Thrombospondin-1 Promotes 3 1 Integrin-Mediated Adhesion And

[CANCER RESEARCH 60, 457–466, January 15, 2000] Thrombospondin-1 Promotes ␣3␤1 Integrin-mediated Adhesion and Neurite-like Outgrowth and Inhibits Proliferation of Small Cell Lung Carcinoma Cells

Neng-hua Guo, Nancy Smyth Templeton, Hebah Al-Barazi, JoAnne Cashel, John M. Sipes, Henry C. Krutzsch, and David D. Roberts1 Laboratory of Pathology, National Cancer Institute, NIH, Bethesda, Maryland 20892 [N-h. G., H. A-B., J. C., J. M. S., H. C. K., D. D. R.], and Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas 77030 [N. S. T.]

ABSTRACT neural cell adhesion molecules. Expression of the latter by SCLC correlates with poor prognosis (1, 7). Although SCLC cell lines Although human small cell lung carcinoma (SCLC) cell lines are typ- generally fail to interact with the adhesive proteins in serum and with ically anchorage-independent and do not attach on most extracellular most extracellular matrix components, some SCLC lines can attach on matrix proteins, OH-1, and several other SCLC cell lines attached on ␤ substrates coated with thrombospondin-1 (TSP1). SCLC cells grew long- laminin substrates (8–10). SCLC cells express some 1 integrins but ␤ ␤ ␤ term as adherent cells on a TSP1-coated substrate. Adhesion of SCLC not 3, 4, or 5 integrins (11–15) and were reported to interact with cells on TSP1 was inhibited by heparin, function-blocking antibodies laminins through ␣3␤1 and ␣6␤1 integrins (12, 16). recognizing ␣3or␤1 integrin subunits, and by soluble ␣3␤1 integrin To better understand the role of cell-matrix interactions in the rapid ligands. SCLC cells extended neurite-like processes on a TSP1 substrate, dissemination of SCLC, we have examined the possible role of TSP1 which was also mediated by ␣3␤1 integrin. Process formation on a TSP1 to mediate interactions of SCLC cells with the extracellular matrix. substrate was specifically stimulated by epidermal growth factor and TSP1 is a major component of the ␣-granules of platelets and is a somatostatin. Adhesion on TSP1 weakly inhibited SCLC cell proliferation, member of the thrombospondin family of matricellular proteins that is but this inhibition was strongly enhanced in the presence of epidermal synthesized by many cell types in response to growth factor stimula- growth factor. TSP1 and an ␣3␤1 integrin-binding peptide from TSP1 tion (reviewed in Ref. 17). also inhibited proliferation when added in solution. High-affinity binding of 125I-labeled TSP1 to OH-1 cells was heparin-dependent and may be In common with other extracellular matrix proteins such as fi- mediated by sulfated glycolipids, which are the major sulfated glycocon- bronectin and laminins, TSP1 plays important roles in regulating jugates synthesized by these cells. Synthesis or secretion of TSP1 by SCLC growth, motility, survival, and adhesion of cells (reviewed in Ref. 18) cells could not be detected. On the basis of these results, the ␣3␤1 integrin and modulating tumor growth and metastasis (reviewed in Ref. 19). and sulfated glycolipids cooperate to mediate adhesion of SCLC cells on TSP1 can directly influence adhesion, growth, and motility of some TSP1. Interaction with TSP1 through this integrin inhibits growth and tumor cell lines in vitro (reviewed in Ref. 19), but its major inhibitory induces neurotypic differentiation, which suggests that this response to effect on tumor growth in vivo is thought to result from the inhibition TSP1 may be exploited to inhibit the progression of SCLC. of angiogenesis (20–25). TSP1 is recognized by several cell surface receptors including ␤3 and ␤1 integrins, CD47, syndecan-1, sulfatides, and CD36 (reviewed INTRODUCTION in Ref. 19). TSP1 also binds to several extracellular matrix compo- SCLC2 accounts for 20–25% of human lung cancers (reviewed in nents (26), which may in turn mediate its binding to cells through Refs. 1–3). SCLC commonly presents as a disseminated disease that additional receptors. Distinct signaling pathways may be induced by is characterized by early metastasis to the lymph nodes, central the binding of TSP1 to each class of receptor (27–30); therefore, nervous system, liver, and bone. SCLC cells have several properties identification of the specific TSP1 receptors used by each cell type is characteristic of neuroendocrine cells, including production of dopa important for understanding the responses that result from these decarboxylase and neuron-specific enolase, secretion of various neu- interactions. ropeptides, and expression of neuronal surface markers such as the We recently observed that OH-1 cells and other nonadherent SCLC neural cell adhesion molecule and HNK-1 (1). Although SCLC often cell lines attached avidly to TSP1 substrates and grew as adherent responds well to chemotherapy when diagnosed at an early stage, cells on this substrate. We have characterized the receptors that disseminated SCLC responds poorly, and the overall 5-year survival mediate this response and demonstrate here that both sulfated glyco- rate is the lowest of all bronchogenic carcinomas. A better under- lipids and ␣3␤1 integrin on SCLC cells function as TSP1 receptors. standing of the molecular mechanisms for dissemination of SCLC is, The activity of this integrin is stimulated by EGF and somatostatin. In therefore, needed to control this disease. addition to mediating adhesion, the ␣3␤1 integrin promotes neurite- Many SCLC cell lines have been established that are typically like outgrowth on TSP1 substrates and modulates SCLC cell growth. nonadherent on tissue culture substrates and grow as tight aggregates in suspension (4, 5). Cell-cell adhesion is, therefore, the dominant interaction for SCLC cells and is mediated by E-cadherin (6) and MATERIALS AND METHODS

