Our study demonstrates that Notch signaling can play important roles in glial tumor development, particularly in promoting nestin expression that may contribute to stem cell potential. We were able to identify Notch receptors and ligands in human glial tumors, suggesting that juxtacrine or autocrine modes of activation are possible—a situation that most often occurs in GBMs, the highest grade of glial tumors. Previous results have indicated higher Notch1 mRNA in grade II to grade III astrocytomas than in GBMs, no significant elevation of Delta-like1 mRNA in astrocytomas and GBMs, and elevation of Jagged1 in only a subset of GBMs [23
]. Our results agree with this previous report on the elevated level of Jagged1 in a subset of GBMs. However, at the protein level, we observed similar levels of Notch1 receptor in human glial tumors and higher ligand levels in GBMs. The lack of correlation between mRNA and protein levels is consistent with previous reports showing that Notch1 is one mRNA that is translationally regulated through its recruitment into polyribosomes by signaling pathways known to be active in GBMs [6
]. In this study, we observed a similar finding in mouse tumors with elevated Notch1 protein in Kras-induced GBMs, where Ras and Akt pathways are significantly elevated. Thus, equivalent mRNA levels may lead to differing amounts of protein. We have also identified Notch2 protein to be expressed in selected GBMs. Thus, our results add to the growing literature supporting the importance of Notch signaling in the formation of human GBMs.
In our mouse glial tumor models, we found that, in Krasinduced GBMs, Notch1 receptor, Delta-like1, and Jagged1 are upregulated, accompanied by increased transcription of HES1, a common Notch transcriptional target. The cooperative nature of activated Notch and Kras signaling was additionally observed with the ability of these two genes combined to generate lesions located in the SVZ, the location of neural stem cells. The cells in these lesions continue to express a proliferation marker and also nestin, much as stem-like cells do. The ability of Notch to sustain the expression of the progenitor marker nestin may have a direct effect on transcription by Notch at the nestin second intron enhancer element. Based on these findings, Notch activation appears capable of promoting or sustaining nestin expression and the stem-like character of SVZ cells.
Given the diverse roles that the Notch pathway plays in normal glial development, parallels between development and tumor formation may be seen. As in development, persistent Notch activation may be a means to keep cells in a more undifferentiated progenitor state. It is believed that cancer stem cells may be the source of tumor cells and that SVZ is thought to be the origin of neural stem cells in the cerebrum [40
]. The periventricular lesions seen with combined Notch and Kras infections in wild-type mice may be a reflection of this aspect of Notch's function. In normal development, nestin-positive cells migrate from the SVZ into the brain on postnatal day 0 and are limited to a small zone of periventricular cells in the adult brain [41
]. By activating the Notch and Ras pathways in nestin-positive progenitors, these cells appear capable of continued proliferation in adult mice in the SVZ as if they have been prevented from proceeding through a normal differentiation pathway.
In Kras-generated GBMs, the activation of the MAP kinase Erk and Akt pathways may affect protein translation. Thus, compared to PDGFB-generated oligodendrogliomas, Kras-induced GBMs produce more Notch proteins from relatively similar amounts of Notch mRNA. The expression of Notch ligands Delta1 and Jagged1 appears to be regulated by transcriptional mechanisms that upregulate expression in Kras tumors and not in PDGFB tumors. The coexpression of ligand and receptor in Kras-induced GBMs may be responsible for juxtacrine Notch signaling that can maintain progenitor characteristics. Therefore, nestin expression is correlated with Kras-induced GBM-like tumors and not with PDGFB-induced oligodendrogliomas. In PDGFB-generated oligodendrogliomas, the Notch1 receptor is overexpressed, but the ligands and Hes1 target are either minimally expressed or at levels slightly higher than that in normal brain. This, however, does not exclude Notch function from these tumors. There are three Delta-like isoforms and two Jagged isoforms, in addition to a newly identified ligand F-contactin that has been shown to have specific effects on oligodendrocytes [42
]. Alternatively, the lower level of receptor and ligand expression seen in Western blot analysis may still be sufficient for signaling.
Inhibition of Notch activity may be useful for glioma therapy. One possible target is presenilin γ secretase, which acts to cleave and release Notch from membranes. The combination of a Notch inhibitor and Ras inhibitor may be particularly effective because these pathways seem to work synergistically to induce lesions and to sustain Ras effects. Further studies that elucidate the molecular mechanisms and specific targets of Notch signaling, particularly ones that distinguish its multiple functions, will also bring greater insight into glioma biology.