Putting the brakes on continued androgen receptor signaling in castration-resistant prostate cancer

Molecular and Cellular Endocrinology 360 (2012) 68–75

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Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce

Review

Putting the brakes on continued androgen receptor signaling in castration-resistant prostate cancer Andrew Eichholz ⇑, Roberta Ferraldeschi, Gerhardt Attard, Johann S. de Bono Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, United Kingdom

a r t i c l e

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Article history: Available online 1 October 2011 Keywords: Abiraterone Orteronel (TAK-700) VN/124-1 (TOK-001) MDV3100 Heat shock proteins OGX-427

a b s t r a c t Patients with advanced prostate cancer initially respond very well to medical or surgical castration. Despite a good initial response, the disease progresses to a castration-resistant state. Castration-resistant prostate cancer (CRPC) remains addicted to androgen receptor signaling. The addition of conventional anti-androgen agents, such as bicalutamide, only provides a transient benefit. This has led to a search for further drug targets. Cytochrome P450 17 (CYP17) is an enzyme that is vital for the adrenal biosynthesis of androgens. The CYP17 inhibitor abiraterone acetate has a proven benefit in a phase III randomized trial and other CYP17 inhibitors are currently being evaluated. The novel antiandrogen MDV3100 is a small molecule androgen receptor antagonist with promising activity. Heat shock proteins (HSPs) bind to the androgen receptor and modify its activity. Several HSP inhibitors are under evaluation in clinical trials. This review explores the role of CYP17 inhibitors, MDV3100, and HSP inhibitors. Ó 2011 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2.

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4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CYP17 inhibition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Abiraterone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Preclinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Orteronel (TAK-700) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Pre-clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. TOK-001 (VN/124-1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. Preclinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. Clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Resistance to CYP17 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Androgen receptor inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Conventional antiandrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. MDV3100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Preclinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Resistance to antiandrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Heat shock protein modulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Preclinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

69 70 70 70 70 71 71 71 71 71 71 71 71 72 72 72 72 72 73 73 73

Abbreviations: AAG, 17-N-allylamino-17-demethoxygeldanamycin; ACTH, adrenocorticotropic hormone; AR, androgen receptor; cAMP, cyclic adenosine monophosphate; CREB, cAMP response element-binding; CRPC, castration-resistant prostate cancer; CYP, cytochrome P450; DHEA, dehydroepiandrosterone; EYFP, enhanced yellow fluorescent protein; GnRH, gonadotropin-releasing hormone; HSF, heat shock factor; HSP, heat shock protein; LH, luteinizing hormone; MAPK, mitogen-activated protein kinase; NCoR, nuclear hormone receptor corepressor; PTEN, phosphatase and tensin homolog; siRNA, small interfering RNA; SMRT, silencing mediator of retinoid. ⇑ Corresponding author. Address: Male Urological Cancer Research Centre, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, United Kingdom. Tel.: +44 20 8722 4366; fax: +44 20 8722 4084. E-mail address: [email protected] (A. Eichholz). 0303-7207/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2011.09.038

A. Eichholz et al. / Molecular and Cellular Endocrinology 360 (2012) 68–75

5.

4.3. Resistance to HSP inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary and future directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The treatment of advanced prostate cancer poses a significant challenge. Seventy years ago, Huggins and Hodges first demonstrated that prostate cancer responds to androgens. Androgen deprivation could cause a decrease in serum acid phosphatase (suggesting regression of bony metastases) whilst administering injections of androgens had the opposite effect (Huggins and Hodges, 1941).

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73 74 74 74

Testosterone and other androgens are produced primarily in the testes. This production is under endocrine control. The hypothalamus produces gonadotropin-releasing hormone (GnRH) that stimulates the pituitary gland to produce luteinizing hormone (LH). This in turn causes the Leydig cells of the testes to produce testosterone and dihydrotestosterone from cholesterol via a biosynthetic pathway (Fig. 1). Cholesterol is converted to progestagens that are then converted to the androgens dehydroepiandrosterone (DHEA), DHEA-sulfate and androstenedione. Testosterone is produced by conversion from DHEA or androstenedione by the action of

Cholesterol

Progesterone

Aldosterone

17α-hydroxyprogesterone

Cortisol

Androstenedione

Testosterone

Pregnenolone

CYP17: 17α-hydroxylase

17α-hydroxypregnenolone CYP17: C17,20-lyase

DHEA

DHEAsulfate

Dihydrotestosterone

Fig. 1. Androgen biosynthesis pathway. CYP17 has 17a-hydroxylase and C17,20-lyase activity which is necessary for androgen production. CYP, cytochrome P450; DHEA, dehydroepiandrosterone.

