Notch3 Activation Promotes Invasive Glioma Formation in a Tissue Site-Specific Manner

Published OnlineFirst January 18, 2011; DOI: 10.1158/0008-5472.CAN-10-0690

Cancer Tumor and Stem Cell Biology Research

Tarran J. Pierfelice1, Karisa C. Schreck1,2, Louis Dang7, Laura Asnaghi3, Nicholas Gaiano1,2,4,5, and Charles G. Eberhart3,5,6

Abstract Although Notch signaling has been widely implicated in neoplastic growth, direct evidence for in vivo initiation of neoplasia by the pathway in murine models has been limited to tumors of lymphoid, breast, and choroid plexus cells. To examine tumorigenic potential in the eye and brain, we injected retroviruses encoding activated forms of Notch1, Notch2, or Notch3 into embryonic mice. Interestingly, the majority of animals infected with active Notch3 developed proliferative lesions comprised of pigmented ocular choroid cells, retinal and optic nerve glia, and lens epithelium. Notch3-induced lesions in the choroid, retina, and optic nerve were capable of invading adjacent tissues, suggesting that they were malignant tumors. Although Notch3 activation induced choroidal tumors in up to 67% of eyes, Notch1 or Notch2 activation never resulted in such tumors. Active forms of Notch1 and Notch2 did generate a few small proliferative glial nodules in the retina and optic nerve, whereas Notch3 was 10-fold more efficient at generating growths, many of which were large invasive gliomas. Expression of active Notch1/Notch3 chimeric receptors implicated the RBPjk-association molecule and transactivation domains of Notch3 in generating choroidal and glial tumors, respectively. In contrast to our findings in the optic nerve and retina, introduction of active Notch receptors, including Notch3, into the brain never caused glial tumors. Our results highlight the differential ability of Notch receptor paralogs to initiate malignant tumor formation, and suggest that glial precursors of the optic nerve, but not the brain, are susceptible to transformation by Notch3. Cancer Res; 71(3); 1115–25. 2011 AACR.

Introduction best described, as activation of the pathway can promote murine T-ALL, similar to that seen in humans (8, 11, 13, 14). Notch signaling plays a critical role in the specification, Notch is thought to play a particularly important role in proliferation, and survival of stem/progenitor cells in a num- poorly differentiated tumor cells, where pathway inhibition ber of tissues, including the central and peripheral nervous may be able to deplete "cancer stem cells," which are resistant systems (1). The pathway is also widely implicated in neopla- to radiation and standard chemotherapies (1, 5, 7, 9, 10, 12, 15– sia, and in most contexts promotes neoplastic growth (2), but 18). Small molecules targeting Notch have shown great pro- can act as a tumor suppressor in some cell types (3–5). Notch mise in preclinical testing of several tumor models. On the is activated in a broad range of hematopoietic and solid basis of such studies, phase I clinical trials for leukemia and tumors (2–4, 6–12). Its role in leukemogenesis is probably breast cancer have been initiated using gamma-secretase inhibitors that block the activation of Notch receptors (Clin- ical Trials.Gov identifiers NCT00106145; NCT00878189). How- ever, for such therapies to be optimally utilized, we must first more fully understand the complexities of Notch signaling in Authors' Affiliations : 1Institute for Cell Engineering, and Departments of 2Neuroscience, 3Pathology, 4Neurology, 5Oncology, and 6Ophthalmology, cancer, as its effects are context-dependent (5, 16). Johns Hopkins University School of Medicine, Baltimore, Maryland; and The role(s) of the various Notch receptors in tumor initia- 7Department of Pediatrics and Communicable Diseases, University of in vivo Michigan, Ann Arbor, Michigan tion are particularly poorly understood. Though struc- turally similar, the 4 mammalian Notch paralogs (Notch1–4) Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). are not functionally equivalent in all contexts. The mechan- K.C. Schreck and L. Dang contributed equally to this work. istic basis of such differences is still being uncovered, but it has been shown that the various Notch receptors can relate N. Gaiano and C.G. Eberhart are the co-senior authors. uniquely to binding site distribution and orientation on target Corresponding Author: Charles G. Eberhart, Johns Hopkins University School of Medicine, Ross Building 558, 720 Rutland Ave., Baltimore, MD gene promoters (19). 21205. Phone: 410-502-5185; Fax: 410-955-9777. In this study, we examined potential functional distinctions E-mail: [email protected] between the Notch paralogs by introducing activated Notch doi: 10.1158/0008-5472.CAN-10-0690 receptors into the brain and eye, and we identified prolifera- 2011 American Association for Cancer Research. tive lesions in the retina, optic nerve, and lens. Importantly, we

