Endocrine-Related Cancer (2002) 9 155–170

Androgen receptors in cancer

Z Culig1, H Klocker1, G Bartsch1 and A Hobisch1,2

1Department of Urology, University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria 2Department of Urology, General Hospital Feldkirch, Austria (Requests for offprints should be addressed to Z Culig; Email: [email protected])

Abstract The receptor (AR), a transcription factor that mediates the action of in target tissues, is expressed in nearly all prostate cancers. Carcinoma of the prostate is the most frequently diagnosed neoplasm in men in industrialized countries. Palliative treatment for non-organ-confined aims to down-regulate the concentration of circulating androgen or to block the transcription activation function of the AR. AR function during endocrine therapy was studied in tumor cells LNCaP subjected to long-term steroid depletion; newly generated sublines could be stimulated by lower concentrations of androgen than parental cells and showed up-regulation of AR expression and activity as well as resistance to apoptosis. Androgenic regulate the expression of key cell cycle regulators, cyclin-dependent kinase 2 and 4, and that of the cell cycle inhibitor p27. Inhibition of AR expression could be achieved by potential chemopreventive agents flufenamic acid, resveratrol, quercetin, polyunsaturated fatty acids and interleukin-1β, and by the application of AR antisense oligonucleotides. In the clinical situation, AR gene amplification and point mutations were reported in patients with metastatic disease. These mutations generate receptors which could be activated by other steroid hormones and non-steroidal antiandrogens. In the absence of androgen, the AR could be activated by various growth-promoting (growth factors, epidermal growth factor receptor-related oncogene HER-2/neu) and pleiotropic (protein kinase Aactivators, interleukin-6) compounds as well as by inducers of differentiation (phenylbutyrate). AR function is modulated by a number of coactivators and corepressors. The three coactivators, TIF-2, SRC-1 and RAC3, are up-regulated in relapsed prostate cancer. New experimental therapies for prostate cancer are aimed to down-regulate AR expression and to overcome difficulties which occur because of the acquisition of agonistic properties of commonly used antiandrogens. Endocrine-Related Cancer (2002) 9 155–170

Androgen receptor structure Residues in the ligand-binding domain are also impli- cated in the regulation of transcription and are designated The (AR) is a transcription factor that reg- AF-2. That part of the AR recruits a group of coregulatory ulates the expression of genes required for normal male proteins, p160 coactivators (e.g. steroid receptor coacti- sexual development and maintenance of the function of vator-1 (SRC-1)) in a -dependent manner (Bevan et accessory sexual organs. In the absence of ligand, AR acti- al. 1999). However, these coactivators which are up- vation is prevented by several heat-shockproteins. The AR regulated in prostate carcinoma interact with the glutamine- is composed of three main parts: well-conserved central DNA- and ligand-binding domains which are separated by a rich region in the N-terminus independently of androgenic hinge region responsible for the translocation of the AR from hormones. AR functional activity is greatly determined by cytoplasm to the nucleus and a less conserved N-terminal interactions between the N-terminal and carboxyl-terminal region, which contains a variable number of polyglutamine (ligand-binding domain) domains (Ikonen et al. 1997, He et (their number varies from 17 to 29) and polyglycine repeats al. 1999). AR agonists and antagonists can be reliably distin- involved in the regulation of transcriptional activity of the guished on the basis of their ability to enhance the N/C inter- AR (Fig. 1). Racial differences in the repeat lengths have actions (Kemppainen et al. 1999). Loss of AR function might been investigated in molecular epidemiological studies. occur because of the presence of mutations that disrupt the Shorter polyglutamine repeats are associated with an N/C interaction although they do not change AR-binding increased transcriptional activity of the AR. N-terminal affinity (Langley et al. 1998, Thompson et al. 2001). AR region residues 141–338 are the main location for the tran- agonists and antagonists differentially regulate receptor phos- scription activation function-1. phorylation. In general, the phosphorylation levels correlate

Endocrine-Related Cancer (2002) 9 155–170 OnlineDownloaded version from via Bioscientifica.com http://www.endocrinology.org at 09/26/2021 08:41:44AM via free access 1351-0088/02/009–155  2002 Society for Endocrinology Printed in Great Britain Culig et al.: Androgen receptors in prostate cancer

Figure 1 Androgen receptor (AR) structural organization. The three main regions, ligand-binding domain (LBD) and DNA-binding domain (DBD), and N-terminal region are depicted. Proteins which interact with the AR and modulate its activity are shown. with the induction of reporter gene activity (Wang et al. treatment of prostate cancer vary in different countries, from 1999). consequent screening and radical prostatectomy to watchful The DNA-binding domain is organized in two zinc fing- waiting. Watchful waiting is an approach in which the treat- ers; each of them being composed of four cysteine residues ment is deferred because of the fact that not all prostate can- bound to a zinc ion. The AR gene consists of nine exons: the cers become clinically manifested (Schmid et al. 2001). Thus N-terminal region is entirely encoded by exon 1, exons 2 and some patients die with their prostate cancer but not because 3 encode the DNA-binding region and the remaining five of prostate cancer. It is hoped that application of new tech- exons encode the ligand-binding domain. The DNA-binding nologies in prostate cancer research will allow identification domain mediates binding to specific sequences on DNA and of markers of more aggressive tumors which could be sub- is involved in receptor dimerization (Wong et al. 1993). jected to eradication therapy. Radical surgery and radiother- Gross deletions in the ligand-binding domain of the AR gen- apy are curative in the early stages of prostate cancer. Hor- erate receptors which are constitutively active (Jenster et al. monal dependence of prostate cancer was first recognized by 1991). Huggins & Hodges (1941). They showed that the removal of Structurally, the human AR is very similar to the human androgens leads to regression of prostate cancer. This recog- glucocorticoid or progesterone receptors. The three receptors nition was a basis for therapy for non-organ-confined prostate recognize the same DNA response element. However, differ- tumors. Androgen ablation could be performed either surgi- ences in hormone-specific action in target tissues occur. For cally by orchiectomy or by administration of gonadotropin example, the probasin gene is induced by androgenic hor- hormone-releasing hormone analogues. In addition, AR func- mones and not by glucocorticoids. This high affinity for the tion could be inhibited by either steroidal (cyproterone probasin–androgen response element is determined by the acetate) or non-steroidal (hydroxyflutamide and three residues in the DNA-binding domain of the AR bicalutamide) compounds which prevent the acquisition of (Schoenmakers et al. 2000). There are also differences the transcriptionally active form of the receptor. One might between the AR, glucocorticoid or progesterone receptors in expect that there is a more efficient inhibition of prostate terms of ligand-independent activation and this issue will be cancer progression by combination of androgen ablation and discussed in detail in the present review. Interestingly, gluco- blockade of AR activity. This combined androgen blockade corticoid hormones are not capable of stimulating growth of could, for example, neutralize the effects of adrenal andro- prostate cancer LNCaP cells even after transfection of gluco- gens which are not eliminated by castration. The question as corticoid receptor cDNA (Cleutjens et al. 1997). In contrast, to whether combined androgen withdrawal has a clinical ben- dexamethasone stimulated the expression of the prostate- efit is still controversially discussed. In this context, it is specific antigen (PSA) gene in LNCaP sublines generated by worthwhile noting that in recently published studies such a stable transfection of glucocorticoid receptor cDNA. These benefit was not observed (Eisenberger et al. 1998). findings point to the complexity of AR functional regulation Prostate growth is regulated by complex interactions by androgens and glucocorticoids respectively. between steroids, peptide growth factors and cytokines, and Expression and function of the AR are frequently studied dysregulation of the expression of these molecules and their in cell lines or tumor specimens obtained from patients with receptors occurs, especially in late tumor stages. The prolifer- androgen sensitivity syndromes or prostate cancer. ation rate of prostate cancer cells is rather low and therefore chemotherapy is of little value in the treatment of advanced Prostate cancer tumor biology prostate cancer (Berges et al. 1995). A characteristic feature of metastatic prostate cancer is the inability of cells to Prostate cancer is the most commonly diagnosed neoplasm undergo programmed cell death, apoptosis. In this context, it in men in the Western world. Policies on the detection and is known that several growth factors (e.g. insulin-like growth

Downloaded from Bioscientifica.com at 09/26/2021 08:41:44AM via free access 156 www.endocrinology.org Endocrine-Related Cancer (2002) 9 155–170 factor-I (IGF-I)) and cytokines (interleukin-6 (IL-6)) act as al. 1991, Van der Kwast et al. 1991). Those findings were survival factors in carcinoma of the prostate (Chung et al. confirmed in prostate cancer metastases obtained from 2000, Sprenger et al. 2002). In addition to conventional anti- patients before and after endocrine treatment (Hobisch et al. androgen therapy, there have been various attempts to intro- 1995, 1996). duce novel experimental treatments for prostate cancer, such as antisense oligonucleotides against the suppressor of AR expression and activity during apoptosis bcl-2 (Miyake et al. 2000a), testosterone-repressed endocrine treatment prostate message-2 (clusterin) (Miyake et al. 2000b), or anti- epidermal growth factor (EGF) antibodies (Ye et al. 1999). It is obvious that either androgen ablation and/or application Prostate cancer is a heterogenous neoplasm and it is therefore of AR antagonists lead to a reduction of tumor volume and believed that only combined therapeutic approaches will be an improvement in symptoms in most of the prostate cancer beneficial in advanced disease. Prostate cancers preferentially patients. It was assumed that alterations which occur during produce metastases in bone and their spread is facilitated by long-term endocrine treatment would be better understood if prostate and bone fibroblasts (Gleave et al. 1991). prostate cancer cells undergo prolonged androgen ablation in vitro. Long-term steroid depletion was performed in human Historical aspects of AR research prostate cancer cells LNCaP which are the most frequently used model in prostate cancer research (Kokontis et al. 1994, There are only a limited number of prostate cancer models Culig et al. 1999, Gao et al. 1999a). An early event after available. This fact greatly hampers research on AR expres- steroid hormone withdrawal of LNCaP cells was a retar- sion and function in carcinoma of the prostate. There have dation of cellular proliferation which was followed by acti- been many attempts to establish a reliable primary prostate vation of various adaptation mechanisms. There are many culture system in which androgenic responsiveness could be similarities between sublines of LNCaP cells generated in studied. For several reasons, it has been difficult to achieve different laboratories after long-term androgen ablation. A this goal. Some primary epithelial cultures yielded only a characteristic feature of those cells is recovery in the basal limited number of passages and others showed a down- proliferation rate and an increased sensitivity to low doses of regulated AR expression. For a long time, rat Dunning androgens. The typical growth curve of parental LNCaP cells tumors were used in different experiments including studies is biphasic; low doses of androgen stimulate proliferation on the AR. Dunning tumor sublines show different histologi- whereas higher concentrations cause a progressive decline in cal patterns, varying from well-differentiated and relatively cell growth (Lee et al. 1995). In contrast, LNCaP secretory slow-growing neoplasms to highly aggressive tumors which function is enhanced by androgenic hormones in a dose- metastasize predominantly to lymph nodes and lungs (Isaacs dependent manner. However, this regulation is still only par- et al. 1986). AR status in Dunning tumor sublines was inves- tially understood; it has been proposed that the proliferation tigated by Quarmby and associates (1990). In general, AR is mediated by IL-6 and inhibition by transforming growth expression decreases in cell lines which show metastatic factor-β (TGF-β)(Kimet al. 1996, Okamoto et al. 1997a). properties. The same phenomenon was seen in the two Both regulatory mechanisms are, however, a matter of human cell lines derived from metastatic lesions, PC-3 and debate; for IL-6 both stimulatory and inhibitory effects on DU-145, in which AR expression is very low or undetectable LNCaP growth have been reported (Degeorges et al. 1996, respectively (Tilley et al. 1990). Early studies on AR in Giri et al. 2001) and contrasting results on the expression of human prostate tissue were carried out using radioligand TGF-β receptors have been published (Guo & Kyprianou binding assays (Gorelic et al. 1987). Because of the afore- 1998). The growth of a subline of LNCaP cells developed in mentioned prostate tumor tissue heterogeneity, the use of the authors’ laboratory was stimulated by the non-steroidal AR-binding assays as a prognostic factor has not been estab- antiandrogen bicalutamide which also enhanced transcrip- lished. In this respect, prostate cancers differ from breast tional activity of the AR (Culig et al. 1999) (Table 1). A tumors in which measurements of and progesterone similar switch of bicalutamide from antagonist to agonist was receptors have been accepted for diagnostic procedures. recently reported after long-term treatment with tumor Considerable progress in investigations on AR expres- necrosis factor-α (Harada et al. 2001). AR expression and sion was achieved after the AR cDNA sequence was pub- activity increase after long-term androgen ablation and this lished (Chang et al. 1988, Trapman et al. 1988, Tilley et al. adaptation mechanism might be very significant in the clini- 1989). A number of monoclonal and polyclonal antibodies cal situation. Although AR activity is higher in long-term have been generated and used for studies on tumor material. steroid-deprived than in parental cells, the AR-regulated PSA In the early 1990s, it was demonstrated that the AR is gene is down-regulated (Gao et al. 1999a) (Fig. 2). This sug- expressed in relapsed prostate cancer and that its expression, gests that there is a cellular dedifferentiation during pro- as determined on semiquantitative evaluation, does not corre- longed steroid depletion. Clearly, it is difficult to generalize late with time to progression after endocrine therapy (Sadi et all these findings which were obtained with one prostate

