SPARC Is a Key Schwannian-Derived Inhibitor Controlling Neuroblastoma Tumor Angiogenesis1

[CANCER RESEARCH 62, 7357–7363, December 15, 2002] SPARC Is a Key Schwannian-derived Inhibitor Controlling Neuroblastoma Tumor Angiogenesis1

Alexandre Chlenski, Shuqing Liu, Susan E. Crawford, Olga V. Volpert, George H. DeVries, Amy Evangelista, Qiwei Yang, Helen R. Salwen, Robert Farrer, James Bray, and Susan L. Cohn2 Departments of Pediatrics [S. L. C.] and Pathology [S. E. C.] and The Robert H. Lurie Comprehensive Cancer Center [A. C., S. L., O. V. V., A. E., Q. Y., H. R. S., J. B.], Northwestern University, Feinberg School of Medicine, Chicago, Illinois 60611, and the Department of Cell Biology, Neurobiology, and Anatomy, Loyola University of Chicago Stritch School of Medicine, Chicago, Illinois 60153 [G. H. D., R. F.]

ABSTRACT NB tumors consist of two main cell populations, neuroblastic/ ganglionic cells and Schwann cells (24). The ratio of these cell types Neuroblastoma (NB), a common pediatric neoplasm, consists of two varies according to tumor maturation. Immature NB tumors are com- main cell populations: neuroblastic/ganglionic cells and Schwann cells. NB posed of undifferentiated neuronal cells and a paucity of Schwannian tumors with abundant Schwannian stroma display a more benign clinical behavior than stroma-poor tumors. Recent studies suggest that Schwann stroma, whereas larger, ganglion-like cells and abundant Schwannian cells influence NB tumor growth via secreted factors that induce differ- stroma are seen in maturing NB tumors. The Schwann cells are entiation, suppress proliferation, and inhibit angiogenesis. Two angiogen- thought to be normal cells that infiltrate the NB tumor (25), although esis inhibitors, pigment epithelium-derived factor and tissue inhibitor of a recent report suggests that the Schwann cells may be malignant (26). metalloproteinase-2, have been detected in Schwann cell secretions. Here, The favorable prognostic impact of the presence of Schwannian we isolated another Schwann cell-derived secreted inhibitor of angiogen- stroma has been emphasized in the pathological classification system esis, a 43-kDa protein identified as SPARC (secreted protein acidic and of Shimada et al. (24) and the International NB Pathology Classifi- rich in cysteine), an extracellular matrix protein. We found SPARC to be cation System (7). It has been speculated that Schwann cells influence critical for the antiangiogenic phenotype of cultured Schwann cells. We NB tumor growth by secreting molecules that serve as antiprolifera- also show that purified SPARC potently inhibits angiogenesis and signif- tive and differentiating factors for neuronal cells (25, 27, 28). icantly impairs NB tumor growth in vivo. SPARC may be an effective candidate for the treatment of children with clinically aggressive, Schwan- Schwann cells also produce a spectrum of angiogenesis inhibitors (29, nian stroma-poor NB tumors. 30), suggesting that Schwann cells may also influence NB growth by restricting angiogenesis. INTRODUCTION The angiogenesis inhibitors PEDF and TIMP-2 have been previously found in media conditioned by Schwann cells, and both factors NB,3 a common pediatric neoplasm that arises from neural crest appear to contribute to the antiangiogenic activity of the SCM (29, tissue, has a broad spectrum of clinical behavior (1–4). Although 30). In this study, we isolated an additional angiogenic inhibitor in numerous factors including stage (5), patient age (6), tumor histology SCM, identified as SPARC. SPARC, also known as osteonectin, (7), molecular markers (8, 9), and genetic abnormalities (10–13) have BM-40, and 43K protein, is a highly conserved calcium-binding been shown to be predictive of outcome in