Abstract
The androgen receptor (AR), ligand-induced transcription factor, is expressed in primary prostate cancer and in metastases. AR regulates multiple cellular events, proliferation, apoptosis, migration, invasion, and differentiation. Its expression in prostate cancer cells is regulated by steroid and peptide hormones. AR downregulation by various compounds which are contained in fruits and vegetables is considered a chemopreventive strategy for prostate cancer. There is a bidirectional interaction between the AR and micro-RNA (miRNA) in prostate cancer; androgens may upregulate or downregulate the selected miRNA, whereas the AR itself is a target of miRNA. AR mutations have been discovered in prostate cancer, and their incidence may increase with tumor progression. AR mutations and increased expression of selected coactivators contribute to the acquisition of agonistic properties of anti-androgens. Expression of some of the coactivators is enhanced during androgen ablation. AR activity is regulated by peptides such as cytokines or growth factors which reduce the concentration of androgen required for maximal stimulation of the receptor. In prostate cancer, variant ARs which exhibit constitutive activity were detected. Novel therapies which interfere with intracrine synthesis of androgens or inhibit nuclear translocation of the AR have been introduced in the clinic.
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1 Introduction
Prostate cancer, the most frequently diagnosed male malignant tumor, is in the focus of interest of basic and clinical researchers. Diagnosis of small prostate cancers, which nowadays is possible because of advancements in diagnostic procedures, opens discussion about the most appropriate treatment of these tumors. There are different approaches available, and the decision to how to treat a patient depends on pathological grading and staging, prostate-specific antigen (PSA) expression, and overall health status. There have been many experimental strategies aimed to identify biomarkers which may allow to distinguish between potentially indolent and aggressive small cancers. Since discrimination between these two groups of cancer is not possible on the basis of current knowledge of biomarkers, many small prostate tumors are removed by radical prostatectomy. This raises questions about benefits and harms of screening programs and possible overtreatment. Tumors which could not be cured by surgery or radiotherapy are treated with therapies based on downregulation of androgen levels in the circulation or blockade of the androgen receptor (AR). Most frequently, therapies with analog of gonadotropin-releasing hormone and/or nonsteroidal anti-androgens hydroxyflutamide and bicalutamide are considered for treatment of non-organ-confined prostate cancer.
For a long period of time, scientists were not aware of the importance of AR signaling during prostate cancer progression. Early research in prostate cancer was performed mostly with a very limited number of human cell lines, some of which do not express the AR. Improved technologies and antibodies allowed the detection of the AR in the tumor material obtained from patients who failed endocrine therapy. Since 1995, there is a consensus that the AR is expressed in relapsed prostate tumors and in lymph node, visceral, and bone metastases [1, 2]. On the basis of those results, the terminology “androgen-independent prostate cancer” was replaced with “castration-therapy-resistant prostate cancer”. Advanced prostate cancer is a heterogenous disease, and the expression of molecular targets may vary between primary tumor and metastases. Pathomorphological studies were followed by investigations of AR function in models which represent advanced prostate cancer. The AR is a transcription factor which becomes translocated to the nucleus following the binding of the active androgen dihydrotestosterone. It binds to hormone response elements on DNA and regulates a variety of genes implicated in regulation of proliferative and differentiation responses. The AR contains three domains: an N-terminal domain with a variable number of polyglutamine and polyglycine repeats, a well-conserved DNA-binding domain, and a ligand-binding domain. The DNA- and ligand-binding domains are separated by a hinge domain which harbors a nuclear localization signal. Transactivation units Tau-1 and -5 located in the N-terminal domain are the main determinants of AR transcriptional activity.
This review will focus on key issues related to AR in prostate cancer. It will include AR function in the regulation of cellular events and will also describe how steroid and peptide hormones influence AR expression and function. Pathophysiological and pharmacological implications of AR mutations in prostate cancer are also a subject of this paper. Importantly, specific action of coactivators at different stages of prostate malignancy will be evaluated. It will be also discussed how AR mutations and the appearance of truncated receptors at different stages of prostate carcinogenesis may affect the natural course of the disease and response to endocrine therapy. Importantly, studies on AR in prostate cancer have a high impact on therapy. Discoveries of abiraterone and enzalutamide are one of the consequences of better understanding the pathways and cellular events regulated by the AR. The achievements in experimental and clinical therapy related to the AR signaling pathway will therefore also be presented in this review.
