Androgen Receptor Nuclear Localization Research Articles

ARN-509: A Novel Antiandrogen for Prostate Cancer Treatment

Continued reliance on the androgen receptor (AR) is now understood as a core mechanism in castration-resistant prostate cancer (CRPC), the most advanced form of this disease. While established and novel AR pathway-targeting agents display clinical efficacy in metastatic CRPC, dose-limiting side effects remain problematic for all current agents. In this study, we report the discovery and development of ARN-509, a competitive AR inhibitor that is fully antagonistic to AR overexpression, a common and important feature of CRPC. ARN-509 was optimized for inhibition of AR transcriptional activity and prostate cancer cell proliferation, pharmacokinetics, and in vivo efficacy. In contrast to bicalutamide, ARN-509 lacked significant agonist activity in preclinical models of CRPC. Moreover, ARN-509 lacked inducing activity for AR nuclear localization or DNA binding. In a clinically valid murine xenograft model of human CRPC, ARN-509 showed greater efficacy than MDV3100. Maximal therapeutic response in this model was achieved at 30 mg/kg/d of ARN-509, whereas the same response required 100 mg/kg/d of MDV3100 and higher steady-state plasma concentrations. Thus, ARN-509 exhibits characteristics predicting a higher therapeutic index with a greater potential to reach maximally efficacious doses in man than current AR antagonists. Our findings offer preclinical proof of principle for ARN-509 as a promising therapeutic in both castration-sensitive and castration-resistant forms of prostate cancer.

Prospective analysis of prostate cancer (PC) circulating tumor cells (CTCs) to predict response to docetaxel (DOC) chemotherapy.

100 Background: Taxane chemotherapy induces cellular microtubule stabilization (bundling) leading to mitotic arrest and apoptotic cell death. We recently reported that in castration-resistant PC, stabilizing of microtubules by taxanes inhibits ligand-induced androgen receptor (AR) nuclear translocation and activation of ARE-gene transcription (Cancer Res 2011;71:6019). This suggests the clinical activity of DOC results from inhibition of AR signaling. A pilot analysis of CTCs from 14 CRPC pts receiving taxanes revealed that predominant cytoplasmic AR and decreased total AR intensity each was associated with increased odds of response (P<0.001). We initiated a prospective trial to determine if CTC analysis could predict DOC response. Methods: Eligibility: Patients (pts) receiving DOC q21 days for CRPC with >5 CTCs determined by CellSearch. On cycle 1 d1 + d8, CTCs are isolated by ficoll tube separation and by CellSearch enrichment. Cells are stained for expression of prostate specific membrane antigen (PSMA), CD45, AR and acetylated tubulin (AcTub) as a marker of microtubule stabilization. AR localization and total fluorescence intensity, and AcTub intensity are quantitated by confocal microscopy and correlated with response to DOC using PCWG2 criteria. 72 pts are planned to provide 80% power to detect an association. Results: 28 of 72 pts have been prospectively enrolled to assess AR and AcTub as a biomarker of response. 20 pts have discontinued DOC. Day 1 samples were obtained from 27 pts and day 8 on 22 (5 pts on days 9 to 21). Immunofluorescence staining and evaluation of PSMA, CD45, and acetylated tubulin was performed successfully in 8 pts analyzed to date. CTCs can be identified in all pts. Cells are scored for AR and AcTub intensity (scale 1- 4) and AR nuclear vs cytoplasmic localization. Conclusions: Our results to date indicate that PC CTCs can be readily isolated from men receiving DOC chemotherapy in a multicenter fashion and assayed for AR localization and AcTub. Preliminary results suggest that monitoring AR subcellular localization and microtubule bundling in CTCs may predict clinical responses to DOC chemotherapy, warranting further study, including our prospective trial.

