From AR to c-Met: androgen deprivation leads to a signaling pathway switch in prostate cancer cells

Abstract

Elucidating the role of androgen deprivation in the transition from androgen-dependence to independence may enable the development of more specific therapeutic strategies against prostate cancer. Our previous in vitro model was employed to further assess the effects of continuous androgen‑deprivation on prostate cancer cells (LNCaP) with respect to both androgen receptor (AR) and c-Met expression. The results indicated that long-term androgen deprivation resulted in a signaling pathway switch from AR to c-Met in androgen-sensitive cells, which was confirmed by immunofluorescence imaging and western blot analysis. This signaling pathway switch may be predictive of a more aggressive disease state following androgen deprivation therapy.

Figure 1.

Downregulation of AR expression in LNCaP cells following androgen deprivation treatment. LNCaP cells cultured in normal growth media, served as AR positive controls; strong fluorescence signals for AR (green) were detected throughout the nuclei. Decreased signals for AR were found in LNCaP cells under androgen deprivation conditions for 2 days and 5 passages. No signals for AR were observed for LNCaP cells proliferated under androgen deprivation conditions by 10 passages or in the AR-negative PC-3 cells. Cell nuclei were counterstained with Hoechst 33342 (blue). Cellular imaging was visualized by confocal microscopy; scale bar, 20 μ m.

Figure 2.

Induced c-Met expression in LNCaP cells following androgen deprivation treatment. PC-3 cells served as c-Met positive controls; fluorescence signals for c-Met (green) were detected on the cell surface. c-Met was found on LNCaP cells proliferated under androgen deprivation conditions by 5 passages and significantly by 10 passages. No signals for c-Met were observed for LNCaP cells cultured in normal growth media. Cell nuclei were counterstained with Hoechst 33342 (blue). Cellular imaging was visualized by confocal microscopy; scale bar, 20 μ m.

Figure 3.

Loss of PSMA expression in androgen-deprived LNCaP cells following androgen deprivation treatment. LNCaP cells cultured in normal growth media, served as PSMA-positive controls; fluorescence signals for PSMA (red) were detected on the cell surface. Decreased signals for PSMA were found in LNCaP cells proliferated under androgen deprivation conditions by 10 passages. No signals for PSMA were observed for either LNCaP cells proliferated under androgen deprivation conditions by 20 passages or PSMA-negative PC-3 cells. Cell nuclei were counterstained with Hoechst 33342 (blue). Cellular imaging was visualized by confocal microscopy; scale bar, 20 μ m.

Figure 4.

Induced morphological changes in LNCaP cells following androgen deprivation treatment. PC-3 cells and LNCaP cells cultured in normal growth medium serve as references. The loss of tight cell-cell contact and a more scattered growth pattern were found in LNCaP cells proliferated under androgen deprivation conditions over time. Cellular imaging was visualized by a light-contrast microscope; scale bar, 100 μ m.

Figure 5.

Western blot analysis detection of AR and PSMA expression. Compared to LNCaP cells cultured in normal growth medium, PSMA expression is significantly reduced in LNCaP cells proliferated under androgen deprivation conditions by 10 passages. No signals for PSMA were observed for either LNCaP cells proliferated under androgen deprivation conditions by 20 passages or PSMA-negative PC-3 cells. Actin expression was detected to serve as a protein loading control.

Figure 6.

Western blot analysis detection of c-Met expression. Strong c-Met expression was detected in LNCaP cells proliferated under androgen deprivation conditions by 10 passages (10P) and 20 passages (20P). PC-3 cells served as c-Met positive controls. Actin expression was detected to serve as a protein loading control.