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Tumor and Stem Cell Biology

Notch3 Activation Promotes Invasive Glioma Formationin a Tissue Site-Specific Manner

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

AbstractAlthough 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, andchoroid plexus cells. To examine tumorigenic potential in the eye and brain, we injected retroviruses encodingactivated forms of Notch1, Notch2, or Notch3 into embryonic mice. Interestingly, the majority of animalsinfected with active Notch3 developed proliferative lesions comprised of pigmented ocular choroid cells, retinaland optic nerve glia, and lens epithelium. Notch3-induced lesions in the choroid, retina, and optic nerve werecapable of invading adjacent tissues, suggesting that they were malignant tumors. Although Notch3 activationinduced 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 opticnerve, whereas Notch3 was 10-fold more efficient at generating growths, many of which were large invasivegliomas. Expression of active Notch1/Notch3 chimeric receptors implicated the RBPjk-association molecule andtransactivation domains of Notch3 in generating choroidal and glial tumors, respectively. In contrast to ourfindings in the optic nerve and retina, introduction of active Notch receptors, including Notch3, into the brainnever caused glial tumors. Our results highlight the differential ability of Notch receptor paralogs to initiatemalignant tumor formation, and suggest that glial precursors of the optic nerve, but not the brain, aresusceptible to transformation by Notch3. Cancer Res; 71(3); 1115–25. �2011 AACR.

Introduction

Notch signaling plays a critical role in the specification,proliferation, and survival of stem/progenitor cells in a num-ber of tissues, including the central and peripheral nervoussystems (1). The pathway is also widely implicated in neopla-sia, and in most contexts promotes neoplastic growth (2), butcan act as a tumor suppressor in some cell types (3–5). Notchis activated in a broad range of hematopoietic and solidtumors (2–4, 6–12). Its role in leukemogenesis is probably

best described, as activation of the pathway can promotemurine T-ALL, similar to that seen in humans (8, 11, 13, 14).

Notch is thought to play a particularly important role inpoorly differentiated tumor cells, where pathway inhibitionmay be able to deplete "cancer stem cells," which are resistantto radiation and standard chemotherapies (1, 5, 7, 9, 10, 12, 15–18). Small molecules targeting Notch have shown great pro-mise in preclinical testing of several tumor models. On thebasis of such studies, phase I clinical trials for leukemia andbreast cancer have been initiated using gamma-secretaseinhibitors 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 firstmore fully understand the complexities of Notch signaling incancer, as its effects are context-dependent (5, 16).

The role(s) of the various Notch receptors in tumor initia-tion in vivo are particularly poorly understood. Though struc-turally similar, the 4 mammalian Notch paralogs (Notch1–4)are not functionally equivalent in all contexts. The mechan-istic basis of such differences is still being uncovered, but it hasbeen shown that the various Notch receptors can relateuniquely to binding site distribution and orientation on targetgene promoters (19).

In this study, we examined potential functional distinctionsbetween the Notch paralogs by introducing activated Notchreceptors into the brain and eye, and we identified prolifera-tive lesions in the retina, optic nerve, and lens. Importantly, we

Authors' Affiliations : 1Institute for Cell Engineering, and Departments of2Neuroscience, 3Pathology, 4Neurology, 5Oncology, and 6Ophthalmology,Johns Hopkins University School of Medicine, Baltimore, Maryland; and7Department of Pediatrics and Communicable Diseases, University ofMichigan, Ann Arbor, Michigan

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

K.C. Schreck and L. Dang contributed equally to this work.

N. Gaiano and C.G. Eberhart are the co-senior authors.

Corresponding Author: Charles G. Eberhart, Johns Hopkins UniversitySchool of Medicine, Ross Building 558, 720 Rutland Ave., Baltimore, MD21205. Phone: 410-502-5185; Fax: 410-955-9777.E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-0690

�2011 American Association for Cancer Research.

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found that active forms of Notch1, Notch2, and Notch3 havediffering abilities to induce tumors, and that glial progenitorsin the optic nerve and retina are particularly susceptible toNotch3-driven transformation as compared with those in therest of the central nervous system. These results highlight thesometimes distinct capacities of various Notch receptors toinduce tumor formation and the context-dependence of glialprogenitor transformation.

