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. 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.
Project description:Expression of a constitutively active Notch-1 intracellular domain (NICD) in MCF-10A cells was found to induce two distinct types of 3D structures: large, hyperproliferative structures and small, growth-arrested structures with reduced cell-to-matrix adhesion. These heterogeneous phenotypes reflect differences in Notch pathway activation levels. High Notch activity caused loss of cell adhesion and inhibition of proliferation, whereas low Notch activity maintained matrix adhesion and provoked a strong hyperproliferative response. In order to gain insight into the dosage-dependent transcriptional events triggered by Notch1 activation, gene expression profiles induced 48 hours after infection of MCF-10A cells with retroviral vectors expressing full-length Notch-1, L1601P+∆P, or NICD were compared. Full-length Notch-1 induced the weakest effect, L1601P+∆P induced an intermediate effect and NICD induced the strongest effect. Results provide insight into the dichotomous activites of Notch during development and tumorigenesis. Overall design: Twelve samples were harvested 48 h after retroviral infection with either vector control, full-length Notch1, Notch1 L1601P+∆P, or NICD. Each condition was performed in triplicate.
Project description:Expression of a constitutively active Notch-1 intracellular domain (NICD) in MCF-10A cells was found to induce two distinct types of 3D structures: large, hyperproliferative structures and small, growth-arrested structures with reduced cell-to-matrix adhesion. These heterogeneous phenotypes reflect differences in Notch pathway activation levels. High Notch activity caused loss of cell adhesion and inhibition of proliferation, whereas low Notch activity maintained matrix adhesion and provoked a strong hyperproliferative response. In order to gain insight into the dosage-dependent transcriptional events triggered by Notch1 activation, gene expression profiles induced 48 hours after infection of MCF-10A cells with retroviral vectors expressing full-length Notch-1, L1601P+∆P, or NICD were compared. Full-length Notch-1 induced the weakest effect, L1601P+∆P induced an intermediate effect and NICD induced the strongest effect. Results provide insight into the dichotomous activites of Notch during development and tumorigenesis. Twelve samples were harvested 48 h after retroviral infection with either vector control, full-length Notch1, Notch1 L1601P+∆P, or NICD. Each condition was performed in triplicate.
Project description:Physiologically, Notch signal transduction plays a pivotal role in differentiation; pathologically, Notch signaling contributes to the development of cancer. Transcriptional activation of Notch target genes involves cleavage of the Notch receptor in response to ligand binding, production of the Notch intracellular domain (NICD), and NICD migration into the nucleus and assembly of a coactivator complex. Posttranslational modifications of the NICD are important for its transcriptional activity and protein turnover. Deregulation of Notch signaling and stabilizing mutations of Notch1 have been linked to leukemia development. We found that the methyltransferase CARM1 (coactivator-associated arginine methyltransferase 1; also known as PRMT4) methylated NICD at five conserved arginine residues within the C-terminal transactivation domain. CARM1 physically and functionally interacted with the NICD-coactivator complex and was found at gene enhancers in a Notch-dependent manner. Although a methylation-defective NICD mutant was biochemically more stable, this mutant was biologically less active as measured with Notch assays in embryos of Xenopus laevis and Danio rerio. Mathematical modeling indicated that full but short and transient Notch signaling required methylation of NICD. Analysis of gene expression upon inactivation and activation of Notch signaling in Beko cells. Beko cells were treated with DAPT or DMSO for 24h. Or Beko cells were infected with a DNMAML-ER or Notch1-ER fusion protein. After Selection cells were treated with 4-OHT (Tamoxifen) or Ethanol for 48h.
Project description:The study deals with the elucidation of potential interaction partners of the intracellular domain of the transmembrane receptor Notch1, termed NICD. The NICD is released from the cytoplasmic tail of the Notch receptor by gamma-secretase treatment and translocated as a transcription factor to the nucleus. Here, virally transduced recombinant NICD constructs (wild-type and deltaEP hyperactive mutant) were employed for AP-MS with the aim of identifying novel NICD interactors in a murine T cell leukemia cell line.
