Androgen receptors beyond prostate cancer: an old marker as a new target.

Androgen receptors (ARs) play a critical role in the development of prostate cancer. Targeting ARs results in important salutary effects in this malignancy. Despite mounting evidence that ARs also participate in the pathogenesis and/or progression of diverse tumors, exploring the impact of hormonal manipulation of these receptors has not been widely pursued beyond prostate cancer. This review describes patterns of AR expression in a spectrum of cancers, and the potential to exploit this knowledge in the clinical therapeutic setting.


INTRODUCTION
Treatments targeting androgen receptors (ARs) have demonstrated efficacy in patients with prostate cancer. Despite a wealth of reports indicating that ARs may contribute to the growth and/or progression of numerous other malignancies, their precise role in cancers beyond those of the prostate is poorly understood. The aim of this review is to illuminate current data in the literature regarding the expression and functional impact of ARs in a range of solid tumors (Table 1), and to discuss the therapeutic implications of AR positivity.

Background
Androgen synthesis and androgen receptors: The goal of androgen deprivation or suppression therapy is to reduce the levels of male hormones (androgens) [1]. The major androgens capable of stimulating the androgen receptors include testosterone and dihydrotestosterone (DHT) [2]. In normal men, androgens are synthesized in the testes (interstitial Leydig cells) and adrenals. Testosterone is the principal circulating androgen in adult men, with >95% being of testicular origin [3] (under the regulatory control of the hypothalamic / pituitary axis), while the remainder originates from peripheral conversion of weaker precursors of adrenal origin: dehydroepiandrosterone (DHEA), DHEA sulfate, and androstenedione [4]. Adrenal androgens are under adrenocorticotropin hormone (ACTH) control [5]. The adrenals and peripheral conversion/synthesis of androgens do not make a major contribution to androgen effects in the normal man, but may become a significant residual source of AR stimulation in men undergoing androgen deprivation/suppression therapy. Skin, fat, liver, and urogenital systems are important peripheral sites of androgen production [6,7].
Prostate cancer as a model for androgen deprivation therapy: Androgens stimulate AR+ cells to proliferate. Since the vast majority of prostate cancers are AR+, androgen deprivation (suppression) treatment was a rational approach for this malignancy, and has become a cornerstone of treatment for advanced prostate cancer ( Table 2) [8]. Androgen deprivation can shrink or stabilize prostate tumors, but is not generally curative. There are protean ways to suppress androgen levels. These include: • Surgical removal of the testes (castration or orchiectomy).
• Luteinizing hormone-releasing hormone (LHRH) analogs (reversible chemical castration). These drugs halt testosterone production by the testicles. www.impactjournals.com/oncotarget Examples of these agents include leuprolide, goserelin, triptorelin, and histrelin [9]. When LHRH analogs are initiated, testosterone levels rise before falling, a phenomenon known as a "clinical flare" [10]. The flare can cause bone pain or spinal cord compression if tumors are present in these areas, as the tumors may have a shortlived growth spurt. The flare can be prevented by giving anti-androgens for a few weeks when LHRN analogs are started.
• Luteinizing hormone-releasing hormone (LHRH) antagonists. LHRH antagonists such as degarelix work like LHRH agonists, but they reduce testosterone levels more rapidly and do not induce a tumor flare like the LHRH agonists do [11].
• Anti-androgens. Anti-androgens impede the body's ability to use androgens, usually by blocking the androgen receptor. Even after orchiectomy or during treatment with LHRH analogs, the adrenal glands still synthesize small amounts of androgens. Drugs of this type include flutamide, bicalutamide, and nilutamide [12].
• Other androgen-suppressing drugs. Estrogens were once the main alternative to orchiectomy for men with advanced prostate cancer. Because of their toxic effects [13] (including blood clots and breast enlargement), oral estrogens have been largely replaced by LHRH analogs and anti-androgens [14]. It has been suggested that parenteral estrogen may avoid long term toxicity [15]. Ketoconazole, first used for treating fungal infections, blocks production of androgens. It offers a quick way to lower testosterone levels, and it is used in countries where abiraterone has yet to be approved [16]. Abiraterone inhibits 17 α-hydroxylase/C17,20 lyase (CYP17A1), an enzyme that is expressed in testicular, adrenal, and prostatic tumor tissues. CYP17 catalyzes key reactions in the testosterone synthesis pathway; inhibition of CYP17 [17] activity by abiraterone thus interferes with the processes in the testes and the adrenals leading to testosterone production.
Androgen deprivation has important side effects [18,19], especially in men: reduced or absent libido, impotence; hot flashes, gynecomastia, osteoporosis and fractures, attenuated mental acuity, loss of muscle mass, depression, and fatigue

