Targeting the Androgen Receptor


Prostate cancer is the most common cancer in men and is unique in that its growth is largely dependent on androgen signaling. This depen- dence was first recognized more than 70 years ago when Huggins and Hodges1,2 observed striking regressions of metastatic prostate cancer in men who underwent either surgical or medical castration. Indeed, in men with metastatic prostate cancer, androgen deprivation improves bone pain, lessens lower urinary tract symptoms, and in- creases overall quality of life.

Despite the impressive clinical improvements observed with androgen deprivation therapy (ADT), androgen suppression is not curative, and all men eventually develop disease growth despite castrate levels of testosterone. Tumor growth in the castrate environment is lethal and accounts for close to 30,000 deaths annually in the United States.3 The median survival for a man with castrate-resistant disease is between 2 and 3 years from the time of diagnosis of castration resistance.

Until recently, the terms androgen-independent prostate cancer (AIPC) or hormone refractory pros- tate cancer (HRPC) were used in the literature to describe this clinical state, implying largely that tumor growth occurred through pathways com- pletely independent of the androgen signaling. Work in recent years, however, has dispelled this misconception and has clearly demonstrated that signaling through the androgen receptor (AR) remains crucial to tumor growth in the castrate tumor environment. With this understanding, the term castration-resistant prostate cancer (CRPC) has gradually emerged to describe this clinical state. The term “CRPC” is especially useful in that it connotes only the clinical state of disease growth despite castrate levels of serum testosterone, while not indicating the potential, one way or another, for response to further androgen manipulation.

As we shall see in this review, castration resistance can develop in many ways, including through the development of AR mutations, through AR ampli- fication, through the production of androgens within tumors (intracrine androgen synthesis), through the emergence of truncated AR proteins (splice variants) capable of ligand-independent signaling, and through other mechanisms. Understanding these mechanisms has led the way for the development of multiple novel agents that target the AR itself. Indeed, enzalutamide, a novel antiandrogen that directly inhibits AR function, and abiraterone acetate (Zytiga), an inhibitor of androgen synthesis that lowers circulating ligand, have both been shown to improve overall survival in well-powered randomized Phase III studies and are discussed later in this article. Additionally, a number of new agents are in clinical development, including agents that directly target the AR, such as ARN-509 and EPI-001, as well as those, like abiraterone acetate, that indirectly target the AR, such as orteronel (TAK-700), galeter- one (TOK-001), and others.
To understand the clinical development of these agents, we first review our current understand- ing of the structure and function of the AR, then explore how castration resistance emerges, focusing specifically on mechanisms that lead to persistent AR signaling in a low testosterone envi- ronment. We then review the clinical development of agents that target the AR, focusing on recently approved agents and those currently in clinical development.


The AR gene is located on chromosome Xq11-12 and is a member of the steroid hormone receptor family that includes, among others, the estrogen receptor, progesterone receptor, and the gluco- corticoid receptor. All share a similar structure. Histologically, the AR is present in benign prostate epithelial cells as well as in all stages and grades of primary and metastatic prostate cancer.5,6 Func- tionally, the AR is a 110-kDa ligand-activated tran- scription factor consisting of 917 amino acids that contain 3 important distinct domains (Fig. 1A): a carboxy-terminal ligand-binding domain (LBD), which binds androgens, a DNA-binding domain (DBD), and a regulatory N-terminal domain (NTD).7 Within the N-terminal domain lies the acti- vation function 1 (AF-1) and AF-5 domains, which contain binding sites for transcriptional coregula- tors and are essential for AR activity.

Under normal conditions, inactive AR is found in the cytoplasm of prostate cancer cells and is stabilized there by various heat-shock proteins (HSPs), which expose the LBD to surrounding proteins and allow for androgen binding. Once bound to androgenic ligands, a conformational change occurs in the AR, causing dissociation of HSPs, AR receptor dimerization, and AR migration to the nucleus (see Fig. 1B).10 Once inside the nucleus, the AR DBD binds to specific androgen response elements (AREs) on the promoter or enhancer regions of androgen-regulated genes. Transcription of genes necessary for prostate cancer growth and survival can then be initiated, and is enhanced by the binding of transcription coregulators to the AF-1 binding site in the AR- NTD.11 Among the many genes under AR tran- scriptional control is prostate-specific antigen (PSA), and hence serum PSA measurement serves as a clinically useful biomarker for AR transcrip- tional activity and, by extension, disease growth.


