[CANCER RESEARCH 64, 7787���7793, November 1, 2004] Notch1 and Notch2 Have Opposite Effects on Embryonal Brain Tumor Growth Xing Fan,1 Irina Mikolaenko,1 Ihab Elhassan,1 XingZhi Ni,1 Yunyue Wang,2 Douglas Ball,2,3 Daniel J. Brat,4 Arie Perry,5 and Charles G. Eberhart1 Departments of 1Pathology, 2Medicine, and 3Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland 4Department of Pathology, Emory University, Atlanta, Georgia and 5Department of Pathology, Washington University School of Medicine, St. Louis, Missouri ABSTRACT The role of Notch signaling in tumorigenesis can vary Notch1 acts as an oncogene in some neoplasms, and a tumor suppressor in others. Here, we show that different Notch receptors can have opposite effects in a single tumor type. Expression of truncated, constitutively active Notch1 or Notch2 in embryonal brain tumor cell lines caused antagonistic effects on tumor growth. Cell proliferation, soft agar colony formation, and xe- nograft growth were all promoted by Notch2 and inhibited by Notch1. We also found that Notch2 receptor transcripts are highly expressed in pro- genitor cell-derived brain tumors such as medulloblastomas, whereas Notch1 is scarce or undetectable. This parallels normal cerebellar devel- opment, during which Notch2 is predominantly expressed in proliferating progenitors and Notch1 in postmitotic differentiating cells. Given the oncogenic effects of Notch2, we analyzed its gene dosage in 40 embryonal brain tumors, detecting an increased copy number in 15% of cases. Notch2 gene amplification was confirmed by fluorescence in situ hybrid- ization in one case with extremely high Notch2 mRNA levels. In addition, expression of the Notch pathway target gene Hes1 in medulloblastomas was associated with significantly shorter patient survival (P 0.01). Finally, pharmacological inhibition of Notch signaling suppresses growth of medulloblastoma cells. Our data indicate that Notch1 and Notch2 can have opposite effects on the growth of a single tumor type, and show that Notch2 can be overexpressed after gene amplification in human tumors. INTRODUCTION Embryonal brain tumors, known as medulloblastomas in the cere- bellum and primitive neuroectodermal tumors (PNETs) elsewhere in the central nervous system (CNS), are among the most frequent causes of cancer death in children (1). Because of their primitive appearance and potential for divergent differentiation, these malignant lesions have long been thought to arise from progenitor cells in germinal epithelia situated periventricularly and on the cerebellar surface (2, 3). Neural stem-like cells have recently been isolated from human medul- loblastoma (4, 5), which supports this proposed histogenesis and which suggests that pathways affecting CNS progenitors might play a role in embryonal brain tumor pathobiology. The Wnt (6), Hedgehog (7, 8), and Notch (9���11) developmental signaling cascades all regulate CNS stem cell dynamics, and both the Wnt and Hedgehog pathways are mutationally activated in a subset of medulloblastomas (1, 12). The role played by Hedgehog signaling is particularly significant, with aberrant activation of this pathway driv- ing proliferation of cerebellar granule cell precursors in vitro and causing tumors in transgenic mice (8, 13). In contrast to Hedgehog and Wnt, the role of Notch signaling in medulloblastoma pathogenesis is unknown. However, in situ hybridization (14, 15), murine reporter knockin (16), and reverse transcription-PCR (RT-PCR ref. 15) anal- yses have all shown that Notch2 is expressed in rodent cerebellar granule cell precursors. In addition, activated Notch2 acts as a mito- gen for cerebellar granule cell precursors (15). Taken together, these data suggest Notch2 signaling may be involved in cerebellar