From HER2/Neu signal cascade to androgen receptor and its coactivators: a novel pathway by induction of androgen target genes through MAP kinase in prostate cancer cells

Abstract

Overexpression of the HER2/Neu protooncogene has been linked to the progression of breast cancer. Here we demonstrate that the growth of prostate cancer LNCaP cells can also be increased by the stable transfection of HER2/Neu. Using AG879, a HER2/Neu inhibitor, and PD98059, a MAP kinase inhibitor, as well as MAP kinase phosphatase-1 (MPK-1), in the transfection assay, we found that HER2/Neu could induce prostate-specific antigen (PSA), a marker for the progression of prostate cancer, through the MAP kinase pathway at a low androgen level. Reporter assays and mammalian two-hybrid assays further suggest this HER2/Neu-induced androgen receptor (AR) transactivation may function through the promotion of interaction between AR and AR coactivators, such as ARA70. Furthermore, we found this HER2/Neu --> MAP kinase --> AR-ARAs --> PSA pathway could not be blocked completely by hydroxyflutamide, an antiandrogen used in the treatment of prostate cancer. Together, these data provide a novel pathway from HER2/Neu to AR transactivation, and they may represent one of the reasons for the PSA re-elevation and hormone resistance during androgen ablation therapy in prostate cancer patients.

Figure 1

The growth curve and the expression of PSA in HER2/Neu stable transfected LNCaP. A HER2/Neu stable transfected LNCaP cell line was established by transfection with HER2/Neu or its parent vector under 400 μg/ml G418 selection. (A and B) Cells were seeded at 2 × 10⁵ on a six-well plate. The cells were counted every 24 h by MTT assay. Each count represents the average of four independent experiments. (C) The blot containing 30 μg of total RNA isolated from cells on each lane was hybridized with a PSA cDNA probe. The 28S RNA shows the equal loading of RNA.

Figure 2

Induction of AR transactivation by HER2/Neu in the presence of androgen in DU145 cells. One microgram of human AR alone or 2.5 μg of pCMVHER2/Neu and/or 2.5 μg of pSG5ARAs was transfected into DU145 cells. PSA-CAT, which contains the 2.8 kb 5′-promoter region of PSA gene, was used as the AR-target reporter gene. Cells were treated with 1 nM DHT after transfection. Cells were harvested after 24 h for CAT assay, and relative CAT activity was normalized by the β-galactosidase activity and quantitated by image- quant software. Data represent an average of three independent experiments.

Figure 3

Effects of androgen concentration on the HER2/Neu-AR-mediated transactivation. The DU145 cells were transfected with or without 1 μg of human pSG5AR, and/or 2.5 μg of pSG5ARA70 or ARA55, and/or 2.5 μg of pCMV-HER2/Neu (lanes 1–5, transfected with AR only; lanes 6–10, AR and ARA55 only; lanes 11–15, AR and ARA70N only; lanes 16–20, AR and HER2/Neu only; lanes 21–25, ARA70N, ARA55, and HER2/Neu only, without AR; lanes 26–30, AR, ARA55, and HER2/Neu; and lanes 31–35, AR, ARA70N, and HER2/Neu). PSA-CAT was applied as the AR-target reporter gene. After transfection the DU145 cells were treated with serial concentrations of DHT from 10⁻¹² M to 10⁻⁹ M for another 24 h (mock: lanes 1, 6, 11, 16, 21, 26, and 31; 10⁻¹² M: lanes 2, 7, 12, 17, 22, 27, and 32; 10⁻¹¹ M: lanes 3, 8, 13, 18, 23, 28, and 33; 10⁻¹⁰ M: lanes 4, 9, 14, 19, 24, 29, and 34; 10⁻⁹ M: lanes 5, 10, 15, 20, 25, 30, and 35). Data represent an average of three independent experiments.

Figure 4

(A) Inhibition of the HER2/Neu-mediated AR transactivation by AG879 or PD98059. (B) Suppression of AR transactivation by CL100. (A) One microgram of human pSG5AR or 3 μg of pSG5ARAs and/or 3 μg of pCMV-HER2/Neu was transfected into DU145 cells. MMTV-CAT was used as the AR-target reporter gene. After transfection, the cells were treated with or without DHT, 10 μM AG879, or 15 μM PD98059 for another 24 h, and cells were then harvested for CAT assay. (B) One microgram of human pSG5AR or 3 μg of pSG5ARA55 and/or 3 μg of pCMV HER2/Neu plus CL100 was transfected into DU145 cells for 24 h, and the cells were mock treated or treated with 10⁻¹¹ or 10⁻⁹ M DHT for 24 h and then harvested for CAT assay. Values represent the mean ± SD of at least three determinations.

Figure 5

Mapping the phosphorylation site on AR. (A) The N terminus of AR, but not the DNA-binding domain–ligand-binding domain (DBD-LBD), was phosphorylated by the MAP kinase. (B) The conserved MAP kinase site on human, rat, and mouse AR. (C) Site-directed mutagenesis of the conserved MAP kinase site on AR. (A) The purified AR peptides, N-DBD (N-terminal AR from amino acid 36 to 643), and DBD-LBD (C-terminal AR from amino acid 153 to 918) were phosphorylated by 0.05 pmol of MAPK2 and subjected to SDS/10% PAGE. (B) The conserved MAP kinase site (PYPSP) on human, rat, and mouse AR (hAR, rAR, and mAR). These five amino acids, PYPSP, correspond to the consensus phosphorylation site [PX_n(S or T)P, where X is a neutral or basic amino acid and n = 1 or 2] for MAP kinase. (C) The nucleotide sequence of wtAR and mtARs514a is shown in Top. DU145 cells were cotransfected with MMTV-CAT with wtAR or mtARs514a in the absence of or in the presence of HER2/Neu. Middle is a chromatogram of CAT reaction products. The results are quantified in Bottom, in which each bar represents the mean of three independent experiments.

Figure 6

Promotion of the interaction of AR and ARAs by HER2/Neu. The DU145 cells were transiently cotransfected with 2.5 μg of reporter plasmids, pG5-LUC, 2.5 μg of Gal4DBD fused to ARA70 amino acids 176–401 (Gal4-ARA70), and 2.5 μg of VP16-AR fusion in the presence or the absence of HER2/Neu. DHT was then added at 1 nM for 24 h before cells were harvested for luciferase (LUC) assay.

Figure 7

Antiandrogen cannot completely block the HER2/Neu-induced AR transactivation. PSA-CAT activity was determined in DU145 cells cotransfected with the expression plasmids of pSG5AR (1 μg) with or without pSG5ARA55 or pSG5ARA70N (3 μg). Cells were then incubated with ethanol (mock) or DHT in the absence or presence of 2 μM HF for 24 h before cells were harvested for CAT assay. The mock treatment was set as 1-fold. Values are the mean (±SD) of at least three determinations.