children with NB, the matricellular glycoprotein (31–33). SPARC is spatially and tempo- mechanisms responsible for the highly variable clinical behavior of rally regulated during development, and it is transiently expressed in NB remain largely unknown. Several recent studies implicate angio- derivatives of the three primitive germ layers in mouse embryos (34). genesis in the regulation of NB growth. In primary NB tumors, high This glycoprotein is highly expressed in bone and in basement mem- vascular index correlates with MYCN amplification, metastases, and branes as well as in a variety of cell types associated with remodeling poor outcome, whereas low tumor vascularity is associated with tissues and high cellular turnover (35). Although its precise function a better prognosis, localized stage, and favorable histology (14). is unclear, SPARC plays a modulatory role in cell-matrix interactions Advanced-stage NB is associated with high levels of angiogenic (36). SPARC induces cell rounding, blocks cell spreading and adhe- ␣ ␤ ␣ ␤ stimuli and v 3 and v 5 integrins, both markers of active angio- sion, and inhibits endothelial cell migration (37–39). SPARC also genesis (15, 16). Overexpression of exogenous MYCN results in appears to contribute to vascular morphogenesis and cellular differ- enhanced malignant growth of NB cells and reduced levels of activin entiation (36). However, the contradictory reports regarding the role A, an inhibitor of angiogenesis (17). Expression of the neurotrophin of SPARC in cell growth and tumor formation (40–42) suggest that receptor TrkA also causes down-regulation of angiogenesis stimula- its effects are cell type specific and may be dependent on concentra- tors and impaired tumorigenicity in a mouse xenograft model (18). tion, extracellular matrix components, and the ability of the cell to Furthermore, preclinical studies have shown effective reduction of NB proteolyze SPARC (42–44). tumor growth in vivo by a variety of antiangiogenesis agents (16, We report that SPARC expression is inversely correlated with the 19–23). degree of malignant progression in NB tumors. We also demonstrate that SPARC is one of the key contributors to the antiangiogenesis Received 7/12/02; accepted 10/10/02. activity of the SCM. Purified SPARC blocks angiogenesis in vitro and The costs of publication of this article were defrayed in part by the payment of page in vivo and significantly impairs NB tumor growth in vivo. Our charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. observations stress the cumulative nature of the angiogenic equilib- 1 Supported in part by the Neuroblastoma Children’s Cancer Society, Friends for rium in NB tumors and suggest that a shift in the angiogenic balance Steven Pediatric Cancer Research Fund, the Elise Anderson Neuroblastoma Research may be prompted by even slight disturbances in the complex array of Fund, the North Suburban Medical Research Junior Board, and the Robert H. Lurie Comprehensive Cancer Center NIH National Cancer Institute Core Grant 5P30CA60553. inhibitors and stimuli in the endothelial cell microenvironment. 2 To whom requests for reprints should be addressed, at Children’s Memorial Hospital, Division of Hematology/Oncology, Box 30, 2300 Children’s Plaza, Chicago, IL 60614. Phone: (773) 880-4562; Fax: (773) 880-3053; E-mail: [email protected]. MATERIALS AND METHODS 3 The abbreviations used are: NB, neuroblastoma; PEDF, pigment epithelium-derived factor; TIMP-2, tissue inhibitor of metalloproteinase-2; SCM, Schwann cell-conditioned Cell Culture and CM Collection. Primary human Schwann cells were media; RA, retinoic acid; BrdUrd, 5-bromo-2Ј-deoxyuridine; CM, conditioned media; MVD, microvascular density; bFGF, basic fibroblast growth factor; RT-PCR, reverse purified from adult nerves or from Schwannian stroma-dominant NB tumors transcription-PCR. and expanded as described previously (29, 30, 45). Tumor-derived Schwann 7357