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17-beta-hydroxysteroid dehydrogenase. Testosterone can be converted to the more potent androgen dihydrotestosterone by the action of 5-alpha reductase. The production of testosterone decreases as men get older. In the European Male Aging Study, a study of 3220 men aged 40–79 years, serum total testosterone concentrations fell by 0.4% per year and the free testosterone concentration fell by 1.3% per year (Wu et al., 2008). The importance of non-testicular sources therefore increases as men get older. The adrenal glands convert progestagens to aldosterone and cortisol. The adrenal glands can also convert progestagens to the androgens DHEA, DHEA-sulfate and androstenedione. It has also been shown that tumor cells are capable of producing their own androgens with conversion of DHEA and androstenedione to testosterone and dihydrotestosterone (Mohler et al., 2004; Nishiyama et al., 2004; Mostaghel et al., 2007). Advanced prostate cancer is treated primarily with androgen deprivation therapy. This can be in the form of medical or surgical castration. Although prostate cancer will initially respond very well to this treatment, patients will generally develop castrationresistant prostate cancer (CRPC) within 12–33 months (Hellerstedt and Pienta, 2002). This is usually accompanied by a rise in PSA. Even when resistance to androgen deprivation therapy develops, further hormonal manipulations may induce a further response. This continued response to hormonal therapy is explained by the finding that there is a 6-fold higher level of androgen receptor in CRPC compared to that found in hormone-sensitive disease (Linja et al., 2001) suggesting that the disease remains driven by AR signaling. Following the development of castration-resistant disease, medical or surgical castration can be combined with antiandrogen therapy (e.g. flutamide or bicalutamide). When the disease progresses again, antiandrogen withdrawal results in a PSA response in 10–30% of patients (Scher and Kelly, 1993; Small and Srinivas, 1995; Figg et al., 1995; Small et al., 2004). Corticosteroids (e.g. prednisolone, dexamethasone or hydrocortisone) cause a decrease in the pituitary production of adrenocorticotropic hormone (ACTH) that leads to reduced production of testicular and adrenal androgens (Kamischke et al., 1998). In a group of 37 men with CRPC treated with relatively low dose prednisone (7.5–10 mg daily), there was a decrease in serum androstenedione and DHEA-sulfate in more than 50% of patients (Tannock et al., 1989). Chemotherapy can also be used to treat patients with CRPC. Docetaxel with low dose prednisone has a proven survival benefit in CRPC patients (Tannock et al., 2004). Docetaxel inhibits microtubule depolymerization causing apoptotic cell death but recent evidence also suggests that docetaxel can also decrease expression of the androgen receptor in prostate cancer cell lines (Kuroda et al., 2009; Gan et al., 2009). Patients whose cancer progresses following docetaxel may then benefit from cabazitaxel. The TROPIC trial has shown that this taxane, which has been developed for use in docetaxel resistance, confers a survival benefit (de Bono et al., 2010b). These agents produce a temporary improvement in the patient’s condition and new agents are needed to help this patient group. This need has prompted the development of novel drugs that act on androgen synthesis, antagonize the androgen receptor directly, and that interact with heat shock proteins.

2. CYP17 inhibition The cytochrome P450 enzyme CYP17 is important in the production of androgenic steroids and estrogens. The 17-alpha-hydroxylase action catalyzes production of the precursors of cortisol. The C17,20-lyase activity then leads to the produc-