THE ROLE OF ANDROGENS IN DIVERSE TUMORS
Although the historic emphasis has been on prosecuting androgen receptors in prostate cancer, it turns out that androgens have a vital role in numerous other cancers as well (Supplemental references 1-119). It is conceivable that interrogation of androgen receptor positivity and treatment with androgen antagonistic agents could therefore be an effective strategy in these cancers.

I. HEAD AND NECK CANCERS
Salivary gland tumors: Remarkable similarities between breast cancer and salivary gland tumors have been documented [S1-S3]. Not only is HER2/neu expression frequent in these tumors, but estrogen receptors (ER) and progesterone receptors (PR) are expressed in 33 reported a partial response after one course of anti-androgen therapy and palliative chemotherapy with paclitaxel in a patient with advanced salivary duct carcinoma.
Thyroid carcinoma: Thyroid cancer is the most frequently occurring endocrine-related malignancy [S9, S10]. The most common form of thyroid cancer

III. SARCOMA
Limited studies of AR in sarcomas have been performed.
Osteosarcoma: AR+ has been demonstrated in 28.5% (8/28) to 50.8% (33/65) of osteosarcomas [S36,S37]. Proliferation of osteosarcoma cell lines in vitro was stimulated by estradiol, progesterone, and 5 alpha-dihydrotestosterone; whereas growth was abrogated by AR antagonists as fulvestrant, mifepristone, and hydroxiflutamide. Interestingly, higher levels of AR correlated with a higher degree of differentiation. In preclinical models, the adrenal androgen synthesis inhibitor ketoconazole prompted apoptosis in human osteosarcoma cells Prostate cancer: The discussion of prostate cancer herein is limited as AR and prostate cancer has been reviewed extensively elsewhere. Briefly, prostate cancer is a prime example of the potential for therapeutically manipulating hormonal levels ( Table 2). This is feasible because strong nuclear AR reactivity is the norm in more than 95% of the samples studied [S58]. There is a high correlation between AR expression and response to hormonal treatment, and anti-androgens are therefore the mainstay of therapy for patients with prostate cancer [S59, S60]. Frequent AR positivity is found in more highly differentiated versus poorly differentiated tumors; further, AR was rarely seen in prostate tumors of patients who had received long-standing hormonal therapy [S61]. Patients with AR-positive serous tumors had improved disease-specific survival [S101]. Additionally, reports of transsexuals who developed ovarian cancer in the setting of androgen supplementation suggest a carcinogenic role for AR [S102]. In regard to treatment, a phase II trial of goserelin and bicalutamide in 35 patients with epithelial ovarian cancer did not prolong PFS [S103, S104]. Because chemotherapy reduces AR expression, anti-androgen therapy may be more effective early in the disease [S104]. One study demonstrated dissimilar AR reactivity within a single ovarian neoplasm, attesting to tumor heterogeneity [S105]. In a phase II trial, 32 patients with advanced ovarian neoplasia received a minimum of two months of flutamide resulting in one complete response for 73 weeks, one partial response for 44 weeks, and partial responses in 28% (9/32) for a median of 24 weeks [S106]. A case report documented a patient with Leydig cell ovarian tumor, an androgen-producing neoplasm, which responded to the GnRH-analogue triptoreline [S107]. Because androgen receptors are frequently expressed in epithelial ovarian www.impactjournals.com/oncotarget cancer, investigation of newer anti-androgens in this disease may be worthwhile [S108].