Depletion of circulating testicular androgens through either orchiectomy or luteinizing hormone–releasing hormone (LHRH) therapy drastically decreases androgenic ligand levels, reduces AR signaling, lowers PSA, and results in regression of disease the vast majority of cases. Both the rate of PSA decline and the nadir PSA on therapy are predictive of overall response to initial ADT, with higher nadir PSA values and shorter times to PSA nadir associated with poor survival.12 This observation further highlights the role that AR-mediated sig- naling has on disease progression and disease aggressiveness.

Antiandrogens, such as bicalutamide (Caso- dex), nilutamide (Nilandron), and flutamide (Eulex- in), have been in clinical use for decades, and observations about their use illustrate one of the first clinically relevant, identifiable steps in AR evolution. It was first noted in the early 1990s that disease progression, despite the combination of an LHRH agonist and an antiandrogen, could be stopped and reversed simply through discontinua- tion of the antiandrogen.13 This antiandrogen with- drawal effect (AAWD) was best characterized in a study by Small and colleagues14 who observed that discontinuation of an antiandrogen in men with a rising PSA leads to sustained PSA declines of more than 75% in more than 10% of men. Further work demonstrated that mutations in the AR can cause AR inhibitors, such as bicalutamide that bind to the LBD, to paradoxically stimulate AR and result in disease growth.15 Although this mechanism remains incompletely understood, this observation illustrates the point that genomic changes occur or arise in the AR in response to therapy and can have important functional and clinical implications. Whether these genomic changes are present in a subpopulation of cells at baseline, or whether they are acquired in response to the selective pressure of AR- targeted therapy, is still unknown.

Targeting the Androgen Receptor

Fig. 1. (A) Structural domains of 2 isoforms of the androgen receptor (AR-A and AR-B). Numbers above the bars refer to the amino acid residues that separate the domains starting from the N-terminus (left) to C-terminus (right). (B) The presence of testosterone (T) or dihydrotestosterone (DHT) causes dissociation of HSP, dimerization, and phosphorylation (P) of the AR and translocation to the nucleus where the AR binds to an ARE, causing recruitment of DNA transcriptional machinery and gene transcription. (Adapted from Li J, Al-Azzawi F. Mecha- nism of androgen receptor action. Maturitas 2009;63:142–8; with permission.)

Further observation about the state of the AR as tumors transition from castration-sensitive to castration-resistant have had important function implications and have helped guide the develop- ment of new therapies. In a landmark study, Stan- brough and colleagues16 profiled the expression signatures of primary and metastatic prostate tumors, including both castration-sensitive and castration-resistant tumors, and found significantly higher levels of AR mRNA in castrate-resistant tumors compared with castration-sensitive tumors. Subsequent studies of metastatic CRPC tumors ob- tained at autopsy showed that the genomic segment of chromosome X encoding the AR is itself amplified, up to 60-fold in some cases, and other studies has shown that the AR protein is overexpressed in metastatic CRPC tumors.17 These observations suggest that depletion of circulating androgens results in a selection for cells that are best able to respond to the low levels of residual ligand.

A second critical laboratory observation is that mRNA encoding enzymes that synthesize hormones are upregulated within tumor cells themselves.16 This novel finding suggested that tumor cells that were deprived of androgens through the use of an LHRH agonist or through receptor inhibition by an antian- drogen could, in addition to increasing AR number, potentially synthesize their own androgens within the local tumor environment and thereby stimulate their own growth. This finding of autocrine stimulation has been lent further support by the finding of increased levels of androgens within CRPC tumors, even despite low levels of circulating androgen.

