ORIGINAL ARTICLE Notch1 signaling promotes survival of glioblastoma cells via EGFR-mediated induction of anti-apoptotic Mcl-1 A Fassl 1 , KE Tagscherer 1 , J Richter 1 , M Berriel Diaz 2 , SR Alcantara Llaguno 3 , B Campos 4 , J Kopitz 5 , C Herold-Mende 4 , S Herzig 2 , MHH Schmidt 6 , LF Parada 3 , OD Wiestler 1 and W Roth 1,5 The Notch1-mediated signaling pathway has a central role in the maintenance of neural stem cells and contributes to growth and progression of glioblastomas, the most frequent malignant brain tumors in adults. Here, we demonstrate that the Notch1 receptor promotes survival of glioblastoma cells by regulation of the anti-apoptotic Mcl-1 protein. Notch1-dependent regulation of Mcl-1 occurs cell type dependent at a transcriptional or post-translational level and is mediated by the induction of epidermal growth factor receptor (EGFR). Inhibition of the Notch1 pathway overcomes apoptosis resistance and sensitizes glioblastoma cells to apoptosis induced by ionizing radiation, the death ligand TRAIL (tumor necrosis factor-related apoptosis- inducing ligand) or the Bcl-2/Bcl-XL inhibitor ABT-737. In conclusion, targeting Notch1 might represent a promising novel strategy in the treatment of glioblastomas. Oncogene (2012) 31, 4698--4708; doi:10.1038/onc.2011.615; published online 16 January 2012 Keywords: glioblastoma; apoptosis; Notch1; Mcl-1; EGFR INTRODUCTION Signaling via Notch receptors is an evolutionarily ancient and highly conserved pathway. It has key roles in embryonic development, homeostasis of adult tissue and maintenance of stem cells. Depending on signal strength and cellular context, the Notch pathway can influence cellular processes including differentiation, proliferation and apoptosis. 1,2 In mammals, the core components of the pathway comprise the cell surface-bound ligands Delta-like 1/3/4 and Jagged 1/2 and four Notch receptors (Notch1--4). Upon ligand binding, the Notch receptor is sequen- tially cleaved by ADAM (a disintegrin and metalloproteinase) and g-secretase resulting in the release of the Notch intracellular domain (NICD) into the cytoplasm. After translocation to the nucleus, NICD activates transcription of target genes by convert- ing the transcriptional repressor C promoter-binding factor 1 (CBF1) into a transcriptional activator. The primary Notch target genes belong to the HES (hairy/enhancer of split) and HEY (HES- related) family of transcriptional repressors. As Notch signaling is involved in cellular processes connected with malignant transformation such as apoptosis, differentiation and proliferation, deregulated Notch signaling can contribute to cancer development. Over the past decade, the tumorigenic potential of aberrant Notch signaling has been demonstrated in a wide variety of human neoplasms. Recent data indicate that Notch1 signaling also has an important role in glioblastomas. 3-6 Glioblastomas are the most common and most malignant primary brain tumors in adulthood. The median survival of patients suffering from these tumors is 14 months after initial diagnosis. The resistance of glioblastoma cells to radiotherapy as well as to chemotherapy contributes to the poor prognosis in patients. One major cause for apoptosis resistance in glioblasto- mas is the overexpression of anti-apoptotic proteins, including Bcl- 2, Mcl-1, Bcl-X L and XIAP. On the other side, Notch signaling has been identified as an activator of several anti-apoptotic proteins in various cellular contexts. 7 - 10 In this study, we investigated the role of Notch1 signaling in the regulation of apoptotic cell death in glioblastoma cells and the molecular mechanisms mediating the anti-apoptotic action of Notch1. RESULTS Elevated Notch1 expression is a feature of glioblastoma Notch1 is known to be a neural stem cell factor. Moreover, previous studies identified Notch1 as an oncogene in human glioblastomas. 3-6 As glioblastomas might originate from cells with stem cell-like features, 11 - 14 we compared the expression levels of Notch1 and the activation of the Notch1 signaling pathway (NICD1) in human neural stem cells (NSC_1, NSC_2), 15 human glioblastoma cells with stem-like features (NCH421k), 16 primary glioblastoma cultures (NCH89, NCH156), glioblastoma cell lines (U251MG, LN229 and U373MG) and human primary astrocytes (Figures 1a and b). Consistent with previous studies, Notch1 was highly expressed in all glioblastoma samples. Glioblastoma cells with stem-like features showed similar Notch1 levels as neural stem cells. In contrast, non-neoplastic astrocytes exhibited significantly lower expression levels. Surgically obtained human glioblastoma samples showed a heterogenic expression pattern for Notch1 with focally strong immunoreactivity (Figure 1c, left panel). In contrast, in normal cortical brain tissue Notch1 was weakly expressed in neuronal cells whereas astrocytes and oligodendrocytes were mostly negative for Notch1 (Figure 1c, right panel). Additionally, we performed an analysis of Notch1 expression in the glioblastoma collection of the TCGA consortium Received 6 May 2011; revised 3 November 2011; accepted 28 November 2011; published online 16 January 2012 1 Molecular Tumor Pathology, DKFZ, Heidelberg, Germany; 2 Joint Division Molecular Metabolic Control, DKFZ, Center for Molecular Biology, University of Heidelberg, and University Hospital Heidelberg, Heidelberg, Germany; 3 Department of Developmental Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA; 4 Department of Neurosurgery, Neurosurgical Research, University of Heidelberg, Heidelberg, Germany; 5 Institute of Pathology, University of Heidelberg, Heidelberg, Germany and 6 Institute of Neurology, Johann Wolfgang Goethe University, Frankfurt, Germany. Correspondence: Dr W Roth, Molecular Tumor Pathology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. E-mail: W.Roth@dkfz.de Oncogene (2012) 31, 4698 - 4708 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc