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Blockade of the Type I Somatomedin Receptor Inhibits Growth of Human Breast Cancer Cells in Athymic Mice

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Blockade of the Type I Somatomedin Receptor Inhibits Growth of Human Breast Cancer Cells in Athymic Mice Blockade of the type I somatomedin receptor inhibits growth of human breast cancer cells in athymic mice. C L Arteaga, … , D C Allred, C K Osborne J Clin Invest. 1989;84(5):1418-1423. https://doi.org/10.1172/JCI114315. Research Article Insulin and insulin-like growth factors (IGIs) stimulate the growth of human breast cancer cells in vitro. The type I somatomedin receptor (SR) expressed in these cells may mediate the growth effects of these peptides. We have examined the role of this receptor on human breast cancer growth with a monoclonal antibody (alpha-IR-3) that blocks the receptor binding domain and inhibits IGF-I-induced growth. alpha-IR-3 inhibited clonal growth in vitro and blocked the mitogenic effect of exogenous IGF-I in both MCF-7 and MDA-231 breast cancer cell lines. Antibody-induced blockade of the type I SR also inhibited the estrogen-independent MDA-231 cells growing in vivo in nude mice, but growth of the estrogen-dependent MCF-7 cells was unaffected. IGIs are important growth regulators of MDA-231 breast cancer cells. Blockade of this growth stimulatory pathway may provide a new treatment strategy. Find the latest version: https://jci.me/114315/pdf Blockade of the Type I Somatomedin Receptor Inhibits Growth of Human Breast Cancer Cells in Athymic Mice Carlos L. Arteaga, Libbey J. Kitten, Ester B. Coronado, Steven Jacobs,* Frederick C. Kull, Jr.,* D. Craig Allred,t and C. Kent Osborne Division ofMedical Oncology and tDepartment ofPathology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284; and * Wellcome Research Laboratories, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709 Abstract cancer cells via the type I somatomedin receptor (SR), also known as the IGF-I receptor (5-7). As shown in other systems, Insulin and insulin-like growth factors (IGIs) stimulate the this receptor may also mediate some of the mitogenic effects of growth of human breast cancer cells in vitro. The type I so- IGF-II and insulin (10-15). Furthermore, breast cancer tissue matomedin receptor (SR) expressed in these cells may mediate from patients exhibits higher IGF-I binding than adjacent the growth effects of these peptides. We have examined the normal tissue (16) suggesting a link between type I SR and role of this receptor on human breast cancer growth with a breast epithelial cell transformation. If these peptides are im- monoclonal antibody (a-IR-3) that blocks the receptor binding portant endocrine or autocrine/paracrine growth regulators, domain and inhibits IGF-I-induced growth. a-IR-3 inhibited then blockade of this receptor might inhibit breast cancer clonal growth in vitro and blocked the mitogenic effect of exog- growth. In the present study we have investigated the effect of enous IGF-I in both MCF-7 and MDA-231 breast cancer cell blockade of the type I SR on cultured human breast cancer lines. Antibody-induced blockade of the type I SR also inhib- cells grown in vitro and in vivo as subcutaneous tumors in the ited the estrogen-independent MDA-231 cells growing in vivo athymic nude mouse. in nude mice, but growth of the estrogen-dependent MCF-7 cells was unaffected. IGIs are important growth regulators of MDA-231 breast cancer cells. Blockade of this growth stimu- Methods latory pathway may provide a new treatment strategy. Cells, hormones, and antibodies. The MCF-7 line was supplied by Dr. Introduction M. Lippman (National Cancer Institute) and the MDA-23 1 line was obtained from the American Type Culture Collection (Rockville, Insulin and insulin-like growth factors (IGFs)' stimulate the MD). Cells were cultured and passaged as previously described (17). proliferation of cultured human breast cancer cells ( 1-6). Re- Human recombinant IGF-I was purchased from Amgen Biologicals cancer have been to and (Thousand Oaks, CA). 