2 Regulation of specific cellular functions by androgen receptor
There is an increasing number of androgen-sensitive cell lines available in prostate cancer research. Understanding of androgen-regulated cellular events is facilitated by the use of cell lines such as VCaP, DUCaP, LAPC-4, or 22Rv1. AR-positive cell lines demonstrate different proliferative responses after treatment with natural or synthetic androgens. LNCaP cells show a typical byphasic regulation which is characterized by induction of cyclin-dependent kinases (cdk) 2 and 4 and cyclins D1 and E stimulating G1 to S progression by lower concentrations of androgens [3]. In contrast, prostate-specific differentiation products such as PSA are regulated by androgenic steroids in a concentration-dependent manner. Higher concentrations of androgens stimulate expression of cell cycle inhibitors p21 and p27. Thus, LNCaP cells are frequently used for preliminary experiments to assess efficacy of novel anti-androgenic compounds. In contrast, 22Rv1 cells show a reduced response to androgens compared to LNCaP cells [4]. Structural changes of the AR responsible for alterations in response to androgens in these cell lines will be discussed in the chapter on AR mutations. Similarly, MDA PCa 2a also demonstrate a diminished proliferative effect after androgenic stimulation, whereas basal PSA levels in the MDA PCa 2b cell line are high [5, 6]. Differences in proliferative versus differentiation responses indicate that AR expression level is not the single determinant of growth of hormone-sensitive cancer cells. Androgenic regulation of programmed cell death depends on the tumor model used. In contrast to cells which express endogenous AR, PC3 cells transfected with AR cDNA are more sensitive to induction of apoptosis [7]. Induction of apoptosis in androgen-sensitive cells was observed by chemoprevention agents which downregulate AR expression, such as resveratrol [8]. One of the growth factors which are upregulated after castration in rats is the transforming growth factor (TGF)-beta [9]. TGF-beta is a negative growth regulator in prostate cancer cells that stimulates apoptosis through increased Smad expression and activation [10]. One of the intermediate signaling molecules, Smad3, downregulates AR activity [11]. It should be mentioned that the effects of TGF-beta in vivo differ from those observed in vitro due to immune suppression, disruption of the basal membrane, and stimulation of angiogenesis. It is not clear whether the AR is required for those in vivo effects of TGF-beta. The TGF-beta receptor is also upregulated by castration [12]. Increase in apoptotic cells was observed after transfection of AR antisense oligonucleotides into parental LNCaP cells and their hypersensitive derivative LNCaP-abl, thus indicating the possibilities for therapeutic intervention and drug development [13]. Interestingly, long-term androgen ablation may lead to downregulation of the cell cycle inhibitor p21 and resistance to apoptosis induced by paclitaxel [14]. These data indicate that several aspects of androgen ablation such as duration and combination with other treatments should be considered in terms of suppression or promotion of malignant growth. However, it should be indicated that p21 may have different pro- and anti-apoptotic effects depending on an experimental context [15]. The treatment of AR-positive prostate cancer cells with histone deacetylase inhibitors in combination with doxorubicine causes p21 degradation in parallel with downregulation of AR expression and increased expression of p53 [16]. Prostate cancer cell survival is also upregulated by the phosphorylated chaperone hsp27 which enhances AR stability, shuttling, and transcriptional activity [17]. Hsp27 displaces hsp90 from a complex with the AR, thus enhancing its genomic function. AR activation is required for the upregulation of the anti-apoptotic FLICE-like inhibitory protein (FLIP) protein, and this process is mediated by the transcription factor Forkhead box protein O3a (FOXO3a) [18]. Downregulation of FLIP or FOXO3a diminishes the anti-apoptotic effect of androgens. Apoptosis is in prostate cancer tissues frequently prevented by the activation of the phoshatidylinositol 3-kinase pathway resulting in increased phosphorylation of Akt. Akt phosphorylation is a consequence of lack of expression of the tumor suppressor phosphatase and tensin homolog (PTEN). Interestingly, the effects of the phosphatidylinositol 3-kinase pathway on the AR may depend on the passage number. For LNCaP cells, it was demonstrated that in low passages, the receptor function is inhibited by Akt, whereas the stimulation is seen at higher passage numbers [19]. Rapamycin, a drug which regulates Akt activity in a biphasic manner, may increase AR activity [20]. Significant apoptosis in prostate cancer cells could be induced by combination of rapamycin and bicalutamide. Akt is a downstream target in the signaling cascades of cytokines, chemokines, and neuropeptides upregulated in prostate cancer. A higher level of AR expression was demonstrated in transgenic mice expressing activated myr-Akt1 [21].
Androgens are important regulators of migration of prostate cancer cells. This process is important to initiate metastasis development. Androgenic hormones upregulate matrix metalloproteinase (MMP)-2 expression, thus facilitating tumor progression [22]. Luciferase assays revealed that androgens stimulate the MMP-2 promoter in cooperation with the phospahtidylinositol 3-kinase pathway. Androgenic steroids also induce phosphorylation of protein ezrin through the activation of the AR [23]. Dominant-negative mutants of ezrin block androgen-induced invasion. The demonstration that the AR is required for prostate cancer progression was provided in a series of experiments with parental MDA PCa 2b cells and their therapy-resistant derivative in which the AR was upregulated [24]. Similar results were obtained with LNCaP cells. In MDA PCa 2b cells, MMP-2 and -9 are regulated by androgen. Thus, it is not surprising that elevated levels of MMP were measured in sera of patients with advanced prostate cancer [25]. Cellular invasion was suppressed by AR shRNA knockdown or bicalutamide [24]. Another mechanism by which androgens induce migration is the enhanced interaction between the AR and the cytoskeleton protein filamin A [26]. The complex AR/filamin A recruits integrin beta 1 and controls expression of focal adhesion kinase, paxilin, and Rac. One of the proteins which promote prostate tumor cell migration is FOXA1, which is overexpressed in castration-resistant prostate cancer [27]. Interestingly, high expression of FOXA1 correlates with lower AR expression, thus suggesting that migration-related cellular events may be AR dependent and AR independent. Downregulation of FOXA1 in LNCaP cells led to the inhibition of migration and proliferation. As mentioned before, PC3 cells stably expressing the AR show a reduced invasion mediated by alpha6beta4 integrin [28]. The reasons why stable transfection has created a less malignant phenotype and the relevance of the PC3-AR model for clinical situation have not been clarified yet.