tion of sex steroid precursors (Fig. 1). Ketoconazole has previously been used as a treatment in CRPC patients as it inhibits CYP17. Unfortunately, ketoconazole causes non-specific inhibition of many cytochrome P450 enzymes and therefore it can affect the metabolism of the patient’s concomitant medications. Ketoconazole has a number of toxic side effects including fatigue, liver toxicity and neurological toxicity (Small et al., 2004). This has limited the use of ketoconazole. Researchers have been searching for and developing more specific CYP17 inhibitors with less toxicity. 2.1. Abiraterone Abiraterone is a high affinity, irreversible inhibitor of both the 17,20-lyase and 17-alpha-hydroxylase activity of CYP17. The prodrug abiraterone acetate is well absorbed after oral administration and deacetylation then occurs rapidly in the liver. 2.1.1. Preclinical studies Preclinical studies in mice showed that abiraterone lowered serum testosterone to castrate levels despite a compensatory three- to four-fold increase in luteinizing hormone (LH) (Barrie et al., 1994). Abiraterone was tested as an alternative to gonadotropinreleasing hormone (GnRH) analogues. Unfortunately, in human prostate cancer patients, sustained suppression of testosterone was not achieved due to the resulting increase in LH (O’Donnell et al., 2004). Abiraterone has therefore been developed for use with concomitant GnRH analogues in the setting of castration-resistant prostate cancer. 2.1.2. Clinical studies In a phase I/II study of abiraterone involving 54 castrate, chemotherapy-naïve patients, a reduction in serum PSA of 50% or greater was observed in 28 (67%) patients and reductions of 90% or more were seen in eight (19%) patients. Decreases in circulating tumor cells (CTCs) and radiologic responses were also observed (Attard et al., 2009a). In a further phase I study involving chemotherapynaïve patients, declines in PSA of 50% or greater were seen in 18 (55%) of 33 patients including nine (47%) of 19 patients who had prior ketoconazole therapy (Ryan et al., 2010). There is a randomized, placebo-controlled phase III study in progress that is examining the role of abiraterone in chemotherapy-naïve patients with metastatic CRPC. The primary outcome measures are overall survival and progression-free survival. The aim is to enroll 1000 patients. This study started in April 2009 and it is estimated that final data collection for the primary outcome measures will be completed by April 2011 (ClinicalTrials.gov identifier: NCT00887198, accessed February 24th, 2011). Abiraterone has also been studied in the setting of castrationresistant prostate cancer previously treated with docetaxel chemotherapy. A phase II study with this patient group enrolled 47 patients. A reduction in PSA of 50% or more was shown in 24 (51%) patients. Circulating tumor cells (CTCs) were enumerated in 34 patients. Of these 34 patients, 27 (79%) had at least 5 CTCs at baseline. There was a reduction from at least 5 to less than 5 CTCs in 12 (41%) of 27 patients. There was a 30% or greater decline in CTCs in 18 (67%) of 27 patients after starting treatment with abiraterone (Reid et al., 2010). A further phase II study evaluated abiraterone in combination with prednisone in patients previously treated with docetaxel chemotherapy. A decline in PSA of 50% or more was demonstrated in 22 (36%) of 58 patients including seven (26%) of 27 patients previously treated with ketoconazole. Little mineralocorticoid-related toxicity was encountered due to the concomitant use of low-dose prednisone (Danila et al., 2010).

A. Eichholz et al. / Molecular and Cellular Endocrinology 360 (2012) 68–75

A randomized, placebo-controlled phase III study using abiraterone with relatively low dose prednisone (10 mg daily) in metastatic CRPC patients has been completed. The results of this trial have been reported at the European Society for Medical Oncology (ESMO) 2010 conference. All patients were previously treated with docetaxel chemotherapy. This study shows that abiraterone improves overall survival which was 14.8 months in the abiraterone-treated patients compared to 10.9 months in the placebo group, HR 0.65 (95% CI 0.54–0.77, P < 0.0001). There were also improvements in time to PSA progression, radiographic progression-free survival, and PSA response rate. The main toxicities noted were fluid retention, hypokalemia, hypertension, liver function test abnormalities and cardiac disorders (de Bono et al., 2010a). 2.2. Orteronel (TAK-700) Orteronel is a non-steroidal imidazole inhibitor of CYP17. Unlike abiraterone, this agent does not have a similar structure to progesterone. Orteronel is being developed by Takeda Pharmaceutical Company Limited and it was previously known by the name TAK-700. 2.2.1. Pre-clinical studies Orteronel was designed using a substrate mimic strategy. This strategy identified groups of imidazoles that were potent C17,20-lyase inhibitors of rat and human enzymes and that caused in vivo suppression of testosterone synthesis in rats. Many analogues were developed and evaluated. The best results were achieved with incorporation of a hydroxy group and an isopropyl group. Orteronel was found to be a more than 260-fold potent inhibitor over 11-beta-hydroxylase for the C17,20-lyase in rats. This compound also demonstrated potent suppression of testosterone synthesis in monkeys after administration of a single oral dose (Matsunaga et al., 2004). 2.2.2. Clinical studies Orteronel has been studied in a phase I/II open label study in patients with metastatic CRPC. The phase I results were presented at the 2010 ASCO Genitourinary Cancers Symposium. Twenty-six patients were treated with orteronel at five different dose levels and a further five patients also received low dose prednisone. All patients treated with an orteronel dose of at least 300 mg twice daily achieved a decrease in PSA. There were no dose-limiting toxicities although 23 out of 26 patients experienced at least one drug-related adverse event. The most common adverse events were fatigue, nausea, constipation, anorexia and vomiting. Patients treated with 400 mg twice daily had a significant decrease in testosterone and DHEA-sulfate and this is the dose that is being used in the phase II portion of the study (Dreicer et al., 2010). Randomized, double-blind phase III studies started in October and November 2010. The first is a study examining orteronel plus prednisone versus placebo plus prednisone in men with metastatic CRPC who are chemotherapy-naïve (ClinicalTrials.gov identifier: NCT01193244). The second study is examining the same treatments in patients that have progressed following taxane-based chemotherapy (ClinicalTrials.gov identifier: NCT01193257). There is also an open label, phase I/II study in progress evaluating the safety and pharmacokinetics of orteronel in combination with docetaxel and prednisone (ClinicalTrials.gov identifier: NCT01084655).

