Expression of the Neurotrophin Receptor Trka Down-Regulates Expression and Function of Angiogenic Stimulators in SH-SY5Y Neuroblastoma Cells1

[CANCER RESEARCH 62, 1802–1808, March 15, 2002] Expression of the Neurotrophin Receptor TrkA Down-Regulates Expression and Function of Angiogenic Stimulators in SH-SY5Y Neuroblastoma Cells1

Angelika Eggert, Michael A. Grotzer, Naohiko Ikegaki, Xing-ge Liu, Audrey E. Evans, and Garrett M. Brodeur2 Division of Oncology, The Children’s Hospital of Philadelphia and Department of Pediatrics, The University of Pennsylvania, Philadelphia, Pennsylvania 19104 [A. E., M. A. G., N. I., X-g. L., A. E. E., G. M. B.]; University Children’s Hospital of Essen, 45122 Essen, Germany [A. E.]; and University Children’s Hospital of Zurich, 8032 Zurich, Switzerland [M. A. G.]

ABSTRACT stood, and the signal transduction pathways that regulate VEGF production have yet to be elucidated. Angiogenesis is essential for tumor growth and metastasis and depends Expression of different members of the Trk family (tyrosine kinase on the production of angiogenic factors. Mechanisms regulating the ex- receptors for neurotrophins) plays an important role in the heteroge- pression of angiogenic factors in tumor cells are largely unknown. High neous biological and clinical behavior of NB, the most common expression of the neurotrophin receptor TrkA in neuroblastomas (NBs) is associated with a favorable prognosis, whereas TrkB is mainly expressed extracranial malignant solid tumor of childhood (11, 12). Observa- on aggressive, MYCN-amplified NBs. To investigate the biological effects tions from several independent studies suggest that high expression of of TrkA and TrkB expression on angiogenesis in NB, we examined the TrkA is present in the subset of NB with favorable biological features expression of angiogenic factors in the human NB cell line SY5Y and its and is highly correlated with patient survival (13, 14), whereas ex- TrkA and TrkB transfectants. In comparison with parental SY5Y cells, pression of TrkB is correlated with unfavorable, aggressive NB (12). mRNA and protein levels of the examined angiogenic factors were signif- Expression of TrkA has been shown to mediate differentiation in vitro icantly reduced in SY5Y-TrkA cells, whereas SY5Y-TrkB cells did not (15, 16), whereas TrkB has been associated with enhanced survival of demonstrate a significant change. Conditioned medium of TrkB transfec- NB cells (12, 17). However, no differences in signal transduction tants and parental SY5Y cells induced endothelial cell proliferation and pathways used by TrkA and TrkB have been reported, and hence the migration, but this effect was completely absent in SY5Y-TrkA cells. molecular events underlying the different behavior of TrkA- and TrkA expression also resulted in severely impaired tumorigenicity in a TrkB-expressing NBs in vivo are still unknown. mouse xenograft model and was associated with reduced angiogenic factor High-level expression of angiogenic factors is associated with expression and vascularization of tumors, as determined by immunohis- advanced tumor stage in human NB (18), and evidence suggests tochemistry and an in vivo Matrigel assay. TrkA expression inhibits that higher vascularity correlates with metastasis, MYCN amplifi- angiogenesis and tumor growth in SY5Y NB cells by down-regulation of angiogenic factors, whereas expression of TrkB does not down-regulate cation, unfavorable histology, and poor outcome of NB (19). The the production of these angiogenic factors. The biologically different purpose of this study was to determine whether expression and behavior of TrkA- and TrkB-expressing NBs may be explained in part by biological activity of important angiogenic factors such as VEGF their effects on angiogenesis. and bFGF were influenced by expression of TrkA or TrkB in NB cells in vitro and in vivo.

INTRODUCTION MATERIALS AND METHODS Neovascularization is essential for tumor growth and metastasis formation (1). The development of new blood vessels in tumors Cell Culture and Transfection. SH-SY5Y is a neuronal subclone from the depends on the production of angiogenic factors released from the SK-N-SH NB cell line and has been described previously (20). The NB cell tumor cells and/or stromal cells. Of the known angiogenic factors, two line NB69 was obtained from the cell line bank of The Children’s Hospital of 3 Philadelphia. Full-length TrkA cDNA and K538N cDNA (a TrkA mutant well-characterized peptides, VEGF (2) and bFGF (3), have been defective in the ATP-binding site of the Trk kinase domain) were a generous implicated in the neovascularization of a wide variety of tumors (4, 5). gift from David Kaplan and were cloned into the retroviral expression vector Other angiogenic factors such as VEGF B, VEGF C, Ang1 and Ang2, pLNCX (21). The TrkB cDNA was cloned into the plasmid vector pcDNA3 ␣ and TGF- have also been shown to induce angiogenesis in vitro and (Invitrogen, Carlsbad, CA). All cells were grown at 5% CO2 in RPMI 1640 in vivo (6, 7). One environmental condition known to enhance VEGF (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, L- expression is hypoxia (8). Recent studies have implicated activation of glutamine, and gentamicin (all from Life Technologies, Inc.). Medium was the tyrosine kinase activities of the src family of proto-oncogenes and changed twice a week. The TrkA-WT and K538N constructs were transfected expression of mutant H- or K-ras oncogenes as important in the into the packaging cell line Bing by electroporation, and virus-containing supernatant (10 ml) from these cells was added to LipoTaxi (Stratagene, La induction of VEGF (9, 10). However, mechanisms that regulate ex- Jolla, CA) and used to infect SH-SY5Y cell cultures (22). Transfection of the pression of angiogenic factors in tumor cells are still not well under- TrkB construct was carried out using N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N- trimethylammoniummethyl sulfate (Sigma Chemical Co., St. Louis, MO). Received 9/7/00; accepted 1/18/02. Transfected cells were subjected to selection with 500 ␮g/ml G418 (Sigma The costs of publication of this article were defrayed in part by the payment of page Chemical Co.). Surviving, drug-resistant SY5Y cells were subcloned by lim- charges. This article must therefore be hereby marked advertisement in accordance with ited dilution to obtain single-cell clonal lines. As negative controls, SY5Y cells 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by grants from the Deutsche Krebshilfe and the Kind-Philipp Stiftung (to were infected with a retrovirus bearing the pLNCX empty retroviral vector A. E.), the Wolfson Young Investigator Fund (A. E.), the Jeffrey Miller Neurooncology (SY5Yvec). For a comparison of different cell lines, the TrkA-WT construct Research Fund (M. A. G.), NIH Grant NS 34514 (to G. M. B.), and the Audrey E. Evans and the empty vector were also transfected into NB69 NB cells as described Endowed Chair (G. M. B.). above. The identity of all transfectants was confirmed by sequencing after 2 To whom requests for reprints should be addressed, at Division of Oncology, ARC, Suite 902D, The Children’s Hospital of Philadelphia, 3516 Civic Center Boulevard, transfection. Philadelphia, PA 19104-4318. RT-PCR. The mRNA Expression of TrkA, TrkB and angiogenic factors 3 The abbreviations used are: VEGF, vascular endothelial growth factor; NB, neuro- was analyzed by RT-PCR using specific biotinylated primers (sequences ␣ blastoma; bFGF, basic fibroblast growth factor; Ang, angiopoietin; TGF- , transforming available upon request). Total RNA was extracted with the RNeasy Kit growth factor ␣; RT-PCR, reverse transcription-PCR; HUVEC, human umbilical vein endothelial cell; CM, conditioned medium (media); NGF, nerve growth factor; EC, (Qiagen, Valencia, CA), reverse transcribed and amplified for 20 cycles on a endothelial cell; GSI, Griffonia simplicifolia I. PTC-100 Programmable Thermal Controller (MJ Research, Inc., Watertown, 1802

MA) using the Superscript amplification system (Life Technologies, Inc.). The RESULTS PCR products were run on a 6% polyacrylamide gel and transferred to a nylon membrane (Hybond Nϩ, Amersham, IL). Biotinylated signals were detected Trk Expression and Autophosphorylation of Stable SY5Y using the Southern Light Detection system (Tropix) and exposed to X-ray film. Transfectants. We stably expressed full-length constructs of TrkA, Target gene expression was normalized to the coamplified housekeeping gene TrkB, a kinase-inactive TrkA mutant (K538N), and the empty vector GAPD as described (23). control (Vec) in the human NB cell line SH-SY5Y. Expression of Determination of VEGF and bFGF Protein Levels in CM. Cell cultures functional Trk receptors was examined by RT-PCR and immunoblot- were grown at 80% confluency for 24 h in serum-free RPMI medium and, ting. The SY5Y-TrkA and SY5Y-TrkB cell lines demonstrated high ␮ where indicated, 100 M desferoxamine mesylate to mimic hypoxia (24). Cell and comparable levels of expression of the inserted receptor on supernatants were collected, centrifuged at 3000 rpm for 10 min and stored at mRNA and protein levels (data not shown). Each receptor was auto- –80°C. Concomitantly, cell pellets were harvested and cell number was phosphorylated at a low level even before the addition of ligand. A determined by counting the cells in a hemocytometer. The amount of VEGF and strong increase in autophosphorylation of the receptor was seen after bFGF protein in the CM was determined with ELISA kits (R&D Systems, Minneapolis, MN). VEGF and bFGF were expressed as pg protein/106cells/24 h. addition of the appropriate ligand NGF or brain-derived neurotrophic Endothelial Proliferation and Migration Assay. HUVECs (Clonetics factor to SY5Y-TrkA and SY5Y-TrkB cells, respectively (data not Corp., Walkersfield, MD) were plated in 24-well tissue culture plates at a shown). In contrast, there was no detectable Trk expression or auto- concentration of 5.0 ϫ 104 cells/well in medium containing optimal concen- phosphorylation in SY5Y-Vec or untransfected SY5Y cells. SY5Y- trations of growth factors (EGM medium; Clonetics Corp.). After starving the K538N showed high expression of the TrkA receptor on mRNA and cells overnight in basic medium containing no growth factors (EBM medium; protein levels, but no autophosphorylation was demonstrated. Clonetics Corp.), HUVECs were incubated at 37°C for 96 h in CM of parental Decrease in Angiogenic Factor mRNA Levels due to Expression and transfected SY5Y cell clones (50-fold concentrated supernatant, diluted of TrkA. We analyzed the effect of TrkA and TrkB on expression of ϩ 1:3 in EBM 1% fetal bovine serum). After incubation, HUVECs were VEGF, VEGF B, VEGF C, bFGF, Ang1, Ang2, and TGF-␣ by stained and counted as described previously (25). To evaluate their capacity to semiquantitative RT-PCR. SY5Y-TrkA cells demonstrated a clear block EC proliferation induced by SY5Y-CM, specific antibodies against decrease in mRNA expression of the most abundant VEGF isoforms, VEGF and bFGF (Santa Cruz Biotechnology, Santa Cruz, CA) were preincubated with SY5Y-CM. Preincubation with normal mouse IgG (Vector Labo- VEGF165 and VEGF121, and Ang1, in comparison with parental ratories, Burlington, CA) was used as control. To determine the ability of NB SY5Y and SY5Y-Vec cells. Even more striking was the effect of CM to activate migration of ECs, we used Transwell inserts (Costar) contain- TrkA transfection on mRNA expression of bFGF and Ang2. Whereas ing a polycarbonate membrane with 8-␮m pores as described previously (26). parental SY5Y and SY5Y-Vec cells expressed high levels of bFGF Migration was quantitated by counting the cells that migrated to the bottom of and Ang2 mRNA, we could