VIII. BREAST CANCER
Peters et al. studied invasive breast ductal adenocarcinomas, finding 56% AR positivity by IHC, and expression was inversely associated with 10-year survival [S109]. In HER2/-positive breast malignancies, AR expression was found in 85.6% (89/104). AR-negative/ estrogen receptor-negative was the most aggressive phenotype and correlated with high-grade tumors [S110]. Similarly, Hu et al.
[S115] studied 2,171 invasive breast malignancies with tissue microarrays, dividing them into luminal-A (64%), luminal-B (15%), basal-like (11%), and HER2 (6%). AR expression was seen in 77% of invasive breast malignancies divided into Once therapeutic circulating androgen blockade ensues, AR-positive luminal cells die, giving origin to the adaptive (selected AR-positive luminal cells will continue to thrive despite lack of androgen exposure) and clonal theory of resistance (pre-existent AR-negative cells develop into a malignant clone). Castration-resistant prostate cancer might develop via ligand-dependent (tissue steroidogenesis, AR mutations, AR amplification) and ligand-independent pathways (heightened AR nuclear translocation, AR cross-talk with additional pathways, disturbing the balance between co-activators and co-repressors).
[S119] report on a multicenter phase II trial bicalutamide for patients with AR+/ER-/PR-breast cancer showed a clinical benefit rate of 19%.

