Mutant Androgen Receptor Research Articles

Hypospadias and Genes Related to Genital Tubercle and Early Urethral Development

We determined whether variants in genes associated with genital tubercle (the anlage for the penis) and early urethral development were associated with hypospadias in humans. We examined 293 relatively common tag single nucleotide polymorphisms in BMP4, BMP7, FGF8, FGF10, FGFR2, HOXA13, HOXD13, HOXA4, HOXB6, SRY, WT1, WTAP, SHH, GLI1, GLI2 and GLI3. The analysis included 624 cases (81 mild, 319 moderate, 209 severe, 15 undetermined severity) and 844 population based nonmalformed male controls born in California from 1990 to 2003. There were 28 single nucleotide polymorphisms for which any of the comparisons (ie overall or for a specific severity) had a p value of less than 0.01. The homozygous variant genotypes for 4 single nucleotide polymorphisms in BMP7 were associated with at least a twofold increased risk of hypospadias regardless of severity. Five single nucleotide polymorphisms for FGF10 were associated with threefold to fourfold increased risks, regardless of severity. For 4 of them the results were restricted to whites. For GLI1, GLI2 and GLI3 there were 12 associated single nucleotide polymorphisms but results were inconsistent by severity and race/ethnicity. For SHH 1 single nucleotide polymorphism was associated with a 2.4-fold increased risk of moderate hypospadias. For WT1 6 single nucleotide polymorphisms were associated with approximately a twofold increased risk, primarily for severe hypospadias. This study provides evidence that single nucleotide polymorphisms in several genes that contribute to genital tubercle and early urethral development are associated with hypospadias risk.

No evidence of cardiomyopathy in spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is a lower motor neuron disease caused by a CAG repeat expansion within the androgen receptor (AR) gene. Toxic nuclear accumulation of mutant AR has been observed in tissues other than nervous system including cardiac muscle. Moreover, CAG polymorphism length within AR has been associated with an increased risk of heart disease. To test the hypothesis of the presence of cardiomyopathy in SBMA, a full cardiac protocol was applied to 25 SBMA patients. Patients' age ranged between 32 and 75years. Cardiologic examination, 12-lead ECG, and echocardiography showed no abnormalities other than those consistent with hypertensive heart disease. One patient showed frequent supraventricular premature beats in absence of other significant arrhythmias at the 24-h ECG Holter. Our findings do not support the hypothesis of a primary cardiomyopathy in SBMA.

A case of complete androgen insensitivity syndrome: genetic analysis of the family.

Besides many other factors, androgens play a role in expression of male phenotype. Androgen activity is mediated by androgen receptor (AR). After binding to androgen, AR translocates to nucleus and adheres to the regulatory regions of specific chromosomal DNA sequences (androgen response elements/ARE), to activate androgen dependent genes. The AR is encoded by the AR gene (Xq11–12). The gene is formed by 8 exons and 7 introns, spanning more than 90 kb. The functional domains are (a) exon 1 encoding the N-terminal domain (NTD) (1,586 bp); (b) exons 2 and 3 encode the DNA-binding domain (DBD) (152 and 117 bp); (c) the “hinge” region which binds the NTD and DBD consists of residues 628–669; (d) exons 4–8 encode the C-terminal ligand-binding domain (LBD), (131–288 bp). The C-terminus of the LBD mediates the hormone-dependent transcription. Mutations in the LBD region of AR perturb the conformation of the helix. The mutant AR is unable to efficiently bind to dihydrotestosterone (DHT) and to transactivate AREs [1]. Mutations in the AR gene leading to disordered AR function results in different forms of X-linked male pseudohermaphroditism, known as androgen insensitivity syndrome (AIS). This affects XY female individuals with normal androgen production and metabolism. AIS is present in 1:20,000–64,000 male births. The presence of variable phenotypic expression allows for the classification of AIS into complete androgen insensitivity (CAIS), partial androgen insensitivity (PAIS), and mild androgen insensitivity (MAIS). Approximately, 90 % of molecular defects in AR gene are single base point mutations, mostly missense mutations [2]. Here, we describe a familial case of CAIS presenting with similar mutations described across three generations. To the best of our knowledge and belief, ours is first such report citing this mutation in an Indian family.

P62/SQSTM1 Differentially Removes the Toxic Mutant Androgen Receptor via Autophagy and Inclusion Formation in a Spinal and Bulbar Muscular Atrophy Mouse Model

Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders that are caused by the expansion of trinucleotide CAG repeats in the causative genes. Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease that is caused by the expansion of a polyQ tract within the androgen receptor (AR). p62 is a ubiquitin- and light-chain 3-binding protein that is known to regulate the degradation of targeted proteins via autophagy and inclusion formation. In this study, we examined the effects of p62 depletion and overexpression on cultured cells and in a transgenic mouse model that overexpressed the mutant AR. Here, we demonstrate that depletion of p62 significantly exacerbated motor phenotypes and the neuropathological outcome, whereas overexpression of p62 protected against mutant AR toxicity in SBMA mice. Depletion of p62 significantly increased the levels of monomeric mutant AR and mutant AR protein complexes in an SBMA mouse model via the impairment of autophagic degradation. In addition, p62 overexpression improved SBMA mouse phenotypes by inducing cytoprotective inclusion formation. Our results demonstrate that p62 provides two different therapeutic targets in SBMA pathogenesis: (1) autophagy-dependent degradation and (2) benevolent inclusion formation of the mutant AR.

Interactions of Abiraterone, Eplerenone, and Prednisolone with Wild-Type and Mutant Androgen Receptor: A Rationale for Increasing Abiraterone Exposure or Combining with MDV3100—Letter

We read with interest the report from Richards and colleagues (1) on the potential interactions of abiraterone, eplerenone, and prednisolone with wild-type and mutant androgen receptor (AR). Abiraterone is a CYP17A1 inhibitor that blocks androgen biosynthesis in the testes, adrenals, and castration-resistant prostate cancer (CRPC) tumors, producing androgen ablation with significant clinical benefit in patients with metastatic CRPC. The current report (1) and other studies (2) suggesting that abiraterone downregulates AR expression and produces antiandrogen activity beyond androgen depletion merit further investigation.The suppression of adrenal steroid biosynthesis via abiraterone inhibition of CYP17A1 reduces circulating cortisol, with compensatory release of adrenocorticotropic hormone (3). This induces increased adrenal secretion of mineralocorticoids that can contribute to hypokalemia and hypertension. These manifestations of mineralocorticoid excess are managed by coadministration of low-dose prednisone (a prodrug of prednisolone) in patients treated with abiraterone acetate.Richards and colleagues reported that prednisolone activated the AR in cells transfected with T877A-mutant AR with an EC50 of 25.1 μmol/L. This suggested that coadministration of prednisolone with abiraterone acetate could contribute to disease progression. However, the EC50 is much higher than the plasma concentrations of prednisolone (4–305 nmol/L) measured in 15 patients with CRPC receiving abiraterone acetate plus prednisolone. Furthermore, the authors extrapolated the reported prednisolone plasma concentrations to a prior publication on the in vitro androgen agonist activity of glucocorticoids at nanomolar concentrations in a cell line bearing an AR ligand-binding domain double mutation (T877A plus L701H) to ascribe clinical significance (4). We feel that this comparison is inappropriate in relation to the current findings and the interpretation of the findings related to abiraterone acetate therapy. The prevalence of this double mutation is unknown without established clinical relevance.The authors also reported findings using spironolactone and eplerenone, which produced androgen agonist activity in vitro in cells transfected with wild-type (spironolactone only) or mutant AR at concentrations relevant to clinical activity. The report suggested that this could contribute to disease progression if these drugs were used in patients with prostate cancer. However, neither agent is proposed for use with abiraterone acetate to ameliorate symptoms of mineralocorticoid excess.Abiraterone acetate was studied in conjunction with low-dose prednisone/prednisolone in registration trials leading to approval (5); only prednisone/prednisolone, not spironolactone or eplerenone, are specified in approved labeling. Given the high concentrations of prednisolone investigated in the reported in vitro studies, the clinical significance of the findings is not obvious.D. End is employed as Director of Johnson and Johnson/Janssen and has ownership interest (including patents) in Johnson and Johnson. A. Molina is the Vice President of Johnson and Johnson and has ownership interest (including patents) in the stock options of Johnson and Johnson. No potential conflicts of interest were disclosed by the other authors.

Abstract 2433: In vitro and in vivo antitumor activity of the next generation HSP90 inhibitor, AT13387, in both hormone-sensitive and castration-resistant prostate cancer models.

