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.     

Androgen receptor (AR) NH2- and COOH-terminal interactions result in the differential influences on the AR-mediated transactivation and cell growth

Early reports showed that androgen receptor (AR) NH2- and COOH-terminal (N-C) interaction was important for full AR function. However, the influence of these interactions on the AR in vivo effects remains unclear. Here we tested some AR-associated peptides and coregulators to determine their influences on AR N-C interaction, AR transactivation, and AR coregulator function. The results showed that AR coactivators such as ARA70N, gelsolin, ARA54, and SRC-1 can enhance AR transactivation but showed differential influences on the N-C interaction. In contrast, AR corepressors ARA67 and Rad9 can suppress AR transactivation, with ARA67 enhancing and Rad9 suppressing AR N-C interaction. Furthermore, liganded AR C terminus-associated peptides can block AR N-C interaction, but only selective peptides can block AR transactivation and coregulator function. We found all the tested peptides can suppress prostate cancer LNCaP cell growth at different levels in the presence of 5alpha-dihydrotestosterone, but only the tested FXXLF-containing peptides, not FXXMF-containing peptides, can suppress prostate cancer CWR22R cell growth. Together, these results suggest that the effects of AR N-C interactions may not always correlate with similar effects on AR-mediated transactivation and/or AR-mediated cell growth. Therefore, drugs designed by targeting AR N-C interaction as a therapeutic intervention for prostate cancer treatment may face unpredictable in vivo effects.     

Proteasome activity is required for androgen receptor transcriptional activity via regulation of androgen receptor nuclear translocation and interaction with coregulators in prostate cancer cells

Upon binding to androgen, the androgen receptor (AR) can translocate into the nucleus and bind to androgen response element(s) to modulate its target genes. Here we have shown that MG132, a 26 S proteasome inhibitor, suppressed AR transactivation in an androgen-dependent manner in prostate cancer LNCaP and PC-3 cells. In contrast, MG132 showed no suppressive effect on glucocorticoid receptor transactivation. Additionally, transfection of PSMA7, a proteasome subunit, enhanced AR transactivation in a dose-dependent manner. The suppression of AR transactivation by MG132 may then result in the suppression of prostate-specific antigen, a well known marker used to monitor the progress of prostate cancer. Further mechanistic studies indicated that MG132 may suppress AR transactivation via inhibition of AR nuclear translocation and/or inhibition of interactions between AR and its coregulators, such as ARA70 or TIF2. Together, our data suggest that the proteasome system plays important roles in the regulation of AR activity in prostate cancer cells and may provide a unique target site for the development of therapeutic drugs to block androgen/AR-mediated prostate tumor growth.     

Functional domain and motif analyses of androgen receptor coregulator ARA70 and its differential expression in prostate cancer

Androgen receptor (AR)-associated coregulator 70 (ARA70) was the first identified AR coregulator. However, its molecular mechanism and biological relevance to prostate cancer remain unclear. Here we show that ARA70 interacts with and promotes AR activity via the consensus FXXLF motif within the ARA70-N2 domain (amino acids 176-401). However, it does not promote AR activity via the classic LXXLL motif located at amino acids 92-96, although this classic LXXLL motif is important for ARA70 to interact with other receptors, such as PPARgamma. The molecular mechanisms by which ARA70 enhances AR transactivation involve the increase of AR expression, protein stability, and nuclear translocation. Furthermore, ARA70 protein is more frequently detected in prostate cancer specimens (91.74%) than in benign tissues (64.64%, p < 0.0001). ARA70 expression is also increased in high-grade prostate cancer tissues as well as the hormone-refractory LNCaP xenografts and prostate cancer cell lines. Because ARA70 can promote the antiandrogen hydroxyflutamide (HF)-enhanced AR transactivation, the increased ARA70 expression in hormone-refractory prostate tumors may confer the development of HF withdrawal syndrome, commonly diagnosed in patients with the later stages of prostate cancer. Because ARA70-N2 containing the AR-interacting FXXLF motif without coactivation function can suppress HF-enhanced AR transactivation in the hormone-refractory LNCaP cells, using the ARA70-N2 inhibitory peptide at the hormone refractory stage to battle the HF withdrawal syndrome may become an alternative strategy to treat prostate cancer.     

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.     

ARA67/PAT1 functions as a repressor to suppress androgen receptor transactivation

The androgen receptor (AR) may recruit multiple coregulators for proper or optimal transactivation. Here we report the identification and characterization of ARA67/PAT1 as an AR coregulator from a prostate cDNA library. ARA67/PAT1 was screened out as an AR N terminus interacting protein. Interaction mapping shows that the cooperation of multiple domains within ARA67/PAT1 may be required for the maximal interaction with AR. ARA67/PAT1 functions as a repressor with better suppressive effects on AR compared to glucocorticoid receptor and estrogen receptor. Further mechanism dissection reveals that the interrupted AR cytoplasmic-nuclear shuttling may play a major role in ARA67/PAT1 mediated suppression on AR. Together, these results suggest that ARA67/PAT1 may function as a novel repressor that can modulate AR function in prostate cancer.     