Coupling these observations of AR gain and in- tratumoral androgen synthesis has drastically changed the current thinking about the develop- ment of CRPC. In older models, CRPC results from the development of mechanisms of growth independent of the androgen axis. The current model of CRPC is one in which tumors are in effect hypersensitive to even low levels of androgens, and one in which tumors are no longer necessarily reliant on ligand produced by outside sources, such as the testis or adrenal glands. These obser- vations, therefore, provide a plausible mechanism by which formerly castration-sensitive tumors re- gain the ability to grow in a castrate environment, and provide targets (ie, inhibition of the androgen synthesis machinery) for drug development. Agents either available or in late-stage clinical trials that specifically target androgen synthesis include abiraterone acetate, orteronel (TAK-700), galeterone (TOK-001), and ketoconazole (Nizoral). Yet hypersensitivity to androgens and upregu- lated intracrine androgen synthesis is not the whole story. Although AR amplification can be seen in up to 70% of tumors, there are still CRPC tumors that retain a wild-type AR copy number.17 Similarly, not all CRPC tumors overpro- duce androgens at a local level. Thus, recent work has explored other mechanisms of autonomous AR activation in men with castrate levels of serum testosterone. Perhaps the most intriguing of these observations is that aberrations can occur in the post-transcriptional splicing of AR mRNA, leading to various forms of a truncated AR protein. Although many forms of these splice-variants are maladaptive and lead to a nonfunctioning AR protein, multiple studies have found the presence, in CRPC tumors, of AR proteins that no longer contains an LBD, but retains the DBD and NTD.19,20 Lacking a binding site for ligand, these AR splice-variant mutants can thereby be ren- dered insensitive to manipulation of circulating or local hormone levels. Despite their insensitivity to ligand, by retaining functional DBDs and NTDs these splice variants nonetheless are able to trans- locate autonomously to the cell nucleus and cause transcription of AR regulated genes, resulting in truly ligand-independent cell growth and survival. Cooperation of other signal transduction pathways with the AR, also known as AR transactivation, has also been shown to enhance AR signaling even in the absence of ligand. Specifically, the SRC kinase is a ubiquitous nonreceptor tyrosine kinase involved tumor cell proliferation, survival, and migration, and has been shown to both be upregulated in CRPC cell lines and to cooperate with the AR to enhance AR signaling.21 Importantly, the SRC kinase can be in- hibited with available agents,22,23 and in preclinical models inhibition of SRC appears to decrease AR transactivation and downstream AR signaling.24 Other proteins, such as the cAMP dependent protein kinase (PKA), interleukin-6, and epidermal growth factor, have been implicated in AR transactivation in the absence of ligand through interaction with the AR-NTD.25–27 To what degree these pathways enhance AR signaling in the absence of ligand in patients, and whether it is possible to identify the emergence of these pathways in patients in real time to target this pathway, is controversial and the subject of current research. Nonetheless, this work points to another druggable nonandrogen-mediated mechanism of AR activation.

The understanding that the AR can lose sensi- tivity to circulating androgens through the devel- opment of AR splice variants or through AR transactivation has guided the development of novel AR inhibitors with mechanisms that go beyond impairing ligand-receptor interactions, but rather focus on preventing the AR from reach- ing target sequences in the cell nucleus. Enzaluta- mide (MDV-3100), ARN-509, and EPI-100 are examples of this new class of and are discussed in further detail later in this article.

To best understand the new agents that have been recently approved or are in clinical develop- ment, it is best to think about these agents as a class. This review, therefore, first explores agents that “indirectly” target the AR by inhibiting androgenic ligand production, then focuses on agents that “directly” inhibit interactions between ligand and the AR LBD, and last focuses on agents that impair the interaction of the AR with target sequences in the DNA.

ADT can be accomplished either through bilateral orchiectomy or medically through the use of gonadotropin-releasing hormone (GnRH) therapy. The GnRH agonists leuprolide (Lupron), goserelin (Zoladex), and triptorelin (Trelstar) are synthetic GnRH analogs that are more than 100 times more potent than natural GnRH and less suscep- tible to enzymatic degradation.28 Binding of these agents to receptors in the pituitary gland causes a burst of LHRH release and an initial rise in testos- terone production by testicular Leydig cells. Persistent GnRH stimulation over time, however, leads to a downregulation in pituitary GnRH recep- tors, leading to declines in LHRH secretion and compensatory falls in testicular testosterone pro- duction to castrate levels.

