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.     

AZGP1 is androgen responsive and involved in AR-induced prostate cancer cell proliferation and metastasis

Alpha-2-glycoprotein 1, zinc-binding (AZGP1), known as zinc-alpha-2-glycoprotein (ZAG), is a multifunctional secretory glycoprotein and relevant to cancer metastasis. Little is known regarding the underlying mechanisms of AZGP1 in prostate cancer (PCa). In the present study, we report that AZGP1 is an androgen-responsive gene, which is involved in AR-induced PCa cell proliferation and metastasis. In clinical specimens, the expression of AZGP1 in PCa tissues is markedly higher than that in adjacent normal tissues. In cultures, expression of AZGP1 is upregulated by the androgen-AR axis at both messenger RNA and protein levels. Furthermore, Chip-Seq assay identifies canonical androgen-responsive elements (AREs) at AZGP1 enhancer; and dual-luciferase reporter assays reveal that the AREs is highly responsive to androgen whereas mutations of the AREs abolish the reporter activity. In addition, AZGP1 promotes G1/S phase transition and cell cycle progress by increasing cyclin D1 levels in PCa cells. Functional studies demonstrate that knocking down endogenous AZGP1 expression in LNCaP and CWR22Rv1 cells largely weaken androgen/AR axis-induced cell migration and invasion. In vivo xenotransplantation tumor experiments also show that AZGP1 involves in androgen/AR axis-mediated PCa cell proliferation. Taken together, our study implicates for the first time that AZGP1 is an AR target gene and is involved in androgen/AR axis-mediated cell proliferation and metastasis in primary PCa.     

Androgen axis in prostate cancer

Endocrine therapy for advanced prostate cancer is based on androgen ablation or blockade of the androgen receptor (AR). AR action in prostate cancer has been investigated in a number of cell lines, their derivatives, and transgenic animals. AR expression is heterogenous in prostate cancer in vivo; it could be detected in most primary tumors and their metastases. However, some cells lack the AR because of epigenetic changes in the gene promoter. AR expression increases after chronic androgen ablation in vitro. In several xenografts, AR upregulation is the most consistent change identified during progression towards therapy resistance. In contrast, the AR pathway may be by-passed during chronic treatment with a nonsteroidal anti-androgen. AR sensitivity in prostate cancer increases as a result of activation of the Ras/mitogen-activated protein kinase pathway. One of the major difficulties in endocrine therapy for prostate cancer is acquisition of agonistic properties of AR antagonists observed in the presence of mutated AR. Enhancement of AR function by associated coactivator proteins has been extensively investigated. Cofactors SRC-1, RAC3, p300/CBP, TIF-2, and Tip60 are upregulated in advanced prostate cancer. Most studies on ligand-independent activation of the AR are focused on Her-2/neu and interleukin-6 (IL-6). On the basis of studies that showed overexpression and activation of the AR in advanced prostate cancer, it was suggested that novel therapies that reduce AR expression will provide a benefit to patients. There is experimental evidence showing that prostate tumor growth in vitro and in vivo is inhibited following administration of chemopreventive drugs or antisense oligonucleotides that downregulate AR mRNA and protein expression.     

PRPF6 promotes androgen receptor/androgen receptor-variant 7 actions in castration-resistant prostate cancer cells

Androgen receptor (AR) and its variants play vital roles in development and progression of prostate cancer. To clarify the mechanisms involved in the enhancement of their actions would be crucial for understanding the process in prostate cancer and castration-resistant prostate cancer transformation. Here, we provided the evidence to show that pre-mRNA processing factor 6 (PRPF6) acts as a key regulator for action of both AR full length (AR-FL) and AR variant 7 (AR-V7), thereby participating in the enhancement of AR-FL and AR-V7-induced transactivation in prostate cancer. In addition, PRPF6 is recruited to cis-regulatory elements in AR target genes and associates with JMJD1A to enhance AR-induced transactivation. PRPF6 also promotes expression of AR-FL and AR-V7. Moreover, PRPF6 depletion reduces tumor growth in prostate cancer-derived cell lines and results in significant suppression of xenograft tumors even under castration condition in mouse model. Furthermore, PRPF6 is obviously highly expressed in human prostate cancer samples. Collectively, our results suggest PRPF6 is involved in enhancement of oncogenic AR signaling, which support a previously unknown role of PRPF6 during progression of prostate cancer and castration-resistant prostate cancers.     

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.     

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.     

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.     

Interleukin-6 and renal cell cancer: Production,regulation, and growth effects

Summary Interleukin-6 (IL-6) is a recently characterized pleiotropic cytokine with antitumor activity. We investigated the production of IL-6 by renal cell cancer (RCC) and the growth effects of IL-6 on RCC. Using immunoperoxidase staining, cytoplasmic IL-6 was detected in four of four renal tumor lines and in tumor cells from freshly nephrectomized RCC. We found that IL-6 mRNA was expressed at basal culture conditions by seven of ten RCC tumor lines tested. Biologically active IL-6, as measured by the B9 assay, was produced by all ten RCC tumor lines. The addition of tumor necrosis factor (TNF) significantly augmented the expression of IL-6 mRNA in five RCC tumor lines (P <0.05). The combination of interferon IFN and TNF further enhanced the augmented IL-6 mRNA accumulation seen with TNF alone (P <0.05). TNF also significantly stimulated the production of biologically active IL-6 (P <0.01). Furthermore, IFN and TNF were found to enhance IL-6 bioactivity synergistically (P <0.05). The growth effects of IL-6 on RCC were also investigated in two experimental systems: IL-6 was found to stimulate proliferative responses in six of six RCC tumor lines as measured by thymidine-uptake assays; however, only one of six tumor lines displayed an increase in proliferative response of greater than 21% (113%). The growth effect of IL-6 was further tested in clonogenic assays. One of the tumor lines tested displayed an enhanced growth response of up to 200%. We conclude that IL-6 is produced by RCC; this production is enhanced by TNF with synergistic effects seen with IFN at both mRNA and protein levels. In turn, IL-6 may have a modest stimulatory growth effect on certain RCC tumor lines.This work was supported in part by National Institutes of Health Grant CA 522 499-01, and the Margaret Early Foundation     

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.     

