activation of neurotrophin-3 receptor trkc induces...

[CANCER RESEARCH 59, 711–719, February 1, 1999]

Activation of Neurotrophin-3 Receptor TrkC Induces Apoptosisin Medulloblastomas1

John Y. H. Kim, Mary E. Sutton, Diane J. Lu, Tracey A. Cho, Liliana C. Goumnerova, Lyuda Goritchenko,Jill R. Kaufman, K. K. Lam, Amy L. Billet, Nancy J. Tarbell, Julian Wu, Jeffrey C. Allen, Charles D. Stiles,Rosalind A. Segal, and Scott L. Pomeroy2

Division of Neuroscience, Department of Neurology [J. Y. H. K., M. E. S., D. J. L., T. A. C., J. R. K., K. K. L., S. L. P.] and Department of Neurosurgery [L. C. G., L. G.],Children’s Hospital; Departments of Neurology [R. A. S.] and Surgery [J. W.], Division of Neurosurgery, The Brain Tumor Center, Beth Israel/Deaconess Medical Center;Departments of Pediatric Hematology-Oncology [J. Y. H. K., A. L. B.] and Microbiology and Molecular Genetics [C. D. S.], Dana-Farber Cancer Institute; and Division ofRadiation Oncology, Massachusetts General Hospital [N. J. T.], Harvard Medical School, Boston, Massachusetts 02115; Department of Neurosurgery, New England MedicalCenter, Tufts School of Medicine, Boston, Massachusetts 02111 [J. W.]; and Division of Neuro-oncology, New York University Medical Center, New York, New York 10016[J. C. A.]

ABSTRACT

Elevated expression of the neurotrophin-3 (NT-3) receptor TrkC bychildhood medulloblastomas is associated with favorable clinical outcome.Here, we provide evidence that TrkC is more than simply a passivemarker of prognosis. We demonstrate that: (a) medulloblastomas undergoapoptosisin vitro when grown in the presence of NT-3; (b) overexpressionof TrkC inhibits the growth of intracerebral xenografts of a medulloblas-toma cell line in nude mice; and (c) trkC expression by individual tumorcells is highly correlated with apoptosis within primary medulloblastomabiopsy specimens. TrkC-mediated NT-3 signaling promotes apoptosis byactivating multiple parallel signaling pathways and by inducing immedi-ate-early gene expression of both c-jun and c-fos. Considered collectively,these results support the conclusion that the biological actions of TrkCactivation affect medulloblastoma outcome by inhibiting tumor growththrough the promotion of apoptosis.

INTRODUCTION

Medulloblastoma, a malignant tumor of the cerebellum, accountsfor ;20% of primary brain tumors in children (1). A high percentageof medulloblastomas express neuron progenitor markers and cerebel-lar granule cell-specific transcription factors, including PAX6, EN2,and Zic, suggesting that they arise by oncogenic transformation ofgranule cell precursors (2–8). Medulloblastomas also express neuro-trophins that regulate granule cell development, including BDNF3 andits specific receptor tyrosine kinase TrkB, as well as NT-3 and itspreferred receptor TrkC (9–12). During normal development, BDNFpromotes survival and differentiation of early postmitotic granulecells (13–16), whereas mature granule cells express increasedamounts of TrkC through which NT-3 promotes axonal maturation(16). The role of these developmentally expressed molecules inmedulloblastoma oncogenesis is not known.

Patients with medulloblastoma have a bimodal outcome. Despiteextensive treatment with surgical excision, external beam irradiationincluding the entire craniospinal axis, and multiple drug chemother-apy, their prognosis is not uniformly favorable (17–19). Sixty to 80%

of children survive 5 years after diagnosis and relapse infrequentlythereafter (20, 21), but the remainder relapse and usually die despitereceiving identical therapy. We have shown that expression of theNT-3 receptor TrkC in medulloblastomas correlates with favorableclinical outcome (12), indicating that they are possibly derived frommore fully differentiated granule cells. Here, we investigate the bio-logical actions of TrkC that may account for the improved prognosisof patients with medulloblastomas that express high levels of thereceptor.

MATERIALS AND METHODS

Patient Data and Human Tumor Bank. All patients treated for medul-loblastoma between June 1993 and September 1997 at Boston Children’sHospital and the Dana-Farber Cancer Institute were included in the study(n 5 28). In addition, 10 samples were obtained from patients at New YorkUniversity Medical School, and 4 were from the New England Medical Center.The samples were immediately snap-frozen in liquid nitrogen and stored at280°C and also transported in medium and processed for tissue culture. In allcases, the diagnosis of medulloblastoma was confirmed by pathological anal-ysis of biopsy samples. At the time of diagnosis, the patients ranged in agefrom 7 to 324 months (mean5 107 months). There were 14 females and 28males. All patients were treated with craniospinal irradiation to 2400–3600cGy with a tumor dose of 5300–7200 cGy. All but three patients were treatedwith chemotherapy consisting of cisplatin or carboplatin and combinations ofvincristine, etoposide, cyclophosphamide, or lomustine. Two patients receivedhigh-dose chemotherapy, one as primary therapy and the other at relapse,including methotrexate and thiotepa, followed by autologous bone marrowtransplantation. The median survival for the entire group was 67 months(follow-up range: 3–102 months).

Cell Culture and Transfections. To test for neurotrophin responsiveness,freshly obtained tumor biopsy samples were minced, triturated, and grown assuspensions in serum-free DMEM (Cellgro; Mediatech, Herndon, VA) con-taining high glucose (6 g/liter) and either NGF, BDNF, or NT-3 (50 ng/ml;from stock solutions containing 0.1 mg/ml BSA; neurotrophins courtesy ofAndrew Welcher, Amgen, Thousand Oaks, CA) or mock-stimulated control(0.1 mg/ml BSA) at 37°C in 5% CO2. Suspension cultures were stimulated forup to 48 h, fixed in 4% paraformaldehyde or formaldehyde in PBS, and driedonto glass microscopy slides. Cells were then stained with the fluorescentnuclear dye bis-benzimide [Hoechst 33342 (10mM in PBS); Sigma ChemicalCo., St. Louis, MO], and the proportion of apoptotic cells was counted in 5–10high-powered fields for each of four replicated coverslips per conditionthrough a fluorescence microscope (Leitz;3100 objective) with the observerblinded to the experimental conditions. Other tumor specimens were preparedand cultivated as monolayers on PLL-coated glass coverslips (Bellco Glass,Vineland, NJ) in MEM (Cellgro) containing 12% heat-inactivated FCS (Sig-ma), high glucose (6 g/liter), 25 mM potassium chloride, sodium pyruvate, andnonessential amino acid supplements (Life Technologies, Inc., Gaithersburg,MD). For monolayer cultures, medium was changed after 24 hin vitro toserum-free MEM containing individual neurotrophins (see above). The cellswere grown for an additional 48 h before fixation in 4% paraformaldehyde inPBS, stained with bis-benzimide, and viewed with a fluorescence microscopeusing the methods noted above.

Received 4/6/98; accepted 11/25/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grant NS27773 (to S. L. P.), Mental Retardation Research CenterGrant HD18655, NIH Training Grant CA09172 (to J. Y. H. K.), Neurological SciencesAcademic Development Award NS01701 (to M. E. S.), the Brain Tumor Society, theElizabeth O’Brien Foundation, the Pediatric Brain Tumor Foundation of the United States,the Kyle Mullarkey Fund, and a generous donation from Dr. and Mrs. Sidney Feldman.

2 To whom requests for reprints should be addressed, at Division of Neuroscience,Enders 260, Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115. Phone:(617) 355-6874; Fax: (617) 738-1542; E-mail: [email protected].

3 The abbreviations used are: BDNF, brain-derived neurotrophic factor; NT-3, neuro-trophin-3; NGF, nerve growth factor; PLL, poly-L-lysine; CHX, cycloheximide; ERK,extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; JNK,c-Jun NH2-terminal protein kinase; SAPK, stress-activated protein kinase; PI39K, phos-phatidylinositol-39-kinase; TUNEL, terminal deoxynucleotide transferase-mediated dUTPnick end labeling; NSE, neuron-specific enolase; IEG, immediate-early gene.

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The medulloblastoma cell line Daoy was obtained from the American TypeCulture Collection and maintained in DMEM with high glucose (6 g/liter), 2mM L-glutamine, and 10% (vol/vol) FCS at 37°C in 5% CO2. Transfected Daoysubclones with increased TrkC expression (Daoy-trkC) were maintained inselective media containing Geneticin (G418, 200mg/ml; GIBCO BRL LifeTechnologies, Inc.) after stable lipid-mediated transfection with an expressionplasmid encoding full-length rat gp145trkC (TrkC; courtesy of Luis Parada,University of Texas; Ref. 22) using Lipofectin, according to vendor recom-mendations (Life Technologies, Inc.). To test for neurotrophin responsiveness,we grew cell lines on PLL-coated glass coverslips that were placed in serum-free DMEM for up to 96 h with individual neurotrophins (50 ng/ml NGF,BDNF, or NT-3) or vehicle control (0.1 mg/ml BSA). For signal transductionand Western blot experiments, transient stimulations were performed for30–60 min and then replaced with serum-free media and incubated untilfixation and staining, as described above.

Growth curves of Daoy and Daoy-trkC cell lines were determined usingtrypan blue exclusion (23). Proliferation of cell lines was measured by deter-mining incorporation of 6-[3H]thymidine (.10 Ci/mmol; New England Nu-clear, Boston, MA; Ref. 24). Briefly, parallel Daoy-trkC cultures were pre-pared, stimulated, and incubated for the last 6 h with [3H]thymidine-containingmedium (60mCi/ml). Cells were then harvested using 0.1% Triton X-100, andtrichloroacetic acid-precipitable3H counts were quantitated with a scintillationcounter.

Pharmacological inhibitors of signal transduction K-252a (Kamiya Bio-chemical, Seattle, WA), wortmannin, PD98059, and SB230850 (Calbiochem,San Diego, CA), or CHX (Sigma) were also added to select cultures inserum-free DMEM or MEM at indicated concentrations for 30 min prior to andduring neurotrophin or mock stimulation.

RNA Isolation and Northern Analysis. Total cellular RNA was isolatedby cesium chloride gradient ultracentrifugation after tissue homogenization inguanidine isothiocyanate-containing lysis buffer. Northern blotting, includinghybridization conditions and mRNA quantification, were performed as de-scribed previously (12). All signals were quantified with a PhosphorImager(Molecular Dynamics, Sunnyvale, CA) and corrected for variations in sampleloading by comparison to internal 28S rRNA expression. RNA expressionindices for cell culture experiments were calculated by normalizing samples totheir 28S rRNA content and by comparing to expression by steady-stateunstimulated cells (25). Trk probes were prepared by random oligonucleotide-primed 32P-labeling of cDNAs encoding the extracellular domains of humanTrkB and TrkC (courtesy of David Shelton, Genentech, San Francisco, CA).The 2.6-kb c-jun cDNA probe was similarly prepared from a previouslydescribed plasmid (26). All other probes have been described previously (12).

Western Analysis, Immunoprecipitation, and Immunohistochemistry.Cellular lysates were prepared from transiently stimulated Daoy-trkC culturesor fresh tissue by lysis in NP40-glycerol lysis buffer and from frozen tissuesamples by rapid boiling in SDS-lysis buffer, as described previously (12, 27).After total protein concentration was determined by Bradford assay (Bio-Rad,Hercules, CA), samples containing equal amounts of protein (50–150mg)were denatured and separated by SDS-polyacrylamide (SDS-PAGE) gel (7.5–10%) electrophoresis, as described previously (28).

Separated proteins were then transferred to nylon membranes (Immo-bilon-P; Millipore, Bedford, MA) for immunoblotting as described previously(29). Specific antibodies included: an antiphosphotyrosine monoclonal (4G10;Upstate Biotechnology Inc., Lake Placid, NY); an anti-p70S6Rskpeptide poly-clonal (courtesy of M. Greenberg and J. Blenis; Ref. 30); an anti-ERK1/2(p44ERK1/p42ERK2) polyclonal generated against a COOH terminus peptide(cMAPK; courtesy of J. Blenis; Ref. 31); an anti-ERK2 monoclonal (1B3B9;courtesy of M. Greenberg and M. Weber; Ref. 32); a phospho-Tyr-204-specificantibody for ERK1/2, a phospho-Thr-183 and phospho-Tyr-185-specific anti-body for JNK/SAPK, and a phospho-Tyr-182-specific antibody for p38MAPK(homologous to yeast HOG kinase; New England Biolabs, Beverly, MA); anda phospho-Ser-73-specific polyclonal for c-Jun (courtesy of M. Greenberg).Membranes were blocked using either 4% BSA or 5% Blotto, incubated, andwashed using standard techniques (29). Secondary antibodies included goatantimouse IgG- and goat antirabbit IgG-conjugated to alkaline phosphatase forchemoluminescent detection with CDP-Star reagents, according to vendorrecommendations (New England Biolabs).

Immunoprecipitations of PI39K and ERK1/2 from cellular lysates wereperformed using antibodies against antiphosphotyrosine (4G10) and the

COOH terminus of ERK1/2 (cMAPK), respectively, with protein A-Sepharosebeads (4CLB; Pharmacia, Piscataway, NJ) for SDS-PAGE analysis, as de-scribed previously (27, 33). Briefly, forin vitro kinase assays of PI39K andERK1/2 activities, cellular lysates were prepared at indicated time points aftertransient stimulation as above and then incubated, as described previously (27,33), with g-[32P]ATP (3000 Ci/mmol; New England Nuclear, Boston, MA),and the appropriate substrate (phospholipid mix: phosphotidylinositol andphosphatidylserine) or rabbit myelin basic protein (Sigma). For PI39K assays,radiolabeled phospholipids were extracted, separated with TLC, and quanti-tated with a PhosphorImager. For ERK assays, radiolabeled myelin basicprotein was precipitated with trichloroacetic acid and immobilized on phos-phocellulose filters (P81; Whatman, Hillsboro, OR), washed in dilute phos-phoric acid, and quantitated with a scintillation counter.