Project description:We asked whether combining Notch and VEGF blockade would enhance suppression of tumor angiogenesis and growth, using the NGP neuroblastoma model. NGP tumors were engineered to express a Notch1 decoy construct (N1D), which restricts Notch signaling, and then treated with either the anti-VEGF antibody bevacizumab or vehicle. Combining Notch and VEGF blockade led to blood vessel regression, increasing endothelial cell apoptosis and disrupting pericyte coverage of endothelial cells. Combined Notch and VEGF blockade did not affect tumor weight, but did additively reduce tumor viability. Our results indicate that Notch and VEGF pathways play distinct but complementary roles in tumor angiogenesis, and show that concurrent blockade disrupts primary tumor vasculature and viability further than inhibition of either pathway alone. 6 neuroblastoma tumors were transfected with Notch1 decoy, 6 with Notch1 decoy and treated with bevacizumab, 6 tumors treated with bevacizumab, and 6 control tumors were profiled by human 133A 2.0 arrays
Project description:The conserved Notch pathway functions in diverse developmental and disease-related processes, requiring mechanisms to ensure appropriate target-selection and gene activation in each context. To investigate, we partitioned Drosophila chromatin into different states, based on histone modifications, establishing the preferred chromatin conditions for binding of CSL, the Notch pathway transcription factor. While most histone modifications were unchanged by CSL binding or Notch activation, rapid changes in H3K56 acetylation occurred at Notch regulated-enhancers. This modification required the histone acetyl-transferase CBP and was independent of transcription. Such rapid changes in H3K56 acetylation are a conserved indicator of enhancer activation, also occurring at mammalian Notch-regulated Hey1 and at Drosophila ecdysone-regulated genes. This core histone modification may therefore underpin the changes in chromatin accessibility needed to promote transcription following signaling activation. H3K56ac profile of control cells (KP) and of NICD overexpression cells (KN). In total 6 samples, with 2 input files (from 2 different conditions) and 2 replicates of H3K56ac ChIP samples of each condition.
Project description:Glioblastoma (GBM) is among the most aggressive cancers. Despite aggressive radiotherapy and treatment with the alkylating agent temozolomide (TMZ), patients ultimately succumb to the disease. Although much interest has focused on highly tumorigenic GBM stem cells (GSCs), adaption of a concept from microbial research proposes that a minor population of dormant “persister” cells in cancer evade current therapies. To separate dormant and treatment-resistant tumor cells in human GBM tumorspheres, we have refined density gradient protocols previously used for separation of neurosphere-forming neural stem cells (NSCs). We find that a minor cell population in human GBM tumorsphere cultures and patient-derived tumor biopsies display increased cell density. These high-density GBM cells (HDGCs) display dormancy, variable expression of proposed GSC markers, and 10-100 fold higher levels of reprogramming gene expression compared to low-density GBM cells (LDGCs). Transcriptional profiling data confirmed the slow-cycling state of HDGCs. As a result, HDGCs show decreased tumorsphere formation capacity in vitro and reduced tumorigenicity in vivo. Using tumorspheres and xenografts, we demonstrated that HDGCs show increased treatment-resistance to ionizing radiation (IR) and temozolomide treatment compared to LDGCs. Similar to the NSC lineage, our data suggest that dormant HDGCs become increasingly sensitive to anti-proliferative therapies as they become activated and further differentiate. In conclusion, density gradients represents a marker-independent approach to separate dormant and treatment-resistant tumor cells in human GBMs and other solid cancers. 12 samples, no replicates, derived from 5 individual patients
Project description:To identify novel down-stream effectors of Notch1 signaling, we have employed cDNA microarray expression profiling as a discovery platform. Human follicualr thyroid carcinoma cell line FTC236 was engineered with tet-on inducible system to establish a stable cell line. The stable cell line FTC236-Notch1 showed robust inducibility with doxycycline, which triggers the expression of Notch1 intracellular domain (NICD). The mRNA samples from FTC236-Notch1 cells with or without doxycline treamtment were analyzed in this assay. Top 50 gene sequences were identified as of the most significance which distinguished between doxycycline treated samples and control samples. Expression of SERPINE1, a down-regulated gene duing Notch1 activation from this signature, was quantified in the same RNA samples by real-time PCR. The protein level was further confirmed by Western blot that restoration of NICD inhibits the expression of SERPINE1. Overall design: NICD induced gene expression in human follicular thyroid cancer cell line was measured at 48 hours after exposure to doses of 0, and 1 ug/mL of doxycycline. Three independent experiments were performed at separate time.
Project description:DNA methylation analysis of T-cell acute lymphoblastic leukemia blast cells from either control mice or Dnmt3a knockout mice carrying a Notch1 Intracellular Domain (NICD) retrovirus Overall design: Comparison of DNA methylation between control and Dnmt3a-KO NICD-driven T-ALL