Increased TGF‐β1‐mediated suppression of growth and motility in castrate‐resistant prostate cancer cells is consistent with Smad2/3 signaling

Abstract

Elevated TGF-β levels are associated with prostate cancer progression. Although TGF-β is a tumor suppressor for normal epithelial and early-stage cancer cells, it may act paradoxically as a tumor promoter in more advanced cancers, although its effects are largely cell and context dependent. This study analyzed prostate cancer responses to TGF-β signaling in an isogenic model of androgen-sensitive and castration-resistant prostate cancer cells. Phosphorylation and nuclear translocation of Smad2 and Smad3 were analyzed using immunoblotting. Proliferation and cell cycle responses to TGF-β1 (5 ng/ml) were assessed using growth assays and flow cytometry for DNA content, as well as Western blot and immunoprecipitation of cell cycle proteins. Both androgen-sensitive (LNCaP) and castration-resistant (C4-2 and C4-2B) prostate cancer cell lines demonstrated TGF-β1-induced phosphorylation and nuclear translocation of Smad2/3 that was robust in metastatic lines. Smad phosphorylation was completely abrogated with inhibition of ALK-5 kinase activity using the kinase inhibitor, SB-431542. Increased sensitivity to TGF-β1-mediated growth inhibition was observed in C4-2 and C4-2B cells, as compared to LNCaP cells. This was paralleled with downregulation of Cyclin D and increased association of p15(Ink4b) or p27(Kip) with CDK's. Additionally, TGF-β1 inhibited motility and invasion of metastatic cell lines. TGF-β-mediated suppression of growth and motility is enhanced in metastatic, castration-resistant prostate cancer cells. Enhanced TGF-β1-induced Smad2 and -3 signaling in prostate cancer cells may correlate with tumor suppressive activity. Therefore, the direct effects of TGF-β1 on prostate cancer cells post-castration may be anti-tumorigenic and growth-suppressive.