Dynamics of coregulator-induced conformational perturbations in androgen receptor ligand binding domain

Extracts from the plant Pygeum africanum are widely used in the therapy of benign prostate hyperplasia (BPH) and in combinational therapy for prostate cancer, the second leading cause of cancer death and the mostly diagnosed form of cancer in men. The androgen receptor (AR) plays a crucial role in the development of the prostate as well as in prostate diseases. Even though the extracts from P. africanum are considered as beneficial for prostate diseases in clinical trials, and some active compounds for treatment of BPH could be identified, compounds responsible for AR inhibition and the molecular mechanism for inhibition of prostatitis need to be identified. Recently, atraric acid and N-butylbenzene-sulfonamide were isolated from a selective dichlormethane extract of P. africanum as two novel AR antagonistic compounds. The molecular mechanisms of AR inhibition were analyzed and are summarized here. Both compounds are the first known natural, complete and specific AR antagonist.     

Estrogen and androgen signaling in the pathogenesis of BPH

Estrogens and androgens have both been implicated as causes of benign prostatic hyperplasia (BPH). Although epidemiological data on an association between serum androgen concentrations and BPH are inconsistent, it is generally accepted that androgens play a permissive role in BPH pathogenesis. In clinical practice, inhibitors of 5α-reductase (which converts testosterone to the more potent androgen dihydrotestosterone) have proven effective in the management of BPH, confirming an essential role for androgens in BPH pathophysiology. To date, multiple lines of evidence support a role for estrogens in BPH pathogenesis. Studies of the two estrogen receptor (ER) subtypes have shed light on their differential functions in the human prostate; ERα and ERβ have proliferative and antiproliferative effects on prostate cells, respectively. Effects of estrogens on the prostate are associated with multiple mechanisms including apoptosis, aromatase expression and paracrine regulation via prostaglandin E2. Selective estrogen receptor modulators or other agents that can influence intraprostatic estrogen levels might conceivably be potential therapeutic targets for the treatment of BPH.     

Androgen receptor is a tumor suppressor and proliferator in prostate cancer

Targeting androgens/androgen receptor (AR) functions via androgen deprivation therapy (ADT) remains the standard treatment for prostate cancer. However, most tumors eventually recur despite ADT. Here we demonstrate that the prostate AR may function as both a suppressor and a proliferator to suppress or promote prostate cancer metastasis. Results from orthotopically recombining stromal WPMY1 cells with epithelial PC3 prostate cancer cells in mice demonstrated that restoring AR in epithelial PC3 cells or knockdown of AR in stromal WPMY1 cells suppressed prostate cancer metastasis. Knockdown of the AR in epithelial CWR22rv1 prostate cancer cells also resulted in increased cell invasion in vitro and in vivo. Restoring AR in PC3 cells (PC3-AR9) results in decreased invasion in bone lesion assays and in vivo mouse models. Mice lacking the prostate epithelial AR have increased apoptosis in epithelial luminal cells and increased proliferation in epithelial basal cells. The consequences of these two contrasting results led to the expansion of CK5/CK8-positive intermediate cells, and mice developed larger and more invasive metastatic tumors in lymph nodes and died earlier than wild-type littermates. Mechanistic dissection suggested that androgens/AR might directly or indirectly modulate metastasis-related genes and suppression of TGFβ1 signals results in the partial inhibition of AR-mediated metastasis. Collectively, our understanding of these opposing roles of prostatic AR may revolutionize the way we combat prostate cancer, and allow the development of new and better therapies by targeting only the proliferative role of AR.     