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Table 1 Induction of CAT activity in LNCaP and LNCaP-abl cells transfected with the androgen-inducible plasmid

ARE2TATA-CAT. (Reprinted from Culig et al. 1999 with permission from the Cancer Research Campaign.)

Compound Cell line (fold induction of CAT activity over basal level ± S.D.) LNCaP LNCaP-abl R1881 (0.01 nM) 1.2 ± 0.1 3.8 ± 0.2 R1881 (0.1 nM) 3.5 ± 0.5 14.4 ± 1.1 R1881 (1 nM) 33.2 ± 2.2 42 ± 1.5 OHF (100 nM) 2.5 ± 0.1 6.3 ± 0.8 OHF (1 µM) 3.8 ± 0.5 9.1 ± 1.2 OHF (10 µM) 4.5 ± 0.3 18.3 ± 2.2 R1881 (1 nM) + OHF (1 µM) 31.6 ± 2.2 35 ± 2.8 Bic (100 nM) 0.9 ± 0.1 1.8 ± 0.2 Bic (1 µM) 0.9 ± 0.2 2.2 ± 0.2 Bic (10 µM) 1.1 ± 0.1 2.8 ± 0.4 R1881 (1 nM) + Bic (1 µM) 3.2 ± 0.6 39.2 ± 5.2 OHF, hydroxyflutamide; Bic, bicalutamide. cancer cell line that expresses a mutated AR. A new AR- et al. 1998). The AR is implicated in the up-regulation of the positive cell line, MDA PCa 2a, was derived from a prostate stromal keratinocyte growth factor (KGF) which binds to the cancer bone (Navone et al. 1997). However, that epithelial receptor and is therefore considered a mediator of AR contains two point mutations whose features will be dis- androgen action (Yan et al. 1992). Similar properties were cussed later. It should be kept in mind that long-term steroid- reported for the related fibroblast growth factor-10 (Lu et al. deprived cells become resistant to induction of apoptosis by 1999). Other examples of how androgenic hormones interact retinoic acid, taxol and adriamycin (Gao et al. 1999a). with growth factors are up-regulations of the EGF receptor Recent research has improved the understanding of the (Schuurmans et al. 1988) and vascular endothelial growth mechanisms by which androgenic hormones regulate pro- factor (Joseph et al. 1997, Levine et al. 1998). Targeted liferation and survival. Transition through the G1 phase of expression of an AR transgene led to the development of the cell cycle is governed by cyclin-dependent kinases (cdk) focal areas of intraepithelial neoplasia which is considered to 4/6-cyclin D complexes and by cdk2-cyclin E complexes be a precursor to prostate cancer (Stanbrough et al. 2001). which peakat the G1 to S transition. Androgenic up- Long-term androgen ablation was also associated with regulation of cdk2 and 4 has been described at mRNA and the down-regulation of the cell cycle inhibitor p21 (Wang et protein levels (Lu et al. 1997). In the prostate cancer xeno- al. 2001a). Treatment of an androgen-independent LNCaP graft CWR-22, the levels of cdk1 and 2, cyclin A and B1 subline with AR antisense oligonucleotides led to re- mRNA decreased after castration and increased after sup- establishment of the expression of p21 and to partial rever- plementation with testosterone propionate (Gregory et al. sion of the androgen-independent phenotype. It is known that 2001a). The levels of those cell cycle regulators in recurrent a number of cytokines and food ingredients down-regulate tumors were high in the absence of testicular androgen, thus expression and/or activity of the AR and its target gene PSA. suggesting that compounds other than androgen activate the Such inhibitory effects of IL-1β were observed in experi- AR. Low androgenic concentrations induce an increase of ments with monocyte-conditioned media. Conditioned media phosphorylation of retinoblastoma protein and the expression caused a reduction of the proliferation of LNCaP cells and of the transcription factor E2F-1 and its target gene product down-regulation of the AR protein (Culig et al. 1998). These cyclin A (Hofman et al. 2001). Consistent with the inhibitory effects were abolished when the media were pretreated with proliferative response, high concentrations of the synthetic a neutralizing anti-IL-1β antibody. Similar to monocyte- androgen R1881 cause retinoblastoma hypophosphorylation, conditioned media, those obtained from cultured activated down-regulation of E2F, and induction of the expression of T-lymphocytes inhibited LNCaP proliferation and AR the cell cycle inhibitor p27 (KIP1). Stimulation of p27 by expression (Hsieh et al. 1995). However, compounds respon- androgen was also reported in long-term androgen-ablated sible for these effects were not identified. Polyunsaturated LNCaP cells (Kokontis et al. 1998). In the case of the fatty acids, such as docosahexanoic acid and eicosapentanoic CWR-22 xenograft, tumor regression was associated with a acid, inhibit AR activity and could be considered for chemo- sustained increase in p27 expression rather than with changes prevention trials (Chung et al. 2001). Other potential chemo- in the expression of regulators of survival (Agus et al. 1999). preventive agents, the anti-inflammatory agent flufenamic In this context, it is important to note that many prostate acid, the red wine compound resveratrol, and the natural fla- cancers lackp27 expression (Guo et al. 1997, Cordon-Cardo vonoid quercetin, cause down-regulation of AR expression