Materials. TSP1 was purified from thrombin-activated human platelets as Received 7/15/99; accepted 11/11/99. described previously (31). TSP1 was labeled with Na125I (ICN Radiochemical, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with Irvine, CA) using Iodogen (Pierce Chemical Co., Rockford, IL) as described 18 U.S.C. Section 1734 solely to indicate this fact. previously (32). Monoclonal antibodies against TSP1 were provided by Dr. 1 To whom requests for reprints should be addressed, at NIH, National Cancer William Frazier (Washington University School of Medicine, St. Louis, MO). Institute, Building 10, Room 2A33, 10 Center Drive, MSC 1500, Bethesda, MD 20892- The integrin-binding peptides Gly-Arg-Gly-Asp-Ser and Gly-Arg-Gly-Glu-Ser 1500. Phone: (301) 496-6264; Fax: (301) 402-0043; E-mail: [email protected]. 2 The abbreviations used are: SCLC, small cell lung carcinoma; EGF, epidermal were purchased from Sigma (St. Louis, MO). Synthetic peptides derived from growth factor; IGF1, insulin-like growth factor-1; mAb, monoclonal antibody; FGF, TSP1 and inactive analogues were synthesized and characterized as described fibroblast growth factor; MBP, maltose-binding protein; peptide 678, FQGVLQNVRFVF previously (32, 33). MBP-invasin 497 expressed in strain SB39 was purified as (TSP1 residues 190–201); peptide 686, FQGVLQAVRFVF; peptide 690, FQGVLQN- described (34). Murine laminin-1 was provided by Dr. Lance Liotta (National VAFVF; peptide 246, KYRFKQDGGWSHWSPWSS (TSP1 residues 412–428); peptide 7N3, FIRVVMYEGKK (TSP1 residues 1102–1112); TSP1, thrombospondin-1; RT-PCR, Cancer Institute, Bethesda, MD). Type IV collagen was obtained from Col- reverse transcription-PCR. laborative Research, Inc. Recombinant human EGF was obtained from R&D 457

Fig. 1. TSP1 specifically promotes SCLC cell adhesion. Bacteriological polystyrene was coated with the indicated concentrations of TSP1 (F), murine laminin-1 (E), or human plasma fibronectin (Œ). SCLC lines H128 (A), OH-1 (C), OH-1 variant (D), and the A2058 melanoma (B) cell lines were allowed to attach on each substrate for 60 min. Adherent cells were counted microscopically and are presented as the mean of triplicate determinations.

Systems. Insulin was from Biofluids, and recombinant human IGF1, bombesin, GCG TAG CCT GTC TGG-3Ј; upstream primer sequence for human THBS3: and somatostatin-14 were from Bachem. 5Ј-GAC ACA GTG CCT GAG GAC TTT GAG-3Ј; downstream primer for The function-blocking CD36 antibody OKM-5 was purchased from Ortho- THBS3:5Ј-TGG CAA TGT GCT GTC ATC TTT CC-3Ј; upstream primer for mune (Raritan, NJ). The integrin ␣v␤3 antibody LM609 was the gift of Dr. glyceraldehyde-3-phosphate dehydrogenase: 5Ј-GCT CTC CAG AAC ATC David Cheresh (Scripps Research Institute, La Jolla, CA; Ref. 35). Rat mono- ATC CCT GCC-3Ј; downstream primer sequence of human glyceraldehyde- ␤ ␣ Ј clonal antibodies to the human 1 integrin (mAb 13) and 5 subunits (mAb 16) 3-phosphate dehydrogenase: 5 -TCC TTG GAG GCC ATG TGG GCC were provided by Dr. Kenneth Yamada (National Institute for Dental and ATG-3Ј. Craniofacial Research, Bethesda, MD; Ref. 36). Integrin function-blocking Cell Culture. The OH-1 cell line (38) was provided by Dr. Joel Shaper antibodies P1B5 (␣3␤1), P4C2 (␣4␤1), and P1D6 (␣5␤1) were obtained from (Johns Hopkins University, Baltimore, MD). Variant OH-1 arose after pro- Life Technologies, Inc. The ␤1 integrin-activating antibody TS2/16 (37) and longed culture of OH-1 and had lost the tight aggregate morphology. The the CD98 antibody 4F2 were prepared from hybridomas obtained from the H128, H69, H82, and H209 cell lines were purchased from the American Type American Type Culture Collection (Rockville, MD). Culture Collection. These cell lines were established from pleural fluids of The following oligonucleotides were synthesized (The Midland Certified SCLC patients (5). N417 and H345 cell lines (39) were provided by Dr. A. Reagent Co., Midland, TX) and used as primers for the RT-PCR: upstream Gazdar (University of Texas Southwestern Medical Center, Dallas, TX). N417 primer for human THBS1:5Ј-CAA CCA CAA TGG AGA GCA CCG-3Ј; originated from a lung metastasis and H345 from a bone marrow metastasis. downstream primer sequence for THBS1:5Ј-TAG TTG CAC TTG GCG TTC All of the cell lines were cultured suspended in RPMI 1640 with 15% FCS Ј Ј TTG TTG-3 ; upstream primer for human THBS2:5-CTC CAC CAG CAA (Biofluids Inc., Rockville, MD) at 37°C in a 5% CO2 incubator. The medium GGT GCC TCG CTG-3Ј; downstream primer for THBS2:5Ј-CCG TCG CCC was changed every 5 days.

Table 1 SCLC cell adhesion to extracellular matrix proteins Adhesion of SCLC cell lines (4 ϫ 105 cells/well) was determined using substrates coated with TSP1, fibronectin, or laminin-1 at the indicated concentrations. Adhesion was quantified microscopically and is presented as cells/mm2, mean Ϯ SD (n ϭ 3). Concentration (␮g/ml)

Cell line Substrate 6.2 12.5 25 50 H69 TSP1 6 Ϯ 146Ϯ 894Ϯ 6 110 Ϯ 14 Fibronectin 1 Ϯ 15Ϯ 113Ϯ 322Ϯ 7 Laminin-1 1 Ϯ 116Ϯ 182Ϯ 8 108 Ϯ 11 H345 TSP1 1 Ϯ 17Ϯ 1 124 Ϯ 17 165 Ϯ 6 Fibronectin 0 6 Ϯ 225Ϯ 517Ϯ 5 Laminin-1 6 Ϯ 28Ϯ 1 151 Ϯ 31 148 Ϯ 9 N417 TSP1 15 Ϯ 230Ϯ 448Ϯ 455Ϯ 10 Fibronectin 19 Ϯ 248Ϯ 486Ϯ 678Ϯ 2 Laminin-1 5 Ϯ 178Ϯ 3 151 Ϯ 6 160 Ϯ 3 458

