A Dominant-negative Mutant of Androgen Receptor Coregulator ARA54 Inhibits Androgen Receptor-mediated Prostate Cancer Growth

Abstract

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.

Transgelin induces apoptosis of human prostate LNCaP cells through its interaction with p53

“…HCT116, harbouring wild-type p53, and HCT116-p53 -/-were kindly provided by Dr ZhiMing Yang (Johns Hopkins University, Baltimore, MD, USA), grown in McCoy's 5A culture medium (Invitrogen) supplemented with 10% FBS. pVP16-transgelin, pG5-LUC and pRL-TK have been described npg previously [17,20]. After digestion of pVP16-transgelin using BamH I and Hind III (Fermentas, Glen Burnie, MD, USA), the product was subcloned into pcDNA 3.1 vector through the Bam I and Hind III sites.…”

“…The proper function of AR requires coregulators for its optimal signalling. Several AR coregulators, including the cAMP response element-binding protein-binding protein, steroid receptor coactivator 1, ARA54, ARA67/PAT1, ARA70, hRad9 and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) have been identified [3,[13][14][15][16][17][18]. ARA54 enhances AR transactivation in a ligand-dependent manner [17,19,20].…”

Identification of steroid derivatives that function as potent antiandrogens

“…Transfections and Luc assays were performed as previously described. 17,20 Briefly, cells seeded to reach a density of 50-60% confluence in 12-well tissue culture plates were transfected with 1.5 lg of DNA according to ''SuperFect transfection'' instructions (Qiagen, Chatsworth, CA). After 2-3 hr incubation, cells were cultured in medium supplemented with charcoal-stripped FBS and either 0.1-0.2% (vol/vol) ETOH or ligands for 24 hr.…”

“…20 An antibody for b-actin (Santa Cruz Biotechnology, Santa Cruz, CA) was used as the internal control. Equal amounts of proteins obtained from cell extracts were electrophoresed on SDS-polyacrylamide gel and blotted onto polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA), using a standard protocol.…”

“…None of these compounds as well as DHT, HF and BC displayed their effects on AR transcriptional activity in PC-3 without transfection of AR. In the LNCaP cell line expressing a mutant AR, HF is known to act as a full agonist 11,20,23 and therefore shows little suppression of DHT-induced AR transcription (Fig. 2e).…”