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Effect of Notch1 pathway activation on high-grade glioma cells

ABSTRACT: In this study, we explored the transcriptomic consequences of strong activation of the Notch pathway in embryonic human neural stem cells and in gliomas. For this we used a forced expression of the Notch intracellular domain (NICD). Glioblastoma multiforms (GBMs) are highly vascularized brain tumors containing a subpopulation of multipotent cancer stem cells. These cells closely interact with endothelial cells in neurovascular niches. In this study we have uncovered a close link between the Notch1 pathway and the tumoral vascularization process of GBM stem cells. We observed that although the Notch1 receptor was activated, the typical target proteins (HES5, HEY1, HEY2) were not or barely expressed in two explored GBM stem cell cultures. Notch1 signalling activation by expression of the intracellular form (NICD) in these cells was found to reduce their growth rate and migration which was accompanied by the sharp reduction of neural stem cell transcription factor expression (ASCL1, OLIG2, SOX2) while HEY1/2, KLF9, SNAI2 transcription factors were upregulated. Expression of OLIG2 and growth were restored after termination of Notch1 stimulation. Remarkably, NICD expression induced the expression of pericyte cell markers (NG2, PDGFRb and a-smooth muscle actin (aSMA)) in GBM stem cells. This was paralleled with the induction of several angiogenesis-related factors most notably cytokines (HB-EGF, IL8, PLGF), metalloprotease (MMP9) and adhesion proteins (VCAM-1, ICAM-1, ITGA9). In xenotransplantation experiments, contrasting with the infiltrative and poorly-vascularized tumors obtained with control GBM stem cells, Notch1 stimulation resulted in poorly-disseminating but highly-vascularized grafts containing large vessels with lumen. Notch1-stimulated GBM cells expressed pericyte cell markers and closely associated with endothelial cells. These results reveal an important role for the Notch1 pathway in regulating GBM stem cell plasticity and angiogenic properties.

ORGANISM(S): Homo sapiens

PROVIDER: GSE44561 | GEO | 2014/06/04

SECONDARY ACCESSION(S): PRJNA190058

REPOSITORIES: GEO

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Project description:In this study, we explored the transcriptomic consequences of strong activation of the Notch pathway in embryonic human neural stem cells and in gliomas. For this we used a forced expression of the Notch intracellular domain (NICD). Glioblastoma multiforms (GBMs) are highly vascularized brain tumors containing a subpopulation of multipotent cancer stem cells. These cells closely interact with endothelial cells in neurovascular niches. In this study we have uncovered a close link between the Notch1 pathway and the tumoral vascularization process of GBM stem cells. We observed that although the Notch1 receptor was activated, the typical target proteins (HES5, HEY1, HEY2) were not or barely expressed in two explored GBM stem cell cultures. Notch1 signalling activation by expression of the intracellular form (NICD) in these cells was found to reduce their growth rate and migration which was accompanied by the sharp reduction of neural stem cell transcription factor expression (ASCL1, OLIG2, SOX2) while HEY1/2, KLF9, SNAI2 transcription factors were upregulated. Expression of OLIG2 and growth were restored after termination of Notch1 stimulation. Remarkably, NICD expression induced the expression of pericyte cell markers (NG2, PDGFRb and a-smooth muscle actin (aSMA)) in GBM stem cells. This was paralleled with the induction of several angiogenesis-related factors most notably cytokines (HB-EGF, IL8, PLGF), metalloprotease (MMP9) and adhesion proteins (VCAM-1, ICAM-1, ITGA9). In xenotransplantation experiments, contrasting with the infiltrative and poorly-vascularized tumors obtained with control GBM stem cells, Notch1 stimulation resulted in poorly-disseminating but highly-vascularized grafts containing large vessels with lumen. Notch1-stimulated GBM cells expressed pericyte cell markers and closely associated with endothelial cells. These results reveal an important role for the Notch1 pathway in regulating GBM stem cell plasticity and angiogenic properties. Embryonic human neural progenitors obtained from Lonza, the U87 glioma cell line, and glioma cancer stem cells (Gb4 and Gb7) characterized in Guichet et al. (Glia, 2013, 61(2), 225-39) were infected with lentiviruses expressing YFP or YFP-IRES-NICD (activated form of Notch receptor). After 48h, RNA were extracted for Affymetrix microarray analysis.

Project description:Somatic NOTCH1 mutations are found in ~60% of T lineage acute lymphoblastic leukemias (T-ALLs). Notch1 is cleaved by γ secretase to generate activated Notch intracellular domain (NICD) proteins. The NOTCH1 mutations found in T-ALL constitutively activate Notch1 signaling by increasing NICD levels. Genetic alterations in components of the Ras/PI3 kinase (PI3K)/Akt pathway are also highly prevalent in T-ALL, and often coexist with NOTCH1 mutations. Exposing a T-ALL cell line to the PI3 kinase (PI3K) inhibitor GDC-0941 generated drug resistant clones that down-regulated NICD expression. To address the in vivo relevance of this unexpected observation, we transplanted primary wild-type (WT) and KrasG12D mutant T-ALLs into recipient mice, and treated them with GDC-0941 alone and in combination with the MEK inhibitor PD0325901 (PD901). Although many leukemias responded dramatically to these targeted agents in vivo, drug-resistant clones invariably emerged. Multiple resistant T-ALLs lost NICD expression through mechanisms that included loss of Notch1 mutations found in the parental T-ALL. These GDC-0941-resistant leukemias exhibited reduced expression of many Notch1 target genes, elevated levels of phosphorylated Akt (pAkt), and displayed cross-resistance to γ secretase inhibitors (GSIs). Consistent with these data, inhibiting Notch1 activity in T-ALL cells enhanced PI3K signaling, providing a likely mechanism for in vivo selection against clones with Notch1 pathway activation. Thus, oncogenic Notch1 mutations that promote clonal outgrowth during malignant transformation unexpectedly “switch” to become deleterious during treatment with a PI3K inhibitor. These data advance our understanding of T-ALL pathogenesis and have implications for implementing new therapeutic regimens. We analyzed 28 mouse T-ALL samples obtained after in vivo treatment with GDC-0941 alone or GDC-0941 + PD0325901. These T-ALL samples are either Kras wild type or harbor a KrasG12D mutations.

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Effect of Notch1 pathway activation on high-grade glioma cells

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