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inhibits AR. This agent is currently being developed by Tokai Pharmaceuticals. It was previously known by the name VN/124-1. 2.3.1. Preclinical studies Studies in LAPC4 tumor xenografts in severe combined immunodeficient mice showed that TOK-001 causes a marked decrease in AR protein expression, unlike bicalutamide or castration that caused an increase in AR expression. Treatment with TOK-001 in this study caused significant regression of LAPC4 tumors and it was also effective in preventing the formation of new tumors (Vasaitis et al., 2008). An in vitro study with a hormone-refractory prostate cancer cell line (high-passage LNCaP) showed that TOK-001 inhibited proliferation in cells that were no longer sensitive to bicalutamide (Schayowitz et al., 2008). 2.3.2. Clinical studies TOK-001 is currently being evaluated in the ARMOR1 study. This is a phase I/II open label trial in chemotherapy-naïve CRPC patients. The primary outcome measures are the incidence of adverse events in phase I and the proportion of patients with a 50% or greater decrease in PSA from baseline in phase II. The aim is to enroll 50 patients. This study started in October 2009 and it is estimated that final data collection for the primary outcome measures will be completed by July 2011 (ClinicalTrials.gov identifier: NCT00959959, accessed January 4th, 2011). 2.4. Resistance to CYP17 inhibitors Patients on abiraterone eventually progress despite an initial response to therapy. The disease progression is normally accompanied by an increase in PSA. This suggests there is reactivation of AR or other steroid receptor signaling pathways. A study of circulating tumor cells in CRPC patients receiving abiraterone showed that these patients have increased AR gene copy number and loss of the phosphatase and tensin homolog (PTEN) tumor suppressor gene with significant intrapatient heterogeneity but were unable to associate resistance to these genetic changes (Attard et al., 2009b). Corticosterone, aldosterone and progesterone are all increased in patients taking CYP17 inhibitors as they are upstream of the CYP17 blockade. It is possible that these steroidal molecules are causing stimulation of AR. In the phase I/II study of abiraterone in chemotherapy-naïve patients, the addition of dexamethasone 0.5 mg reversed resistance in 25% of patients regardless of prior dexamethasone treatment (Attard et al., 2009a) and it is possible that this response was mediated by reduction of ACTH-mediated stimulation of these steroidal molecules. Abiraterone and TOK-001 have a steroidal backbone and structural similarity to progesterone so it is possible that they could stimulate an altered AR however there have been no reports so far of a withdrawal response to these drugs.There are other possible mechanisms of resistance which are discussed further in Section 3.3. The drugs discussed above all function by decreasing the production of androgenic steroids that act on the androgen receptor. An alternative approach is to target the binding of all ligands to the androgen receptor. 3. Androgen receptor inhibition

2.3. TOK-001 (VN/124-1) Further agents are being developed that inhibit CYP17. One of the most promising of these agents is a 17-benzoimidazole called TOK-001. Apart from inhibiting CYP17, TOK-001 also directly

Antiandrogens are agents that compete with endogenous androgens for the ligand-binding pocket of the androgen receptor. The first antiandrogens to be developed can be subdivided into two groups: steroidal and non-steroidal antiandrogens. The steroidal

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compounds include the progesterone analogue cyproterone acetate. The non-steroidal compounds are flutamide and its derivatives nilutamide and bicalutamide.

bicalutamide and MDV3100. Compared to vehicle-treated mice, bicalutamide slowed tumor growth and MDV3100 treatment led to tumor regression (Tran et al., 2009).