cells were maintained at 5% CO2 in DMEM (Life Technologies, Inc., Grand magnification, and the average MVD counted in the 10 fields was reported as Island, NY) supplemented with 10% fetal bovine serum (US Biotechnologies, MVD/mm2. Inc., Parkerford, PA), 50 ng/ml recombinant human heregulin ␤1 (R&D In Vitro Angiogenic Assay. Migration assays were performed with human Systems, Minneapolis, MN), 1% penicillin/streptomycin, 2.5 ␮g/ml ampho- umbilical vein endothelial cells [National Cancer Institute Preclinical Repos- tericin, 0.5 ␮M isobutylmethylxanthine (Sigma, St. Louis, MO), and 0.5 ␮M itory (Bethesda, MD) and VEC Technologies (Rensselaer, NY)] as described forskolin (Sigma). NB cell lines used in this study have been described previously (29). Test substances were assayed in Opti-MEM media (Life previously (46–49), with the exception of NBL-L and NBL-R, MYCN-ampli- Technologies, Inc.) with or without 3 ng/ml bFGF (National Cancer Institute fied lines established in our laboratory from clinically aggressive NB tumors. Preclinical Repository). Purified human platelet osteonectin (referred to hereafter as SPARC) was obtained from Calbiochem (San Diego, CA). To generate NB cells were grown at 5% CO2 in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and antibiotics. For some experi- dose-response curves, the data were normalized as percentage of maximum ments, 10 ␮M all-trans-RA (Sigma) or 6 ␮M BrdUrd (Sigma) was added where migration using the difference between bFGF/Opti-MEM-induced migration indicated, and the cells were harvested at the indicated time intervals. CM from and background migration in Opti-MEM alone as 100% control. For some NB cell lines and SCM were collected as described previously (29, 30). assays, neutralizing anti-SPARC antibody (Zymed) or isotype-matched control ␮ Isolation of SPARC from SCM. Concentrated SCM were dialyzed against antibody was added to the media at 10 g/ml after dialysis against PBS. PBS and fractionated on a HiTrap Q-Sepharose column (Amersham, Piscat- Control mouse IgG was obtained from Lab Vision (Fremont, CA). away, NJ) with a 0.1–1.0 M NaCl gradient in 20 mM Tris-HCl (pH 8.0). Endothelial Cell Apoptosis Assay. Bovine adrenal capillary endothelial Fractions that blocked endothelial cell chemotaxis or induced endothelial cell cells were treated overnight with SCM or control Opti-MEM. Dialyzed anti- ␮ apoptosis were dialyzed against loading buffer, fractionated using a HiTrap SPARC antibody (Zymed) and control antibody were added at 10 g/ml in some assays. Apoptotic cells were visualized using the ApopTag in situ heparin-Sepharose column (Amersham; 0.1–1.0 M NaCl gradient), and subjected to identical functional assays. Fractions were analyzed by SDS-PAGE Apoptosis Detection kit (InterGen, Gaithersburg, MD). Each assay was per- followed by silver staining. A common 43-kDa band in the inhibitory flow- formed in triplicate, and the percentage of apoptotic cells was calculated as the through fractions was cut from a Coomassie Blue-stained gel and submitted for number of green terminal deoxynucleotidyl transferase-mediated nick end sequence analysis at the Harvard University Microchemistry Facility. labeling-positive cells with DNA fragmentation divided by the total number of Hoechst-counterstained nuclei. SPARC Expression Analysis. To examine SPARC mRNA expression In Vivo Angiogenesis Assay. Female Fischer 344 rats (Harlan, Madison, levels, total RNA was isolated from cultured cells using Trizol reagent (Life WI) were used to perform rat corneal assays using