Calmodulin protects androgen receptor from calpain-mediated breakdown in prostate cancer cells

Although inactivation of the androgen receptor (AR) by androgen-ablation or anti-androgen treatment has been frontline therapy for disseminated prostate cancer for over 60 years, it is not curative because castration-resistant prostate cancer cells retain AR activity. Therefore, curative strategy should include targeted elimination of AR protein. Since AR binds to calmodulin (CaM), and since CaM-binding proteins are targets of calpain (Cpn)-mediated proteolysis, we studied the role of CaM and Cpn in AR breakdown in prostate cancer cells. Whereas the treatment of prostate cancer cells individually with anti-CaM drug or calcimycin, which increases intracellular Ca(++) and activates Cpn, led to minimal AR breakdown, combined treatment led to a precipitous decrease in AR protein levels. This decrease in AR protein occurred without noticeable changes in AR mRNA levels, suggesting an increase in AR protein turnover rather than inhibition of AR mRNA expression. Thus, CaM inactivation seems to sensitize AR to Cpn-mediated breakdown in prostate cancer cells. Consistent with this possibility, purified recombinant human AR (rhAR) underwent proteolysis in the presence of purified Cpn, and the addition of purified CaM to the incubation blocked rhAR proteolysis. Together, these observations demonstrate that AR is a Cpn target and AR-bound CaM plays an important role in protecting AR from Cpn-mediated breakdown in prostate cancer cells. These observations raise an intriguing possibility that anti-CaM drugs in combination with Cpn-activating agents may offer a curative strategy for the treatment of prostate cancer, which relies on AR for growth and survival.     

Inhibition of Plk1 represses androgen signaling pathway in castration-resistant prostate cancer

Prostate cancer (PCa) is the second leading cause of cancer-related death in males in the United States. Majority of prostate cancers are originally androgen-dependent and sensitive to androgen-deprivation therapy (ADT), however, most of them eventually relapse and progress into incurable castration-resistant prostate cancer (CRPC). Of note, the activity of androgen receptor (AR) is still required in CRPC stage. The mitotic kinase polo-like kinase 1 (Plk1) is significantly elevated in PCa and its expression correlates with tumor grade. In this study, we assess the effects of Plk1 on AR signaling in both androgen-dependent and androgen-independent PCa cells. We demonstrate that the expression level of Plk1 correlated with tumorigenicity and that inhibition of Plk1 caused reduction of AR expression and AR activity. Furthermore, Plk1 inhibitor BI2536 down-regulated SREBP-dependent expression of enzymes involved in androgen biosynthesis. Of interest, Plk1 level was also reduced when AR activity was inhibited by the antagonist MDV3100. Finally, we show that BI2536 treatment significantly inhibited tumor growth in LNCaP CRPC xenografts. Overall, our data support the concept that Plk1 inhibitor such as BI2536 prevents AR signaling pathway and might have therapeutic potential for CRPC patients.     

c-Myc is a novel target of cell cycle arrest by honokiol in prostate cancer cells

Honokiol (HNK), a highly promising phytochemical derived from Magnolia officinalis plant, exhibits in vitro and in vivo anticancer activity against prostate cancer but the underlying mechanism is not fully clear. This study was undertaken to delineate the role of c-Myc in anticancer effects of HNK. Exposure of prostate cancer cells to plasma achievable doses of HNK resulted in a marked decrease in levels of total and/or phosphorylated c-Myc protein as well as its mRNA expression. We also observed suppression of c-Myc protein in PC-3 xenografts upon oral HNK administration. Stable overexpression of c-Myc in PC-3 and 22Rv1 cells conferred significant protection against HNK-mediated growth inhibition and G₀-G₁ phase cell cycle arrest. HNK treatment decreased expression of c-Myc downstream targets including Cyclin D1 and Enhancer of Zeste Homolog 2 (EZH2), and these effects were partially restored upon c-Myc overexpression. In addition, PC-3 and DU145 cells with stable knockdown of EZH2 were relatively more sensitive to growth inhibition by HNK compared with control cells. Finally, androgen receptor overexpression abrogated HNK-mediated downregulation of c-Myc and its targets particularly EZH2. The present study indicates that c-Myc, which is often overexpressed in early and late stages of human prostate cancer, is a novel target of prostate cancer growth inhibition by HNK.     

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.     

The impact of cell adhesion changes on proliferation and survival during prostate cancer development and progression

In the normal prostate epithelium, androgen receptor (AR) negative basal epithelial cells adhere to the substratum, while AR expressing secretory cells lose substratum adhesion. In contrast, prostate cancer cells both express AR and adhere to a tumor basement membrane. In this review, we describe the differential expression of integrins, growth factor receptors (GFRs), and AR in normal and cancerous epithelium. In addition, we discuss how signals from integrins, GFRs, and AR are integrated to regulate the proliferation and survival of normal and malignant prostate epithelial cells. While cell adhesion is likely of great importance when considering therapeutic approaches for treatment of metastatic prostate cancer, no data on integrin expression are available from tissues of prostate cancer metastasis. However, several drug targets that are upregulated after androgen ablative therapy regulate cell adhesion and thus novel targeted therapies indirectly interfere with cell adhesion mechanisms in prostate cancer cells.