GLI1, a crucial mediator of sonic hedgehog signaling in prostate cancer, functions as a negative modulator for androgen receptor

Sonic hedgehog (SHH) signaling, acting in a combinatorial manner with androgen signaling, is essential for prostate patterning and development. Recently, elevated activation of SHH signaling has been shown to play important roles in proliferation, progression and metastasis of prostate cancer. In this report, we demonstrate for the first time, that GLI1, which has been shown to play a central role in SHH signaling in prostate cancer, can act as a co-repressor to substantially block androgen receptor (AR)-mediated transactivation, at least in part, by directly interacting with AR. Our observations suggest that the SHH-GLI pathway might be one of determinants governing the transition of prostate cancer from an androgen-dependent to an androgen-independent state by compensating, or even superseding androgen signaling.     

The androgen receptor: a biologically relevant vaccine target for the treatment of prostate cancer

The androgen receptor (AR) plays an essential role in the development and progression of prostate cancer. However, while it has long been the primary molecular target of metastatic prostate cancer therapies, it has not been explored as an immunotherapeutic target. In particular, the AR ligand-binding domain (LBD) is a potentially attractive target, as it has an identical sequence among humans as well as among multiple species, providing a logical candidate for preclinical evaluation. In this report, we evaluated the immune and anti-tumor efficacy of a DNA vaccine targeting the AR LBD (pTVG-AR) in relevant rodent preclinical models. We found immunization of HHDII-DR1 mice, which express human HLA-A2 and HLA-DR1, with pTVG-AR augmented AR LBD HLA-A2-restricted peptide-specific, cytotoxic immune responses in vivo that could lyse human prostate cancer cells. Using an HLA-A2-expressing autochthonous model of prostate cancer, immunization with pTVG-AR augmented HLA-A2-restricted immune responses that could lyse syngeneic prostate tumor cells and led to a decrease in tumor burden and an increase in overall survival of tumor-bearing animals. Finally, immunization decreased prostate tumor growth in Copenhagen rats that was associated with a Th1-type immune response. These data show that the AR is as a prostate cancer immunological target antigen and that a DNA vaccine targeting the AR LBD is an attractive candidate for clinical evaluation.     

Calpain-mediated androgen receptor breakdown in apoptotic prostate cancer cells

Since androgen receptor (AR) plays an important role in prostate cancer development and progression, androgen-ablation has been the frontline therapy for treatment of advanced prostate cancer even though it is rarely curative. A curative strategy should involve functional and structural elimination of AR from prostate cancer cells. We have previously reported that apoptosis induced by medicinal proteasome-inhibitory compound celastrol is associated with a decrease in AR protein levels. However celastrol-stimulated events contributing to this AR decrease have not been elucidated. Here, we report that a variety of chemotherapeutic agents, including proteasome inhibitors, a topoisomerase inhibitor, DNA-damaging agents and docetaxel that cause cell death, decrease AR levels in LNCaP prostate cancer cells. This decrease in AR protein levels was not due to the suppression of AR mRNA expression in these cells. We observed that a proteolytic activity residing in cytosol of prostate cancer cells is responsible for AR breakdown and that this proteolytic activity was stimulated upon induction of apoptosis. Interestingly, proteasome inhibitor celastrol- and chemotherapeutic drug VP-16-stimulated AR breakdown was attenuated by calpain inhibitors calpastatin and N-acetyl-L-leucyl-L-leucyl-L-methioninal. Furthermore, AR proteolytic activity pulled down by calmodulin-agarose beads from celastrol-treated PC-3 cells showed immunoreactivity to a calpain antibody. Taken together, these results demonstrate calpain involvement in proteasome inhibitor-induced AR breakdown, and suggest that AR degradation is intrinsic to the induction of apoptosis in prostate cancer cells.     

Role for 18:1 lysophosphatidic acid as an autocrine mediator in prostate cancer cells

Lysophosphatidic acid (LPA) is a lipid mediator that may play an important role in growth and survival of carcinomas. In this study, LPA production and response were characterized in two human prostate cancer (CaP) cell lines: PC-3 and Du145. Bombesin, a neuroendocrine peptide that is mitogenic for CaP cells, stimulated focal adhesion kinase phosphorylation and activated the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Similar responses were elicited by 18:1 LPA (oleoyl-LPA). Studies using radioisotopic labeling revealed that both PC-3 and Du145 generate LPA and that LPA production is increased by bombesin. The kinetics of bombesin-induced phospholipase D activation and LPA production were similar. Using electrospray ionization mass spectrometry, 18:1 LPA was found to be an abundant LPA species in CaP cell medium. Structure activity studies of acyl-LPAs revealed that 18:1 LPA is most efficacious for activation of extracellular signal-regulated kinase and phospholipase D in CaP cells. Incubation with 18:1 LPA caused homologous desensitization of LPA response, whereas bombesin caused heterologous desensitization. LPA was present at nanomolar levels in medium from bombesin-treated cells. LPA extracted from the medium induced calcium mobilization in CaP cells. These results demonstrate that bioactive LPA is generated by CaP cells in response to a mitogen and suggest that 18:1 LPA can act as an autocrine mediator.     

Lovastatin-induced RhoA modulation and its effect on senescence in prostate cancer cells

Lovastatin inhibits a 3-hydroxy 3-methylglutaryl coenzyme A reductase and prevents the synthesis of cholesterol precursors, such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), responsible for important cell signaling in cell proliferation and migration. Recently, the anti-cancer effect of lovastatin has been suggested in various tumor types. In this study, we showed that a low dose lovastatin induced senescence and G1 cell cycle arrest in human prostate cancer cells. Addition of GGPP or mevalonate, but not FPP, prevented the lovastatin-induced G1 phase cell cycle arrest and cell senescence. We found that constitutively active RhoA (caRhoA) reversed lovastatin-induced senescence in caRhoA-transfected PC-3 cells. Thus, we postulate that modulation of RhoA may be critical in lovastatin-induced senescence in PC-3 cells.     

Androgens as therapy for androgen receptor-positive castration-resistant prostate cancer

Prostate cancer is the most frequently diagnosed non-cutaneous tumor of men in Western countries. While surgery is often successful for organ-confined prostate cancer, androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, this therapy is associated with several undesired side-effects, including increased risk of cardiovascular diseases. Shortening the period of androgen ablation therapy may benefit prostate cancer patients. Intermittent Androgen Deprivation therapy improves quality of life, reduces toxicity and medical costs, and delays disease progression in some patients. Cell culture and xenograft studies using androgen receptor (AR)-positive castration-resistant human prostate cancers cells (LNCaP, ARCaP, and PC-3 cells over-expressing AR) suggest that androgens may suppress the growth of AR-rich prostate cancer cells. Androgens cause growth inhibition and G1 cell cycle arrest in these cells by regulating c-Myc, Skp2, and p27^Kip via AR. Higher dosages of testosterone cause greater growth inhibition of relapsed tumors. Manipulating androgen/AR signaling may therefore be a potential therapy for AR-positive advanced prostate cancer.     

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.     

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.     

Androgen receptor down regulation by small interference RNA induces cell growth inhibition in androgen sensitive as well as in androgen independent prostate cancer cells