For immunohistochemical detection of c-Fos, Daoy-trkC cultures weregrown in PLL-coated coverslips and stimulated as above. Following fixationand incubation with an anti-c-Fos polyclonal antibody (courtesy of M. Green-berg), a peroxidase-antiperoxidase system was used according to the manu-facturer’s recommendations (Vectastain; Vector Laboratories, Burlingame,CA).

In Situ Hybridization. Paraffin-embedded 10-mm-thick tumor sectionswere hybridized with35S-labeled antisense and sense human Trk riboprobes(see above) according to a previously described protocol (34). The tissuesections were hybridized at 45°C for 18–20 h, dipped in photographic emul-sion (NTB; Kodak, Rochester, NY), exposed at 4°C for 7 days, developed,counterstained with hematoxylin, and viewed with bright- and dark-fieldmicroscopy.

Apoptosis and Proliferation Analysis. Hematoxylin-stained biopsy sec-tions were also examined to define regions with dense infiltration of tumorcells. Comparable fields in adjacent serial sections were examined for apop-tosis and for proliferation. The proportion of proliferating cells was determinedin tissue sections stained with a Ki-67 antibody (DAKO, Carpinteria, CA) andvisualized using peroxidase-antiperoxidase techniques, per vendor recommen-dations. For detection of apoptosis, the paraffin sections from the same patientsand fixed monolayer cell cultures were assessed for nucleosomal degradationby TUNEL using digoxigenin-labeled nucleotide incorporation, a fluorescein-labeled antidigoxigenin antibody, or peroxidase-antiperoxidase techniques,according to the manufacturer’s recommendations (Apoptog; Oncor, Gaithers-burg, MD). The sections were counterstained with propidium iodide or bis-benzimide. Nuclei were counted through a fluorescence microscope under highpower (Leitz,3100 objective). In a separate set of observations, the proportionof pyknotic, condensed, and fragmented nuclei in propidium iodide-stainedsections were counted (340 objective). Nuclei were counted in 10 high-powered fields per tumor biopsy sample by blinded observers to determine thefraction of nuclei undergoing apoptosis. For the purposes of statistical analysis,we calculated an apoptosis index by normalizing the proportion of apoptosisfor each condition tested to the percentage observed in a parallel mock-stimulated control performed in each experiment.

Medulloblastoma Cell Line Intracranial Xenografts in Nude Mice.Equal numbers (13 105 cells in 5ml) of the trkC-overexpressing Daoy cells(Daoy-trkC) and control parental Daoy cells were each injected into oppositecerebral hemispheres of nude mice (nu/nu Balb/C adult males anesthetizedwith Avertin, 0.02 ml/g body weight) using a Hamilton syringe guided by astereotaxic apparatus (Stoelting, Wood Dale, IL). Injections were made 3 mmcaudal to Bregma, 2.5 mm lateral of midline, and 3 mm below the surface ofthe brain. The animals were then allowed to recover, and after 7–9 weeks wereanesthetized and sacrificed by intracardiac perfusion with PBS followed by 4%paraformaldehyde in PBS. Serial 15-mm sections were cut on a cryostat,mounted on glass slides, stained, and viewed with a bright-field microscope.To measure tumor volumes, the tumors in each section were traced, andcross-sectional area was calculated on a videomicroscopy image analysissystem (Image-1; Universal Imaging, West Chester, PA). The volume persection was calculated by multiplying the area by section thickness, and thetotal tumor volume was calculated by summation of the tumor section volumesin each hemisphere.

Statistical Analysis. Kaplan-Meier survival analysis was calculated withSAS (SAS Institute, Inc., Cary, NC) using the log-rank test for comparisons(35). Cell counting data were tested for normality with the Kolmogorov-Smirnov test and evaluated by ANOVA (Stat-View; Abacus Concepts, Inc.,Berkeley, CA) using the Bonferroni procedure for multiple post hoc pairwise

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TrkC ACTIVATION INDUCES APOPTOSIS IN MEDULLOBLASTOMA



comparisons. A least squares model was used to generate correlation coeffi-cients. Two-tailed tests of significance were used. The investigations werecompleted with approval of the Committee on Clinical Investigation and theAnimal Care and Use Committee at Children’s Hospital (Boston, MA).

RESULTS

trkC Overexpression Is Associated with Favorable Clinical Out-come in Medulloblastoma.We accrued medulloblastoma specimensand patient data, expanding our earlier findings to a larger cohort(n 5 42). All tumor samples demonstrated expression oftrkC (Fig.1A). A trkC expression index for each tumor sample was calculated

by normalizing the 14.0-kbtrkC transcript signal to a standardizedmouse brain RNA control (25). The expression oftrkC was highlyvariable in medulloblastoma specimens (expression index range, 0.1–45.7; median5 0.80; mean5 3.9). trkC expression in normal humancerebellum was determined to be 2.0 times higher than mouse brain.The cohort of patients was dichotomized into groups expressing highor low levels oftrkC by dividing at the median index value (overalltrends are similar using the mean index). Survival data compiled inFig. 1 and Table 1 reveal that patients with medulloblastomas ex-pressing high levels oftrkC had more favorable progression-freesurvival (Fig. 1B) and overall survival (Fig. 1C; median survival5 92months) compared to those with lowtrkC expression (median sur-vival 5 39 months). Fewer patients with hightrkC-expressing tumorshad evidence of metastatic disease at the time of diagnosis (2 of 20patients) than those (10 of 22 patients) with lowtrkC expression,although the trend was not statistically significant (Fisher’s exact test,P 5 0.09).

The expression of other neurotrophins and their receptors was notpredictive of outcome (Table 1). Neither patient age nor the extent ofsurgical resection correlated withtrkC expression. Moreover, age,degree of surgical resection, sex, and treatment center were notpredictive of survival. Considered collectively, these data indicate thattrkC expression was the only parameter studied that served as asignificant predictor of clinical outcome.

NT-3-stimulated TrkC Activation Induces Cell Death in Pri-mary Medulloblastomas in Vitro. We initially addressed whetherTrkC activation alters medulloblastoma growth by adding NT-3 toprimary cultures of medulloblastomasin vitro. Medulloblastoma cul-tures from two patients with hightrkC expression and another withlow trkC expression were grown under serum-free conditions in thepresence of NT-3, BDNF, or NGF (each at 50 ng/ml) for 18–48 h.Upon microscopic examination, some of the cells in each culturerevealed nuclear pyknosis, condensation, or fragmentation indicativeof cell death. Cultures from all three medulloblastomas displayed amarked increase of apoptosis when grown in the presence of NT-3(Fig. 2B) compared to mock-stimulated controls (Fig. 2A), but not inBDNF or NGF (P, 0.0001, ANOVA; Fig. 2D), indicating that NT-3binding to its preferred receptor, TrkC, increased cell death. Thiseffect was blocked by CHX in the one tumor tested (Fig. 2C). Thus,medulloblastomas grown in culture respond to TrkC activation byundergoing apoptosis.