neopla- sia. Interestingly, Notch1 is not expressed in proliferating cerebellar precursors (14, 15) but is, instead, found in differentiated internal granule layer neurons (17). Although Notch was initially described as a gene regulating epi- dermal and neuronal cell fate decisions in Drosophila (18), over time it has become clear that the effects of Notch signaling are dependent on cellular context (19). This is particularly true for vertebrates, in which the presence of four Notch receptors and multiple ligands in the Jagged and Delta families leads to a complex array of possible receptor-ligand combinations (20���22). Signaling is initiated when ligands bind a Notch receptor and permit the -secretase���mediated proteolytic release of the Notch intracellular domain (NICD). NICD then translocates into the nucleus, in which it interacts with the transcriptional cofactor CBF1 and transactivates gene targets such as those in the Hes and Hey families (22). Notch signaling has been implicated in several neoplasms arising outside the CNS. Dysregulated expression of Notch receptors or other pathway components has been demonstrated in both hematopoietic tumors and in cervical, pancreatic, and colon carcinomas (19, 21, 23). In human T-cell lymphoblastic leukemias, Notch1 can be activated by 9 7 chromosomal translocations resulting in expression of a truncated, constitutively active receptor (24). Bone marrow cells transduced with activated Notch1 cause similar leukemias when transplanted into lethally irradiated mice (25). Another pathway member, MAML2, is activated by translocations in mucoepidermoid carcinoma (26). Whereas genetic alterations activating Notch2 have not yet been identified in human neoplasms, truncated forms of the Notch2 recep- tor induce thymic lymphomas in cats (27). Notch2 can also transform rat kidney cells in vitro in combination with E1A (28). Given this oncogenic potential, and the mitogenic effects of Notch2 in cerebellar granule cells progenitors, we investigated whether Notch2 signaling regulates the growth of medulloblastomas and other CNS embryonal tumors, hypothesizing it would act as an oncogene. MATERIALS AND METHODS Clinical Material and Immunohistochemistry. 3,3 -diaminobenzidine (DAB)-immunoperoxidase staining was performed with the DAKO Vectastain Elite system with Notch2 (M20 1:200) from Santa Cruz Biotechnology (Santa Cruz, CA) Hes1 (1:3,000) from Dr. Tesuo Sudo (Toray Industries, Kanagawa, Japan), and Ki67 (1:1,000) from DAKO (Carpinteria, CA). Peptide competi- tion eliminated immunoreactivity for Notch2. Medulloblastoma/PNET surgi- cal specimens were from The Johns Hopkins Hospital Department of Pathol- ogy. Frozen tissue from normal fetal cerebella was obtained from the Brain and Tissue Bank for Developmental Disorders (University of Maryland, Balti- more). The medulloblastoma tissue array was constructed as described previ- ously (29). These studies were approved by the Internal Review Board of the Johns Hopkins University School of Medicine. Immunocytochemistry on DAOY cells was performed with standard techniques. In brief, cells were seeded in 6-well tissue culture plate and were allowed to adhere overnight. Received 4/23/04 revised 9/9/04 accepted 9/10/04. Grant support: Supported by grants from the Children���s Brain Tumor Foundation and NINDS K08NS43279 (C. Eberhart) and National Cancer Institute R01CA70244 (D. Ball). C. Eberhart is the recipient of a Burroughs Wellcome Career Award. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: X. Ni is currently in the Department of Surgery, Shanghai Second Medical University, Shanghai, People���s Republic of China. Requests for reprints: Charles Eberhart, Johns Hopkins University School of Med- icine, Department of Pathology, 720 Rutland Avenue, Ross Building 558, Baltimore, MD 21205. Phone: (410) 502-5185 Fax: (410) 955-9777 E-mail: ceberha@jhmi.edu. ��2004 American Association for Cancer Research. 7787

After infection with AdNICD2 or Ad gal for 48 hours, cells were fixed with 4% paraformaldehyde, permeabilized with 0.4% Triton X-100, blocked in PBS/5% bovine serum albumin, then incubated with anti-Notch2 antibody [C651.6DbHN originally developed by Dr. Artavanis-Tsakonas (30) and ob- tained from the University of Iowa Developmental Hybridoma Studies Bank, (1:10 dilution)] for 2 hours. Cy3-conjugated secondary antibody incubations and 4 ,6-diamidino-2-phenylindole (DAPI) counterstains were performed with appropriate wash steps before mounting and visualization with a fluorescence microscope (Zeiss, Germany). Polymerase Chain Reaction. Quantitative RT-PCR was performed as described previously (31), with all reactions normalized to actin (Applied Bio- systems, Foster City, CA). The primers and probes for Notch1, Notch2, and Hes1 are: Notch1 forward (F) 5 -CCGCAGTTGTGCTCCTGAA-3, Notch1 reverse (R) 5 -ACCTTGGCGGTCTCGTAGCT-3 , Notch1 probe 5 -AACAAAGATATG- CAGAACAACAGGGAGGAGACA-3 Notch2 F 5 -GGCATTAATCGCTA- CAGTTGTGTCT-3 , Notch2 R 5 -GGAGGCACACTCATCAATGTCA-3 , Notch2 probe 5 -CACCAGGATTCACAGGGCAGAGATGTAACA-3 Hes1F 5 -AGCGGGCGCAGATGAC-3 , Hes1R 5 -CGTTCATGCACTCGCTGAA-3 , Hes1 probe 5 -CTGCGCTGAGCACAGACCCAAGTGT-3 . Assays-on-Demand TaqMan probes (Applied Biosystems) were used to measure Hey1, Hey2, PTCH, Gli1, and Gli2 expression. In tumor material, Notch and Hes1 expression was normalized to the median levels in three fetal cerebella of gestational ages 19 to 20 weeks. Differential PCR quantitation of Notch2 gene dosage was performed as described previously (31) with the following primers for three Notch2 exons and a 1q sequence-tagged site (STS) reference sequence: Ex12 F 5 -GGGTTAATT- GTGAAATTAATT-3 , Ex12 R 5 -CGATTAATGCCATCCATACAGA-3 Ex26 F 5 -ATGACAAATACTGTGCAGACCA-3 , Ex26 R 5 -AGCATCCTG- GAGCAGTTGTT-3 Ex30 F 5 -TGAAGCTGCAGACATCCGTA-3 Ex30 R: 5 -AACACATCCACCTCCTGCTC-3 1qSTS F 5 -ATTACTGCTTTC- CCCAGTTCC-3 , 1qSTS R 5 -CTTGTTTTGTTTTCCACACCG-3 . DNA ex- tracted from eight nonneoplastic brain samples was used to calibrate the assay and calculate a threshold for significant copy number gains (2.7 fold increase). Cases in which the mean ratio of Notch2 to 1qSTS exceeded this threshold for all three exons, were considered to be amplified. Fluorescence In situ Hybridization. Fluorescence in situ hybridization (FISH) with probes generated by using bacterial artificial chromosomes (BACs) CTD-2574B15 (Notch2), and 260I23 [1p32 reference probe (a refer- ence sequence on 1p32) Research Genetics, Pasadena, CA] was performed as described previously (32). The cytogenetic localization of the Notch2 BAC clone (1p11���13) was confirmed on metaphase preparations. Cell Culture and Xenograft Experiments. DAOY and PFSK cell lines were obtained from the American Type Culture Collection and maintained in Richter���s zinc option media supplemented with 10% fetal bovine serum unless otherwise noted UW228 cells were the kind gift of Dr. John Silber (Depart- ment of Neurological Surgery, University of Washington, Seattle, WA) and were grown in DMEM/F-12 with 10% fetal bovine serum. The Notch intra- cellular domain (NICD) adenovirus construction and procedures for infection have been described previously (33). Stable transfectants were established by selection with G418 with standard techniques. Flow cytometric analysis of S-phase fraction and cell cycle kinetics was performed after fixation and staining with propidium iodide with a FACSCalibur (Becton Dickinson, San Jose, CA) with CELL Quest, version 3.3 software. Small interfering RNAs (siRNAs) for Notch1 (sequence available from L. Miele, Department