Altered endocytosis of epidermal growth factor receptor in androgen receptor positive prostate cancer cell lines

Although androgens and the androgen receptor (AR) are involved in tumorigenesis of prostate cancer (PC) in initial phases, less clear is the role played in advanced androgen-independent (AI) stages of the disease. Several recent reports indicated that re-expression of AR in PC-derived cell lines determines a less aggressive phenotype of the cells. We have previously demonstrated that re-expression of AR decreases the invasion ability of PC3 cells in vitro by affecting signalling and internalization processes of epidermal growth factor receptor (EGFR). Here, we show that reduced EGFR internalization is also a characteristic of AR positive PC cell lines LNCaP and 22Rv1. Reduced internalization in PC3-AR cells is associated to a defective interaction between the EGFR and two adaptor proteins which mediate the endocytotic process, Grb2 and c-Cbl. As a consequence of such reduced interaction, ubiquitination of the receptor, which is mainly mediated by c-Cbl, is also altered. In addition, we show that internalized EGFR co-localizes with early endosome antigen-1, a marker of clathrin-mediated endocytosis, in PC3-Neo cells but not in AR positive cell lines. Conversely, EGFR maintains co-localization with caveolin-1 after EGF stimulation in PC3-AR cells. These data suggest that expression of AR affects clathrin-mediated endocytosis pathway of EGFR, which, according to recent findings, plays an essential role in the completeness of signalling of the receptor. Taken together, these data emphasize the role of AR in the regulation of EGFR endocytotic trafficking and active signalling in PC cells. In view of the role of EGFR signalling in invasion of carcinoma cells, our data may explain the lower invasive phenotype observed in AR-positive cell lines.     

Growth hormone (GH) receptors in prostate cancer: gene expression in human tissues and cell lines and characterization, GH signaling and androgen receptor regulation in LNCaP cells

Various hormones and growth factors have been implicated in progression of prostate cancer, but their role and the underlying molecular mechanism(s) involved remain poorly understood. In this study, we investigated the role of human growth hormone (GH) and its receptor (GHR) in human prostate cancer. We first demonstrated mRNA expression of GHR and of its exon 9-truncated isoform (GHR_tr) in benign prostate hyperplasia (BPH) and prostate adenocarcinoma patient tissues, as well as in LNCaP, PC3 and DU145 human prostate cancer cell lines. GHR mRNA levels were 80% higher and GHR_tr only 25% higher, in the carcinoma tissues than in BPH. Both isoforms were also expressed in LNCaP and PC3 cell lines and somewhat less so in DU145 cells. The LNCaP cell GHR protein was further characterized, on the basis of its M_r of 120 kDa, its binding to two different GHR monoclonal antibodies, its high affinity and purely somatogenic binding to ¹²⁵I-hGH and its ability to secrete GH binding protein, all characteristic of a functional GHR. Furthermore, GH induced rapid, time- and dose-dependent signaling events in LNCaP cells, including phosphorylation of JAK2 tyrosine kinase, of GHR itself and of STAT5A (JAK2-STAT5A pathway), of p42/p44 MAPK and of Akt/PKB. No effect of GH (72 h) could be shown on basal or androgen-induced LNCaP cell proliferation nor on PSA secretion. Interestingly, however, GH caused a rapid (2–12 h) though transient striking increase in immunoreactive androgen receptor (AR) levels (≤5-fold), followed by a slower (24–48 h) reduction (≤80%), with only modest parallel changes in serine-phosphorylated AR. In conclusion, the GH-induced activation of signaling pathways, its effects on AR protein in LNCaP cells and the isoform-specific regulation of GHR in prostate cancer patient tissues, suggest that GH, most likely in concert with other hormones and growth factors, may play an important role in progression of human prostate cancer.     

Expression of GnRH type II is regulated by the androgen receptor in prostate cancer

GnRH II has important functional effects in steroid hormone-dependent tumours. Here we investigated the expression and regulation of GnRH II in prostate cancer. GnRH II protein was equally expressed in benign (73%) and malignant (78%) biopsies studied in a prostate tissue microarray (P = 0.779). There was no relationship between expression and clinical parameters in the cancer cohort. GnRH II was, however, significantly reduced in tumour biopsies following hormone ablation. This was further investigated in a prostate xenograft model where androgens increased GnRH II levels, while their withdrawal reduced it. In cell lines, we confirmed high levels of GnRH II in androgen receptor (AR)-positive LNCaP cells but low levels in AR-negative PC3 cells. In LNCaP cells, GnRH II induction by androgens was blocked by the AR inhibitor casodex, but not by cycloheximide treatment. Sequence analysis subsequently revealed a putative androgen response element in the upstream region of the GnRH II gene and direct interaction with the AR was confirmed in chromatin immunoprecipitation experiments. Finally, to test whether the effects of GnRH II were dependent on AR expression, LNCaP and PC3 cells were exposed to exogenous peptide. In both cell lines, GnRH II inhibited cell proliferation and migration, suggesting that its function is independent of AR status. These results provide evidence that GnRH II is widely expressed in prostate cancer and is an AR-regulated gene. Further studies are warranted to characterise the effects of GnRH II on prostate cancer cells and investigate its potential value as a novel therapy.     

