The molecular events responsible for the progression of prostate cancer (CaP) from a relatively indolent disease to one that can be life threatening are unclear. Bmi-1, a transcriptional repressor belonging to the polycomb-group family of proteins is known to be involved in regulation of proliferation, senescence and is overexpressed in a variety of human cancers. Here we establish the clinical significance of Bmi-1 gene in human CaP and delineate its mechanism of action. Expression patterns of Bmi-1 was determined in normal and virally transformed human prostate epithelial cells PrEC and RWPE-1, human CaP cells LNCaP, DU-145 and PC-3 and in tissue specimens of 125 CaP patients with normal prostate and adenocarcinoma of tumor grades I-III. The protein level of Bmi-1 was significantly higher in all CaP cells as compared with PrEC and RWPE-1. Based on immunohistochemical staining pattern, a progressive increase in the protein levels of Bmi-1 was observed with increasing tumor grade in CaP specimens as compared to normal tissue specimens. Next, we investigated the mechanism through which Bmi-1 gene regulates the proliferation of CaP cells. Bmi-1 gene was knocked down in DU-145 cells using a transient transfection of siRNA and a gene micro-array was performed. We observed that Bmi-1 gene-knockdown decreased the expression pattern of several genes including ERBB2, Src, Myc, Jun, H-Ras, Akt1, PI3K, Urokinase, Bcl-2, VEGF, Cyclin D1, CDK4, MMP-9, NFκB1, HIF-1, IGF1, IGF2, TNF, IL2, GGT1, PKC delta and PKC epsilon, all of which are known to participate in proliferation, cell cycle and survival of CaP cells. Further, Bmi-1 gene-knockdown increased the expression of several genes including p16, p15, p57, CD164, Cadherin-9, TIMP-3, PIAS2 and SHBG which are known to negatively regulate proliferation, cell cycle and survival of cancer cells. Suppression of Bmi-1 gene resulted in (i) decreased cyclin D1 protein expression, (ii) increased p16 protein expression and (iii) decreased proliferative and clonogenic potential of CaP cells as assessed by colony formation assay. Next, we investigated the effect of Bmi-1 overexpression by transfecting pbabe-Bmi-1 plasmid in LNCaP, DU-145 and PC-3 cells which resulted in (i) increased expression levels of Bmi-1 and Cyclin D1 proteins and (ii) decreased p16protein expression. Finally, we observed that transfection of pbabe- Bmi-1 plasmid abolished the senescence of PrEC which generally enters into a stage of senescence after 4-5 divisions. To summarize, we provide evidence that over-expression of Bmi-1 in cells and tissue specimens correlates with the clinical stages of human CaP and that Bmi-1 regulates the proliferation by regulating several oncogenes and tumor suppresser genes. We advocate a role for Bmi-1 in staging human CaP and suggest its potential use as a biomarker for establishing the efficacy of therapeutic and chemopreventive interventions.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA