NATURE MEDICINE • VOLUME 8 • NUMBER 9 • SEPTEMBER 2002 979 ARTICLES Notch genes encode heterodimeric transmembrane receptors that regulate differentiation, proliferation and apoptosis 1,2 . Mammals have four known Notch genes and two families of Notch ligands designated ‘Delta’ and ‘Jagged’, respectively. Upon receptor-ligand interaction, Notch proteins are cleaved by a presenilin-1 (PS-1)-dependent γ-secretase activity 3 . This re- leases a cytoplasmic subunit (N IC ), which migrates to the nu- cleus. This ‘active’ form of Notch regulates the function of several transcription factors. The best-characterized Notch downstream effectors are known as CSL factors (C BF-1 in mam- mals, s uppressor of hairless in Drosophila and L AG-1 in Caenorhabditis elegans). Several reports suggest that Notch signal- ing participates in neoplastic transformation. Extracellular dele- tions of Notch-1 have been implicated in T-cell acute lymphoblastic leukemia 4 . Similar truncated Notch receptors have transforming activity in vitro 5–7 and in animal models 8–10 . However, truncated Notch receptors are uncommon in human malignancies. Deregulated expression of wild-type Notch recep- tors, ligands and downstream targets has been described in cervi- cal, lung, colon, head and neck and renal carcinomas 11–14 , acute myeloid leukemia 15 as well as Hodgkin and large-cell lym- phomas 16 . However, it is unknown whether wild-type Notch sig- naling has a role in spontaneous tumor progression and/or in maintaining the neoplastic phenotype. Ras proto-oncogenes carry activating mutations in approxi- mately 30% of human malignancies 17 . Overexpression of wild- type Ras proteins and activation of Ras signaling by other oncogenes further increase the number of malignancies in which Ras signaling is deregulated. The Ras signaling network is considered a prime target for the development of novel anti- neoplastic agents. Consequently, the identification of key Ras signaling mediators in human neoplastic cells is of considerable significance. Here we show that Notch-1 is a downstream effector of onco- genic Ras, and that downregulation of Notch-1 in Ras-trans- formed human cells is sufficient to abolish key elements of the neoplastic phenotype in vitro and in vivo. Notch-1 is upregulated in Ras-transformed cells We investigated the role of Notch-1 in two human tumor mod- els, human foreskin fibroblasts (BJ) and human embryonic kid- ney epithelial cells (HEK), expressing the human telomerase reverse transcriptase subunit (hTERT), SV40 oncoproteins and oncogenic H-RasV12 18 . Western blots with antibodies to intra- Activation of Notch-1 signaling maintains the neoplastic phenotype in human Ras-transformed cells SANNE WEIJZEN 1 , PAOLA RIZZO 2 , MIKE BRAID 1 , RADHIKA VAISHNAV 1 , SUZANNE M. JONKHEER 1 , ANDREI ZLOBIN 1 , BARBARA A. OSBORNE 3 , SRIDEVI GOTTIPATI 3 , JON C. ASTER 4 , WILLIAM C. HAHN 4,5 , MICHAEL RUDOLF 1 , KALLIOPI SIZIOPIKOU 7 ,W. MARTIN KAST 1 & LUCIO MIELE 2 1 Cancer Immunology Program, Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood, Illinois, USA 2 Department of Biopharmaceutical Sciences and Cancer Center, University of Illinois at Chicago, Chicago, Illinois, USA 3 Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts, USA 4 Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA 5 Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA 6 Department of Adult Oncology, Dana-Farber Cancer Institute and Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA 7 Department of Pathology, Loyola University Chicago, Maywood, Illinois, USA Correspondence should be addressed to L.M.; email: lmiele@uic.edu Published online: 19 August 2002, doi:10.1038/nm754 Truncated Notch receptors have transforming activity in vitro and in vivo. However, the role of wild-type Notch signaling in neoplastic transformation remains unclear. Ras signaling is deregu- lated in a large fraction of human malignancies and is a major target for the development of novel cancer treatments. We show that oncogenic Ras activates Notch signaling and that wild- type Notch-1 is necessary to maintain the neoplastic phenotype in Ras-transformed human cells in vitro and in vivo. Oncogenic Ras increases levels and activity of the intracellular form of wild- type Notch-1, and upregulates Notch ligand Delta-1 and also presenilin-1, a protein involved in Notch processing, through a p38-mediated pathway. These observations place Notch signaling among key downstream effectors of oncogenic Ras and suggest that it might be a novel thera- peutic target. © 2002 Nature Publishing Group http://www.nature.com/naturemedicine