Abstract HSP90 is a molecular chaperone involved in the conformational maturation and function of a large number of ‘client’ proteins that have been implicated in oncogenesis. The androgen receptor (AR), a key driver of prostate cancer growth and treatment resistance, is an HSP90 client and its function is dependent on HSP90 chaperone activity. The aim of this study was to evaluate the anti-tumour activity of AT13387, a novel second generation non-ansamycin HSP90 inhibitor, in prostate cancer models. AT13387 is currently being investigated in the clinic in GIST and NSCLC. It is a potent inhibitor of HSP90 (Kd 0.71 nM) and has previously been shown to inhibit proliferation and bring about the depletion of client proteins in a wide range of cell lines as well as inhibiting tumor growth in a number of xenograft models. In prostate cancer cell lines (VCaP, LNCaP, 22Rv1), AT13387 treatment depleted HSP90 clients such as AKT, HER-2 and CRAF, along with the induction of HSP72, confirming target inhibition. AT13387 treatment also resulted in depletion of mutant and wild-type AR, as well as the constitutively active truncated AR splice variant 7 (AR-V7). HSP90 inhibition disrupted AR nuclear localisation and AR transcriptional activity and resulted in down-regulation of AR-regulated genes in vitro. Inhibition of HSP90 translated into proliferative inhibition, with GI50 values in the range of 15-70 nM, and induction of apoptosis. AT13387 showed substantially greater potency than the first generation, natural product-based HSP90 inhibitor 17-AAG in modulating HSP90 client proteins, inhibiting cell growth and inducing cell death. The anti-tumor activity of AT13387 was demonstrated in vivo in a human xenograft model of castration-resistant prostate cancer. Our data suggest that HSP90 inhibition, through the use of more potent and better tolerated HSP90 inhibitors, will be active in multi drug resistant prostate cancer. These led to the design of a Phase I/II clinical trial of AT13387 with and without abiraterone acetate in castration-resistant prostate cancer no longer responding to abiraterone that is now accruing patients (NCT01685268). Citation Format: Roberta Ferraldeschi, Somaieh Hedayat, Tomoko Smyth, Nicola Wallis, John Lyons, Ruth Riisnaes, Mateus Crespo, Daniel Nava Rodrigues, Susana Miranda, Swee Sharp, Gerhardt Attard, Suzanne Eccles, Paul Workman, Johann de Bono. In vitro and in vivo antitumor activity of the next generation HSP90 inhibitor, AT13387, in both hormone-sensitive and castration-resistant prostate cancer models. [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 2433. doi:10.1158/1538-7445.AM2013-2433

Abstract 2482: Development of novel small-molecule inhibitors of androgen receptor variants lacking the ligand-binding domain.

Abstract Over 30,000 men die from prostate cancer in Canada and USA each year. Current therapeutic strategy for advanced prostate cancer is to suppress androgen receptor (AR) signaling by reducing level of androgens (via castration) and by antiandrogens to compete with endogenous androgens for binding in the AR ligand-binding domain (LBD). Despite of initial response, lethal ‘castration-resistant’ prostate cancer (CRPC) arises as a result of oncogenic reactivation of the AR. Current treatments for CRPC provide only a modest improvement in overall survival (a few months). It is now firmly established that reactivation of the AR signaling in CRPC is due to multiple mechanisms, including point mutations in the AR LBD and expression of AR variants lacking the LBD. Recently, a series of AR splice variants lacking the LBD, such as AR-v7, were identified from tissue specimen of CRPC patients. Significantly, AR-v7 is constitutively active even in the absence of androgens and it is thus no longer possible to be inhibited by conventional LBD-targeting antiandrogens (such as bicalutamide) or by androgen-depleting agents (such as Abirateron). This potentially provides a novel mechanism for the development of CRPC. Our recent work has identified a novel chemical compound that inhibits AR-v7 in reporter assays. We found this compound is also active against full-length ARs, including the wild type and multiple clinically-relevant mutant ARs, such as the T877A, H874Y and W741C mutants. Our study indicated this compound targets the N-terminal domain of the AR. We have obtained structure-activity relationship (SAR) of this compound. Citation Format: Xiaohong Tian, Weiguo Liu, Guoyan Geng, Jian Hui Wu. Development of novel small-molecule inhibitors of androgen receptor variants lacking the ligand-binding domain. [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 2482. doi:10.1158/1538-7445.AM2013-2482

Abstract 1321: Castration-resistant prostate cancer: BAY 1024767 blocks function of mutated androgen receptor.