Androgen-independent prostate cancer cells acquire the complete steroidogenic potential of synthesizing testosterone from cholesterol

The proliferation and differentiation of normal prostate epithelial cells depends upon the action of androgens produced by the testis. Prostate cancers retain the ability to respond to androgens in the initial stages of cancer development, but progressively become independent of exogenous androgens in advanced stages of the disease while maintaining the expression of functional androgen receptor (AR). In the present study, we have determined the potential of prostate cancer cells to synthesize androgens from cholesterol which may be involved in intracrine regulation of AR in advanced stages of the disease. Established androgen-independent prostate cancer cell lines, PC3 and DU145 cells, expressed mRNA and proteins for scavenger receptor type B1 (SRB1), steroidogenic acute regulatory (StAR) protein, cytochrome P450 cholesterol side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and other enzymes involved in androgen biosynthesis. Expression of all these proteins and enzymes was significantly higher in the androgen-independent derivative of LNCaP prostate cancer cells (C81) than in the androgen-dependent cell line (C33). In serum-free cultures, the androgen-independent C81 cells secreted approximately 5-fold higher testosterone than C33 cells as determined in the conditioned media by immunoassays. These cells could also directly convert radioactive cholesterol into testosterone which was identified by thin layer chromatography. These results for the first time show that prostate cancer cells in advanced stages of the disease could synthesize androgens from cholesterol and hence are not dependent upon testicular and/or adrenal androgens.     

Gadd45gamma is androgen-responsive and growth-inhibitory in prostate cancer cells

In our previous microarray analysis searching for genes differentially regulated by androgens in the rat ventral prostate, we identified GADD45gamma (growth arrest and DNA damage inducible, gamma) as one of the genes up-regulated by androgens. GADD45gamma was initially identified to be a gene involved in negative growth control and its overexpression induced cycle arrest and apoptosis in vitro. In this study, we showed that GADD45gamma was transiently up-regulated by androgens in the androgen-responsive human prostate cancer cell line LNCaP. The GADD45gamma up-regulation was blocked by an androgen receptor (AR) antagonist, bicalutamide, suggesting the involvement of the androgen receptor. However, this up-regulation was inhibited by cycloheximide, indicating that GADD45gamma induction by androgens requires new protein synthesis. Overexpression of GADD45gamma inhibited cell growth of LNCaP and PC3 cells and resulted in dramatic morphological changes in both cell lines, arguing that GADD45gamma is likely to participate in the differentiation program induced by androgens in the prostate. The above observations provide evidence that GADD45gamma is an androgen-responsive gene with growth-inhibitory activity in human prostate cancer cells.     

Targeting 5α-reductase for prostate cancer prevention and treatment

Testosterone is the most abundant circulating androgen, and can be converted to dihydrotestosterone (DHT), a more potent androgen, by the 5α-reductase enzymes in target tissues. Current treatments for prostate cancer consist of reducing androgen levels by chemical or surgical castration or pure antiandrogen therapy that directly targets the androgen receptor (AR). Although these therapies reduce tumor burden and AR activity, the cancer inevitably recurs within 18-30 months. An approach targeting the androgen-AR axis at different levels could, therefore, improve the efficacy of prostate cancer therapy. Inhibition of 5α-reductase is one such approach; however, the two largest trials to investigate the use of the 5α-reductase inhibitors (5ARIs) finasteride and dutasteride in patients with prostate cancer have shown that, although the incidence of cancer was reduced by 5ARI treatment, those cancers that were detected were more aggressive than in patients treated with placebo. Thus, the best practice for using these drugs to prevent and treat prostate cancer remains unclear.     