The role of androgens in regulation of epithelial to mesenchymal transition (EMT) in prostate cancer is under debate. EMT is characterized by reduced expression of E-cadherin and increased N-cadherin and vimentin. EMT is a condition for metastatic spread. Contrasting data were published whether androgens or androgen ablation are stimulatory to EMT. According to the research of Zhu et al., androgens stimulate migration, invasion, and EMT through activation of the transcription factor Snail in a TGF-beta-independent manner [29]. Cells showing mesenchymal properties expressed low levels of ARs. In contrast, Sun et al. reported that mesenchymal changes were observed in benign and malignant prostate cells undergoing androgen deprivation [30]. The differences between the results of those experimental studies require further investigation because of possible implications on endocrine therapies.
3 Regulation of AR expression and transcriptional function
To understand the regulation of AR expression, it is important to distinguish between the effects of short-term and long-term AR regulation. Androgens can downregulate AR mRNA after a short time treatment; however, the protein is stabilized and therefore expressed at a higher level in LNCaP cells [31]. When LNCaP cells are cultured in the steroid-depleted medium over a long time period, AR mRNA and protein levels gradually increase [32, 33]. This leads to hypersensitivity of the AR, i.e., to efficient receptor stimulation by low concentrations of androgens and other steroids. In one of the sublines derived in this manner, LNCaP-abl, it was observed that the nonsteroidal anti-androgen bicalutamide induces transcriptional activity of the AR, stimulates cellular proliferation, and increases tumor volume [33]. LNCaP-abl and similar models such as C4-2 cells are useful for studies of AR-mediated prostate cancer progression. The AR in C4-2 cells could be targeted by AR-neutralizing antibodies or AR mRNA hammerhead ribozyme to decrease proliferation [34]. An increased AR expression in relapsed prostate cancer may also be a consequence of AR gene amplification [35]. It is estimated that about one third of the patients with recurrent cancer present an AR gene amplification. The presence of AR gene amplification may have dual implications on therapeutic strategies: (i) enhanced AR expression may indicate that the tumor is differentiated and will respond to complete androgen ablation, however, (ii) higher drug doses may be needed to interfere with the androgen signaling pathway. AR expression is downregulated by a number of chemopreventive agents, such as resveratrol, silmyarin, and oligomeric proanthocyanidin complexes [36–38]. These natural compounds can be found in fruits and vegetables. Although the experiments with those agents have been performed with prostate cancer cell lines representing advanced stage of the cancer, they are used to promote the concept of prostate cancer chemoprevention. Interestingly, AR transcriptional activity is enhanced by vitamin D or phenylbutyrate, compounds which show an anti-proliferative response in prostate cancer [39, 40]. Taken together, the results summarized in this chapter indicate that AR expression does not solely determine whether the cells will respond to dihydrotestosterone by growth stimulation or inhibition. One possibility which has not been sufficiently investigated is that a specific set of coactivators is recruited by compounds which stimulate AR activity, but not proliferation.
Noncoding RNA, micro-RNA (miRNA), may be stimulated or inhibited by androgens; however, miRNA also regulate the expression of the AR itself. miRNAs are involved in many regulatory processes in prostate cancer, in particular, in the regulation of tumor-initiating cells, apoptosis, and metastasis. Expression and function of multiple oncogenic and tumor-suppressive miRNA are reviewed elsewhere [41]. It was shown that androgens display different effects on miRNA [42]. Dihydrotestosterone treatment upregulated 17 miRNAs, whereas castration caused an increase in expression of 42 miRNAs. AR expression is under control of miRNA-130, -203, and -205, which are tumor suppressors expressed at a lower level in prostate cancer [43]. These miRNAs also have an inhibitory role on the mitogen-activated protein kinase signaling pathway. Downregulation of multiple signaling cascades by miRNA could represent a new way for a rationale disease therapy. miRNA-221/222 expression has increased in progressive prostate cancer [44]. These miRNAs lowered the level of androgens required for maximal activation of the AR. In contrast, hypersensitive features of the LNCaP-abl subline were reversed when the two miRNAs were downregulated. In contrast, Spahn et al. reported that miRNA-221 is downregulated in aggressive prostate cancer and in metastases [45]. Thus, further studies are required to clarify the role of miRNA-221 in prostate carcinogenesis and progression. Another oncogenic miRNA in prostate cancer is miRNA-21 which is regulated by androgen and is required for androgen-dependent and -independent prostate cancer growth [46]. Its expression is elevated in cancer compared to benign prostate tissue. On the contrary, AR-regulated miRNA-101 inhibits histone methyltransferase EzH2, thus reducing invasiveness of prostate cancer cells [47].
The levels of AR are regulated by 71 unique miRNAs in prostate cancer cells [48]. Inverse correlation between miRNA-34a and -34c and AR levels was observed. miRNA-34a and -34c are downregulated by androgen which caused p53 suppression, thus inhibiting doxorubicin-induced apoptosis [49]. Those studies are complemented by the findings of Kashat et al. who found that miRNA-34 also downregulates the Notch pathway, which is important for the maintenance of a stem cell phenotype in several human cancer [50]. AR is a target for miRNA-488 which has anti-proliferative and pro-apoptotic properties in prostate cancer [51]. Tumor-suppressive miRNA-31 is epigenetically downregulated in aggressive prostate tumors with higher AR expression [52]. Mutual repression of the AR and miRNA-31 has been proposed. This miRNA is important for the downregulation of several cell cycle regulators such as E2F1, E2F2, and FOXM1. Similarly, tumor-suppressive miRNA let-7c negatively regulates AR expression through regulation of Myc expression in carcinogenesis [53]. Lin28 is a repressor of let-7, and its levels are inversely correlated with those of the tumor suppressor miRNA.
