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.     

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.     

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.     

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.     

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.     

The significance of galectin-3 as a new basal cell marker in prostate cancer

Prostate cancer may originate from distinct cell types, resulting in the heterogeneity of this disease. Galectin-3 (Gal-3) and androgen receptor (AR) have been reported to play important roles in the progression of prostate cancer, and their heterogeneous expressions might be associated with different cancer subtypes. Our study found that in various prostate cancer cell lines Gal-3 expression was always opposite to AR expression and other luminal cell markers but consistent with basal cell markers including glutathione S-transferase-π and Bcl-2. This expression pattern was confirmed in human prostate cancer tissues. Our results also showed that prostate cancer cells positive with basal cell markers were more aggressive. Downregulation of Gal-3 expression resulted in increased apoptotic potential and decreased metastasis potential of prostate cancer cells. Our findings demonstrate for the first time that Gal-3 may serve as a new marker for basal characteristics of prostate cancer epithelium. This study helps us to better understand the heterogeneity of prostate cancer. The clinical significance of this study lies in the application of Gal-3 to distinguish prostate cancer subtypes and improve treatment efficacy with designed personalized therapy.     

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.     

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.     

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 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.     

The androgen receptor (AR) in syndromes of androgen insensitivity and in prostate cancer

The actions of androgens, principally testosterone and 5alpha-dihydrotestosterone, are mediated by a specific receptor protein, the androgen receptor (AR), which is encoded by a single-copy gene located on the human X-chromosome. This receptor protein is a prototypical member of the nuclear receptor family and modulates a range of processes during embryogenesis and in the adult. During embryogenesis, normal AR function is critical to the development of the male phenotype and defects of the AR cause a range of phenotypic abnormalities of male sexual development. Complete loss of AR function has been traced to a number of distinct types of genetic events, including abnormalities of mRNA splicing, the introduction of premature termination codons, and amino acid substitution mutations. An interesting subset of mutations is that in which the AR is completely undetectable using sensitive immunoassays. In all instances, these functional abnormalities are associated with a phenotype of complete androgen insensitivity (complete testicular feminization). By contrast, partial defects of AR function are almost invariably caused by amino acid substitutions within the DNA- and hormone-binding domains of the receptor protein. Such partial defects of receptor function may be caused by changes in either receptor function or receptor abundance.The alterations of AR function and expression that have been characterized in clinical prostatic cancers and in prostate cancer cell lines differ in several important respects. A number of studies have documented the emergence of considerable heterogeneity of AR expression at early stages in the development of prostate cancer. Despite these early changes of AR expression, a substantial body of information suggests that the AR is expressed in advanced forms of prostate cancer, in some cases as the result of amplification events. While infrequent in localized tumors, mutations of the AR have been identified in a number of advanced prostatic cancers and in some instances appear to alter the ligand specificity of the AR. Finally, it appears that other signaling pathways can act to influence AR function.