Androgen Receptor Antagonists for Prostate Cancer Therapy

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C Helsen et al. Role of AR antagonists in 21:4 T105–T118 Thematic Review treatment of PCa

Androgen receptor antagonists for prostate cancer therapy

Christine Helsen1, Thomas Van den Broeck1,2, Arnout Voet3, Stefan Prekovic1, Hendrik Van Poppel2, Steven Joniau2 and Frank Claessens1 Correspondence 1Laboratory of Molecular Endocrinology, Department of Cellular and Molecular Medicine, should be addressed Katholieke Universiteit Leuven, Herestraat 49, B-3000 Leuven, Belgium to F Claessens 2Urology, Department of Development and Regeneration, University Hospitals Leuven, Herestraat 49, Email 3000 Leuven, Belgium frank.claessens@med. 3Laboratory for Structural Bioinformatics, Center for Life Science Technologies, RIKEN, Yokohama, Japan kuleuven.be

Abstract

Androgen deprivation is the mainstay therapy for metastatic prostate cancer (PCa). Key Words Another way of suppressing androgen receptor (AR) signaling is via AR antagonists or " androgen receptor antiandrogens. Despite being frequently prescribed in clinical practice, there is conﬂicting antagonist evidence concerning the role of AR antagonists in the management of PCa. In the " antiandrogen castration-resistant settings of PCa, docetaxel has been the only treatment option for " enzalutamide decades. With recent evidence that castration-resistant PCa is far from AR-independent, " bicalutamide there has been an increasing interest in developing new AR antagonists. This review gives " abiraterone a concise overview of the clinically available antiandrogens and the experimental AR " prostate cancer antagonists that tackle androgen action with a different approach.

Endocrine-Related Cancer Endocrine-Related Cancer (2014) 21, T105–T118

Introduction

Prostate cancer (PCa) remains a major healthcare problem prostate specific antigen (PSA) levels) and patients’ overall and it is expected that the prevalence will only increase health status. Low-risk, localized PCa is generally managed due to aging of the population. In 2013, it is estimated that by active surveillance. For intermediate- and especially PCa will account for 28% of all cancers in the USA with high-risk disease, more invasive therapy is required, for 29 720 expected PCa-related deaths (Siegel et al. 2013). which surgery or radiotherapy are currently the standard Epidemiological studies from Europe show comparable treatment options whether or not within the context of a data with an estimated incidence of 416 700 new PCa cases multimodal approach. In the metastatic setting, androgen in 2012, representing 22.8% of cancer diagnoses in men. deprivation therapy (ADT) is the mainstay treatment, In total, 92 200 PCa-specific deaths are expected, making targeting androgen receptor (AR) signaling. The first it one of the three cancers men are most likely to die therapies, aimed at blocking AR activity, were already from, with a mortality rate of 9.5% (Ferlay et al. 2013). being used in the 1940s by Huggins (1942). He proposed Due to its heterogeneity, the treatment of PCa has castration and high doses of estrogens to create an been shown to be an important challenge, for which androgen-deprived state of the tumor. This revolutionary careful selection of the most suitable treatment regimen method was refined during the years and has led to the use is required (Fig. 1). of luteinizing hormone-releasing hormone (LHRH) ago- Currently, therapy selection is based on clinical nists that interfere with the hypothalamic–pituitary–testis staging (including TNM staging, Gleason scoring, and axis, leading to androgen deprivation. This initially leads

http://erc.endocrinology-journals.org q 2014 Society for Endocrinology This paper is one of 12 papers that form part of a thematic review section on DOI: 10.1530/ERC-13-0545 Published by Bioscientiﬁca Ltd. Androgens and the AR in Breast and Prostate Cancer. The Guest Editors for this section Printed in Great Britain were Wayne Tilley and Frank Claessens.Downloaded F Claessens from wasBioscientifica.com not involved in at the 09/24/2021 handling 04:08:59PM of this paper, on which he is listed as an author. via free access Thematic Review C Helsen et al. Role of AR antagonists in 21:4 T106 treatment of PCa

Organ-confined Localized a number of novel AR antagonists. Other promising non-

Active Radical AR-targeted therapies such as cabazitaxel (de Bono et al. Radiotherapy surveillance prostatectomy 2010), sipuleucel-T (Higano et al. 2009), alpharadin (Harrison et al. 2013), and tasquinimod (Armstrong et al. Locally advanced Extra-capsular 2013) are currently under development but are not within Radiotherapy Radical + prostatectomy HT the scope of this review.