We investigated the effects of androgen receptor (AR) down regulation with a small interference RNA molecule (siRNA_AR(start)) on androgen sensitive LNCaP and androgen independent LNCaPabl prostate cancer cells, the latter representing an in vitro model for the development of therapy resistance in prostate cancer. Although LNCaPabl cells express increased levels of AR in comparison with androgen sensitive LNCaP cells, the protein was significantly down regulated in response to siRNA_AR(start) treatment. This AR down regulation resulted in a marked cell growth inhibition in both cell lines. By contrast, DU-145 prostate cancer cells, which lack AR expression, were not inhibited by the siRNA_AR(start). In consequence to AR down regulation, both cell lines, LNCaP and LNCaPabl, shared a highly similar gene expression profile in terms of major changes in cell cycle regulatory genes. The cell cycle inhibitor p21(Waf1/Cip1) as well as cyclin D1 were significantly up regulated by siRNA_AR(start) treatment, considering a switch in cyclin expression towards cell cycle retardation. Control molecules had moderate effects on cell proliferation and gene expression, respectively. In summary, we found that AR inhibition with siRNA induces cell growth retardation in androgen sensitive as well as in androgen independent prostate cancer cells and thus may represent an interesting approach to combat hormone-refractory prostate cancer.     

Effects of androgen receptor and androgen on gene expression in prostate stromal fibroblasts and paracrine signaling to prostate cancer cells

The androgen receptor (AR) is expressed in a subset of prostate stromal cells and functional stromal cell AR is required for normal prostate developmental and influences the growth of prostate tumors. Although we are broadly aware of the specifics of the genomic actions of AR in prostate cancer cells, relatively little is known regarding the gene targets of functional AR in prostate stromal cells. Here, we describe a novel human prostate stromal cell model that enabled us to study the effects of AR on gene expression in these cells. The model involves a genetically manipulated variant of immortalized human WPMY-1 prostate stromal cells that overexpresses wildtype AR (WPMY-AR) at a level comparable to LNCaP cells and is responsive to dihydrotestosterone (DHT) stimulation. Use of WPMY-AR cells for gene expression profiling showed that the presence of AR, even in the absence of DHT, significantly altered the gene expression pattern of the cells compared to control (WPMY-Vec) cells. Treatment of WPMY-AR cells, but not WPMY-Vec control cells, with DHT resulted in further changes that affected the expression of 141 genes by 2-fold or greater compared to vehicle treated WPMY-AR cells. Remarkably, DHT significantly downregulated more genes than were upregulated but many of these changes reversed the initial effects of AR overexpression alone on individual genes. The genes most highly effected by DHT treatment were categorized based upon their role in cancer pathways or in cell signaling pathways (transforming growth factor-β, Wnt, Hedgehog and MAP Kinase) thought to be involved in stromal-epithelial crosstalk during prostate or prostate cancer development. DHT treatment of WPMY-AR cells was also sufficient to alter their paracrine potential for prostate cancer cells as conditioned medium from DHT-treated WPMY-AR significantly increased growth of LNCaP cells compared to DHT-treated WPMY-Vec cell conditioned medium.     

Adiponectin as a growth inhibitor in prostate cancer cells

Prostate cancer is associated with obesity. However, the molecular basis of this association is not well known. Adiponectin is a major adipose cytokine that decreases in circulation in obesity and ameliorates obesity. Here, we identify adiponectin as a novel inhibitor in prostate cancer cell growth. Adiponectin occurs in non-proteolytic (full-length adiponectin: f-adiponectin) and proteolytic (globular adiponectin) forms in various oligomeric states (trimer, hexamer, and high molecular weight complex). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay demonstrates that f-adiponectin inhibits prostate cancer cell growth drastically at subphysiological concentrations. Furthermore, velocity sedimentation analysis shows that the high molecular weight complex of f-adiponectin is the inhibitory form. Moreover, f-adiponectin suppresses leptin- and/or insulin-like growth factor-I (IGF-I)-stimulated, androgen-independent DU145 cell growth, and dihydrotestosterone-stimulated, androgen-dependent LNCaP-FGC cell growth. In addition, f-adiponectin enhances doxorubicin inhibition of prostate cancer cell growth. Therefore, f-adiponectin is a molecular mediator between prostate cancer and obesity, and may be therapeutic to prostate cancer.     