3.1. Conventional antiandrogens

3.2.2. Clinical studies A phase I/II study of MDV3100 involved 140 men with progressive, metastatic CRPC. They had all progressed on at least one prior hormonal therapy and 54% had received chemotherapy. Antitumor effects were noted at all doses used and there was a decrease in serum PSA of 50% or more in 78 (56%) patients (Scher et al., 2010). MDV3100 is currently being evaluated in two placebo-controlled, randomized phase III trials. One of these studies will determine the overall survival benefit in patients with castrationresistant prostate cancer who have been previously treated with docetaxel-based chemotherapy (ClinicalTrials.gov identifier: NCT00974311). The other phase III study is examining the overall survival and progression-free survival benefits in patients with progressive metastatic prostate cancer who have failed androgen deprivation therapy but have not yet received chemotherapy (ClinicalTrials.gov identifier: NCT01212991).

Cyproterone acetate is no longer commonly used as it is a partial AR agonist (Labrie et al., 1987) and there are concerns regarding its safety and efficacy. The Prostate Cancer Trialists’ Collaborative Group meta-analysis in 2000 demonstrated that cyproterone acetate may worsen survival in advanced prostate cancer patients. The 5-year survival for patients treated with androgen deprivation therapy alone was 18.1% and when this was combined with cyproterone acetate, the 5-year survival was 15.4% (Prostate Cancer Trialists’ Collaborative Group, 2000). In contrast to cyproterone acetate, flutamide was initially shown to have no androgenic activity and was therefore acting a pure antiandrogen (Labrie et al., 1987). The 2000 meta-analysis showed a 3% improvement in 5-year overall survival when flutamide or its derivative nilutamide was added to medical or surgical castration. A further flutamide derivative, bicalutamide, was developed. Bicalutamide has a higher affinity for AR, a longer half-life (allowing once daily dosing) and less toxicity, in particular hepatotoxicity. A non-inferiority study of medical castration with either flutamide or bicalutamide showed less toxicity and a trend towards longer median survival in the patients treated with bicalutamide (Schellhammer et al., 1997). Due to these advantages, bicalutamide has become the most popular antiandrogen medication. Bicalutamide is currently commonly used in the treatment of advanced prostate cancer, either alone or in combination with medical or surgical castration. 3.2. MDV3100 MDV3100 is a small molecule AR antagonist. It was discovered from a screen for non-steroidal antiandrogens that retained activity in LNCap, a human prostate cancer cell line with AR gene amplification. MDV3100 binds to the human androgen receptor with high affinity and it is well absorbed after oral administration (Tran et al., 2009). These properties made it an ideal candidate for further investigation. 3.2.1. Preclinical studies MDV3100 blocks the binding of testosterone to the androgen receptor. It has five- to eightfold greater binding affinity for AR than bicalutamide (Tran et al., 2009). MDV3100 reduces translocation of the androgen receptor to the nucleus of the prostate cancer cell. This has been shown in vitro with confocal microscopy in live LNCaP cells with AR that has been tagged with enhanced yellow fluorescent protein (EYFP). The ratio of nuclear to cytoplasmic AR in MDV3100-treated cells was reduced fivefold compared to bicalutamide-treated cells (Tran et al., 2009). MDV3100 inhibits binding of DNA. In LNCaP cell lines overexpressing AR, bicalutamide causes expression of the AR target genes PSA and transmembrane protease serine 2 (TMPRSS2). MDV3100 does not have this effect. Also, bicalutamide activates transcription when AR is fused to the VP16 transactivation domain. MDV3100 does not cause such transcription (Tran et al., 2009). These results suggest that MDV3100 does not have any AR agonist activity in a castration-resistant setting, unlike the partial agonist activity of bicalutamide. MDV3100 was shown to have antitumor activity in xenograft models of CRPC. Castrate male mice with tumors with LNCaP with stably transfected AR were treated with vehicle (used as a control),

3.3. Resistance to antiandrogens It has been well documented that withdrawal of conventional antiandrogens can cause a reduction in PSA. This withdrawal response demonstrates that conventional antiandrogens can act as AR agonists. It is possible that AR gene amplification occurs, causing an increase in AR protein levels. A study of 102 matched, paired hormone-sensitive and hormone-resistant samples from 51 patients examined X chromosome copy number and locus-specific AR gene amplification using fluorescent in situ hybridization. More tumors exhibited AR amplification after the development of hormone resistance (20%) compared to the matched hormone-sensitive samples (2%). The rate of AR gene amplification is too low to fully explain the development of androgen resistance (Edwards et al., 2003). It is possible that there is an increase in AR protein expression without an increase in AR gene amplification. Xenograft models of androgen-dependent and corresponding androgen-independent sublines did not show any difference in AR copy number (Laitinen et al., 2002). Microarray-based profiling of xenograft models has shown that a modest increase in AR mRNA is the only change consistently associated with resistance to antiandrogen treatment. This increase in mRNA converts hormone-sensitive prostate cancer to a hormone-refractory state (Chen et al., 2004). A number of different primary molecular events could occur to cause this increase in mRNA. Studies of AR transcription regulation in the human prostate cancer cell line PC3 suggest that AR is a selfregulating transcription factor causing increased mRNA levels (Grad et al., 1999). Alternatively, increased kinase pathway signaling (e.g. mitogen-activated protein kinase (MAPK), ErbB2 or Ras pathways) could explain this (Chen et al., 2004). Several different forms of androgen receptor mutation can also explain resistance to antiandrogens. Mutations of the ligand-binding domain of the androgen receptors have been shown to produce AR that can be stimulated by a wider range of ligands than usual, e.g. estradiol and DHEA (Elo et al., 1995; Tan et al., 1997). Mutations of the N-terminal domain of AR affect interactions with coregulators including coactivators and corepressors. The coactivators include p300 and cyclic adenosine monophosphate (cAMP) response element-binding (CREB) protein, which link transcriptional molecules to AR, and the p160 family of coactivators, which locally modify chromatin structure. Many corepressors have been shown to impact AR activity including the silencing mediator

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of retinoid and thyroid hormone receptors (SMRT)/nuclear hormone receptor corepressor (NCoR) which serve as docking platforms for histone deacetylases on promoter sites (Bergerat and Ceraline, 2009). Constitutive activation of AR can occur by truncated androgen receptors that have been produced by exon splice variants. These truncated AR molecules have lost their carboxy-terminal end region. They promote the action of endogenous AR-dependent genes in prostate cancer cell lines and in xenograft models (Dehm et al., 2008; Guo et al., 2009). Constitutively active androgen receptor splice variants expressed in CRPC in some human and murine prostate cancer models require a full-length androgen receptor. These constitutively active, ligand-independent splice variants cause anchorageindependent ( in vitro) and castration-resistant ( in vivo) growth. Interestingly, this growth is blocked by MDV3100 or small interfering RNA (siRNA) silencing of full-length AR mRNA. MDV3100 may therefore prove a useful agent to treat patients with constitutively active AR splice variants (Watson et al., 2010). Changes in the expression of co-activators alone may cause resistance to antiandrogen drugs. Overexpression of the coactivators transcriptional intermediary factor 2 (TIF2) and steroid receptor 1 (SRC1) has been shown to cause bicalutamide to act as an AR agonist (Gregory et al., 2001; Feng et al., 2009). The antiandrogen drugs discussed in this section all depend on blocking ligand binding to the androgen receptor. An alternative therapeutic approach is to target downstream signaling by means of modulating heat shock proteins. 4. Heat shock protein modulation Molecular chaperones are involved in the processes of folding, activation, trafficking, and transcriptional activity of most steroid receptors, including AR. A number of chaperone proteins have been identified as being of interest in CRPC, including HSP90 and HSP27. 4.1. Preclinical studies HSP90 is an ATP-dependent chaperone that accounts for the maturation and functional stability of a plethora of proteins termed HSP90 client proteins. HSP90 interacts with several key proteins that are involved in prostate cancer progression, including AR, Src, Raf and Akt (Mahalingam et al., 2009). HSP90 modulation is a particularly attractive therapeutic strategy in CRPC as the inhibition of HSP90 offers the prospect of simultaneously inhibiting (a) multiple kinase-dependent signaling pathways that control cell growth, resistance to apoptosis and post-translational modification of AR; and (b) the stability of AR protein. HSP90 is essential for the maintenance of the functionality of the AR (Vanaja et al., 2002; Georget et al., 2002). In its unbound state, the AR is stabilized in the cytoplasm in a conformation that permits androgen binding by a complex containing several chaperones including HSP70, HSP90, co-chaperones, and tetratricopeptide repeat (TPR)-containing proteins (Prescott and Coetzee, 2006). Androgen binding to the AR induces a conformational change that causes it to dissociate from HSPs complex. This leads to receptor dimerization and translocation to the nucleus (Lee and Chang, 2003). Preclinical observations suggested that CRPC might respond favorably to HSP90 inhibitor therapy (Saporita et al., 2007; Tsui et al., 2008; Solit et al., 2002). HSP27 is a stress-inducible, ATP-independent, cytoprotective chaperone that is now emerging as the key chaperone involved in AR function in the nucleus (Zoubeidi et al., 2007). A feed-forward loop involving cooperative interactions between ligand-activated AR and HSP27 phospho-activation has been demonstrated (Zoube-