previously described Technologies, Inc.) according to the manufacturer’s instructions, and 1 ␮gof methods (29, 52). Briefly, 5 ␮l Hydron pellets (IFN Sciences, New Brunswick, total RNA was reversed transcribed using Superscript II (Life Technologies, NJ) prepared with 25 ␮g/ml SPARC (Calbiochem) with or without 50 ng/ml Inc.). To detect the SPARC transcripts, semiquantitative RT-PCR was per- bFGF were implanted into the corneas of anesthetized rats. Control studies formed using template diluted 1:100 and the sense primer 5Ј-CTGCCTGC- were performed with pellets containing PBS with or without bFGF. Additional CACTGAGGGTTCC-3Ј and antisense primer 5Ј-TCCAGGCAGAACAA- experiments were performed with pellets also containing 50 ␮g/ml anti- CAAACCATCC-3Ј. ␤-Actin was used as a loading control with template SPARC antibody (Zymed). After 7 days, the animals were sacrificed and diluted 1:1000 and sense primer 5Ј-TGTTGGCGTACAGGTCTTTGC-3Ј and perfused with waterproof drawing ink (Sanford, Bellwood, IL) by intracardiac antisense primer 5Ј-GCTACGAGCTGCCTGACGG-3Ј. All targets were am- injection. The eyes were fixed in 10% neutralized buffered formalin overnight. plified for 30 cycles at an annealing temperature of 60°C. SPARC mRNA The corneas were examined, and dense capillaries reaching the pellet were levels were also analyzed using real-time RT-PCR as described previously scored as positives. Animals were treated according to NIH guidelines for (50). For the quantitative real-time RT-PCR experiments, the primer pair animal care and use, and protocols were approved by the Animal Care and Use Ј Ј Ј 5 -TCTTCCCTGTACACTGGCAGTTC-3 (sense) and 5 -AGCTCGGTGT- Committee at Northwestern University. Ј GGGAGAGGTA-3 (antisense) was used with the probe Fam-CAGCTGGAC- In Vivo Inhibition of NB Growth. NB xenografts were grown in female CAGCACCCCATTGA-QSY7. SPARC protein levels in CM were examined 4–6-week-old homozygous athymic nude mice (Harlan) after s.c. inoculation by Western blots. Briefly, CM were concentrated 50-fold using 5K cutoff of 5 ϫ 106 SMS-KCNR NB cells into the right flank. Once tumors were ␮ centrifugal filter devices (Millipore, Bedford, MA). Total protein (10 g) was palpable, animals were anesthetized, and ALZET osmotic pumps (Durect, electrophoresed in a 4–20% SDS-PAGE gradient gel and transferred to a Cupertino, CA) containing purified SPARC (Calbiochem; n ϭ 3) or PBS nitrocellulose membrane (Bio-Rad, Richmond, VA) using standard techniques (n ϭ 3) were implanted s.c. SPARC was released s.c. by the pump at a rate of (51). After transfer, the blots were stained with Ponceau S (Sigma) to confirm 62.5 ng/h. Tumor volume was measured weekly using the formula: tumor equal loading. Membranes were blocked with 5% nonfat dry milk for1hand volume ϭ (length ϫ width2)/2 (53). Mice were sacrificed after 3 weeks of then incubated for 2 h with anti-osteonectin antibody (referred to hereafter as treatment, and tumors were resected for histological analysis. Tumor volume anti-SPARC antibody; Zymed, San Francisco, CA) at a 1:2000 dilution. The was analyzed using Student’s t test to compare control and treatment groups. membranes were washed three times with PBT (PBS with 0.1% Tween 20) and then incubated for 2 h with a 1:2000 dilution of horseradish peroxidase- conjugated secondary antibody (Kirkegaard and Perry Laboratories, Gaithers- RESULTS burg, MD). The bound antibody complexes were detected using the LumiGLO chemiluminescence reagent (KPL). Schwann Cells Secrete Antiangiogenic SPARC. We have previ- Immunohistochemistry Studies. Histological sections of human NB and ously reported antiangiogenic activity in SCM collected from normal ganglioneuromas (mature Schwannian stroma-rich NB) were immunostained and NB tumor-derived