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found that active forms of Notch1, Notch2, and Notch3 have described previously (20). All viruses used for injection had differing abilities to induce tumors, and that glial progenitors titers of 1 108 to 4 108. The animals were sacrificed at 30 to in the optic nerve and retina are particularly susceptible to 60 days of age, and ocular and brain tissues were harvested. Notch3-driven transformation as compared with those in the rest of the central nervous system. These results highlight the Staining of tissue sections and analysis sometimes distinct capacities of various Notch receptors to Eye and brain tissue obtained from virally injected mice was induce tumor formation and the context-dependence of glial fixed in 4% paraformaldehyde for frozen sections or formalin progenitor transformation. for paraffin-embedded sections. Sectioning, PLAP staining (22), hematoxylin and eosin (H&E) staining, and immunohis- Materials and Methods tochemistry were carried out using standard methods. We blindly scored multiple H&E stained adult eye sections from NICD1/NICD3 chimeric constructs virally injected animals. Immunohistochemistry on paraffin The NICD1/NICD3 chimeric human proteins were sections was carried out using the Vectastain ABC kit and expressed using the retroviral vector pCLE (20).The chimeric DAB Peroxidase Substrate kit (Vector Laboratories) as speci- cDNAs were made using standard PCR and ligation protocols, fied by the manufacturer's protocol. Primary antibodies used and were confirmed by DNA sequencing. Briefly, we amplified were a-Notch3 (rabbit, 1:1,000; Santa Cruz Biotechnology), 3 different segments of Notch1 intracellular domain (NICD1) a-Pax6 (mouse, 1:400, Millipore Corporation), a-Chx10 (sheep, and 3 of NICD3, each product varying in length to include 1:100, Exalpha Biologicals), a-GFAP (rabbit, 1:1,000; DAKO), different regions. For NICD1 these were called DN1xxx, a-Nestin (chicken, 1:250; Aves Labs, Inc.), and Hes5 (rabbit, DN11xx, and DN111x, and included the RBPjk-association Gaiano Laboratory, only recognizes overexpression). Alexa molecule (RAM) domain and adjacent downstream residues, Fluor Dye conjugated secondary antibodies used were RAM, RAM/Ank1–2, and RAM/Ank1–5, respectively (Ank, obtained from Invitrogen. ankyrin repeat). For NICD3, these were called DNxxx3, DNxx33, DNx333, which included TADD (a proximal portion Luciferase assay of the transactivation domain), Ank3–5/TADD, Ank1–5/ Luciferase assays were conducted on E14.5 mouse cortical TADD, respectively. We already possessed the human neural progenitors or NIH3T3 cells transfected with the DN1111 and DN3333 constructs in the retroviral vector pCLE Hes5promoter (Hes5p)- or CBF1RE [4xCBF1 (C promoter- (DN1CLE and DN3CLE; ref. 21). All primers were designed to binding factor 1)-responsive elements]–luciferase constructs include endogenous restriction enzyme sites, or sites created along with one of the Notch-expressing constructs, according by silent point mutations, to permit generation of chimeric to the manufacturer's protocol (Lonza). The Hes5 promoter constructs by ligation (e.g., DN11xx was ligated to DNxx33). was a gift from Toshiyuki Ohtsuka at Kyoto University and the DN1113 encodes residues 1761–2093 of Notch1 and 2006–2098 CBF1RE-luciferase construct was a gift from Diane Hayward at of Notch3. DN1133 encodes residues 1761–1993 of Notch1 and Johns Hopkins University. Cells were harvested 48 hours after 1905–2098 of Notch3. DN1333 encodes residues 1761–1858 of transfection and luciferase activity was measured (Promega). Notch1 and 1767–2098 of Notch3. See Supplementary Figure Each sample was normalized to beta-galactosidase expression S1 for the amino acid sequence of the chimeric proteins. (Clontech).