The expression and function of the AR during chemotherapy are a subject of recent investigations. Importantly, AR downregulation and nuclear depletion were observed after treatment with taxanes [29]. AR interaction with tubulin is targeted by paclitaxel, thus leading to a reduced receptor nuclear accumulation. Taxanes may therefore have a previously unrecognized effect on the expression of AR target genes in the clinic. Moderate effects of the inhibitor of intracrine androgen synthesis abiraterone were observed in patients who failed docetaxel therapy, thus indicating the importance of the androgen signaling in patients who failed endocrine therapy [54].
4 AR mutations and their implications in prostate cancer
Initial studies on AR mutations in prostate cancer were performed with LNCaP cells, in which a Thr877Ala mutation was discovered. This mutation leads to an increased AR binding affinity for estrogenic and progestagenic steroids and by hydroxyflutamide and to a higher activation of the AR by those compounds [55]. Cells infected with mutated AR acquired a growth advantage in medium deprived of androgen [56]. This finding reflects the clinical situation because the cell line was derived from a patient who failed endocrine therapy. Two AR mutations (L701H and T877A) have been identified in the MDA PCa 2b cell line [5]. In this constellation, there is a reduced responsiveness to androgenic stimulation in that cell line. An in-frame tandem duplication of exon 3 of the AR gene was discovered in the CWR22 xenograft [57]. In vitro, AR could be stimulated by ligands to a lower level compared to the LNCaP AR. CWR22Rv1 cells express low PSA mRNA levels. The first AR mutation functionally characterized in a patient's tissue is the substitution of Val715 with Met [58]. The mutated receptor was discovered in a specimen derived from a patient who failed anti-androgen treatment with hydroxyflutamide. Functional studies revealed that this is a gain of function mutation, characterized by increased activation of the AR by progesterone, adrenal androgens, and dihydrotestosterone metabolites. Different results as to the frequency of AR mutations in prostate cancer were published in the literature [59, 60]. It is possible that some of the previous studies carried out before the introduction of laser capture microdissection did not include pure epithelial population. Higher frequency of mutations in detected bone metastases reflects the genetic instability and heterogeneity of the disease [61]. AR mutations were discovered in about one third of the patients who received therapy with flutamide [62]. Interestingly, second-line treatment with bicalutamide caused a time-limited response in these patients. However, without appropriate studies with a large number of patients, it could not be concluded with confidence that secondary treatment with bicalutamide should be considered a general strategy for patients who failed hydroxyflutamide treatment. Haapala et al. found that AR alterations occur in patients whose tumors relapsed after treatment with orchiectomy and bicalutamide [63]. The same mutations as those found in the patients in the above mentioned study were detected in LNCaP sublines generated after prolonged treatment with bicalutamide [64]. Bicalutamide also promoted the growth of the KUCaP xenograft established from a liver metastasis of a patient who was treated with bicalutamide [65]. Mutated AR W741C was discovered in those cells. An agonistic effect of bicalutamide was confirmed in experiments in which this anti-androgen promoted tumor growth and PSA expression. The use of patient-derived xenografts may further improve the understanding of the androgenic signaling pathway in advanced prostate cancer. For some of the AR mutations in prostate cancer, functional analysis was not performed, and their significance remains unclear. It could be concluded that most mutations in the well-conserved DNA- and ligand-binding domains of the AR lead to dramatic changes in the activation spectrum of the AR. Since AR mutations may alter agonist/antagonist properties of anti-androgens, the results obtained in those studies stimulated the search for novel drugs which inhibit AR signal transduction. Mutations in the N-terminal domain were found in patients who were treated with orchiectomy and estramustine [66]. These mutations did not show an increased response to androgenic hormones or estradiol. Because of considerable side effects caused by estramustine, this treatment is not a current standard in patients' care.
5 AR coactivators in prostate cancer
Initial studies on AR coactivator and corepressor expression were hampered because of a lack of antibodies for protein detection. That situation has been changed, and specific functions of AR coactivators have been investigated. One of the most interesting coactivators of AR in prostate cancer is ARA70, which exists in two isoforms with differential biological functions [67]. ARA70alpha is a tumor suppressor, which is particularly important for anti-invasive properties. The beta isoform is expressed at a higher level in prostate cancer and is regarded as an oncogene. Stimulatory effects of ARA70 on AR transcriptional activity in prostate cancer were reported, in particular, in cells treated with androgen metabolites and hydroxyflutamide [68, 69]. Thus, several molecular mechanisms and especially gain of function mutations and increased expression of selected coactivators may at least in part explain the anti-androgen withdrawal syndrome, a temporary PSA decline, and the reduction of metastatic spots upon cessation of hydroxyflutamide or bicalutamide. Although ARA70 is not specific for AR activation stimulation, it is an important coactivator with multiple functions.