Materials and Methods

NICD1/NICD3 chimeric constructsThe NICD1/NICD3 chimeric human proteins were

expressed using the retroviral vector pCLE (20).The chimericcDNAs were made using standard PCR and ligation protocols,and were confirmed by DNA sequencing. Briefly, we amplified3 different segments of Notch1 intracellular domain (NICD1)and 3 of NICD3, each product varying in length to includedifferent regions. For NICD1 these were called DN1xxx,DN11xx, and DN111x, and included the RBPjk-associationmolecule (RAM) domain and adjacent downstream residues,RAM, RAM/Ank1–2, and RAM/Ank1–5, respectively (Ank,ankyrin repeat). For NICD3, these were called DNxxx3,DNxx33, DNx333, which included TADD (a proximal portionof the transactivation domain), Ank3–5/TADD, Ank1–5/TADD, respectively. We already possessed the humanDN1111 and DN3333 constructs in the retroviral vector pCLE(DN1CLE and DN3CLE; ref. 21). All primers were designed toinclude endogenous restriction enzyme sites, or sites createdby silent point mutations, to permit generation of chimericconstructs by ligation (e.g., DN11xx was ligated to DNxx33).DN1113 encodes residues 1761–2093 of Notch1 and 2006–2098of Notch3. DN1133 encodes residues 1761–1993 of Notch1 and1905–2098 of Notch3. DN1333 encodes residues 1761–1858 ofNotch1 and 1767–2098 of Notch3. See Supplementary FigureS1 for the amino acid sequence of the chimeric proteins.

Constructs and virus preparationRetroviral constructs expressing the activated forms of

Notch1–3 (human Notch intracellular domain (NICD1, NICD2,NICD3) or the NICD1/NICD3 chimeras and human alkalinephosphatase (placental alkaline phosphatase; PLAP) reporterwere generated using standard protocols (20). The DN1CLE(22), DN2CLE, DN3CLE, DN1113CLE, DN1133, andDN1333CLE-expressing viruses encode truncated NICD thatlacks a portion of the C terminus, a modification required toobtain high viral titer. DN1CLE encodes residues 1761–2196;DN2CLE encodes residues 1703–2146; and DN3CLE encodesresidues 1663–2098. The N3CLE-expressing virus encodes full-length NICD3, residues 1663–2321 (21). Virus expressingempty vector (CLE) was used as a control.

Animals and in utero viral injectionsThe CD1 (Charles River) and Black Swiss (Taconic) mice

used in this study were maintained in accordance with theInstitutional Animal Care and Use Committee at Johns Hop-kins University School of Medicine. Embryos were injected inutero at E9.5 or E10.5 with virus using ultrasound guidance as

described previously (20). All viruses used for injection hadtiters of 1� 108 to 4� 108. The animals were sacrificed at 30 to60 days of age, and ocular and brain tissues were harvested.

Staining of tissue sections and analysisEye and brain tissue obtained from virally injected mice was

fixed in 4% paraformaldehyde for frozen sections or formalinfor paraffin-embedded sections. Sectioning, PLAP staining(22), hematoxylin and eosin (H&E) staining, and immunohis-tochemistry were carried out using standard methods. Weblindly scored multiple H&E stained adult eye sections fromvirally injected animals. Immunohistochemistry on paraffinsections was carried out using the Vectastain ABC kit andDAB Peroxidase Substrate kit (Vector Laboratories) as speci-fied by the manufacturer's protocol. Primary antibodies usedwere a-Notch3 (rabbit, 1:1,000; Santa Cruz Biotechnology),a-Pax6 (mouse, 1:400, Millipore Corporation), a-Chx10 (sheep,1:100, Exalpha Biologicals), a-GFAP (rabbit, 1:1,000; DAKO),a-Nestin (chicken, 1:250; Aves Labs, Inc.), and Hes5 (rabbit,Gaiano Laboratory, only recognizes overexpression). AlexaFluor Dye conjugated secondary antibodies used wereobtained from Invitrogen.

Luciferase assayLuciferase assays were conducted on E14.5 mouse cortical

neural progenitors or NIH3T3 cells transfected with theHes5promoter (Hes5p)- or CBF1RE [4xCBF1 (C promoter-binding factor 1)-responsive elements]–luciferase constructsalong with one of the Notch-expressing constructs, accordingto the manufacturer's protocol (Lonza). The Hes5 promoterwas a gift from Toshiyuki Ohtsuka at Kyoto University and theCBF1RE-luciferase construct was a gift fromDiane Hayward atJohns Hopkins University. Cells were harvested 48 hours aftertransfection and luciferase activity was measured (Promega).Each sample was normalized to beta-galactosidase expression(Clontech).