DISCUSSION
Therapeutic implications of AR across malignancies: Lessons from prostate cancer: During the last several decades, androgen deprivation (Table 2, Figure 1) has been standard of care for metastatic prostate cancer; however, most patients eventually develop disease progression despite pharmacologic suppression of testosterone levels [20][21][22], denoted as castrationresistant prostate cancer (Figure 2) perhaps mediated by androgen independence (Figure 1) [23,24]. The goals of hormonal deprivation therapy for prostatic cancer are to decrease circulating plasma testosterone to castration levels and to block residual androgen at the cellular level ( Figure 3). While orchiectomy is very effective at achieving some of these goals, it does not halt conversion of residual adrenal androgens to dihydrotestosterone (DHT). Certainly, the common examples of potent androgens include testosterone and DHT, which can directly stimulate androgen receptors. Subsequent therapies were added to the hormonal armamentarium including, but not limited to, bicalutamide, a competitive Figure 2: The fate of testosterone in prostatic tissues. Testosterone circulates in the blood and is bound to albumin, whereas free testosterone is introduced into prostate cells and is subsequently converted to DHT by 5-alpha-reductase. Binding of DHT to the AR induces dissociation from HSPs and receptor phosphorylation. The AR dimerizes and can bind to androgen-response elements in the promoter regions of target genes, leading to growth, survival and production of PSA. Enzalutamide, formerly called MDV3100, exerts its mechanism of action during several steps in the AR signaling pathway including inhibition of AR binding to androgens, inhibition of nuclear translocation of AR, inhibition of AR association to DNA, and AR amplification. As some of those aberrations may occur late in the disease, it is unknown at this point if enzalutamide will have a role upfront in the management of prostate cancer. Abbreviations: AR, androgen receptor; DHT, dihydrotestosterone; GTA, general transcription activation; HSP, heat-shock protein; SHBG, sex-hormone-binding globulin; AKT, akt serine/threonine kinase; DHEA, dihydroepiandrosterone; ERK, extracellular signal-regulated kinase; P, phosphorylated residues; PI3K, phosphoinositide 3-kinase; PTEN, phoshatase and tensin homolog. non-steroidal androgen receptor antagonist; flutamide, a non-steroidal anti-androgen; and luteinizing hormonereleasing hormone (LHRH) or gonadotropin-releasing hormone (Gn-RH) [25]. Leydig cells, which are located in the testes, are dependent on LH to produce and secrete testosterone. Gn-RH is a hypothalamic decapeptide that governs the synthesis of pituitary LH; thus synthetic Gn-RH analogues were initially developed to treat infertility in cases of endogenous Gn-RH deficiency. Long-term administration of supra-physiologic Gn-RH produce the paradoxical effect of pituitary overstimulation including pituitary desensitization to Gn-RH, breakdown of physiological feedback systems, down-regulation of Gn-RH receptors, depletion of releasable LH content, and reduction of testosterone secretion to castrate levels. It is not, therefore, surprising that high-dose Gn-RH analogues became a major therapeutic option in prostate cancer, as they bypass the need for orchiectomy [26]. Exogenous estrogens indirectly affect the prostate by disturbing the hypothalamus-pituitary-testes axis as estrogens inhibit the release of Gn-RH from the hypothalamus [27]. Recently, the realization that non-gonadal sources of androgens, such as the adrenal glands and intra-tumoral production, might be critical in the progression of prostate cancer led to the development of abiraterone acetate, an oral specific inhibitor of CYP17A1, which is a rate-limiting enzyme in androgen (and estrogen) synthesis that, when targeted, specifically suppresses adrenal androgens [28]. A novel alternative is the use of intermittent androgen suppression which holds the promise of decreasing toxicity while maintaining efficacy, although the role of intermittent therapy in the metastatic setting remains a challenge [29]. Hussain et al. [30] has studied intermittent versus continuous androgen deprivation in hormone sensitive metastatic prostate cancer patients in a phase III trial and their published final results are eagerly awaited. In the non-metastatic setting, Crook et al. [31] confirmed that intermittent androgen deprivation showed non-inferior overall survival when compared to continuous therapy after prostate irradiation.  /AKT/AR pathway (red arrows) leads to tumor cell proliferation. Rapamycin combined with bicalutamide has an apoptosisinducing effect in prostate cancer. Rapamycin inhibits both mTOR complexes, mTORC1 with raptor and mTORC2 with rictor; nevertheless abrogation of mTORC2, a kinase for AKT phosphorylation, further inhibits the AR transcription cascade in an AKT-dependent manner. As a counterpoint, abrogation of mTORC1 produces AKT/AR-independent apoptosis, though it continues to stimulate the AR transcriptional cascade and AKT phosphorylation. According to the suggested cross-talk, it would take a combination of androgen blockade plus mTOR inhibitors to fully abrogate the mTORC2/AKT/AR pathway. Barnett et al. [44] found that, out of 47 tumors evaluable by immunohistochemistry, 36% had PTEN loss which was associated with an increased relapse in high risk prostate cancer treated with chemotherapy followed by surgery. PTEN loss activates the AKT/mTOR pathway [45] thus supporting the use of mTOR inhibitors in this condition. Abbreviations: AR, androgen receptor; AKT, aKT serine/threonine kinase.