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idi et al., 2007). This enhances AR stability, shuttling, and transcriptional activity, thereby increasing prostate cancer cell survival. HSP27 expression is induced by hormone therapy or chemotherapy and inhibits treatment-induced apoptosis through multiple mechanisms (Rocchi et al., 2004, 2005). Recent evidence supports the hypothesis that increased HSP27 after androgen ablation is an adaptive response induced by castration to enhance cell survival and tumor growth and that HSP27 knockdown using an antisense oligonucleotide (OGX-427) or siRNA delays progression postcastration (Zoubeidi et al., 2007). 4.2. Clinical studies The first-in-class HSP90 inhibitor 17-AAG showed promising antitumor properties in preclinical studies. In a phase I trial involving patients with advanced prostate cancer one patient achieved a PSA response (Goetz et al., 2005). However a two-stage phase II trial of 17-AAG in metastatic CRPC patients failed to meet the primary end point of PSA response and was closed prematurely (Heath et al., 2008). Retaspimycin (IPI-504), the hydroquinone hydrochloride derivative of 17-AAG, has been tested as monotherapy in CRPC in a single-arm Phase II trial. One of four patients without bony metastases had a PSA decline of 48% from baseline after 9 cycles of treatment (Oh et al., 2009). A durable response in a patient with advanced prostate cancer was also reported in a phase I trial of Alvespimycin (17-DMAG), a water soluble analog of 17AAG (Pacey et al., 2009). Ganetespib (STA-9090), a potent, second-generation, small-molecule HSP90 inhibitor, is currently being evaluated in a single-arm phase II study in men with CRPC who have received prior docetaxel based therapy. The primary endpoint for this study is progressionfree survival. This trial will involve 51 patients and it is estimated that data collection for the primary endpoint will be completed by March 2013 (ClinicalTrials.gov identifier: NCT01270880, accessed January 24th, 2011). OGX-427, a second generation antisense drug targeting HSP27, has recently advanced into phase II clinical trials for treatment of a variety of cancers including CRPC. OGX-427 was well tolerated as a monotherapy in a phase I trial and demonstrated declines in circulating tumor cells as well as reduction in PSA levels in three patients with CRPC (Hotte et al., 2009). OGX-427 is currently being evaluated in a randomized, open-label phase II study in combination with low dose prednisone in patients with CRPC who have not previously received chemotherapy. The primary endpoint for this study is the proportion of patients without disease progression after 12 weeks. This trial will involve 72 patients and it is estimated that data collection for the primary endpoint will be completed by March 2011 (ClinicalTrials.gov identifier: NCT01120470, accessed January 4th, 2011). 4.3. Resistance to HSP inhibitors HSP90 inhibitors such as geldanamycin induce AR degradation by directly binding to the ATP-binding pocket of HSP90 to inhibit its function (Solit et al., 2002). Nevertheless, the geldanamycin derivative Tanespamycin (17AAG) did not show significant clinical activity in CRPC (Heath et al., 2008). Although inhibitors of HSP90 disrupt chaperone machinery, they can also activate the heat shock response pathway mediated through heat shock factor 1 (HSF1) transcription factor activation (Powers and Workman, 2007). This inadvertent induction of HSPs may attenuate the beneficial effects of HSP90 inhibitors. Studies have found that the expression of HSP27 and HSP72 (Maloney et al., 2007) are increased following HSP90 inhibition and that silencing of HSP27 and HSP72 increases cancer cell sensitivity to 17-AAG treatment (Powers et al., 2008). In