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these studies that stable expression of the AR leads to a retar- dation of growth of the cells (Yuan et al. 1993, Heisler et al. 1997, Bonaccorsi et al. 2000, Shen et al. 2000). Moreover, in PC-3 cells which stably express the AR there was a down- regulation of molecules responsible for the invasive pheno- type, namely α6β4 integrins (Bonaccorsi et al. 2000). Andro- gen treatment of PC-3/AR cells resulted in an increased expression of neutral endopeptidase, an enzyme that cleaves and inactivates neuropeptides which are implicated in the growth of advanced prostate cancers (Shen et al. 2000). Results on AR expression and function obtained with long-term androgen-ablated LNCaP cells are interesting because of the clinical evidence of AR gene amplification (Visakorpi et al. 1995, Cher et al. 1996). A subgroup of patients with therapy-resistant prostate cancer present with an increased number of copies of the AR gene and, conse- quently, these tumors have a higher content of the AR protein (Linja et al. 2001). Thus, it is likely that AR gene amplifica- tion is one of the mechanisms to explain the development of hypersensitivity to stimulation by low androgenic concentra- tions. Mechanisms which are related to structural alterations will be discussed in detail in the next section. In long-term androgen-ablated cells, AR expression increases either because of up-regulation at the mRNA level (Kokontis et al. 1994) or increased stability of the protein, as evidenced in the prostate cancer xenograft CWR 22 (Gregory et al. 2001b). In the LNCaP tumor model, amplification of the AR gene was not observed in hypersensitive sublines (Kokontis et al. 1998, Culig et al. 1999). While prostate cancers show significant heterogeneity, some tumor cells show a loss of AR expression because of methylation of CpG sites in the AR promoter (Jarrard et al. 1998). AR promoter contains hot spots (131 to −121 and +44 Figure 2 Northern blot analysis of steady-state levels of to +54) that are markers for gene silencing (Kinoshita et al. prostate-specific antigen (PSA), prostate-specific membrane 2000). antigen (PSM), cytokeratin 8 (CK8) and AR mRNAs in AD and AI cells grown in a defined medium (MEGM). AD and AI cells were plated at 5×105 per 100 mm dish in RPMI with AR structural alterations in prostate 10% fetal bovine serum and switched to MEGM. After 30 h of cancer incubation, the cells were harvested, the total RNAwas extracted, and 15 µg of each sample was separated on a 1% As mentioned previously, the AR is mutated in the LNCaP agarose. After blotting, the filter was sequentially hybridized cell line and the discovery of the mutation has greatly influ- with 32P-labeled PSA, PSM, CK8, AR, and glyceraldehyde-3- enced research in that field (Harris et al. 1990, Veldscholte phosphate dehyrogenase (GAPDH) cDNA probes. (Reprinted from Gao et al. (1999). Reprinted by permission of et al. 1990a). The mutation is located in exon H of the AR Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.) gene and leads to an exchange of the wild-type threonine (position 877) to alanine. It has been demonstrated in several studies that hormones that bind to the mutated LNCaP AR and activity (Mitchell et al. 1999, Xing et al. 2001, Zhu et with increased affinity stimulate cellular proliferation and al. 1999). enhance transcriptional activation function of that AR However, the antiproliferative activity on LNCaP cells (Veldscholte et al. 1990a,b, 1992, Montgomery et al. 1992, is also observed in conditions in which AR expression is Tan et al. 1997). These hormones are estrogenic and proges- up-regulated, as evidenced after treatment with 1α,25- tagenic steroids, adrenal androgens, and the non-steroidal dihydroxyvitamin D3 (Zhao et al. 1997). Human prostate antiandrogens hydroxyflutamide and nilutamide. The proges- cancer cells PC-3 have been stably transfected with AR tagenic compound cyproterone acetate was one of the first cDNA in several laboratories. There is a consensus from agents introduced in prostate cancer treatment. For some

