Transgelin functions as a suppressor via inhibition of ARA54-enhanced androgen receptor transactivation and prostate cancer cell growth

The androgen receptor (AR) requires coregulators for its optimal function. However, whether AR coregulators further need interacting protein(s) for their proper function remains unclear. Here we describe transgelin as the first ARA54-associated negative modulator for AR. Transgelin suppressed ARA54-enhanced AR function in ARA54-positive, but not in ARA54-negative, cells. Transgelin suppressed AR transactivation via interruption of ARA54 homodimerization and AR-ARA54 heterodimerization, resulting in the cytoplasmic retention of AR and ARA54. Stable transfection of transgelin in LNCaP cells suppressed AR-mediated cell growth and prostate-specific antigen expression, whereas this suppressive effect was abolished by the addition of ARA54-small interfering RNA. Results from tissue surveys showing decreased expression of transgelin in prostate cancer specimens further strengthened the suppressor role of transgelin. Our findings reveal the novel mechanisms of how transgelin functions as a suppressor to inhibit prostate cancer cell growth. They also demonstrate that AR coregulators, like ARA54, might have dual in vivo roles functioning as both a direct coactivator and as an indirect mediator in AR function. The finding that a protein can modulate AR function without direct interaction with AR might provide a new therapeutic approach, with fewer side effects, to battle prostate cancer by targeting AR indirectly.     

Dominant-negative androgen receptor inhibition of intracrine androgen-dependent growth of castration-recurrent prostate cancer

Background

Prostate cancer (CaP) is the second leading cause of cancer death in American men. Androgen deprivation therapy is initially effective in CaP treatment, but CaP recurs despite castrate levels of circulating androgen. Continued expression of the androgen receptor (AR) and its ligands has been linked to castration-recurrent CaP growth.

Principal Finding

In this report, the ligand-dependent dominant-negative ARΔ142–337 (ARΔTR) was expressed in castration-recurrent CWR-R1 cell and tumor models to elucidate the role of AR signaling. Expression of ARΔTR decreased CWR-R1 tumor growth in the presence and absence of exogenous testosterone (T) and improved survival in the presence of exogenous T. There was evidence for negative selection of ARΔTR transgene in T-treated mice. Mass spectrometry revealed castration-recurrent CaP dihydrotestosterone (DHT) levels sufficient to activate AR and ARΔTR. In the absence of exogenous testosterone, CWR-R1-ARΔTR and control cells exhibited altered androgen profiles that implicated epithelial CaP cells as a source of intratumoral AR ligands.

Conclusion

The study provides in vivo evidence that activation of AR signaling by intratumoral AR ligands is required for castration-recurrent CaP growth and that epithelial CaP cells produce sufficient active androgens for CaP recurrence during androgen deprivation therapy. Targeting intracrine T and DHT synthesis should provide a mechanism to inhibit AR and growth of castration-recurrent CaP.     

Growth inhibition of androgen-responsive prostate cancer cells with brefeldin A targeting cell cycle and androgen receptor

Background  

Androgen ablation is one of the viable therapeutic options for patients with primary hormone (androgen)-dependent prostate cancer. However, an antibiotic brefeldin A (BFA) has been shown to exhibit the growth inhibitory effect on human cancer cells. We thus investigated if BFA might inhibit proliferation of androgen-responsive prostate cancer LNCaP cells and also explored how it would be carried out, focusing on cell cycle and androgen receptor (AR).     

Membrane androgen receptor sensitive Na+/H+ exchanger activity in prostate cancer cells

Membrane androgen receptors (mAR) are expressed in several tumors. mAR activation by testosterone albumin conjugates (TAC) suppresses tumor growth and migration. mAR signaling involves phosphoinositide-3-kinase (PI3K) and Rho-associated protein kinase (ROCK). PI3K stimulates serum- and glucocorticoid-inducible kinase SGK1, which in turn activates Na⁺/H⁺-exchangers (NHE). In prostate cancer cells cytosolic pH (pH_i) was determined utilizing 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-fluorescence and NHE-activity utilizing Na⁺-dependent cytosolic realkalinization following an ammonium pulse. TAC (100 nM) significantly increased pH_i and NHE-activity, effects abrogated by NHE1-inhibitor cariporide (10 μM), SGK1-inhibitors EMD638683 (50 μM) and GSK650349 (10 μM) and ROCK-inhibitors Y-27632 (10 μM) and fasudil (100 μM). TAC treatment rapidly and significantly increased cell volume and actin polymerization, effects abolished in the presence of cariporide. Thus, mAR-activation activates cariporide-sensitive Na⁺/H⁺-exchangers, an effect requiring SGK1 and ROCK activity.     

The androgen receptor associates with the epidermal growth factor receptor in androgen-sensitive prostate cancer cells

Many recent evidences indicate that androgen-sensitive prostate cancer cells have a lower malignant phenotype that is in particular characterized by a reduced migration and invasion. We previously demonstrated that expression of androgen receptor (AR) by transfection of the androgen-independent prostate cancer cell line PC3 decreases invasion and adhesion of these cells (PC3-AR) through modulation of alpha6beta4 integrin expression. The treatment with the synthetic androgen R1881 further reduced invasion of the cells without, however, modifying alpha6beta4 expression on the cell surface, suggesting an interference with the invasion process in response to EGF. We investigated whether the presence of the AR could affect EGF receptor (EGFR)-mediated signaling in response to EGF by evaluating autotransphosphorylation of the receptor as well as activation of downstream signalling pathways. Immunoprecipitation studies demonstrated a reduction of EGF-induced tyrosine phosphorylation of EGFR in PC3-AR cells. In addition, EGF-stimulated PI3K activity, a key signalling pathway for invasion of these cells, was decreased in PC3-AR cells and further reduced by treatment with R1881, indicating decreased functionality of EGFR. An interaction between EGFR and AR has been demonstrated by immunoconfocal and co-immunoprecipitation analysis in PC3-AR cells, suggesting a possible interference of AR on EGFR signalling by interaction of the two proteins. In conclusion, our results suggest that the expression of AR by transfection in PC3 cells confers a less malignant phenotype by interfering with EGFR autophosphorylation and signalling in response to EGF leading to invasion through a mechanism involving an interaction between AR and EGFR.     

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.     

Suppression of androgen receptor signaling in prostate cancer cells by an inhibitory receptor variant

There is increasing evidence that sensitization of the androgen receptor (AR) signaling pathway contributes to the failure of androgen ablation therapy for prostate cancer, and that direct targeting of the AR may be a useful therapeutic approach. To better understand how AR function could be abrogated in prostate cancer cells, we have developed a series of putative dominant-negative variants of the human AR, containing deletions or mutations in activation functions AF-1, AF-5, and/or AF-2. One construct, AR inhibitor (ARi)-410, containing a deletion of AF-1 and part of AF-5 of the AR, had no intrinsic transactivation activity but inhibited wild-type AR (wtAR) in a ligand-dependent manner by at least 95% when transfected at a 4:1 molar ratio. ARi-410 was an equally potent inhibitor of gain-of-function AR variants. Ectopic expression of ARi-410 inhibited the proliferation of AR-positive LNCaP cells, but not AR-negative PC-3 cells. Whereas ARi-410 also marginally inhibited progesterone receptor activity, this was far less pronounced than the effect on AR (50% vs. 95% maximal inhibition, respectively), and there was no inhibition of either vitamin D or estrogen receptor activity. In the presence of ligand, ARi-410 interacted with wtAR, and both receptors translocated into the nucleus. Whereas the amino-carboxy terminal interaction was not necessary for optimal dominant-negative activity, disruption of dimerization through the ligand binding domain reduced the efficacy of ARi-410. In addition, although inhibition of AR function by ARi-410 was not dependent on DNA binding, the DNA binding domain was required for dominant-negative activity. Taken together, our results suggest that interaction between ARi-410 and the endogenous AR in prostate cancer cells, potentially through the DNA binding and ligand binding domains, results in a functionally significant reduction in AR signaling and AR-dependent cell growth.     

Androgen receptor activation by G(s) signaling in prostate cancer cells

The androgen receptor (AR) is activated in prostate cancer patients undergoing androgen ablative therapy and mediates growth of androgen-insensitive prostate cancer cells, suggesting it is activated by nonandrogenic factors. We demonstrate that activated alpha subunit of heterotrimeric guanine nucleotide-binding G(s) protein activates the AR in prostate cancer cells and also synergizes with low concentration of androgen to more fully activate the AR. The G alpha(s) activates protein kinase A, which is required for the nuclear partition and activation of AR. These data suggest a role for G alpha(s) and PKA in the transactivation of AR in prostate cancer cells under the environment of reduced androgen levels.     

A role of androgen receptor protein in cell growth of an androgen-independent prostate cancer cell line

Prostate cancer, which develops due to androgen and is initially responsive to androgen deprivation therapy, often comes to acquire androgen deprivation therapy resistance in short order. We investigated the role of androgen receptor (AR) protein in an androgen-independent prostate cancer cell line using AR ligands and AR siRNA. Although the androgen-independent cell line scarcely responded to AR ligands, their growth was attenuated by ablation of AR protein by siRNA.     

Mutually antagonistic effects of androgen and activin in the regulation of prostate cancer cell growth

Activin, a member of the TGFbeta superfamily, is expressed in the prostate and inhibits growth. We demonstrate that the effects of activin and androgen on regulation of prostate cancer cell growth are mutually antagonistic. In the absence of androgen, activin induced apoptosis in the androgen-dependent human prostate cancer cell line LNCaP, an effect suppressed by androgen administration. Although activin by itself did not alter the cell cycle distribution, it potently suppressed androgen- induced progression of cells into S-phase of the cell cycle and thus inhibited androgen-stimulated growth of LNCaP cells. Expression changes in cell cycle regulatory proteins such as Rb, E2F-1, and p27 demonstrated a strong correlation with the mutually antagonistic growth regulatory effects of activin and androgen. The inhibitory effect of activin on growth was independent of serine, serine, valine, serine motif phosphorylation of Smad3. Despite their antagonistic effect on growth, activin and androgen costimulated the expression of prostate-specific antigen through a Smad3-mediated mechanism. These observations indicate the existence of a complex cross talk between activin and androgen signaling in regulation of gene expression and growth of the prostate.