Preparation of rat astrocyte cell culturesThe cortex and optic nerves were isolated from postnatal

day 2 Sprague-Dawley rats as described previously (23, 24).The optic nerve and retinal cultures were 90% glial fibrillaryacidic protein (GFAP)–immunopositive, and the cortical cul-tures were 60% GFAP-immunopositive (data not shown).Murine DN1CLE (22) caused the same incidence of oculartumors as DN1CLE and was used to infect these cultures.

Quantitative PCRRNA was obtained from NIH3T3 cells or rat optic nerve

and cortical astrocyte cultures infected with the Notch-expres-sing viruses and real-time polymerase chain reaction (RT-PCR)was conducted using SYBR Green reagent (Applied Biosystems)withall samples run in triplicate onan I-Cycler (Bio-Rad). Valueswere normalized to actin or GAPDH using the standard curvemethod. The following primer sequences were used. MouseHes1 Forward (F): 50-AAAGCCTATCATGGAGAAGAGGCG andHes1 Reverse (R): 50-GGAATGCCGGGAGCTATCTTTCTT-30;mouse Hey1F: 50- ACGACATCGTCCCAGGTTTTG-30 andHey1R: 50-GGTGATCCACAGTCATCTGCAAG-30; mouse

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GAPDHF: 50-AAACCCATCACCATCTTCCA-30 and GAPDHR: 50-AGGGGCCATCCACAGTCTTC-30; human PLAP (23); rat Hes1,Hes5, Hey1, and Hey2 (24).

Results

Constitutive Notch3 signaling generates retinal lesionsthat express retinal progenitor markersAt E9.5 to E10.5 the developing forebrain and optic cup form

a continuous open structure, allowing viral infection of anlagegiving rise to the retina (Fig. 1A). PLAP staining of the opticcup neuroepithelium in animals sacrificed 6 days post viralinjection confirmed infection of these cells (Fig. 1Bi, arrow).Animals injected with N3CLE retrovirus encoding constitu-tively active Notch3 developed retinal lesions in 62 of 94 adulteyes (66%) examined (Table 1). N3CLE-induced retinal lesionswere focal and varied in size. Some disrupted only the innerretinal layer (Fig. 1Bii) whereas others affected all 3 retinallayers (Fig. 1Biii). The retinal lesions coincided with PLAPstaining and nuclear Notch3 immunoreactivity (Fig. 1Biv andv) indicating that they were induced by Notch3 activity.To identify the cell types contained within the N3CLE-

induced retinal lesions, we used immunofluorescence todetect various markers of retinal differentiation. The disorga-nized regions contained some cells expressing the glial markerglutamine synthetase (GS) and the early neuronal marker BIII-tubulin (Fig. 1Biv and v). Interestingly, within the lesions wealso identified numerous cells-expressing Pax6 or Chx10(Fig. 1C), and a subpopulation coexpressing both of thesemarkers (Fig. 1C, arrowheads). Occasional cells were Ki67-positive, indicating a degree of proliferation. However, thelesions did not have microscopic features of a retinocytoma ora retinoblastoma. Previous studies have shown that retinalstem/progenitor cells coexpress Pax6 and Chx10 during earlyeye development; their expression later segregates andbecomes cell specific (25–27). Pax6/Chx10 coexpressionwithin the retinal lesions therefore suggests the presence ofstem/progenitor-like cells in which Notch pathway deregula-tion has inhibited normal differentiation. Prior reports haveshown that Notch1 activity promotes progenitor cell characterin the developing retina (28–30), and our findings suggest thatNotch3 can play a similar role.

Constitutive Notch3 signaling causes overproliferationof lens epitheliumMany mice injected at E9.5 or E10.5 with N3CLE developed

white, cataractous eyes by 1 month of age (Fig. 1C). Lensformation is initiated early in fetal development and at E10.5the ectodermal lens placode is still located on the surface ofthe embryo (31). In the normal mature lens, the only remain-ing cells are an anterior lens epithelium (ALE) monolayerbelow the capsule. On microscopic examination, we observedthat 22 of 94 eyes (23%) from animals injected with N3CLEshowed overproliferation of lens epithelium with adjacentdeposition of capsular material (Fig. 1Dii; Table 1). In some,almost all of the epithelium was multilayered, whereas inothers normal-looking epithelium (Fig. 1Dii, arrowhead) waspresent next to regions 10 or more cells thick (Fig. 1Dii, arrow).