Constructs and virus preparation Preparation of rat astrocyte cell cultures Retroviral constructs expressing the activated forms of The cortex and optic nerves were isolated from postnatal Notch1–3 (human Notch intracellular domain (NICD1, NICD2, day 2 Sprague-Dawley rats as described previously (23, 24). NICD3) or the NICD1/NICD3 chimeras and human alkaline The optic nerve and retinal cultures were 90% glial fibrillary phosphatase (placental alkaline phosphatase; PLAP) reporter acidic protein (GFAP)–immunopositive, and the cortical cul- were generated using standard protocols (20). The DN1CLE tures were 60% GFAP-immunopositive (data not shown). (22), DN2CLE, DN3CLE, DN1113CLE, DN1133, and Murine DN1CLE (22) caused the same incidence of ocular DN1333CLE-expressing viruses encode truncated NICD that tumors as DN1CLE and was used to infect these cultures. lacks a portion of the C terminus, a modification required to obtain high viral titer. DN1CLE encodes residues 1761–2196; Quantitative PCR DN2CLE encodes residues 1703–2146; and DN3CLE encodes RNA was obtained from NIH3T3 cells or rat optic nerve residues 1663–2098. The N3CLE-expressing virus encodes full- and cortical astrocyte cultures infected with the Notch-expres- length NICD3, residues 1663–2321 (21). Virus expressing sing viruses and real-time polymerase chain reaction (RT-PCR) empty vector (CLE) was used as a control. was conducted using SYBR Green reagent (Applied Biosystems) with all samples run in triplicate on an I-Cycler (Bio-Rad). Values Animals and in utero viral injections were normalized to actin or GAPDH using the standard curve The CD1 (Charles River) and Black Swiss (Taconic) mice method. The following primer sequences were used. Mouse used in this study were maintained in accordance with the Hes1 Forward (F): 50-AAAGCCTATCATGGAGAAGAGGCG and Institutional Animal Care and Use Committee at Johns Hop- Hes1 Reverse (R): 50-GGAATGCCGGGAGCTATCTTTCTT-30; kins University School of Medicine. Embryos were injected in mouse Hey1F: 50- ACGACATCGTCCCAGGTTTTG-30 and utero at E9.5 or E10.5 with virus using ultrasound guidance as Hey1R: 50-GGTGATCCACAGTCATCTGCAAG-30; mouse

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GAPDHF: 50-AAACCCATCACCATCTTCCA-30 and GAPDHR: 50- In contrast, injection of control CLE virus was never asso- AGGGGCCATCCACAGTCTTC-30; human PLAP (23); rat Hes1, ciated with gross or microscopic lens abnormalities. This was Hes5, Hey1, and Hey2 (24). an ongoing proliferative process, as evidenced by the presence of mitotic figures and Ki67-positive cells (Fig. 1Dii inset and Results iii), with 11% of cells expressing this proliferative marker in the depicted cataract. The thickened epithelial layers stained Constitutive Notch3 signaling generates retinal lesions positive for PLAP (Fig. 1Diii), indicating that viral infection that express retinal progenitor markers was directly associated with the hyperproliferative lesions. At E9.5 to E10.5 the developing forebrain and optic cup form Another group recently reported that active Notch1 signaling a continuous open structure, allowing viral infection of anlage also induced ALE hyperproliferation (32). Our N3CLE-induced giving rise to the retina (Fig. 1A). PLAP staining of the optic lesions seem very similar to human anterior subcapsular cup neuroepithelium in animals sacrificed 6 days post viral cataracts (ASC), which are also characterized by a multi- injection confirmed infection of these cells (Fig. 1Bi, arrow). layered lens epithelium and abnormal deposition of capsular Animals injected with N3CLE retrovirus encoding constitu- material (33). tively active Notch3 developed retinal lesions in 62 of 94 adult eyes (66%) examined (Table 1). N3CLE-induced retinal lesions Pigmented choroidal tumors are generated by Notch3 were focal and varied in size. Some disrupted only the inner signaling retinal layer (Fig. 1Bii) whereas others affected all 3 retinal N3CLE-injected animals also developed invasive tumors layers (Fig. 1Biii). The retinal lesions coincided with PLAP arising in the choroidal layer of the eye, a loose fibrovascular staining and nuclear Notch3 immunoreactivity (Fig. 1Biv and tissue containing scattered melanocytes. The invasive chor- v) indicating that they were induced by Notch3 activity. oidal neoplasms, comprised of oval and spindled cells, were To identify the cell types contained within the N3CLE- present in 63 of the 94 eyes (67%) examined in N3CLE-injected induced retinal lesions, we used immunofluorescence to animals (Table 1). Such tumors were never observed in control detect various markers of retinal differentiation. The disorga- CLE-injected eyes (n ¼ 22). Tumors were identified in all nized regions contained some cells expressing the glial marker regions of the choroid, from the periphery (Fig. 2A, arrow) to glutamine synthetase (GS) and the early neuronal marker BIII- the posterior pole, and some eyes contained multiple separate tubulin (Fig. 1Biv and v). Interestingly, within the lesions we lesions in various regions. The sheets and clusters of tumor also identified numerous cells-expressing Pax6 or Chx10 cells were highly proliferative, as evidenced by the presence of (Fig. 1C), and a subpopulation coexpressing both of these numerous mitotic figures and Ki67-positive cells (Fig. 2Biii). markers (Fig. 1C, arrowheads). Occasional cells were Ki67- PLAP staining (Fig. 2A and Biii) and nuclear Notch3 immu- positive, indicating a degree of proliferation. However, the nopositivity (Fig. 2Bii) indicated that the choroidal lesions lesions did not have microscopic features of a retinocytoma or arose from cells from N3CLE infection with increased Notch a retinoblastoma. Previous studies have shown that retinal activity. Many of the larger choroidal tumors invaded through stem/progenitor cells coexpress Pax6 and Chx10 during early transscleral canals into the orbital space (Fig. 2Bi). A few eye development; their expression later segregates and tumors extensively infiltrated the periocular tissues and were becomes cell specific (25–27). Pax6/Chx10 coexpression grossly apparent on external examination as exophytic lesions within the retinal lesions therefore suggests the presence of around the eye. The fact that not all eyes injected with N3CLE stem/progenitor-like cells in which Notch pathway deregula- developed these neoplasms suggests that additional genetic tion has inhibited normal differentiation. Prior reports have alterations may be needed to promote tumorigenesis, shown that Notch1 activity promotes progenitor cell character although limited sampling may have also affected the number in the developing retina (28–30), and our findings suggest that of lesions detected. Notch3 can play a similar role. The precise pathological classification of these neoplasms is not clear. They did not seem embryonal, and were not Constitutive Notch3 signaling causes overproliferation immunopositive for Nestin, synaptophysin, Tuj1, GS, PAX6, of lens epithelium or GFAP, suggesting that they were not neuronal or glial. In Many mice injected at E9.5 or E10.5 with N3CLE developed humans, uveal melanoma is the most common malignant white, cataractous eyes by 1 month of age (Fig. 1C). Lens neoplasm of the choroid. A subset of injections was performed formation is initiated early in fetal development and at E10.5 into the pigmented Swiss Black mouse line, and choroidal the ectodermal lens placode is still located on the surface of tumors in these animals always contained pigmented cells the embryo (31). In the normal mature lens, the only remain- (Fig. 2Bi). The tumors clearly arose from choroid rather than ing cells are an anterior lens epithelium (ALE) monolayer retinal pigment epithelium, as the latter pigmented cell layer below the capsule. On microscopic examination, we observed was intact in all of the smaller lesions examined (Fig. 2Bi, that 22 of 94 eyes (23%) from animals injected with N3CLE arrow and data not shown). Choroidal melanocytes derive showed overproliferation of lens epithelium with adjacent from neural crest, a tissue that would be at least partially deposition of capsular material (Fig. 1Dii; Table 1). In some, exposed to virus at the time of injection (34). The pigmenta- almost all of the epithelium was multilayered, whereas in tion we observe suggests that these may represent murine others normal-looking epithelium (Fig. 1Dii, arrowhead) was melanomas arising from choroid, and the oval to spindled present next to regions 10 or more cells thick (Fig. 1Dii, arrow). morphology of the tumor cells is similar to that seen in human

