Stem Cell Feature in EMT Cells Figure 7. Sox2, Nanog, Oct4 and Lin28B regulated Prostasphere-forming ability of PC3 PDGF-D cells. (A) Single cell suspensions of PC3 PDGF-D transfected with Sox2, Nanog, Oct4 and Lin28B siRNA and incubated for 24 h were plated on ultra low adherent wells of 6-well plate at 2000 cells/well. After 3 days, the numbers of prostaspheres were counted under microscope. Transfection of Sox2, Nanog, Oct4 or Lin28B siRNA reduced the prostasphere-forming capacity (B) Prostaspheres were photographed and the size of prostaspheres in diameter was measured using software image analysis program Scion Image. (C). Western blot analysis showed that levels of Sox2, Nanog, Oct4 and Lin28B in PC3 PDGF-D cells transfected with Sox2, Nanog, Oct4 and Lin28B siRNA compared with transfection with control siRNA (*, p ,0.05, **, p,0.01 compared to control, Con: control). doi:10.1371/journal.pone.0012445.g007 Taken together, EMT cells such as PC3 PDGF-D and ARCaPM cells shared stem-like cell signatures characterized by increased colonogenic and prostasphere-forming capacity. The miR-200, especially miR-200b and miR-200c were identified to be mechanically linked with EMT phenotype and stem cell signatures in these cells via regulation of Notch1 and/or Lin28B expression. Lin28B inhibited accumulation of mature let-7, which resulted in the increased expression of Sox2, Nanog and Oct4 (Fig. 8D), leading to increased self-renewal and tumor growth. Thus, miR- 200b and miR-200c appear to play a central role in linking EMT with stem cell features in prostate cancer progression. Discussion Epithelial to mesenchymal transition is a physiological process during embryonic development and functions in adults to promote organ morphogenesis and tissue regeneration as well as wound healing. Emerging evidence suggest that the acquisition of EMT is strongly associated with cancer cell invasion and tumor metastasis. Recently, studies have shown that cells with EMT phenotype share characteristics that are consistent with the signatures of cancer stem-like cells, which are associated with tumor recurrence and drug resistant phenotype and contribute to the demise of patients diagnosed with cancers [12,22,38]. In the current study, we found that over-expression of PDGF-D in PC3 cells induced cellular morphological changes that were consistent with the acquisition of EMT phenotype as characterized by the loss of expression of epithelial markers and the gain or increased expression of mesenchymal markers. More importantly, PC3 PDGF-D cells displayed an increased clonogenic and prosta- sphere-forming capacity, the characteristics that are known to be associated with cancer stem-like cell characteristics. ARCaPM cells were derived from ARCaP cells showing mesenchymal morphol- ogy and increased expression of mesenchymal markers such as ZEB1 and vimentin as well as decreased expression of epithelial markers such as E-cadherin. Interestingly, ARCaPM cells also displayed stem-like cell phenotype characterized by increased clonogenic and prostasphere-forming capacity compared with ARCaPE cells having epithelial phenotype. Our results suggest that EMT-type cells show stem-like cells or cancer stem-like cell characteristics. It is well known that co-expression of Sox2, Nanog, Oct4 and Lin28 or Oct4, Sox2, Klf4 and c-myc in human or mouse somatic cells can reprogram these somatic cells into pluripotent embryonic stem-like cells [4,5,39]. These results are consistent with data from Dr. Ben-porath, et al. who demonstrated that histologically poorly differentiated tumors showed preferential over-expression of Nanog, Oct4, Sox2 and c-Myc genes that are normally enriched in embryonic stem cells (ES cells). Moreover, they also found that activation targets of Nanog, Oct4, Sox2 and c-Myc were more frequently over-expressed concomitant with repression of poly- comb-regulated genes in poorly differentiated tumors than in well- differentiated tumors [7]. However, high-grade tumors with ES signatures failed to demonstrate CD44high/CD24low population of cells in these tumors [7], suggesting the complexity of stem cell characteristics. In our study, we found that the expressions of Sox2, Nanog, Oct4 and Lin28B were dramatically up-regulated in PC3 PDGF-D cells. More importantly, knock-down of Sox2, Nanog, Oct4 and Lin28B by siRNA transfection repressed prostasphere-forming capacity of PC3 PDGF-D cells. Moreover, we also found enhanced expression of Stat3, Sall2, Sall4, Zic2 and Zic3, downstream targets of Nanog, Oct4 and Sox2, and up- regulation of EZH2, Suz12 and EED (which consisted polycomb repressor complex 2) in PC3 PDGF-D cells. Polycomb repressor complex 2 occupies developmental genes in ES cells to maintain pluripotency of ES cells. Target genes of polycomb repressor complex are known to be repressed in ES cells, and activated during the ES cell differentiation, and are regulated by Nanog, Oct4 and Sox2 [32,33]. Taken together, PC3 PDGF-D cells with EMT signatures showed stem-like cell characteristics through over-expression of Nanog, Oct4 and Sox2, Lin28B and activation of polycomb repressor complex 2. The miR-200 family plays a critical role in mediating EMT phenotype induced by various factors including PDGF-D PLoS ONE | www.plosone.org 9 August 2010 | Volume 5 | Issue 8 | e12445