AR coactivator expression may either be stimulated or inhibited by androgen ablation. Importantly, the transcriptional integrator p300 and the related protein CREB-binding protein (CBP) are repressed by androgenic steroids [70, 71]. Androgen ablation enhances their expression in prostate cancer cells, and the relevance of those findings is reflected by their presence in tissue specimens obtained from patients with castration-therapy-resistant prostate cancer. Besides its role in the stimulation of ligand-independent AR activation by IL-6, p300 upregulates AR target genes in cells with a diminished expression of AR [72]. In patients with enhanced expression of CBP, there is an increased agonistic activity of hydroxyflutamide as evidenced in experiments with wild-type and mutated AR [73]. Inhibition of CBP expression in vitro was observed in the presence of IL-6 [71]. It should also be mentioned that gelsolin is another coactivator which potentiates agonistic effects of hydroxyflutamide in prostate cancer cells [74]. Interestingly, nuclear overexpression of filamin A in nuclei of prostate cancer cells enhances bicalutamide-induced growth inhibition in prostate cancer cells C4-2 [75]. This effect is mediated by decreased Akt phosphorylation, thus confirming that Akt regulates the androgen signaling pathway in multiple ways. Little is known about the coactivators which are specifically involved in the acquisition of agonistic properties of bicalutamide. The effects of p300 are not restricted only to AR-positive cells. It also regulates the expression of the nuclear factor kappaB subunit p65 and MMP-2 and -9, thus promoting cellular migration [76]. Another example of an androgen-suppressed coactivator is Tip60, which is implicated in prostate cancer progression [77]. Nuclear localization of Tip60 is increased in models representing therapy-resistant prostate cancer. It was demonstrated that Tip60 knockdown downregulates proliferation by inducing a G1 growth arrest [78]. NU9056 is a potent Tip60 antagonist recently identified in prostate cancer [79]. It downregulated proliferation and induced apoptosis in a variety of prostate cancer cell lines. Androgens also downregulate the expression of TIF2, whose levels are elevated in castration-therapy-resistant prostate cancer [80]. TIF2 is implicated in synergistic activation of the AR by low doses of androgen and epidermal growth factor (EGF) and diminishes the antagonistic effect of bicalutamide in the presence of IL-6 [81, 82]. Specific effects in potentiation of agonistic effects of the anti-androgen cyproterone acetate were reported for the BAG-1 coactivator [83].
There has been an extensive investigation of the role of steroid receptor coactivators (SRC) in prostate cancer. The expression of SRC-1 correlates with prostate cancer progression. It was found that SRC-1 especially potentiates the stimulation of the AR in the presence of low-androgen concentration and mediates the ligand-independent activation of the receptor by IL-6 [84, 85]. Its knockdown leads to the inhibition of proliferation of AR-positive prostate cancer cells with no effect on androgen-insensitive cell lines. Inhibition of SRC-1 is also relevant to the regulation of proliferation in castration-therapy-resistant prostate cancer models, such as the C4-2 derivative of LNCaP. Expression of SRC-3, which also potentiates AR activity, is enhanced in prostate cancer of a high grade and stage [86]. The SRC-3 coactivator is involved in potentiation of effects of insulin-like growth factor-I and the Akt pathway [87]. Recently, results of studies in mice in which PTEN and SRC-3 were inactivated were reported [88]. In control animals, the deletion of PTEN increased the tumor aggressiveness. These properties were reversed by the deletion of SRC-3, thus pointing to the importance of the coactivator for the development of castration-therapy-resistant prostate cancer.
Other coactivators' function has also been investigated and specific cellular alterations affected by their overexpression identified. Prostate cancer invasion is stimulated by leupaxin, a coactivator highly expressed in cancer tissue, by AR-dependent and -independent mechanisms [89]. The activation of the AR by androgen and EGF is potentiated by the Vav3 coactivator [90]. This protein was also found to upregulate nuclear factor kappaB activity and affect the prostatic inflammatory response [91]. Specific potentiation of migration and differentiation was attributed to p68, which acts as RNA helicase [92]. Prostate cancer specimens express higher levels of p68, as evidenced on a tissue microarray.
Alterations in expression of chaperones may affect cellular events in prostate cancer. Chaperones control folding trafficking and transcriptional activity of the AR and other steroid receptors. AR activity is potentiated by the chaperone hsp27 [17]. This coactivator is a target for novel therapies in prostate cancer and it was demonstrated that the use of the antisense drug complementary to hsp27 OGX-427 enhances degradation of the AR and inhibits tumor growth in vivo. More recently, the role of hsp27 in EMT and metastasis was revealed in prostate cancer [93]. Hsp27 is a part of the sigaling of interleukin (IL)-6, whose interaction with the AR is discussed in detail in this review. A cochaperone of Hsp90, FK506-binding protein, augments AR activity in LAPC4 cells [94].
In contrast to coactivators whose expression is elevated in prostate cancer, AR-trapped clone-27 (ART-27) is highly express in luminal cells in the benign tissue [95]. In cancer tissue, ART-27 levels are lowered, consistent with possible tumor-suppressive function.
In summary, there are numerous reports in the literature about the specific action of selected coactivators in prostate cancer. Selective targeting of coactivators by newly designed small molecules in tumors in which these proteins are overexpressed may be a reasonable step in the development of personalized therapy approaches in prostate cancer. Coactivators which have multiple effects in AR-dependent and -independent manner may be particularly the appropriate targets for new therapeutics such as small molecules.
6 Truncated AR in prostate cancer
AR variants have been discovered in prostate cancer cell lines and clinical specimen. Their common feature is a C-terminal deletion which leads to constitutive activity of the receptor. Ligand-independent AR activation was for the first time observed with a receptor in which glutamine 640 is replaced by a premature stop codon. The mutated AR may acquire the ability to stimulate neighboring cells in which it causes receptor nuclear localization in the absence of androgen [96]. Interestingly, another AR splice variant discovered in prostate cancer, AR 23, resulting from alternative splicing of intron 2, exclusively exhibits cytoplasmic action [97]. This receptor could decrease AP-1 transcriptional activity, thus behaving similarly as the full-length AR. Constitutively active ARs were identified in the 22Rv1 model in which the novel exon 2b is expressed at the 3′ end.