Fig. 1. Expression oftrkC in medulloblastoma correlates with patient outcome.A,Northern blot demonstrating variable expression of the 14.0-kb alternate splice variant oftrkC in mouse whole brain control and four primary medulloblastoma specimens. Totalcellular RNA was prepared from primary tumor samples, separated in formaldehyde-agarose gels, transferred to nylon membranes, probed withtrkC cDNA or 28S rRNAprobes, and quantitated using a PhosphorImager, as described in “Materials and Methods.”B andC, Kaplan-Meier plots demonstrating that hightrkC expression is linked to a morefavorable outcome of medulloblastoma (n 5 42). Improved progression-free (B) andoverall survival (C) are associated with highertrkC expression in medulloblastomasamples.OO, results for patients hightrkC-expressing tumors;z z z z z, patients with lowtrkC-expressing tumors. AtrkC expression index for each sample was calculated bydividing the tumor 14.0-kbtrkC transcript signal by that of a standard mouse brain RNAcontrol. The trkC expression index distribution was highly skewed (range, 0.1–45.7;median5 0.80; mean5 3.9). For Kaplan-Meier analysis, the cohort was dichotomizedinto groups expressing high or low levels oftrkC by dividing at the median index value.Comparisons were made with the log-rank test.

Table 1 Expression of neurotrophin and trk mRNAs and outcome of medulloblastomas

mRNAtranscripta

No. of tumorsexpressingmRNA/no.tested (%)

Significance of analysiscomparing expressionwith progression-free

survivalb

Significance of analysiscomparing expressionwith overall survivalb

14.0-kbtrkC 42/42 (100) 0.02 0.00019.0-kb trkB 18/35 (51) 0.43 0.217.0-kb trkB 26/35 (74) 0.64 0.94trkA 0/35 (0) NAc NANT-3 33/40 (82) 0.50 0.50BDNF 12/35 (34) 0.79 0.62p75NTR 11/35 (31) 0.48 0.33

a As measured by Northern analysis. Briefly, total cellular RNA samples from snap-frozen primary tumors were separated in formaldehyde-agarose gels, transferred to nylonmembranes, probed with radiolabeled cDNA, and quantitated using a PhosphorImager.

b P values are based on Northern analysis of primary medulloblastoma samples, eachof which demonstrated expression of the 14.0-kbtrkC mRNA. An index oftrkC expres-sion was calculated for each specimen by normalizing to its 28S rRNA expression anddividing its 14.0-kbtrkC signal intensity by that of mouse brain as a standard control. ForKaplan-Meier analysis, the patients were dichotomized into groups expressing high or lowlevels of 14.0-kbtrkC mRNA (encoding the full-length gp145trkC, 7.0-kbtrkB (encodinga truncated tyrosine kinase negative receptor), and 9.0-kbtrkB (encoding full lengthgp145trkB), by dividing at the median expression index for each. The patients weredichotomized for expression of NT-3, BDNF, and p75NTR by whether the molecules weredetected in the samples. Survival comparisons were made with the log-rank test.

c NA, not applicable.

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TrkC Activation Induces Apoptosis of the Daoy Medulloblas-toma Cell Line in Vitro. To test whether we could induce TrkC-mediated apoptosis in tumor cells that normally do not respond toNT-3, we up-regulated the expression of the TrkC receptor in amedulloblastoma cell line that normally has no detectable expressionof full-length p145trkC (TrkC). The human medulloblastoma-derivedDaoy cell line normally expresses very low levels of the 5.5-kbtrkCalternate splice variant and no detectable 14-kbtrkC, p75NTR, trkA, ortrkB mRNA (12). We stably transfected Daoy cells with an expressionplasmid encoding full-length TrkC and tested subclones (designatedDaoy-trkC) for increased TrkC protein expression by immunoprecipi-tation and for TrkC receptor activity using immunocytochemistry todetect NT-3-induced c-Fos expression (see below; Ref. 36). Thelevels of trkC mRNA expression of two Daoy-trkC subclones were4.6 and 6.6 times greater than mouse brain and, therefore,;2–3 timesgreater than normal human cerebellum.

When grown in serum-free conditions, the Daoy-trkC subclonesoverexpressingtrkC underwent extensive cell death in the presence ofNT-3 (Fig. 3B) compared to mock-stimulated controls (Fig. 3A) butnot BDNF or NGF (50 ng/ml each;P , 0.0001; Fig. 3E). Apoptosis

was morphologically evident within 24 h of exposure to NT-3 andincreased significantly by the 48- and 72-h time points. By 96 h, theremaining adherent monolayer cells in the NT-3 conditions had;10-fold greater nuclear pyknosis and fragmentation [51.76 0.4%(mean6 SE); Fig. 3B] compared to mock-stimulated control condi-tions (5.16 0.3%; Fig. 3A), which was statistically significant [ap-optosis index5 10.16 0.08 (mean6 SE); P , 0.0001; Fig. 3E].

Fig. 2. NT-3 induces apoptosis in primary medulloblastoma cultures, which is blockedby CHX. A and B, dissociated primary human medulloblastoma cells were plated onPLL-coated glass coverslips in complete medium, then stimulated in serum-free condi-tions with BSA control (A) or individual neurotrophin (50 ng/ml) for 48 h, and then fixedand stained with bis-benzimide. Pyknotic and fragmented nuclei (indicated byarrows;B)were observed in significantly higher proportions when cultivated in NT-3 compared tomock-stimulated control conditions, shown inA. BDNF or NGF were indistinguishablefrom control.Scale bar, 10mm. C, parallel medulloblastoma cultures stimulated by NT-3but in the presence of CHX (1mg/ml) revealed protection from apoptosis, with similaroverall survival as in control conditions, suggesting that NT-3-induced apoptosis requiresprotein synthesis.Scale bar, 10mm. D, bar graph summarizing the results of the aboveexperiment, represented as a percentage of nuclei with apoptotic changes. In primarymedulloblastoma cultures, NT-3 induced significantly increased apoptosis (19.96 1.2%)compared to other conditions (control, 4.36 0.7%, P , 0.0001, ANOVA; BDNF,6.5 6 1.5%; and NGF, 6.16 1.4%) and was blocked by concurrent exposure to CHX.These data (from blinded examination of ten high-powered fields from each of four toeight replicated coverslips) were representative of those obtained from similar experi-ments with two other primary tumors.Columns, means;bars, SE.

