Androgen Receptors in Prostate Cancer

0022-5347/03/1704-1363/0 THE JOURNAL OF UROLOGY® Copyright © 2003 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 170, 1363–1369, October 2003 Printed in U.S.A.

DOI: 10.1097/01.ju.0000075099.20662.7f

Review Article ANDROGEN RECEPTORS IN PROSTATE CANCER ZORAN CULIG,* HELMUT KLOCKER, GEORG BARTSCH, HANNES STEINER AND ALFRED HOBISCH From the Departments of Urology, University of Innsbruck (ZC, HK, GB, HS, AH), Innsbruck and General Hospital Feldkirch (AH), Feldkirch, Austria

ABSTRACT

Purpose: Androgen receptor (AR) is expressed in the majority of human prostate cancers. For a better understanding of prostate carcinoma events it is necessary to present findings on the regulation of AR target genes, AR interaction with associated proteins, ligand independent activation and point mutations. Materials and Methods: A comprehensive literature review of manuscripts published on AR in prostate cancer was performed using PubMed. Results: AR regulates the expression of genes involved in the proliferation and differentiation of prostate cancer cells. Due to differential interactions with coactivators and corepressors AR activation results in the stimulation of a mitogenic response or in the expression of secretory proteins. AR is functional in advanced carcinoma of the prostate, as evidenced in studies of mutant receptors and ligand independent activation. AR point mutations appear in advanced prostate cancer more frequently than in organ confined disease. Conclusions: Current therapy options aimed to inhibit AR function in prostate cancer are limited. Antiandrogenic drugs frequently acquire agonistic properties in the presence of mutated ARs. In addition, androgen signaling pathway activity increases during long-term androgen ablation. AR coactivator complexes might be a target for novel therapies for prostate cancer. KEY WORDS: prostate, prostatic neoplasms, androgens, gene expression

In the last decade research on androgen receptor (AR) in prostate cancer led to discoveries that significantly changed our understanding of tumor biology. This research field was previously considered less important because of a lack of correlation between AR expression in cancer tissue and disease outcome. AR binding assays were not routinely used in prostate cancer diagnosis or prognostic studies, in contrast to determinations of estrogen and progesterone receptor levels in breast cancer. The classic concept that prostate cancers progress toward therapy resistance because of the loss of AR expression has been revisited. AR basic science studies have implications in ongoing chemoprevention and clinical trials. An important issue that should be considered is that, at clinics prostate cancers are increasingly detected at earlier stages. Early detection may in some cases lead to over treatment because there are no molecular markers available that allow the detection of clinically indolent or potentially aggressive cancers. Thus, AR function should also be studied during the early stages of prostate carcinogenesis. In this context it is worthwhile to note that the AR is implicated in the regulation of proliferative responses and differentiation. Growth and differentiation are induced by different concentrations of androgen, and intermediary factors involved in this regulation are not known. For better understanding the AR role in prostate cancer it is important to develop models that resemble the human situation that could be used to study integrated signaling pathways.

AR MOLECULE

AR belongs to the superfamily of nuclear hormone receptors and it shows a similarity to progesterone and glucocorticoid receptors. These receptors are composed of well conserved DNA and ligand binding domains, and the less conserved N-terminal region, and they share the same response element on DNA. Tissue specific differences in gene induction by androgen and glucocorticoid hormones could be explained by the existence of a subclass of repeated response elements that are recognized exclusively by AR and not by other steroid receptors. In addition to androgens, the prostate specific antigen (PSA) gene could be induced by glucocorticoids.1 However, dexamethasone did not influence the growth of prostate cancer cells stably transfected with glucocorticoid receptor cDNA. The AR N-terminal region is important to determine the extent of receptor transcriptional activity. It contains a variable number of polyglutamine and polyglycine repeats, and it was shown in experimental studies that a decreased number of polyglutamine stretches is associated with higher receptor activity. In epidemiological studies it was reported that black Americans, which is the population with the highest prostate cancer incidence, have shorter polyglutamine repeats than white or Asian individuals.2 It has been suggested that racial variations in CAG repeat length explain in part the excess risk of prostate cancer. In a study of 587 men with newly diagnosed prostate cancer cases it was shown that those with shorter repeats are at higher risk for metastatic prostate cancer.3 In a low incidence Chinese population a shorter polyglutamine repeat length conferred an increased risk for clinically significant prostate cancer.4 However, analysis of