Casein kinase 2 inhibition attenuates androgen receptor function and cell proliferation in prostate cancer cells

Casein kinase 2 (CK2) is constitutively active with dual specificity and exists as a hetero-tetrameric complex of α, α', and β subunits. Its aberrant expression and elevated activity have been linked to many human cancers, including prostate cancer. As an effort to develop new chemotherapy for prostate cancers, in this study, we tested the effects of tetra-bromo-cinnamic acid (TBCA), a newly synthetic CK2-selective CK2 inhibitor, on androgen receptor (AR) transactivation, cell proliferation, and viability in multiple prostate cancer cell lines. We utilized a comprehensive approach of a newly synthetic CK2-selective inhibitor TBCA, plus gene-specific siRNAs in multiple cell-based assays to further understand the role of CK2 in AR signaling. Alamar-blue-based cell growth assay, flow cytometry for cell cycle distribution, Luciferase report gene assay for AR transactivation, and immuno-fluorescent approach for AR nuclear localization as well as quantitative PCR assay for AR-mediated gene expression were utilized. The significance of the differences between treatment and control was analyzed using the SPSS software (SPSS, Chicago, IL). Our data revealed that TBCA reduced cell proliferation and caused G2/M cell cycle arrest in a dose-dependent manner. Further analysis demonstrated that TBCA blocked AR nuclear translocation and gene expression. To confirm the target specificity, we used gene-specific siRNAs for both CK2α and CK2α' subunits, and the results suggested that both CK2 catalytic subunits are involved in androgen-stimulated AR nuclear translocation and AR-mediated gene expression in prostate cancer cells. CK2 subunits α and α' are likely involved in AR signaling, and TBCA might be useful in the management of prostate cancers as a chemo-preventive agent in the future.

Hsp90 inhibitor 17‐AAG inhibits progression of LuCaP35 xenograft prostate tumors to castration resistance

Advanced prostate cancer is currently treated with androgen deprivation therapy (ADT). ADT initially results in tumor regression; however, all patients eventually relapse with castration-resistant prostate cancer. New approaches to delay the progression of prostate cancer to castration resistance are in desperate need. This study addresses whether targeting Heat shock protein 90 (HSP90) regulation of androgen receptor (AR) can inhibit prostate cancer progression to castration resistance. The HSP90 inhibitor 17-AAG was injected intraperitoneally into nude mice bearing LuCaP35 xenograft tumors to determine the effect of HSP90 inhibition on prostate cancer progression to castration resistance and host survival. Administration of 17-AAG maintained androgen-sensitivity, delayed the progression of LuCaP35 xenograft tumors to castration resistance, and prolonged the survival of host. In addition, 17-AAG prevented nuclear localization of endogenous AR in LuCaP35 xenograft tumors in castrated nude mice. Targeting Hsp90 or the mechanism by which HSP90 regulates androgen-independent AR nuclear localization and activation may lead to new approaches to prevent and/or treat castration-resistant prostate cancer.

Antiandrogenic and growth inhibitory effects of ring‐substituted analogs of 3,3′‐diindolylmethane (Ring‐DIMs) in hormone‐responsive LNCaP human prostate cancer cells

Cruciferous vegetables protect against prostate cancer. Indole-3-carbinol (I3C) and its major metabolite 3,3'-diindolylmethane (DIM), exhibit antitumor activities in vitro and in vivo. Several synthetic ring-substituted dihaloDIMs (ring-DIMs) appear to have increased anticancer activity. Inhibition of LNCaP prostate cancer cell growth was measured by a WST-1 cell viability assay. Cytoplasmic and nuclear proteins were analyzed by immunoblotting and immunofluorescence. Androgen receptor (AR) activation was assessed by measuring prostate-specific antigen (PSA) expression and using LNCaP cells containing human AR and an AR-dependent probasin promoter-green fluorescent protein (GFP) construct. Like DIM, several ring-substituted dihaloDIM analogs, namely 4,4'-dibromo-, 4,4'-dichloro-, 7,7'-dibromo-, and 7,7'-dichloroDIM, significantly inhibited DHT-stimulated growth of LNCaP cells at concentrations ≥1 µM. We observed structure-dependent differences for the effects of the ring-DIMs on AR expression, nuclear AR accumulation and PSA levels in LNCaP cells after 24 hr. Both 4,4'- and 7,7'-dibromoDIM decreased AR protein and mRNA levels, whereas 4,4'- and 7,7'-dichloroDIM had minimal effect. All four dihaloDIMs (10 and 30 µM) significantly decreased PSA protein and mRNA levels. Immuofluorescence studies showed that only the dibromoDIMs increased nuclear localization of AR. All ring-DIMs caused a concentration-dependent decrease in fluorescence induced by the synthetic androgen R1881 in LNCaP cells transfected with wild-type human AR and an androgen-responsive probasin promoter-GFP gene construct, with potencies up to 10-fold greater than that of DIM. The antiandrogenic effects of ring-DIMs suggest they may form the basis for the development of novel agents against hormone-sensitive prostate cancer, alone or in combination with other drugs.