Endocrine mechanisms of disease: Expression and degradation of androgen receptor: mechanism and clinical implication

The androgen-androgen receptor (AR) signaling pathway plays a key role in proper development and function of male reproductive organs, such as prostate and epididymis, as well as nonreproductive organs, such as muscle, hair follicles, and brain. Abnormalities in the androgen-AR signaling pathway have been linked to diseases, such as male infertility, Kennedy's disease, and prostate cancer. Regulation of AR activity can be achieved in several different ways: modulation of AR gene expression, androgen binding to AR, AR nuclear translocation, AR protein stability, and AR trans-activation. This review covers mechanisms implicated in the control of AR protein expression and degradation, and their potential linkage to the androgen-related diseases. A better understanding of such mechanisms may help us to design more effective androgens and antiandrogens to battle androgen-related diseases.     

Evaluation of Plasma Interleukin-8 Concentration in Patients with Prostate Cancer and Benign Prostate Hyperplasia

Background: Prostate specific antigen (PSA) has been used as a screening test for the early detection of prostate cancer (PC) for many years. Although the introduction of PSA test led to a considerable increase in reported prostate cancer cases, there is still some controversy over the sensitivity and specificity of this marker in distinguishing PC patients from those with benign prostate hyperplasia (BPH), the most common benign prostate condition. Objective: An attempt is made to elucidate if the plasma level of Interleukin 8 (IL-8) could be used effectively as a marker for the detection of prostate cancer. Methods: Plasma levels of IL-8 and PSA were measured in two groups of 40 BPH and PC patients using enzyme-linked immunosorbent (ELISA) and radioimmunoassay (RIA) techniques, respectively. In addition IL-8 levels in PC3 and DU145 cell line supernatants were measured by ELISA technique. Results: The concentration of IL-8 in the plasma of PC patients was not significantly higher than the BPH subjects. Although, a correlation between plasma IL-8 concentration and the Gleason score of PC patients was found, no indicated correlation was detected between the concentration of IL-8 or PSA and age of the patients in both groups. DU145 and PC3 cell lines produced and secreted IL-8 in the media. Conclusion: Data of this investigation collectively conclude no correlation between IL-8 concentration in PC and BPH patients.     

Androgen receptor corepressors and prostate cancer

The androgen receptor (AR) mediates the effects of male steroid hormones (androgens) and contributes to a wide variety of physiological and pathophysiological conditions. As such, the regulatory mechanisms governing AR activity are of high significance. Concerted effort has been placed on delineating the mechanisms that control AR activity in prostate cancer, as AR is required for survival and proliferation in this tumor type. Moreover, AR is the central therapeutic target for metastatic prostate cancers, and recurrent tumors evade therapy by restoring AR activity. It is increasingly apparent that AR cofactors which modulate receptor activity can contribute to prostate cancer growth or progression, and this has been particularly well established for AR coactivators. The present review is focused on the role of AR corepressors in governing androgen action, with a specific emphasis on their activities in prostate cancer.     