* Corresponding author: Department of Urology, University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria (telephone: 43–512-504 – 4818; FAX: 43–512-504 – 4817 or 43–512-504 – 8365; e-mail: [email protected]). 1363

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AR repeats in a French-German population revealed a similar distribution in controls, individuals with sporadic prostate cancer and prostate cancer families comprising affected and nonaffected family members.5 The issues of whether CAG repeat length correlates with the early onset of prostate cancer, response to endocrine therapy or overall survival within a select population are also a matter of debate that is beyond the scope of this review. The ligand binding domain is also involved in the regulation of transcription and respective amino acids are designated activation function-2. Interactions between the N-terminal and carboxyl-terminal domains are important for the regulation of AR functional activity. AR agonists could be distinguished from antagonists based on their ability to promote these N/C interactions. In the AR DNA binding domain there are 2 zinc fingers, each composed of 4 cysteine residues bound to a zinc ion. The AR gene located on the X chromosome is structurally organized in 8 exons. The N-terminal region is encoded by exon 1, the DNA binding domain is encoded by exons 2 and 3, and the ligand binding domain is encoded by exons 4 to 8. In addition to its action as a classic nuclear transcription factor, AR also mediates rapid effects of androgens, such as the activation of intracellular kinases and up-regulation of calcium levels.6 To our knowledge the impact of these cellular events for prostate cancer has yet to be determined. AR COACTIVATORS AND COREPRESSORS RELEVANT TO PROSTATE CARCINOGENESIS

AR is expressed in the overwhelming majority of prostate cancers, including metastases in patients in whom endocrine therapy failed, in contrast to findings obtained in several cell lines of human and rat origin.7 Those immunohistochemical studies greatly enhanced research on AR expression and function in advanced prostate carcinoma. In prostate tissue epithelial and stromal cells contain AR. It has been suggested that AR in the stroma is a primary androgenic target. Upon hormonal stimulation it up-regulates the expression of soluble growth factors that bind to membrane receptors on epithelial cells. Candidate mediators of androgenic action between prostate stroma and epithelium are keratinocyte growth factor and fibroblast growth factor-10. The lack of AR expression in some cell lines and patient material could be explained by methylation of the AR gene promoter CpG island.8 The AR N-terminal is involved in hormone independent interactions with AR associated coactivator proteins of the p160 group, to which SRC1 and TIF2 belong. AR coactivators augment functional activity of the receptor by interaction with histones or by recruitment of chromatin modifying complexes. Proteins that interact with AR have been identified in 2-hybrid assays. Some proteins that enhance transcription activation function are required for other cellular processes since they are associated with cell cycle regulators, tumor suppressors and other steroid receptors. For the development of new therapy concepts in prostate cancer it is important to understand better the expression and function of coactivators. Reporter gene assays have some usefulness in these studies but they do not provide information on the regulation