(2.5%; Life Technologies, Inc.) was added for 5 min and triturated as above. The single cell suspension was washed once with the same medium, centrifuged, and suspended in fresh medium. For adhesion assays, cell aggregates were washed once with RPMI 1640 and centrifuged at 200 ϫ g for 2 min. The pellet was suspended in PBS (pH 7.4), containing 2.5 mM EDTA and incubated for 10 min at 37°C. After trituration three times and centrifugation for 2 min at 400 ϫ g, the cells were resuspended in RPMI 1640 containing 0.1% BSA (Sigma). Trypan blue staining showed greater than 90% cell viability. Adhesion of SCLC Cells to Extracellular Matrix Proteins. Extracellular matrix proteins or peptides in Dulbecco’s PBS were adsorbed onto polystyrene by incubating overnight at 4°C. Adsorption isotherms of TSP1 on plastic have been reported previously (40). The supernatant fluid was removed, and the dishes were incubated with Dulbecco’s PBS with 1% BSA for 30 min to minimize nonspecific adhesion. The dishes were washed twice with cold PBS (pH 7.2) and overlaid with dissociated SCLC cells, prepared as described above, at a density of 5 ϫ 104/cm2. For inhibition, inhibitors or antibodies were added and incubated with SCLC cells at the indicated concentrations. After incubation for 60–90 min at 37°C, the dishes were washed three times with PBS (pH 7.2), fixed with 1% glutaraldehyde in PBS (pH 7.2), and stained with Diff-Quik. Attached cells were counted microscopically. Neurite outgrowth was assessed in SCLC cells after incubation for 75–90 min on a TSP1 substrate. Neurites extending more than one-cell-diameter from the central cell body were counted microscopically in four adjacent 0.25-mm2 fields for each triplicate analysis. Immunoprecipitation and Western Analysis. OH-1 cells were surface- labeled using EZ-Link Sulfo-NHS-LC-Biotin (Pierce) as suggested by the manufacturer. The cells were then lysed in 50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 1 mM EGTA, 1 mM NaF supplemented with 10 ␮g/ml each of the following protease inhibitors: antipain, pepstatin A, chymostatin, leupeptin, aprotinin, soybean trypsin inhibitor, and 1 mM phen- ylmethylsulfonyl fluoride. A total of 90 ␮g of proteins were immunoprecipitated using 0.75 ␮g mouse monoclonal anti-EGF receptor antibody (Trans- duction Laboratories) prebound to antimouse IgG agarose (Sigma). After washing, the immune complex was eluted with sample buffer. Immunopre- cipitated proteins were fractionated on a 10% SDS gel along with 5 ␮gof A431 carcinoma total proteins as a control. The proteins were transferred to polyvinylidene difluoride membrane and blotted with mouse anti-EGF receptor antibody. The membrane was washed and incubated with a mixture of horseradish peroxidase-conjugated goat antimouse IgG antibody (Kirkegaard and Perry, Gaithersburg, MD) and horseradish peroxidase-streptavidin (Pierce) and was visualized using chemiluminescent substrate (Pierce). Proliferation. Effects of soluble and substrate-bound TSP1 or TSP1 peptides on cell proliferation were quantified using a tetrazolium proliferation assay (CellTiter Assay, Promega). Treatment with soluble TSP1 was performed in 96-well tissue culture plates, and proliferation was determined after 72 h in RPMI containing 15% FCS. Proteins and peptides were immobilized on Nunc Maxisorp 96-well plates by overnight incubation with the proteins or peptides dissolved in 50 ␮l of sterile Dulbecco’s PBS. The supernatant fluid was removed, and the wells were incubated for 30 min. in Dulbecco’s PBS Fig. 2. SCLC cell adhesion on TSP1 is mediated by the ␣3␤1 integrin. A, OH-1 cell containing 1% BSA. OH-1 cells (1 ϫ 104/well) were added in RPMI contain- adhesion on a substrate coated with 40 ␮g/ml TSP1 (mean Ϯ SD; n ϭ 3) was determined ing 15% FCS and incubated for 72 h at 37° in 5% CO . For assessing inhibition in RPMI containing 1 mg/ml BSA (control) or the same medium containing 25 ␮g/ml 2 heparin, 5 ␮g/ml mAb13 (anti-␤1), mAb13 and heparin (anti-␤1 ϩ heparin), or 40 ␮g/ml by soluble proteins or peptides, OH-1 cells were grown in suspension in MBP-invasin fusion protein and 25 ␮g/ml heparin (invasin ϩ heparin). B, OH-1 SCLC Nunclon 96-well tissue culture plates using the same medium supplemented cell adhesion on substrates coated using 40 ␮g/ml TSP1 (f), 5 ␮M TSP1 peptide 678 ( ), with the indicated inhibitors and growth factors. or 0.2 ␮g/ml MBP-invasin (Ⅺ) was determined in the presence of 5 ␮g/ml antibody P1B5 Cell-binding Assay. A 0.2-ml cell suspension, dissociated using EDTA as (anti-␣3), 5 ␮g/ml antibody P4C2 (anti-␣4), 5 ␮g/ml antibody P1D6 (anti-␣5), 5 ␮g/ml above, was transferred into 12 ϫ 75-mm polypropylene tubes (PGC Scientific antibody mAb13 (anti-␤1), 20 ␮M TSP1 peptide 678 (p678), or 40 ␮g/ml MBP-invasin (invasin). Results are presented as a percent of control adhesion determined for each Inc., Gaithersburg, MD). Iodinated proteins (final concentration of 0.2 ␮g/ml) protein without inhibitors (mean Ϯ SD; n ϭ 3). C, OH-1 cell adhesion to substrates coated were added and incubated for1honicewith rotary shaking. Bound radioac- with 25 ␮g/ml TSP1 or 5 ␮M of TSP1 peptides that bind to ␣3␤1 integrin (p678), CD36 tivity was quantified after centrifugation of the cells through oil as described (Mal II), or heparin (p246) was determined in the absence (f) or presence of the ␤1 integrin-activating antibody TS2/16 at 5 ␮g/ml (o). Results are presented as mean Ϯ SD previously (32). For inhibition studies, inhibitors were added first and incu- (n ϭ 3) bated with the cells for 15 min. Iodinated proteins, peptides, or fragments were then added and incubated as above. To test the effect of divalent cations on binding of TSP1 to OH-1 cells, the cells were treated and suspended in HBSS Cells were passaged every 9- 11 days. In brief, cells were centrifuged at or HBSS containing EDTA or divalent cations. After incubation for1hat4°C, 400 ϫ g for 2 min, and the medium was aspirated. Cell pellets were washed cells were centrifuged and separated from the unbound ligand by centrifuga- once with RPMI 1640 containing 5 mM MgCl2 and treated for 5 min with 50 tion through oil and counted in a gamma counter. Using the same procedure, ␮g/ml DNase-1 (Biofluids Inc.) in RPMI 1640 containing 5 mM magnesium other media (including DMEM, RPMI 1640, or RPMI 1640 without phos- chloride. The cells were triturated three times, and 0.1 volume of trypsin phate) were tested but had no effect on the binding of TSP1 to the cells. 459