An Interdomain Interaction of the Androgen Receptor Is Required for Its Aggregation and Toxicity in Spinal and Bulbar Muscular Atrophy

Polyglutamine expansion within the androgen receptor (AR) causes spinal and bulbar muscular atrophy (SBMA) and is associated with misfolded and aggregated species of the mutant AR. We showed previously that nuclear localization of the mutant AR was necessary but not sufficient for SBMA. Here we show that an interdomain interaction of the AR that is central to its function within the nucleus is required for AR aggregation and toxicity. Ligands that prevent the interaction between the amino-terminal FXXLF motif and carboxyl-terminal AF-2 domain (N/C interaction) prevented toxicity and AR aggregation in an SBMA cell model and rescued primary SBMA motor neurons from 5α-dihydrotestosterone-induced toxicity. Moreover, genetic mutation of the FXXLF motif prevented AR aggregation and 5α-dihydrotestosterone toxicity. Finally, selective androgen receptor modulators, which prevent the N/C interaction, ameliorated AR aggregation and toxicity while maintaining AR function, highlighting a novel therapeutic strategy to prevent the SBMA phenotype while retaining AR transcriptional function.

Abstract 4076: Taxanes inhibit the microtubule-dependent androgen receptor trafficking and signaling in prostate cancer

Abstract Androgens (testosterone, DHT) control the development, differentiation and function of the prostate. Their principal action is to regulate gene expression through the androgen receptor (AR), a nuclear steroid hormone receptor, which translocates to the nucleus upon binding to its ligand and activates androgen response elements of target genes. This drives the growth and progression of prostate cancer (PC), the most commonly diagnosed cancer in males. Initial treatment involves androgen ablation, yet, these tumors eventually progress to a castrate-resistant state and taxanes represent the most effective class of anti-tumor agents to improve survival of CRPC patients. However, the molecular basis of taxane activity in prostate cancer is not fully elucidated. Preliminary data from out lab has shown that taxane treatment of PC cells inhibits the ligand-induced nuclear localization of AR and subsequent activation of ARE-containing genes including PSA. We further show that AR colocalizes with the microtubule (MT) cytoskeleton by immunofluorescence followed by confocal microscopy. This result is further supported by co-immunoprecipitation and microtubule co-sedimentation assays that revealed the preferential association of the WT-AR to the MT polymer rather than tubulin dimers. Dynein, a minus-end directed MT motor protein involved in moving cargoes towards the nucleus, co-precipitated with AR and tubulin in untreated PC cells and at higher amounts following ligand induction, further implicating microtubule dynamics in AR trafficking and activity. To investigate whether AR binds MTs directly or via accessory proteins we plan to perform in vitro tubulin binding assays using purified MT protein and purified recombinant AR. We further plan on identifying the minimum AR domain that mediates microtubule binding. To that end we have generated deletion mutants of the AR, exogenously expressed them in PC3 cells stably expressing mCherry-tubulin and are in the process of testing their MT binding ability. To assess the role of microtubule dynamics in AR trafficking and nuclear translocation we performed live-cell confocal microscopy using PC3-mCherry-tubulin cells transfected with full length wild-type GFP-AR. This study revealed AR trafficking along the MTs and rapid AR nuclear accumulation within 30 min of R1881 (synthetic AR ligand) treatment. In contrast, taxol pre-treatment at concentrations that stabilized MTs attenuated the ligand-induced AR nuclear accumulation as well as the rate of AR's translocation to the nucleus, resulting in increased cytoplasmic sequestration of the receptor. Taken together we have identified a new mechanism of action for taxanes that involves the microtubule-dependent regulation of AR trafficking and downstream signalling. This mechanism may help us better understand the molecular basis of clinical response to taxane treatment in CRPC. 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 4076.