Abstract Analysis of biopsies from prostate cancer metastases and of circulating tumor cells shows that androgen receptor (AR) mutations are often found in patients developing resistance to antiandrogen therapy, i.e. at the castration-resistant prostate cancer (CRPC) stage. Here we report on preclinical studies with a novel androgen receptor suppressor compound BAY 1024767 that exhibits strong antagonism for wild-type and mutated AR, both in vitro and in vivo. Mutations located in the AR ligand-binding domain (LBD) and potentially involved in the resistance to the standard of care bicalutamide were generated and tested in cell-based transactivation assays, using 0.1 nM R1881 for stimulation. Bicalutamide showed reduced antagonistic activity for AR E709Y and no antagonism up to 10 μM for AR W741C. In comparison, BAY 1024767 exhibited strong antagonism for these mutants. Another mechanism leading to CRPC is the elevation of intratumoral androgen levels. To mimick this situation, the transactivation assays were performed at a higher (1 and 10 nM R1881) androgen concentration. Bicalutamide, and also the novel antiandrogen MDV3100, showed much reduced antagonism for wild-type and W741C or E709Y mutant AR. Conversely, antagonism was still observed for BAY 1024767, especially at 1 nM R1881. The KuCaP-1 model, which is derived from a CRPC patient and harbors the W741C mutation, was obtained from the group of O. Ogawa (Kyoto University). Mice bearing the KuCaP-1 xenograft subcutaneously were treated with bicalutamide (60 mg/kg, PO, qdx20) or BAY 1024767 (50 mg/kg, PO, q2dx10). No effect on tumor growth was observed with bicalutamide (T/C=103%) whereas BAY 1024767 was able to inhibit tumor growth with a T/C of 36% on day 54 post-implantation. In another study we found that MDV 3100 (100 mg/kg, PO) and abiraterone acetate (200 mg/kg, PO) were inactive in the KuCaP-1 model (100% and 73% T/C, respectively). Citation Format: Bernard Haendler, Tatsuo Sugawara, Pascale Lejeune, Silke Koehr, Hortensia Faus, Matthias Busemann, Arwed Cleve, Ulrich Luecking, Franz von Nussbaum, Michael Brands, Dominik Mumberg. Castration-resistant prostate cancer: BAY 1024767 blocks function of mutated androgen receptor. [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 1321. doi:10.1158/1538-7445.AM2013-1321

A multi‐parameter imaging assay identifies different stages of ligand‐induced androgen receptor activation

Androgens exert their key function in development and maintenance of the male phenotype via the androgen receptor (AR). Ligand-activated ARs also play a role in prostate cancer. Despite initial success of treatment by testosterone depletion or blocking of androgen binding to the AR using antiandrogens, eventually all tumors escape to a therapy resistant stage. Development of novel therapies by other antagonistic ligands or compounds that target events subsequent to ligand binding is very important. Here, we validate a fluorescence resonance energy transfer (FRET) based imaging assay for ligand-induced AR activity, based on the conformational change in the AR caused by interaction between the FQNLF motif in the N-terminal domain and the cofactor binding groove in the ligand-binding domain (N/C-interaction). We test the assay using known agonistic and antagonistic ligands on wild type AR and specific AR mutants. Our data show a strong correlation between the ligand-induced AR N/C-interaction and transcriptional activity in wild type AR, but also in AR mutants with broadened ligand responsiveness. Moreover, we explore additional readouts of this assay that contribute to the understanding of the working mechanism of the ligands. Together, we present a sensitive assay that can be used to quantitatively assess the activity of agonistic and antagonistic AR ligands.

A role for androgen reduction treatment in kennedy disease?