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Figure 1. Retinal lesions and ASCs are caused by Notch3 signaling. A, virus is microinjected into the ventricles of an E10.5 mouse embryo. At the time of injection, the forebrain and optic cup are a continuous structure, allowing viral infection of the anlage giving rise to the retina and optic nerve. Viral particles are released into the amniotic sac near the injection site on needle withdrawal and infect the developing lens. B, E17.5 retina in a N3CLE-injected animal with a PLAP-positive clone (i). H&E staining showed that N3CLE-induced retinal lesions can span 1 (ii) or all 3 layers (iii) of the retina. Retinal lesions contained BIII- tubulin–positive (iv) and GS-positive (v) cells that were often double positive for nuclear Notch3 (N3). C, Pax6/Chx10 double-positive cells in retinal lesions (arrowheads). D, i, white cataractous lenses developed in N3CLE-injected animals. ii, the ALE (arrowhead) expanded into multiple epithelial layers (arrow) and contained mitotic bodies in the N3CLE-induced ASCs (inset). iii, areas of hyperproliferation were PLAP positive and contained Ki67-positive cells (DAPI counterstain). Original magnifications: 20 (Bi, Biv, and C), 40 (Bv and Dii), 200 (Bii, Biii, and Di).

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Table 1. Ocular tumor and proliferative cataract occurrence in animals injected with the Notch-expressing or NICD1/NICD3 chimeric viruses

Virus injected Number Occurrence (%) of eyes Choroidal Retinal Retinal Episcleral Optic nerve– Proliferative tumors disorganizations glial lesions glial lesions associated cataracts glial lesions

CLE 22 0 0 0 0 0 0 N3CLE 94 67 66 24 50 21 23 DN3CLE 22 18 63 18 50 45 36 DN1333CLE 54 2 76 19 30 26 50 DN1133CLE 38 3 50 5 18 11 32 DN1113CLE 26 0 54 12 23 23 15 DN1CLE 46 0 20 2 4 2 7