Because GnRH agonists cause an initial serum testosterone flare, novel GnRH antagonists, which avoid raising serum testosterone levels, have been developed. Degarelix (Firmagon) was approved by the Food and Drug Administration (FDA) in 2008 and works by inhibiting the interac- tion of endogenous GnRH with receptors on pituitary gonadotropin-producing cells. Although useful for patients with newly diagnosed widespread metastatic disease in whom a testos- terone flare should be avoided, the higher inci- dence of local injection site reactions (40% vs <1% for men receiving leuprolide) and the need for monthly injections make long-term treat- ment with this agent less desirable for most practictioners. Regardless of the method, ADT to achieve a serum testosterone level lower than 50 ng/dL is the mainstay of the treatment of metastatic pros- tate cancer, and is beneficial when combined with radiation therapy for patients with high-risk localized disease.30–32 Recent work has shown that the degree of AR suppression achieved by initial ADT, as measured by serum PSA produc- tion, is significantly correlated with the duration of response to hormone therapy. In a study of men receiving ADT for metastatic disease, a time to nadir PSA of less than 6 months was associated with shorter overall survival on univariate anal- ysis.12 Similarly, the median survival for men with a PSA nadir of 0.2 ng/mL or less was 75 months, compared with 44 months for men with a nadir PSA between 0.2 to 4.0 ng/mL, and only 13 months for men who never nadired below 4.0 ng/ mL. These findings support the notion that incom- plete suppression of AR signaling leads to AR- mediated disease growth and faster disease progression. Ketoconazole Ketoconazole is a synthetic oral imidazole anti- fungal designed to disrupt fungal cell membranes through inhibition of ergosterol synthesis. Because of the homology between specific fungal and human enzymes, ketoconazole also impairs an- drogen synthesis in humans, specifically though inhibition of CYP51A, CYP11A1, CYP11B1, CYP11B2, CYP17, and CYP19.33 Because it also suppresses mineralocorticoid and glucocorticoid synthesis, ketoconazole is given with a replace- ment dose of corticosteroid, usually oral hydrocor- tisone. In the largest randomized study to date, ketoconazole was given to men with CRPC at the time of AAWD.14 PSA responses were ob- served in 32% of patients taking ketoconazole compared with 10% of men undergoing AAWD alone (P<.001), with twice as many patients having objective responses in the ketoconazole arm. Although crossover of patients randomized to AAWD alone to ketoconazole likely obscured any overall survival benefit, 2 important observations to come out of this study were that patients who had a more than 50% PSA decline had a 41-month survival, compared with 13 months in those who did not (P<.001), and that patients with high base- line circulating androstenedione levels were more likely to benefit from therapy than those with low circulating levels.34 Both of these observations suggest that androgens remain important even after the development of castration-resistance, and show that decreasing the levels of other androgenic ligands in tumors still reliant on ligand-receptor stimulation can greatly slow the growth of the CRPC. Whether baseline circulating androgen levels can be used to identify a popula- tion of patients with CRPC more likely to benefit from ketoconazole is still the subject of debate. Abiraterone Acetate The recognition that inhibition of androgen syn- thesis by ketoconazole could result in both PSA and sustained objective responses led the way for the search for better inhibitors of androgen synthesis. Abiraterone acetate is the prodrug of abiraterone, a potent and selective inhibitor of CYP17 (17alpha-hydroxylase/C17,20-lyase), an enzyme that catalyzes key steps in the synthetic pathway of androgens (Figs. 2 and 3). Abiraterone acetate plus prednisone was tested against placebo plus prednisone in a randomized Phase III study of 1195 men with metastatic CRPC who had previously received docetaxel. After a median follow-up of 12.8 months, a statistically significant survival benefit was observed in the abiraterone/ prednisone arm with median survival of 14.8 months in this arm, compared with 10.9 months in the placebo/prednisone arm (hazard ratio, 0.65; P<.001).35 This study was the first ever to show an overall survival benefit to a “secondary” hormonal therapy, as well as the first ever to show a survival benefit for patients with docetaxel-treated CRPC. With the results of this study, abiraterone acetate received FDA approval in 2011 and has become a standard of care for men with docetaxel-treated CRPC. Fig. 2. Androgen synthesis cascade. CYP17A1 is inhibited by abiraterone, orteronel, and galeterone. Blockade of downstream synthesis is hypothesized to deprive tumors of androgenic ligand and thereby impair tumor growth. Fig. 3. Molecular structure of 6 novel agents that target the AR. A second randomized, placebo-controlled Phase III study of abiraterone acetate in men with doce- taxel-na¨ıve CRPC with