14-3-3η Amplifies Androgen Receptor Actions in Prostate Cancer

PURPOSE: Androgen receptor abundance and androgen receptor-regulated gene expression in castration-recurrent prostate cancer are indicative of androgen receptor activation in the absence of testicular androgen. Androgen receptor transactivation of target genes in castration-recurrent prostate cancer occurs in part through mitogen signaling that amplifies the actions of androgen receptor and its coregulators. Herein we report on the role of 14-3-3eta in androgen receptor action. Experimental Design and RESULTS: Androgen receptor and 14-3-3eta colocalized in COS cell nuclei with and without androgen, and 14-3-3eta promoted androgen receptor nuclear localization in the absence of androgen. 14-3-3eta interacted with androgen receptor in cell-free binding and coimmunoprecipitation assays. In the recurrent human prostate cancer cell line, CWR-R1, native endogenous androgen receptor transcriptional activation was stimulated by 14-3-3eta at low dihydrotestosterone concentrations and was increased by epidermal growth factor. Moreover, the dihydrotestosterone- and epidermal growth factor-dependent increase in androgen receptor transactivation was inhibited by a dominant negative 14-3-3eta. In the CWR22 prostate cancer xenograft model, 14-3-3eta expression was increased by androgen, suggesting a feed-forward mechanism that potentiates both 14-3-3eta and androgen receptor actions. 14-3-3eta mRNA and protein decreased following castration of tumor-bearing mice and increased in tumors of castrate mice after treatment with testosterone. CWR22 tumors that recurred 5 months after castration contained 14-3-3eta levels similar to the androgen-stimulated tumors removed before castration. In a human prostate tissue microarray of clinical specimens, 14-3-3eta localized with androgen receptor in nuclei, and the similar amounts expressed in castration-recurrent prostate cancer, androgen-stimulated prostate cancer, and benign prostatic hyperplasia were consistent with androgen receptor activation in recurrent prostate cancer. CONCLUSION: 14-3-3eta enhances androgen- and mitogen-induced androgen receptor transcriptional activity in castration-recurrent prostate cancer. (Clin Cancer Res 2009;15(24):7571-81).

HDAC6 Regulates Androgen Receptor Hypersensitivity and Nuclear Localization via Modulating Hsp90 Acetylation in Castration-Resistant Prostate Cancer

The development of castration-resistant prostate cancer (PCa) requires that under castration conditions, the androgen receptor (AR) remains active and thus nuclear. Heat shock protein 90 (Hsp90) plays a key role in androgen-induced and -independent nuclear localization and activation of AR. Histone deacetylase 6 (HDAC6) is implicated, but has not been proven, in regulating AR activity via modulating Hsp90 acetylation. Here, we report that knockdown of HDAC6 in C4-2 cells using short hairpin RNA impaired ligand-independent nuclear localization of endogenous AR and inhibited PSA expression and cell growth in the absence or presence of dihydrotestosterone (DHT). The dose-response curve of DHT-stimulated C4-2 colony formation was shifted by shHDAC6 such that approximately 10-fold higher concentration of DHT is required, indicating a requirement for HDAC6 in AR hypersensitivity. HDAC6 knockdown also inhibited C4-2 xenograft tumor establishment in castrated, but not in testes-intact, nude mice. Studies using HDAC6-deficient mouse embryonic fibroblasts cells showed that inhibition of AR nuclear localization by HDAC6 knockdown can be largely alleviated by expressing a deacetylation mimic Hsp90 mutant. Taken together, our studies suggest that HDAC6 regulates AR hypersensitivity and nuclear localization, mainly via modulating HSP90 acetylation. Targeting HDAC6 alone or in combination with other therapeutic approaches is a promising new strategy for prevention and/or treatment of castration-resistant PCa.