NOTE: Virally infected eyes were blindly scored. The percent occurrence was rounded to the nearest whole number.

and into the orbit. Because of this highly invasive behavior and melanomas at this site. However, we were unable to verify the presence of scattered mitotic figures, we believe these are melanocytic differentiation in the lesions using several immu- glial neoplasms rather than reactive gliotic lesions. nohistochemical markers (data not shown). The multilayered Glial tumors of similar appearance were also identified in concentric growth of the tumors in focal regions also shows the optic nerves of 20 of 94 N3CLE-injected animals (21%; some similarity to myopericytoma, although in humans such Table 1). These tumors were only moderately cellular, with tumors are benign and are not known to occur in the eye. abundant cytoplasm and some elongated cytoplasmic pro- Thus, the classification, grade, and relationship of these cesses (Fig. 3A). Occasional mitotic figures and Ki67-positive choroidal tumors to a specific type of human neoplasm is cells were present (Fig. 3Aii inset and Ci). In some tumors, unclear at this point. neoplastic cells were localized under the meninges and focally distended the coverings of the optic nerve (Fig. 3A, arrow- Notch3 signaling induces glial tumors in the retina and heads); in others, the optic nerve was more diffusely infiltrated optic nerve by tumor. In 16 of the 20 eyes with optic nerve tumors (80%), Histological examination revealed that 23 of 94 eyes (24%) the neoplasm invaded into the soft tissues surrounding the from the N3CLE-injected animals developed GFAP-positive nerve. This most commonly occurred in the region of the optic glial lesions in the retina (Table 1; Fig. 2C and D). Some of disk, but in some optic nerves greatly distended by tumor, these were relatively small (Fig. 2C), but most extended along strands of neoplastic cells exited through more posterior large portions of the retina, and these often invaded through channels penetrating the nerve sheath (Fig. 3Bi). Other glial the retinal pigment epithelium and into the choroid (Fig. 2Di). lesions involved both the optic nerve and retina. In total, we In 2 cases, retinal glial tumors invaded through scleral canals identified invasive glial tumors in the orbital tissues of 47 of 94

Figure 2. Invasive choroidal tumors and retinal glial lesions arise following Notch3 activation. N3CLE-induced cellular choroidal tumors in the adult eye were PLAP positive (A), pigmented, and arose in the choroidal layer beneath an intact retinal pigment epithelium (Bi, arrow). Many choroidal tumors invaded outward through breaks in the sclera (Bi, arrowheads) and could diffusely infiltrate periocular tissues. Choroidal tumors contained nuclear N3 and Ki67-positive cells (Bii and Biii). C and D, N3CLE-injected animals contained PLAP-positive retinal glial lesions (C), GFAP-positive (Dii), and GS-positive (Diii). Original magnifications: 10 (A), 20 (Bi), 40 (Bii and Diii), 63 (Biii, Di, and Dii), 64 (Bii), 200 (C).

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Figure 3. N3CLE induced formation of glial tumors along the optic nerve. A, glial tumors (arrowheads) formed along the optic nerve (arrow) in N3CLE-injected animals and contained mitotic bodies (ii, inset). B, the optic nerve tumors expressed GFAP and Notch3 and invaded orbital tissues surrounding the optic nerve. Some glial tumors involved both the retina (arrowhead) and optic nerve (arrow). C, i, glial tumors contained Ki67-positive proliferating cells. Nestin-positive and Nestin/GFAP-positive cells within the lesions coexpressing Hes5 or Notch3, respectively (Cii and D). Original magnifications: 63 (C and D), 100 (Ai and Bi), 200 (Ai, Aii, and Bii), 400 (Aii, inset).