Human checkpoint protein hRad9 functions as a negative coregulator to repress androgen receptor transactivation in prostate cancer cells

Positive responses to combined androgen elimination therapy and radiation therapy have been well documented in the treatment of prostate cancer patients. The detailed mechanisms how androgen-androgen receptor (AR) cross talks to the radiation-related signal pathways, however, remain largely unknown. Here we report the identification of hRad9, a key member of the checkpoint Rad protein family, as a coregulator to suppress androgen-AR transactivation in prostate cancer cells. In vivo and in vitro interaction assays using Saccharomyces cerevisiae two-hybrid, mammalian two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation methods prove that AR can interact with the C terminus of hRad9 via its ligand binding domain. The FXXLF motif within the C terminus of hRad9 interrupts the androgen-induced interaction between the N terminus and C terminus of AR. This interaction between AR and hRad9 may result in the suppression of AR transactivation, demonstrated by the repressed AR transactivation in androgen-induced luciferase reporter assay and the reduced endogenous prostate-specific antigen expression in Western blot assay. Addition of small interfering RNA of hRad9 can reverse hRad9 suppression effects, which suggests that hRad9 functions as a repressor of AR transactivation in vivo. Together, our data provide the first linkage between androgen-AR signals and radiation-induced responses. Further studies of the influence of hRad9 on prostate cancer growth may provide potential new therapeutic approaches.     

Identification and characterization of androgen receptor associated coregulators in prostate cancer cells

The androgen receptor (AR) is a member of the nuclear receptor (NR) superfamily that mediates the effects of androgens on target tissues. Over the last decade, it has become apparent that NRs require accessory factors for optimal activation of target gene expression. Numerous NR coregulators have been identified, with diverse structures and potential mechanisms of coregulation, creating an increasingly complicated picture of NR action. Due to the expanding complexity of the coregulator field, this review will focus on the AR ligand-binding domain (LBD) and N-terminal interacting proteins identified by our lab. The LBD-interacting proteins ARA70, ARA55 and ARA54 were first characterized and ARA70 was found to have a relatively higher specificity for the AR in human prostate cancer DU145 cells. Characterization of the functional relationship between the AR and these coregulators indicated that ARA70 and ARA55 could enhance the androgenic effects of 17beta-estradiol (E2) and hydroxyflutamide (HF), an antiandrogen commonly used in the treatment of prostate cancer. ARA160, an AR N-terminal interacting protein also known as TATA element modulatory factor (TMF), was subsequently shown to cooperate with ARA70 in enhancing AR activity. Another AR N-terminal interacting protein, ARA24, interacted with the poly-Q tract, a region within the N-terminus of the AR linked to Kennedy's disease (X-linked spinal and bulbar muscular atrophy). More recently, our lab has identified ARA267, a SET domain containing protein, and supervillin, an F-actin binding protein, as AR coregulators. Collectively, the data from these studies indicate that these coregulators are necessary for optimal AR transactivation. Interruption of the interaction between AR and these proteins may serve as a new therapeutic target in the treatment of prostate cancer.     

Alterations of androgen receptor in prostate cancer

The significance of androgens in the development of prostate cancer has been known for more than half century. During the last decade, a lot of effort has been put to study the significance of the specific nuclear receptor of the hormone, androgen receptor (AR). It has been suggested that polymorphisms, especially the length of CAG repeat in exon 1 of the gene, are associated with the risk of prostate cancer. However, not all studies have confirmed the association. Most surprisingly, it has now become clear that prostate carcinomas emerging during the androgen withdrawal therapy (i.e. hormone-refractory tumors) are capable of reactivating the AR-mediated signalling despite of the low levels of androgens. In addition, it has been shown that AR gene itself is genetically targeted. One-third of the hormone-refractory prostate carcinomas contains amplification of the gene. In addition, 10-30% of prostate carcinomas treated by antiandrogens acquire point mutation in the AR gene. The genetic alterations in AR indicate that receptor should be considered as putative treatment target. Evidently, the currently available antiandrogens are not capable to abolish the AR-mediated signalling efficiently enough.