Androgen receptor: a key molecule in the progression of prostate cancer to hormone independence

Despite earlier detection and recent advances in surgery and radiation, prostate cancer is second only to lung cancer in male cancer deaths in the United States. Hormone therapy in the form of medical or surgical castration remains the mainstay of systemic treatment in prostate cancer. Over the last 15 years with the clinical use of prostate specific antigen (PSA), there has been a shift to using hormone therapy earlier in the disease course and for longer duration. Despite initial favorable response to hormone therapy, over a period of time these tumors will develop androgen‐independence that results in death. The androgen receptor (AR) is central to the initiation and growth of prostate cancer and to its response to hormone therapy. Analyses have shown that AR continues to be expressed in androgen‐independent tumors and AR signaling remains intact as demonstrated by the expression of the AR regulated gene, PSA. Androgen‐independent prostate cancers have demonstrated a variety of AR alterations that are either not found in hormone naïve tumors or found at lower frequency. These changes include AR amplification, AR point mutation, and changes in expression of AR co‐regulatory proteins. These AR changes result in a “super AR” that can respond to lower concentrations of androgens or to a wider variety of agonistic ligands. There is also mounting evidence that AR can be activated in a ligand independent fashion by compounds such as growth factors or cytokines working independently or in combination. These growth factors working through receptor tyrosine kinase pathways may promote AR activation and growth in low androgen environments. The clinical significance of these AR alterations in the development and progression of androgen‐independent prostate cancer remains to be determined. Understanding the changes in AR signaling in the evolution of androgen‐independent prostate cancer will be key to the development of more effective hormone therapy. © 2003 Wiley‐Liss, Inc.     

5alpha-androstane-3alpha,17beta-diol supports human prostate cancer cell survival and proliferation through androgen receptor-independent signaling pathways: implication of androgen-independent prostate cancer progression

Androgen and androgen receptor (AR) are involved in growth of normal prostate and development of prostatic diseases including prostate cancer. Androgen deprivation therapy is used for treating advanced prostate cancer. This therapeutic approach focuses on suppressing the accumulation of potent androgens, testosterone and 5alpha-dihydrotestosterone (5alpha-DHT), or inactivating the AR. Unfortunately, the majority of patients with prostate cancer eventually advance to androgen-independent states and no longer respond to the therapy. In addition to the potent androgens, 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), reduced from 5alpha-DHT through 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs), activated signaling may represent a novel pathway responsible for the progression to androgen-independent prostate cancer. Androgen sensitive human prostate cancer LNCaP cells were used to compare 5alpha-DHT and 3alpha-diol activated androgenic effects. In contrast to 5alpha-DHT, 3alpha-diol regulated unique patterns of beta-catenin and Akt expression as well as Akt phosphorylation in parental and in AR-silenced LNCaP cells. More significantly, 3alpha-diol, but not 5alpha-DHT, supported AR-silenced LNCaP cells and AR negative prostate cancer PC-3 cell proliferation. 3alpha-diol-activated androgenic effects in prostate cells cannot be attributed to the accumulation of 5alpha-DHT, since 5alpha-DHT formation was not detected following 3alpha-diol administration. Potential accumulation of 3alpha-diol, as a result of elevated 3alpha-HSD expression in cancerous prostate, may continue to support prostate cancer growth in the presence of androgen deprivation. Future therapeutic strategies for treating advanced prostate cancer might need to target reductive 3alpha-HSD to block intraprostatic 3alpha-diol accumulation.     