of specific cellular events. For this reason stable transfections of prostate cancer cells with coactivator cDNA should be performed. It is believed that the identification of AR coregulatory proteins that potentiate mitogenic and/or antiapoptotic effects of androgen provide the basis for more rational therapy. Research on the clinical significance of coregulatory proteins is of general interest because it is relevant to the action of WT and mutated receptors. Efforts of researchers in this field were hampered because of a lack of antibodies for detecting some coregulatory proteins. The 2 coactivators SRC1 and TIF2 are expressed at a higher level in the tissues of patients with therapy resistant prostate cancer (table 1).9 In cells that express high levels of nuclear receptor coactivators AR is more sensitive to stimulation by adrenal androgens. Adrenal steroids were also shown to enhance the activity of mutated AR detected in prostate cancer.10 Expression of the coactivator RAC3 correlates with tumor stage, grade and patient survival.11 Prostate cancer cell growth was inhibited by a dominant negative mutant of the cofactor ARA 54.12 The fact that elimination of a single cofactor causes growth retardation is intriguing because of a redundancy among coactivators. It has become evident that AR coactivators are implicated in ligand independent activation of the receptor. A functional homologue of the cyclic adenosine monophosphate response element binding protein binding protein, p300, is critical for ligand independent activation of the AR by interleukin-6 (IL-6).13 In cells transfected with small interference RNA that targeted p300 there was no induction of reporter gene activity by IL-6. AR activation by IL-6 was also augmented in the presence of SRC1, a cofactor phosphorylated by mitogen activated protein kinase (MAPK).14 Protein-protein interaction between AR and SRC1 is induced by treatment with ligand or IL-6. In some cases mutated AR discovered in prostate cancer shows decreased activation by androgenic hormones. This lack of activation could be explained by inappropriate interaction with coactivators. In case of mutated AR 619 cys3tyr it was shown that inability to transactivate androgen responsive genes is associated with SRC1 sequestration.15 It was suggested that the protein ARA70 is an AR specific coactivator. However, that observation remains controversial.16 In studies done elsewhere it was demonstrated that the presence of ARA70 also leads to enhancement of the functional activity of progesterone and glucocorticoid receptors.17 In addition, the level of augmentation of AR activity by ARA70 did not differ from that caused by other coactivators. More recently, it was shown that the interaction between the AR and ␤-catenin is AR specific.18 ␤-catenin, of which the expression is regulated by the cell adhesion molecule E-cadherin, does not interact with estrogen receptor-␣, progesterone receptor-␤ or glucocorticoid receptor. Due to the absence of E-cadherin in cancer tissues there is increased expression of ␤-catenin in nuclei and consequently enhanced AR activation. The observation that ␤-catenin stabilization induces prostate intraepithelial neoplasms might be partly related to interaction with the androgen signaling pathway. ARA70 is implicated in the acquisition of agonistic properties of AR antagonists and AR activation by other steroids.19, 20 There were no major differences between reporter gene ac-

TABLE 1. AR cofactors implicated in prostate cancer pathogenesis and progression Co-Activator

Prostate Ca Expression

ARA 70 ARA 54 ARA 55 p300 SRC-1 TIF2 PIAS RAC3

Down-regulated Up-regulated Up-regulated Up-regulated Up-regulated Up-regulated, correlation with grade ⫹ stage

Function Decreases colony formation, enhances AR activities Enhances AR activation (estradiol ⫹ hydroxyflutamide) Enhances AR activation (estradiol ⫹ hydroxyflutamide) Ligand independent activation (IL-6) Enhances AR activation (adrenal androgens) Enhances AR activation (adrenal androgens)

ANDROGEN RECEPTORS IN PROSTATE CANCER

tivities induced by antiandrogen (hydroxyflutamide or bicalutamide) in the presence of ARA70. Interestingly prostate cancer cells stably transfected with ARA70 cDNA slowed proliferation and colony formation was also decreased.21 In situ hybridization of AR cofactors performed in 43 primary prostate cancers revealed alterations in their expression, in that the levels of protein inhibitor of signal transducers and activators of transcription signaling and ARA 54 increased, whereas the levels of ARA 70 were down-regulated.21 These recent provide the interpretation that ARA 70 in fact has tumor suppressor properties. In this context it is important to note that retinoblastoma (Rb) tumor suppressor also acts as an AR coactivator.22 The potentiation of AR activity by breast cancer susceptibility gene 1 enhances androgen induced cell death.23 In contrast to AR coactivators, there are no studies of alterations of AR corepressors in prostate cancer. However, it seems that the corepressor SMRT modulates the action of various antiandrogens in a different manner. SMRT binds to N-terminus AR only after treatment with the progestagenic antiandrogen cyproterone acetate but not in the presence of nonsteroidal antagonists hydroxyflutamide or bicalutamide.24 SMRT dissociates from the receptor when ligand independent activation by protein kinase A activators is initiated. Briefly, although a large number of AR interacting proteins have been identified, there is still limited information on their individual contributions to prostate cancer development and progression.