In contrast, injection of control CLE virus was never asso-ciated with gross or microscopic lens abnormalities. This wasan ongoing proliferative process, as evidenced by the presenceof mitotic figures and Ki67-positive cells (Fig. 1Dii inset andiii), with 11% of cells expressing this proliferative marker in thedepicted cataract. The thickened epithelial layers stainedpositive for PLAP (Fig. 1Diii), indicating that viral infectionwas directly associated with the hyperproliferative lesions.Another group recently reported that active Notch1 signalingalso induced ALE hyperproliferation (32). Our N3CLE-inducedlesions seem very similar to human anterior subcapsularcataracts (ASC), which are also characterized by a multi-layered lens epithelium and abnormal deposition of capsularmaterial (33).

Pigmented choroidal tumors are generated by Notch3signaling

N3CLE-injected animals also developed invasive tumorsarising in the choroidal layer of the eye, a loose fibrovasculartissue containing scattered melanocytes. The invasive chor-oidal neoplasms, comprised of oval and spindled cells, werepresent in 63 of the 94 eyes (67%) examined in N3CLE-injectedanimals (Table 1). Such tumors were never observed in controlCLE-injected eyes (n ¼ 22). Tumors were identified in allregions of the choroid, from the periphery (Fig. 2A, arrow) tothe posterior pole, and some eyes contained multiple separatelesions in various regions. The sheets and clusters of tumorcells were highly proliferative, as evidenced by the presence ofnumerous mitotic figures and Ki67-positive cells (Fig. 2Biii).PLAP staining (Fig. 2A and Biii) and nuclear Notch3 immu-nopositivity (Fig. 2Bii) indicated that the choroidal lesionsarose from cells from N3CLE infection with increased Notchactivity. Many of the larger choroidal tumors invaded throughtransscleral canals into the orbital space (Fig. 2Bi). A fewtumors extensively infiltrated the periocular tissues and weregrossly apparent on external examination as exophytic lesionsaround the eye. The fact that not all eyes injected with N3CLEdeveloped these neoplasms suggests that additional geneticalterations may be needed to promote tumorigenesis,although limited sampling may have also affected the numberof lesions detected.

The precise pathological classification of these neoplasms isnot clear. They did not seem embryonal, and were notimmunopositive for Nestin, synaptophysin, Tuj1, GS, PAX6,or GFAP, suggesting that they were not neuronal or glial. Inhumans, uveal melanoma is the most common malignantneoplasm of the choroid. A subset of injections was performedinto the pigmented Swiss Black mouse line, and choroidaltumors in these animals always contained pigmented cells(Fig. 2Bi). The tumors clearly arose from choroid rather thanretinal pigment epithelium, as the latter pigmented cell layerwas intact in all of the smaller lesions examined (Fig. 2Bi,arrow and data not shown). Choroidal melanocytes derivefrom neural crest, a tissue that would be at least partiallyexposed to virus at the time of injection (34). The pigmenta-tion we observe suggests that these may represent murinemelanomas arising from choroid, and the oval to spindledmorphology of the tumor cells is similar to that seen in human

Notch3 Promotes Glioma

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A

B

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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 ofinjection, 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 arereleased 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 aPLAP-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) andcontained mitotic bodies in the N3CLE-induced ASCs (inset). iii, areas of hyperproliferation were PLAP positive and contained Ki67-positive cells (DAPIcounterstain). Original magnifications: �20 (Bi, Biv, and C), �40 (Bv and Dii), �200 (Bii, Biii, and Di).

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melanomas at this site. However, we were unable to verifymelanocytic differentiation in the lesions using several immu-nohistochemical markers (data not shown). The multilayeredconcentric growth of the tumors in focal regions also showssome similarity to myopericytoma, although in humans suchtumors are benign and are not known to occur in the eye.Thus, the classification, grade, and relationship of thesechoroidal tumors to a specific type of human neoplasm isunclear at this point.

Notch3 signaling induces glial tumors in the retina andoptic nerveHistological examination revealed that 23 of 94 eyes (24%)

from the N3CLE-injected animals developed GFAP-positiveglial lesions in the retina (Table 1; Fig. 2C and D). Some ofthese were relatively small (Fig. 2C), but most extended alonglarge portions of the retina, and these often invaded throughthe retinal pigment epithelium and into the choroid (Fig. 2Di).In 2 cases, retinal glial tumors invaded through scleral canals

and into the orbit. Because of this highly invasive behavior andthe presence of scattered mitotic figures, we believe these areglial neoplasms rather than reactive gliotic lesions.

Glial tumors of similar appearance were also identified inthe optic nerves of 20 of 94 N3CLE-injected animals (21%;Table 1). These tumors were only moderately cellular, withabundant cytoplasm and some elongated cytoplasmic pro-cesses (Fig. 3A). Occasional mitotic figures and Ki67-positivecells were present (Fig. 3Aii inset and Ci). In some tumors,neoplastic cells were localized under the meninges and focallydistended the coverings of the optic nerve (Fig. 3A, arrow-heads); in others, the optic nerve was more diffusely infiltratedby tumor. In 16 of the 20 eyes with optic nerve tumors (80%),the neoplasm invaded into the soft tissues surrounding thenerve. This most commonly occurred in the region of the opticdisk, but in some optic nerves greatly distended by tumor,strands of neoplastic cells exited through more posteriorchannels penetrating the nerve sheath (Fig. 3Bi). Other gliallesions involved both the optic nerve and retina. In total, weidentified invasive glial tumors in the orbital tissues of 47 of 94

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

Virus injected Numberof eyes

Occurrence (%)

Choroidaltumors

Retinaldisorganizations

Retinalglial lesions

Episcleralglial lesions

Optic nerve–associatedglial lesions

Proliferativecataracts

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

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

A B

C D

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






eyes injected with N3CLE (50%; Table 1). Although many ofthese were clearly associated with retinal or optic nervegliomas, others were not contiguous with these neural struc-tures in the sections examined. We believe that most or all ofthese were associated with optic nerve or retinal gliomas thatwere not identified in limited tissue examined microscopi-cally, but some could be distinct lesions arising from orbitalneural tissues.

As was true for the choroidal tumors induced by NICD3,both PLAP and nuclear Notch3 (Fig. 3Ci and D) were identifiedin all optic nerve and retinal tumors examined, and no gliallesions were identified in animals injected with control CLEvirus. We believe these tumors are astrocytic because theystrongly express GFAP when examined using immunohisto-chemistry (Figs. 2Dii, 3Bi and D), and frequently containedelongated cellular processes similar to those in human astro-cytoma. Nestin, an intermediate filament often expressed inneural progenitor cells and glial tumors, was diffusely presentin the proliferative lesions (Figs. 3Cii and D). The Nestin- andGFAP-positive cells within the glial lesions also coexpressednuclear Notch3 and the downstream target Hes5 (Fig. 3Cii andD), and PLAP (not shown) on immunofluorescent analyses.

The choroidal tumors, which were sometimes encountered inthe same eyes as invasive gliomas, were negative for GFAP andNestin (data not shown).

Notch1 and Notch2 activation does not efficientlygenerate ocular tumors

The mammalian genome contains 4 Notch receptors, 3 ofwhich (Notch1–3) are very similar in sequence, althoughNotch1 and Notch2 are more similar to each other than toNotch3 (35, 36). Despite their high level of identity, the effectsof the various Notch receptors are not always the same, and insome contexts they can even have opposing effects (4). Wetherefore sought to determine whether introduction of eitheractivated Notch1 or Notch2 would produce proliferative ocu-lar lesions similar to those described above.

To generate high-titer virus expressing constitutively activeNotch1 and Notch2, we found that we needed to truncate aportion of the NICD C terminus. Because N3CLE expressesfull-length NICD3, we also generated a shortened NICD3-expressing virus (DN3CLE) that contained a comparable C-terminal truncation (Fig. 4A). The resulting viruses all infectedthe retina and optic nerve. The overall extent of infection as

A

B

C D

Figure 3. N3CLE induced formation of glial tumors along the optic nerve. A, glial tumors (arrowheads) formed along the optic nerve (arrow) inN3CLE-injected animals and contained mitotic bodies (ii, inset). B, the optic nerve tumors expressed GFAP and Notch3 and invaded orbital tissuessurrounding the optic nerve. Some glial tumors involved both the retina (arrowhead) and optic nerve (arrow). C, i, glial tumors contained Ki67-positiveproliferating cells. Nestin-positive and Nestin/GFAP-positive cells within the lesions coexpressing Hes5 or Notch3, respectively (Cii and D). Originalmagnifications: �63 (C and D), �100 (Ai and Bi), �200 (Ai, Aii, and Bii), �400 (Aii, inset).

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measured by PLAP staining was equivalent for DN1CLE,DN3CLE, and N3CLE (Fig. 4B). Some animals infected withDN2CLE also showed comparable levels of PLAP in theseregions (Fig. 4B), but a significant percentage had less exten-sive staining despite injection with the same viral titers.All constructs were capable of inducing the CBF1-depen-

dent Notch targets Hes1 and Hey1 to a similar level as N3CLEin NIH3T3 cells 3T3 cells (Fig. 4Ci), and all could also induce aHes5p-luciferase Notch reporter construct in E14.5 mousecortical neural progenitors (Fig. 4Cii). We have also previouslyshown similar effects for the activated Notch1 and Notch3constructs in neural progenitors in the developing brain (21,22). Importantly, DN3CLE-injected animals developed retinallesions, cataracts, and glial tumors with a similar incidence asthose observed in the N3CLE-injected animals (Table 1),

suggesting that the truncation does not affect overall Notchactivity or tumor initiation capacity in vivo.

Retinal disorganizations with an appearance and immuno-phenotype identical to those induced by activated Notch3were present in 20% of eyes injected with DN1CLE, indicatingthat the construct was biologically active in vivo. Both DN1CLEand DN2CLE could also induce proliferative cataracts in thelens, but in contrast to N3CLE and DN3CLE they were not ableto generate large tumors in the eye. The most pronounceddifferences following Notch activation were in the choroid, asanimals injected with DN1CLE, and DN2CLE never developedtumors in this tissue. Glial lesions were also decidedly lesscommon in the DN1CLE-injected animals. Only very small,noninvasive retinal, and optic nerve glial proliferations wereidentified, and these were limited to 2% to 4% of the eyes

A C

B

Figure 4. The Notch-expressing retroviruses diffusely infect the optic nerve. A, the NICD1–3 and chimeric protein constructs. B, PLAP staining ofthe optic nerve of virally infected animals (5� magnification). C, i, RT-PCR showed that the Notch-expressing constructs increased Hes1 and Hey1expression to similar levels in NIH3T3 cells when compared with CLE. ii, Notch-expressing constructs increased reporter expression from theHes5promoter(Hes5p)-luciferase construct in E14.5 cortical neural progenitors. n ¼ 3; *, P < 0.05.






examined (Table 1). The generation of small glial prolifera-tions by DN1CLE is consistent with a previous study showingthat active Notch1 signaling promotes a glial cell fate inthe developing retina, but failed to induce tumor formationwithin a 60-day postnatal period (28). DN2CLE caused theformation of cataracts, retinal disorganizations, and gliallesions in only 1% to 4% of the 113 eyes examined, but thismay be due in part to the lesser extent of cells showing signs ofviral infection.

Analysis of receptor chimeras indicates a role forNotch3 TAD in glial tumor formation

To address the mechanism underlying the dramatic phe-notypic differences between the various Notch receptors, wegenerated a series of chimeras between NICD1 and NICD3.In brief, either the Notch3 TAD alone, or the Notch3 TADwith varying numbers of Ank were used to replace those ofNotch1 (Fig. 4A). Interestingly, introducing the Notch3 TADalone into NICD1 was sufficient to greatly enhance thenumber of glial lesions identified in vivo, suggesting thatthis region plays a key role in the differences between the 2receptors (Table 1). It was also notable that none of ourchimeric constructs were able to efficiently induce choroidaltumors. The only part of Notch3 lacking in all of these wasthe RAM domain, which mediates binding interactionsbetween NICD and CBF1, thus this region may play animportant role in the induction of this tumor type (Table 1).As in the experiments above, infection of the optic nerve,retina, and forebrain, as visualized using the PLAP marker,was similar between chimeric constructs, suggesting thatalterations in the number of infected clones did notaccount for the observed differences in phenotype(Fig. 5A). Additionally, the chimeric constructs were cap-able of activating the Hes5p- and CBF1RE-luciferase con-structs in NIH3T3 cells, indicating these constructs werefunctionally capable of activating canonical Notch signal-ing (Fig. 5B).

Notch activation does not drive glial tumor formationoutside the optic nerve and retina

We have previously shown that DN1CLE and N3CLE canpromote astroglial fates in the postnatal forebrain (21, 22).Consistent with these prior observations, we found that thebrains of DN1CLE-, DN2CLE-, DN3CLE-, and N3CLE-injectedanimals contained numerous PLAP-positive cells with anastrocytic morphology that were S100-immunopositive(Fig. 6A and B). Although some variation in the number ofPLAP-positive cells was seen from animal to animal, theoverall density of infected cells in the DN1CLE-, DN2CLE-,DN3CLE-, and N3CLE-injected cohorts was similar, althoughthe affected region in DN2CLE-injected animals seemed morerestricted to the ventral forebrain. As observed previously inN3CLE-injected animals, DN2CLE was able to induce choroidplexus hyperplasias or papillomas in 23 of 67 animals exam-ined, and DN1CLE in 5 of 7 (Fig. 6C). The induction ofpapillomas by DN1CLE contrasts with our prior work, likelydue to the use of the human receptor in this study ascompared with the murine receptor previously (37).

Apart from ocular glial tumors, we did not identify glialtumors of the brain in any of the animals injected with activeNotch1, Notch2, or Notch3. This implies that glial precursorsin the optic nerve and retina have different tumorigenicpotentials than those in the brain. To address whether thiswas due to clear differences in Notch pathway induction in thedifferent tissues, we isolated neural progenitors from the opticnerve and cortex of newborn rat pups. Analysis of a variety ofdirect Notch pathway targets following infection with the CLE,DN1CLE, DN2CLE, DN3CLE, and N3CLE viruses revealed thatsignaling was induced in cells derived from both sites, buthighlighted greater induction of Hes5 and Hey2 in the cortexas compared with the optic nerve (Fig. 6D).

Discussion

In this study, we show that the introduction of activatedNotch3 into developing ocular tissues induced a number ofaggressive neoplasms, including invasive pigmented tumors ofthe choroid of uncertain classification, and invasive gliomasarising from the optic nerve and retina. The neoplastic phe-notypes we observed in the eye are consistent with the knownroles of Notch in ocular development. Notch2 and Notch3, andother pathway members, are expressed in the developing lens(29, 38). In the retina, Notch promotes progenitor cell char-acter (30, 39), but it can also drive gliogenesis (40, 41). In bothrodents and humans, astrocytic progenitor cells migrate dur-ing fetal and early postnatal life from the optic nerve into theretina and extend outward, forming a glial scaffold on whichthe retinal vasculature develops (42, 43). It has previously been

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fera

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Figure 5. NICD1/NICD3 chimeras infect the optic nerve, retina, andforebrain. A, PLAP staining of the optic nerve and forebrain of animalsinjected with the chimeric viruses (�5, �2.5, respectively). B, chimericconstructs increased reporter expression from the Hes5p- or CBF1RE-luciferase constructs in NIH3T3 cells. DN1333 increased CBF1RE, but notHes5p, reporter expression. n ¼ 3; *, P < 0.05.

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shown that Hedgehog signaling promotes the proliferation ofoptic nerve astrocytes (44), and our findings suggest thatNotchmay also play a role in this process and in the malignanttransformation of astrocytes in these tissues.A second significant finding in our study is that different

Notch receptors do not have equivalent abilities to inducetumors in the optic nerve and eye. Only activated Notch3 wasable to efficiently drive the formation of choroidal tumors andinvasive glial lesions, whereas signaling by Notch1 and Notch2generated no choroidal tumors and only very small gliallesions that did not have the capacity for diffuse invasion.These data are consistent with prior studies in which activa-tion of Notch1 in the retina altered cell fates but failed to resultin tumor formation (28, 30, 40, 41). Less is known about theroles of Notch2 and Notch3 in the developing eye and brain,although it seems in loss of function studies that Notch1 andNotch3 may play distinct roles in rod and cone differentiation(45).To further investigate the molecular basis of the differences

in tumor induction between Notch receptors, we generatedand analyzed a series of chimeras using the intracellulardomains of Notch1 and Notch3. These studies showed thatreplacement of the Notch1 TAD with that of Notch3 issufficient to dramatically increase the formation of invasiveglial tumors in vivo, and suggests that this domain plays a keyrole in specifying their disparate effects. Indeed, the largestdifference between Notch1 and Notch3 is in the C terminus(21% amino acid identity), a region containing the TAD andPEST (Proline, Glutamine, Serine, Threonine rich motif)