Interaction of IGF signaling and the androgen receptor in prostate cancer progression

The insulin-like growth factor type I receptor (IGF-IR) has been suggested to play an important role in prostate cancer progression and possibly in the progression to androgen-independent (AI) disease. The term AI may not be entirely correct, in that recent data suggest that expression of androgen receptor (AR) and androgen-regulated genes is the primary association with prostate cancer progression after hormone ablation. Therefore, signaling through other growth factors has been thought to play a role in AR-mediated prostate cancer progression to AI disease in the absence of androgen ligand. However, existing data on how IGF-IR signaling interacts with AR activation in prostate cancer are conflicting. In this Prospect article, we review some of the published data on the mechanisms of IGF-IR/AR interaction and present new evidence that IGF-IR signaling may modulate AR compartmentation and thus alter AR activity in prostate cancer cells. Inhibition of IGF-IR signaling can result in cytoplasmic AR retention and a significant change in androgen-regulated gene expression. Translocation of AR from the cytoplasm to the nucleus may be associated with IGF-induced dephosphorylation. Since fully humanized antibodies targeting the IGF-IR are now in clinical trials, the current review is intended to reveal the mechanisms of potential therapeutic effects of these antibodies on AI prostate cancers.     

Androgen receptor action in hormone-dependent and recurrent prostate cancer

The importance of androgens and androgen receptors (AR) in primary prostate cancer is well established. Metastatic disease is usually treated with some form of androgen ablation, which is effective for a limited amount of time. The role of AR in prostate cancers that recur despite androgen ablation therapy is less certain. Most of these tumors express prostate specific antigen (PSA), an androgen-regulated gene; moreover, AR is generally highly expressed in recurrent prostate cancer. We propose that AR continues to play a role in many of these tumors and that it is not only the levels of AR, ligands, and co-regulators, but also the changes in cell signaling that induce AR action in recurrent prostate cancer. These pathways are, therefore, potential therapeutic targets.     

Amino-terminus domain of the androgen receptor as a molecular target to prevent the hormonal progression of prostate cancer

Prostate cancer has a propensity to metastasize to the bone. Currently the only effective systemic treatment for these patients is androgen ablation therapy. However, the tumor will invariably progress to an androgen-independent stage and the patient will succumb to his disease within approximately 2 years. The earliest indication of hormonal progression is the rising titer of serum prostate specific antigen. Current evidence implicates the androgen receptor (AR) as a key factor in maintaining the growth of prostate cancer cells in an androgen-depleted state. Under normal conditions, binding of ligand activates the receptor, allowing it to effectively bind to its respective DNA element. However, AR is also transformed in the absence of androgen (ligand-independent activation) in prostate cells via multiple protein kinase pathways and the interleukin-6 (IL-6) pathway that converge upon the N-terminal domain of the AR. This domain is the main region for phosphorylation and is also critical for normal coregulator recruitment. Here we discuss evidence supporting the role of the AR, IL-6 and other protein kinase pathways in the hormonal progression of prostate cancer to androgen independence and the mechanisms involved in activation of the AR by these pathways. Receptor-targeted therapy, especially potential drugs targeting the N-terminal domain, may effectively prevent or delay the hormonal progression of AR-dependent prostate cancer.     

Mechanisms of prostate cancer cell survival after inhibition of AR expression

Recent reports have shown that the AR is the key determinant of the molecular changes required for driving prostate cancer cells from an androgen‐dependent to an androgen‐independent or androgen depletion‐independent (ADI) state. Several recent publications suggest that down‐regulation of AR expression should therefore be considered the principal strategy for the treatment of ADI prostate cancer. However, no valid data is available about how androgen‐dependent prostate cancer cells respond to apoptosis‐inducing drugs after knocking down AR expression and whether prostate cancer cells escape apoptosis after inhibition of AR expression. This review will focus on mechanisms of prostate cancer cell survival after inhibition of AR activity mediated either by androgen depletion or by targeting the expression of AR by siRNA. We have shown that knocking down AR expression by siRNA induced PI3K‐independent activation of Akt, which was mediated by calcium/calmodulin‐dependent kinase II (CaMKII). We also showed that the expression of CaMKII genes is under AR control: active AR in the presence of androgens inhibits CaMKII gene expression whereas inhibition of AR activity results in an elevated level of kinase activity and in enhanced expression of CaMKII genes. This in turn activates the anti‐apoptotic PI3K/Akt pathways. CaMKII also express anti‐apoptotic activity that is independent from the Akt pathway. This may therefore be an important mechanism by which prostate cancer cells escape apoptosis after androgen depletion or knocking down AR expression. In addition, we have found that there is another way to escape cell death after AR inhibition: DNA damaging agents cannot fully activate p53 in the absence of AR and as a result p53 down stream targets, for example, microRNA‐34, cannot be activated and induce apoptosis. This implies that there may be a need for re‐evaluation of the therapeutic approaches to human prostate cancer. J. Cell. Biochem. 106: 363–371, 2009. © 2008 Wiley‐Liss, Inc.     