251I-IGF-I (sp act 2,125 Ci/mmol) was obtained cently, breast cells reported synthesize from the Amersham Corp. (Arlington Heights, IL). secrete IGF activities into the culture medium (5-7), suggest- a-IR-3 antibody is a mouse monoclonal IgG, prepared after immu- ing that these polypeptides could function as both endocrine nization with partially purified insulin receptors from human placenta and autocrine/paracrine growth regulators (8). In estrogen re- (18). This antibody specifically blocks type I SR binding (18, 19). The ceptor (ER)-positive cells, the expression and secretion of both mouse monoclonal a-IR- 1 antibody, which recognizes a nonbinding IGF type I- and II-like proteins are increased by estrogen domain of the insulin receptor and has no biologic activity (12, 19), leading to the possibility that they contribute to estrogen-in- was used as a control. Purified antibody preparations contained no duced growth (6, 9). detectable endotoxin (< 0.3 pg/fug by the Limulus ameba lysate test). It is assumed that IGF-I stimulates the growth of breast Binding studies. Breast cancer cells were grown in 24-well tissue culture plates (Corning Glass Works, Corning, NY). Once confluent, the monolayers were washed with binding buffer (improved minimum essential medium [IMEM], 0.1% BSA, 25 mM Hepes [Gibco Labora- This work was presented in part at the 70th Annual Meeting of the tories, Grand Island, NY], pH 7.4) and incubated with 40 pM '25I- Endocrine Society in New Orleans, LA, 1988, and published in ab- IGF-I plus various concentrations of unlabeled a-IR-3 or IGF-I, for 90 stract form in the 1988 Meeting Proceedings, p. 191. min at room temperature. Monolayers were then washed three times Dr. Arteaga's current address is Division of Medical Oncology, with cold PBS/0. 1% BSA and solubilized with 0.5 N NaOH, and cell- Vanderbilt University, VA Medical Center, 1310 24th Avenue South, associated radioactivity was measured in a gamma counter. Nashville, TN 37212. In vitro growth experiments. For anchorage-independent growth Address reprint requests to Dr. Osborne, Department of Medicine, experiments, a single-cell suspension of 5 X 103 cells was cloned in Division of Oncology University of Texas Health Science Center at 0.8% agarose as previously described (17) with different concentrations San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7884. of a-IR-3 or a-IR-l. The dishes were incubated in a 5% CO2 atmo- Receivedfor publication 16 March 1989 and in revisedform 17 July sphere at 37°C and colonies (measured 2 50 um) were counted using 1989. an image analyzer (Omnicon model II, Bausch & Lomb, Inc., Roches- ter, NY). For monolayer growth experiments, 30 X 103 cells per well 1. Abbreviations used in this paper: EGF, epidermal growth factor; ER, were plated in 24-well tissue culture plates in IMEM supplemented estrogen receptor; IGF, insulin-like growth factor; IMEM, improved with 5% fetal calf serum (FCS). After 24 h, the medium was removed minimum essential medium; SR, somatomedin receptor. and replaced with phenol red-free serum-free IMEM alone or with IGF-I, a-IR-3, or both. Medium with hormone and/or antibody was J. Clin. Invest. exchanged every 2 d. Cell counts were determined after suspending the © The American Society for Clinical Investigation, Inc. cells with 1 mM EDTA 5 d after plating. 0021-9738/89/11/1418/06 $2.00 In vivo experiments. 3-wk-old female athymic mice (Harlan Volume 84, November 1989, 1418-1423 Sprague-Dawley, Madison, WI) were inoculated in the flank just cau- 1418 Arteaga et al. Table L Effect ofa-IR-3 and a-IR-i on Anchorage-independent percent inhibition of binding. Each sample of mouse serum was tested Growth ofBreast Cancer Cells individually in triplicate wells. MCF-7 MDA-23 1 Results PBS 194±24 1098±28 a-IR-3 Binding experiments. In both breast cancer cell lines competi- 1.0 nM 115±38 827±91 tive binding studies and Scatchard analysis demonstrated that 10 nM 55±7 550±37 a-IR-3 was more potent than the native hormone in inhibiting 100 nM 34±10 488±57 binding of '25I-IGF-I (data not shown). These binding data a-IR-I revealed a Kd of 0.6 nM (MCF-7) and 2.1 nM (MDA-23 1) for 100 nM 205±21 1160±141 the receptor antibody compared with 2.9 and 4.2 nM for IGF-I. a-IR- 1, at a concentration of 130 nM, had no effect on A single-cell suspension of 5 X 103 cells was cloned in 0.8% agarose labeled IGF-I binding. containing IMEM, 10% calf serum, and 10 mM Hepes. Colonies In vitro growth experiments. Since a-IR-3 was an effective measuring 2 50 lsm were counted as described in Methods after inhibitor of binding to the type I SR, we next determined 10-14 d. Values are the mean±SE of triplicate determinations. whether this antibody-mediated receptor blockade could in- hibit the growth of breast cancer cells in vitro. Anchorage-in- dependent growth of both lines was inhibited reproducibly by dal to the forelimb with 107 log phase MCF-7 or MDA-231 cells. Twice a-IR-3 in a dose-dependent fashion (Table I). A maximal ef- weekly intraperitoneal injections of PBS, a-IR-3, or a-IR- 1 were fect was observed with 100 nM a-IR-3, the concentration at started simultaneously and continued for 4-5 wk. All mice inoculated which maximal inhibition of receptor binding was also ob- with MCF-7 cells and some with the MDA-231 cells received a 0.25- served. The a-IR- 1 control antibody had no inhibitory effect mg 17f3-estradiol pellet (Innovative Research, Toledo, OH) implanted on colony growth.
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