LIGAND INDEPENDENT ACTIVATION OF AR IN THE REGULATION OF PROSTATE CANCER GROWTH AND DIFFERENTIATION

The recognition that AR could be activated in a ligand independent and synergistic manner has significant implications for prostate tumor biology.25, 26 Clearly AR activation by growth factors and growth factor receptor related molecules could significantly enhance tumor growth in conditions in which androgen levels are suppressed. Growth factors insulin-like growth factor-I, epidermal growth factor or keratinocyte growth factor signaling depends on their binding to membrane receptors.25 The receptors of most polypeptide growth factors are widely expressed in prostate cancer. The significance of ligand independent activation of AR by the epidermal growth factor receptor related molecule HER-2/ neu was confirmed by Craft et al26 That group investigated ligand independent AR activation in the prostate cancer xenograft LAPC-4 and noted that over expression of Her-2/ neu promotes tumor growth and enhances PSA expression. They stimulated cyclin dependent kinase 2 activity and decreased p27 levels. These findings are of particular importance for understanding prostate tumor biology in patients who receive endocrine therapy, in that minimal AR activation by residual androgens is potentiated by nonsteroidal hormones. For AR activation by growth promoting compounds a functional MAPK signaling pathway is required. Clinical studies of antibodies that recognize phosphorylated MAPK revealed a correlation between MAPK activity and higher Gleason score.27 AR activation by HER-2/neu also involves protein kinase B, Akt.28 However, an inhibitory effect of Akt on AR and androgen induced apoptosis was also reported and these divergent findings require further investigation.29 Castration of animals bearing prostate cancer xenografts leads to time limited retardation of tumor growth in vivo. Tumor relapse is associated with an increased expression of AR regulated genes.30 The most likely explanation of this phenomenon is that ligand independent activation of AR has physiological significance. In most cases the levels of AR activation by a nonsteroidal protein kinase activator reach about 50% of those measured in the presence of high concen-

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trations of androgenic hormones but they seem to be sufficient to induce gene expression in vivo. Several studies provide evidence that AR is activated by nonsteroidal compounds that inhibit cell growth or cause pleiotropic effects.31⫺33 It seems that the protein kinase A pathway has a central role in regulation of AR activity. This signaling pathway is implicated in ligand independent and ligand dependent AR activation.31 In cells treated with protein kinase A inhibitor there was substantially decreased androgen induced reporter gene activity.31 For several reasons there is considerable interest in AR activation by IL-6. IL-6 serum levels increase in patients with therapy resistant prostate cancer and recent studies showed that IL-6 is elevated in tissue extracts from patients with organ confined disease.34 Divergent results of the regulation of LNCaP cell proliferation by IL-6 were reported in the literature. Some researchers observed paracrine inhibition,35 whereas in other publications a growth stimulatory effect caused by this cytokine was reported.34 It was demonstrated that IL-6 activates AR in a ligand independent and synergistic manner, and its effect could be suppressed by AR antagonists.32 In contrast, AR blockers were ineffective in the presence of the IL-6-related cytokine oncostatin M.36 The reasons for these differences have not yet been clarified, although it is known that in other cancers oncostatin M induces a more persistent signal that could not be blocked by kinase inhibitors. It could be speculated that there are different requirements for adaptor proteins by IL-6 and oncostatin M. Inhibitors of signaling pathways were efficient in blocking AR activation by IL-6 but they failed to block AR activity induced by oncostatin M. In LNCaP cells that express endogenous AR receptor function enhancement by IL-6 is associated with stimulation of the promoter of the AR gene and elevation in the levels of AR mRNA and protein.32 In contrast, changes in AR expression levels were not reported in cells transfected with AR cDNA and treated with nonsteroidal activator.31, 36 Activation of the AR by IL-6 leads to increased expression of PSA mRNA and protein.37 These findings strongly suggest activation of AR by IL-6 in association with cellular differentiation. As a consequence of prolonged treatment with IL-6, there are changes in signal transduction pathways that lead to acquisition of a growth advantage. Phenylbutyrate, a compound that induces AR expression and activity, and PSA expression, causes cellular proliferation retardation.33 As mentioned for IL-6-type cytokines, there is a different behavior of nonsteroidal antiandrogens in the presence of various AR activators. In some experiments reporter gene activity was completely down-regulated by these compounds but there are also reports showing that antiandrogens fail to down-regulate AR activity induced in a ligand independent manner. These differences might occur because of different coactivator/corepressor requirements for AR activations by various nonsteroidal components. For example, antiandrogen efficiency was decreased in activation processes caused by ␤-catenin or HER-2/neu.26, 38 The tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) is involved in bidirectional communication with AR, in that it represses AR transcriptional activity.39 On the other hand, androgens antagonized PTEN induced apoptosis in an AR dependent manner.39 AR is also activated by products of neuroendocrine cells that are expressed during progression toward therapy resistance.40 These studies revealed that the 3 nonreceptor tyrosine kinases focal adhesion kinase, Src and Etk/BMX are involved in ligand independent activation by the neuropeptide bombesin. AR activation by caveolin, a scaffold protein of several signal transduction pathways, might be relevant to tumor progression.41 Colocalization of AR and caveolin was demonstrated in low density membrane fractions and the interaction between AR and caveolin is enhanced by androgen. Furthermore, there is an enhancement of androgenic