Androgen Receptor Phosphorylation and Activity Are Regulated by an Association with Protein Phosphatase 1

Androgen receptor (AR) is phosphorylated at multiple sites in response to ligand binding, but the functional consequences and mechanisms regulating AR phosphorylation remain to be established. We observed initially that okadaic acid, an inhibitor of the major PPP family serine/threonine phosphatases PP2A and protein phosphatase 1 (PP1), had cell type-dependent effects on AR expression. More specific inhibitors of PP2A (fostriecin) and PP1 (tautomycin and siRNA against the PP1alpha catalytic subunit) demonstrated that PP1 and protein phosphatase 2A had opposite effects on AR protein and transcriptional activity. PP1 inhibition enhanced proteasome-mediated AR degradation, while PP1alpha overexpression increased AR expression and markedly enhanced AR transcriptional activity. Coprecipitation experiments demonstrated an AR-PP1 interaction, while immunofluorescence and nuclear-cytoplasmic fractionation showed androgen-stimulated nuclear translocation of both AR and PP1 in prostate cancer cells. Studies with phosphospecific AR antibodies showed that PP1 inhibition dramatically increased phosphorylation of Ser-650, a site in the AR hinge region shown to mediate nuclear export. Significantly, PP1 inhibition caused a marked decrease in nuclear localization of the wild-type AR, but did not alter total or nuclear levels of a S650A mutant AR. These findings reveal a critical role of PP1 in regulating AR protein stability and nuclear localization through dephosphorylation of Ser-650. Moreover, AR may function as a PP1 regulatory subunit and mediate PP1 recruitment to chromatin, where it can modulate transcription and splicing.

AR inhibitors identified by high-throughput microscopy detection of conformational change and subcellular localization.

Signaling via the androgen receptor (AR) plays an important role in human health and disease. All currently available anti-androgens prevent ligand access to the receptor, either by limiting androgen synthesis or by competitive antagonism at the ligand binding domain. It is unknown to what extent various steps of receptor activation may be separable and distinctly targeted by inhibitors. We have previously described the use of fluorescent protein fusions to AR to monitor its subcellular distribution and ligand-induced conformational change by fluorescence resonance energy transfer (FRET). We have now used a microscopy-based screen to identify inhibitors that prevent AR conformational change or nuclear accumulation after ligand activation. Hits were secondarily selected on the basis of their ability to inhibit AR transcription at a PSA-luciferase promoter and were tested for effects on (3)H-DHT binding to AR in cells. We find a strong correlation between compounds that block DHT binding and those that inhibit nuclear accumulation. These compounds are structurally distinct from known antagonists. Additional compounds blocked AR conformational change but did not affect DHT binding or nuclear localization of AR. One compound increased ligand-induced FRET yet functioned as a potent inhibitor. These results suggest that multiple inhibitory conformations of AR are possible and can be induced by diverse mechanisms. The lead compounds described here may be candidates for the development of novel antiandrogens and may help identify new therapeutic targets.

A Novel Mutation in the Human Androgen Receptor Suggests a Regulatory Role for the Hinge Region in Amino-Terminal and Carboxy-Terminal Interactions

The androgen insensitivity syndrome (AIS) is caused by molecular defects in the androgen receptor (AR). Clinically, the partial AIS has a variable phenotype. Many mechanisms explain the phenotype in the AIS. A crucial step in AR action is the interaction of the N and C termini. The role of the hinge region of the AR is not as well understood as other parts of the receptor. We aim to study the role of this region in the N/C-termini interaction. We report a patient with severe undermasculinization and poor response to exogenous androgens. Androgen binding was performed, and the AR gene was sequenced. The mutation was recreated and transfected in COS-1 cells. Transactivation was studied. N/C-termini interaction was studied using a mammalian two-hybrid assay. A nuclear localization study was performed. Androgen binding was normal, and a novel mutation (Arg629Trp) in the AR hinge region was identified. Mutant AR transactivation was 40% higher compared with wild type (WT). A 3-fold increase in transcription occurred when both WT N and C-terminal domains were cotransfected; no response occurred when the mutated region of the AR was included (P < 0.001). Cells with mutant AR showed a comparable nuclear localization to the WT AR. A mutation in the hinge region impaired N/C-domain interaction in the presence of normal AR binding and nuclear localization. It resulted in severe undermasculinization at birth and resistance to androgens. The findings confirm a unique regulatory role for the hinge region in AR function.