Schwann cells (29) and demonstrated the using a mouse anti-SPARC monoclonal antibody (Zymed). Briefly, paraffin- presence of several angiogenic inhibitors including PEDF and embedded NB tumor tissue fixed in 10% buffered formalin was sliced into TIMP-2 (29, 30). Seeking additional angiogenesis inhibitors in SCM, 4-␮m-thick sections, rehydrated in graded alcohols, and rinsed in PBS. Anti- we subjected it to multiple-step chromatography and tested fractions gen retrieval was performed with 0.01 M citrate buffer (pH 6.0) in a boiling for the ability to block bFGF-induced endothelial cell migration and steamer (20 min). Sections were incubated overnight with primary antibody cause endothelial cell apoptosis. Fractions capable of both activities (1:1600 dilution) at 4°C and developed with peroxidase labeled-dextran poly- contained a 43-kDa protein (Fig. 1) that was identified as SPARC mer followed by diaminobenzidine (Envision Plus System; DAKO Corp., using sequence analysis of the tryptic peptide fragments. No contam- Carpinteria, CA). Sections were counterstained with Gill’s hematoxylin. A human Schwannoma sample was included in each assay as a positive control, inating sequence was detected in the SPARC band. and staining without primary antibody was used as a negative control. Cyto- SPARC Was Expressed by Schwann Cells and Differentiated plasmic patches of brown color were scored as SPARC positive. Rat antimouse NB Cells in Vitro and in Vivo. SPARC expression was evaluated by monoclonal CD31 (PECAM-1) antibody (1:100 dilution; Research Diagnostics semiquantitative RT-PCR in Schwann cells, a panel of NB cell lines, Inc., Flanders, NJ) was used to highlight endothelial cells on frozen tumor and in phenotypically distinct subclones of NB cell lines [neuroblastic sections. MVD was quantified by counting 10 consecutive fields at ϫ200 (N-type) and substrate adherent (S-type)] that exhibit different malig- 7358

tected not only in Schwann cells (Fig. 2E) but also in differentiating neuroblasts/ganglion cells (Fig. 2F). SPARC in SCM Inhibited Migration and Induced Apoptosis of Endothelial Cells. The previously reported inhibition of bFGF- induced endothelial cell migration by SCM (29) was SPARC dependent because migration was largely restored in the presence of neutralizing antibody against SPARC (Fig. 3A). We also showed that SPARC-dependent induction of endothelial cell apoptosis by SCM was effectively neutralized by the same antibody (Fig. 3B). Consistent Fig. 1. Silver-stained gel of SCM fractions collected after heparin-Sepharose chromatography. The 43-kDa protein detected in Lane 3 was identified as SPARC. Fraction 3 with earlier studies (38), purified SPARC blocked bFGF-induced potently inhibited bFGF-induced endothelial cell migration as indicated at the bottom of endothelial cell migration in a dose-dependent manner at concentra- the gel. tions ranging from 0.05 to 5 ␮g/ml (Fig. 3C). However, endothelial cell migration inhibition was not observed at higher concentrations of SPARC. Biphasic responses have similarly been reported with the nant potentials (Refs. 54 and 55; Fig. 2A). Although SPARC mRNA angiogenesis inhibitor thrombospondin-1 (60, 61). SPARC also trig- was detected in all NB cell lines with the exception of NBL-W-N, gered endothelial cell apoptosis, with maximal induction at 20 ␮g/ml mRNA levels were significantly higher in the Schwann cells and (Fig. 3D). nontumorigenic S-type subclones than in tumorigenic N-type subclones and NB cell lines. SPARC protein levels in CM collected from SPARC Inhibited Angiogenesis and Impaired Tumor Growth the cells paralleled the mRNA levels (Fig. 2B). NB cells can be in Vivo. Purified SPARC blocked bFGF-induced angiogenesis in vivo induced to