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action by triiodothyronine, as revealed in studies of the transcriptional regulation of PSA promoter.42 Studies of ligand independent and synergistic activation of AR have a considerable impact on prostate cancer biology since they clearly show that AR is functional in an environment with low androgen supply. DIFFERENT EFFECTS OF AR ON GROWTH OF PROSTATE CANCER CELLS

Not only nonsteroidal AR activators, but also androgenic hormones cause different effects on tumor cell proliferation and cell cycle regulation. A classic example of this dual role of androgens is a biphasic regulation of the growth of LNCaP cells derived from lymph node metastasis from a patient who did not respond to endocrine therapy. Most studies of AR in prostate cancer were done in that cell line. Prostate specific genes are not induced after treatment with low concentrations of androgen that enhance tumor growth. Findings in LNCaP cells could be explained by different effects on cell cycle regulators by low or high doses of androgenic hormones. A critical cell cycle regulator that prevents uncontrolled entry into the S phase of the cell cycle is a product of the Rb gene (pRb). Its phosphorylation increases after treatment with low androgen doses.43 Hyperphosphorylated pRb does not bind the transcription factor E2F. The growth promoting effect of androgens is associated with the up-regulation of cyclin dependent kinase 2 and 4 expression and activity.44 Experiments in probasin-large T antigen transgenic mice revealed that progressive histopathological changes ranging from dysplasia to prostate cancer occur, thus, strongly suggesting a role for androgens in mitogenic responses and prostate cancer pathogenesis.45 In contrast, in cells treated with high doses of androgen there was increased expression of the cell cycle inhibitor p27. Interestingly p27 expression increases in long-term androgen ablated cells.46 These LNCaP sublines were inhibited by androgen in vitro and in vivo. There is a consensus that AR transcriptional activity increases during long-term androgen deprivation (table 2).46, 47 In these sublines there is a marked decrease in PSA expression, for which the most likely explanation is methylation of its promoter.48 There was strong down-regulation of the androgen regulated cell cycle inhibitor p21 in a LNCaP derivative established in the absence of androgen.48 After treatment with AR antisense oligonucleotides p21 expression increased. AR antisense administration reestablished androgen dependence of these tumor cells. PC-3 prostate cancer cells that do not express AR were stably transfected with AR cDNA at several laboratories. Reexpression of AR led to the establishment of a less malignant phenotype, as evidenced by the induction of growth arrest and apoptosis by dihydrotestosterone.49 The growth inhibitory action of androgenic hormones in PC-3/AR cells was mediated by neutral endopeptidase, an enzyme that cleaves and inactivates neuropeptides implicated in prostate cancer growth.50 In addition, ␣6␤4integrins responsible for the invasive phenotype are down-regulated in PC-3 cells transfected with full length AR cDNA.51 Those observations are

TABLE 2. Characteristic features of sublines of androgen sensitive LNCaP prostate cancer cells developed during chronic steroid deprivation References Kokontis et al46

Phenotype

AR mediates growth inhibition, p27 upregulation Increased basal ⫹ hormone induced receptor Culig et al47 activity, bicalutamide agonism, growth stimulation p21 Protein down-regulation, apoptosis resisWang et al48 tance AR expression was up-regulated.

