carcinomas toward malignancy is associated with the loss of epithelial differentiation and by switching toward mesenchymal phenotype, which is accompanied by increased cell motility and invasion. Recent studies have demonstrated that EMT plays a critical role not only in tumor metastasis but also in tumor recurrence that is believed to be tightly linked with the biology of cancer stem-like cells or cancer-initiating cells [11–13]. Howev- er, the mechanisms by which EMT cells generate the stem-like cells remain to be elucidated. MicroRNAs (miRNAs) are emerging as master regulators of cell differentiation and involved in the acquisition of EMT phenotype during tumor progression [14]. Two evolutionary conserved families, miR-200 and let-7 have been shown to regulate the differentiation processes during the development. Interestingly, recent studies have also shown that miR-200 family not only could regulate the processes of EMT by targeting E-box binding protein ZEB1 and ZEB2 [15] but was also associated with stem-like cell signatures by regulating the expression of Bmi1 [16,17]. Let-7 family of miRNAs has been shown to act as a tumor suppressor through targeting Ras, high mobility group A2 (HMGA2) and c-myc, and decreased let-7 expression has been linked with increased tumorigenicity and poor patient prognosis. More importantly, Let-7 family members regulate the self-renewal of breast cancer cells [18], which is associated with stem cell phenotype. Recent studies have also documented that lin28B could block the accumulation of mature let-7 [19], which in turn regulates ‘‘stemness’’ by inhibiting self-renewal, the cellular characteristics of cancer stem-like cells associated with tumor recurrence. The recurrence of PCa is believed to be strongly linked with the biology of prostate cancer stem cells or cancer-initiating cells [11,20,21], while increasing evidence have shown that cells with EMT induced by different factors are rich source of cancer stem- like cells [12,13,22,23]. Therefore, it is important to identify which factors could induce EMT and how the EMT cells could become a resource for cancer stem-like cells, which further underscores the mechanistic role of such factors toward the development of novel and targeted therapies for CRPC. Studies have shown that platelet derived growth factor (PDGF) signaling contributes to EMT [24,25]. Recently, we and others have shown that PDGF-D could regulate cancer cell invasion and angiogenesis, which was consistent with the acquisition of EMT phenotype [26–30]. Interestingly, increased expression of PDGF-D has also been found in human PCa [31], suggesting that PDGF-D could play important roles in the progression of human PCa contributed by EMT-phenotypic or cancer stem-like cells. In this study, we found that PDGF-D over-expressing PC3 cells acquired EMT phenotype and shared stem-like cell features characterized by increased expression of stem cell markers such as Notch1, Sox2, Nanog, Oct4 and Lin28B, which was consistent with enhanced clonogenic, prosta- sphere-forming ability as well as increased tumorigenicity in mice. Concomitantly, we found ARCaPM cells with EMT phenotype also shared stem-like cell signatures characterized by increased expression of transcription factor Notch1 and enhanced clono- genic, prostasphere-forming ability compared with control cells (ARCaPE cells) with epithelial phenotype. These EMT-type cells also showed decreased expression of miR-200 or let-7, and reversal of EMT by forcing expression of miR-200 significantly inhibited clonogenic and prostasphere-forming ability, which was consistent with the inhibition of Notch1 and Lin28B expression. Moreover, knock-down of Lin28B markedly increased let-7 expression, suggesting that miR-200 and let-7 could act as a target for the prevention of tumor recurrence and metastasis in PCa. Stem Cell Feature in EMT Cells Results Clonogenic ability was increased in PC3 PDGF-D and ARCaPM cells having EMT phenotype The results from Western blot analysis and cell morphology as well as our published data have demonstrated that over-expression of PDGF-D induced EMT phenotype in PC3 cells [26,28] (Fig. 1A), which was consistent with higher levels of PDGF-D in cell lysates and conditioned medium (CM) from PC3 PDGF-D cells compared with PC3 Neo cells (Fig. S1A and S1B). ARCaPM cells having mesenchymal morphology showed an increased expression of mesenchymal markers and decreased expression of epithelial markers compared to ARCaPE cells with epithelial phenotype (Fig. 1B). To determine whether cells with EMT phenotype could have stem-like cell characteristics, we performed clonogenic assay. We found that clonogenic ability was signifi- cantly increased in PC3 PDGF-D cells compared with PC3 Neo cells (Fig. 1C, left panel). Fig. 1C, right panel, further indicated that PC3 PDGF-D cells showed an 11-fold increase in colony numbers relative to PC3 Neo cells. ARCaPM cells also displayed an increased clonogenic capacity showing 3-fold increase in colony numbers compared to ARCaPE cells (Fig. 1D). Soft agar assay, also known as anchorage independent growth assay, was performed. PC3 PDGF-D cells showed dramatic increase in clonogenic potential compared to PC3 Neo cells (Fig. 1E, left panel). Fig. 1E, right panel, clearly showed the colony numbers generated from PC3 Neo and PC3 PDGF-D cells per microscopic field (40 X). These results demonstrated an increase in clonogenic ability of EMT cells. Self-renewal capacity was increased by EMT cells To further determine whether cells with EMT phenotype could show stem-like cell characteristics, we tested sphere-forming (termed as prostasphere) ability of PC3 PDGF-D cells compared to PC3 Neo as well as ARCaPM cells compared to ARCaPE cells when grown in suspension cultures, which is an in vitro measure of stem-like cell characteristics. We found that PC3 PDGF-D cells showed significantly increased ability to form prostaspheres relative to PC3 Neo cells (Fig. 2A and 2B). The prostaspheres from PC3 Neo were less than 100 mm in diameter after 6 days, while 40% of the prostaspheres from PC3 PDGF-D cells were 100–200 mm, 20% of the prostaspheres from PC3 PDGF-D cells were greater than 200 mm (Fig. 2C). In order to further confirm whether the prostaspheres are the progeny of individual cells rather than the aggregates of multiple cells, the prostaspheres (P1) were collected and the cells were plated at one cell per well in 96- well plate with ultra low attachment. After 12 days, we found that one to two new prostaspheres were generated from single PC3 PDGF-D cells, while only 20% single cells of PC3 Neo generated new prostaspheres (P2, Fig. 2D). ARCaPM cells also exhibited an increased prostasphere-forming capacity (Fig. 2E and 2F), suggesting that cells with EMT phenotype have acquired increased self-renewal capacity. Based on these results, further mechanistic experiments were focused using PC3 PDGF-D cells compared to PC3 Neo cells and compared with ARCaPM and ARCaPE cells wherever warranted. Gene expression profiling of stem cell markers in cells with EMT phenotype To discover the genes regulating and maintaining the stem cell phenotype of PC3 PDGF-D cells having EMT signatures, we performed microarray to determine gene expression profiling of PC3 PDGF-D cells compared to PC3 Neo cells. We found that PC3 PDGF-D cells showed dramatic changes in expression of PLoS ONE | www.plosone.org 2 August 2010 | Volume 5 | Issue 8 | e12445