Notch activation promotes cell proliferation and the formation of neural stem cell-like colonies in human glioma cells

Abstract

Since Notch signaling plays a critical role in stem cells and oncogenesis, we hypothesized that Notch signaling might play roles in cancer stem cells and cancer cells with a stem cell phenotype. In this study, we accessed potential functions of the Notch pathway in the formation of cancer stem cells using human glioma. Using RT-PCR, we found that most human astrogliomas of different grades expressed moderate to high level of Notch receptors and ligands. mRNA of Hes5 but not Hes1, both of which are major downstream molecules of the Notch pathway, was also detected. In human glioma cell lines BT325, U251, SHG-44, and U87, mRNA encoding different types of Notch receptors were detected, but active form of Notch1 (NIC) was only detected in SHG-44 and U87 by Western blot. Interestingly, proliferation of these two glioma cell lines appeared faster than that of the other two lines in which NIC was not detected. We have over-expressed NIC of Notch1 in SHG-44 cells by constitutive transfection to evaluate the effects of Notch signaling on glioma cells. Our results showed that over-expression of NIC in SHG-44 cells promoted the growth and the colony-forming activity of SHG-44 cells. Interestingly, over-expression of NIC increased the formation neurosphere-like colonies in the presence of growth factors. These colonies expressed nestin, and could be induced to cells expressing neuron-, astrocyte-, or oligodendrocyte-specific markers, consistent with phenotypes of neural stem cells. These data suggest that Notch signaling promote the formation of cancer stem cell-like cells in human glioma.

c-Jun N-terminal kinase has a pivotal role in the maintenance of self-renewal and tumorigenicity in glioma stem-like cells

“…5 In a similar way to neural precursor cell culture, those glioma stem-like cells expressing CD133 can be enriched in serum-free medium supplemented with growth factors, in which a fraction of glioma cells continue to proliferate and form spheres instead of monolayer. 10,29 Here, we also cultured U87 and U373 glioma cells in sphere forming conditions (Supplementary Figure S1A) as described before 34,35 and found the increase of CD133, Nestin-expressing cells, compared with monolayer-cultured cells (Figure 1b and Supplementary Figure S1B). Sphere-cultured glioma cells also enhanced mRNA and protein expression of neural stem cell markers such as CD133, Nestin and Musashi-1, compared with monolayer-cultured glioma cells (Supplementary Figure S1B).…”

“…Inhibition of Notch pathway exhausts CD133 þ stem-like tumor cells and suppresses sphere formation in vitro and tumor formation in vivo. 29,33,48 Furthermore, Notch signaling pathway directly upregulates the stem cell marker Nestin in glioma. 49 Consistent with these previous studies, our findings show that Notch-2 expression is upregulated in spherecultured cells, compared with monolayer-cultured cells.…”

“…28 Notch activation through introduction of the notch intracellular domain stimulates growth and sphere formation in glioma cell lines and endows radioresistance to CD133 þ glioma stem cells. 29,30 In contrast, Notch inhibition by small interference RNAs (siRNAs) or treatment with g-secretase inhibitors induces cell death and differentiation of glioma cell lines, depletes CD133 þ cell population and suppresses intracranial tumor formation of glioma stem-like cells. 31 --33 In this study, we attempted to identify specific signaling pathways involved in the maintenance of stem-like properties in glioma cells.…”

The brain tumor microenvironment

“…Notch signaling is a critical developmental pathway that controls neural stem cell fate and suppresses radial glia differentiation (Ehebauer et al, 2006). The notch signaling pathway is constitutively active in many high-grade gliomas and has been associated with progression of gliomas (Hulleman et al, 2009;Jeon et al, 2008;Shih and Holland, 2006;Zhang et al, 2008). Notch signaling from endothelial cells to hematopoetic stem cells (HSC) via expression of Notch ligands has been shown to drive the self-renewal characteristics and in vivo repopulation of long-term HSCs that lie adjacent to the vasculature (Butler et al, 2010).…”

z-Leucinyl-Leucinyl-Norleucinal Induces Apoptosis of Human Glioblastoma Tumor–Initiating Cells by Proteasome Inhibition and Mitotic Arrest Response

“…Increased activation of the NOTCH signaling has been reported in several tumor types (18). Recent studies showed that this pathway induces the survival and/or proliferation in GBM and glioma cells (19)(20)(21)(22), and the expression of stem cell markers in glioma cells (21,22). According to these findings, the inhibition of this pathway leads to depletion of stem-like cells and to the block of the engraftment in embryonal brain tumors (23).…”