Isosilybin B causes androgen receptor degradation in human prostate carcinoma cells via PI3K-Akt-Mdm2-mediated pathway

The identification and development of novel nontoxic phytochemicals that target androgen and androgen receptor (AR) signaling remains a priority for prostate cancer (PCA) control. In the present study, we assessed the antiandrogenic efficacy of isosilybin B employing human PCA LNCaP (mutated AR), 22Rv1 (mutated AR) and LAPC4 (wild-type AR) cells. Isosilybin B (10-90 microM) treatment decreased the AR and prostate specific antigen (PSA) levels in LNCaP, 22Rv1 and LAPC4 cells, but not in non-neoplastic human prostate epithelial PWR-1E cells. Isosilybin B treatment also inhibited synthetic androgen R1881-induced nuclear localization of AR, PSA expression and cell growth, and caused G(1) arrest. In mechanistic studies identifying AR degradation, isosilybin B caused increased phosphorylation of Akt (Ser-473 and Thr-308) and Mdm2 (Ser-166), which was linked with AR degradation as pretreatment with PI3K inhibitor (LY294002)-restored AR level. Further, overexpression of kinase-dead Akt largely reversed isosilybin B mediated-AR degradation suggesting a critical role of Akt in AR degradation. Antibody pull-down results also indicated that isosilybin B treatment enhances the formation of complex between Akt, Mdm2 and AR, which promotes phosphorylation-dependent AR ubiquitination and its degradation by proteasome. Together, present findings identify a novel mechanism for isosilybin B-mediated anticancer effects in human PCA cells.

Hemodynamic Regulation of Androgen Receptor‐dependent Inflammatory Signaling

The correlation between timing of vascular disease in women and decreased production of estrogen after menopause suggests that circulating androgens promote vascular inflammation in the absence of protective estrogens. We hypothesized that androgen (R1881) stimulates androgen receptor (AR) in human endothelial cells to induce inflammatory signaling in a manner that is modulated by an atheroprotective shear stress (SS) profile. R1881 treatment for 24 h caused 2‐fold upregulation of AR protein expression and increased AR nuclear localization, whereas steady unidirectional SS (15 dyn/cm2) for 24 h caused 1.2‐fold downregulation of expression and nearly 3‐fold decrease in nuclear localization. Atheroprotective SS prevented AR upregulation regardless of whether cells were pretreated with or sheared in the presence of R1881. R1881 treatment for 48 h induced a nearly 2‐fold increase in VCAM‐1 expression, suggesting that ligand‐activated AR promotes inflammatory signaling. SS abrogated the effects of R1881. These data are the first evidence that SS and ligand differentially regulate AR to modulate inflammatory signaling in human endothelial cells. Supported by NIH grant HL071958 and a UVa CVRC Partners Award.

Lycopene inhibits IGF-I signal transduction and growth in normal prostate epithelial cells by decreasing DHT-modulated IGF-I production in co-cultured reactive stromal cells

Prostate stromal and epithelial cell communication is important in prostate functioning and cancer development. Primary human stromal cells from normal prostate stromal cells (PRSC) maintain a smooth muscle phenotype, whereas those from prostate cancer (6S) display reactive and fibroblastic characteristics. Dihydrotestosterone (DHT) stimulates insulin-like growth factor-I (IGF-I) production by 6S but not PSRC cells. Effects of reactive versus normal stroma on normal human prostate epithelial (NPE or PREC) cells are poorly understood. We co-cultured NPE plus 6S or PRSC cells to compare influences of different stromal cells on normal epithelium. Because NPE and PREC cells lose androgen receptor (AR) expression in culture, DHT effects must be modulated by associated stromal cells. When treated with camptothecin (CM), NPE cells, alone and in stromal co-cultures, displayed a dose-dependent increase in DNA fragmentation. NPE/6S co-cultures exhibited reduced CM-induced cell death with exposure to DHT, whereas NPE/PRSC co-cultures exhibited CM-induced cell death regardless of DHT treatment. DHT blocked CM-induced, IGF-I-mediated, NPE death in co-cultured NPE/6S cells without, but not with, added anti-IGF-I and anti-IGF-R antibodies. Lycopene consumption is inversely related to human prostate cancer risk and inhibits IGF-I and androgen signaling in rat prostate cancer. In this study, lycopene, in dietary concentrations, reversed DHT effects of 6S cells on NPE cell death, decreased 6S cell IGF-I production by reducing AR and beta-catenin nuclear localization and inhibited IGF-I-stimulated NPE and PREC growth, perhaps by attenuating IGF-I's effects on serine phosphorylation of Akt and GSK3beta and tyrosine phosphorylation of GSK3. This study expands the understanding of the preventive mechanisms of lycopene in prostate cancer.