Downloaded from Bioscientifica.com at 09/26/2021 08:41:44AM via free access www.endocrinology.org 159 Culig et al.: Androgen receptors in prostate cancer unclear reasons, antiandrogen regulation of LNCaP AR activity does not parallel that of the PSA protein. It was observed that hydroxyflutamide is still capable of down- regulating PSA although it increases AR-mediated reporter gene activity (Young et al. 1991, Montgomery et al. 1992). Interestingly, agonistic effects of hydroxyflutamide were not evident in LNCaP cells which overexpressed the proapop- totic molecule Bax (Andriani et al. 2001). This raises the possibility that Bax acts as an AR corepressor in prostate cancer. After elucidation of the crystal structure of the human AR, the reasons why the LNCaP AR responds to androgen in a manner similar to that of the wild-type AR became clear (Matias et al. 2000). Due to the mutation, a hydrogen-bound partner for the 17β hydroxyl group in R1881 would be 705 missing. However, the amino acid residue Asn could still Figure 3 AR mutation in the hormone-binding domain. Total orientate the ligand in the ligand-binding pocket. Threonine RNAwas purified from a fine-needle biopsy obtained from a 877 which contacts the ligand directly is mutated in LNCaP patient with a therapy-resistant prostatic carcinoma. RNAwas cells and the mutation changes the stereochemistry of the reverse transcribed to cDNA, and the AR fragments were binding pocket, thus leading to a broadened ligand specificity amplified by PCR, isolated, and directly sequenced. This (McDonald et al. 2000). In MDA PCa 2a cells, which were figure shows the section around the mutated base 2671. (Top) Normal AR sequence (N). (Bottom) Mutant AR found in derived from bone metastases of prostate cancer, the AR con- a specimen from a therapy-resistant, metastatic carcinoma tains the LNCaP mutation and the second one which is a (PC). (Reprinted from Culig et al. (1993). Reprinted by substitution of amino acid leucine 701 with histidine (Zhao permission of The Endocrine Society.) et al. 1999). Transcription activation function of the MDA PCa 2a AR is strongly induced by the glucocorticoid hor- mones, cortisol and cortisone (Zhao et al. 2000), which pro- either cannot bind androgen or show a reduced transcrip- mote the growth of that cell line. In the prostate cancer xeno- tional activity, AR mutations are, in most prostate cancer graft CWR 22, there is an exchange of histidine at position cases, non-inherited and promiscuous. Several well- 874 with tyrosine (Tan et al. 1997). Histidine 874 does not characterized AR point mutations in the ligand-binding contact ligand and the mutation found in the prostate cancer domain are located in the region responsible for recognition xenograft most probably affects binding of AR coactivators and specificity of ligand (Wurtz et al. 1996). The function of and their regulation of AR function (McDonald et al. 2000). the AR in human prostate cancer has frequently been investi- There is an agreement in the scientific community that gated by cotransfection-transactivation assays in heterolog- AR point mutations are not common in the early stages of ous cells in which a high transfection efficacy could be prostate cancer (Culig et al. 1993a,b, Elo et al. 1995, Evans achieved, such as monkey kidney COS and CV-1 cells, as et al. 1996, Marcelli et al. 2000). They could be detected in well as in prostate cancer cells PC-3 or DU-145. Aberrant prostatic specimens obtained from patients with metastatic activation of the AR might be associated with an increased disease and in samples from bone metastases (Culig et al. ligand-binding affinity, as described for the LNCaP AR 1993b, Taplin et al. 1995) (Fig. 3). Analysis of tissue speci- (Veldscholte et al. 1990a), or occurs without appreciable mens from the stable phase of the disease and ones obtained changes in binding characteristics (Culig et al. 1993b). after the onset of tumor progression in the case of the Among substances which stimulate the activity of mutated mutated AR 715 ValǞMet has revealed the mutation solely AR in prostate cancer, testosterone precursors (adrenal in the latter tissue (Culig et al. 1993b). To exclude the pos- androgens) and metabolites, as well as antiandrogens, are sibility that AR mutations remain undetected because of their particularly important. AR activation by naturally occurring presence in a very small percentage of cells, Marcelli and androgenic precursors and metabolites is described for the associates (2000) analyzed the mutations in microdissected LNCaP AR, AR 715 ValǞMet, 730 ValǞMet, and 874 material. They discovered AR structural changes in 21% of HisǞTyr (Culig et al. 1993b, Peterziel et al. 1995, Tan et al. patients with metastatic disease whereas no mutation was 1997). Hydroxyflutamide and bicalutamide were considered detectable in samples obtained from patients who did not more promising antiandrogenic drugs than cyproterone acet- present with metastases. Those results are in a good agree- ate because of their non-steroidal structure. They both bind ment with the findings of Taplin and associates (1995) who to the AR with a low affinity and prevent androgen-induced first reported a high frequency of AR structural alterations in conformational change of the receptor (Culig et al. 1993b, bone metastases. In contrast to AR mutations in androgen- Kallio et al. 1994). However, hydroxyflutamide acts as a insensitivity syndromes which generate receptors which partial agonist even in the presence of the wild-type AR as