not detect these mRNAs in SY5Y-TrkA the membrane in 10 high power fields at ϫ320 magnification. cells (Fig. 1). VEGF C was not expressed in any of the cell clones, and In Vivo Tumor Growth. SY5Y, SY5Y-TrkA, and SY5Y-TrkB cells were VEGF B was expressed at similar moderate levels in all of the cell cultured to 80% confluence, harvested, and resuspended in Matrigel (Collab- clones examined (data not shown). SY5Y-TrkB cells expressed orative Biomedical Products, Bedford, MA). Four-week-old female athymic slightly decreased mRNA levels of Ang2 and slightly increased NCR (nu/nu) mice (National Cancer Institute, Frederick, MD) were inoculated mRNA levels of VEGF and Ang1 (Fig. 1), but no other changes in s.c. in the flank with 2.5 ϫ 107 cells/inoculate as described previously (27). 121 angiogenic factor expression were observed. Transfection of SY5Y Tumors were measured two times a week, and calculations of tumor volume were made by multiplying the product of three dimensions by ␲/6. Harvested with the kinase-inactive TrkA mutant K538N resulted in decreased tumors were cut into two fragments: one part was fixed in formalin for immunohistochemistry; and one part was snap-frozen in liquid nitrogen to obtain RNA. Immunohistochemistry. Three different randomly chosen xenografts of SY5Y and SY5Y-TrkB as well as the two available TrkA xenografts were analyzed by immunohistochemistry. Tumor specimens were sectioned (5 ␮m) and processed for standard immunohistochemical staining. Anti-VEGF rabbit polyclonal antibody A-20 (Santa Cruz Biotechnology) was used at a 1:400 dilution, anti-bFGF rabbit polyclonal antibody (Sigma Chemical Co.) was used at a 1:200 dilution, and anti-Ki-67 rabbit polyclonal antibody NCLki67p (Novocastra Laboratories, New Castle, United Kingdom) was used at a 1:1600 dilution. After incubation with an antirabbit secondary antibody, the immune complex was visualized with the StrAviGen Super Sensitive kit (BioGenex Laboratories, San Ramon, CA) using diaminobenzidine tetrahydrochloride as a chromogen. Sections were counterstained with hematoxylin. Microvessel staining was performed by EC labeling with GSI lectin (Sigma Chemical Co.) at a 1:10 dilution as described previously (28). Evaluation of immunostained sections was carried out independently by two observers. The intensity of VEGF, bFGF, and GSI lectin staining in representative tissue sections was graded on a scale of 0–3ϩ. The proliferation index (number of Ki-67-positive cells/100 neoplastic cells) was determined by counting the percentage of Ki-67-positive cells in at least 1000 tumor cells at ϫ400 magnification in the most densely stained region. Matrigel Assay for Angiogenesis. In vivo angiogenesis assays were car- Fig. 1. Expression of TrkA down-regulates angiogenic factors on mRNA levels in ried out by injecting athymic NCR (nu/nu) mice with 0.5 ml of Matrigel SH-SY5Y cells. A representative example of semiquantitative RT-PCR showing expres- 7 (Collaborative Biochemical Products) alone or mixed on ice with 10 SY5Y, sion levels of the angiogenic factors VEGF (VEGF165 and VEGF121), bFGF, Ang1, Ang2, SY5Y-TrkA, or SY5Y-TrkB cells as the angiogenic stimulus. Matrigel was and TGF-␣ in the indicated cell clones is shown. Expression of the housekeeping gene injected s.c. in the abdominal midline on day 0 in all experiments as described GAPD was used as internal control. Lower expression levels were observed of all analyzed angiogenic factors in TrkA-transfected SY5Y cells. Expression levels shown are previously (29). Matrigel plugs were harvested on day 11 for hemoglobin the representative results of three independent experiments with at least two different cell assay. Quantitation of hemoglobin was performed by the Drabkin method (30). clones. 1803