Steroid hormones, polypeptide growth factors, hormone refractory prostate cancer, and the neuroendocrine phenotype

The growth, development, and differentiation of the prostate gland is largely dependent on the action of androgens and peptide growth factors that act differentially at the level of the mesenchymal and epithelial compartments. It is our premise that to understand the emergence of metastatic and hormone refractory prostate cancer we need to investigate: (1) how androgen action at the level of the mesenchyme induces the production of peptide growth factors that in turn can facilitate the growth and development of the epithelial compartment; (2) how androgen action at the level of the epithelium induces and maintains cellular differentiation, function, and replicative senescence; and (3) how transformation of the prostate gland can corrupt androgen and growth factor signaling homeostasis. To this end, we focus our discussion on how deregulation of the growth factor signaling axis can cooperate with deregulation of the androgen signaling axis to facilitate transformation, metastasis, and the emergence of the hormone refractory and neuroendocrine phenotypes associated with progressive androgen-independent prostate cancer. Finally, we suggest a working hypothesis to explain why hormone ablation therapy works to control early disease but fails to control, and may even facilitate, advanced prostate cancer.     

Survey of genetically engineered mouse models for prostate cancer: analyzing the molecular basis of prostate cancer development, progression, and metastasis

Genetically engineered mouse models have been generated to study the molecular basis of prostate cancer (PCa) development, progression, and metastasis. Selection of a prostate-specific promoter, such as the probasin (PB) and prostate specific antigen (PSA) promoters, is critical for generating sufficient levels of transgene expression to elicit a phenotypic response. To date, target genes have included growth factors, cell cycle regulators, pro- and anti-apoptotic proteins, steroid hormone and growth factor receptors, oncogenes, tumor suppressors, and homeobox genes. The experimental approaches used to generate these mouse models include overexpression of the transgene, knock-out/knock-in of transgene expression and conditional regulation of expression using Cre/lox technology. This review summarizes the promoters, which have been utilized to create genetically engineered mouse models for PCa. Furthermore, the effects of gene disruption on promoting low- and high-grade intraepithelial neoplasia (LGPIN and HGPIN, respectively), locally invasive carcinoma and metastatic lesions will be discussed. To date, the PB-Cre4 x PTENloxp/loxp model appears to be the only model that represents the entire continuum of prostate adenocarcinoma development, tumor progression, and metastasis, although models that develop prostatic neuroendocrine (NE) cancer can be generated by disrupting one genetic event. Indeed, analysis of bigenic mouse models indicates that two genetic events are generally required for progression from HGPIN to locally invasive adenocarcinoma and that two to five genetic events can promote metastasis to distant sites. Studying the effects of genetic perturbation on PCa biology will increase our understanding of the disease process and potentially provide targets for developing novel therapeutic approaches.     

