br Materials and methods br Results br

Materials and methods
Results
Discussion We have previously shown that cellular phenotype is an important determinant of the malignant potential in human neuroblastoma Dynasore cost and tumors (Walton et al., 2004). In particular, in those experiments, we identified a stem cell phenotype in human neuroblastoma which was at least 5-fold more tumorigenic than either of the other two predominant cell types. Moreover, we showed that the frequency of this stem cell type in N-myc non-amplified neuroblastoma tumors correlated with a poor clinical outcome (Ross and Spengler, 2007). In the present study, we sought to determine which genes were overexpressed in this cell type which might promote and/or maintain the stemness and/or the malignancy of human neuroblastoma cancer stem cells. For this study, we used a microarray platform which compared the steady-state expression levels of mRNAs from 13 human neuroblastoma cell lines representing the three predominant cellular phenotypes. We identified seven genes whose expression was significantly and consistently elevated in I-type cells — CD133, KIT, GPRC5C, NOTCH1, PlGF2, TRKB, and LNGFR. Several previous studies, including our own (Walton et al., 2004) have identified CD133 as both a stem cell marker as well as a marker for a poor clinical outcome in cancers, including neuroblastoma and other nervous system cancers (Tong et al., 2008). Two different mechanisms have been proposed for mediating these effects. Sartelet and colleagues have shown that overexpression of CD133 is associated with poor clinical outcome in neuroblastoma and is associated with increased chemoresistance (Sartelet et al., 2012). More recently, studies have revealed that CD133 enhances radioresistance in glioblastoma stem-like cells (Jamal et al., 2012). By contrast, Takenobu et al. have reported that CD133 suppresses neuroblastoma cell differentiation via suppression of RET signaling (Takenobu et al., 2011). In many tissues, including fetal neural tissue, cells with elevated CD133 are capable of self-renewal (Uchida et al., 2000). Likewise, c-kit has been identified as a marker of normal stem cells, such as hematopoietic and neural crest cells, as well as in several cancers, including glioma and neuroblastoma (Cohen et al., 1994; Serfozo et al., 2006). In neural crest stem cells, from which neuroblastomas arise, c-kit has been shown to support stem cell survival and has been suggested to play a role in the growth regulation of neuroblastoma (Sieber-Blum and Zhang, 2002). Other studies have delineated a role for c-kit in glioma cell migration/metastasis and radioresistance (Serfozo et al., 2006; Perez-Losada et al., 2003). G-coupled protein receptor C5C (GPRC5C) is a member of the G protein-coupled receptor superfamily, characterized by a signature 7-transmembrane domain motif. The specific function of this protein is not known. However, examination of the GEO database in mice revealed that this specific G-coupled protein receptor is more highly expressed in dorsal root ganglia (derived from neural crest) compared to spinal cord and is significantly higher in differentiated embryonic stem cells when compared to undifferentiated stem cells (Edgar et al., 2002). Thus, the elevated expression and higher protein amounts of GPRC5C in neuroblastoma stem cells may signify its peripheral neuroectodermal origin and its differentiated embryonic stem cell state. A primary function of Notch signaling in the nervous system is to restrict further differentiation of the stem cell. Several studies in neuroblastoma cells have shown that Notch1 expression is required for the maintenance of neural stem cells by the suppression of proneural genes (Imayoshi et al., 2010). In neuroblastoma cells, Notch1 inhibition has been shown to lead to neuronal/neuroendocrine differentiation (Pahlman et al., 2004). Thus, elevated levels of Notch1 suppress differentiation and keep the cells in an undifferentiated “stem cell” state. In the present study, we show that suppression of NOTCH1 leads to neuronal differentiation and reduced malignant potential. One proposed candidate for negative regulation of the Notch1 gene is the Krüppel-like factor 4 (Klf4) tumor suppressor gene. Klf4 binds to the Notch1 promoter and its overexpression in normal cells is sufficient to down-modulate NOTCH1 gene transcription (Lambertini et al., 2010). Klf4 suppresses neuroblastoma cell growth and determines non-tumorigenic lineage differentiation and lower Klf4 expression is associated with unfavorable neuroblastoma outcome in patients (Shum et al., 2013).