SY5Y-TrkA Cells Have Diminished Ability to Stimulate HUVEC Proliferation and Migration in Vitro. One of the initial steps of tumor-induced neovascularization is EC proliferation and movement toward the chemoattractive stimuli produced by the tumor cells. Therefore we sought to determine whether the expression of TrkA affected the ability of SY5Y cells to activate endothelial proliferation and migration by factors secreted into the CM. The data shown in Fig. 3, A and B, indicate that CM from the parental cell line SY5Y, SY5Y-Vec, SY5Y-K538N, or SY5Y- TrkB were highly stimulatory for HUVEC proliferation, whereas CM from TrkA-expressing SY5Y cell clones demonstrated mini- mal stimulation, regardless of the presence of NGF. For a comparison of different cell lines, we also analyzed the effect of CM obtained from TrkA-transfected NB69 NB cells on EC prolifera- Fig. 2. Expression of TrkA in SY5Y cells reduces VEGF protein levels in the supernatant. The indicated cell lines were cultured with serum-free medium for 24 h under tion. Whereas CM of this cell line transfected with the empty normoxic and hypoxic conditions. TrkA- and TrkB-transfected SY5Y cells were cultured vector control was highly stimulatory for EC proliferation, CM of in the absence and presence of their specific ligands (NGF and brain-derived neurotrophic NB69-TrkA cells did not stimulate EC proliferation (data not factor) as indicated. VEGF concentrations in the CM were determined by specific VEGF ELISA and are expressed as pg/106 cells/24 h. Shown are the representative results of one shown). The notion that VEGF is the major element in the CM that of three different clones of transfected cells tested. Data points are the mean of triplicates. promoted HUVEC proliferation was tested by including a VEGF- Ͻ p f SDs were 10%. , normoxic conditions; , hypoxic conditions. neutralizing polyclonal antibody in the CM of SY5Y cells during the proliferation assay (Fig. 3B). This antibody completely blocked the stimulation of EC proliferation elicited by CM of SY5Y. In expression of Ang2, but expression of all other angiogenic factors was contrast, a bFGF-neutralizing monoclonal antibody added to the comparable with that seen in parental SY5Y cells. CM of SY5Y cells only reduced the stimulatory effect on HUVEC Significant Reduction of VEGF and bFGF Protein Levels in proliferation to 50%. No effect was seen by the addition of normal Cell Supernatants of SY5Y-TrkA Cells. Parental SY5Y and mouse IgG. These data suggest that VEGF is the major component SY5Y-Vec cells produced high quantities of VEGF (ϳ700 pg/106 in the CM of NB cells that stimulates HUVEC proliferation and cells/24 h) but less bFGF protein (ϳ3 pg/106 cells/24 h). SY5Y- migration. An endothelial migration assay produced results similar TrkB cells produced increased amounts of VEGF and bFGF pro- to those of the proliferation assay (data not shown). tein in their supernatants in comparison with parental cells. Inter- TrkA Mediates Inhibition of in Vivo Tumor Growth and Vas- estingly, a 4.6-fold decrease in the production of VEGF protein cularity. Having demonstrated that down-regulation of angiogenic was observed in the supernatants of SY5Y-TrkA cells compared factor production by expression of TrkA had substantial in vitro with parental SY5Y cells (Fig. 2). Secretion of bFGF protein into effects on HUVEC proliferation and migration, we next sought to CM of TrkA transfectants was virtually undetectable (data not determine the effects on in vivo behavior. We implanted parental shown). The presence of exogenous NGF did not significantly SY5Y cells as well as TrkA- and TrkB-transfected SY5Y cells s.c. change the protein levels of VEGF or bFGF produced by SY5Y- in the flank of athymic nude mice. Cells from parental SY5Y and TrkA cells, suggesting that activation of TrkA by exogenous NGF SY5Y-TrkB clones formed tumors with similar kinetics and of might not be required for the effect of TrkA on angiogenic factor similar size 15–20 days after injection, whereas tumor growth was expression. Corresponding to the mRNA expression levels, VEGF remarkably suppressed for SY5Y-TrkA cells (Fig. 4). Injection of up to 5 ϫ 108 and bFGF protein levels in the kinase-inactive TrkA mutant SY5Y-TrkA cells into 24 mice resulted in the growth of only 2 small tumors (tumor take, 8%), and these started to grow K538N were comparable with the levels seen in parental SY5Y only after a prolonged latency period of about 90 days (data not cells, indicating that a functional TrkA receptor molecule is re- shown). One of these tumors (TrkA1) demonstrated a very slow quired for the inhibiting effect on angiogenic factor production. growth behavior, remained small for several weeks, and appeared Because hypoxic stimulation of VEGF has been described previ- macroscopically less vascular than TrkB and SY5Y tumors when it ously in NB (31), we examined the response to hypoxia in parental was harvested after 120 days. Four additional SY5Y-TrkA xe- and transfected SY5Y cell clones. Hypoxia-like conditions (mimicked nografts formed small nodules that were barely palpable and re- by the addition of desferoxamine mesylate) resulted in a 6–10-fold mained in a dormant state for Ͼ6 months. All other TrkA xe- up-regulation of VEGF protein production in all cell clones (Fig. 2) nografts failed to form tumors altogether (18 of 24 mice). In including the TrkA transfectants. In SY5Y-TrkA cells, the hypoxia- comparison, SY5Y and SY5Y-TrkB cells had a tumor take of 83% induced up-regulation of the very low basal VEGF protein levels and 100%, respectively. resulted in measurable amounts of VEGF, but the corresponding CM Fig. 5 shows the results of immunostaining for VEGF and bFGF of SY5Y and SY5Y-TrkB cells still contained about 6-fold more and staining for ECs in representative tumor sections of SY5Y-, immunodetectable VEGF. Up-regulation of protein production was TrkA-, and TrkB-transfected cells. SY5Y and TrkB transfectants are even more striking for bFGF. Whereas SY5Y, SY5Y-Vec, SY5Y- highly positive for VEGF and bFGF protein. In contrast, the slowly TrkB, and SY5Y-K538N demonstrated a 20–30-fold induction under growing TrkA1 tumor is largely negative for VEGF and bFGF. hypoxia-like conditions, SY5Y-TrkA cells only produced an 8–10- Consistent with this pattern, a reduction in the number of microvessels fold induction of basal bFGF protein levels (data not shown). This in the TrkA1 tumor versus the SY5Y and TrkB tumors was also resulted in 20–25-fold reduced bFGF protein levels in TrkA super- observed (Fig. 5). The proliferation index was determined by Ki-67 natants under hypoxic conditions. Thus, hypoxic CM can also induce staining. Ki-67-immunoreactive cells were scattered throughout the VEGF and bFGF protein secretion in SY5Y-TrkA cells, but the tumors, and the Ki-67 proliferation indices were similar in SY5Y, magnitude of the effect is markedly reduced in comparison with that SY5Y-TrkA, and SY5Y-TrkB xenografts (data not shown). To con- seen in parental SY5Y cells. firm the results obtained by immunostaining, an alternative method 1804

