A dominant-negative mutant of androgen receptor coregulator ARA54 inhibits androgen receptor-mediated prostate cancer growth.

The ligand-bound androgen receptor (AR) regulates target genes via a mechanism involving coregulators such as androgen receptor-associated 54 (ARA54). We investigated whether the interruption of the AR coregulator function could lead to down-regulation of AR activity. Using in vitro mutagenesis and a yeast two-hybrid screening assay, we have isolated a mutant ARA54 (mt-ARA54) carrying a point mutation at amino acid 472 changing a glutamic acid to lysine, which acts as a dominant-negative inhibitor of AR transactivation. In transient transfection assays of prostate cancer cell lines, the mt-ARA54 suppressed endogenous mutated AR-mediated and exogenous wild-type AR-mediated transactivation in LNCaP and PC-3 cells, respectively. In DU145 cells, the mt-ARA54 suppressed exogenous ARA54 but not other coregulators, such as ARA55-enhanced or SRC-1-enhanced AR transactivation. In the LNCaP cells stably transfected with the plasmids encoding the mt-ARA54 under the doxycycline inducible system, the overexpression of the mt-ARA54 inhibited cell growth and endogenous expression of prostate-specific antigen. Mammalian two-hybrid assays further demonstrated that the mt-ARA54 can disrupt the interaction between wild-type ARA54 molecules, suggesting that ARA54 dimerization or oligomerization may play an essential role in the enhancement of AR transactivation. Together, our results demonstrate that a dominant-negative AR coregulator can suppress AR transactivation and cell proliferation in prostate cancer cells. Further studies may provide a new therapeutic approach for blocking AR-mediated prostate cancer growth.     

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.     

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.     

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.     

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.     

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.     

Hic-5/ARA55: a prostate stroma-specific AR coactivator

Growth factors and cytokines mediate communication between the epithelial and stromal compartments of the prostate. In prostate cancer (PCa), changes in the spatial arrangements of the two compartments (i.e. basement membrane invasion), DNA mutations, or cellular dedifferentiation (i.e. myofibroblasts) leads to significant changes in gene expression within both compartments. This results in altered cytokine and/or growth factor signaling in PCa. Recently, a stromal-specific androgen receptor (AR) coactivator, Hic-5/ARA55, has been identified that may play a role in regulating expression of the growth factor and/or cytokine expression in the prostate. Specifically, Hic-5/ARA55 expression influences androgen-induced keratinocyte growth factor (KGF) expression in WPMY-1 prostate stromal cells. Because Hic-5/ARA55's expression is also altered in PCa, it may play a role in the differential cellular signaling events that occur during tumor progression.     

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.