Downloaded from Bioscientifica.com at 09/26/2021 08:41:44AM via free access 160 www.endocrinology.org Endocrine-Related Cancer (2002) 9 155–170 evidenced in reporter gene assays and conformational studies paracrine (KGF) (Yan et al. 1992) and/or both autocrine and (Peterziel et al. 1995, Wong et al. 1995). Micromolar con- paracrine manner (EGF, IGF-I) (Connolly & Rose 1994, centrations of hydroxyflutamide which are efficient in Nickerson et al. 2001). However, it should be emphasized reporter gene assays were measured in sera of prostate cancer that ligand-independent activation of the AR is not neces- patients (Belanger et al. 1988). In patient material, mutations sarily associated with tumor cell proliferation. Compounds which could be aberrantly activated by hydroxyflutamide that activate the AR include peptide hormones, which were detected more frequently than those activated by bicalu- increase intracellular cAMP and IL-6 which are pleiotropic tamide (Taplin et al. 1999). Moreover, some patients in regulators of cell growth (Nazareth & Weigel 1996, Hobisch whom hydroxyflutamide acts as an agonist show a response et al. 1998), and phenylbutyrate, which is a prodifferentiation to the second-line treatment with bicalutamide. Recently, agent used in experimental therapies for prostate cancer three missense mutations were discovered in patients who (Sadar & Gleave 2000). Vitamin D also caused inhibition of received a combined androgen blockade by orchiectomy and cellular proliferation in association with the induction of PSA bicalutamide (Haapala et al. 2001). Interestingly, there was expression, an event which was blocked by bicalutamide no evidence of AR gene amplification in patients whose (Zhao et al. 1997). In the same series of experiments in tumors relapsed after endocrine treatment. which the activator of the protein kinase A pathway forskolin AR alterations are occasionally germ-line mutations. The up-regulated AR activity in a ligand-independent manner, the mutation Arg726ǞLeu was detected in 2% of Finnish pros- human progesterone receptor was activated by progesterone tate cancer patients whereas only 0.3% of healthy blood and forskolin in a synergistic fashion (Nazareth & Weigel donors have that mutated AR (Elo et al. 1995, Mononen et 1996). However, the outcome of experiments on ligand- al. 2000). There is a possibility that enhanced stimulation of independent activation of the AR depends on the cell type that receptor with androgenic hormones and estradiol leads and promoter used. For example, Reinikainen and associates to overexpression of genes involved in proliferation and/or (1996) have demonstrated an enhancement of the androgenic inhibition of apoptosis. effect on reporter gene activity by EGF in non-prostatic cells. In clinics, the frequency and implications of the anti- In primary cells of the developing mouse reproductive tract, androgen withdrawal syndrome have been frequently both ligand-independent and ligand-dependent effect of EGF debated. According to an initial observation by Scher & on AR activity were reported (Gupta 1999) (Figs 4 and 5). Kelly (1993), improvement of clinical symptoms and decline Consistent with previous results by Culig et al. (1994) and of PSA after discontinuation of hydroxyflutamide was seen Gupta (1999), EGF was less potent than androgen in the in about one-third of patients. This improvement is, however, modulation of reporter gene activity (Gupta 1999). In cells only temporary. Similar observations were reported for other which were transfected with AR cDNA and reporter gene, drugs used in endocrine therapy for prostate cancer (Small & there was no change in AR expression by cAMP derivatives Carroll 1994, Akakura et al. 1995, Dawson & McLeod 1995, (Nazareth & Weigel 1996). This is in contrast to the regu- Nieh 1995, Gomella et al. 1997, Sella et al. 1998, Laufer et lation of AR by IL-6 in LNCaP cells which involves up- al. 1999). Unfortunately, there is no AR structural analysis regulation of AR mRNA and protein, and stimulation of available in a large number of patients who experience the reporter gene activity in experiments in which the AR pro- withdrawal syndrome and therefore it is difficult to judge moter is coupled to a reporter gene (Lin et al. 2001a). AR which mechanisms are responsible for the occurrence of the activation by the EGF receptor-related molecule HER-2/neu antiandrogen withdrawal syndrome. was described by Craft and colleagues (1999). The experi- ments carried out in the LAPC-4 prostate cancer xenograft, Cross-talk with other signaling pathways which expresses the wild-type AR, clearly demonstrated that the overexpression of HER-2/neu leads to the promotion of The AR is structurally similar to the human progesterone and tumor growth and PSA expression. In concordance with glucocorticoid receptors. The two latter receptors exhibit these findings, it was shown that the mitogen-activated pro- ligand-dependent synergistic activation by ligand and low tein kinase (MAPK) pathway is required for AR activation doses of non-steroidal compounds, such as growth factors, by HER-2/neu and that serine 514 in the N-terminal part is peptide hormones that increase intracellular cAMP, or neuro- a target for MAPK phosphorylation (Yeh et al. 1999). transmitters. For this reason, it was not expected that the AR Involvement of the MAPK pathway in AR activation was would show a ligand-independent activation. After several also demonstrated for IL-6 (Hobisch et al. 1998). Besides years of intensive research in this area, it became clear that AR, estrogen receptor-β is an important regulator of the the AR could be activated in a ligand-independent manner growth of prostate epithelium. Androgenic and estrogenic by different substances. Activation of the AR in the absence steroids induced a complex between the AR, estrogen recep- of ligand was first demonstrated in DU-145 cells for growth tor-β and the Src protein, which provides a linkto the MAPK factors IGF-I, KGF, and EGF (Culig et al. 1994). These pathway (Migliaccio et al. 2000). AR activation in the growth factors are thought to stimulate prostate growth in a absence of ligand was demonstrated for MAPK kinase kinase

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Figure 4 Effect of testosterone (T) and epidermal growth factor (EGF) on the luciferase reporter activity of the reproductive tract cells transfected androgen response element (ARE) vector in the presence and absence of AR vector. −AR represents the experiments performed in the absence of the AR vector. The results were normalized against β-galactosidase reporter activity in each assay and the data represent means ± S.D. from four different sets of experiments. *P<0.05 compared with vehicle-treated control cells by Student’s t-test. RLU, relative luciferase units. (Reprinted from Gupta (1999). Reprinted by permission of Elsevier Science.)