supported by studies in androgen repressed invasive human prostate cancer cell ARCaP derived from metastatic prostate cancer.52 As a consequence of the stable transfection of ARCaP cells with AR cDNA, there was decreased growth, invasion and migration. Together these studies emphasize that AR has also an important role in the promotion of prostate cell differentiation. For this reason down-regulation or elimination of AR would not necessarily lead to a substantial improvement in prostate cancer clinical treatment. Short-term experiments in androgen responsive prostate cancer cells revealed that various compounds that downregulate AR expression cause proliferation inhibition. For example, AR expression is down-regulated by interleukin-1␤, a cytokine present in monocyte conditioned medium.53 Retardation of LNCaP growth in association with AR downregulation was observed after treatment with the nonsteroidal anti-inflammatory agent flufenamic acid and the red wine ingredient resveratrol.54, 55 In an experimental therapy approach a hammerhead ribozyme was used to decrease AR expression at the mRNA level.56 The ribozyme was applied without causing toxic effects to prostate cancer cells. Antisense oligonucleotides directed against polyglutamine repeats were used to down-regulate AR in LNCaP cells.57 Their proliferation was inhibited as well as PSA expression. Microinjection of an antiAR antibody or administration of a hammerhead ribozyme suppressed the growth of prostate cancer cells that up-regulate the AR thus confirming importance of AR activation in progressive tumor cell growth.58 Not only AR, but also HER2/neu and Akt require the chaperone Hsp 90 for folding. A therapy approach based on the inhibition of Hsp 90 function by 17-allylamino-17-demethoxygeldanamycin appears reasonable because it targets multiple growth promoting agents.59 In prostate cancer this drug inhibited the growth of a xenograft. Vitamin E inhibited the expression of AR but not that of other steroid receptors.60 It slowed LNCaP cell growth and PSA expression. On the other hand, AR protein is down-regulated by basic fibroblast growth factor, a potent mitogen.61 Thus, proliferative responses in prostate cancer cells are not determined only by AR expression. AR stimulation by androgenic hormones also leads to increased expression of fatty acid synthase, an enzyme that is over expressed in prostate cancer clinical specimens and required for endogenous lipogenesis.62 This effect of androgens could be prevented by bicalutamide administration. At several laboratories LNCaP cells were subjected to longterm androgen ablation in vitro (table 2). Those studies yielded a consensus that prostate cancer cells adapt to steroid deprivation by up-regulating AR signaling.46, 47 If LNCaP cells are continuously cultured in the presence of low doses of androgen, there is no up-regulation of AR expression.63 In one of the sublines developed after chronic androgen ablation bicalutamide was able to stimulate transcriptional activity of the mutated receptor.47 In concordance with this observation tumor growth in vitro and in vivo was enhanced by that AR blocker. Although AR gene amplification occurs in a subgroup of patients who receive endocrine therapy for prostate cancer, that mechanism was not operative in long-term androgen ablated cells.64 AR protein expression is increased in patient tissues and in xenografts with an amplified AR gene.47, 64 Furthermore, AR hypersensitivity in the steroid depleted subline was not associated with the appearance of a new mutation. It could be hypothesized that alterations occur in the function of AR coactivators. However, it should be kept in mind that patients with AR amplification show a more favorable response to complete androgen ablation than patients with a nonamplified AR gene.65 Tumors with amplified AR might have a higher degree of differentiation than their counterparts. From the studies detailed in this section it is clear that the growth of prostate cancer cells

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is regulated by androgenic induction of AR downstream molecules involved in the G1/S transition. IMPACT OF AR MUTATIONS ON PROSTATE CANCER