coprimary end points of radiographic progression-free survival (rPFS) and overall survival (OS) was unblinded in March 2011 after the second of 3 pre-planned interim analyses performed after 43% of events revealed a statisti- cally significant improvement in rPFS. rPFS for patients treated with placebo plus prednisone was 8.3 months, compared with an approximate doubling for patients treated with abiraterone plus prednisone. Additionally, a trend to improvement in overall survival was observed.36 Based on these data, it is possible that abiraterone will become an option for all men with metastatic CRPC, regardless of prior docetaxel use. Inhibition of CYP17 blocks androgen and cor- tisol production; however, does not impair miner- alocorticoid synthesis. Reductions in cortisol levels are sensed by the pituitary, which responds by increasing serum adrenocorticotropic hormone (ACTH) levels; as abiraterone blocks further cor- tisol and androgen synthesis, the net effect of increased ACTH is to increase mineralocorticoid synthesis. Thus, most side effects of abiraterone are a result of mineralocorticoid excess, with any grade of hypertension, hyperkalemia, and fluid retention observed in 10%, 17%, and 31% of patients, respectively, treated on the post- docetaxel study. Data from early-phase testing showed that the addition of prednisone, 5 mg twice a day, can abrogate many of these effects, and therefore prednisone should be given concur- rently with abiraterone therapy. Although rare, Grade 3 and 4 elevations in liver enzymes have also been observed, and frequent liver function test monitoring, especially at the initiation of therapy, is recommended. Work is under way to better understand which patients are likely to respond to abiraterone. In accordance with the mechanism of action of abir- aterone, higher baseline tumor CYP17 expression and higher baseline circulating androgen levels both correlate with response to therapy.37 This finding reinforces the idea that patients harboring tumors that still depend on AR signaling stand to benefit from ligand-targeted therapy. Similarly, how resistance to abiraterone develops is still unknown; whether resistance is due to upregula- tion of androgen synthesis by the host adrenal gland, upregulation of intracrine androgen synthesis within tumors, impairment of abiraterone transport within cancer cells, or evolution of the AR to a ligand-independent state will need to be addressed in future studies. Similar to abiraterone acetate, orteronel is a ratio- nally designed inhibitor of CYP17 with increased selectivity for inhibition of the 17,20-lyase activity over the 17-hydroxylase activity of the enzyme (see Figs. 2 and 3). The differential selectivity has been hypothesized to result in less overall mineral- ocorticoid toxicity and has allowed for the omis- sion of concurrent replacement steroids in early clinical trials. Doses above 300 mg twice daily, however, do appear to suppress cortisol synthesis and therefore concomitant prednisone has been incorporated in the ongoing studies. In a phase I/II study, 26 patients received ortero- nel in dosages ranging between 200 and 600 mg twice daily with or without concurrent prednisone, and an additional 65 received 400 mg twice a day in the dose-expansion portion.38 All patients who received more than 300 mg twice a day in the Phase I study had PSA declines, including 12 patients with a more than 50% PSA decline and 4 patients with a greater than 90% decline. Efficacy was similar in the Phase II portion, with 54% of patients achieving a more than 50% PSA response by week 12. The most common treatment-related adverse events were fatigue, nausea, constipation, headache, and diarrhea, and although Grade 3 hypokalemia, hyponatremia, and hyperglycemia was seen in 2 patients each, there were no dose- limiting toxicities. Consistent with preclinical data, both testosterone and dehydroepiandrostenedione sulfate (DHEA-S) decreased dramatically, and blunted responses to ACTH stimulation were observed in patients receiving more than 300 mg twice a day dosing, indicative of impaired cortisol synthesis in patients not receiving concurrent prednisone. Based on these results, 2 concurrent Phase III studies are under way exploring the efficacy of orteronel plus prednisone versus prednisone alone in men with either chemotherapy-naı¨ve or docetaxel-treated metastatic CRPC. The primary end point of these studies is overall survival, with a co-primary end point of rPFS in the chemo- therapy-naı¨ve study. If positive, orteronel plus prednisone would represent an alternative to abir- aterone or ketoconazole to impair androgen synthesis for men with metastatic CRPC. Although there are data to suggest that abiraterone is effec- tive in some men who have previously received ketoconazole,39 it is unknown if patients who have developed disease progression while on ketoconazole or abiraterone will respond to orteronel. Galeterone (TOK-001) Galeterone is another inhibitor of CYP17. In preclinical testing, it was shown to have a higher specificity for CYP17 inhibition, with a half- maximal inhibitory