1 whose overexpression caused apoptosis in prostate cancer cells (Abreu-Martin et al. 1999). The physiological significance of AR activation by a non-steroidal compound is more convincing if expression of an endogenous AR target gene is up-regulated. Examples of such an effect include stimulation of PSA gene expression by IGF-I and IL-6 in LNCaP cells (Culig et al. 1994, Hobisch et al. 1998, Chen et al. 2000) or by forskolin in prostate explants (Nakhla et al. 1997). Thus, non-steroidal activation of the AR is pathophysiologically relevant and it is not a simple reflection of overexpression of AR cDNA. In previous sections of this review, it was emphasized that AR antagonists could act as agonists because of AR hyp- ersensitivity or the presence of mutated receptors. The ability of these drugs to antagonize non-steroidal AR activation was demonstrated with peptide growth factors, forskolin, IL-6, butyrate, and luteinizing hormone-releasing hormone (Culig Figure 5 Effect of EGF on the luciferase reporter activity in et al. 1994, 1997, Nazareth & Weigel 1996, Hobisch et al. the presence and absence of testosterone (T). −AR 1998, Sadar & Gleave 2000). However, they were less effi- represents the experiments performed in the absence of AR cient in the presence of HER-2/neu and phorbol ester which vector. The conditions of the experiments are the same as suggests that they cannot down-regulate AR activity in con- ± = described in Fig. 4. The results represent means S.D., n 4. ditions in which the MAPK pathway is hyperactive (Darne *P<0.05 compared with the vehicle-treated control; **P<0.005 compared with testosterone response by Student’s t-test. et al. 1998, Craft et al. 1999, Yeh et al. 1999). (Reprinted from Gupta (1999). Reprinted by permission of Among the regulators of AR activity, IL-6 has a particu- Elsevier Science.) larly important role in prostate tumor biology. Its levels are

Downloaded from Bioscientifica.com at 09/26/2021 08:41:44AM via free access 162 www.endocrinology.org Endocrine-Related Cancer (2002) 9 155–170 elevated in the sera of patients with advanced carcinoma of enhancement of AR activity by different coregulatory pro- the prostate and the cytokine’s expression is up-regulated teins are similar and (b) there is a certain redundancy in their even in tissue extracts obtained from patients with non- action so that inhibition of function of one coregulator could, metastatic prostate cancer (Twillie et al. 1995, Adler et al. most probably, be compensated. 1999, Giri et al. 2001). Divergent results on IL-6 regulation The role of the first coactivator which was discovered of proliferation of LNCaP cells have been reported and these in prostate cancer cells DU-145, ARA 70, is controversially differences most probably occur because of the use of differ- discussed (Yeh & Chang 1996). The same group has pro- ent passages of LNCaP cells and subtle differences in cell posed that ARA 70 is implicated in AR stimulation by estra- culture conditions (Degeorges et al. 1996, Okamoto et al. diol and non-steroidal antiandrogens (Miyamoto et al. 1998, 1997b, Chung et al. 1999, Giri et al. 2001). In the authors’ Yeh et al. 1998). Initial reports that this protein specifically laboratory, parental LNCaP cells were constantly inhibited enhances activity of the AR but not that of other steroid by IL-6 (Hobisch et al. 1998, 2001). After long-term treat- receptors were disputed by Alen et al. (1999) and Gao et al. ment with IL-6, the cells conferred a growth advantage in (1999b). According to those reports, the up-regulation of AR vitro and could not be growth inhibited by exogenous IL-6. activity by ARA 70 was not substantially higher than the Interestingly, AR activation was preserved in the newly gen- enhancement of activity caused by other coactivators. An erated LNCaP-IL-6+ cell line, most probably because of the important issue for studies on AR coactivators is determi- up-regulation of AR expression. IL-6 and its receptor are nation of AR expression in cells in which a coactivator expressed in benign and malignant prostate and therefore it cDNA has been introduced. Hofman and associates (2000) was concluded that IL-6 autocrine and paracrine loops occur have recently shown that some of the apparent coactivators, (Hobisch et al. 2000). The cross-talkbetween the pathways in fact, cause the up-regulation of AR protein expression of IL-6 and AR is bidirectional: it was reported that andro- (Fig. 6). A role for ARA 70 has been recently suggested in genic hormones are able to enhance the expression of acute an autochthonous transgenic carcinoma of the prostate (Han phase response genes (Matsuda et al. 2001). Consistent with et al. 2001). ARA 70 enhanced activation of the mutated these findings, expression of the protein inhibitor of STAT3 murine AR containing the substitution Glu231ǞGly in signaling (PIAS) reduced AR transcriptional activity response to androgen and estradiol, whereas another coacti- (Junicho et al. 2000). STAT3 is the major intermediary IL-6 vator ARA 160 was not effective with estradiol. As expected, signal transducer. Recent findings indicate that the PIAS pro- cotransfection of two AR coactivators frequently yields teins differentially affect AR activity and may cause both its further additive or synergistic effect on AR functional enhancement or its inhibition (Gross et al. 2001). In AR- activity (Yeh et al. 2000, Wang et al. 2001b). negative prostate cancer cells, IL-6 acts as a survival factor An unexpected effect of D-type cyclins on AR transcrip- through the phosphatidyl inositol 3-kinase pathway (Chung tional activity was reported. Cyclin D1 acts as a transcrip- et al. 2000). tional coactivator for estrogen receptor (Zwijsen et al. 1998) The spectrum of AR-activating compounds is even but in the case of the AR it forms a specific complex with brighter than that of other steroid receptors. Activation of the the receptor and inhibits its functional activity (Knudsen et AR was reported for β-catenin (Truica et al. 2000), caveolin al. 1999). This action might be specific for the D1 cyclin (Lu et al. 2001), thyroid hormone (Zhang et al. 1999), and because activation of the AR was enhanced by cyclin E cadmium (Ye et al. 2000). Caveolin is a component of caveo- lae membranes which is clearly implicated in prostate cancer progression; administration of caveolin antisense oligo- nucleotides led to the re-establishment of the androgen dependency of tumors (Nasu et al. 1998). Caveolin enhances ligand-dependent receptor activity and associates with the AR in a ligand-dependent manner.