Finally, there are novel therapeutic agents including the once-daily androgen receptor signaling inhibitor enzalutamide, previously called MDV3100 [32,33] (Figure 3), which significantly prolonged overall survival in a randomized phase III trial that involved 1,199 men with castration-resistant prostate cancer [34]. Such results triggered the Food and Drug Administration to approve enzalutamide in August of 2012 for the treatment of patients with metastatic castration-resistant prostate cancer who have previously received docetaxel. The recent wave of novel anti-androgen agents, as abiraterone and enzalutamide, serves as proof-of-principle that AR signaling continues even in the so-called castrationresistant prostate cancer population [35].
Tolerance of Anti-Androgens: Hormonal therapy is not devoid of side effects, and depending on the agent used, toxicity can include increased risk of fractures, increased lipoproteins, decreased insulin sensitivity, increased cardiovascular disease, low libido and emasculation [36]; and such toxicity must be kept in mind during the design of future clinical trials.
Overcoming resistance to AR targeted therapy: Relevant molecular pathways: The mTOR (mammalian target of rapamycin) pathway has been hailed as a possible target for suppressing prostate malignancies ( Figure  3), as the PI3K/AKT components of the pathway are upregulated in hormone-refractory prostate neoplasia, and because the combination of rapamycin and bicalutamide produces apoptosis in prostate cell lines [37][38][39]. Indeed, this pathway is deregulated in up to 65% of prostate cancers, most commonly due to PTEN loss, and less commonly related to PIK3CA amplification or mutation [40]. Wang et al. described the regulation of androgen receptor transcriptional activity by rapamycin in prostate cancer cell proliferation and survival [38]. Furthermore, there seems to be cross-talk between AR and the epidermal growth factor receptor (EGFR) (Figure 3), which in turn activates the expression of mTOR [41]. Gonzalez-Angulo et al. [42] found significantly higher AR levels in breast cancer patients with kinase domain PIK3CA mutations versus wild-type PIK3CA. Wang et al. [43] hypothesized that AR halts PTEN transcription in the prostate in contrast to the breast, in which AR promotes PTEN transcription. Barnett et al. [44] found that, out of 47 tumors evaluable by IHC, 36% had PTEN loss which was associated with an increased relapse in high risk prostate cancer treated with chemotherapy followed by surgery. PTEN loss activates the PI3K/AKT/mTOR pathway [45] thus supporting the use of mTOR inhibitors in this condition.
While both EGFR and AR directly stimulate cell growth, AR apparently indirectly exerts stimulation of EGFR synthesis by paracrine or autocrine mechanisms [46]. In addition to AR-EGFR crosstalk, Src kinase is implicated in EGFR phosphorylation [47]. Meanwhile, interaction between AR and the MAPK pathway has been demonstrated with HER2-AR-ERK feedback loops in breast cancer [48]. Naderi et al. has documented synergy between flutamide and the HER2 inhibitor AG825; and subsequently, synergy between flutamide and the MEK inhibitor CI-1040 [49,50]. In a preclinical model, DHT enhanced IL-6 and IL-8 expression and flutamide abrogated IL-6 and IL-8 expression [51]. Darshan et al. suggested that taxanes, microtubule stabilization agents, are active in castrate-resistant prostate cancer and act by inhibiting AR trafficking and the downstream cascade of transcriptional events, including AR target genes such as prostate-specific antigen [52]. Testing this concept, Kuroda et al.
[S8] treated a patient with metastatic AR-positive salivary duct carcinoma with combined anti-androgen treatment and paclitaxel, achieving a partial response.
Other mechanisms promoting resistance may include genetic and epigenetic adaptation, clonal selection, and evolution of the tumor microenvironment in prostate cancer. The synthesis of constitutively active AR variants lacking the canonical ligand-binding domain may also promote resistance [53].

FUTURE DIRECTIONS
AR is ubiquitously expressed across malignancies. Drawing definitive conclusions about rates of expression is however challenging, mainly due to the lack of standardized AR measurement methods and the relatively small number of patients tested for AR expression. Importantly, there is a paucity of studies of androgen manipulation in AR+ tumors other than prostate cancer. Importantly, some tumors such as salivary gland ductal tumors have AR positivity rates approaching 100%. Anecdotal reports and small studies in a variety of malignancies suggest that AR-positive tumors may respond to hormonal manipulation. Of interest, breast and gynecologic tumors also have high rates of AR positivity ( Table 1). The latter may have special clinical relevance [54], especially since aromatase inhibitors, used to suppress estrogen levels in patients with ER+ breast tumors, can raise testosterone levels. As an example, anastrozole increases testosterone levels by decreasing serum estradiol levels [55]. Finally, PIK3CA/AKT/mTOR signaling is ubiquitously deregulated in cancer, and it is apparent that there is significant crosstalk between this pathway and that related to androgens (Figure 3). Further exploration of androgen modulation in patients with diverse cancers merits investigation.