Down‐regulation of Notch‐1 is associated with Akt and FoxM1 in inducing cell growth inhibition and apoptosis in prostate cancer cells

Although many studies have been done to uncover the mechanisms by which down‐regulation of Notch‐1 exerts its anti‐tumor activity against a variety of human malignancies, the precise molecular mechanisms remain unclear. In the present study, we investigated the cellular consequence of Notch‐1 down‐regulation and also assessed the molecular consequence of Notch‐1‐mediated alterations of its downstream targets on cell viability and apoptosis in prostate cancer (PCa) cells. We found that the down‐regulation of Notch‐1 led to the inhibition of cell growth and induction of apoptosis, which was mechanistically linked with down‐regulation of Akt and FoxM1, suggesting for the first time that Akt and FoxM1 are downstream targets of Notch‐1 signaling. Moreover, we found that a “natural agent” (genistein) originally discovered from soybean could cause significant reduction in cell viability and induced apoptosis of PCa cells, which was consistent with down‐regulation of Notch‐1, Akt, and FoxM1. These results suggest that down‐regulation of Notch‐1 by novel agents could become a newer approach for the prevention of tumor progression and/or treatment, which is likely to be mediated via inactivation of Akt and FoxM1 signaling pathways in PCa. J. Cell. Biochem. 112: 78–88, 2011. © 2010 Wiley‐Liss, Inc.     

The androgen receptor antagonist enzalutamide induces apoptosis,dysregulates the heat shock protein system,and diminishes the androgen receptor and estrogen receptor β1 expression in prostate cancer cells

Enzalutamide's accepted mode of action is by targeting the androgen receptor's (AR) activity. In clinical practice, enzalutamide demonstrates a good benefit-risk profile for the treatment of advanced prostate cancer (PC), even after poor response to standard antihormonal treatment. However, since both, well-established antiandrogens and enzalutamide, target AR functionality, we hypothesized that additional unknown mechanisms might be responsible for enzalutamide's superior anticancer activity. In the current study, PC cells were incubated with enzalutamide and enzalutamide-dependent modulation of apoptotic mechanisms were assessed via Western blot analysis, TDT-mediated dUTP-biotin nick end-labeling assay, and nuclear morphology assay. Alterations of heat shock protein (HSP), AR, and estrogen receptor (ER) expression were examined by Western blot analysis. Enzalutamide attenuated the proliferation of PC cells in a time- and dose-dependent manner. In the presence of enzalutamide, apoptosis occurred which was shown by increased BAX expression, decreased Bcl-2 expression, nuclear pyknosis, and genomic DNA fragmentation. Moreover, enzalutamide inhibited the expression of HSPs primarily involved in steroid receptor stabilization and suppressed AR and ERβ1 expression. This study demonstrates for the first time that enzalutamide treatment of PC cells triggers varying molecular mechanisms resulting in antiproliferative effects of the drug. In addition to the well-characterized antagonistic inhibition of AR functionality, we have shown that enzalutamide also affects the intracellular synthesis of steroid receptor-associated HSPs, thereby diminishing the expression of AR and ERβ1 proteins and inducing apoptotic pathways. According to an indirect attenuation of HSP-associated factors such as steroid receptors, endometrial carcinoma, uterine leiomyosarcoma, and mamma carcinoma cells also demonstrated inhibited cell growth in the presence of enzalutamide. Our data, therefore, suggest that enzalutamide's high efficacy is at least partially independent of AR and p53 protein expression, which are frequently lost in advanced PC.     

1alpha,25-Dihydroxyvitamin D3-3beta-(2)-bromoacetate,an affinity labeling derivative of 1alpha,25-dihydroxyvitamin D3 displays strong antiproliferative and cytotoxic behavior in prostate cancer cells

