancer.

Impaired Dihydrotestosterone Catabolism in Human Prostate Cancer: Critical Role of AKR1C2 as a Pre-Receptor Regulator of Androgen Receptor Signaling

We previously reported the selective loss of AKR1C2 and AKR1C1 in prostate cancers compared with their expression in paired benign tissues. We now report that dihydrotestosterone (DHT) levels are significantly greater in prostate cancer tumors compared with their paired benign tissues. Decreased catabolism seems to account for the increased DHT levels as expression of AKR1C2 and SRD5A2 was reduced in these tumors compared with their paired benign tissues. After 4 h of incubation with benign tissue samples, (3)H-DHT was predominantly catabolized to the 5alpha-androstane-3alpha,17beta-diol metabolite. Reduced capacity to metabolize DHT was observed in tumor samples from four of five freshly isolated pairs of tissue samples, which paralleled loss of AKR1C2 and AKR1C1 expression. LAPC-4 cells transiently transfected with AKR1C1 and AKR1C2, but not AKR1C3, were able to significantly inhibit a dose-dependent, DHT-stimulated proliferation, which was associated with a significant reduction in the concentration of DHT remaining in the media. R1881-stimulated proliferation was equivalent in all transfected cells, showing that metabolism of DHT was responsible for the inhibition of proliferation. PC-3 cells overexpressing AKR1C2 and, to a lesser extent, AKR1C1 were able to significantly inhibit DHT-dependent androgen receptor reporter activity, which was abrogated by increasing DHT levels. We speculate that selective loss of AKR1C2 in prostate cancer promotes clonal expansion of tumor cells by enhancement of androgen-dependent cellular proliferation by reducing DHT metabolism.

Inhibition of Androgen-Independent Prostate Cancer by Estrogenic Compounds Is Associated with Increased Expression of Immune-Related Genes

The clinical utility of estrogens for treating prostate cancer (CaP) was established in the 1940s by Huggins. The classic model of the anti-CaP activity of estrogens postulates an indirect mechanism involving the suppression of androgen production. However, clinical, preclinical studies have shown that estrogens exert growth-inhibitory effects on CaP under low-androgen conditions, suggesting additional modes whereby estrogens affect CaP cells and/or the microenvironment. Here we have investigated the activity of 17β estradiol (E2) against androgen-independent CaP, identified molecular alterations in tumors exposed to E2. E2 treatment inhibited the growth of all four androgen-independent CaP xenografts studied (LuCaP 35V, LuCaP 23.1AI, LuCaP 49, LuCaP 58) in castrated male mice. The molecular basis of growth suppression was studied by cDNA microarray analysis, which indicated that multiple pathways are altered by E2 treatment. Of particular interest are changes in transcripts encoding proteins that mediate immune responses, regulate androgen receptor signaling. In conclusion, our data show that estrogens have powerful inhibitory effects on CaP in vivo in androgendepleted environments, suggest novel mechanisms of estrogen-mediated antitumor activity. These results indicate that incorporating estrogens into CaP treatment protocols could enhance therapeutic efficacy even in cases of advanced disease.

Regulator of G‐protein Signaling 2 (RGS2) inhibits androgen‐independent activation of androgen receptor in prostate cancer cells

Hormones acting through G protein-coupled receptors (GPCRs) can cause androgen-independent activation of androgen receptor (AR) in prostate cancer cells. Regulators of G-protein Signaling (RGS) proteins, through their GTPase activating protein (GAP) activities, inhibit GPCR-mediated signaling by inactivating G proteins. Here, we identified RGS2 as a gene specifically down-regulated in androgen-independent prostate cancer cells. Expression of RGS2, but not other RGS proteins, abolished androgen-independent AR activity in androgen-independent LNCaP cells and CWR22Rv1 cells. In LNCaP cells, RGS2 inhibited Gq-coupled GPCR signaling. Expression of exogenous wild-type RGS2, but not its GAP-deficient mutant, significantly reduced AR activation by constitutively activated GqQ209L mutant whereas silencing endogenous RGS2 by siRNA enhanced GqQ209L-stimulated AR activity. RGS2 had no effect on RGS-insensitive GqQ209L/G188S-induced AR activation. Furthermore, extracellular signal-regulated kinase 1/2 (ERK1/2) were found to be involved in RGS2-mediated regulation of androgen-independent AR activity. In addition, RGS2 functioned as a growth suppressor for androgen-independent LNCaP cells whereas androgen-sensitive LNCaP cells with RGS2 silencing had a growth advantage under steroid-reducedconditions. Finally, RGS2 expression level was significantly decreased in human prostate tumor specimens. Takentogether, our results suggest RGS2 as a novel regulator of AR signaling and its repression may be an important step during prostate tumorigenesis and progression.