Variant ARs with different truncations of the DNA- or ligand-binding domain have been described in prostate cancer. A detailed review describing the structures of the variant receptors was published elsewhere [98]. Some of the truncated receptors such as V7 are upregulated during prostate cancer progression [99]. The V7 receptor is regulated by Akt, which is one of the main anti-apoptotic molecules in prostate cancer, and its phosphorylation is increased in tumors with high Gleason grade. The overexpression of PTEN in prostate cancer cells diminished constitutive activity of the AR V7 [100]. The issue of interaction between full-length and variant AR may have implications on prostate cancer therapy. It was suggested that variant AR requires the presence of the wild-type AR for their activity [101]. However, there was no coimmunoprecipitation of the full-length AR and variant AR in the CWR22R model [99]. There are different expression signatures of cell cycle genes induced by variant AR compared to the full-length receptor [102]. Recent work in which the authors have investigated the mechanisms of anti-androgen therapy resistance revealed that variant ARs act independently of the wild-type receptor [103]. Those results imply that novel means for the inhibition of variant AR in the late stages of prostate cancer remain to be discovered. The application of the inhibitor of intracrine androgen synthesis abiraterone in prostate cancer research will be discussed in detail below. However, it should be mentioned that AR variant expressions during abiraterone treatment increase, thus contributing to therapy failure [104]. The presence of AR variants in bone metastases correlates with therapy resistance and short survival [105].
7 Ligand-independent AR activation
In castration-therapy-resistant prostate cancer, concentrations of circulating androgens are reduced. It has been therefore of major interest to investigate how peptide hormones regulate AR activity. Cross-talk between steroid and peptide signaling pathways has repercussions on cellular events in various hormone-dependent tumors. Ligand-independent and synergistic effects of androgens and a growth factor were reported in prostate cancer cells transfected with AR cDNA and reporter gene as well as in those which express endogenous AR [106]. Amplification of a signal induced by low doses of androgen by a growth factor may be relevant in patients who undergo androgen deprivation therapy. In such a situation, full activation of AR may be achieved by low steroid doses and a nonsteroidal compound. EGF induces phosphorylation of the AR at tyrosine residues 267 and 534 [107]. In vivo importance of ligand-independent AR activation was observed with the EGF receptor-related oncogene ErbB2 [108]. The growth of androgen-sensitive cells which overexpress ErbB2 was inhibited by an anti-androgen. Ligand-independent activation of the AR is not necessarily associated with prostate cancer cell proliferation. This is particularly important for compounds which increase intracellular effects of cAMP and activate protein kinase A [109]. They may cause multifunctional responses in prostate cancer cells to which terminal neuroendocrine differentiation belongs. For nonsteroidal activation of the AR, it is important to evaluate the effects of a nonsteroidal compound on AR endogenous target genes. Such analysis was performed and, surprisingly, it was revealed that there is a relatively low percentage of overlapping genes activated by both androgens and forskolin [110]. Thus, there should be a caution when translating the results of reporter gene assays in vivo. The relevance of nonsteroidal activation of AR was investigated for neuropeptides. These compounds have a paracrine effect on the growth of prostate cancer cells. An increased expression of neuroendocrine cells in prostate cancer is associated with bad prognosis [111]. Androgen withdrawal in vitro leads to a high expression of several neuropeptides. Bombesin is a neuropeptide which stimulates transcriptional function of the AR and the growth of prostate cancer cells [112]. This latter effect could be effectively inhibited by bicalutamide, thus confirming the involvement of the AR. In general, anti-androgens do not acquire agonistic properties in the presence of nonsteroidal AR antagonists. Several studies were focused on the role of pro-inflammatory cytokines IL-6 and -8, and anti-inflammatory interleukin-4 in AR activation. There is no doubt that these cytokines cause ligand-independent and synergistic activation of the AR. IL-6 expression in prostate cancer tissue is increased in the tissue of patients treated with radical prostatectomy [113]. Several mechanisms such as androgen deprivation, loss of tumor suppressor retinoblastoma, or upregulation of the AP-1 complex may contribute to the elevation of IL-6 expression in prostate cancer [114]. Expression of several pro-inflammatory cytokines in prostate cancer is stimulated by nuclear factor kappaB, which promotes AR nuclear localization and activity in several cancer cell lines [115]. After binding to the IL-6 receptor which contains two subunits, ligand-binding gp80 and ligand-transducing gp130, signaling pathways of Janus kinases/signal transduction and transcription factors (STAT) 3, mitogen-activated protein kinases, and phosphoinositol 3-kinase may be activated. AR amino acids 234-558 interact with STAT3 [116]. The Rous sarcoma virus-related kinase Src may also be involved in IL-6 signaling in prostate tumors through phosphorylation of the tyrosine 534 residue of the AR [117]. Src inhibition by protein phosphatase 2 reduced recruitment of the AR to the PSA gene enhancer region and diminished PSA luciferase reporter activation [118]. In addition, serum levels of IL-6 are high during metastatic progression of prostate cancer. IL-6 and its receptor were detected in the tumor cells on pathohistological examination and in the supernatants of the cell lines [119, 120]. IL-6 also acts by a mechanism known as transsignaling which depends on the presence of the soluble receptor in circulation [121]. AR activation by IL-6 is of particular interest because of a variety of cellular events influenced by this pro-inflammatory cytokine. It was found that IL-6 inhibits the proliferation of LNCaP cells, and at the same time, it induces neuroendocrine differentiation. Elevation of PSA mRNA and protein by IL-6 reflects the importance of differentiation induced by IL-6 [122]. A similar growth inhibitory effect was observed