eyes injected with N3CLE (50%; Table 1). Although many of The choroidal tumors, which were sometimes encountered in these were clearly associated with retinal or optic nerve the same eyes as invasive gliomas, were negative for GFAP and gliomas, others were not contiguous with these neural struc- Nestin (data not shown). tures in the sections examined. We believe that most or all of these were associated with optic nerve or retinal gliomas that Notch1 and Notch2 activation does not efficiently were not identified in limited tissue examined microscopi- generate ocular tumors cally, but some could be distinct lesions arising from orbital The mammalian genome contains 4 Notch receptors, 3 of neural tissues. which (Notch1–3) are very similar in sequence, although As was true for the choroidal tumors induced by NICD3, Notch1 and Notch2 are more similar to each other than to both PLAP and nuclear Notch3 (Fig. 3Ci and D) were identified Notch3 (35, 36). Despite their high level of identity, the effects in all optic nerve and retinal tumors examined, and no glial of the various Notch receptors are not always the same, and in lesions were identified in animals injected with control CLE some contexts they can even have opposing effects (4). We virus. We believe these tumors are astrocytic because they therefore sought to determine whether introduction of either strongly express GFAP when examined using immunohisto- activated Notch1 or Notch2 would produce proliferative ocu- chemistry (Figs. 2Dii, 3Bi and D), and frequently contained lar lesions similar to those described above. elongated cellular processes similar to those in human astro- To generate high-titer virus expressing constitutively active cytoma. Nestin, an intermediate filament often expressed in Notch1 and Notch2, we found that we needed to truncate a neural progenitor cells and glial tumors, was diffusely present portion of the NICD C terminus. Because N3CLE expresses in the proliferative lesions (Figs. 3Cii and D). The Nestin- and full-length NICD3, we also generated a shortened NICD3- GFAP-positive cells within the glial lesions also coexpressed expressing virus (DN3CLE) that contained a comparable C- nuclear Notch3 and the downstream target Hes5 (Fig. 3Cii and terminal truncation (Fig. 4A). The resulting viruses all infected D), and PLAP (not shown) on immunofluorescent analyses. the retina and optic nerve. The overall extent of infection as

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Figure 4. The Notch-expressing retroviruses diffusely infect the optic nerve. A, the NICD1–3 and chimeric protein constructs. B, PLAP staining of the optic nerve of virally infected animals (5 magnification). C, i, RT-PCR showed that the Notch-expressing constructs increased Hes1 and Hey1 expression to similar levels in NIH3T3 cells when compared with CLE. ii, Notch-expressing constructs increased reporter expression from the Hes5promoter(Hes5p)-luciferase construct in E14.5 cortical neural progenitors. n ¼ 3; *, P < 0.05. measured by PLAP staining was equivalent for DN1CLE, suggesting that the truncation does not affect overall Notch DN3CLE, and N3CLE (Fig. 4B). Some animals infected with activity or tumor initiation capacity in vivo. DN2CLE also showed comparable levels of PLAP in these Retinal disorganizations with an appearance and immuno- regions (Fig. 4B), but a significant percentage had less exten- phenotype identical to those induced by activated Notch3 sive staining despite injection with the same viral titers. were present in 20% of eyes injected with DN1CLE, indicating All constructs were capable of inducing the CBF1-depen- that the construct was biologically active in vivo. Both DN1CLE dent Notch targets Hes1 and Hey1 to a similar level as N3CLE and DN2CLE could also induce proliferative cataracts in the in NIH3T3 cells 3T3 cells (Fig. 4Ci), and all could also induce a lens, but in contrast to N3CLE and DN3CLE they were not able Hes5p-luciferase Notch reporter construct in E14.5 mouse to generate large tumors in the eye. The most pronounced cortical neural progenitors (Fig. 4Cii). We have also previously differences following Notch activation were in the choroid, as shown similar effects for the activated Notch1 and Notch3 animals injected with DN1CLE, and DN2CLE never developed constructs in neural progenitors in the developing brain (21, tumors in this tissue. Glial lesions were also decidedly less 22). Importantly, DN3CLE-injected animals developed retinal common in the DN1CLE-injected animals. Only very small, lesions, cataracts, and glial tumors with a similar incidence as noninvasive retinal, and optic nerve glial proliferations were those observed in the N3CLE-injected animals (Table 1), identified, and these were limited to 2% to 4% of the eyes

