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Thrombospondin-1 Promotes 3 1 Integrin-Mediated Adhesion And

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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 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2000 American Association for Cancer Research. SCLC REGULATION BY TSP-1 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).
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