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view of the induction of HSF1-mediated activation of the stress response observed with HSP90 inhibitor treatment, the potential therapeutic benefit of modulating HSF1 or HSP70 has become attractive to overcome the self-limiting mechanism that may attenuate the anticancer effects of HSP90 inhibitors (de Billy et al., 2009; Powers et al., 2010). 5. Summary and future directions Although initially most patients with advanced prostate cancer respond well to androgen deprivation therapy, they will eventually develop castration-resistant disease. Inhibition of CYP17 reduces the production of adrenal and intratumoral androgens. Abiraterone has a proven clinical benefit and it has been shown to improve survival in CRPC. Conventional antiandrogen medications and the novel antiandrogen MDV3100 target the ligand-binding domain of the androgen receptor. Phase III randomized, controlled trials with MDV3100 are in progress. Targeting heat shock proteins provides an alternative therapeutic approach and a phase II trial of OGX427 is on-going. Further research will need to focus on identifying mechanisms of resistance and how to overcome these mechanisms. Furthering our knowledge of androgen receptor signaling will help us to develop the next generation of drugs and consider rational combinations of existing drugs that may act synergistically. Acknowledgements We would like to thank our prostate cancer patients and their carers and Professor Mike Jarman, Dr. Elaine Barrie and Dr. Gerry Potter who first designed and synthesized abiraterone acetate. References Attard, G., Reid, A.H., A’hern, R., Parker, C., Oommen, N.B., Folkerd, E., Messiou, C., Molife, L.R., Maier, G., Thompson, E., Olmos, D., Sinha, R., Lee, G., Dowsett, M., Kaye, S.B., Dearnaley, D., Kheoh, T., Molina, A., De Bono, J.S., 2009a. Selective inhibition of CYP17 with abiraterone acetate is highly active in the treatment of castration-resistant prostate cancer. J. Clin. Oncol. 27, 3742–3748. Attard, G., Swennenhuis, J.F., Olmos, D., Reid, A.H., Vickers, E., A’hern, R., Levink, R., Coumans, F., Moreira, J., Riisnaes, R., Oommen, N.B., Hawche, G., Jameson, C., Thompson, E., Sipkema, R., Carden, C.P., Parker, C., Dearnaley, D., Kaye, S.B., Cooper, C.S., Molina, A., Cox, M.E., Terstappen, L.W., De Bono, J.S., 2009b. Characterization of ERG, AR and PTEN gene status in circulating tumor cells from patients with castration-resistant prostate cancer. Cancer Res. 69, 2912– 2918. Barrie, S.E., Potter, G.A., Goddard, P.M., Haynes, B.P., Dowsett, M., Jarman, M., 1994. Pharmacology of novel steroidal inhibitors of cytochrome P450(17) alpha (17 alpha-hydroxylase/C17–20 lyase). J. Steroid. Biochem. Mol. Biol. 50, 267–273. Bergerat, J.P., Ceraline, J., 2009. Pleiotropic functional properties of androgen receptor mutants in prostate cancer. Hum. Mutat. 30, 145–157. Chen, C.D., Welsbie, D.S., Tran, C., Baek, S.H., Chen, R., Vessella, R., Rosenfeld, M.G., Sawyers, C.L., 2004. Molecular determinants of resistance to antiandrogen therapy. Nat. Med. 10, 33–39. Danila, D.C., Morris, M.J., De Bono, J.S., Ryan, C.J., Denmeade, S.R., Smith, M.R., Taplin, M.E., Bubley, G.J., Kheoh, T., Haqq, C., Molina, A., Anand, A., Koscuiszka, M., Larson, S.M., Schwartz, L.H., Fleisher, M., Scher, H.I., 2010. Phase II multicenter study of abiraterone acetate plus prednisone therapy in patients with docetaxel-treated castration-resistant prostate cancer. J. Clin. Oncol. 28, 1496–1501. De Billy, E., Powers, M.V., Smith, J.R., Workman, P., 2009. Drugging the heat shock factor 1 pathway Exploitation of the critical cancer cell dependence on the guardian of the proteome. Cell Cycle 8, 3806–3808. De Bono, J.S., Logothetis, C.J., Fizazi, K., North, S., Chu, L., Chi, K.N., Kheoh, T., Haqq, C., Molina, A., Scher, H.I., 2010a. Abiraterone acetate (AA) plus low dose prednisone (P) improves overall survival (OS) in patients (PTS) with metastatic castrationresistant prostate cancer (MCRPC) who have progressed after docetaxel-based chemotherapy (CHEMO): Results of COU-AA-301, a randomized double-blind placebo-controlled phase III study. Ann. Oncol., 21, viii2. De Bono, J.S., Oudard, S., Ozguroglu, M., Hansen, S., Machiels, J.P., Kocak, I., Gravis, G., Bodrogi, I., Mackenzie, M.J., Shen, L., Roessner, M., Gupta, S., Sartor, A.O., 2010b. Prednisone plus cabazitaxel or mitoxantrone for metastatic castration-resistant prostate cancer progressing after docetaxel treatment: a randomised open-label trial. Lancet 376, 1147–1154.

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