460

Extraction of Sulfatide and Interaction with TSP1. Sulfatides were extracted from SCLC cells, desalted by Sephadex G-25, and separated into neutral and acidic fractions by DEAE-Sepharose ion-exchange chromatogra- phy according to the previously described method (41). Fractions were evap- orated to dryness and dissolved in chloroform-methanol (1:1) for analysis. The extracts were chromatographed on aluminum-backed high-performance TLC plates and incubated with 125I-labeled TSP1 according to the previously described method (41). RT/PCR. Total RNAs from SCLC cell lines were extracted using RNAzol B according to the supplier’s protocol. RNA pellets were washed with 75% ethanol, briefly dried under vacuum for 2 min, and stored in Ϫ70°C. RT-PCR was performed according to the manufacturer’s procedure (Perkin-Elmer Ce- tus). Aliquots (2 ␮l) containing 250 ng of human SCLC total RNA were brought to a final volume of 20 ␮l with reverse transcription buffer containing rTth polymerase and downstream primer. The samples were heated to 70° for 15 min. PCR buffer with the same amount of sense primers was added to a final volume of 100 ␮l. The reaction mixture was heated to 95° for 1 min and amplified for 30–50 cycles: at 95° for 10 s and at 60° for 15 s. Finally, the sample was incubated at 60° for 5 min. The PCR products were analyzed by agarose gel electrophoresis. Metabolic Labeling of Sulfatides and Proteoglycans. SCLC cells were cultured in 5 ml of RPMI 1640 without methionine containing 4% Ultroser HY (IBF Biotechnics) for 12 h in 25-cm2 culture flask before the addition of 125 ␮Ci of [35S]sulfate (ICN Radiochemicals). After 48 h, the cells were harvested, and glycolipids or proteoglycans were extracted according to the previously described method (41). For some experiments, cells were grown in sulfate- depleted medium supplemented with sodium chlorate to inhibit sulfation as described previously (42).

RESULTS TSP1 Specifically Promotes Adhesion of SCLC Cells. Several SCLC lines were tested for adhesion on substrates coated with TSP1, laminin, or fibronectin (Fig. 1; Table 1). All of the SCLC lines tested grew as aggregates in suspension with no adhesion to the substratum when cultured in serum-based media. The cell lines H128 (Fig. 1A), OH-1 (Fig. 1C), and a variant of OH-1 (Fig. 1D) that had lost the classic tight aggregate morphology (38), all attached avidly on TSP1 but failed to attach on murine laminin-1 or human plasma fibronectin. All three of these proteins were functional to promote comparable levels of adhesion for A2058 melanoma cells (Fig. 1B). OH-1 SCLC cells also failed to attach on substrates coated with vitronectin, fibrinogen, type IV collagen, or gelatin (data not shown). Thus, the OH-1 SCLC line lacks adhesion receptors for all of the matrix proteins tested except TSP1. Several additional SCLC cell lines attached on TSP1 but also exhibited some adhesion to laminin-1 or fibronectin Fig. 4. EGF and somatostatin stimulate adhesion and neurite outgrowth on TSP1. A, (Table 1). H345 and H69 cells attached on TSP1 and laminin-1 at neurite outgrowth on a substrate coated with 40 ␮g/ml TSP1 was determined as in Fig. 3. higher levels than on fibronectin substrates, whereas N417 cells Cells were plated in RPMI containing 1 mg/ml BSA (control) or the same medium adhered preferentially on laminin-1. containing 5 ␮g/ml antibody TS2/16, 3 ng/ml EGF, 10 nM IGF1, 100 ng/ml basic FGF2, 1 ␮M insulin, 10 ␮M TSP1 peptide 7N3, 0.5 ␮M bombesin, or 1 ␮M somatostatin. A, inset, Adhesion of SCLC on TSP1 is Mediated by ␣3␤1 Integrin. OH-1 cells were surface labeled with biotin and immunoprecipitated using an EGF Because TSP1 was the only extracellular matrix protein recognized by receptor antibody. After SDS gel electrophoresis and transfer to polyvinylidene difluoride membrane, immunoprecipitated EGF receptor was detected using streptavidin-peroxidase OH-1 cells, we used this cell line to identify the specific TSP1 and chemiluminescent detection. A lysate of A431 epidermoid carcinoma cells was used receptor expressed on SCLC cells. Several function-blocking antibod- as a positive control. B,: EGF-stimulated SCLC outgrowth on TSP1 is mediated by the ␣ ␤ ␮ ies that recognize known TSP1 receptors were examined. Antibodies 3 1 integrin. OH-1 cells were plated on a substrate coated with 40 g/ml TSP1 in ␣ ␤ medium without EGF (ϪEGF) or in medium containing 10 ng/ml EGF alone or with 5 against the TSP1 receptors, v 3 integrin (LM609) and CD36 ␮g/ml of the integrin function-blocking antibodies P1B5 (EGFϩanti-␣3) or mAb13 (OKM5), had no effect on adhesion to TSP1 (results not shown). A ϩ ␤ ␮ (EGF anti- 1) or with 10 g/ml heparin to inhibit the heparin-binding sites of TSP1. C, function-blocking antibody recognizing ␤1 integrin was a dose- neurite outgrowth on a substrate coated with 0.2 ␮g/ml of MBP-invasin was determined as in A for untreated OH-1 cells (control) or in the presence of 5 ␮g/ml antibody TS2/16, dependent inhibitor of OH-1 cell adhesion on TSP1 but inhibited 3 ng/ml EGF, 10 nM IGF1, or 100 ng/ml basic FGF2. adhesion only by half at saturating concentrations (Fig. 2A and data

