Abstract 497: Loss of Notch-1 expression in human prostate epithelial cells results in reduced PTEN expression and altered differentiative capacity

Abstract

Abstract Notch-1 is part of an evolutionarily ancient cell surface receptor signaling pathway that functions in the determination of stem/progenitor cell fate. The human genome encodes four Notch-receptors (Notch-1, -2, -3, and -4) and at least five Notch-ligands (Delta-1, -3, -4, Jagged-1, and-2). Signaling is initiated upon ligand binding to the Notch receptor, which results in a series of proteolytic cleavages that release constitutively active intracytoplamsic Notch (ICN). Nuclear translocation of ICN results in activation of the CBF-1 transcription factor which in turn increases expression of developmentally regulated transcription factors such as Hes-1 and Hey-1. The PTEN tumor suppressor gene is a recently identified Notch-1 target. We have reported that Notch-1 signaling increases expression of the PTEN tumor suppresser gene in prostate tumor cell lines and that Notch-1 signaling is lost in the tumor foci of prostate adenocarcinoma patients. To test the hypothesis that loss of Notch-1 will result in deregulation of normal prostate epithelial cell differentiation and promote tumorigenesis, a lentiviral vector bearing a Notch-1 shRNA (shN1) was used to stably knockdown Notch-1 expression in the benign human prostate epithelial cell line, RWPE-1. Western blotting revealed that RWPE-1/shN1 cells lacked Notch-1 expression, and exhibited reduced expression of Hes-1, confirming that loss of Notch-1 expression resulted in loss of Notch signaling. PTEN protein expression was also reduced, consistent with our previous studies demonstrating that Notch-1 signaling regulated PTEN expression. RT-PCR experiments verified that loss of PTEN expression was mediated at the level of transcription. RWPE-1/shN1 cells lacking Notch-1 expression exhibited a more rounded morphology, as compared with the parental cell line. Rhodamine-phalloidin staining revealed that RWPE-1/shN1 cells had less numerous and shorter cell surface actin projections. RWPE-1/shN1 cells lacking Notch-1 expression exhibited a 2-fold increase in acinar differentiation upon 3-D culture in matrigel as compared with the parental cell line. Flow cytometric analysis revealed that RWPE-1 cells without Notch-1 expression had reduced surface expression of TROP2 and CD149f. Preliminary RT-PCR results indicate that expression of the basal cell marker keratin-5 is reduced in RWPE-1/shN1 cells as compared with parental RWPE-1 cells. Our data suggest that loss Notch-1 expression, and hence loss of signaling, may promote developmental plasticity in prostate epithelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 497. doi:10.1158/1538-7445.AM2011-497