concentration (IC50) of 300 nM compared with 800 nM for abiraterone (see Figs. 2 and 3).40 In addition to its activity against CYP17, it also competitively blocks androgen binding at the AR and downregulates AR expres- sion in cell lines, thus functioning in some respects as both an indirect and a direct inhibitor of the AR.40 Importantly, galeterone retains activity in cell lines resistant to bicalutamide expressing higher levels of AR. In the phase I ARMOR-1 study, 49 patients with chemotherapy-naı¨ve nonmetastatic CRPC received single or split daily dosages ranging from 650 mg to 2600 mg for a minimum of 12 weeks without concurrent prednisone.42 PSA reductions of 30% or greater were seen in 49% of patients, with 22% of patients having a 50% or greater decline in PSA. The most common side effects were fatigue, liver function test (LFT) abnormalities, pruritis, nausea, and diarrhea. Although Grades 2 and 3 LFT abnormalities were observed, most were as- ymptomatic and resolved with discontinuation of the drug. Six of 7 patients holding drug for LFT abnormalities were successfully rechallenged with galeterone without recurrent grade 3 LFT eleva- tions. One case of grade 4 rhabdomyolysis was observed in a patient taking concurrent simvastatin; however, no maximum tolerated dose was reached. A Phase II study is expected to open in late 2012. The nonsteroidal antiandrogens bicalutamide, flu- tamide, and nilutamide block androgen-mediated signaling by binding to the AR LBD. Addition of an antiandrogen at the time of CRPC in men treated with androgen deprivation alone can lower PSA by 50% or more in more than a third of patients, and response to an antiandrogen in this setting has been associated with improved cause-specific survival.43 Although several studies of high-dose bicalutamide monotherapy have been performed in men with CRPC, PSA declines of 50% or more occur in only approximately one- quarter of patients,44–46 and in light of a number of new therapies, this strategy is not commonly used. There are no data supporting the superiority of one antiandrogen over another; however, bicalu- tamide has an increased affinity for both the wild- type and mutated AR,47 and therefore may work in cases in which AR antagonism with flutamide have failed. Similarly nilutamide has been shown to induce PSA declines in men who have developed resistance to AR inhibition with flutamide or bicalutamide. Long-term treatment with any of the nonste- roidal antiandrogens may select for cells with AR mutations that allow for paradoxic stimulation of the AR by the antiandrogen. Stopping the antian- drogen in men with a rising PSA, termed AAWD, can result in PSA declines in between 10% and 20% of men.14,49 Withdrawal responses should be seen within 4 weeks following discontinuation of flutamide or nilutamide, and up to 6 weeks following discontinuation of bicalutamide. The recognition that persistent AR signaling is still important at the time of castration resistance led to the search for more potent inhibitors of the AR, including the search for compounds that could inhibit binding of the AR to nuclear response elements (NREs) and thereby act independently of the LBD. One of the most significant findings came from the identification of enzalutamide, a compound that has a more than fivefold greater affinity for the AR than bicalutamide (see Fig. 3).50 Enzalutamide was subsequently shown to both impair AR nuclear translocation and induce a con- formational change in the AR that impairs DNA binding and cofactor recruitment. Promising results from Phase I and II studies led the way for the Phase III study, a double-blinded random- ized study of 1199 men with CRPC previously treated with docetaxel. Patients were randomized 2:1 to receive enzalutamide or placebo, and continuation of corticosteroids was allowed but not required. The study was unblinded after a pre-planned interim analysis after 520 deaths showed a significant overall survival benefit (P<.001; hazard ratio 0.631), with median overall survival of 18.4 months in the enzalutamide arm compared with 13.6 months in the placebo.21 Secondary end points, including rPFS, soft tissue response rate, and time to PSA progression, all favored enzalutamide. Impressively, 54% of patients treated experienced a more than 50% PSA decline compared with 1.5% of placebo- treated patients and 25% of patients treated expe- rienced a more than 90% PSA decline compared with 1% of placebo-treated patients. Improvement in quality of life was significantly higher as assessed by the Functional Assessment of Cancer Therapy – Prostate (FACT-P) questionnaire in enzalutamide- treated patients compared with those treated with placebo (43.2% vs 18.3%, P<.0001). Common side effects included fatigue, diarrhea, and hot flashes. Seizures, which had been a concern in the early-phase testing were reported in 5 patients (<1%) treated with enzalutamide. Enzalutamide is currently being evaluated in a Phase III study of men with chemotherapy-naı¨ve metastatic CRPC. This study has enrolled 1680 patients and has co-primary end points of overall survival and progression-free survival. Results of this study and a decision regarding FDA approval of enzalutamide are expected in late 2012 or early 2013. ARN-509 ARN-509 is a structural analog of enzalutamide, which has been shown to have similar in vitro activity, but greater in vivo activity (see Fig. 3).51 ARN-509 binds the AR with a 7- to 10-fold greater affinity than bicalutamide and specifically inhibits the growth of cells overexpressing the androgen receptor. Similar to enzalutamide, ARN-509 im- pairs AR nuclear localization and binding to NREs, and unlike bicalutamide has no intrinsic AR-agonist activity. ARN-509 showed improved efficacy in preclinical models at lower steady- state plasma concentrations than enzalutamide, implying potentially a higher therapeutic index. ARN-509 is currently being evaluated in Phase I and Phase II studies in multiple different CRPC populations, including in men with nonmetastatic CRPC, as well as in men previously treated with chemotherapy and/or abiraterone acetate. EPI-001 The clinical efficacy of enzalutamide supports the hypothesis that inhibition of interaction between the AR and AREs in the nucleus can reduce PSA and slow disease growth in men with CRPC. Simi- larly, there is increasing evidence that AR-splice variants and transactivated ARs still rely on intact AR DBDs and AR NTDs for function in the absence of ligand. Therefore, it is hypothesized that pure inhibitors of the DBD or NTD should be able to block interaction of the AR with nuclear response elements regardless of the presence of ligand. This hypothesis led to the identification of the molecule EPI-001 though a screen of extracts from marine sponges looking specifically for mole- cules that could block the AR-NTD. EPI-001 was found to inhibit AR activity in vitro regardless of the presence of ligand, both in cells bearing wild- type AR as well as in those with a constitutively active AR splice variant lacking an AR LBD (see Fig. 3).52 EPI-001 blocks the expression of TMPRSS2, PSA, and other AR-mediated mRNAs though inhibition of binding of the AR to nuclear response elements in the promoters of these genes. EPI-001 is specific for the AR and does not inhibit the progesterone or glucocorticoid receptors. Importantly, EPI-001 does not inhibit ligand binding and is thought to work exclusively through inhibition of the AR-NTD. In mouse models, EPI-001 decreases tumor growth and reduces PSA expression without significant toxicity, including in castrated mice bearing castration-resistant LNCaP xenografts. Phase I–II clinical trials with EPI-001 are currently in planning, including combinatorial ap- proaches with androgen synthesis inhibitors and other novel AR inhibitors. SUMMARY Much has been learned about the pivotal role of the AR in the 701 years since androgens were first identified as the major drivers of prostate cancer. Recent work has shown that the vast majority of castration-resistant tumors still rely on AR activity and recent clinical trials using agents that either directly or indirectly target the AR have shown clear efficacy. Understanding of the molecular structure and function of the AR has allowed for the development of therapies that block necessary and specific receptor domains. Given the clinical efficacy of abiraterone acetate, orteronel, and galeterone, it seems likely that inhibi- tion of AR ligand production is sufficient in most tumors to slow AR signaling through deprivation of ligand. None of these therapies are curative, however, and more work will need to be performed to understand the exact mechanisms of resistance to these agents. It is possible that multiple different mechanisms may be at work, including but not limited to selection for cells capable of further AR amplification, the emergence of cells bearing constitutively active AR splice variants, and selec- tion for cells capable of ligand-independent AR transactivation. Enzalutamide, ARN-509, and EPI-001 represent an exciting new class of agents that are capable of targeting the AR regardless of the presence of ligand, and are able to impair downstream AR functions through inhibition of interactions bet- ween the AR and nuclear response elements. Beyond establishing efficacy and safety of each of these agents, further research will be needed to better understand the optimal sequence of use of these novel agents and to better evaluate whether combinatorial approaches will be more effective at suppressing AR-mediated cell growth than sequen- tial therapy. Will sequential use of the novel androgen synthesis inhibitors have clinical benefit, or will resistance to one predict resistance to the entire class? Similarly, will agents like abiraterone have efficacy in cells resistant to direct AR inhibi- tors, such as enzalutamide? Finally, will resistance to AR-targeted therapy involve the emergence of pathways that are completely independent of the AR, and if so, will we be able to identify and target these mechanisms? We eagerly await the answers to these questions. REFERENCES 1. Huggins C, Hodges CV. Studies on prostatic cancer. I. 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