Over the past 4 years, there have been 3 published reports of randomized, placebo-controlled trials of androgen reduction therapy in patients with spinal and bulbar muscular atrophy (SBMA, Kennedy disease). 1–3 Despite clear evidence of efficacy of androgen reduction in transgenic mouse models, 4,5 the clinical trials, which studied 2 different drugs, leuprorelin and dutasteride, and included 50–200 subjects followed for up to 2 years, did not show significant benefits compared with placebo in the primary outcome measures [the amyotrophic lateral sclerosis (ALS) functional rating scale, barium swallow, and quantitative muscle assessment, respectively]. There was evidence for efficacy in secondary outcome measures in an open-label extension and with post hoc subgroup analysis of patients symptomatic for less than 10 years; this left open the possibility of unproven benefit that might emerge with longer treatment. Now, a small, open-label study reported in this issue indicates that androgen reduction with leuprorelin does not prevent disease progression over a longer period (3.5 years).6 The primary outcome measure in this study was leg strength. There are shortcomings of such a small-scale, uncontrolled study: Would patients on placebo have fared worse? Would a different, perhaps more sensitive and clinically meaningful outcome measure have shown benefit? Would patients closer to disease onset, or even before the onset of weakness, have done better? Here again, a subset analysis indicates that patients early in the disease course, with higher baseline testosterone levels, may do better with the treatment. Secondary outcome measures such as the 10 m timed walk and ALS Functional Rating Scale did not show a significant decline, and serum creatine kinase levels decreased with treatment. It is still possible that androgen reduction has some benefit in SBMA, particularly in younger patients with higher testosterone levels before treatment. However, based on the results of these studies, it is unlikely that such a benefit, if present, amounts to much for the typical patient. It is clear that this is not the treatment that patients and clinicians would like, one that improves muscle function or stops the disease progression over time. One problem may be that androgen reduction, while perhaps preventing the toxicity of the mutant androgen receptor protein, exacerbates the loss of androgen receptor function that also occurs in SBMA, and deprives patients of the anabolic benefits of endogenous androgens on muscle. The positive correlation of testosterone levels with muscle strength found in this and previous studies supports this conjecture.6,7 Other therapeutic approaches, currently under investigation and also beneficial in mouse models, may do better in patients.8 Further well-designed clinical trials are needed to address this possibility.

Enhanced Aggregation of Androgen Receptor in Induced Pluripotent Stem Cell-derived Neurons from Spinal and Bulbar Muscular Atrophy

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motor neuron disease caused by a CAG repeat expansion in the androgen receptor (AR) gene. Ligand-dependent nuclear accumulation of mutant AR protein is a critical characteristic of the pathogenesis of SBMA. SBMA has been modeled in AR-overexpressing animals, but precisely how the polyglutamine (polyQ) expansion leads to neurodegeneration is unclear. Induced pluripotent stem cells (iPSCs) are a new technology that can be used to model human diseases, study pathogenic mechanisms, and develop novel drugs. We established SBMA patient-derived iPSCs, investigated their cellular biochemical characteristics, and found that SBMA-iPSCs can differentiate into motor neurons. The CAG repeat numbers in the AR gene of SBMA-iPSCs and also in the atrophin-1 gene of iPSCs derived from another polyQ disease, dentato-rubro-pallido-luysian atrophy (DRPLA), remain unchanged during reprogramming, long term passage, and differentiation, indicating that polyQ disease-associated CAG repeats are stable during maintenance of iPSCs. The level of AR expression is up-regulated by neuronal differentiation and treatment with the AR ligand dihydrotestosterone. Filter retardation assays indicated that aggregation of ARs following dihydrotestosterone treatment in neurons derived from SBMA-iPSCs increases significantly compared with neurological control iPSCs, easily recapitulating the pathological feature of mutant ARs in SBMA-iPSCs. This phenomenon was not observed in iPSCs and fibroblasts, thereby showing the neuron-dominant phenotype of this disease. Furthermore, the HSP90 inhibitor 17-allylaminogeldanamycin sharply decreased the level of aggregated AR in neurons derived from SBMA-iPSCs, indicating a potential for discovery and validation of candidate drugs. We found that SBMA-iPSCs possess disease-specific biochemical features and could thus open new avenues of research into not only SBMA, but also other polyglutamine diseases.