Fig. 3. NT-3 induces apoptosis in a medulloblastoma cell line expressingtrkC.Monolayer cultures were grown on PLL-coated coverslips, stimulated in serum-freeconditions, fixed, and stained with bis-benzimide.Scale bars, 10mm. A andB, cultures ofthe subcloned medulloblastoma cell line Daoy with up-regulated TrkC expression (Daoy-trkC) undergo increased nuclear condensation and fragmentation, as indicated byarrowsin B, in the presence of NT-3 (50 ng/ml) for 96 h, compared to mock-stimulated controlconditions, as shown inA. C andD, TUNEL labeling of NT-3-treated Daoy-trkC culturesconfirm that these observed changes in nuclear morphology are due to apoptotic nucleo-somal degradation (arrow in D) compared to negative TUNEL results in control condi-tions (C). Cultures were grown and stimulated as above, harvested at 24 h, and processedusing fluorescein-conjugated antidigoxigenin antibody. These results were confirmed infour separate experiments using two different Daoy-trkCsubclones.E, bar graph sum-marizing Daoy-trkCdata from three separate experiments. An index of apoptosis wascalculated from the proportion of stained nuclei displaying characteristic condensation andfragmentation under each experimental condition normalized to the proportion of apop-totic nuclei in mock-stimulated control. These results indicate that a significantly higherpercentage of TrkC-expressing Daoy cells undergo apoptosis when grown for 96 h in thepresence of NT-3 (apoptosis in 51.76 0.4%; apoptosis index5 10.16 0.08), comparedto BDNF (apoptosis index5 0.86 6 0.08), NGF (apoptosis index5 0.82 6 0.04), ormock-stimulated control (apoptosis in 5.16 0.3%; apoptosis index5 1.00;P , .0001,ANOVA), whereas FCS prevented NT-3-induced apoptosis.Columns, averaged data fromfive high-powered fields from each of four replicated coverslips per experimental condi-tion; bars, SE.F, growth and [3H]thymidine incorporation curves of mock-versusNT-3-stimulated Daoy-trkCcultures. Total cell numbers were counted for parallel culturesby trypan blue exclusion. NT-3-stimulated Daoy-trkCcultures (‚) revealed decreasingcell numbers with increasing duration of NT-3 stimulation, in contrast to stable cellnumbers in parallel mock-stimulated Daoy-trkCcultures (E). Proliferation, as quantitatedby [3H]thymidine incorporation from parallel cultures using trichloroacetic acid precipi-tation methods, reflected lower rates of [3H]thymidine uptake in NT-3-stimulated Daoy-trkC cultures (f), compared to parallel mock-stimulated Daoy-trkC cultures (u). Thedecreased growth and proliferation rates in NT-3-stimulated Daoy-trkC cultures indicatethat NT-3 induces apoptosis without increasing proliferation.Data points, means oftriplicate samples, representative of multiple experiments;bars, SD.

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To demonstrate that NT-3-induced cell death was due to apoptoticmechanisms, we examined Daoy-trkCcultures for nucleosomal deg-radation characteristic of apoptosis. The proportion of TUNEL-posi-tive nuclei was significantly higher in Daoy-trkC cultures grown withNT-3 for 24 h [0.086 0.03; (mean6 SE); Fig. 3D] compared tocontrol mock-stimulated conditions in which no TUNEL-positivecells were present (P , 0.03; Fig. 3C).

NT-3/TrkC-induced apoptosis was not observed in the presence ofFCS in culture media (Fig. 3E), suggesting that serum containsprotective factor(s). In Daoy-trkC cells, neither FCS nor NT-3 in-duced morphological changes suggestive of differentiation. Similarly,Western blot analysis of NT-3-stimulated Daoy-trkC cell lysates didnot reveal changes in the expression of markers such as NSE ormicrotubule-associated protein-2 isoforms (data not shown).

Growth curves of NT-3-stimulated Daoy-trkC cultures (Fig. 3F)revealed decreasing cell numbers (by approximately one log unit)with increasing duration of NT-3 stimulation, in contrast to stable cellnumbers in parallel mock-stimulated Daoy-trkC cultures (Fig. 3F).Proliferation, as determined by [3H]thymidine incorporation rates,also progressively decreased in NT-3-stimulated Daoy-trkC cultures,compared to parallel mock-stimulated Daoy-trkC cultures (Fig. 3F).The decreased growth and proliferation rates in NT-3-stimulatedDaoy-trkC cultures indicate that NT-3 induces apoptosis withoutincreasing proliferation. These results support apoptosis rather thanproliferation or differentiation as the primary effect of NT-3/TrkCsignaling on Daoy cell growthin vitro.

TrkC Signal Transduction Activates PI3*K, ERK1/2,p38MAPK, and JNK/SAPK and Expression of the IEGs c-fosandc-jun. Because NT-3-induced apoptosis might have been due to de-fective signal transduction by overexpressed TrkC, we sought toconfirm the integrity of NT-3/TrkC signaling pathways. The tyrosinekinase function of activated TrkC receptors in Daoy-trkCcells ap-pears necessary for apoptosis induction because there was no increasein cell death over mock-stimulated cultures when the Trk tyrosinekinase inhibitor K252a (200 nM) was added along with NT-3 (P. 0.4for both subclones; data not shown).

Further studies of NT-3-stimulated TrkC transduction were thenundertaken to confirm that downstream signaling cascades were func-tionally intact. Daoy-trkC cultures were prepared as described abovebut were only transiently stimulated with NT-3 for 30–60 min; thenthe NT-3 was replaced with serum-free medium, and cultures wereharvested at regular intervals. Using phosphospecific antibodies inWestern blots of these Daoy-trkC lysates, we detected activation(phosphorylation) of known signaling intermediates by PI39K. Nor-mally, PI39K phosphorylates the serine-threonine protein kinase, Akt,and ribosomal S6 subunit kinase, p70S6Rsk. Using p70S6Rsk-specificantibody in Western blots of NT-3-stimulated Daoy-trkC cell lysates,mobility shifts of p70S6Rsk were detectable for 6 h after transientstimulation (Fig. 4A), consistent with phosphorylation by PI39K.

Western blots of Daoy-trkC lysates also revealed phosphorylationpatterns indicating NT-3-induced activation of three parallel MAPKsignaling cascades: ERK (p44/p42), JNK/SAPK, and p38MAPK. Anantibody specific for phospho-Tyr-204 of ERK1/2 demonstrated pro-longed activation of ERK1/2 (Fig. 4B), whereas no changes in theoverall quantity of ERK was detected by a phosphorylation-indepen-dent monoclonal antibody (1B3B9; Fig. 4C). As shown by a phospho-Thr-183-/Tyr-185-specific JNK/SAPK antibody, JNK/SAPK is phos-phorylated for several h when transiently activated by NT-3/TrkC(Fig. 5B). p38MAPK is more transiently activated by NT-3/TrkC, asindicated by phospho-Tyr182-specific antibody (lasting;1 h; Fig.5A). The PI39K and ERK1/2 Western blot results were verifieddirectly usingin vitro kinase assays. After 1 h ofNT-3 stimulation,PI39K and ERK1/2 activity increased 2.31- and 2.66-fold, respec-

tively, in immunoprecipitated Daoy-trkC lysates compared to mock-stimulated controls.

For a variety of neural cells, activation of PI39K, ERK1/2, JNK/SAPK, or p38MAPK have been linked to cell death or survival(37–43). Blocking either PI39K signaling with wortmannin (100–200nM) or ERK1/2 signaling by inhibiting upstream MEK1/2 withPD98059 (10–50mM) attenuated but did not completely block NT-3-induced apoptosis (Fig. 6,E and F). In both cases, successfulinhibition of the kinases was shown by Western blot analysis (data notshown). In contrast, specific inhibition of p38MAPK with SB230850(25–75 mM) completely prevented apoptosis in NT-3-stimulatedDaoy-trkCcells (Fig. 6,D andF), indicating that p38MAPK activityis required for NT-3-induced apoptosis (Fig. 6,A andB).

Both p38MAPK and JNK/SAPK pathways have been shown toconverge on the IEGsc-jun and c-fos (44). We found by probingNorthern blots that TrkC activation induced sustained up-regulation ofc-jun transcription (Fig. 5D). Moreover, mobility shifts of c-Jun (Mr

44,000–45,000) bands were evident on Western blots probed with aphospho-Ser-73-specific c-Jun antibody (Fig. 5C), indicating activa-tion by posttranslational phosphorylation of serine residue 73 byJNK/SAPK (45, 46). Finally, NT-3-induced nuclear expression ofc-Fos in Daoy-trkC cells was demonstrated by immunohistochemicalmethods using c-Fos-specific antibodies (Fig. 5,E andF).

CHX Blocks NT-3/TrkC-induced Apoptosis in Medulloblas-toma. To test for the possible requirement for protein translation inNT-3-/TrkC-stimulated apoptosis, Daoy-trkC cells were incubatedwith CHX at concentrations (1–5mg/ml) sufficient to reduce proteinsynthesis approximately 50%, as determined by [35S]methionine/cysteine incorporation (data not shown). In the presence of CHX (1mg/ml), NT-3-stimulation did not increase apoptosis (Fig. 6,C andF).Cytotoxic effects of CHX in dose ranges used were not evident duringthese experiments, and Western blots of Daoy-trkC lysates did notreveal CHX-induced changes in signaling intermediates (data notshown).

Fig. 4. NT-3 activated TrkC signals by PI39K and ERK1/2. Western blots wereprepared from DAOY-trkC lysates harvested after transient (60 min) NT-3- (Lanes1) ormock- (Lanes2) stimulation after times indicated, separated by SDS-PAGE, transferredto Immobilon-P, blocked with BSA or Blotto, and then incubated with primary antibodiesincluding rabbit anti-p70S6Rsk(A) and anti-phospho-ERK1/2 (B) and anti-ERK2 mono-clonal (C); then the appropriate alkaline phosphatase-conjugated secondary antibodies anddeveloped using chemiluminescent reagents. Transient rather than prolonged NT-3 stim-ulation (as in apoptosis experiments) was used to expose the regulation of signalingintermediates.Open arrowheads, phosphorylated isoforms and closed arrowheads hy-pophosphorylated forms.A, PI39K is activated by NT-3 stimulation. The shifted bands(open arrowheads) in NT-3-stimulated lysates are indicative of p70S6Rskisoforms phos-phorylated by activated PI39K.Closed arrowheads, indicate hypophosphorylated iso-forms. B, ERK1/2 (p44/p42) are phosphorylated by NT-3 stimulation. Phosphospecificantibody reveals NT-3 induced phosphorylation of ERK1/2 at Tyr-204, which decreasesover several hours (open arrowheads). Nonspecific phosphorylation can be detected atlater time points in control mock-stimulated lysates.C, monoclonal antibody staining fortotal ERK2 (p42) reveals consistent levels throughout the experimental time courseindependent of NT-3 stimulation, indicating that NT-3 induces phosphorylation of pre-existing ERK2 without up-regulating overall levels of ERK2 protein.

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TrkC Expression Inhibits the Growth of Daoy Medulloblas-toma Cell Line in Vivo. We tested the biological significance ofNT-3-/TrkC-induced apoptosisin vivo by examining the growth ofintracerebral xenografts of Daoy and Daoy-trkC cells in nude mice.We first tested whether there are inherent differences of proliferationrates of the Daoy-trkCsubclones and the parent cell line. Shown inFig. 7A, the growth curve of parental Daoy cellsin vitro is indistin-guishable from Daoy-trkC cells.

Equal numbers of Daoy and Daoy-trkC cells were injected intoopposite hemispheres of the brains of anesthetized nude mice and theanimals were allowed to recover. After 7–9 weeks, marked differ-ences in the size of the xenografts were evident. In 8 of 10 animals,the intracerebral control Daoy tumors were substantially larger com-pared to Daoy-trkCtumors from matched opposite cerebral hemi-

spheres (P, 0.03, Wilcoxon rank sum test; Fig. 7,B andC). Thesedifferences were most notable after 9 weeks of xenograft growth; theparent Daoy tumors were 7–220-fold larger than the Daoy-trkCtu-mors. Histologically, both Daoy and Daoy-trkCtumors were highlycellular and without morphological evidence of differentiation. Thesesections were also examined for apoptosis by TUNEL staining, whichrevealed dramatically greater proportions of apoptotic nuclei in theDaoy-trkC tumors compared to parental Daoy tumors (Fig. 7,D andE). These results demonstrate that, in ourin vivo nude mouse model,trkC overexpression inhibited tumor growth in association with in-creased apoptosis.

trkC Expression by Medulloblastoma Tumor Cells Is Associatedwith Apoptosis. We then addressed whether TrkC-induced apoptosismight have growth-inhibiting effects on human medulloblastomasin

Fig. 5. NT-3/TrkC signaling in Daoy-trkC activates p38MAPK and JNK/SAPK, c-Junphosphorylation, c-juntranscription, and c-Fos nuclear translocation. Western blots wereprepared from Daoy-trkClysates harvested after transient NT-3- (Lanes1) or mock(Lanes2) stimulation after times indicated, separated by SDS-PAGE, transferred to nylonmembranes, blocked with BSA or Blotto, and incubated with primary antibodies includingphosphospecific rabbit anti-p38MAPK, anti-JNK/SAPK, or anti-c-Jun primary antibodies.Open arrowheads, phosphorylated isoforms;closed arrowheads, hypophosphorylatedforms.A, p38MAPK is transiently activated in NT-3-stimulated lysates by phosphoryla-tion at Tyr-182, as revealed by phosphospecific antibody.B, NT-3 induces sustainedphosphorylation of JNK/SAPK species (p46 and p54) at Thr-183 and Tyr-185, as detectedby phosphospecific antibody.C, the substrate of JNK/SAPK, c-Jun, is also phosphorylated(at Ser-73) in response to NT-3 stimulation, as revealed by phosphospecific antibodystaining of mobility shifted upper bands (open arrowheads). Lesser degree of nonspecificphosphorylation is also detected in mock-stimulated lysates.D, TrkC activation inducesup-regulation of c-jun mRNA expression. Northern blots of total cellular RNA preparedfrom transiently stimulated (60 min) Daoy-trkC cultures were hybridized with32P-labeledc-jun probe revealing splice variants (arrows). An index of expression was calculated bycorrecting for 28S rRNA content and normalizing to baseline unstimulated levels (har-vested at time 0). These results were replicated in four separate experiments.E and F,Daoy-trkC cultures grown on PLL-coated coverslips, stimulated, and processed forimmunohistochemical staining with c-Fos-specific antibody reveal NT-3 induction ofc-Fos (F) but no induction in mock-stimulated Daoy-trkCcontrols (E).Scale bar, 10mm.

Fig. 6. NT-3-induced apoptosis in Daoy-trkCcells is blocked by SB230850 or CHX.Daoy-trkCcells were grown, stimulated, fixed, stained with bis-benzimide, and examinedusing fluorescence microscopy.Scale bars, 10mm. A andB, mock-stimulated Daoy-trkCcontrol cultures (A) show minimal nuclear changes compared to NT-3-stimulation (50ng/ml) of Daoy-trkC cells (B), which caused a marked increase of pyknotic nuclei after72 h (nuclei indicated byarrows). C, NT-3-induced apoptosis was blocked by CHX (1mg/ml) in Daoy-trkC cultures.D, incubation of NT-3 (50 ng/ml) stimulated Daoy-trkCcultures with p38MAPK inhibitor SB230850 (25mM) significantly reduced apoptosiscompared to NT-3 alone (B).E, addition of PD98059 (10mM), which blocks MEK1/2upstream of ERK1/2, to NT-3 (50 ng/ml) stimulated Daoy-trkC did not block NT-3-induced cell death compared to NT-3 alone (B).F, bar graph summarizing four experi-ments with Daoy-trkC cells showing the effects of various signal transduction inhibitors[Wort, wortmannin (100 nM); PD, PD98059 (10mM); SB, SB230850 (25mM)] and CHX.An apoptosis index was calculated by normalizing data (from five high-powered fieldsfrom each of four replicated coverslips per experimental condition) to the proportion ofapoptotic nuclei in mock-stimulated controls (3.46 0.3%). The apoptosis index fromNT-3-stimulated Daoy-trkC cells (10.716 0.45) was significantly higher than that ofmock-stimulated controls (P , 0.0001; ANOVA), and the effect was completely blockedby the presence of CHX (apoptosis index5 1.08 6 0.08) or SB230850 (apoptosisindex 5 1.536 0.20;P , 0.0001 for each) but not by wortmannin (apoptosis index56.886 1.57) or PD98059 (apoptosis index5 6.536 0.83).Columns, means;bars, SE.

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situ. First, we determined which cells within medulloblastomas ex-presstrkC, by microscopically examining biopsy sections from ourinitial 12 patients usingin situ hybridization (12).trkC was expressedpredominantly by neoplastic cells throughout each tumor section (Fig.8, AandB). The level oftrkC expression detected byin situ hybrid-

ization corresponded well with the overall level of receptor expressionmeasured by Northern analysis (Mann-WhitneyU test,P 5 0.002).

We then asked whethertrkC expression correlates with apoptosis orproliferation in these human tumor biopsy specimens. Tissue sectionsfrom our 12 initial tumor samples (12) were examined for nucleoso-mal degradation by TUNEL labeling (Fig. 8,D and E) and forpyknosis by propidium iodide staining. We found thattrkC expres-sion, as measured by Northern analysis, was correlated with apoptosisas measured both by the TUNEL method (r 5 0.80,P 5 0.002; Fig.8F) and by nuclear pyknosis (r 5 0.77, P 5 0.02). Neither theexpression of the 9-kbtrkB alternate splice variant (r5 0.21;P 5 0.80) nor that of the 7.0-kb splice variant (r5 0.16;P 5 0.80)

Fig. 7. Daoy-trkC xenografts demonstrate diminished growth and increased apoptosisin vivo compared to the parental Daoy cell line.A, growth curves reveal similar growthkinetics of parental Daoy (E) and Daoy-trkC(3) cell lines cultivatedin vitro. B, scatterplot summarizing data from a total of 10 animals, sacrificed at 7 and 9 weeks postinjec-tion, shows the range of differential xenograft growth.Data points, ratios of the volumeof the parental Daoy xenograft compared to the volume of the contralateral Daoy-trkCxenograft from a single animal (i.e., a ratio of 1.0 represents equal-sized xenografts).Equal numbers of Daoy-trkC and control parental Daoy cells were each injected intoopposite cerebral hemispheres of anesthetized nude mice using a Hamilton syringe guidedby a stereotaxic apparatus. The animals were then allowed to recover, and after 7–9 weekswere anesthetized and sacrificed by intracardiac perfusion with PBS followed by 4%paraformaldehyde in PBS. Serial 15-mm cryostat sections were mounted, stained, andviewed with a bright-field microscope. The volume of tumors in each section werecalculated on a videomicroscopy image analysis system, and the total tumor volumecalculated by summation of the tumor section volumes in each hemisphere.C, hematox-ylin-stained section reveal significantly greater growth of a parental Daoy xenograft in theleft hemisphere (indicated byclosed arrowheads) compared to a Daoy-trkCxenograft inthe contralateral (right) hemisphere (indicated byopen arrowheads) of a homozygousnude mouse. (32.5 objective;scale bar, 500mm). D andE, TUNEL staining of Daoy (D)and Daoy-trkC(E) xenografts (visible at thebottom portion of each panel) and lessdensely cellular cerebral parenchyma (at thetop of each panel;p) reveals significantlymore apoptotic nuclei in the Daoy-trkCxenograft (arrowheadsin E) compared to theparental cell line Daoy xenograft (D), which does not express TrkC. The TUNEL stainingwas visualized using peroxidase-antiperoxidase methods, and the sections were counter-stained with methyl green.320 objective;scale bar, 50mm.

Fig. 8. Apoptosis correlates withtrkC expression in primary human medulloblastomas.Primary human tumor specimens were prepared as described in “Materials and Methods”and processed forin situ hybridization (A–C) and TUNEL staining (D and E). A lowtrkC-expressing tumor is shown inA and D, in comparison to a hightrkC-expressingtumor in B andE. Cells from adjacent normal cerebellum are shown inC. A–C, in situhybridization of medulloblastoma biopsy samples with a riboprobe (RNA) complemen-tary to trkC sequences. A section from a primary tumor expressing low levels oftrkC (A),as determined by Northern analysis, had a significantly lower density of silver grains inthe emulsion above above the neoplastic cells compared to a section from a tumorexpressing high levels oftrkC (B). Expression oftrkC in cerebellar parenchyma (C) isshown within the same tissue section as the hightrkC-expressing tumor.3100 objective;scale bars, 10mm. D and E, TUNEL staining of cells from low- (D) and high (E)trkC-expressing tumors reveals increased apoptotic nuclei in the tumor expressing highlevels oftrkC (arrows in E). 340 objective,scale bars, 25mM. F, the scatter plot displaysthe association of TUNEL staining withtrkC expression (as measured by Northernanalysis). The average proportion of TUNEL positive nuclei in each tumor is highlycorrelated with expression oftrkC (r 5 0.8;P 5 0.002). The proportion of apoptotic cellsin 12 medulloblastoma samples was calculated by counting TUNEL-positive nuclei anddividing by the total number of nuclei (propidium iodide counterstained) in 10 high-powered fields (340 objective) per tumor.