In this report we describe that 1,25(OH)(2)D(3)-3-BE, a VDR-affinity labeling analog of 1,25(OH)(2)D(3), showed strong and dose-dependent growth-inhibitory effect in several epithelial cells, i.e., keratinocytes (primary cells), MCF-7 breast cancer, PC-3, and LNCaP prostate cancer and PZ-HPV-7 immortalized normal prostate cell-lines. Furthermore, 10(-6) M of 1,25(OH)(2)D(3)-3-BE induced apoptosis specifically in LNCaP and PC-3 cells; and the effect was much less pronounced at lower doses. We also showed that the effect (of 1,25(OH)(2)D(3)-3-BE) was not due to probable degradation (hydrolysis) of 1,25(OH)(2)D(3)-3-BE or random interaction of this molecule with cellular proteins. Tissue- or cell-specific action of 1,25(OH)(2)D(3) and its mimics is not common due to the ubiquitous nature of VDR. Furthermore, variable effects of 1,25(OH)(2)D(3) and its analogs in various cell-lines potentially limits their application as anticancer agents. We showed that 1,25(OH)(2)D(3)-3-BE displayed similar growth-inhibitory and cytotoxic activities towards androgen sensitive LNCaP and androgen-independent PC-3 cell-lines. Therefore, these results raise the possibility that 1,25(OH)(2)D(3)-3-BE or similar VDR-cross linking analogs of 1,25(OH)(2)D(3) might be considered for further development as potential candidates for prostate cancer.     

A transcriptional target of androgen receptor,miR-421 regulates proliferation and metabolism of prostate cancer cells

Prostate cancer is one of the most common malignancies, and microRNAs have been recognized to be involved in tumorigenesis of various kinds of cancer including prostate cancer (PCa). Androgen receptor (AR) plays a core role in prostate cancer progression and is responsible for regulation of numerous downstream targets including microRNAs. This study identified an AR-repressed microRNA, miR-421, in prostate cancer. Expression of miR-421 was significantly suppressed by androgen treatment, and correlated to AR expression in different prostate cancer cell lines. Furthermore, androgen-activated AR could directly bind to androgen responsive element (ARE) of miR-421, as predicted by bioinformatics resources and demonstrated by ChIP and luciferase reporter assays. In addition, over-expression of miR-421 markedly supressed cell viability, delayed cell cycle, reduced glycolysis and inhibited migration in prostate cancer cells. According to the result of miR-421 target genes searching, we focused on 4 genes NRAS, PRAME, CUL4B and PFKFB2 based on their involvement in cell proliferation, cell cycle progression and metabolism. The expression of these 4 downstream targets were significantly repressed by miR-421, and the binding sites were verified by luciferase assay. Additionally, we explored the expression of miR-421 and its target genes in human prostate cancer tissues, both in shared microarray data and in our own cohort. Significant differential expression and inverse correlation were found in PCa patients.     

The Raf/MEK/extracellular signal-regulated kinase 1/2 pathway can mediate growth inhibitory and differentiation signaling via androgen receptor downregulation in prostate cancer cells

Upregulated ERK1/2 activity is correlated with androgen receptor (AR) downregulation in certain prostate cancer (PCa) that exhibits androgen deprivation-induced neuroendocrine differentiation, but its functional relevance requires elucidation. We found that sustained ERK1/2 activation using active Raf or MEK1/2 mutants is sufficient to induce AR downregulation at mRNA and protein levels in LNCaP. Downregulation of AR protein, but not mRNA, was blocked by proteasome inhibitors, MG132 and bortezomib, indicating that the pathway regulation is mediated at multiple points. Ectopic expression of a constitutively active AR inhibited Raf/MEK/ERK-mediated regulation of the differentiation markers, neuron-specific enolase and neutral endopeptidase, and the cyclin-dependent kinase inhibitors, p16^INK4A and p21^CIP1, but not Rb phosphorylation and E2F1 expression, indicating that AR has a specific role in the pathway-mediated differentiation and growth inhibitory signaling. However, despite the sufficient role of Raf/MEK/ERK, its inhibition using U0126 or ERK1/2 knockdown could not block androgen deprivation-induced AR downregulation in an LNCaP neuroendocrine differentiation model, suggesting that additional signaling pathways are involved in the regulation. We additionally report that sustained Raf/MEK/ERK activity can downregulate full length as well as hormone binding domain-deficient AR isoforms in androgen-refractory C4-2 and CWR22Rv1, but not in LAPC4 and MDA-PCa-2b. Our study demonstrates a novel role of the Raf/MEK/ERK pathway in regulating AR expression in certain PCa types and provides an insight into PCa responses to its aberrant activation.