domains (36, 46). The TAD is associated with different post-translational modifications of the Notch receptors that mayallow NICD to interact with other signaling pathways (47, 48).Early reports suggested that Notch3 lacks a TAD (46, 49) andthat Notch1 is a much stronger activator of downstreamsignaling than Notch3 (46). More recent studies, however,have indicated that the spacing and orientation of bindingelements in target promoters mediates some of these differ-ences, and that the Notch3 TAD can efficiently activate theHes5 promoter (19). Therefore, differences in Notch1 andNotch3 TAD structure and promoter binding element require-ments may confer their differential tumorigenic potentials.

In addition to TAD, the RAM domain of Notch3 was alsoidentified as a key region in the formation of choroidaltumors. The RAM domain mediates the binding of NICDto CBF1 (RBPjk in mice) and brings the Ank repeats close totheir CBF1 binding sites. These protein–protein interactionsare thought to recruit coactivator proteins, such as Mas-termind (50), and displace corepressor proteins at the CBF1-transcriptional complex (51). Notch1 and Notch3 share lowsimilarity in this region (41% amino acid identity; refs. 36, 46)and differences in the RAM domain may selectively mod-ulate Notch activity.

Our final major finding is that glioma induction by Notchis dependent on the spatial location of neural stem/progenitorcells in which the pathway is activated. Despite roughlyequivalent levels of viral infection in the optic nerve, retina,and brain, we only detected invasive gliomas in the first 2structures. This was not due to a lack of virally infected

A

B

D

C

Figure 6. DN1CLE, DN2CLE, DN3CLE, and N3CLE do not induce glial tumors in the brain. A, Notch-expressing viruses were capable of infecting thedeveloping forebrain (PLAP stain). B, the Notch-expressing viruses promote an S100b-positive astroglial fate in the adult forebrain. C, the Notch-expressingand 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, andHey2 expression in rat cortical and optic nerve cultures infected with Notch-expressing viruses. Gray shaded boxes indicate a 2-fold or higherchange as compared with CLE.






glial precursors outside the optic nerve, as numerous PLAP-positive, S100b-positive astrocytic cells were present in brainsinjected with all 4 Notch-expressing viruses. In addition, noglioma induction in the brain was observed following intro-duction of NICD1 and NICD3 in earlier studies (21, 22, 52).Interestingly, Chambers and colleagues (53) have also shownthat mammalian forebrain precursors have a variableresponse to Notch1 activation depending on spatial andtemporal context.

These data raise the question of why glial precursors in theoptic nerve and retina have a selective competence to formNotch3-induced tumors as compared with those in the brain.We examined the ability of the various Notch receptors toinduce canonical downstream targets in optic nerve andbrain-derived cultures, but did not find a lesser degree ofactivation in the brain-derived cells explaining their lack ofneoplastic response (Fig. 6D).

In summary, our data expand the spectrum of solid cancersin which Notch activation alone is sufficient to induce tumor-igenesis in vivo. They also support the concept that the sametypes of tumor in different locations along the neuroaxis mighthave distinct biologies due to inherently different properties ofthe cells of origin, a hypothesis recently advanced by a numberof other groups (54–56). Finally, they indicate that the recep-tors Notch1, Notch2, and Notch3 are not equivalent in their

ability to induce tumor formation in the brain, and suggest arole for the TAD in mediating these differences. Becauseselective targeting of individual Notch receptors in tumorsmight ameliorate treatment side effects, a better understand-ing of which receptor paralogs play critical roles in theinitiation and growth of various cancer types is of clinicalsignificance.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. Masoud Aghsaei-Fard and Dr. Katayoon Baradaran Ebrahimifor their help in isolating and characterizing rat neural progenitors and glia.

Grant SupportThis work was supported by RO1 NS055089 (to C.G. Eberhart), Research to

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

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 26, 2010; revised October 29, 2010; accepted November 24,2010; published OnlineFirst January 18, 2011.

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2011;71:1115-1125. Published OnlineFirst January 18, 2011.Cancer Res Tarran J. Pierfelice, Karisa C. Schreck, Louis Dang, et al. Tissue Site-Specific MannerNotch3 Activation Promotes Invasive Glioma Formation in a

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