differentiate in vitro with a number of agents including in the rat corneal neovascularization assay (Fig. 4; Table 1). Further- all-trans-RA or BrdUrd (56–59), and real-time quantitative RT-PCR more, the addition of the anti-SPARC antibody fully restored angio- demonstrated up to a 10-fold increase in SPARC mRNA in differen- genesis by bFGF, indicating that this inhibitory effect was indeed due tiated NB cells (Fig. 2C). to SPARC. Angiogenesis was not observed when SPARC was tested To investigate whether SPARC was expressed within NB tumors, alone (Fig. 4). To our knowledge, the ability of SPARC to inhibit histological sections from NB tumors displaying varying degrees of angiogenesis has not previously been tested in a rat corneal model. differentiation and abundance of Schwannian stroma and from gan- The effect of SPARC on NB growth in vivo was tested in a mouse glioneuromas were stained with antibody against human SPARC. xenograft model where SPARC was delivered continuously for 3 Schwannian stroma-poor tumors were composed predominantly of weeks using osmotic pumps. During the first 2 weeks, tumor growth neuroblasts and showed minimal or no staining for SPARC (Fig. 2D). was completely arrested in the SPARC-treated group, whereas in the Conversely, in maturing and mature tumors, SPARC could be de- control animals carrying PBS-charged pumps, the volume of the

Fig. 2. SPARC expression in NB and Schwann cells. A, SPARC mRNA was measured in NB cell lines and Schwann cells by semiquantitative RT- PCR. Higher levels of expression were observed in nontumorigenic S-type NB subclones and Schwann cells than in tumorigenic NB cell lines and N-type subclones. B, SPARC protein was measured in media conditioned by NB cell lines, normal Schwann cells (N), and tumor-derived Schwann cells (T) by Western blot analysis. Higher levels of secreted protein were detected in the nontumorigenic S-type subclones and Schwann cells compared with tumorigenic NB cell lines. C, SPARC expression, measured by real-time quantitative RT- PCR, was up-regulated in SMS-KCNR NB cells induced to differentiate with RA or BrdUrd. D, SPARC is not detected by immunoperoxidase staining in an undifferentiated, Schwannian stroma- poor NB (magnification, ϫ400; inset, ϫ600). Im- munoperoxidase staining of a ganglioneuroma (Schwannian stroma-dominant tumor; E) and differentiating NB (F) demonstrates SPARC expression in Schwann cells (arrows) and differentiating neuroblasts/ganglion cells [arrowheads; (magnification, ϫ200; insets, ϫ600)].

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Fig. 3. SPARC inhibits angiogenesis in vitro. A, neutralizing anti-SPARC antibody reverses SCM inhibition of bFGF-induced endothelial cell migration. No effect is seen with control antibody. bFGF-induced endothelial cell migration is observed in the Opti-MEM control. B, terminal deoxynucleotidyl transferase-mediated nick end labeling-positive cells (top panels) with Hoechst-counterstained nuclei (bottom panels). Protection of endothelial cells from SCM-induced apoptosis was observed in the presence of anti-SPARC neutralizing antibody but not control antibody. The percentage of endothelial cell apoptosis for each experiment is shown in the bar graph. C, purified SPARC inhibits bFGF-induced endothelial ϳ ␮ cell migration with an ED50 of 2nM. D, purified SPARC triggers endothelial cell apoptosis in a dose-dependent manner with maximal response at 20 g/ml.

animals (152 Ϯ 44 mm3 versus 919 Ϯ 317 mm3; P ϭ 0.03). Histo- logical comparison revealed decreased vascularity in SPARC-treated tumors (MVD ϭ 23/mm2) compared with control tumors (MVD ϭ 47/mm2) as assessed by the number of structures that stained positively with an anti-CD31 antibody (Fig. 5, C and D).