Metastatic prostate cancer

Metastases Complete AR-targeted therapies LHRH- Orchiectomy androgen agonists blockade As androgens have a pivotal role in the development of the LHRH- antagonists prostate gland and prostate carcinogenesis, the AR is the fundamental target of systemic therapy for PCa (Taplin & Balk 2004). The AR is a ligand-dependent transcription Addition of a non-steroidal anti-androgen (NSAA) factor that is activated when androgens are present (Helsen et al.2012a). In response to androgen binding, it can initiate Disease progression

Withdrawal of NSAA expression of genes that contain response elements, which are recognized by the AR (Denayer et al.2010). Indeed, in prostate cells, the AR controls the balance between cell Castration-resistant differentiation and proliferation. In normal prostate tissue,

Castration- Abiraterone Sipuleucel T Docetaxel resistant the scale is tipped toward differentiation, while during progression of PCa from early to advanced cancer the scale gradually tips in favor of proliferation. This shift is accompanied by a downregulation of genes that support Docetaxel-resistant differentiation and upregulation of genes that promote Docetaxel- Enz Abiraterone Cabazitaxel Alpharadin resistant (MDV3100) proliferation (Hendriksen et al.2006, Marques et al.2011). Thus, the uncontrolled cell growth that is associated with PCa is reﬂected by a shift of the transcriptional program of Figure 1 AR toward a proliferative gene set (Wang et al.2009). It is Endocrine-Related Cancer Overview of prostate cancer therapy. This scheme gives the therapeutic options assigned to each stage of prostate cancer disease. Based on therefore crucial to inhibit androgen signaling either by tumor characteristics, patients’ health status, biological age, and depriving the tumor from androgens or by blocking the personal preference, the optimal therapeutic regimen can be chosen. receptor activity. AR activity can be enhanced not only by Treatments with a dotted frame are not considered as standard therapy according to the EAU guidelines. HT, hormone therapy; NSAA, steroids produced in the testis and adrenal glands but non-steroidal antiandrogen. Adapted from Higano & Crawford (2011) also by androgens that were synthesized by the tumor itself and Massard & Fizazi (2011). (Montgomery et al.2008). In that way, the tumor supports its own uncontrolled AR activity leading to CRPC. to tumor regression, but eventually the tumor adapts to the low androgen levels by developing an alternative way Androgen deprivation therapy of activating AR or by bypassing AR (Higano & Crawford 2011, Massard & Fizazi 2011). This stage of the disease is Inhibition of testicular androgen synthesis ADT referred to as castration-resistant PCa (CRPC). leads to a systemic decrease in the level of circulating In the castration-resistant setting, docetaxel has been androgens resulting in an androgen-depleted environ- the only treatment option that could prolong life for ment that prevents activation of the AR (Fig. 2). ADT, decades. The recent understanding that the AR remains an achieved by chemical or surgical castration, still is the important target, even in the castration-resistant setting, mainstay of treatment for patients with metastatic PCa. has led to the development of a plethora of treatment Due to convincing evidence showing comparable efﬁcacy, options targeting the AR. surgical castration is generally replaced by chemical The goal of this review is to give an insight into castration in the Western world (Soloway et al. 1991, the available therapeutic options for the treatment of Vogelzang et al. 1995, Seidenfeld et al. 2000). metastatic PCa, directly or indirectly targeting the AR. Chemical castration is achieved by blocking the Secondly, we will review the preclinical development of hypothalamic–pituitary–testis feedback system with

http://erc.endocrinology-journals.org q 2014 Society for Endocrinology Published by Bioscientiﬁca Ltd. DOI: 10.1530/ERC-13-0545 Printed in Great Britain Downloaded from Bioscientifica.com at 09/24/2021 04:08:59PM via free access Thematic Review C Helsen et al. Role of AR antagonists in 21:4 T107 treatment of PCa

testosterone production (Schally et al. 1992). Due to the Hypothalamus initial release of luteinizing hormone (increase by up to tenfold), there is an initial rise in testosterone levels LHRH (approximately twofold), occurring during the initial 72 h LHRH LHRH of treatment, which leads to a transient increase in PSA antagonist Pituitary gland agonist levels (Thompson 2001). Testosterone levels are being FSH ACTH suppressed at 10–20 days from administration, however, LH at a lower efﬁcacy compared with orchidectomy, as a Testis Adrenal glands signiﬁcant number of patients treated with the LHRH

