Peptide Growth Factors Factor Expression by Human Breast Carcinoma Cell Lines and Regulation of Fibroblast Hepatocyte Growth Factor/Scatter

Hepatocyte growth factor/scatter factor (HGF/SF) is a stromallv de rived modulator of epithelial cell proliferation and morphology. To better assess the potential role of HGF/SF in tumor progression we sought to identify factors and biological conditions which regulate its expression. We show that several adult human primary fibroblast cultures from breast and prostate produce HGF/SF. HGF expression in the MRC-5 human fetal lung fibroblast cell line is stimulated by conditioned media harvested from human breast tumor cell lines (MCF-7, T47D, and MDA-MB-231). In contrast, both indirect and direct coculture of each of these tumor lines with MRC-5 fibroblasts down-regulates HGF/SF expression. Finally, we show that MRC-5 HGF expression is inhibited by several known peptide growth factors, including transforming growth factor ß, epidermal growth factor, and transforming growth factor a. INTRODUCTION Recent findings concerning HGF' suggest that our concept of this molecule and its potential biological role must be enlarged. HGF was initially characterized as a potent stimulator of hepatocyte prolifera tion ( 1). Independently, SF was discovered in media conditioned by certain cultured fibroblast cell lines based on its ability to "scatter" colonies of epithelial cells (2). Recently, protein sequencing, comple mentary DNA analysis, immunological data, and functional assays revealed that these two factors are identical (3-5). In addition, char acterization of a broad-spectrum lung fibroblast-derived mitogen re vealed that it too was a highly related to HGF (6). To reflect the functional duality of this molecule as both a mitogen and a motogen, we will refer to it henceforth as HGF/SF. HGF/SF is synthesized as a single polypeptide chain with a mo lecular weight of 87,000. This form is then processed into a dimeric protein composed of light (M, 30,000) and heavy (M, 60,000) chains linked by a disulfide bond ( 1, 7). The receptor for HGF/SF is a tyrosine kinase transmembrane protein encoded by the c-met protoon cogene (8, 9). Despite detailed characterization of HGF/SF (10-12), the definition of its role in vivo remains incomplete. Although several studies have established HGF/SF as a humoral mediator of liver regeneration [reviewed by Nakamura (13), Michalopoulos and Zarnegar (14), Zarnegar et al. (15). and Gohdaef al. (16)], it is clear that this definition alone is insufficient. Increasing evidence suggests that HGF/SF may have a more global impact on stromal epithelial inter actions in processes such as: wound healing (17), carcinogenesis [reviewed by Gherardi and Stoker (18)], and morphogenesis (19). Because HGF/SF affects both epithelial cell proliferation and motility, the potential role of HGF/SF in tumor progression is particularly Received 8/21/92; accepted 1/8/93. 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 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported in part by a grant from the National Kidney Foundation. To whom requests for reprints should be addressed, at Department of Anatomy and Cell Biology. Georgetown University. Medical-Dental Building. 39(X) Reservoir Rd.. Washington. DC 20007. ' The abbreviations used are: HGF. hepatocyte growth factor; SF. scatter factor; PCF. peptide growth factor; EGF. epidermal growth factor; TGF-a. transforming growth factor a; TGF-ß,transforming growth factor ß;bFGF. basic fibroblast growth factor; aFGF. acidic fibroblast growth factor; DMEM. Dulbecco's modified Eagle's medium; FBS. fetal bovine serum; BPH. benign prostatic hyperplasia. compelling. To better assess this possibility, we sought to identify the factors and biological conditions which regulate its expression. Here we show that HGF/SF expression is regulated in a paracrine fashion by human breast tumor cell lines and show that several PGFs inhibit HGF/SF expression. MATERIALS AND METHODS Cell Culture. The MRC-5 human fetal lung fibroblast cell line was pur chased from the American Type Culture Collection (Rockville, MD). The human breast carcinoma cell lines including MCF-7, T47D. and MDA-MB231 were donated by the laboratory of Dr. Marc E. Lippman (Lombardi Cancer Center. Washington. DC). All cell lines were maintained in DMEM (Irving Scientific. Santa Ana, CA) plus 10% FBS and antibiotics (Sigma Chemical Co., St. Louis. MO). Conditioned Media Experiments. Breast carcinoma cell lines MCF-7. T47D, and MDA-MB-231 were plated at a density of 300.000 cells/cm2. These cultures were left overnight in DMEM plus 10% FBS to attach. Cells were then washed with serum free DMEM and pretreated for 4 h to remove residual serum. Following pretreatment cells were placed in fresh DMEM and left for 24 h prior to the collection of conditioned medium. MRC-5 cells were plated at 50,000 cells/cmand left overnight in DMEM plus 10% FBS to attach. Cells were then washed with serum free DMEM and pretreated for 4 h to remove residual serum. Following pretreatment in DMEM, conditioned media from the breast carcinoma cultures were added directly to MRC-5 cultures. Treatments were left for 24 h, at which time, the media and MRC-5 RNA were harvested. For some experiments, breast carcinoma cell line conditioned media samples were either frozen, heat treated to 80°Cfor 5 min. or dialyzed in an Amicon Centricon-30 microconcentrator (Danvers. MA) prior to being added to MRC-5 cultures. Coculture Experiments. In coculture experiments, the overall cell num bers and the ratio of carcinoma cells to MRC-5 fibroblasts were identical to those in conditioned media experiments (i.e., breast cell lines, 300.000 cells/ cm-; MRC-5 cells, 50,000 cells/cm2; ratio, 6:1). Coculture experiments were done in direct or indirect fashion. For direct coculture, both cell types were plated simultaneously in six-well plates and left overnight in DMEM plus 10% FBS to attach. For indirect coculture. the breast carcinoma cell lines were plated indepen dently in Millipore (Bedford, MA) culture inserts (30 mm diameter; tissue culture treated polycarbonate membranes. 0.4 urn pore size). MRC-5 cells were seeded in a six-well plate, and each cell type was allowed to attach overnight in DMEM plus 10% FBS. Culture inserts containing the human breast carci noma cell lines were then placed into the wells containing MRC-5 fibroblasts. Both direct and indirect cocultures were washed in DMEM and pretreated for 4 h prior to beginning the 24-h conditioning period. Following the 24-h conditioning period coculture conditioned media were harvested. For indirect coculture experiments, RNAs from breast carcinoma cell lines and the MRC-5 cell line were harvested separately. For coculture experiments involving the use of blocking antibodies to TGFß,.the indirect coculture format was used. Antibodies were added following the 4-h pretreatment in DMEM, at levels sufficient to block 4 ng/ml TGF-ß,. Treatments were left for 24 h prior to Western blot analysis. Growth Factor Experiments. MRC-5 cells were prepared for treatment as in the conditioned media experiments. Following pretreatment in DMEM for 4 h. cells were placed in fresh DMEM and growth factors were added. EGF, TGF-a, aFGF. and bFGF (Sigma) were added in 10-ng/ml doses. TGF-ß, (a gift from Dr. Anita B. Roberts) was added in doses of 10-1000 pg/ml. Treatments were left for 24 h at which time conditioned media and MRC-5