Epidermal growth factor induces bladder cancer cell proliferation through activation of the androgen receptor

Abstract

Androgen receptor (AR) signals have been suggested to contribute to bladder tumorigenesis and cancer progression. Activation of epidermal growth factor receptor (EGFR) also leads to stimulation of bladder tumor growth. However, crosstalk between AR and EGFR pathways in bladder cancer remains uncharacterized. We have recently shown that androgens activate the EGFR pathway in bladder cancer cells. The purpose of this study was to investigate the effects of EGF on AR activity in bladder cancer. EGF increased AR transcriptional activity by 1.2-, 1.9- and 2.0-fold in UMUC3, 5637-AR and J82-AR cell lines, respectively, over mock treatment and a specific EGFR inhibitor, PD168393, antagonized the EGF effect. Combined treatment of EGF and dihydrotestosterone (DHT) further induced AR transactivation while an AR antagonist, hydroxyflutamide (HF), abolished the effect of not only DHT but also EGF. In growth assays, EGF alone/DHT alone/EGF+DHT increased cell numbers by 16/12/19%, 6/14/18% and 30/12/38% in UMUC3-control-shRNA, 5637-AR and J82-AR, respectively, whereas the effects of EGF were marginal or less significant in UMUC3-AR-shRNA (8%) or AR-negative 5637-V (<1%) and J82-V (17%) cells. HF treatment at least partially counteracted the EGF effect on the growth of AR-positive cells. Western blotting demonstrated that EGF, especially in the presence of DHT, upregulated the expression of the p160 coactivator TIF2 and HF again blocked this stimulation. Co-immunoprecipitation revealed the association between AR and estrogen receptor (ER)-β or Src in UMUC3 cells and stronger associations with EGF treatment, implying the involvement of the AR/ER/Src complex in EGF-increased AR transactivation and cell growth. Current results, thus, suggest that EGF promotes bladder cancer cell proliferation via modulation of AR signals. Taken together with our previous findings, crosstalk between EGFR and AR pathways can play an important role in the progression of bladder cancer.

Figure 1.

Effects of EGF on AR transactivation. Bladder cancer cells (A, UMUC3; B, 5637-AR; C, J82-AR) were transfected with MMTV-Luc and were then cultured for 24 h in the presence of ethanol (mock), 100 ng/ml EGF and/or 1 μM PD168393, as indicated. Luciferase activity analyzed in a luminometer is presented relative to that of mock treatment in each cell line (first lanes; set as 1-fold). Each value represents the mean + SD from at least three independent experiments. ^*p<0.05.

Figure 2.

Effects of androgen and antiandrogen on EGF-mediated AR transactivation. Bladder cancer cells (A, UMUC3; B, 5637-AR; C, J82-AR) were transfected with MMTV-Luc and were then cultured for 24 h in the presence of ethanol (mock), 100 ng/ml EGF, 10 nM DHT and/or 10 μM HF, as indicated. Luciferase activity analyzed in a luminometer is presented relative to that of mock treatment in each cell line (first lanes; set as 1-fold). Each value represents the mean + SD from at least three independent experiments. ^*p<0.05; ^**p<0.01.

Figure 3.

Effects of EGF on cell viability. Bladder cancer cells (A, UMUC3-control-shRNA/AR-shRNA; B, 5637-AR/vector; C, J82-AR/vector) were cultured for 4 days in the presence of ethanol (mock), 100 ng/ml EGF, 10 nM DHT and/or 10 μM HF, as indicated. Cell viability was assayed with MTT and growth induction is presented relative to cell number with mock treatment estimated by measuring the absorbance at a wavelength of 570 nm with a background subtraction at 655 nm (first lanes; set as 1-fold). Each value represents the mean + SD from at least three independent experiments. ^*p<0.05; ^**p<0.01.

Figure 4.

Effects of EGF on AR and TIF2 protein expression. Bladder cancer cells (A, UMUC3; B, 5637-AR; C, J82-AR) were cultured for 24 h in the presence of ethanol (mock), 100 ng/ml EGF, 10 nM DHT and/or 10 μM HF, as indicated. Equal amounts of protein extracted from each cell line were immunoblotted for AR (110 kDa, upper), TIF2 (160 kDa, middle), or GAPDH (37 kDa, lower) as indicated. Densitometry values for specific bands standardized by GAPDH that are relative to those of mock treatment (first lanes; set as 1-fold) are included below the lanes.

Figure 5.

Effects of EGF on AR/ER/Src association. UMUC3 cells were cultured for 24 h in the presence of ethanol (mock) or 100 ng/ml EGF. Cell lysates were immunoprecipitated with anti-AR antibody or normal rabbit IgG and were then immunoblotted for AR (110 kDa), ERβ (56 kDa), or Src (60 kDa), as indicated.