Negative feedback CYP17A1 agonists fail to reach castrate levels of testosterone (Wex inhibitor et al. 2013). It has been suggested that this ‘biochemical flare’, Testosterone occurring in 4–63% of patients, could cause clinical ‘flares DHEA of disease’, with reports of increased pain from bone metastases, spinal cord compression, pathological frac- Prostate tures, bladder outlet obstruction, and even death (Bubley 2001, Heidenreich et al. 2011). Besides the initial flare, DHT miniflares are observed within 12 h after the second or CYP17A1 subsequent administration of the LHRH agonist (Sarosdy inhibitor et al. 1999, Sharifi & Browneller 2002). To prevent these AR flares, antiandrogens (see below) are being combined with LHRH agonistic treatment, before and during the first CoR weeks, to reach a total androgen blockade. While the AF1 Function antiandrogens will block the action of testosterone and adrenal androgens on the AR, they will not inhibit the hormonal surge and some of the potentially harmful Inactive AR effects that it produces (Brawer 2001). To circumvent the flares, LHRH antagonists have been Endocrine-Related Cancer developed, resulting in an equally effective, but more rapid chemical castration as LHRH agonists (Van Poppel Figure 2 et al. 2008, Crawford et al. 2011, Garnick & Mottet 2012, Androgen deprivation therapy. Depriving the tumor from androgens is one strategy to tackle uncontrolled androgen signaling by the AR. LHRH Ozono et al. 2012). Moreover, the LHRH antagonists do analogues, both agonists and antagonists, are able to inhibit the synthesis not have increased risk for cardiovascular diseases as is the of testosterone in the testis and of DHEA in the adrenal glands by affecting case for LHRH agonists (Levine et al. 2010). the hypothalamic–pituitary–gonadal/adrenal axis. CYP17A1 inhibitors cause androgen deprivation by inhibiting the intracellular biosynthesis of Despite the lack of evidence on the contribution of androgens in the testis and adrenals starting from cholesterol. Besides antiandrogens, the current EAU Guidelines recommend reduced testosterone, DHT, and DHEA levels, some CYP17A1 inhibitors ADT therapy in all (symptomatic and asymptomatic) (e.g., abiraterone and TOK-001) are also able to directly bind to the androgen receptor (AR) and block its activity as a ligand-dependent metastatic patients combined with short-term adminis- transcription factor. LHRH, luteinizing hormone-releasing hormone; FSH, tration of antiandrogen therapy to reduce the risk of the follicle-stimulating hormone; LH, luteinizing hormone; ACTH, adrenocor- ‘flare’ phenomenon in patients receiving LHRH agonists. ticotropic hormone; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; AF1, activation function 1; CoR, corepressor complexes. They emphasize that, especially in patients at high risk of ‘clinical flare’, LHRH agonists should not be administered as monotherapy, even though there is no evidence of LHRH analogues (Fig. 2). Currently, the available LHRH long-term effects (Heidenreich et al. 2011). agonists are leuprolide, goserelin, triptorelin, and histre- lin, which need to be administered, monthly to yearly, by Inhibition of adrenal and intracellular androgen injection or implantation. There is an initial release of synthesis Despite the initial good response to ADT, PCa luteinizing hormone, causing a downregulation of LHRH progresses toward a castration-resistant stage after a median receptors at the hypothalamus, disrupting the hypo- time of 2–3 years (Knudsen & Kelly 2012). This means that thalamic–pituitary–testicular axis and therefore reducing the tumor has adapted to survive the castrate levels of

androgens or the presence of antiandrogens. Frequently, resulting in castration resistance is an increase of the AR signaling has been restored not only by an upregulation intracellular androgen levels (Montgomery et al. 2008). of the AR, by mutation of the AR,byanimbalanceof CYP17A1, which is an enzyme involved in the androgen AR co-regulatory proteins but also by the synthesis of biosynthesis pathway, is speciﬁcally inhibited by abirater- androgens in the tumor cells itself (Taplin & Balk 2004). one. By suppressing testosterone synthesis in the adrenals Several compounds, such as ketoconazole and abir- and intratumorally, administration of abiraterone results aterone, are able to inhibit the intracellular biosynthesis in decreased intraprostatic testosterone levels. of androgens (CYP17A1) and thereby prevent activation After the efﬁcacy had been proved in clinical trials of the AR (Attard et al. 2009; Fig. 2). Compared with on both chemotreated and chemonaive mCRPC patients ketoconazole, abiraterone acetate is a potent, selective, (de Bono et al.2011, Ryan et al.2013), the drug was and safe drug. It inhibits the androgen synthesis not only approved by the FDA and EMA for use in patients with in the testis but also in the adrenal glands and the prostate mCRPC. Recently, Richards et al.(2012)demonstrated that (Attard et al. 2009). Ketoconazole and abiraterone have part of the mechanism of action of abiraterone (steroidal) to be administered together with prednisone to avoid can be attributed to AR binding and inhibition (Fig. 2). symptoms of mineralocorticoid excess, caused by an Novel CYP17A1 inhibitors Other CYP17A1 inhibitors are under excess of ACTH secretion (Danila et al. 2010). development (Vasaitis et al. 2008, Yamaoka et al. 2012; Abiraterone acetate Abiraterone acetate was developed to Fig. 3). Indeed, abiraterone is an inhibitor of both the manage metastatic PCa, progressing after chemotherapy. C17,20-lyase activity and the 17a-hydroxylase activity of One of the observed adaptive mechanisms in PCa cells CYP17A1 and inhibition of the latter causes symptoms

Steroidal antiandrogens

DHT CPA MPA

O O OH O O Endocrine-Related Cancer O H H H H H O H H H H H H O O O H CI CYP17A1 inhibitor

TAK700 Abiraterone N TOK-001 N

O N

N OH N H H H N H H H H OH OH

Figure 3 Steroidal antiandrogens and CYP17 inhibitors. The CYP17 inhibitors with a steroidal structure also display antiandrogenic effects by binding directly to the AR. DHT, dihydrotestosterone; CPA, cyproterone acetate; MPA, medroxyprogesterone acetate.

of mineralocorticoid excess. Therefore, compounds with DHT higher speciﬁcity against the C17,20-lyase activity of AR CYP17A1 have been developed to prevent the need for prednisone. TAK700, a non-steroidal CYP17A1 inhibitor, CoA

is an example of more selective CYP17A1 inhibitor; AF1 Function however, the phase 3 clinical trial with TAK700 has ended preliminary because of the observation that Active AR TAK700 plus prednisone would not likely meet the primary endpoint of improved overall survival compared with placebo plus prednisone. For TOK-001, it is known that it is not only a CYP17 inhibitor, but also a competitive DHT AR antagonist that results in the downregulation of the Bic AR (Vasaitis et al. 2008, Bruno et al. 2011). AR

CoR Antiandrogens AF1 Function AR antagonists or antiandrogens prevent androgens from carrying out their biological activity by directly binding Inactive AR and blocking the AR LBD, and by inducing repressive activity (Fig. 4). The current roles of antiandrogen therapy are limited DHT to preventing the ﬂare-up of LHRH-agonistic therapies Enz (see above) and usage in combination with LHRH- AR agonistic or -antagonistic therapy to achieve complete androgen blockade (Fig. 1). CoR Despite the fact that chemical or surgical castration AF1 Function reduces 95% of testosterone levels, an intraprostatic

Endocrine-Related Cancer androgen stimulus is still present as a result of circulating Inactive AR androgens and androgen precursors of adrenal origin. Adding an antiandrogen to castration blocks the action of these adrenal androgens, resulting in complete androgen blockade. However, as the clinical advantage of this Figure 4 combined treatment is still questionable, the current Mechanism of action of AR antagonists. After binding to the ligand- EAUguidelinesdonotrecommendthisasstandard binding pocket of AR and nuclear translocation of AR, dihydrotestosterone (DHT) is able to induce the formation of a coactivator-binding platform, treatment (Heidenreich et al. 2011). called AF1, which leads to the recruitment of coactivators (CoA). These In the next section, we will discuss the current coactivators will induce the formation of an active transcriptional complex evidence on the role of (steroidal and non-steroidal) containing RNA polymerase that leads to transcription of androgen- regulated genes. AR antagonists can interfere with all of these required antiandrogens in treating advanced PCa. events for activation of AR gene expression by an androgen. Bicalutamide (bic) binds to the ligand-binding pocket of AR but fails to induce the correct Steroidal antiandrogens In analogy with the conformational change. The platform that is formed cannot recruit coactivators, but corepressors (CoR) leading to an inactive AR–DNA structure of androgens, the ﬁrst antiandrogens contained complex. Enzalutamide (Enz) has a similar mechanism of action as bic, a steroidal skeleton to ensure binding to the receptor but can prevent nuclear translocation of the AR and the binding of AR (Fig. 3). Several steroidal antiandrogens (cyproterone to DNA as well, making it a stronger AR antagonist. acetate (CPA), megestrol acetate, and medroxyprogesterone acetate) were initially used for obtaining maximal Pavone-Macaluso et al.1986, 1989, Moffat 1990, Patel androgen blockade in patients; however, severe et al.1990, Dawson et al.2000, Schroder et al.2004). drawbacks such as hepatotoxicity, interference with libido The side effects that occur for these drugs are related and potency, cardiovascular side effects, and low efﬁcacy to their effects on other steroid receptors such as the have limited their clinical use (Jacobi et al.1980, progesterone receptor and the glucocorticoid receptor, for