Endocrine prevention and treatment of prostate cancer

The major androgen within the prostate is dihydrotestosterone (DHT). DHT and 5α-reductase are highly associated with prostate cancer. It has been hypothesised that inhibition of 5α-reductase activity might reduce the risk of prostate cancer development, slow tumour progression and even treat the existing disease. The basis for endocrine treatment of prostate cancer is to deprive the cancer cells of androgens. Every type of endocrine treatment carries adverse events which influence quality of life in different ways. 5α-Reductase inhibitors (5-ARI) reduce risk of being diagnosed with prostate cancer but they do not eliminate it. By suppressing PSA from BPH and indolent prostate cancers 5-ARI enhances the ability of a rising PSA to define a group of men at increased risk of clinically significant prostate cancer. Also fewer high-grade cancers are missed because biopsy is more accurate in smaller prostates. Androgen deprivation is an effective treatment for patients with advanced prostate cancer. However, it is not curative, and creates a spectrum of unwanted effects that influence quality of life. Castration remains the frontline treatment for metastatic prostate cancer, where orchiectomy, oestrogen agonists, GnRH agonists and antagonists produce equivalent clinical responses. MAB is not significantly more effective than single agent GnRH agonist or orchiectomy. Nonsteroidal antiandrogen monotherapy is as effective as castration in treatment of locally advanced prostate cancer offering quality of life benefits. Neoadjuvant endocrine treatment has its place mainly in the external beam radiotherapy setting. Increasing data suggest IAD is as effective as continuous ADT. The decision regarding the type of androgen deprivation should be made individually after informing the patient of all available treatment options, including watchful waiting, and on the basis of potential benefits and adverse effects. There are new promising secondary or tertiary forms of endocrine therapies under evaluation, like CTP17A1 inhibitors and more potent antiandrogens including MDV3100, which give new hope for patients developing castration resistant prostate cancer.     

A role for the androgen-receptor in clinically localized and advanced prostate cancer

Earlier studies of androgen-receptor (AR) expression using frozen prostate tissue, and later studies using archived specimens, produced the consensus that ligand-stabilized AR is nuclear, AR expression is similar in benign epithelia and stroma, AR expression is greater in secretory epithelia than basal cells, and AR expression is more variable in prostate cancer (CaP) than in benign prostatic hyperplasia (BPH). Accurate measurement of AR expression remains technically challenging but necessary to evaluate the relevance of AR to clinical CaP. Recent studies demonstrated that AR expression in epithelia and stroma may be prognostic in clinically localized CaP, and AR expression may play a role in racial differences in CaP mortality and predict response to androgen deprivation therapy. High levels of AR and AR-regulated gene expression indicate a central role for AR in growth regulation of castration-recurrent CaP. New treatments for the lethal phenotype of CaP require better understanding of AR transactivation during androgen deprivation therapy.     

Androgen receptor signalling in prostate: effects of stromal factors on normal and cancer stem cells

The prostate gland is the most common site for cancer in males within the developed world. Androgens play a vital role in prostate development, maintenance of tissue function and pathogenesis of prostate disease. The androgen receptor signalling pathway facilitates that role in both the epithelial compartment and in the underlying stroma. Stroma is a key mediator of androgenic effects upon the epithelium and can regulate both the fate of the epithelial stem cell and potentially the initiation and progression of prostate cancer. Different groups of growth factors are expressed by stroma, which control proliferation, and differentiation of prostate epithelium demonstrating a critical role for stroma in epithelial growth and homeostasis. Paracrine stromal proteins may offer the possibility to control tumour stem cell growth and could permit prostate specific targeting of both therapies and of androgen responsive proteins. The effect of 5alpha-dihydrotestosterone, the more potent metabolite of testosterone, on expression of androgen-regulated genes in stroma from benign prostatic hyperplasia is a key mediator of epithelial cell fate. Global gene expression arrays have recently identified new candidate genes in androgen responsive stroma, some of which have androgen receptor binding sites in their promoter regions. Some of these genes have direct androgen receptor binding ability.     

Mechanisms of prostate cancer progression to androgen independence

Prostate cancer is a major health problem in the United States and worldwide. In 2007, more than 27,000 men were estimated to have died from prostate cancer in the United States alone. Although important advances have been made in the diagnosis and treatment of prostate cancer, therapies focused on the removal or inhibition of androgen action remain the most important components of therapy for individuals with metastatic disease. Despite the application of such modalities, the vast majority of patients with metastatic disease progress with a median survival of less than 2 years. A number of different mechanisms have been identified that may potentially contribute to the progression of prostate cancer. These insights suggest that signaling via the androgen receptor (AR) -- either via alternate signaling pathways impinging on the AR or through the in situ formation of androgens within progressive tumors -- is an important contributor to such progressive disease. It is anticipated that such mechanistic insights will lead to the development of useful new therapies in the future.