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examined (Table 1). The generation of small glial prolifera- Apart from ocular glial tumors, we did not identify glial tions by DN1CLE is consistent with a previous study showing tumors of the brain in any of the animals injected with active that active Notch1 signaling promotes a glial cell fate in Notch1, Notch2, or Notch3. This implies that glial precursors the developing retina, but failed to induce tumor formation in the optic nerve and retina have different tumorigenic within a 60-day postnatal period (28). DN2CLE caused the potentials than those in the brain. To address whether this formation of cataracts, retinal disorganizations, and glial was due to clear differences in Notch pathway induction in the lesions in only 1% to 4% of the 113 eyes examined, but this different tissues, we isolated neural progenitors from the optic may be due in part to the lesser extent of cells showing signs of nerve and cortex of newborn rat pups. Analysis of a variety of viral infection. direct Notch pathway targets following infection with the CLE, DN1CLE, DN2CLE, DN3CLE, and N3CLE viruses revealed that Analysis of receptor chimeras indicates a role for signaling was induced in cells derived from both sites, but Notch3 TAD in glial tumor formation highlighted greater induction of Hes5 and Hey2 in the cortex To address the mechanism underlying the dramatic phe- as compared with the optic nerve (Fig. 6D). notypic differences between the various Notch receptors, we generated a series of chimeras between NICD1 and NICD3. Discussion In brief, either the Notch3 TAD alone, or the Notch3 TAD with varying numbers of Ank were used to replace those of In this study, we show that the introduction of activated Notch1(Fig.4A).Interestingly, introducing the Notch3 TAD Notch3 into developing ocular tissues induced a number of alone into NICD1 was sufficient to greatly enhance the aggressive neoplasms, including invasive pigmented tumors of number of glial lesions identified in vivo, suggesting that the choroid of uncertain classification, and invasive gliomas this region plays a key role in the differences between the 2 arising from the optic nerve and retina. The neoplastic phe- receptors (Table 1). It was also notable that none of our notypes we observed in the eye are consistent with the known chimeric constructs were able to efficiently induce choroidal roles of Notch in ocular development. Notch2 and Notch3, and tumors. The only part of Notch3 lacking in all of these was other pathway members, are expressed in the developing lens the RAM domain, which mediates binding interactions (29, 38). In the retina, Notch promotes progenitor cell char- between NICD and CBF1, thus this region may play an acter (30, 39), but it can also drive gliogenesis (40, 41). In both important role in the induction of this tumor type (Table 1). rodents and humans, astrocytic progenitor cells migrate dur- As in the experiments above, infection of the optic nerve, ing fetal and early postnatal life from the optic nerve into the retina, and forebrain, as visualized using the PLAP marker, retina and extend outward, forming a glial scaffold on which was similar between chimeric constructs, suggesting that the retinal vasculature develops (42, 43). It has previously been alterations in the number of infected clones did not account for the observed differences in phenotype (Fig.5A).Additionally,thechimericconstructswerecap- A able of activating the Hes5p- and CBF1RE-luciferase constructs in NIH3T3 cells, indicating these constructs were functionally capable of activating canonical Notch signaling (Fig. 5B).

Notch activation does not drive glial tumor formation outside the optic nerve and retina We have previously shown that DN1CLE and N3CLE can promote astroglial fates in the postnatal forebrain (21, 22). Consistent with these prior observations, we found that the brains of DN1CLE-, DN2CLE-, DN3CLE-, and N3CLE-injected B

animals contained numerous PLAP-positive cells with an 20 30 astrocytic morphology that were S100-immunopositive 20 (Fig. 6A and B). Although some variation in the number of 10 PLAP-positive cells was seen from animal to animal, the 10 D D overall density of infected cells in the N1CLE-, N2CLE-, 0 0

DN3CLE-, and N3CLE-injected cohorts was similar, although change fold Luciferase change fold Luciferase the affected region in DN2CLE-injected animals seemed more restricted to the ventral forebrain. As observed previously in N3CLE-injected animals, DN2CLE was able to induce choroid plexus hyperplasias or papillomas in 23 of 67 animals exam- Figure 5. NICD1/NICD3 chimeras infect the optic nerve, retina, and ined, and DN1CLE in 5 of 7 (Fig. 6C). The induction of forebrain. A, PLAP staining of the optic nerve and forebrain of animals D injected with the chimeric viruses (5, 2.5, respectively). B, chimeric papillomas by N1CLE contrasts with our prior work, likely constructs increased reporter expression from the Hes5p- or CBF1RE- due to the use of the human receptor in this study as luciferase constructs in NIH3T3 cells. DN1333 increased CBF1RE, but not compared with the murine receptor previously (37). Hes5p, reporter expression. n ¼ 3; *, P < 0.05.