which they are (weak/partial) agonists (Pridjian et al.1987, was not the case in men with advanced PCa. Their serum Schroder 1993). Interference with libido and potency is the LH and serum T levels were significantly increased due to result of their antigonadotropic effects leading to reduced bic therapy (Mahler & Denis 1990). secretion of LH and follicle-stimulating hormone (FSH) and Enzalutamide was first introduced in 2009 as a as a consequence decreased plasma levels of T and E. Unlike second-generation antiandrogen with several advantages the steroidal antiandrogens, flutamide (flut) increases T over bic (Tran et al. 2009). In contrast to the ‘classical and E levels which could lead to gynecomastia (Schroder antiandrogens’, enz does not only block androgen 1993). In concert with LHRH treatment, the steroidal binding, but it also inhibits translocation of the AR to antiandrogens seem to reduce gynecomastia and hot the nucleus and impairs AR binding to DNA (Tran et al. flushes. The effectiveness of the LHRH therapy, however, 2009). It has been approved by the FDA and EMA for cannot be increased as was shown in a prospective, treating patients with chemo-resistant CRPC (Ning et al. randomized study comparing goserelin acetate vs CPA vs 2013). Further structural optimization for inhibition of a combination of the two, and this study concluded that proliferation, pharmacokinetics, and in vivo efficacy CPA as monotherapy was inferior and the combined resulted in the development of a compound called ARN- treatment was not superior to goserelin acetate alone 509 (Fig. 5). It has a higher efficacy than enz, as 30 mg/kg (Thorpe et al.1996). With regard to reducing hot flushes, per day has the same therapeutic effect as 100 mg/kg per Irani et al. (2010) performed a randomized double-blind day enz (Clegg et al. 2012). Moreover, due to the lower trial, comparing efficacies of these drugs, concluding that required dosage of ARN-509, it has a lower tendency to medroxyprogesterone acetate could be considered as the induce seizures, a typical side effect of antiandrogens from standard treatment for hot flushes. the bic class (Foster et al. 2011).

Non-steroidal antiandrogens Non-steroidal antian- Clinical use Several studies have been conducted to drogens (NSAAs) were first introduced in 1989 in clinical determine the efficacy and to assess whether NSAAs in practice as treatment for advanced and metastatic PCa. monotherapy have an advantage over LHRH analogues Currently, they are mainly used in combination with regarding side effects such as erectile dysfunction, loss of LHRH agonists in preventing clinical ‘flare-up’ and in libido, fatigue, etc. Both the first- and second-generation combination with LHRH agonists/antagonists to achieve NSAAs demonstrate an identical off-target on GABAA ‘complete androgen blockade’ (see above). Available drugs

Endocrine-Related Cancer receptors in the brain, leading to seizures (Foster et al. are the first-generation antiandrogens flut, bicalutamide 2011). In light of this, ARN-509 has been developed, which (bic), and nilutamide (nil), and the second-generation has a reduced passage through the blood–brain barrier. compound enzalutamide (enz) (previously known as We will briefly discuss the clinical use of the first- and MDV3100) (Fig. 5). They antagonize the actions of second-generation antiandrogens. androgens at the receptor level and thereby inhibit tumor growth. The first-generation antiandrogens bic Flutamide Flut was the first NSAA available for clinical use, and nil were derived from flut and thus have a similar approved by the FDA in 1989. It is indicated to use in non-steroidal, chemical structure (Fig. 5). The non- combination with LHRH agonists for the management steroidal structure avoids the typical constraints associ- of locally confined/advanced and metastatic PCa. ated with the previous steroidal antiandrogens. Of them, Its role as a monotherapeutic agent is still unclear, with bic is the best tolerated and most stable antiandrogen only one randomized controlled trial (RCT) published, currently used in clinical practice. It has a half-life of seven comparing the therapeutic efficacy of flut with surgical days compared with 6–8 h for flut and 2 days for nil which castration in patients with metastatic PCa. However, the allows once-a-day dosing (McLeod 1997). The efficacy of results remained inconclusive on its efficacy (Boccon- bic in clinical trials was reported to be at least equivalent Gibod et al. 1997). Furthermore, the EORTC group could to the efficacy of flut (Schellhammer et al. 1996) and it is not show a difference in outcome between flut and CPA better tolerated in terms of diarrhea, a common adverse either (Schroder et al. 2004). The same EORTC trial 30892 effect in flut-treated patients (Sarosdy 1999). No beneficial showed that only 20% of men treated with flut remained effects were observed for nil over flut and nil has sexually active for 7 years (Schroder et al. 2004). Due to the least favorable safety profile (Sarosdy 1999). While the poor evidence for its efficacy and its side effects on peripheral selectivity was observed in intact rats due to a sexual activity in men, flut is currently not indicated as low passage across the blood–brain barrier (Furr 1996), this a monotherapeutic drug in patients with metastatic PCa.

Non-steroidal antiandrogens

First-generation Second-generation

Flutamide Enzalutamide

F F O H F F N F N N F F O O2N S O N NH

Bicalutamide ARN-509

F F O O H HO O N S F F F N N F F O F N S O N N NH

Nilutamide CH O 3 CH3 F F N NH F

Endocrine-Related Cancer O2N

Figure 5 Non-steroidal antiandrogens. Both the ﬁrst- and second-generation group in meta-position and a nitro- or cyano-group in para-position. In antiandrogens that are currently being used in clinical practice share a nilutamide, enzalutamide, and ARN-509, the amide of the anilide is a part structural motif consisting of an anilide substituted with a triﬂuoromethyl of (thio)hydantoin.

Bicalutamide Bic is currently the most investigated and most monotherapy as an alternative for the gold standard (ADT) widespread NSAA in clinical practice, with recommended in well-informed patients with favorable clinical para- doses of 50 mg/day in complete androgen blockage and meters (Loblaw et al. 2007). 150 mg/day for monotherapy. The latter has been inves- Nilutamide There are no comparative trials of nil monotherapy tigated most extensively, with several prospective RCTs with castration or with other antiandrogens, because of being performed. When comparing bic in monotherapy which nil is currently not licensed for monotherapy. with castration, bic was associated with worse survival rates, although the difference in median survival was only Enzalutamide In patients who progressed during docetaxel 6 weeks (Tyrrell et al. 1998). Two small RCTs, comparing therapy, enz has been shown to increase overall survival bic with complete androgen blockade, remained incon- in comparison with placebo in mCRPC patients in the clusive, although Boccardo et al. showed an increased risk AFFIRM trial, leading to its FDA approval (Scher et al. 2012, of death in poorly differentiated tumors (Fourcade et al. Ning et al. 2013). A recent report from the AFFIRM trial 1998, Boccardo et al. 2002). showed comparable efﬁcacy, safety, and tolerability in Despite these inconclusive results, the American patients aged O75 years as well, suggesting its possible Society of Clinical Oncology (ASCO) has suggested bic future widespread use (Sternberg et al. 2014). Two small

retrospective studies demonstrated only a limited activity Each step is important for the execution of the normal of enz in the post-docetaxel and post-abiraterone patient function of AR, and each step is therefore a potential target population, suggesting the existence of cross-resistance for antiandrogen therapy. The precise mechanism of action (Bianchini et al. 2013a, Schrader et al. 2013). These studies of bic and enz is described below (Fig. 4). are not RCTs and lack power with only 35–39 patients Bicalutamide When bic binds to the ligand-binding pocket being investigated, limiting its clinical value. However, the molecular basis of such cross-resistance should be of the AR, a displacement of helix 12 and consequently further investigated because it will provide information on a distortion of the coactivator platform were suggested the sequence in which these treatments should be used. by molecular dynamics-based simulations (Osguthorpe & To test its efficacy in the pre-chemotherapy setting, Hagler 2011). Furthermore, it leads to the assembly of the PREVAIL trial was started (NCT01212991). However, a transcriptionally inactive complex due to the inability it has recently been stopped early after meeting its of the bic-bound AR to recruit coactivators and/or the co-primary endpoints of overall survival and radiographic preferential recruitment of corepressors, NCoR and SMRT progression-free survival, making it possible for patients (Masiello et al. 2002). By fluorescence recovery after from the placebo group to switch treatment arms. photobleaching (FRAP) analysis with a GFP-tagged AR, the immobile fraction of nuclear AR disappeared when bic Mechanism of action of antiandrogens Antiandro- was present, meaning that the receptor was unable to bind gens bind to the ligand-binding pocket of the AR and DNA (Farla et al. 2005). Moreover, the AR was shown to thereby prevent its activation. When androgens such as be destabilized in the presence of bic (Waller et al. 2000). dihydrotestosterone (DHT) bind to the AR, a very specific All these observations explain why bic can decrease conformational change occurs in the ligand-binding androgen-induced gene expression and reduce the weight domain, leading to an active receptor (Pereira de Jesus-Tran of rat ventral prostate and seminal vesicles after oral et al.2006; Fig. 4). Crystal structures of steroid receptor administration (Furr & Tucker 1996). ligand binding domains (LBDs) with an agonist have shown Enzalutamide As mentioned earlier, enz is considered as a that after binding, helix 12 closes off the pocket and forms a more effective inhibitor of AR compared with bic due to platform for the binding of coactivators, called activation a higher binding affinity, the inhibition of AR nuclear function 1 (AF1; Shiau et al.1998, Williams & Sigler 1998). translocation and DNA binding by AR (Tran et al.2009, This specific position of helix 12 is required and can only be Endocrine-Related Cancer Guerrero et al.2013). FoxA1 is a PCa-involved pioneering induced by agonists. When antagonists bind, they induce factor that makes DNA more accessible for binding by the a distinct inactive LBD conformation in which helix 12 is AR. Enz prevented the AR from binding to DNA response repositioned from its active position or helix 12 is forced into a flexible position (Kauppi et al.2003, Hodgson et al. elements in presence of FoxA1, while bic could not (Belikov 2008, Lusher et al. 2011). This disables the binding et al.2012). Further optimization of enz is ongoing and has of coactivators and/or enables the formation of another led to the development of ARN-509, a compound with platform to which corepressors can bind. Moreover, optimized pharmacokinetics, phase 1 and 2 clinical studies antiandrogens can recruit corepressors via other domains of which are currently running (Clegg et al.2012, Rathkopf such as the amino terminal domain (NTD), which was et al.2013). Due to its structural similarities (Fig. 5), a demonstrated for CPA (Dotzlaw et al.2002). For the AR, similar binding mode to AR and thus a similar working the carboxy-terminal end of helix 12 is anchored by the mechanism are attributed to ARN-509 (Clegg et al.2012, formation of a b-sheet leading to a less flexible helix 12. Balbas et al.2013). Unfortunately, so far no antagonist- In absence of antagonist-bound AR crystals, molecular containing WT AR LBD crystal structures are available modeling techniques have predicted that, for the AR, a (Balbas et al.2013, Joseph et al.2013, Korpal et al.2013). displacement of helix 12 could occur in the presence of The discovery of the AR W741C/L mutation in bic-resistant antiandrogens, although involvement of residues in helix 5 cells, AR T877A mutation in flut-resistant cells, and AR and helix 11 cannot be excluded (Georget et al.2002, Bohl F876L mutation in enz-resistant cells has given some et al.2005a, Bisson et al.2008, Osguthorpe & Hagler 2011). insights. A model for the binding of enz to the ligand- The conformation of the receptor induced by antagonists binding pocket of WT AR has been proposed (Fig. 6; Balbas results in the inhibition of many required actions of the AR, et al.2013). A focused chemical screen, based on this in silico such as inhibition of entry to the cell nucleus, binding to model, has led to a compound that circumvents this escape DNA response elements, recruitment of coactivators, etc. mutation and remains antagonistic (Balbas et al.2013).