Although AR mutations in prostate cancer have been studied since 1990, there are still some controversies concerning their frequency in patient material. Interestingly the 2 prostate cancer cell lines that respond to androgenic stimulation, LNCaP and MDA PCa, express mutated AR.66, 67 In clinical specimens there is a low rate of mutations in organ confined disease. In most studies the search for mutations was done in the ligand and DNA binding domains.68 In a report of Tilley et al it was proposed that there is a high percent of AR mutations in the N-terminal region even before the initiation of endocrine therapy.69 Recently AR mutations in the N-terminal region were reported in tumors derived from patients who progressed after treatment with orchiectomy and estramustine phosphate.70 It seems that there is no major difference in the transcriptional activity of N-terminal region mutant versus WT AR. Investigations of AR point mutations are hampered because the availability of material, especially metastatic lesions from patients, is limited. Several mutated receptors were re-created in expression vectors and transiently expressed in heterologous or in prostate cancer cell lines. In most cases AR point mutations broadened the activation spectrum, so that these mutated receptors could be more efficiently activated by various steroids and antihormones than WT AR. Estrogenic and progestagenic steroids, and hydroxyflutamide bind LNCaP AR with increased affinity and cause its strong activation and dissociation of heat shock proteins from the receptor, which is normally observed in the presence of androgens.66 In addition, those compounds stimulated the proliferation of LNCaP cells in vitro.71 However, the agonistic activity of hydroxyflutamide was not reflected by the induction of PSA gene expression. It should be pointed out that hydroxyflutamide is an agonist not only in LNCaP cells, but also with mutated AR 874 His3 Tyr from the xenograft CWR22. Mutated AR were also found in tissue specimens from patients who progressed after therapy with hydroxyflutamide.72 Interestingly those patients showed a temporary response after bicalutamide treatment. AR activation by adrenal androgens and dihydrotestosterone metabolites demonstrated in studies performed with several mutated ARs may facilitate prostate cancer growth. A mechanism relevant to prostate cancer progression in the presence of a mutated receptor was proposed based on studies in MDA prostate cancer cells. The doubly mutated AR (AR 701 Leu3 His and 877 Thr3 Ala) detected in MDA prostate cancer cells was efficiently stimulated by the glucocorticoids cortisol and cortisone, hormones that bind to the mutated receptor with high affinity.73 A mutation that was not detectable in an original tumor was recently discovered in a relapsed CWR22 xenograft.74 The mutation is an in-frame tandem duplication of exon 3 that encodes the second zinc finger of the AR DNA binding domain. The mutated receptor is trancriptionally less active than one in the parental cell line. AR mutation at codon 726 (AR 726 Arg3 Leu) is a rare example of a germline mutation present in 2% of Finnish patients with prostate cancer.75 Briefly, AR mutations are frequently colocalized in the 3 regions. They are 1) codons 701 to 730, which encompass the highly conserved loop between helices 3 and 4 of nuclear receptors, 2) codons 874 to 910, which flank activation function-2, the primary binding site for the p160 coactivators, and 3) codons 670 to 678, which are located at the boundary of the hinge and ligand binding domain, and may define a protein-protein interaction site. In most cases AR mutations are implicated in the progression of prostate cancer through enhanced activation by adrenal androgens, nonandrogenic steroids and antiandrogens.

CONCLUSIONS

Prostate cancers progress toward therapy resistance in most cases in the presence of functional AR. There are indications that the expression and function of some AR coactivators (SRC1, TIF2, RAC3 and p300/cyclic adenosine monophosphate response element binding protein binding protein) are altered in prostate cancer. It is important to continue research in this field by identifying other complexes that might be relevant to prostate cancer. High throughput approach tissue arrays may be useful in these experiments. Androgen ablation and drugs that block AR signaling are widely used in endocrine therapy for prostate cancer. During long-term treatment cells adapt to an environment with a low androgen supply by increasing AR expression and activity. Activation of mutated AR by glucocorticoids, adrenal androgens, dihydrotestosterone metabolites, estrogens, progestagenic steroids and antiandrogens also represents a mechanism by which tumor growth in late stages is facilitated. It is assumed that increased AR activity and aberrant stimulation by antiandrogens are associated with the antiandrogen withdrawal syndrome observed in a subgroup of patients with prostate cancer. AR activation by growth factors, cytokines and neuropeptides occurs in a ligand independent or synergistic manner and it is important in conditions in which androgen levels are lowered. Although our understanding of AR mediated events in prostate cancer is improved, progress in the achievement of prolonged stabilization of the disease is still missing. Novel prostate cancer therapies based on the inhibition of AR coactivators overexpressed in prostate cancer and involved in the regulation of mitogenic and antiapoptotic responses should be developed out. In early stages prostate carcinogenesis AR could be targeted in chemoprevention trials since it is known that various food ingredients down-regulate its expression. REFERENCES

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