Fig. 3. TSP1 promotes ␣3␤1-dependent neurite-like process outgrowth by OH-1 SCLC cells. OH-1 SCLC cells were incubated for 75 min on a substrate coated with 40 ␮g/ml TSP1 alone (A) or in the presence of 5 ␮g/ml mAb13 (B)or20␮M TSP1 peptide 678 (C). Scale bar (A), 10 ␮m for A-C. D, OH-1 cells were plated on TSP1 alone (control), in the presence of 5 ␮g/ml indicated integrin function-blocking antibodies, in the presence of 20 ␮M of peptide 678, or in the presence of 40 ␮g/ml MBP-invasin. Neurite outgrowth was quantified 2 by counting four 0.25-mm grids for each triplicate determination (mean Ϯ SD). E, neurite outgrowth was determined as in D on substrates coated with 40 ␮g/ml TSP1, 5 ␮M TSP1 peptide 678 or the control peptides 690 or 686, 5 ␮M TSP1 heparin-binding peptide 246, or 1 ␮g/ml MBP invasin. F and G, OH-1 cells attached on TSP1 in the presence of 10 ng/ml EGF were stained for F-actin using BODIPY TR-X phallacidin and for ␤1 integrin using TS2/16 and BODIPY FL antimouse IgG. Scale bar (F), 20 ␮m for F and G. 461