A of RU59063, a non-steroidal arylthiohydantoin AR agonist that binds AR with the highest known afﬁnity (in the nanomolar range) (Jung et al. 2010). Holding on to this structural element, however, diminishes the structural diversity of AR ligands and could therefore be the cause of R752 cross-resistance in case of sequential therapy. Molecular modeling of bic and enz in the ligand-binding pocket of AR shows a highly conserved interaction required for binding to AR (Voet et al. 2013; Fig. 6). Structurally more diverse AR LBD antagonists could thus provide an innovative way of inhibiting AR, by introducing a B different conformational change in AR and hence also a different helix 12 position. From the latest studies on enz resistance and the much earlier reports of bic and ﬂut resistance, it is almost certain that eventually every LBD inhibitor, which has been and R752 will be developed, will lead to the introduction of a somatic mutation that causes a switch to agonist. There- fore, it seems that the future of AR antagonists lies in the development of compounds with different targets or different strategies. For instance, compounds that do not act via the ligand-binding pocket but via other sites on the

Figure 6 LBD, the NTD or compounds that could affect AR protein Similar binding modes of bic and enz to the AR ligand-binding pocket. levels will be of interest. Furthermore, the combination In silico model of bic (A) and enz (B) binding as an antagonist in the ligand- of compounds with a complementary action mechanism binding domain of the human AR according to Voet et al. (2013). Both molecules show key conserved interactions in the deeper end of the pocket could lead to a more efficient inhibitory action of AR and where the common trifluoromethyl cyano benzyl group is accommodated thus a better control of the disease. in a highly hydrophobic area with the exception of one hydrogen bridge Endocrine-Related Cancer with arginine 752 (R752). Experimental AR-targeted therapies Inhibitors binding to the ligand-binding pocket We and others This was confirmed not only by transfection experiments have screened for novel AR antagonists belonging to with AR F876L (where ARN-509 also performed as an classes that are structurally different from those that are agonist), but also by the detection of the AR F876L currently used in the clinic (Helsen et al. 2012b, Voet et al. mutation in the plasma DNA of ARN-509-treated CRPC 2013). Although such molecules have the advantage that patients (Balbas et al.2013, Joseph et al.2013). they might still work in case of resistance to bic, flut, or enz, they still need to be developed further before they Structural similarities for the current anti- could be tested in a clinical trial setting. androgens Strikingly, the available first- and second- ODM-201 is a novel AR antagonist that binds to AR generation antiandrogens share a common structural with a higher affinity than enz. Antiproliferative effects element, an anilide core motif (Fig. 6). For some of ODM-201 have been demonstrated in subcutaneous compounds, the amide of the anilide can be a part of VCaP xenografts and, due to its unique pharmacological (thio)hydantoin. The benzene ring of the anilide has a properties, it has also been shown to have superior trifluoromethyl group in meta-position and a nitro- or preclinical efficacy in phase 1 studies with no side effects cyano-group in para-position. The chemical scaffold of on the CNS. The most common adverse effects were Bic has been studied well by means of structure–activity asthenia, diarrhea, and nausea (Fizazi et al. 2012). relationships (Kirkovsky et al. 2000, Bohl et al. 2005b) and has also served as a template for the development of Inhibitors of AR that do not bind to the ligand-binding pocket EPI-001 is several selective AR modulators (SARMs), such as Ostarine a bisphenol A diglycidyl ether-like compound that has and LGD-4033 (Dalton et al. 2013). The structures of been reported to affect the transactivation mediated by the RD162, MDV3100 (enz), and ARN-509 are based on that AR NTD. It inhibits the AR regardless of ligand and inhibits