Isolation and characterization of ARA160 as the first androgen receptor N-terminal-associated coactivator in human prostate cells.

The androgen receptor (AR) is a member of the steroid receptor superfamily that may require coactivators for proper or maximal transactivation. Using a purified AR N-terminal peptide as a probe to screen the human testis expression library, we identified an androgen-enhanced AR N-terminal-associated protein ARA160, which consists of 1,093 amino acids with an apparent molecular mass of 160 kDa. Sequence comparison in GenBank(TM) reveals that ARA160 shares an identical sequence with a HIV-1 TATA element modulatory factor, TMF. The far-Western blotting and co-immunoprecipitation assays demonstrate that the AR can interact directly with ARA160/TMF. Affinity gel pull-down and mammalian two-hybrid assays further suggest androgen can enhance significantly the interaction between AR and ARA160. Transient transfection assays demonstrated that ARA160 might function as a coactivator for AR-mediated transactivation in human prostate cancer PC-3 cells. Our data further suggest that this AR N-terminal coactivator can function cooperatively with AR C-terminal coactivator, ARA70, in PC-3 cells. Together, our data demonstrate that ARA160 might represent the first identified androgen-enhanced N-terminal coactivator for the AR.     

A competitive inhibitor that reduces recruitment of androgen receptor to androgen-responsive genes

The androgen receptor (AR) has a critical role in the growth and progression of androgen-dependent and castration-resistant prostate cancers. To identify novel inhibitors of AR transactivation that block growth of prostate cancer cells, a luciferase-based high-throughput screen of ~160,000 small molecules was performed in cells stably expressing AR and a prostate-specific antigen (PSA)-luciferase reporter. CPIC (1-(3-(2-chlorophenoxy) propyl)-1H-indole-3-carbonitrile) was identified as a small molecule that blocks AR transactivation to a greater extent than other steroid receptors. CPIC inhibited AR-mediated proliferation of androgen-sensitive prostate cancer cell lines, with minimal toxicity in AR-negative cell lines. CPIC treatment also reduced the anchorage-independent growth of LAPC-4 prostate cancer cells. CPIC functioned as a pure antagonist by inhibiting the expression of AR-regulated genes in LAPC-4 cells that express wild-type AR and exhibited weak agonist activity in LNCaP cells that express the mutant AR-T877A. CPIC treatment did not reduce AR levels or alter its nuclear localization. We used chromatin immunoprecipitation to identify the site of action of CPIC. CPIC inhibited recruitment of androgen-bound AR to the PSA promoter and enhancer sites to a greater extent than bicalutamide. CPIC is a new therapeutic inhibitor that targets AR-mediated gene activation with potential to arrest the growth of prostate cancer.     

The use of phage display technique for the isolation of androgen receptor interacting peptides with (F/W)XXL(F/W) and FXXLY new signature motifs

Early studies suggested that the signature motif, LXXLL, within steroid hormone receptor p160 coregulators may play important roles for the mediation of receptor-coregulator interaction. Interestingly, several androgen receptor (AR) coregulators, such as ARA70 and ARA55, may not use such a unique motif to mediate their coregulator activity. Here we apply the phage display technique to identify some new signature motifs, (F/W)XXL(F/W) and FXXLY (where F is phenylalanine, W is tryptophan, L is leucine, Y is tyrosine, and X is any amino acid) that can influence the interaction between AR and AR coregulators. Sequence analyses found that several AR coregulators, such as ARA70, ARA55, ARA54, and FHL2, contain FXXL(F/Y) motifs. Both glutathione S-transferase pull-down assays and transient transfection reporter assays demonstrate that these AR coregulators may use the FXXL(F/Y) motif to interact with AR and exert their AR coregulator activity. Exchanging the amino acid of Phe, Trp, or Tyr in this newly identified signature motif cluster may influence these peptides to interact with AR. The motif-containing peptides, as well as ARA70 or ARA54, may require selective flanking sequences for the better interaction with AR. In addition to influencing the AR transactivation, these motifs in AR-interacting peptides/proteins were also able to influence the AR N-/C-terminal interaction. Together, our data suggest that AR interacting peptides and/or AR coregulators may utilize the (F/W)XXL(F/W) and FXXLY motifs to mediate their interaction with AR and exert their influences on the AR transactivation.     

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.