after treatment of LAPC-4 cells by IL-6. However, in contrast, a modest growth stimulation in vitro of another AR-positive cell line, MDA PCa 2b, after IL-6 treatment was reported [123]. These observations were confirmed in vivo in experiments in which animals with MDA PCa 2b tumors were either castrated or remained intact. Castrated animals were treated with IL-6 alone or IL-6 and bicalutamide. The volumes of tumors measured after IL-6 treatment were similar to those measured in non-castrated animals. Treatment with bicalutamide caused the inhibition of IL-6-induced tumor growth. At present, it seems that several unknown factors may determine the proliferative or inhibitory effect of IL-6 in cells which express AR. Although STAT3 activation was discussed in the context of IL-6-induced AR regulation, STAT3 activation by IL-6 is not evident in LAPC-4 or MDA PCa 2b cells. Thus, signaling pathways of mitogen-activated protein kinases or Akt may play a role in the process of IL-6 regulation of the AR. Protein inhibitors of activated STAT (PIAS) are implicated in coactivation of the AR [124]. A member of this family, PIAS1, is expressed in prostate cancer at a higher level and causes cell cycle proliferation through the inhibition of p21 [125]. In addition to previously mentioned p300, SRC-1 is another important AR coactivator implicated in AR activity regulation by IL-6 [85]. It was also demonstrated that IL-6 may increase intracrine synthesis of androgenic steroids in the prostate [126]. Experimental treatment with the anti-IL-6 antibody siltuximab was found to have a pro-apoptotic effect in both androgen-insensitive and -sensitive cellular models such as PC-3 or LUCaP 35 xenografts [127, 128]. In the latter model, that antibody delayed progression of the tumor toward therapy resistance. A phase I study demonstrated the potential of the antibody in inhibition of steroidogenic enzymes [129]. Kinase pathways were studied also with other nonsteroidal activators of the AR. As mentioned above, Akt exhibits different effects on the AR, dependent on cell passage number. The role of Akt in regulation of AR is of primary importance because of an increased phosphorylation of this kinase in prostate cancer, in particular, in tumor progression [130]. Constitutive activation of the Ras/MAPK pathway contributes to hypersensitivity of the AR as observed in models of castration-therapy-resistant prostate cancer [131]. AR activity is stimulated by Aurora A kinase which also supports tumor cell proliferation [132]. The mechanism by which tyrosine kinase Ack1 activates the AR may include the degradation of the tumor suppressor Wwox, thus leading to activation of Akt [133].
IL-8 is an important anti-apoptotic and pro-angiogenic cytokine in prostate cancer. Its stimulatory effect on AR activity may contribute to malignant progression [134]. However, little is known about the mechanisms and pathways responsible for the effect of IL-8 on the regulation of AR activity. IL-4 effects on AR are relevant to the inhibition of apoptosis. In addition, IL-4 interaction with the AR was demonstrated to upregulate PSA expression [135].
8 Novel experimental and clinical therapies
Reactivation of AR signaling has been recognized as a mechanism driving castration resistance. Thus, the AR axis is not only the target of choice in androgen-sensitive prostate cancer, but it as well remains a valuable target in castration-resistant prostate cancer (CRPC). A number of novel experimental and clinical therapies take into account the resistance mechanisms leading to the reactivation of AR signaling.
Medical or surgical castration for the treatment of prostatic cancers prevents androgen production by the testes. However, the adrenals are also capable of releasing dehydroepiandrosterone and androstenedione, two precursors that are conversed by prostate cancer cells to testosterone [136]. Moreover, overexpression of enzymes of the so-called “backdoor pathway” enables intratumoral de novo synthesis of dihydrotestosterone from progesterone [137]. In both cases, CYP17A1 is a key enzyme in androgen biosynthesis and has, thus, been ascribed as a therapeutic potential. The CYP17A1 inhibiting capability of abiraterone acetate (CB7630) was firstly identified in 1994 [138], evaluated in clinical trials [139–141], and finally approved in 2011 for the treatment of patients with metastatic CRPC who have received prior chemotherapy with docetaxel in combination with prednisone. Recent results show also a benefit of abiraterone acetate treatment for patients that did not receive previous chemotherapy [142]. Although abiraterone acetate is efficient in prolonging the patients' overall survival, resistances to the drug may occur. The molecular mechanisms on the basis of abiraterone acetate resistance include overexperession of CYP17A1 and the upregulation of AR and AR splice variants [104]. Interestingly, cells expressing the AR mutant T877A are not dependent on CYP17A1 [143]. This AR mutant that is for instance being expressed by the LNCaP cell line model can be promiscuously activated by progesterone [144].
Enzalutamide (MDV3100) is a second-generation, nonsteroidal anti-androgen that binds the AR in the ligand-binding domain with greater relative affinity than bicalutamide [145]. The drug's mechanisms of action are the blocking of testosterone binding to the AR, the reduction of AR nuclear translocation, and the impairment of DNA binding to androgen response elements and of AR coactivator binding. Unlike bicalutamide and flutamide, no agonistic activity of enzalutamide has been observed. In the AFFIRM study, enzalutamide could prolong the overall survival of patients with metastatic CRPC after chemotherapy by 18.4 months in the enzalutamide group versus 13.6 months in the placebo group [146]. However, also de novo resistance to enzalutamide was observed in a number of patients, and some initial responders developed resistance to enzalutamide [146, 147]. Moreover, abiraterone acetate has only modest activity in patients that have progressed after docetaxel and enzalutamide treatment [148]. This leads to the hypothesis that resistance mechanisms against enzalutamide could involve constitutive active AR splice variants lacking the ligand-binding domain, and hence, the target for enzalutamide. The first evidence for this hypothesis was presented by Li et al., who could show that cells expressing full length and truncated AR grow androgen-independent and are resistant to enzalutamide treatment [103].