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2000 American Association for Cancer Research. SCLC REGULATION BY TSP-1 not shown). The residual integrin-independent adhesion of OH-1 cells these growth factors had no significant effect on the function of the on TSP1 may be mediated by the heparin-binding sites of TSP1 same integrin in SCLC cells to promote adhesion on TSP1 or neurite because heparin also partially inhibited adhesion of OH-1 cells on outgrowth (Fig. 4A and data not shown). Basic FGF also had no effect, TSP1, and a combination of heparin with either the ␤1-blocking but EGF was a potent inducer of OH-1 neurite outgrowth on TSP1 and antibody or the ␤1 integrin ligand invasin (43) completely inhibited moderately increased cell adhesion on TSP1 or peptide 678 (Fig. 4A adhesion (Fig. 2A). and data not shown). Expression of EGF receptor by OH-1 cells was Function-blocking integrin ␣ subunit antibodies were used to define verified by immunoprecipitation using an EGF receptor antibody, the specific ␤1 integrin that recognized TSP1 (Fig. 2B). An ␣3␤1 which comigrated with the EGF receptor from A431 epidermoid ␣ ␣ integrin function-blocking antibody (P1B5) but not anti- 4 or anti- 5 carcinoma cells (inset in Fig. 4A). integrin antibodies, which have been reported to recognize TSP1 in EGF-stimulated outgrowth of neurite-like processes on a TSP1 other cell types (44, 45), partially inhibited adhesion on TSP1. The ␣3 substrate required the ␣3␤1 integrin because both ␣3- and ␤1-specific and ␤1 function-blocking antibodies also partially inhibited adhesion function-blocking antibodies reversed the stimulation by EGF (Fig. of OH-1 cells on an immobilized TSP1 peptide (peptide 678), which 4B). Blocking the heparin-binding sites of TSP1 using soluble hepa- is recognized by the ␣3␤1 integrin on breast carcinoma cells (33, 45), rin, in contrast, had no effect on EGF-stimulated outgrowth (Fig. 4B). and on immobilized invasin (Fig. 2B). Invasin binds to several ␤1 EGF also specifically induced neurite outgrowth on the ␣3␤1 ligand integrins, including ␣3␤1, ␣4␤1, and ␣5␤1 (43); therefore. the failure invasin, confirming that the stimulation of neurite outgrowth by EGF of the ␣4␤1 and ␣5␤1 antibodies to significantly inhibit adhesion to was ␤1 integrin-dependent (Fig. 4C). immobilized invasin combined with their lack of adhesion on the Several neuropeptides have also been reported to promote neurite well-characterized ligand for these integrins, fibronectin (Fig. 1), outgrowth in other neurectoderm-derived cell lines (46, 47). Repre- indicates that OH-1 cells do not express functional ␣4␤1 nor ␣5␤1 sentatives of two major neuropeptide families were tested. Somatosta- integrins. The consistently greater inhibition by the integrin antago- tin-14, a member of the somatostatin family, stimulated outgrowth on nists of adhesion on invasin relative to that on TSP1 is also consistent TSP1 (Fig. 4A) and slightly stimulated adhesion on TSP1 (results not with involvement of a heparin-sensitive receptor in adhesion to TSP1. shown). In contrast, bombesin, a member of the bombesin/gastrin- The ␤1 integrin-activating antibody TS2/16 enhanced adhesion on releasing peptide family that are secreted by many SCLC cell lines, TSP1 and on the TSP1 peptide 678 but not on a CD36-binding peptide was inactive (Fig. 4A). (Mal II) or a heparin-binding peptide (p246) from TSP1 (Fig. 2C). CD47-binding peptides from the carboxyl-terminal domain of This further confirmed that the recognition of TSP1 peptide 678 by TSP1 activate the function of several integrins in other cell types (27, OH-1 cells is ␤1 integrin-mediated and suggested that this integrin 29, 48) but did not stimulate ␣3␤1 integrin function in breast carci- exists in a partially inactive state on OH-1 cells. noma cells (45). Consistent with the latter results, the CD47-binding TSP1 Promotes Neurite-like Outgrowth of SCLC. The attached peptide 7N3 did not significantly stimulate neurite outgrowth or OH-1 cells generally retained rounded cell bodies, but many cells adhesion of OH-1 cells on TSP1 (Fig. 4A and results not shown). rapidly extended neurite-like processes on the TSP1 substrate (Fig. Therefore, TSP1 cannot stimulate a neurite-like outgrowth response of 3A). ␣3␤1 integrin binding was necessary for neurite outgrowth on SCLC cells to itself by binding to CD47. TSP1 because function-blocking ␣3 and ␤1 integrin antibodies inhib- TSP1 and an ␣3␤1 Integrin-binding Peptide from TSP1 Inhibit ited the response (Fig. 3, B and D). In contrast, function-blocking SCLC Proliferation. TSP1 is known to modulate the growth of antibodies that recognized ␣4␤1or␣5␤1 integrins did not inhibit several cell types (reviewed in Ref. 19). The addition of soluble TSP1 neurite outgrowth (Fig. 3D). The ␣3␤1 integrin-binding peptide 678 to nonadherent OH-1 cells markedly inhibited their proliferation, with ␣ ␤ from TSP1 (Fig. 3, C and D) and the 3 1 ligand invasin also an IC50 of 40 nM (Fig. 5A). This inhibition may result from ligation of inhibited neurite outgrowth on a TSP1 substrate (Fig. 3D). Ligation of the ␣3␤1 integrin because two additional ligands for this integrin, ␣ ␤ ϭ ϭ 3 1 integrin also was sufficient to promote formation of neurite-like MBP-invasin (IC50 80 nM) and the TSP1 peptide 678 (IC50 6 processes, in that substrates coated with immobilized TSP1 peptide ␮M), also inhibited OH-1 cell proliferation (Fig. 5A). The activity of 678 or invasin both promoted neurite formation (Fig. 3E). The control peptide 678 was specific in that the analogue 686, in which the peptide 690, in which the essential Arg residue was replaced by Ala, essential Asn residue was replaced by Ala (33), was inactive. A was inactive, and an analogue with decreased integrin binding, pep- heparin-binding peptide from the type 1 repeats (peptide 246) only tide 686 (33), was a weaker stimulator of neurite outgrowth. The weakly inhibited OH-1 cell proliferation at the same concentrations TSP1 heparin-binding peptide 246 only weakly promoted neurite (data not shown), further indicating that the inhibition by the integrin- outgrowth, which indicated that the heparin-binding activity of TSP1 binding peptide from TSP1 is specific. is not sufficient to stimulate the neurite outgrowth response. Activa- When OH-1 cells were plated on a TSP1 substrate, the attached tion of the ␣3␤1 integrin by antibody TS2/16 increased neurite SCLC cells continued to grow and formed extended flattened colonies formation on TSP1 (Fig. 4A). on the TSP1 substrate. In the absence of TSP1, the cells remained as The neurites induced by a TSP1 substrate contained F-actin, as floating aggregates with no substrate adhesion. Adhesion of OH-1 visualized by phallacidin staining, but no organization of actin was cells on a substrate coated with TSP1 only weakly inhibited the observed in the rounded cell bodies (Fig. 3F). ␤1 Integrin staining was proliferation of OH-1 cells in their growth medium (Fig. 5B). On the also prominent in the neurites formed on TSP1 (Fig. 3G). The integrin basis of the observation that EGF stimulated the neurite outgrowth antibody also visualized long processes extending from the neurites response to TSP1 (Fig. 4), we also examined the effect of immobilized that lacked detectable actin staining. This localization of the ␤1 TSP1 on the proliferation of OH-1 cells in the presence of this growth integrin supports the evidence from antibody blocking studies which factor. indicates that the ␣3␤1 integrin binding mediates neurite-like out- Surprisingly, OH-1 cell proliferation was much more sensitive to growth on TSP1. inhibition by immobilized TSP1 in the presence of EGF (Fig. 5B). The EGF and Somatostatin Stimulate Integrin-mediated Outgrowth addition of EGF alone had no significant effect on the proliferation of of SCLC on TSP1. Although we previously demonstrated that insu- OH-1 cells, but, in the presence of immobilized TSP1, it produced a lin and insulin-like growth factor-1 specifically stimulated the ␣3␤1 dose-dependent inhibition of proliferation (Fig. 5C). The inhibition of integrin-mediated spreading of breast carcinoma cells on TSP1 (45), proliferation on a TSP1 substrate by EGF was specific in that IGF1 462

and bombesin did not display synergism with TSP1 to inhibit proliferation (data not shown). The inhibition of proliferation by a TSP1 substrate in the presence of EGF may also be mediated by the ␣3␤1 integrin because substrates coated with TSP1 peptide 678 or MBP- invasin showed similar cooperative effects with EGF to inhibit OH-1 cell proliferation (Fig. 5D). TSP1 peptides that bind to CD47 (7N3) or heparin (p246) did not synergize with EGF, which indicated that the activity of TSP1 peptide 678 is specific (Fig. 5D). Thus, EGF specifically and synergistically suppressed proliferation of SCLC cells attached on TSP1 or an ␣3␤1-binding sequence from TSP1. Sulfatides Mediate High-affinity Binding of Soluble TSP1 to SCLC Cells. Although integrins mediate these biological responses of OH-1 cells to TSP1, heparin-inhibitable binding accounted for most of the high-affinity binding of soluble TSP1 to OH-1 cells. TSP1 bound saturably to OH-1 cells with a dissociation constant of 72 Ϯ 16 5 nM and 2.6 ϫ 10 sites/cell (Fig. 6A). This is comparable to the binding constant of 50 nM for TSP1 reported for TSP1 binding to resting platelets (49) and of 22 nM for keratinocytes (50). The dissociation constant for H128 cells was slightly higher than for OH-1 cells ϭ Ϯ ϫ 5 (Kd 92 21 nM) with 2.1 10 binding sites/cell (data not shown). Additional low-affinity sites may be present on both cell lines but were not detected using the accessible TSP1 concentrations. The binding of soluble TSP1 was almost completely inhibited by heparin (Fig. 6B). Of the TSP1 antibodies tested, only A2.5, which blocks heparin and sulfatide binding to TSP1 (51), significantly inhibited high-affinity TSP1 binding to OH-1 cells (Fig. 6B). To con- firm the role of sulfated glycoconjugates in high-affinity binding of soluble TSP1 to OH-1 cells, the cells were cultured in medium containing chlorate to inhibit sulfation. Incorporation of [35S]sulfate in macromolecules was inhibited by 86% at 10 mM chlorate, and binding of TSP1 was inhibited by 55% for these cells (Fig. 6B). Binding of soluble TSP1 was not significantly inhibited in the presence of 2.5 mM EDTA (67.4 Ϯ 25.0 of control; P, 0.19 by a 2-tailed t test), which provided further evidence that most high-affinity binding of TSP1 to OH-1 cells was not mediated by the ␣3␤1 integrin.