androgen-induced gene expression and proliferation both Future perspectives in vitro and in LNCaP xenograft mice models (Andersen A new era has started in treating mCRPC patients thanks et al. 2010, Myung et al. 2013). EPI-001 is an alternative to the development of multiple new potent AR-targeted drug candidate for PCa as it does not act via the LBD. therapies. These agents first have to prove their efficacy in EPI-001 might circumvent escape mechanisms such as the post-chemotherapeutic setting, as have abiraterone and LBD mutation or truncation and should therefore still be active in many forms of CRPC. enzalutamide, leading to their FDA and EMA approval. For An alternative way of action compared with enz and bic ARN-509, a safety, pharmacokinetic, and proof-of-concept was discovered for ASC-J9, an AR-binding compound that study is currently ongoing (NCT01171898). The efficacy of destabilizes full-length AR as well as the AR splice variants these AR-targeted compounds in these advanced stages has involved in castration resistance of PCa (Yamashita et al. confirmed that CRPC is not an AR-independent disease 2012). They all inhibit PCa proliferation and reduce PSA and these novel treatments are even being considered to be levels, but only ASC-J9 was able to suppress cell invasion used at an earlier stage during the course of the disease. and thus the formation of metastases in in vitro and in vivo When resistance to these novel agents will arise, the eluci- models (Lin et al.2013a). A different modulation of the dation of the underlying mechanisms will tell us what STAT3–CCL2 signaling pathway was suggested to lie at the the targets of the next generation of compounds should be. base of this observed difference (Lin et al.2013b). The emergence of AR splice variants and the antagonist- to-agonist switching mutation AR F876L indicate that the Affecting AR localization SNARE-1 binds WT and mutant ARs AR can also circumvent the new LBD inhibitors, which via the LBD. It not only inhibits nuclear translocation of are being developed and might necessitate the develop- the AR, but also facilitates nuclear export leading to ment of structurally or strategically different AR inhibitors. reduced nuclear AR levels. Furthermore, it promotes Will the AR keep its critical role in PCa treatment or will degradation of the AR via the ubiquitination pathway. it be replaced by completely different targets? Its mechanism of action is completely different compared with the clinically available antiandrogens and its activity hasbeendemonstratedonPCaxenograftsinmice Declaration of interest (Narayanan et al. 2010). The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the review.

Endocrine-Related Cancer Affecting AR mRNA half-life EZN-4176 is a 16-mer antisense oligonucleotide that decreases full-length AR protein AR expression by binding to exon 4 of mRNA (the Funding hinge). Both in vitro and in animal models, EZN-4176 This work was supported by the fund from the KU Leuven (OT08-11) and can inhibit the proliferation of androgen-sensitive and the FWO (G.0684-12/N and G.0830-13N), and the authors gratefully acknowledge the financial support from the company Ravago Distribution CRPC cells (Zhang et al. 2011). A small study with 22 CRPC Center NV (LUOR fonds). patients who were progressive (prior abiraterone or enzalutamide treatment was allowed) demonstrated only little biochemical and soft tissue response and only three Acknowledgements out of eight patients showed a reduction in circulating The authors would like to acknowledge Servier for producing Figs 2 and 4 tumor cells at the doses used (Bianchini et al. 2013b); side using Servier Medical Art (www.servier.com). effects such as fatigue and liver toxicity prevented further dose escalation (Bianchini et al. 2013b). References Inhibitors binding to BF3 of AR Next to the ligand-binding Andersen RJ, Mawji NR, Wang J, Wang G, Haile S, Myung JK, Watt K, Tam T, pocket, a newly discovered surface pocket of AR, called Yang YC, Banuelos CA et al. 2010 Regression of castrate-recurrent binding function 3 (BF3), could be targeted to influence prostate cancer by a small-molecule inhibitor of the amino-terminus domain of the androgen receptor. Cancer Cell 17 535–546. (doi:10.1016/ coactivator binding (Estebanez-Perpina et al. 2007). Several j.ccr.2010.04.027) structural scaffolds have been identified through virtual Armstrong AJ, Haggman M, Stadler WM, Gingrich JR, Assikis V, Polikoff J, screening as binders to this BF3 region and were confirmed Damber JE, Belkoff L, Nordle O, Forsberg G et al. 2013 Long-term to have antagonistic effects not only on AR transcriptional survival and biomarker correlates of tasquinimod efficacy in a multicenter randomized study of men with minimally symptomatic activity (Lack et al. 2011), but also on proliferation of metastatic castration-resistant prostate cancer. Clinical Cancer Research LNCaP and enz-resistant cells (Munuganti et al. 2013). 19 6891–6901. (doi:10.1158/1078-0432.CCR-13-1581)

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Received in ﬁnal form 21 February 2014 Accepted 13 March 2014 Made available online as an Accepted Preprint 17 March 2014