Identification of a new androgen receptor (AR) co-regulator BUD31 and related peptides to suppress wild-type and mutated AR-mediated prostate cancer growth via peptide screening and X-ray structure analysis

Abstract

Treatment with individual anti-androgens is associated with the development of hot-spot mutations in the androgen receptor (AR). Here, we found that anti-androgens-mt-ARs have similar binary structure to the 5α-dihydrotestosterone-wt-AR. Phage display revealed that these ARs bound to similar peptides, including BUD31, containing an Fxx(F/H/L/W/Y)Y motif cluster with Tyr in the +5 position. Structural analyses of the AR-LBD-BUD31 complex revealed formation of an extra hydrogen bond between the Tyr+5 residue of the peptide and the AR. Functional studies showed that BUD31-related peptides suppressed AR transactivation, interrupted AR N-C interaction, and suppressed AR-mediated cell growth. Combination of peptide screening and X-ray structure analysis may serve as a new strategy for developing anti-ARs that simultaneously suppress both wt and mutated AR function.

Keywords: Androgen receptor; Anti-androgen withdrawal syndrome; BUD31; Crystallography; FxxLF.

Figure 1

(A). Superposition of the overall structures of wt‐AR‐LBD (red), T877A‐AR‐LBD (green), and W741L (blue)‐AR‐LBD, drawn as ribbon models. (B). Overlapping crucial residues in the active site. Residues are shown as sticks. Nitrogen and oxygen atoms are colored blue and red, respectively. DHT, HF, and CDX are illustrated as red, green, and blue, respectively. Fluorine, nitrogen, oxygen, and phosphorous atoms are depicted as light green, blue, red, and orange, respectively.

Figure 2

Interactions in the AF2 site. Interactions between the peptides and proteins in front‐end (A) and back‐end (B) areas. Peptides are color‐coded as follow: unbound, gray; FQNLF, green, FDLFY, cyan; FSRLY, magenta; FSQHY, yellow, and FSRYY, wheat. Residues are shown as sticks; carbon, nitrogen, and oxygen atoms are shown in gray, blue, and red, respectively. Hydrogen bond contacts are illustrated with black dashed lines. The lengths of the hydrogen bonds are summarized in the tables.

Figure 3

Structural profiles of AR peptide‐binding interfaces among variants. Cα atoms of the peptides, illustrated as thin stick structures, were superposed onto the AF2 site of the (A) wt‐AR, (B) T877A‐AR, and (C) W741L‐AR proteins, shown as ribbon diagrams. Hydrophobic side chains of the peptides were superposed (right). (D) Various peptides were superposed in the AF2 site. The protein and peptides are shown in surface representation and as thin sticks, respectively. Positively and negatively charged areas are blue and red, respectively. The atoms of nitrogen and oxygen are colored blue and red, respectively.

Figure 4

Interaction between AR and BUD31. (A) Co‐IP of Flag‐BUD31 with endogenous AR in the prostate cancer cell line LNCaP. Extracts of LNCaP cells overexpressing 3xFlag‐BUD31 were treated with 1 μM DHT. IP was performed using an anti‐AR (C19) or anti‐Flag antibody, or normal rabbit serum (negative control), followed by immunoblotting (IB) with antibodies to AR or BUD3. (B) BUD31 interacted with full‐length AR and N‐ and C‐terminal regions of the AR in GST pull‐down assays; mutation of the FxxFY motif to AxxAA in BUD31 reduced BUD31 interactions with the AR. BUD31 enhanced AR transactivation and blocked AR N–C interactions. (C) PC‐3 prostate cancer cells transfected with BUD31. PC‐3 cells in 24‐well plates were co‐transfected with 300 ng of MMTV‐LUC reporter plasmid and 0.5 ng of SV40‐Renilla luciferase plasmid together with 100 ng of pCMV‐Flag‐AR, 100–500 ng of p3xFLAG‐BUD31‐wt‐FxxFY or 100–500 ng p3xFLAG‐BUD31‐mt‐AxxAA; plasmid DNA was brought to a total of 1 μg with pCMV. After 16 h, ethanol or 10 nM DHT was added and cells were incubated for an additional 16 h. Relative LUC activity was determined using the dual luciferase system. ARA70 served as a positive control. (D) BUD31 interacts with AR in promoter regions of target genes. LNCaP cells were transfected with P3xflag‐BUD31 plasmid and cultured overnight. Soluble chromatin was prepared from LNCaP cells treated with or without DHT, and ChIP assays were performed using an antibody to AR (AR), BUD31 (BUD31), Flag (Flag), and RNA polymerase II. The final DNA extracts were amplified using the primers for ARE I/II and ARE III, described in Materials and methods. (E) BUD31 blocked AR N–C interactions. PC‐3 cells in 24‐well plates were transfected with 200 ng of pG5‐LUC reporter plasmid and 0.5 ng of SV40‐Renilla luciferase plasmid together with various combinations of 200 ng of Gal‐4‐AR‐C (amino acids 1–501), 200 ng of VP16‐AR‐N (amino acids 556–919), 200–400 ng of p3xFLAG‐BUD31, as indicated; pCMV was added as necessary to bring the total to 1 μg plasmid DNA. After 16 h, ethanol or 10 nM DHT was added and cells were incubated for an additional 16 h. Luciferase activity was assessed using a dual luciferase assay system. (F) BUD31 is expressed in benign prostatic hyperplasia tissue. Human prostate gland tissues were immunostained for AR and BUD31. The arrows indicate ARs (left panel) and BUD31 (right panel). The figures are representative of three benign prostatic gland hyperplasia tissues.

Figure 5

BUD31 peptide potently suppresses AR‐mediated function and growth of AR‐harboring prostate cancer cells, and blocks AR N–C interactions. Short peptides of wt‐BUD31, C320 (FxxLY motif), 3–18 (FxxLF motif) (Hsu et al., 2003), and 3–14 (LxxLL motif) peptides conjugated with TAT were synthesized and tested in LNCaP cells (B and C) and PC‐3 cells (D) in the presence and absence of 0.2 nM DHT. (A) Peptide sequences. (B–D) Test peptides suppress AR transactivation in the presence of DHT or HF (B), suppress LNCaP cell growth in vitro (C), and block AR N–C interactions (D).