Fig. 5. TSP1 inhibits SCLC cell proliferation. A, soluble TSP1 and ␣3␤1 integrin ligands inhibit SCLC cell proliferation. OH-1 cells (1 ϫ 104/well) were incubated for 72 h in growth medium containing the indicated concentrations of TSP1 (F), MBP-invasin (E), TSP1 peptide 678 (Œ), or the inactive peptide analogue 686 (‚). Net proliferation was determined by the CellTiter assay (Promega) and is presented as mean Ϯ SD (n ϭ 3). B, growth on immobilized TSP1 inhibits proliferation in the presence of EGF. OH-1 cell Fig. 6. Sulfated glycoconjugates mediate high-affinity binding of TSP1 to SCLC cells. proliferation in growth medium (F) or medium supplemented with 10 ng/ml EGF (E) was A, scatchard plot for binding of 125I-labeled TSP1 to OH-1 cells. Each point is the mean determined after 72 h on substrates coated with the indicated concentrations of TSP1, of triplicate determinations. B, inhibition of binding of thrombospondin to OH-1 cells by mean Ϯ SD (n ϭ 3). C, cell proliferation was determined in the presence of the indicated antibodies and heparin. Binding of 125I-labeled TSP1 to untreated OH-1 cells (2 ϫ 106/ F ␮ E ␣ ␤ ␮ concentrations of EGF in wells coated with BSA ( ) or with 50 g/ml TSP1 ( ). D, 3 1 ml) in the presence of 20 g/ml TSP1 antibodies A2.5 (NH2-terminal heparin-binding integrin ligands cooperate with EGF to inhibit OH-1 cell proliferation. Proliferation in the domain), C6.7 (COOH-terminal CD47-binding domain), D4.6 (calcium-dependent absence (f) or presence of 10 ng/ml EGF (o) was determined in wells coated with 10 ␮M epitope), A4.1 (central stalk of TSP1), or 25 ␮g/ml heparin, and to OH-1 cells grown in TSP1 peptides 678 (␣3␤1 ligand), 246 (heparin-binding peptide), 7N3 (CD47 ligand), or the presence of 10 mM chlorate to inhibit sulfation (86 Ϯ 4% inhibition of sulfation ␮ ␣ ␤ 35 1 g/ml MBP-invasin ( 3 1 ligand). Net proliferation is presented as a percent of the assessed by SO4 incorporation) is presented as a percent of binding for untreated ϪEGF control (mean Ϯ SD; n ϭ 3 for treated groups; n ϭ 6 for control groups). controls (mean Ϯ SD; n ϭ 3). 463

only on substrates coated with TSP1. SCLC cell adhesion on TSP1 is mediated by interactions of TSP1 with both ␣3␤1 integrin and sulfated glycolipids. Interaction of the ␣3␤1 integrin on SCLC cells with immobilized TSP1 stimulates formation of neurite-like processes and inhibits SCLC cell growth. Both the induction of neurite-like processes and the inhibition of proliferation by TSP1 are enhanced in the presence of EGF, which suggests that TSP1 and EGF cooperate to reduce SCLC growth by inducing neurotypic differentiation. These results demonstrate that the ␣3␤1 integrin is an important receptor in SCLC cells for transducing signals from TSP1. On the basis of the activity of a TSP1 peptide, these signals are initiated by