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B C

Figure 6. DN1CLE, DN2CLE, DN3CLE, and N3CLE do not induce glial tumors in the brain. A, Notch-expressing viruses were capable of infecting the developing forebrain (PLAP stain). B, the Notch-expressing viruses promote an S100b-positive astroglial fate in the adult forebrain. C, the Notch-expressing and NICD1/NICD3 chimeric-injected animals develop choroid plexus papillomas. Original magnifications: 2.5 (A), 40 (B and C). D, fold change in Hes1, Hes5, Hey1, and Hey2 expression in rat cortical and optic nerve cultures infected with Notch-expressing viruses. Gray shaded boxes indicate a 2-fold or higher change as compared with CLE. shown that Hedgehog signaling promotes the proliferation of domains (36, 46). The TAD is associated with different post- optic nerve astrocytes (44), and our findings suggest that translational modifications of the Notch receptors that may Notch may also play a role in this process and in the malignant allow NICD to interact with other signaling pathways (47, 48). transformation of astrocytes in these tissues. Early reports suggested that Notch3 lacks a TAD (46, 49) and A second significant finding in our study is that different that Notch1 is a much stronger activator of downstream Notch receptors do not have equivalent abilities to induce signaling than Notch3 (46). More recent studies, however, tumors in the optic nerve and eye. Only activated Notch3 was have indicated that the spacing and orientation of binding able to efficiently drive the formation of choroidal tumors and elements in target promoters mediates some of these differ- invasive glial lesions, whereas signaling by Notch1 and Notch2 ences, and that the Notch3 TAD can efficiently activate the generated no choroidal tumors and only very small glial Hes5 promoter (19). Therefore, differences in Notch1 and lesions that did not have the capacity for diffuse invasion. Notch3 TAD structure and promoter binding element require- These data are consistent with prior studies in which activa- ments may confer their differential tumorigenic potentials. tion of Notch1 in the retina altered cell fates but failed to result In addition to TAD, the RAM domain of Notch3 was also in tumor formation (28, 30, 40, 41). Less is known about the identified as a key region in the formation of choroidal roles of Notch2 and Notch3 in the developing eye and brain, tumors. The RAM domain mediates the binding of NICD although it seems in loss of function studies that Notch1 and to CBF1 (RBPjk in mice) and brings the Ank repeats close to Notch3 may play distinct roles in rod and cone differentiation their CBF1 binding sites. These protein–protein interactions (45). are thought to recruit coactivator proteins, such as Mas- To further investigate the molecular basis of the differences termind (50), and displace corepressor proteins at the CBF1- in tumor induction between Notch receptors, we generated transcriptional complex (51). Notch1 and Notch3 share low and analyzed a series of chimeras using the intracellular similarity in this region (41% amino acid identity; refs. 36, 46) domains of Notch1 and Notch3. These studies showed that and differences in the RAM domain may selectively mod- replacement of the Notch1 TAD with that of Notch3 is ulate Notch activity. sufficient to dramatically increase the formation of invasive Our final major finding is that glioma induction by Notch glial tumors in vivo, and suggests that this domain plays a key is dependent on the spatial location of neural stem/progenitor role in specifying their disparate effects. Indeed, the largest cells in which the pathway is activated. Despite roughly difference between Notch1 and Notch3 is in the C terminus equivalent levels of viral infection in the optic nerve, retina, (21% amino acid identity), a region containing the TAD and and brain, we only detected invasive gliomas in the first 2 PEST (Proline, Glutamine, Serine, Threonine rich motif) structures. This was not due to a lack of virally infected

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glial precursors outside the optic nerve, as numerous PLAP- ability to induce tumor formation in the brain, and suggest a positive, S100b-positive astrocytic cells were present in brains role for the TAD in mediating these differences. Because injected with all 4 Notch-expressing viruses. In addition, no selective targeting of individual Notch receptors in tumors glioma induction in the brain was observed following intro- might ameliorate treatment side effects, a better understand- duction of NICD1 and NICD3 in earlier studies (21, 22, 52). ing of which receptor paralogs play critical roles in the Interestingly, Chambers and colleagues (53) have also shown initiation and growth of various cancer types is of clinical that mammalian forebrain precursors have a variable significance. response to Notch1 activation depending on spatial and temporal context. Disclosure of Potential Conflicts of Interest These data raise the question of why glial precursors in the optic nerve and retina have a selective competence to form No potential conflicts of interest were disclosed. Notch3-induced tumors as compared with those in the brain. We examined the ability of the various Notch receptors to Acknowledgments induce canonical downstream targets in optic nerve and brain-derived cultures, but did not find a lesser degree of We thank Dr. Masoud Aghsaei-Fard and Dr. Katayoon Baradaran Ebrahimi activation in the brain-derived cells explaining their lack of for their help in isolating and characterizing rat neural progenitors and glia. neoplastic response (Fig. 6D). In summary, our data expand the spectrum of solid cancers Grant Support in which Notch activation alone is sufficient to induce tumor- This work was supported by RO1 NS055089 (to C.G. Eberhart), Research to igenesis in vivo. They also support the concept that the same Prevent Blindness (to C.G. Eberhart), and RO1 NS046731 (to N. Gaiano). The costs of publication of this article were defrayed in part by the payment types of tumor in different locations along the neuroaxis might of page charges. This article must therefore be hereby marked advertisement in have distinct biologies due to inherently different properties of accordance with 18 U.S.C. Section 1734 solely to indicate this fact. the cells of origin, a hypothesis recently advanced by a number – of other groups (54 56). Finally, they indicate that the recep- Received February 26, 2010; revised October 29, 2010; accepted November 24, tors Notch1, Notch2, and Notch3 are not equivalent in their 2010; published OnlineFirst January 18, 2011.

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Notch3 Activation Promotes Invasive Glioma Formation in a Tissue Site-Specific Manner

Tarran J. Pierfelice, Karisa C. Schreck, Louis Dang, et al.

Cancer Res 2011;71:1115-1125. Published OnlineFirst January 18, 2011.

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