DISCUSSION Schwann cells secrete substances that promote NB cell survival and differentiation and inhibit angiogenesis (25, 27, 29, 30). It is thought that cross-talk between neuroblasts and Schwann cells is responsible for the more benign nature of Schwannian stroma-rich/stroma-dominant NB tumors (25, 29). Seeking factors produced by Schwann cells that could contribute to this clinically less aggressive tumor phenotype, we identified a factor present in SCM that was capable of inhibiting angiogenesis. Fig. 4. SPARC inhibits angiogenesis in vivo. Top panels, SPARC inhibits bFGF- induced angiogenesis in the rat corneal assay. Addition of anti-SPARC antibody restored angiogenesis by bFGF. Bottom panels, in the absence of bFGF, control media, purified SPARC, or purified SPARC plus anti-SPARC neutralizing antibody did not induce Table 1 In vivo antiangiogenic activity of SPARC angiogenesis in rat corneal assays. Positive corneas/ Sample bFGF total implanted PBS No 0/6 tumors doubled every 5–6 days (Fig. 5, A and B). During the third PBS Yes 9/9 SPARC No 1/7 week of treatment, a slight increase in tumor size was observed in the SPARC Yes 0/8 treatment group. After 3 weeks of treatment with SPARC, the average SPARC antibody No 0/5 tumor volume was significantly smaller than that observed in control SPARC antibody Yes 7/7 7360

Fig. 5. SPARC inhibits NB tumor growth in vivo. A and B, short-term delivery of SPARC by osmotic pumps impairs the rate of NB xenograft tumor growth compared with control animals. C and D, immunohistochemistry staining of the SMS-KCNR NB xenograft tumors with anti-CD31 antibody demonstrates decreased numbers of blood vessels in NB xenografts from the SPARC-treated mice compared with control-treated animals (magnification, ϫ200).

SPARC, a known member of the matricellular protein family, appears to Our study revealed an inverse correlation between SPARC expres- be a key contributor to the cumulative antiangiogenic activity produced sion levels and the degree of malignant progression in NB tumors. by Schwann cells. We confirmed a known ability of SPARC to block Whereas tumorigenic cell lines and N-type subclones showed low or chemotaxis of endothelial cells induced by angiogenic stimuli (38) and no detectable levels of SPARC mRNA and secreted protein, non- report its previously unknown function to induce endothelial cell apo- tumorigenic S-type NB subclones and NB cells induced to differen- ptosis. tiate in vitro expressed SPARC at high levels. In maturing and mature Previous studies have indicated that SPARC contributes to the regu- NB tumors rich in Schwannian stroma, Schwann cells and differen- lation of tumor formation, although its role appears to be cell type tiated neuroblasts/ganglion cells showed strong positive staining for specific. SPARC expression is down-regulated in rat and chick embryo SPARC, whereas little to no staining was detected in undifferentiated, fibroblasts transformed with c-Jun and v-Src (40, 62), and its reintroduc- Schwannian stroma-poor tumors. Furthermore, we demonstrated an- tion counteracts tumorigenesis by these cells (41). In addition, SPARC tiangiogenic activity of SPARC in a mouse NB xenograft model. slows in vitro growth, induces apoptosis, and reduces tumorigenicity of The mechanisms underlying these highly variable cell type-specific ovarian cancer cells (44). In contrast, suppression of SPARC expression activities of SPARC remain unknown. However, similar to throm- abrogates the tumorigenicity of melanoma cells (42). bospondin-1 (60, 61), concentration-dependent activity of SPARC SPARC is also involved in angiogenesis. SPARC blocks the G1 to may be explained if two distinct receptors are present on vascular S transition of endothelial cells manifested by decreased proliferation endothelium, where a low-affinity receptor activated by higher ligand rates (63, 64) and antagonizes the activity of the potent angiogenic concentrations conveys a proangiogenic function of SPARC, and a activators bFGF and VEGF (38, 39). SPARC-null mice display sub- high-affinity receptor is antiangiogenic. SPARC function may also be stantially higher tumor invasion and angiogenesis compared with their altered via posttranslational modifications. SPARC subspecies