the binding of residues 190–201 in the NH2-terminal pentraxin mod- ule of TSP1 to this integrin. The ␣3␤1 integrin also plays a major role in the adhesion of breast carcinoma and endothelial cells to TSP1 (45),4 promotes neurite outgrowth in rat neurons on TSP1 (52), and Fig. 7. Sulfated glycolipids synthesized by OH-1 cells bind TSP1. Glycolipids extracts was recently shown to mediate interactions of neuroblastoma cells from OH-1 cells were resolved on silica gel high-performance TLC plates developed in ␣ ␤ ␣ ␤ chloroform:ethanol:0.25% aqueous CaCl2 (60:35:7). Lane a, acidic lipids from OH-1 cells with TSP1 (53). Although the 4 1 and 5 1 integrins are TSP1 metabolically labeled with [35S]sulfate (20 mg wet weight of cells) were separated by receptors on other cell types (44, 45, 53), these integrins do not play high-performance TLC and detected by autoradiography. Lanes b and c, acidic glycolipids from OH-1 cells (Lane b) and purified bovine brain sulfatide (50 ng, Lane c) were a significant role in SCLC cell adhesion on TSP1. The high-affinity resolved by high-performance TLC and incubated with 0.2 ␮g/ml 125I-labeled TSP1. binding of TSP1 to SCLC cells, however, is not mediated by this TSP1 binding was detected by autoradiography. integrin. This observation is consistent with previous reports that the affinity of TSP1 binding to ␣3␤1 integrin is relatively low (34) and that high-affinity binding of TSP1 to both breast carcinoma cells and 35 OH-1 cells incorporated SO4 into acidic glycolipids, glycopro- SCLC cells is mediated by sulfated glycoconjugates rather than the teins, and proteoglycans. In contrast to the predominant labeling of ␣3␤1 integrin (45). proteoglycans in most cell types examined previously (Ref. 41 and Sulfated glycolipids on cell membranes play a role in the interac- 3 35 unpublished results), the majority (89%) of [ S]-incorporation was tions of several cell types with TSP1 (54). In melanoma (41), carci- recovered in the lipid fraction extracted using chloroform/methanol. 5 125 noma, and endothelial cells, sulfated glycolipids typically account for No binding of I-labeled TSP1 was detected to the glycoprotein or 35 only 0.5–10% of the total SO4 incorporation at steady state, but proteoglycan fractions (results not shown); therefore, these fractions these are the major sulfated glycoconjugate on OH-1 cells. These were not further characterized. The sulfated glycolipids identified by 35 glycolipids mediate most high-affinity binding of soluble TSP1 to [ S]-labeling in extracts of OH-1 cells are shown in Fig. 7, Lane a. OH-1 cells and significantly contribute to their adhesion on immobi- Galactosyl sulfatide was the major sulfated lipid based on comigration lized TSP1 but do not mediate neurite outgrowth. of the labeled glycolipid with authentic bovine brain sulfatide in two Formation of neurite-like processes has been observed previ- developing solvents. When the total acidic glycolipids separated on ously when SCLC cells were plated on substrates coated with TLC were incubated with 125I-labeled TSP1 (Lane b), a major band laminin-1 (10), polyethyleneimine or the extracellular matrix pro- comigrating with authentic galactosyl sulfatide from bovine brain duced by PC-9 lung carcinoma cells (55). The present results (Lane c) was strongly labeled. Several more complex glycolipids also identify TSP1 as a matrix protein that also induces this response in incorporated [35S]sulfate (Lane a) but were not present in sufficient SCLC cells and is mediated by the ␣3␤1 integrin. This is consist- concentration to detect TSP1 binding (Lane b). ent with the observations that the ␣3␤1 integrin mediated neurite SCLC Cells Do Not Express TSP1. Initial attempts to detect outgrowth of rat sympathetic neurons on TSP1 (52) and that central synthesis of TSP1 by SCLC cell lines using a TSP1 RIA or metabolic and peripheral neurons (56) and neuroblastoma cells (53) formed labeling and immunoprecipitation were unsuccessful (data not neurites when plated on TSP1. EGF-induced neurotypic differen- shown). On the basis of RIA of conditioned medium from OH-1 cells tiation of thymic epithelial cells induced TSP1 expression, and grown in 4% Ultroser HY, which lacked detectable TSP1 immuno- TSP1 also induced neurite outgrowth in these cells (57). Therefore, reactivity, the cells secreted less than 100 ng of TSP1/106 cells/72 h. TSP1 may inhibit the growth of SCLC cells by triggering signaling Total RNAs were extracted from the SCLC cell lines, and mRNA for ␣ ␤ three thrombospondin genes were detected using the RT-PCR. THBS1 through the 3 1 integrins that induce the cells to differentiate mRNA expression was not detected in any SCLC cell lines (Fig. 8, along a neuronal pathway. Enhancement of this differentiation Lane a, and data not shown), but the predicted PCR product was signal by EGF may explain the synergism of these two proteins to readily detected using endothelial cell RNA (Lane e). THBS2 expres- inhibit SCLC cell proliferation. EGF is known to have both sion was detected at low levels in some SCLC cell lines but only after growth-stimulating and -inhibitory activities in other cell types 50 cycles of amplification (data not shown). However, all six of the (58), but inhibition by EGF has not been observed in SCLC. 4 human SCLC cell lines examined expressed significant levels of As was observed in breast carcinoma and endothelial cells (45), ␣ ␤ THBS3 mRNA (Fig. 8, Lane c, and data not shown). the activation state of 3 1 integrin in SCLC cells to recognize TSP1 is regulated. The signals that mediate this regulation, however, seem to be different for each cell type. The ␣3␤1 integrin in SCLC cells is DISCUSSION activated by EGF and somatostatin but not by CD47 ligation or by TSP1 is an adhesive protein for all of the SCLC lines we examined. FGF or IGF1 receptor ligands, whereas IGF1 but not EGF activates Although some SCLC lines attached on other extracellular matrix components, the two classic SCLC lines, OH-1 and H128, attached 4 Chandrasekaran, L., He, C-Z., Krutzsch, H. C., Iruela-Arispe, M. L., and Roberts, D. D. Modulation of endothelial cell behavior and angiogenesis by an ␣3␤1 integrin- binding peptide from thrombospondin-1, submitted for publication. 3 Roberts, D. D., unpublished observations. 5 Roberts, D. D., unpublished observations. 464

suppress growth and increase neurotypic differentiation of SCLC. Inducing neuroendocrine differentiation of SCLC by a combination of TSP1 (or the integrin-binding peptide from TSP1) and EGF may facilitate treatment of this cancer because neuroendocrine differentiation is correlated with increased sensitivity of SCLC to radiotherapy (38).

ACKNOWLEDGMENTS

We thank Drs. Steven Akiyama (NIH, Research Triangle Park, NC), David Cheresh, William Frazier, Adi Gazdar, Ralph Isberg (Tufts University School Fig. 8. RT-PCR analysis of THBS mRNA expression. Total mRNA was prepared from OH-1 SCLC (Lanes a-d) and aortic endothelial cells (Lane e). Specific primers were used of Medicine, Boston, MA), Lance Liotta, Joel Shaper, and Kenneth Yamada to reverse transcribe and amplify 192-, 258-, and 291-bp fragments of THBS1 (Lanes a, for providing reagents. e), THBS2 (Lane b), and THBS3 mRNA (Lanes c), respectively. Control primers amplified a 400-bp fragment of glyceraldehyde 3-phosphate dehydrogenase cDNA from positions 664 to 1064 (Lane d). Numbers in margins indicate migration of ␾␹174 HaeIII digested markers. All of the primers amplified the expected size products using RNA from the REFERENCES human breast carcinoma cell line MDA-MB-435, which was used as a positive control 1. Cook, R. M., Miller, Y. E., and Bunn, P. A. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2000 American Association for Cancer Research. Thrombospondin-1 Promotes α3β1 Integrin-mediated Adhesion and Neurite-like Outgrowth and Inhibits Proliferation of Small Cell Lung Carcinoma Cells

Neng-hua Guo, Nancy Smyth Templeton, Hebah Al-Barazi, et al.

Cancer Res 2000;60:457-466.

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