Androgen receptor auto-regulates its expression by a negative feedback loop through upregulation of IFI16 protein

Expression of androgen receptor (AR) in prostate epithelial cells is thought to regulate cell proliferation, differentiation, and survival. However, the molecular mechanisms remain unclear. We report that re-expression of AR in PC-3 human prostate cancer cell line resulted in upregulation of IFI16 protein, a negative regulator of cell growth. We found that the IFI16 protein bound to AR in a ligand-dependent manner and the DNA-binding domain (DBD) of the AR was sufficient to bind IFI16. Furthermore, re-expression of IFI16 protein in LNCaP prostate cancer cells, which do not express IFI16 protein, resulted in downregulation of AR expression and an inhibition of the expression of AR target genes. Our observations identify a role for IFI16 protein in AR-mediated functions.     

Requirement of androgen-dependent activation of protein kinase Czeta for androgen-dependent cell proliferation in LNCaP Cells and its roles in transition to androgen-independent cells

A cell line that we designed, AILNCaP, proliferated in androgen-depleted medium after emerging from long-term androgen-depleted cultures of an androgen-sensitive prostate cancer cell line, LNCaP. Using this cell line as a model of progression to androgen independence, we demonstrated that the activity of the mammalian target of rapamycin/p70 S6 kinase transduction pathway is down-regulated after androgen depletion in LNCaP, whereas its activation is related to transition of this cell line to androgen-independent proliferation. Kinase activity of protein kinase Czeta is regulated by androgen stimulation in LNCaP cells, whereas it is activated constitutively in AILNCaP cells under androgen-depleted conditions. Treatment with a protein kinase Czeta pseudosubstrate inhibitor reduced p70 S6 kinase activity and cell proliferation in both cell lines. We identified that both protein kinase Czeta and p70 S6 kinase were associated in LNCaP cells and this association was enhanced by the androgen stimulation. We examined the expression of phospho-protein kinase Czeta and phospho-p70 S6 kinase in hormone-naive prostate cancer specimens and found that the expression of both kinases was correlated with each other in those specimens. Significant correlation was observed between the expression of both kinases and Ki67 expression. Most of the prostate cancer cells that survived after prior hormonal treatment also expressed both kinases. This is the first report that shows the significance of this pathway for both androgen-dependent and -independent cell proliferation in prostate cancer. Our data suggest that protein kinase Czeta/mammalian target of rapamycin/S6 kinase pathway plays an important role for the transition of androgen-dependent to androgen-independent prostate cancer cells.     

Androgen receptor down regulation by small interference RNA induces cell growth inhibition in androgen sensitive as well as in androgen independent prostate cancer cells

We investigated the effects of androgen receptor (AR) down regulation with a small interference RNA molecule (siRNA_AR(start)) on androgen sensitive LNCaP and androgen independent LNCaPabl prostate cancer cells, the latter representing an in vitro model for the development of therapy resistance in prostate cancer. Although LNCaPabl cells express increased levels of AR in comparison with androgen sensitive LNCaP cells, the protein was significantly down regulated in response to siRNA_AR(start) treatment. This AR down regulation resulted in a marked cell growth inhibition in both cell lines. By contrast, DU-145 prostate cancer cells, which lack AR expression, were not inhibited by the siRNA_AR(start). In consequence to AR down regulation, both cell lines, LNCaP and LNCaPabl, shared a highly similar gene expression profile in terms of major changes in cell cycle regulatory genes. The cell cycle inhibitor p21(Waf1/Cip1) as well as cyclin D1 were significantly up regulated by siRNA_AR(start) treatment, considering a switch in cyclin expression towards cell cycle retardation. Control molecules had moderate effects on cell proliferation and gene expression, respectively. In summary, we found that AR inhibition with siRNA induces cell growth retardation in androgen sensitive as well as in androgen independent prostate cancer cells and thus may represent an interesting approach to combat hormone-refractory prostate cancer.