An Antibody Targeting the Type I Insulin-like Growth Factor Receptor Enhances the Castration-Induced Response in Androgen-Dependent Prostate Cancer

To determine the effect of inhibition of insulin-like growth factor-IR (IGF-IR) signaling with an antibody to the IGF-IR, A12, in conjunction with androgen withdrawal on prostate cancer progression in a human prostate xenograft model, LuCaP 35. LuCaP 35 was implanted s.c. in severe combined immunodeficient mice. At the time of castration, mice were randomized to one of three groups. Group 1 was castrate only; group 2 received A12 40 mg/kg i.p. for 2 weeks beginning 1 week after castration; and group 3 received A12 40 mg/kg i.p. for 2 weeks beginning 2 weeks after castration. In group 1, tumor volume decreased to 60% of the starting volume 4 weeks post-castration. In groups 2 and 3, tumor volumes nadired 6 weeks after castration at <10% of the volume at time of castration (P < 0.01). Tumor regrowth was not seen in groups 2 or 3 until 15 weeks after castration. Androgen receptor (AR) localization in tumors showed a decrease in nuclear staining in groups 2 and 3 compared with group 1 (P < 0.001). Tumor volume correlated with nuclear AR intensity. AR-regulated genes increased early in group 1, but did not increase in groups 2 and 3. Thus, tumor-specific survival was prolonged by the addition of A12 to castration. This study shows that the inhibition of IGF-IR enhances the effects of castration in prostate cancer. These effects are associated with a decrease in AR signaling and nuclear AR localization, and recurrence is associated with an increase in AR-regulated gene expression.

A novel synthetic compound that interrupts androgen receptor signaling in human prostate cancer cells

The purpose of this study was to determine the effects of 6-amino-2-[2-(4-tert-butyl-phenoxy)-ethylsulfonyl]-1H-pyrimidine-4-one (DL3), a novel synthetic compound with small-molecule drug properties, on androgen-regulated gene expression and cell growth in human prostate cancer cells. LNCaP, 22Rv1, and LAPC-4 cells were used in the studies. Expression of prostate-specific antigen (PSA) and androgen receptor (AR) was determined by ELISA, Western blotting, real-time reverse transcription-PCR, nuclear run-on, and/or promoter luciferase reporter assays. Effects of DL3 on cell growth were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide staining. DL3 inhibited dihydrotestosterone (DHT)-induced PSA expression in a dose-dependent fashion. The inhibitory effects of DL3 were more potent than those of flutamide, nilutamide, and bicalutamide. Moreover, DL3 blocked the stimulatory effects of nilutamide on PSA expression in LNCaP cells. Unlike the three classic antiandrogens, DL3 did not show intrinsic AR agonist activity. Nuclear run-on and PSA promoter reporter assays revealed that DL3 blocked DHT-induced PSA gene transcription. Consistent with its effects on PSA expression, DL3 inhibited DHT-stimulated cell growth with a potency significantly superior to flutamide, nilutamide, or bicalutamide. Furthermore, cells resistant to flutamide or nilutamide were as susceptible as their parental counterparts to the inhibitory effects of DL3 on both PSA expression and cell growth. DL3 did not inhibit AR nuclear localization and the NH(2)- and COOH-terminal interaction of AR induced by DHT. These data show that DL3 is a novel inhibitor of the AR signaling axis and a potentially potent therapeutic agent for the management of advanced human prostate cancer.