AR coactivators and corepressors in Figure 6 AR expression levels in cotransfection experiments. prostate cancer The AR concentration was estimated in the lysates from COS-7 cells used to measure luciferase activity after AR accomplishes its action in target tissues by interaction cotransfection (see Fig. 1) and treatment with (+) or without with molecules which have histone acetylase and deacetylase (−) 10 nM R1881 (3×10 µl were pooled from each triplicate). activity, namely coactivators and corepressors. The progress Proteins were separated by SDS-PAGE and electroporated onto a nitrocellulose membrane, which was probed with a in this field is reflected mainly in discoveries of a number of polyclonal antiserum against the AR. Immunoreactive proteins new molecules which associate with the AR. It is not easy were visualized by chemiluminescence. (Reprinted from to assess the importance of these new findings for prostate Hofman et al. (2000). Reprinted by permission of Elsevier cancer because of the two main reasons: (a) the levels of Science.)

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(Yamamoto et al. 2000). Hydroxyflutamide lacked antagon- on TGF-β, divergent effects of Akt on the regulation of AR istic properties in those experiments. AR-mediated gene tran- activity have been reported and repetition of those experi- scription was enhanced by a cdk-activating kinase which ments by other researchers will perhaps clarify these issues interacts with the N-terminal part of the AR (Lee et al. 2000). (Wen et al. 2000, Lin et al. 2001b). On the other hand, tumor suppressor gene 101 protein has an In two recent reports, coactivator expression was linked inhibitory effect on AR transactivation (Sun et al. 1999). to prostate cancer progression. Association between the over- Again, all these findings point to a complex regulation of expression of coactivators TIF-2 and SRC-1 and recurrence proliferative responses by AR coregulatory proteins. of prostate cancer was described (Gregory et al. 2001c). The Enhancement of p21 expression by the AR coactivator breast expression of the coactivator RAC3 was highest in LNCaP cancer susceptibility gene 1 reversed the apoptosis-resistant cells and lowest in DU-145 cells (Gnanapragasam et al. phenotype of prostate cancer cells (Yeh et al. 2000). 2001). RAC3 levels correlated significantly with tumor grade Repression of AR activity was reported for the tumor sup- and stage and the coactivator expression was associated with pressor gene PTEN (Li et al. 2001). PTEN is an inhibitor poorer disease-specific survival. One of the obstacles in of the phosphoinositide 3-kinase which exerts proapoptotic research on coactivator expression is the fact that these pro- effects. Its inhibitory effect on AR transcriptional activity is teins are present in a limited amount in prostate tissue and paralleled by an induction of apoptosis and suppression of therefore detection problems might occur. For this reason, production of PSA. Inactivation of PTEN occurs in prostate expression of some of the proposed coactivators has been cancer cell lines and xenografts mainly because of mutations studied at mRNA level (Tekur et al. 2001). It was reported and deletions (Vlietstra et al. 1998). Another repressor of the that the levels of ARA 70 decrease in immortalized prostate AR is the p21-activated kinase PAK6 (Yang et al. 2001). cancer cell lines compared with primary cultures and that Little is known, however, about alterations of PAK6 in androgenic up-regulation of ARA 70 is inhibited by hydroxy- advanced carcinoma of the prostate. TGF-β is a pleiotropic flutamide. growth factor which is considered inhibitory for prostate cells β in vitro.TGF- has effects on immunosuppression and angi- Conclusions and directions for future ogenesis and therefore promotes growth of prostate tumors research in vivo (Steiner 1995). The nature of its effect on the AR remains unclear since two groups have published conflicting In the last decade there has been a considerable change in results. Either coactivator or corepressor function for the the understanding of the role of the AR in prostate cancer. TGF-β intermediary molecule Smad3 has been proposed The recognition that the AR is expressed in metastases of (Kang et al. 2001, Hayes et al. 2001). Similar to the findings therapy-resistant prostate cancers has greatly stimulated

Figure 7 Key mechanisms for AR involvement in prostate cancer progression. The AR which is in some cases overexpressed could be activated in a hypersensitive manner by ligands, non-androgenic steroids, and non-steroidal regulators of protein kinase activity.

Downloaded from Bioscientifica.com at 09/26/2021 08:41:44AM via free access 164 www.endocrinology.org Endocrine-Related Cancer (2002) 9 155–170 research on its function. It is clear that the AR is implicated with distinct regions of SRC1. Molecular and Cellular Biology in the cross-talkwith signaling pathways of growth factors, 19 8383–8392. peptide hormones, cytokines, and various inducers of differ- Bonaccorsi L, Carloni V, Muratori M, Salvadori A, Giannini A, Carini M, Serio M, Forti G & Baldi E 2000 Androgen receptor entiation (Fig. 7). The most important mechanisms by which expression in prostate carcinoma cells suppresses alpha6beta4 the AR facilitates prostate cancer progression are an increase integrin-mediated invasive phenotype. Endocrinology 141 3172– in sensitivity and aberrant activation due to structural altera- 3182. tions. New approaches in therapy are aimed to down-regulate Chang CS, Kokontis J & Liao S 1988 Structural analysis of AR expression and to overcome the difficulties associated complementary DNA and amino acid sequences of human and with the use of common antiandrogens (Eder et al. 2000). rat androgen receptors. PNAS 86 9534–9538. Chen T, Wang LH & Farrar WL 2000 Interleukin 6 activates The AR, however, has a role in the maintenance of differen- androgen receptor-mediated gene expression through a signal tiation function in the prostate and therefore the timing of transducer and activator of transcription 3-dependent pathway in such new therapies should be appropriate. LNCaP prostate cancer cells. Cancer Research 60 2132–2135. 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