Regulator of G-protein signaling 2 (RGS2) inhibits androgen-independent activation of androgen receptor in prostate cancer cells

Hormones acting through G protein-coupled receptors (GPCRs) can cause androgen-independent activation of androgen receptor (AR) in prostate cancer cells. Regulators of G-protein signaling (RGS) proteins, through their GTPase activating protein (GAP) activities, inhibit GPCR-mediated signaling by inactivating G proteins. Here, we identified RGS2 as a gene specifically downregulated in androgen-independent prostate cancer cells. Expression of RGS2, but not other RGS proteins, abolished androgen-independent AR activity in androgen-independent LNCaP cells and CWR22Rv1 cells. In LNCaP cells, RGS2 inhibited G(q)-coupled GPCR signaling. Expression of exogenous wild-type RGS2, but not its GAP-deficient mutant, significantly reduced AR activation by constitutively activated G(q)Q209L mutant whereas silencing endogenous RGS2 by siRNA enhanced G(q)Q209L-stimulated AR activity. RGS2 had no effect on RGS-insensitive G(q)Q209L/G188S-induced AR activation. Furthermore, extracellular signal-regulated kinase 1/2 (ERK1/2) was found to be involved in RGS2-mediated regulation of androgen-independent AR activity. In addition, RGS2 functioned as a growth suppressor for androgen-independent LNCaP cells whereas androgen-sensitive LNCaP cells with RGS2 silencing had a growth advantage under steroid-reduced conditions. Finally, RGS2 expression level was significantly decreased in human prostate tumor specimens. Taken together, our results suggest RGS2 as a novel regulator of AR signaling and its repression may be an important step during prostate tumorigenesis and progression.

A type I DnaJ homolog, DjA1, regulates androgen receptor signaling and spermatogenesis

Two type I DnaJ homologs DjA1 (DNAJA1; dj2, HSDJ/hdj-2, rdj1) and DjA2 (DNAJA2; dj3, rdj2) work similarly as a cochaperone of Hsp70s in protein folding and mitochondrial protein import in vitro. To study the in vivo role of DjA1, we generated DjA1-mutant mice. Surprisingly, loss of DjA1 in mice led to severe defects in spermatogenesis that involve aberrant androgen signaling. Transplantation experiments with green fluorescent protein-labeled spermatogonia into DjA1(-/-) mice revealed a primary defect of Sertoli cells in maintaining spermiogenesis at steps 8 and 9. In Sertoli cells of DjA1(-/-) mice, the androgen receptor markedly accumulated with enhanced transcription of several androgen-responsive genes, including Pem and testin. Disruption of Sertoli-germ cell adherens junctions was also evident in DjA1(-/-) mice. Experiments with DjA1(-/-) fibroblasts and primary Sertoli cells indicated aberrant androgen receptor signaling. These results revealed a critical role of DjA1 in spermiogenesis and suggest that DjA1 and DjA2 are not functionally equivalent in vivo.

Regulation of androgen receptor signaling in prostate cancer

Prostate cancer is a significant cause of morbidity and mortality worldwide. Normal prostate tissue is regulated by androgens, which activate the androgen receptor, a nuclear receptor transcription factor. Most prostate tumors retain androgen dependence, therefore, current therapies for advanced prostate cancer either reduce androgen levels or prevent binding to the androgen receptor. Despite this regimen, prostate cancer invariably progresses to a fatal, androgen-refractory state. Although these relapsed tumors are androgen independent, they are still dependent on the androgen receptor for their growth and survival. The focus of this review will be to highlight our current understanding of the mechanisms of androgen receptor activation in androgen-refractory prostate cancer. How these mechanisms of androgen receptor activation could be targeted in this advanced stage of the disease is also discussed.

Regulation of androgen receptor signaling by PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor through distinct mechanisms in prostate cancer cells.

Defects in the PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor gene have been found in many human cancers including breast and prostate. Here we show that PTEN suppresses androgen receptor (AR) activity via a phosphatidylinositol-3-OH kinase/Akt-independent pathway in the early passage numbers prostate cancer LNCaP cells. We provide the direct links between PTEN and androgen/AR signaling by demonstrating that AR directly interacts with PTEN. The interaction between PTEN and AR inhibits the AR nuclear translocation and promotes the AR protein degradation that result in the suppression of AR transactivation and induction of apoptosis. The minimum interaction peptide within AR (amino acids 483-651) disrupts the interaction of PTEN with AR and reduces the PTEN effect on AR transactivation and apoptosis. Genetic approaches using PTEN-null mouse embryonic fibroblasts (MEFs) further demonstrate that both AR expression and AR activity were much higher in PTEN-null MEFs than wild-type MEFs, and reintroducing PTEN into PTEN-null MEFs dramatically reduced AR protein levels and AR activity. Interestingly, we also found that PTEN could suppress AR activity via the phosphatidylinositol-3-OH kinase/Akt-dependent pathway in the higher passage number LNCaP cells, because restoration of Akt activity blocks the effect of PTEN on AR activity. Together, these contrasting PTEN effects on AR activity in the same prostate cancer cell line with different passage numbers suggest that PTEN, via distinct mechanisms, differentially regulates AR in various stages of prostate cancers.

Protein Inhibitor of Activated STAT3 Regulates Androgen Receptor Signaling in Prostate Carcinoma Cells

Protein inhibitor of activated STAT3 (PIAS3) is a specific inhibitor of signal transducer and activator of transcription 3 (STAT3). PIAS3 binds to STAT3 and inhibits its DNA-binding activity, and thereby STAT3-mediated gene activation. PIAS1, another member of the PIAS family, was recently shown to interact with the androgen receptor (AR), a nuclear hormone receptor that has an important role in both physiological and pathological processes, and acts as a cofactor for AR. Here we demonstrate that PIAS3 is expressed in prostate cancer cells and its expression is induced in response to dihydrotestosterone (DHT) treatment. Ectopic overexpression of PIAS3 suppressed AR-mediated gene activation induced by DHT-stimulation in LNCaP cells. We provide evidence that these activities were due to direct physical interactions between PIAS3 and AR. These results indicate that PIAS3 acts as a coregulator of AR signaling pathway in prostate cancer cells.