of Pharmceutical Sciences and Cancer Center, University of Illinios, Chicago, IL), Notch2 (5 -GUCUCAGAAGCUAACCUAAtt-3 ), Hes1 (5 -AGACGAA- GAGCAAGAAUAAtt-3 ), or scrambled control siRNA were transfected with siPORT amine (Ambion, Austin, TX). Cell growth was measured by using celltiter 96 cell proliferation assay (Promega, Madison, WI), and plotted either as growth curves or as relative growth [(A490 nmDay3 to A490 nmDay1 )/A490Day1]. Soft agar clonogenicity assays were performed in triplicate as previously described (34) in 2% fetal bovine serum. Subcutaneous xenografts were established in athymic (nude) mice by using three million cells in 100 L of culture medium mixed with 100 L of Matrigel (BD Bioscience, Bedford, MA). Mitoses were counted by a neuropathologist (I. M.) blinded with respect to xenograft line. Western blots contained 10 or 20 g of protein per lane, and were hybridized overnight with the following primary antibodies: for Notch1, Santa Cruz Biotechnology C20, 1:200 for Notch2, Santa Cruz Biotechnology M20, 1:200 or with the monoclonal antibody C651.6DbHN, 1:10, and Hes1 (polyclonal from T. Sudo) 1:2000. DFK-167 (Enzyme Systems, Livermore, CA) was diluted in DMSO. RESULTS Differential Expression of Notch1 and Notch2 in Normal and Neoplastic Cerebellar Cells. We first sought to define the expression pattern of Notch transcripts in embryonal brain tumors derived from the external germinal layer (EGL) and ventricular zone, predicting lower expression of Notch1 than of Notch2 in cerebellar tumors. To obtain quantitative data on gene expression, we used real-time RT- PCR to measure Notch1 and Notch2 levels in mRNA extracted from snap-frozen brain tumors. We compared Notch expression in tumors to that in rapidly proliferating fetal cerebellum in which the pathway should be active, based on developmental studies in rodents. Notch2 mRNA was present in all 30 medulloblastomas examined, and 25 expressed higher levels than did fetal cerebellum (Fig. 1A). In parallel with its absence from the proliferating outer EGL in normal develop- ment, Notch1 was undetectable in 9 of 22 medulloblastomas, and only 3 of the 13 positive cases had expression equal to or higher than that in fetal cerebellum. Hes1 message was also detected in all cases, although only five had levels higher than fetal cerebellum (data not shown). In medulloblastoma, Notch2 and Hes1 expression (Fig. 1B) showed a significant positive correlation (P 0.04), whereas Notch1 and Hes1 did not, which suggests that Notch2 may regulate Hes1 Fig. 1. Notch mRNA expression in medulloblastoma/PNET. In A, Notch1 mRNA expression, measured by quantitative RT-PCR and expressed as fold change in relation to levels in nonneoplastic fetal cerebella (n 3), was low or undetectable in 19 of 22 medulloblastomas. In contrast, Notch2 was detected in all 30 medulloblastomas examined and, in 25, was more highly expressed than in normal cerebellum. In B, Notch2 mRNA expression correlated significantly with that of Hes1 in medulloblastomas. In C, Signif- icantly higher expression of Notch1 was present in extra-cerebellar PNET as compared with medulloblastoma (MB). In D, a trend toward higher Notch2 expression in PNET was seen as well. A PNET shown by FISH to be amplified at the Notch2 locus had especially high mRNA levels (arrow). In E, expression of Notch receptors and Hes1 was also detected in medulloblastoma (DAOY, UW228) and PNET (PFSK) cell lines. In F, the goat polyclonal Notch2 antibody used in our immunohistochemical studies identified the same Mr 120,000 transmembrane Notch2 band in DAOY protein extracts as the anti- Notch2 monoclonal C651. An appropriately sized Mr 31,000 Hes1 band was also identified in these extracts. 7788 NOTCH1 AND NOTCH2 IN EMBRYONAL BRAIN TUMOR GROWTH