Genistein, a natural product derived from soybeans, ameliorates polyglutamine‐mediated motor neuron disease

Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of a polyglutamine (polyQ) tract within the androgen receptor (AR) gene. The pathologic features of SBMA are motor neuron loss in the spinal cord and brainstem, and diffuse nuclear accumulation and nuclear inclusions of mutant AR in residual motor neurons and certain visceral organs. AR-associated coregulator 70 (ARA70) was the first coregulator of AR to be identified, and it has been shown to interact with AR and increase its protein stability. Here, we report that genistein, an isoflavone found in soy, disrupts the interaction between AR and ARA70 and promotes the degradation of mutant AR in neuronal cells and transgenic mouse models of SBMA. We also demonstrate that dietary genistein ameliorates behavioral abnormalities, improves spinal cord and muscle pathology, and decreases the amounts of monomeric AR and high-molecular-weight mutant AR protein aggregates in SBMA transgenic mice. Thus, genistein treatment may be a potential therapeutic approach for alleviating the symptoms of SBMA by disrupting the interactions between AR and ARA70.

The Discovery of Novel Human Androgen Receptor Antagonist Chemotypes Using a Combined Pharmacophore Screening Procedure

Unraveling the mechanisms involved in castration- and therapy-resistant prostate cancer has led to a renewed interest in androgen receptor (AR)-targeted therapeutics. Anti-androgens that block the activity of the AR therefore remain a valid therapeutic option. However, they must be more effective than, or display a distinct mechanism of action or binding mode from those of bicalutamide and hydroxyflutamide, which are currently in clinical use. For that reason, the second-generation anti-androgen MDV3100 was developed. MDV3100, however, shares its 4-cyano-3-(trifluoromethyl)phenyl group with bicalutamide and hydroxyflutamide required for binding to the AR. In this work, we used a combined strategy to find new antagonist structures distinct from the 4-cyano-3-(trifluoromethyl)phenyl group to avoid cross-resistance for these compounds and to find structures without agonist activity on mutant ARs (AR W741C and AR T877A). We found two novel chemotypes with AR-antagonistic activity (IC(50): 3-6 μM) by virtual screening and confirmed their biological activity in an androgen-responsive reporter assay. The design of our computational approach was validated by the observation of strongly decreased or absence of agonistic activity on the two mutant ARs. Further structural derivatization to optimize the potency of these compounds can render these chemotypes into very promising, alternative AR antagonists for prostate cancer therapy.

New Routes to Therapy for Spinal and Bulbar Muscular Atrophy

Spinal and bulbar muscular atrophy (SBMA), also known as Kennedy's disease, is a genetically inherited neuromuscular disorder characterized by loss of lower motor neurons in the brainstem and spinal cord and skeletal muscle fasciculation, weakness, and atrophy. SBMA is caused by expansion of a polyglutamine (polyQ) tract in the gene coding for the androgen receptor (AR). PolyQ expansions cause at least eight other neurological disorders, which are collectively known as polyQ diseases. SBMA is unique in the family of polyQ diseases in that the disease manifests fully in male individuals only. The sex specificity of SBMA is the result of the interaction between mutant AR and its natural ligand, testosterone. Here, we will discuss emerging therapeutic perspectives for SBMA in light of recent findings regarding disease pathogenesis.

Melanoma Antigen-A11 (MAGE-A11) Enhances Transcriptional Activity by Linking Androgen Receptor Dimers

Prostate cancer growth and progression depend on androgen receptor (AR) signaling through transcriptional mechanisms that require interactions with coregulatory proteins, one of which is the primate-specific steroid receptor coregulator melanoma antigen-A11 (MAGE-A11). In this report, we provide evidence how increased expression of MAGE-A11 during prostate cancer progression enhances AR signaling and prostate cancer growth. MAGE-A11 protein levels were highest in castration-recurrent prostate cancer. The cyclic AMP-induced increase in androgen-dependent and androgen-independent AR transcriptional activity correlated with an increase in MAGE-A11 and was inhibited by silencing MAGE-A11 expression. MAGE-A11 mediated synergistic AR transcriptional activity in LAPC-4 prostate cancer cells. The ability of MAGE-A11 to rescue transcriptional activity of complementary inactive AR mutants and promote coimmunoprecipitation between unlike forms of AR suggests that MAGE-A11 links transcriptionally active AR dimers. A model for the AR·MAGE-A11 multidimeric complex is proposed in which one AR FXXLF motif of the AR dimer engages in the androgen-dependent AR NH(2)- and carboxyl-terminal interaction, whereas the second FXXLF motif region of the AR dimer interacts with dimeric MAGE-A11. The AR·MAGE-A11 multidimeric complex accounts for the dual functions of the AR FXXLF motif in the androgen-dependent AR NH(2)- and carboxyl-terminal interaction and binding MAGE-A11 and for synergy between reported AR splice variants and full-length AR. We conclude that the increased expression of MAGE-A11 in castration-recurrent prostate cancer, which is enhanced by cyclic AMP signaling, increases AR-dependent growth of prostate cancer by MAGE-A11 forming a molecular bridge between transcriptionally active AR dimers.

Phosphorylation of the androgen receptor by PIM1 in hormone refractory prostate cancer

Integration of cellular signaling pathways with androgen receptor (AR) signaling can be achieved through phosphorylation of AR by cellular kinases. However, the kinases responsible for phosphorylating the androgen receptor at numerous sites and the functional consequences of AR phosphorylation are only partially understood. Bioinformatic analysis revealed AR serine 213 (S213) as a putative substrate for PIM1, a kinase overexpressed in prostate cancer. Therefore, phosphorylation of AR serine 213 by PIM1 was examined using a phosphorylation site-specific antibody. Wild type PIM1, but not catalytically inactive PIM1, specifically phosphorylated AR but not an AR serine to alanine mutant (S213A). In vitro kinase assays confirmed that PIM1 can phosphorylate AR S213 in a ligand independent manner and cell type specific phosphorylation was observed in prostate cancer cell lines. Upon PIM1 overexpression AR phosphorylation was observed in the absence of hormone and was further increased in the presence of hormone in LNCaP, LNCaP-abl, and VCaP cells. Moreover, phosphorylation of AR was reduced in the presence of PIM kinase inhibitors. An examination of AR mediated transcription showed that reporter gene activity was reduced in the presence of PIM1 and wild type AR, but not S213A mutant AR. Androgen mediated transcription of endogenous PSA, Nkx3.1, and IGFBP5 was also decreased in the presence of PIM1 whereas IL6, cyclin A1, and caveolin 2 were increased. Immunohistochemical analysis of prostate cancer tissue microarrays showed significant P-AR S213 expression that was associated with hormone refractory prostate cancers, likely identifying cells with catalytically active PIM1. In addition, prostate cancers expressing a high level of P-AR S213 were twice as likely to be from biochemically recurrent cancers. Thus, AR phosphorylation by PIM1 at S213 impacts gene transcription and is highly prevalent in aggressive prostate cancer.

48IN - Integrating Novel Endocrine Therapies: Sequential or Concomitant Treatment?

ABSTRACT Prostate cancer progression is associated with continued androgen receptor (AR) activation despite treatment with castration and/or currently available anti-androgens. Abiraterone, a rationally- designed inhibitor of CYP17A1 has been recently approved for the treatment of docetaxel-treated castration-resistant prostate cancer (CRPC), is often effective, but requires co-administration with glucocorticoids to curtail side effects. Enzalutamide (MDV3100) is a novel AR antagonist that effectively blocks AR signaling when currently available AR antagonists are unable to do this and has shown impressive antitumor activity and a similar impact on overall survival as abiraterone. We now report that these two drugs in combination may be superior to either drug alone. We have hypothesized that progressive disease on abiraterone may occur secondary to glucocorticoid-induced activation of mutated AR. We also found that prednisolone plasma levels in CRPC patients were sufficiently high to activate mutant AR. Mineralocorticoid receptor antagonists, such as spironoloactone and eplerenone that are used to treat side-effects related to mineralocorticoid excess, also bound to and activated signaling through both wild-type and mutant AR. Activation of mutant AR was inhibited by MDV3100, bicalutamide or greater concentrations of abiraterone. Moreover, higher androgenic steroid concentrations resulted in decreased enzalutamide antitumour activity. Together, our findings provide a strong rationale for the clinical evaluation of combined CYP17A1 inhibition and AR antagonism by enzalutamide. Disclosure J.S. de Bono: I am an employee of The Institute of Cancer Research that has a commercial interest in abiraterone. I have served as an advisor for J&J, Medivation, Astellas and Takeda.