se- wild-type littermates (65), indicating that SPARC may interfere with creted by normal fibroblasts and by melanoma tumors differ in size tumor angiogenesis. However, other studies point to proangiogenic and glycosylation pattern (69). It is tempting to speculate that turnover activity of SPARC (66): it was found at high levels in breast cancer rates and receptor affinity of SPARC produced by malignant mela- and colon cancer (67, 68), in metastatic melanoma (69), and in noma cells may be disparate from those of SPARC from normal invasive meningiomas (70). Our studies suggest that disparate data stroma. Specific cleavage of SPARC by tumor cells may be glycosy- regarding the role of SPARC in tumor growth and angiogenesis may lation dependent and lead to an altered function. This hypothesis be explained by its biphasic effect on the endothelial cells, where appears more feasible because the peptides from distinct structural higher concentrations appear inactive or even stimulatory. Such domains of SPARC affect diverse cell phenotypes including growth biphasic effects are not uncommon for antiangiogenic factors and rate, cell shape, matrix attachment, and angiogenic potential (66, 69, are shared by at least two more inhibitors, thrombospondin-1 (60, 61) 71). Changes in any of these functions alone may lead to decreased and PEDF.4,5

5 R. Schodlu et al., Augmentation of choroidal neovascularization in a laser induced 4 D. W. Dawson, unpublished data. mouse model by PEDF, in preparation. 7361

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2002 American Association for Cancer Research. SPARC INHIBITS NEUROBLASTOMA ANGIOGENESIS angiogenesis and slow down tumor progression. However, it is likely Speleman, F. Gain of chromosome arm 17q and adverse outcome in patients with that it is the ability of SPARC, shared with the majority of the neuroblastoma. N. Engl. J. Med., 340: 1954–1961, 1999. 13. Look, A. T., Hayes, F. A., Shuster, J. J., Douglass, E. C., Castleberry, R. P., Bowman, angiogenesis inhibitors (reviewed in Refs. 72 and 73), to induce L. C., Smith, E. I., and Brodeur, G. M. Clinical relevance of tumor cell ploidy and apoptosis in the endothelial cells of remodeling vasculature that N-myc gene amplification in childhood neuroblastoma: a Pediatric Oncology Group Study. J. Clin. Oncol., 9: 581–591, 1991. allows it to interrupt angiogenesis and to delay tumor progression. 14. Meitar, D., Crawford, S. E., Rademaker, A. W., and Cohn, S. L. Tumor angiogenesis Our data point to SPARC as a key contributor to the antiangiogenic correlates with metastatic disease, N-myc amplification, and poor outcome in human activity of factors secreted by the Schwann cells. However, other neuroblastoma. J. Clin. Oncol., 14: 405–414, 1996. 15. Eggert, A., Ikegaki, N., Kwiatkowski, J., Zhao, H., Brodeur, G. M., and Himelstein, inhibitors of angiogenesis including PEDF and TIMP-2 also appear to B. P. High-level expression of angiogenic factors is associated with advanced tumor be involved in the cross-talk between Schwann cells and the neuronal stage in human neuroblastomas. Clin. Cancer Res., 6: 1900–1908, 2000. 16. Erdreich-Epstein, A., Shimada, H., Groshen, S., Liu, M., Metelitsa, L. S., Kim, K. S., component of NB tumors (29, 30, 74). Our data clearly demonstrate ␣ ␤ ␣ ␤ Stins, M. F., Seeger, R. C., and Durden, D. L. Integrins v 3 and v 5 are expressed that the angiogenic balance is complex: it is not a result of a single by endothelium of high-risk neuroblastoma and their inhibition is associated with inducer-inhibitor combination but a composite value determined by increased endogenous ceramide. Cancer Res., 60: 712–721, 2000. 17. Breit, S., Ashman, K., Wilting, J., Rossler, J., Hatzi, E., Fotsis, T., and Schweigerer, all of the inducers and inhibitors present (reviewed in Ref. 75). L. The N-myc oncogene in human neuroblastoma cells: down-regulation of an Changes in angiogenic phenotype may occur gradually (76) or in a angiogenesis inhibitor identified as activin A. Cancer Res., 60: 4596–4601, 2000. single step (77, 78). In Schwannian stroma-rich/stroma-dominant NB 18. Eggert, A., Grotzer, M. A., Ikegaki, N., Liu, X. G., Evans, A. E., and Brodeur, G. M. 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