Subcellular Localization Modulates Activation Function 1 Domain Phosphorylation in the Androgen Receptor

Although the steady-state distribution of the androgen receptor (AR) is predominantly nuclear in androgen-treated cells, androgen-bound AR shuttles between the nucleus and the cytoplasm. In the present study we have addressed how nucleocytoplasmic shuttling contributes to the regulation of AR. Nuclear transport signal fusions were used to force AR localization to the nucleus or cytoplasm of prostate cancer cells, and the effect of localization on shuttling, transcription, androgen binding, and phosphorylation was determined. Fusing the simian virus 40 nuclear localization signal or c-Abl nuclear export signal to AR resulted in androgen-independent localization to the nucleus or cytoplasm, respectively. AR forced to the nucleus was transcriptionally active on prostate-specific antigen and mouse mammary tumor virus promoters driving reporter genes. AR forced to the cytoplasm was largely inactive on the prostate-specific antigen promoter, but, surprisingly, AR was active on the mouse mammary tumor virus promoter and on two endogenous genes examined. Thus, highly transient nuclear localization of AR is sufficient to activate transcription. Androgen dissociation rates and the dissociation constant (K(D)) of AR for androgen were similar whether AR was localized to the cytoplasm or the nucleus, suggesting the ligand-binding cycle of AR is not strictly linked to its compartmentalization. Using phosphosite antibodies, we found that compartmentalization influences the phosphorylation state of AR. We show there is a bias for androgen-dependent phosphorylation of Ser81, Ser256, and Ser308 in the nucleus and androgen-independent phosphorylation of Ser94 in the cytoplasm. We propose that one function of nucleocytoplasmic shuttling is to integrate the signaling environment in the cytoplasm with AR activity in the nucleus.

The Hsp90 inhibitor, 17‐AAG, prevents the ligand‐independent nuclear localization of androgen receptor in refractory prostate cancer cells

Androgen receptor (AR) is the key molecule in androgen-refractory prostate cancer. Despite androgen ablative conditions, AR remains active and is necessary for the growth of androgen-refractory prostate cancer cells. Nuclear localization of AR is a prerequisite for its transcriptional activation. We examined AR localization in androgen-dependent and androgen-refractory prostate cancer cells. We demonstrate increased nuclear localization of a GFP-tagged AR in the absence of hormone in androgen-refractory C4-2 cells compared to parental androgen-sensitive human prostate cancer LNCaP cells. Analysis of AR mutants impaired in ligand-binding indicates that the nuclear localization of AR in C4-2 cells is truly androgen-independent. The hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), inhibits basal PSA expression and disrupts the ligand-independent nuclear localization of AR at doses much lower than required to inhibit androgen-induced nuclear import. Hsp90 is a key regulator of ligand-independent nuclear localization and activation of AR in androgen-refractory prostate cancer cells.

A novel function of caspase-8 in the regulation of androgen-receptor-driven gene expression

Transcriptional regulation by the androgen receptor (AR) is critical for male sexual development and prostate cancer. In this study, we used an expression cloning strategy to identify molecules that regulate AR-driven transcription. Screening of a human cDNA library resulted in isolation of caspase-8 (Casp8), an initiator caspase that mediates death-receptor-induced apoptosis. Casp8 repressed AR-dependent gene expression independently of its apoptotic protease activity by disrupting AR amino-terminal and carboxy-terminal (N/C) interaction and inhibiting androgen-induced AR nuclear localization. Protein-protein interaction analysis revealed that three motifs in Casp8 specifically interacted with the motifs that are known to be involved in AR N/C interaction. Substitutions of the amino-acid residues critical for AR-Casp8 interactions abolished the Casp8-mediated inhibition of AR transactivation. In addition, knockdown of Casp8 by RNA interference specifically affected the androgen-dependent expression of AR-targeting genes in LNCaP cells. These results indicate that Casp8 has a novel function beyond its known role in the mediation of apoptosis.