Fig. 3. Effect of NB cell supernatant on the proliferation of HUVECs. Parental and transfected SY5Y cells were cultured in serum-free media for 24 h under normoxic conditions. Supernatants were concentrated 50-fold and diluted 1:3 with basic EBM medium. HUVECs were cultured in the indicated CM, and cells were counted after 96 h. A, representative example of HUVECs cultured with SY5Y CM (bottom left) and CM of TrkA transfectants (bottom right) in comparison with full EGM medium (top left; containing EC growth factors) and basic EBM medium (top right; containing no growth factors). Phase-contrast microscopy, ϫ320. B, HUVEC proliferation in relation to control media. HUVEC counts cultured in full EGM medium as positive control were determined to be 100%, and HUVEC counts cultured in basic EBM medium were determined to be 0%. HUVEC proliferation of the tested CM is expressed as a percentage of HUVEC proliferation in full medium. For additional positive controls, recombinant human VEGF (100 ng/ml) or recombinant human bFGF (10 ng/ ml) was added to basic EBM medium. SY5Y CM was preincubated with specific anti-VEGF or anti- bFGF monoclonal antibodies to test their neutralizing effect on HUVEC proliferation.

was used to assess tumor angiogenesis. Matrigel implants were used Inclusion of SY5Y or SY5Y-TrkB cells in the Matrigel implant as an in vivo assay to quantitate vascularization by measuring the provided an angiogenic stimulus that made the implant visibly vas- hemoglobin content of the Matrigel plugs. Matrigel implants without cularized and resulted in a hemoglobin content of 8–12 mg/g Matrigel additives were pale and unvascularized 11 days after implantation. (Fig. 6). On the other hand, inclusion of SY5Y-TrkA cells in Matrigel implants resulted in significantly less vascularization (ϳ4 mg hemoglobin/g Matrigel), indicating that SY5Y-TrkA cells are less angiogenic in vivo. Revertant Cells Regain Their Angiogenic and Tumorigenic Properties after Loss of TrkA Expression. One mouse in the group that had been inoculated with the TrkA-transfected cells developed a tumor (TrkA2) that grew with kinetics similar to SY5Y and TrkB tumors after a prolonged latency period of 90 days. RT-PCR revealed an absence of TrkA expression in this tumor, comparable with parental SY5Y tumors, indicating that the cells had genetically reverted and lost their TrkA expression. Interestingly, VEGF and bFGF mRNA Fig. 4. Tumor growth in nude mice. SY5Y, SY5Y-TrkA, and SY5Y-TrkB cells levels (Fig. 7) and protein levels (Fig. 5) had returned to levels (2.5 ϫ 107) in Matrigel were injected s.c. in the flank of nude mice (6 mice each for SY5Y comparable with those seen in parental SY5Y cells (Fig. 7). Also, and TrkB; 24 mice for TrkA). Tumor volumes were estimated at the indicated times after implantation, and data are shown as the mean of each group. The experiment was microvessel density was similar to SY5Y and TrkB tumors in this terminated when tumors reached a volume of 300–400 mm3. revertant TrkA tumor. Thus, these tumor cells recovered their tumor- 1805

Fig. 5. Immunohistochemistry of NB xenografts. Immunohistochemistry of xenografts formed in nude mice by parental SY5Y cells (a, e, and i), SY5Y-TrkB cells (b, f, and j), SY5Y-TrkA1 cells (c, g, and k) and revertant SY5Y-TrkA2 cells (d, h, and l). Staining with anti-VEGF (a–d), anti-bFGF (e–h), and GSI lectin (i–l) is shown. Magnification, ϫ100.

igenicity and angiogenic capacity concomitantly with the loss of TrkA expression.

DISCUSSION Expression of TrkA and TrkB is generally thought to contribute to NB tumor biology primarily through effects on cell differentiation and proliferation (12, 13). However, the molecular events underlying the different behaviors of TrkA- and TrkB-expressing NBs in vivo are largely unknown. Aggressive NBs with poor outcomes are associated with higher vascularity in the tumors, and therefore angiogenesis is presumably controlled by genetic alter- Fig. 6. Vascularization of Matrigel implants. Mice were injected s.c. with 0.5 ml of Matrigel either alone or mixed with 107 tumor cells of SY5Y, TrkA transfectants, or TrkB ations in oncogene and tumor suppressor gene expression. We transfectants, as indicated. Eleven days later, the Matrigel implants were harvested, and hypothesized that expression of TrkA or TrkB might not only their hemoglobin content was assayed. Each group contained five mice whose implants were assayed separately. Bars, SDs. The hemoglobin content of Matrigel plugs with TrkA affect differentiation and proliferation of NB cells but might also transfectants is significantly less than the hemoglobin content of Matrigel plugs with be involved in the regulation of tumor angiogenesis. parental SY5Y cells or TrkB transfectants. 1806

to be elucidated, is the existence of a different, NGF-independent mechanism by which TrkA down-regulates expression of angiogenic factors. Is a 4-6-fold Suppression of VEGF a Sufficient Explanation for the Profound Antitumor Effect of TrkA? We demonstrated a 4-6- fold suppression of VEGF protein in TrkA transfectants and observed a profound antitumor effect. Studies in VEGF knockout mice have shown that disruption of only a single VEGF allele, equivalent to 50% reduction of VEGF protein, is sufficient to block vasculogenesis and angiogenesis to such an extent that embryos die between days 11 and 12 of gestation (32). Induced suppression of VEGF protein expression by antisense methods in a human glioblastoma by only 3-fold can almost completely obliterate the tumorigenic ability of such cells in nude mice (33). Thus, relatively small reductions in VEGF can lead to rather profound suppression of angiogenesis. However, we cannot rule out mechanisms other than down-regulation of angiogenic factors by which TrkA might influence angiogenesis and invasiveness of NBs. Changes in the net balance of angiogenic inhibitors and activa- Fig. 7. Expression of TrkA and angiogenic factors on mRNA levels in mouse tors directly affect vascularity, tumor growth, and metastasis. The fact xenografts. Representative example of semiquantitative RT-PCR shows expression levels that the inverse correlation of TrkA expression and expression of of TrkA in comparison with the angiogenic factors VEGF (VEGF165 and VEGF121) and angiogenic factors such as VEGF and bFGF was not significant in 37 bFGF in mouse xenografts of parental SY5Y cells, TrkB transfectants, and TrkA transfectants. Expression of the housekeeping gene GAPD was used as internal control. Low primary NBs (18) also suggests that mechanisms other than down- expression levels of VEGF and bFGF were observed in the xenograft with high TrkA regulation of angiogenic factors are involved in the effect of TrkA on expression. Lost TrkA expression in the second TrkA xenograft corresponds to restored high mRNA expression levels of VEGF and bFGF. angiogenesis. We are currently analyzing the expression of angiogenic inhibitors in the CM of TrkA-expressing cells. Additional events involved in tumor progression include secretion of matrix-degrading Expression of TrkA Inhibits Angiogenesis in Human SH-SY5Y enzymes by tumor (25); therefore, this should be examined further. NB Xenografts. The results of our study indicate that TrkA overex- Another mechanism by which TrkA might affect tumor growth is to pression in human NB cells markedly inhibits angiogenesis. We influence rates of tumor cell proliferation, as shown previously in provide several lines of evidence for this TrkA-mediated effect on some in vitro studies. However, this effect of Trk receptor expression angiogenesis. We demonstrate (a) a decrease in the mRNA expression on proliferation is not evident in vivo, as shown by similar Ki-67 of several angiogenic factors, (b) a corresponding significant reduc- proliferation indices in SY5Y, SY5Y-TrkB, and SY5Y-TrkA tumors. tion of VEGF and bFGF protein secretion under normoxic and hy- This suggests that the different effect of TrkA and TrkB on angio- poxic conditions, (c) a diminished ability of TrkA transfectants to genesis might be more important than proliferation effects for the stimulate EC proliferation and migration in vitro,(d) a diminished tumor biology and prognosis of NB. ability to induce angiogenesis in vivo, and (e) reversion of these Comparison with TrkA Data Obtained in a Different Cell Line effects after loss of TrkA expression in one tumor. However, there are and in Primary NBs. We demonstrated previously that high-level several additional observations and questions raised by our study. expression of angiogenic factors is associated with advanced tumor Quantification of Tumor Angiogenesis. There was only one stage in NB (18). To examine whether the phenomena observed in TrkA tumor available in our experiments that was not revertant and SY5Y cells also occur in primary NB tumors, we compared expres- suitable for immunohistochemistry. However, this tumor is most sion levels of angiogenic factors with expression levels of TrkA in likely not representative because it is the only TrkA-expressing tumor that study. Although there was a tendency toward inverse correlation growing beyond a size of 2 mm3 of 24 injected xenografts. To of TrkA expression and angiogenic factor expression, the correlation complement the studies regarding quantification of angiogenesis, we did not reach statistical significance. This might be due to the small used Matrigel implants as an in vivo assay for tumor angiogenesis and number of primary tumors examined (37) in the study panel. Another quantitated vascularization by measuring hemoglobin content (29). explanation may be that not only down-regulation of angiogenic The results of this assay indicated that significant inhibition of angio- stimulators but also up-regulation of angiogenic inhibitors might be genesis was indeed the cause of different tumorigenicity of SY5Y- involved in the inhibitory effect of TrkA on angiogenesis. This hy- TrkA cells in comparison with SY5Y and SY5Y-TrkB cells. pothesis is supported by preliminary data suggesting that EC prolif- Addition of Exogenous NGF Is Not Required for the Inhibitory eration in the CM of SY5Y-TrkA cells is still not stimulated in the Effect of TrkA on Angiogenesis. The presence of exogenous NGF presence of recombinant VEGF or bFGF (data not shown). We are was not required for the reduction of angiogenic factors in SY5Y- currently trying to identify angiogenic inhibitors in the CM of SY5Y- TrkA cells. SY5Y cells express low amounts of endogenous NGF as TrkA cells. The comparable effects of CM obtained from SY5Y-TrkA determined by RT-PCR (data not shown), and we observed a low level and NB69 NB cells on EC proliferation indicate that the observed of autophosphorylation in TrkA- and TrkB-transfected cells in the results are not just phenomena seen in a single cell line. absence of exogenous ligands even in serum-free medium. A func- VEGF Is the Major Angiogenic Factor Produced by NB Cells. tional, activated TrkA receptor molecule is required for the reduction VEGF and bFGF have a potent and synergistic effect on induction of of angiogenic factor production because the kinase-inactive TrkA angiogenesis, but VEGF is currently regarded as the major angiogen- mutant K538N was not capable of mediating an inhibitory effect on esis stimulator for most types of human cancer (34). Our data suggest angiogenesis. This fact supports the hypothesis that the low level of that VEGF is also the major angiogenic factor in NB cells because the autophosphorylation seen in TrkA-transfected cells even in the ab- angiogenic effect of NB CM was completely blocked by anti-VEGF, sence of exogenous NGF might be sufficient to cause the inhibitory but not by anti-bFGF. These results are in agreement with the obser- effects of TrkA on angiogenesis. Another possibility, which remains vations of Ribatti et al. (25), who demonstrated the in vivo angiogenic 1807

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2002 American Association for Cancer Research. TrkA INHIBITS ANGIOGENESIS IN NEUROBLASTOMA capacity of CM from two NB cell lines, which could also be blocked and favorable outcome in human neuroblastomas. N. Engl. J. Med., 328: 847–854, by a neutralizing anti-VEGF antibody. However, these results need to 1993. 14. Kogner, P., Barbany, G., Dominici, C., Castello, M., Raschella, G., and Persson, H. be confirmed in a larger panel of NB cell lines and tumors. Coexpression of messenger RNA for Trk proto-oncogene and low affinity nerve Further Evidence for the Involvement of Tyrosine Kinase Re- growth factor receptor in neuroblastomas with favorable prognosis. Cancer Res., 53: ceptors in Angiogenesis. Results obtained in other cell systems also 2044–2050, 1993. 15. Matsushima, H., and Bogenmann, E. Expression of TrkA cDNA in neuroblastomas are consistent with our data. 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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2002 American Association for Cancer Research. Cancer Correction Research Correction: Expression of the Neurotrophin Receptor TrkA Down-Regulates Expression and Function of Angiogenic Stimulators in SH-SY5Y Neuroblastoma Cells

In this article (Cancer Res 2002;62:1802–8), which was published in the March 15, 2002, issue of Cancer Research (1), the authors did not cite a previously published article from Medical and Pediatric Oncology (2) as the original source of Fig. 2 as well as portions of the Introduction. The Medical and Pediatric Oncology article, which was published as part of a Meeting Proceeding for the 2000 Advances in Neuro- blastoma Research meeting, was not peer reviewed and the authors considered it to be an extended abstract. The authors are correcting this oversight here.

References 1. Eggert A, Grotzer MA, Ikegaki N, Liu X, Evans AE, Brodeur GM. Expression of the neurotrophin receptor TrkA down-regulates expression and function of angiogenic stimulators in SH-SY5Y neuroblastoma cells. Cancer Res 2002;62:1802–8. 2. Eggert A, Grotzer MA, Ikegaki N, Liu X, Evans AE, Brodeur GM. Expression of neurotrophin receptor TrkA inhibits angiogenesis in neuroblastoma. Med Pediatr Oncol 2000;35:569–72.

Published OnlineFirst February 27, 2012. doi: 10.1158/0008-5472.CAN-12-0556 Ó2012 American Association for Cancer Research.

1588 Cancer Res; 72(6) March 15, 2012 Expression of the Neurotrophin Receptor TrkA Down-Regulates Expression and Function of Angiogenic Stimulators in SH-SY5Y Neuroblastoma Cells

Angelika Eggert, Michael A. Grotzer, Naohiko Ikegaki, et al.

Cancer Res 2002;62:1802-1808.

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