A number of novel, experimental CYP17A1 inhibitors and anti-androgens have been described in literature that eventually target the carboxyterminal, ligand-binding domain of AR. One of the most promising agents is galeterone (TOK-001; VN/124-1) that has initially been synthesized as CYP17A1 inhibitor [149]. It soon became evident that galeterone is also able to act as anti-androgen by displacing androgens from the AR ligand-binding domain and by increasing the degradation rate of AR resulting in low AR expression levels [150]. Galeterone is currently in phase II clinical trials (ARMOR2 study). The recently described ARN-509 is an anti-androgen with greater affinity to the AR ligand-binding domain than enzalutamide [151]. Its mechanism of action is via impairment of AR nuclear localization and DNA binding. Unlike bicalutamide, it does not have agonistic properties on the AR. Currently, ARN-509 is being tested in a phase I/II study. The third experimental anti-androgen, that has made its way into clinical trials, is BMS-641988, although with limited success [152, 153]. The clinical development of BMS-641988 was discontinued due to partial agonism of the compound and its insufficient anti-tumor activity. A company-developed CYP17A1 inhibitor is orteronel (TAK-700) which is currently in phase III clinical examination for patients with CRPC [154]. Proof of concept for orteronel was previously done in cynomolgus monkeys and rats [155, 156]; however, no data comparing the anti-tumor efficiency of orteronel and abiraterone acetate are available to date.
An interesting small molecule is EPI-001 that was isolated from a library of marine sponge extracts [157]. Unlike other anti-androgens, it binds to the aminoterminal AF1 domain of AR, a region that is the major contributor of AR transcriptional activity [158]. EPI-001 is able to inhibit the binding of AR coactivators, antiparallel dimer formation, and interaction with androgen response elements in a ligand-independent manner. Furthermore, it has high specificity for AR on other nuclear steroid receptors and may also inhibit AR splice variants and carboxyterminal mutants. An overview of anti-androgens and inhibitors of intracrine androgen synthesis is provided in Table 1.
Other possibilities to target the AR axis is by inhibiting the function of AR cointegrators and/or interacting proteins. HSP90 is a molecular chaperone that is essential to stabilize AR in a ligand-prone conformation [159]. Inhibition of HSP90 function leads to proteasomal degradation of its clients, including AR [160]. However, the clinically tested HSP90 inhibitors tanespimycin (17-AAG) and retaspimycin (IPI-504) failed to show a positive response in CRPC patients [161, 162]. A reason for this could be the fact that HSP90 inhibitors like 17-AAG and ganetespib are not able to decrease the expression levels of truncated AR variants [163, 164]. On the other hand, ganetespib could exert anti-tumor activity in both AR-positive and -negative cells by interfering with survival pathways and cell cycle regulators [164]. A number of studies have reported on the use of histone deacetylase inhibitors (HDACi) for prostate cancer [165–167]. HDACi may impair recruitment of polymerase II by the AR, thus blocking transcription of AR downstream genes in androgen-sensitive and CRPC models [168]. However, a phase II clinical study reported only minimal anti-tumor activity of romidepsin [169], a HDACi that is approved for treatment of lymphomas. A more rational approach could be the inhibition of AR coactivators possessing histone acetyltransferase (HAT) activity. We could show that inhibition of p300 by the small molecule C646 leads to a decreased AR activity and induces apoptosis in prostate cancer cell lines [76]. However, apoptosis was also induced in AR-negative cell lines, showing that p300 is essential in other survival pathways such as NF-κB. Ideally, targeting of AR coactivators should involve coactivators that interact with the aminoterminal AR domain to ensure that also truncated AR variants are targeted. A candidate would be the p160 family of steroid receptor coactivators (SRC). Downregulation of SRC-1 reduced the growth of AR positive cell lines, while AR-negative cell lines were not affected [84]. The inhibition of SRC-1 and -3 expression by the small molecule gossypol resulted in a selective cytotoxicity for cancer cells over normal cells [170].
9 Conclusions
Investment in prostate cancer research worldwide has improved our understanding of the mechanisms by which the AR promotes oncogenic events. Importantly, many studies have investigated how agonist/antagonist balance of compounds used to block the AR is regulated. It is established that long-term androgen ablation or prolonged treatment with nonsteroidal drugs may enhance their agonistic properties. Important discoveries on intracrine androgen synthesis were followed by clinical development of abiraterone, a drug which inhibits the CYP17A1 enzyme responsible for androgen biosynthesis. AR antagonist enzalutamide has been introduced in therapy on the basis of its ability to prevent nuclear translocation of the AR. Although clinical improvements with these new drugs have been achieved, the resistance develops also in these patients. Current and future studies focus on the role of variant AR and their role during endocrine therapy. In addition to the AR, small-molecule inhibitors and other drugs are being developed for prostate cancer experimental treatment. Translational and clinical investigations should be aimed to design appropriate combination treatments for castration-therapy-resistant prostate cancer, in contrast to unsuccessful monotherapies performed so far, in order to achieve multiple target inhibition which is tolerated in clinical settings.
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Culig, Z., Santer, F.R. Androgen receptor signaling in prostate cancer. Cancer Metastasis Rev 33, 413–427 (2014). https://doi.org/10.1007/s10555-013-9474-0
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DOI: https://doi.org/10.1007/s10555-013-9474-0