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was correlated with apoptosis. Furthermore, proliferation as measuredwith the Ki-67 antibody did not correlate withtrkC expression(r 5 0.21; P 5 0.51). These data from human tumors indicate thattrkC overexpression is associated with apoptosis but not proliferationin primary medulloblastomasin vivo.

DISCUSSION

Although trkC expression serves as a marker for prognosis, ourcurrent data suggest a deterministic role for TrkC in medulloblastomagrowth. We have observed that: (a) NT-3 stimulation influencestumor growthin vitro by inducing apoptosis, (b) spontaneous apop-tosis is highly correlated withtrkC-expression in primary medullo-blastoma specimens, and (c) TrkC-expression inhibits the growth ofintracerebral xenografts of the Daoy cell line medulloblastomain vivo.We have detectednt-3 transcripts in;83% of patient tumor RNAspecimens. Although the precise source of NT-3 has not been defined(Table 1), its coexpression withtrkC suggests that endogenous acti-vation of TrkC may regulate tumor growth.

We hypothesize that TrkC activity directly influences tumor growthby inducing apoptosis that promotes more favorable clinical out-comes. Because medulloblastomas do not spontaneously regress butcontinue to grow despite increased cell death in tumors with hightrkCexpression, the rate of apoptosis induced by activated TrkC presum-ably does not exceed the rate of tumor cell proliferationin vivo.Moreover, medulloblastomas are cured only in the context of treat-ment with surgery, radiation, and chemotherapy, indicating that theeffects of TrkC activation act in concert with conventional cancertherapies in determining clinical outcome.

The growth of medulloblastomas, like many childhood tumors,reflects dysregulated responses to environmental cues and develop-mental signals such as neurotrophins. Most evidence available to dateindicates that neurotrophins promote the differentiation or survival ofcerebellar granule cells (12–15). Our data from the medulloblastomacell line Daoy support the conclusion that NT-3-induced apoptosis ofmedulloblastomas occurs in the context of intact TrkC signaling. Wehave shown that NT-3 stimulation of the Daoy-trkC cell line activatesmultiple parallel signal transduction pathways involving PI39K,ERK1/2, JNK/SAPK, and p38MAPK. Of these, p38MAPK activityappears necessary for NT-3-/TrkC-induced apoptosis, based on ex-periments using pharmacological inhibitors. NT-3-/TrkC-induced ap-optosis also appears to require the expression of downstream targetgenes as shown by the protective effects of CHX. We have identifiedthe IEGs c-junand c-fosamong the intermediates activated by TrkCsignaling, which, in turn, may promote the expression of target genesresponsible for apoptosis. Although our evidence argues that apopto-sis in medulloblastomas occurs with appropriate TrkC signal trans-duction, this unusual response may arise because constitutively pro-liferating cancer cells are incapable of executing an appropriatedifferentiation program in an abnormal, transformed intracellular con-text.

A report by Muragakiet al.(47) supports the apoptotic function ofneurotrophin/Trk signaling in medulloblastoma-derived cell lines(47). However, they reported that transfected medulloblastoma celllines undergo apoptosis in response to TrkA activation, whereas thoseexpressing TrkC undergo differentiation when grown in the presenceof NT-3, similar to neuroblastomas with TrkA-activation (48). Thesedifferences probably arise from details in experimental design (suchas our use of serum-free medium), or possibly from differences in thelevel of receptor protein expression or from clonal variation withincell lines. Regardless of the divergence of our results, the physiolog-ical relevance of these different studies lies in the common ability ofTrk receptor kinase activity (of either TrkA or TrkC) to induce

apoptosis in medulloblastomas, in contrast to their canonical survival-promoting effects in normal neurons. Neither morphological evidenceof differentiation nor changes in marker expression were appreciatedin our NT-3-stimulated Daoy-trkC cultures. Similarly, activation ofgrowth factor receptors in other tumor cell lines, including neuronaland glial phenotypes, have been shown to induce or enhance apopto-sis, independent of differentiation (49–52). Our experimental obser-vations of NT-3-induced apoptosis in primary medulloblastoma cul-tures and our correlation of apoptosis withtrkC expression in primarymedulloblastomas are most consistent with the conclusion that apo-ptosis is the predominant biological response to TrkC activation inthese poorly differentiated tumors.

Although our evidence implicates TrkC activation as a modulatorof medulloblastoma growth and survival, other molecules must beconsidered as the initiators of tumorigenesis. One candidate primarydefect in medulloblastoma oncogenesis is mutation ofPTCH, thehuman homologue of theDrosophilasegment polarity gene,patched,which has been identified as the genetic lesion in individuals withGorlin’s syndrome (nevoid basal cell carcinoma syndrome), an auto-somal dominant disorder associated with increased incidence ofmedulloblastomas through loss of heterozygosity in tumors (53–55).Site-directed mutation ofPTCHpromotes the development of medul-loblastomas in mice (56). These data identifyPTCH as a cerebellartumor suppressor gene. Furthermore, theAPC (adenomatous polypo-sis coli) gene has emerged as another candidate tumor suppressor genein medulloblastoma oncogenesis (57).

Together with this report, the evidence to date implies that growth-promoting mutations of genes such asPTCH or APC act in combi-nation with growth-inhibiting actions of TrkC to determine the be-havior of medulloblastomas. This model of medulloblastomaprogression is consistent with other studies, which reveal that apo-ptosis profoundly slows the growth of central nervous system tumors(58, 59). Further investigation will be required to explore the tanta-lizing possibility of using physiological growth factors to enhanceapoptosis in medulloblastomas without the short-term side effects orthe long-term sequelae of conventional treatments.

ACKNOWLEDGMENTS

We thank A. O’Neill and T. Leong of the Dana-Farber Cancer Institute andDr. D. Zurakowski of Children’s Hospital for help with the statistical analysis;A. Carter, S. R. Datta, S. Khoxayo, and J. Kornhauser for advice and technicalassistance; P. Black, J. Madsen, and R. M. Scott for tumor samples; M.Greenberg, J. Blenis, M. Weber, and A. Welcher of Amgen, Inc., and D.Shelton of Genentech, Inc. for reagents; and M. Greenberg and J. Volpe foradvice and reading the manuscript.

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1999;59:711-719. Cancer Res John Y. H. Kim, Mary E. Sutton, Diane J. Lu, et al. in MedulloblastomasActivation of Neurotrophin-3 Receptor TrkC Induces Apoptosis

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