Androgen receptor dependent and independent signal transduction pathways leading to proliferation in androgen-independent prostate cancer

934 Prostate cancer is initially dependent on androgens for growth and thus responsive to androgen ablation. However, many patients treated with hormonal therapy ultimately progress to an androgen independent (AI) state. The LNCaP cell line has been widely used as a model for androgen-dependent (AD) prostate cancer. This cell line can be induced to progress to androgen independence by prolonged androgen deprvation and two AI prostate tumor cell lines cloned from LNCaP cells, LNCaP-AI (developed in vitro) and C4-2 (developed in vivo), are often used as models to study androgen independence. Both these LNCaP clones express the androgen receptor (AR), and hence, in this study, we evaluate the role of the AR in their proliferation. Proliferation was determined by MTT assay and by flow cytometry in propidium iodide stained ethanol fixed cells. Protein expression was determined by immunoblotting while AR transcriptional activity was determined by luciferase assay in cells transfected with a human prostate specific antigen (PSA) promoter tagged to a luciferase construct (hPSA-luc). We used siRNA technology and a pharmacological inhibitor to downregulate the expression and activation of the AR, respectively. Both LNCaP-AI and C4-2 cells showed increased proliferation rates compared to LNCaP cells. LNCaP cells were growth arrested by 10 μM Casodex, a competitive inhibitor of androgen binding to the AR, whereas LNCaP-AI and C4-2 cells were not, reflecting their AI nature. C4-2 cells exhibited lower AR expression and higher endogenous express of the AR target gene PSA compared to LNCaP. In contrast, LNCaP-AI cells showed higher AR expression compared to LNCaP but did not express endogenous PSA. Despite this, in LNCaP-AI cells, AR transcriptional activity on hPSA-luc was increased compared to LNCaP and C4-2 cells and was androgen-sensitive, showing that the AR in these cells remained active. Next, we determined whether AR expression mediated AI cell proliferation. Transfection with AR-specific siRNA duplexes downregulated the AR and inhibited proliferation in LNCaP and LNCaP-AI, but not in C4-2 cells. These results indicated that the AR mediated the growth of LNCaP-AI but not C4-2 cells. Despite AR-independent proliferation in C4-2 cells, the AR in this cell line was capable of regulating cell cycle proteins. However, we provide evidence that proliferation in C4-2 cells exhibited increased dependence on an alternate signaling pathway involving Akt, a serine/threonine kinase downstream of phosphatidylinositol 3-kinase. Our data suggest that LNCaP-AI and C4-2 cells represent two different forms of AI prostate cancer - while the LNCaP-AI cells are AR-dependent, the C4-2 cells are AR-independent.