Insulinlike Growth Factor (IGF)-1 Administration Ameliorates Disease Manifestations in a Mouse Model of Spinal and Bulbar Muscular Atrophy

Spinal and bulbar muscular atrophy is an X-linked motor neuron disease caused by polyglutamine expansion in the androgen receptor. Patients develop slowly progressive proximal muscle weakness, muscle atrophy and fasciculations. Affected individuals often show gynecomastia, testicular atrophy and reduced fertility as a result of mild androgen insensitivity. No effective disease-modifying therapy is currently available for this disease. Our recent studies have demonstrated that insulinlike growth factor (IGF)-1 reduces the mutant androgen receptor toxicity through activation of Akt in vitro, and spinal and bulbar muscular atrophy transgenic mice that also overexpress a noncirculating muscle isoform of IGF-1 have a less severe phenotype. Here we sought to establish the efficacy of daily intraperitoneal injections of mecasermin rinfabate, recombinant human IGF-1 and IGF-1 binding protein 3, in a transgenic mouse model expressing the mutant androgen receptor with an expanded 97 glutamine tract. The study was done in a controlled, randomized, blinded fashion, and, to reflect the clinical settings, the injections were started after the onset of disease manifestations. The treatment resulted in increased Akt phosphorylation and reduced mutant androgen receptor aggregation in muscle. In comparison to vehicle-treated controls, IGF-1-treated transgenic mice showed improved motor performance, attenuated weight loss and increased survival. Our results suggest that peripheral tissue can be targeted to improve the spinal and bulbar muscular atrophy phenotype and indicate that IGF-1 warrants further investigation in clinical trials as a potential treatment for this disease.

Specific morphogenetic events in mouse external genitalia sex differentiation are responsive/dependent upon androgens and/or estrogens

The objective of this study was to perform a comprehensive morphologic analysis of developing mouse external genitalia (ExG) and to determine specific sexual differentiation features that are responsive to androgens or estrogens. To eliminate sex steroid signaling postnatally, male and female mice were gonadectomized on the day of birth, and then injected intraperitoneally every other day with DES (200ng/g), DHT (1μg/g), or oil. On day-10 postnatal male and female ExG were dissected, fixed, embedded, serially sectioned and analyzed. We identified 10 sexually dimorphic anatomical features indicative of normal penile and clitoral differentiation in intact mice. Several (but not all) penile features were impaired or abolished as a result of neonatal castration. Those penile features remaining after neonatal castration were completely abolished with attendant clitoral development in androgen receptor (AR) mutant male mice (XTfm/Y and X/Y AR-null) in which AR signaling is absent both pre- and postnatally. Administration of DHT to neonatally castrated males restored development of all 10 masculine features to almost normal levels. Neonatal ovariectomy of female mice had little effect on clitoral development, whereas treatment of ovariectomized female mice with DHT induced partial masculinization of the clitoris. Administration of DES to neonatally gonadectomized male and female mice elicited a spectrum of development abnormalities. These studies demonstrate that the presence or absence of androgen prenatally specifies penile versus clitoral identity. Differentiated penile features emerge postnatally and are sensitive to and dependent upon prenatal or pre- and postnatal androgen. Emergence of differentiated clitoral features occurs postnatally in either intact or ovariectomized females. It is likely that each penile and clitoral feature has a unique time-course of hormonal dependency/sensitivity.

Design, Synthesis, and Biological Evaluation of Nonsteroidal Cycloalkane[d]isoxazole-Containing Androgen Receptor Modulators

We report here the design, preparation, and systematic evaluation of a novel cycloalkane[d]isoxazole pharmacophoric fragment-containing androgen receptor (AR) modulators. Cycloalkane[d]isoxazoles form new core structures that interact with the hydrophobic region of the AR ligand-binding domain. To systematize and rationalize the structure-activity relationship of the new fragment, we used molecular modeling to design a molecular library containing over 40 cycloalkane[d]isoxazole derivatives. The most potent compound, 4-(3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazol-3-yl)-2-(trifluoromethyl)benzonitrile (6a), exhibits antiandrogenic activity significantly greater than that of the most widely used antiandrogenic prostate cancer drugs bicalutamide (1) and hydroxyflutamide (2) in reporter gene assays measuring the transcriptional activity of AR (decreasing approximately 90% of the total AR activity) and in competitive AR ligand-binding assays (showing over four times higher potency to inhibit radioligand binding in comparison to bicalutamide). Notably, 6a maintains its antiandrogenic activity with AR mutants W741L and T877A commonly observed and activated by bicalutamide and hydroxyflutamide, respectively, in prostate cancer patients.