0013-7227/90/1261-0596$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society

Vol. 126, No. 1 Printed in U.S.A.

Expression of the Transforming Growth Factor-a/Epidermal Growth Factor Receptor Pathway in Normal Human Breast Epithelial Cells SUSAN E. BATES, EVA M. VALVERIUS, BRUCE W. ENNIS, DIANE A. BRONZERT, JAMES P. SHERIDAN, MARTHA R. STAMPFER*, JOHN MENDELSOHN, MARC E. LIPPMAN, AND ROBERT B. DICKSON Medicine Branch, National Cancer Institute, National Institutes of Health (S.E.B., D.A.B.), Bethesda, Maryland 20892; Lombardi Cancer Research Center, Georgetown University (E.M. V., B. W.E., J.P.S., M.E.L., R.B.D.J Washington, D.C. 20007; Lawrence Berkeley Laboratory (M.R.S.), Berkeley, California 92720; and the Laboratory of Receptor Biology, Memorial Sloan Kettering Cancer Center and Cornell University Medical College (J.M.), New York, New York 10021

184) failed to reduce the expression of either TGFa or the EGF receptor. Likewise, cessation of growth associated with both senescence and confluence of the 184 cells did not result in reduced expression. However, regulation of TGFa mRNA could be demonstrated by withdrawal of EGF from the medium or by antibody-mediated blockade of the EGF receptor in 184 cells. Antibody-mediated EGF receptor blockade also results in inhibition of growth and [3H]thymidine labeling. An autoregulatory autocrine loop appears operant in proliferating breast epithelial cells. Both growth and levels of TGFa mRNA expression are controlled by binding of ligand to the EGF receptor. These studies suggest a role for the TGFa/EGF receptor pathway in normal breast cell physiology. (Endocrinology 126: 596-607, 1990)

ABSTRACT. To better understand the possible roles and interactions of transforming growth factor-a (TGFa) and its receptor, the epidermal growth factor (EGF) receptor in human breast epithelium, we have studied the expression of TGFa and the EGF receptor in a series of normal human mammary epithelial cells derived from reduction mammoplasty before in vitro propagation, during short term proliferation in vitro, and after immortalization. Increased TGFa mRNA expression coincided with conversion of the cells to a proliferative state in vitro. After establishment, propagation, and proliferation in vitro, the cells expressed high levels of both TGFa and EGF receptor mRNAs. Addition of diverse growth inhibitory agents, including 12-0tetradecanoylphorbol-13-acetate (TPA), TGFjS, and sodium butyrate, to one of these rapidly proliferating cell populations (no.

T

HE INTERACTION of transforming growth factor-a (TGFa) with the epidermal growth factor (EGF) receptor has been proposed to be an integral part of cellular transformation or proliferation in a number of systems (1). Its first description involved the phenotypic transformation of normal rat kidney (NRK) cells, an immortal rat fibroblast line (2). Similarly, transformation of both the NRK cells and second immortal rodent fibroblast line, Rat 1, has been induced by transfection with cDNAs expressing TGFa (3, 4). While TGFa expression has been noted in a spectrum of malignancies (5), the relationship of the phenotypic alterations in immortal fibroblast lines to its real role in malignancy has been obscure.

The presence of TGFa in an increasing variety of normal human cell types has been noted, including keratinocytes, macrophages, and ovarian, colonic, liver, and breast cells (6-11). Additionally, TGFa-like activity has been found in breast milk and fluids (12,13). The significance of TGFa in these systems, as in malignancy, is unknown. Its putative role in stimulating human cell growth in vivo remains unproven. We have studied normal human breast epithelial cells derived from reduction mammoplasty. Pure epithelial cell clusters, termed organoids, can be isolated from these tissues. Placement of these cells in culture in serum-free medium results in the outgrowth of rapidly proliferating normal mammary epithelial cells (14, 15). After 2-3 passages in vitro the majority of cells die, while the remaining cells continue to rapidly proliferate in tissue culture for 12-22 passages before senescence. Rapid cellular proliferation is marked by doubling times ranging from 18-24 h, while during senescence a steady state number of cells is maintained without increases in cell

Received August 8,1989. Address all correspondence and requests for reprints to Susan E. Bates, Medicine Branch, Building 10, Room 12N226, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892. * Supported by NIH Grant CA-24844 and the Office of Energy Research, Office of Health and Environmental Research of the U.S. Department of Energy under Contract DE-AC03-76SF00098.

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TGFa/EGF RECEPTOR IN NORMAL BREAST CELLS

number, and eventually cell death occurs. Cells from 1 individual specimen donor, identified as 184, have been extensively characterized (15). A near diploid nonmalignant immortal line known as 184A1 was obtained after treatment of 184 cells with benzo-a-pyrene (16). A subclone of the 184A1 line, termed A1N4, was isolated on the basis of less stringent medium requirements. The proliferating normal human breast epithelial cells contain high levels of TGFa and EGF receptor mRNA, secrete TGFa into their medium, and average 5 x 105 EGF receptor-binding sites/cell (9, 10). The cells depend on EGF for growth at clonal density and lose that requirement at higher density (9) suggesting that crossfeeding of endogenous TGFa is occurring between cells at normal seeding densities. Taken together, these data suggest that maintenance of rapid proliferation in the normal mammary cells in vitro (at densities above clonal) is occurring via an autocrine pathway. By comparison, the A1N4 cells also have high levels of TGFa and EGF receptor mRNA and protein, but are much more dependent upon exogenous EGF for growth in monolayer culture. The mechanism underlying this increased dependence in A1N4 cells compared to the 184 parental cell population is not known. This dependence occurs despite secretion of a biologically active form of TGFa and expression of apparently normal receptors by binding characteristics. After the observation that the proliferating normal 184 cells were making large quantities of TGFa in culture, we sought to examine regulation of TGFa and EGF receptor gene expression to provide insight into their involvement in cellular proliferation. If TGFa and the EGF receptor were linked in an autocrine loop to provide a proliferative stimulus for the cell, it seemed plausible that their expression would be tightly coupled to the growth state of the cell. We, therefore, examined TGFa and EGF receptor expression in normal mammary epithelial cells before growth in tissue culture, during rapid proliferation, during growth inhibition induced by a variety of pathways, and during senescence. We found that TGFa/EGF receptor mRNA expression was clearly induced by conversion to the proliferative state after the cells adapted to tissue culture. Once induced, gene expression was not suppressed by the addition of exogenous growth inhibitors. However, effective withdrawal of EGF from the cells resulted in a decrease in TGFa mRNA expression. Addition of EGF receptor-blocking antibodies resulted in decreased growth, distinct morphological alterations, and decreased DNA synthesis. Thus, TGFa or EGF seems to be the primary regulator of the system. Taken together, the data suggest the existence of an autoregulatory autocrine pathway in normal breast epithelial cells.

597

Materials and Methods Three human breast cancer cell lines were used. MDA-MB231 and MDA-MB-468, obtained from the American Type Culture Collection (Rockville, MD), served as positive controls for TGFa and EGF receptor expression in Northern blots (17). Hs578T, kindly provided by Helene Smith (18), served as a negative control for TGFa: mRNA expression (17). The cell lines were propagated in T-175 flasks in Improved Modified Eagle's Medium (Gibco, Grand Island, NY), supplemented with 10% fetal bovine serum (FBS; Gibco). Organoids prepared according to previously described methods were stored over liquid nitrogen until use (14, 15). Normal human mammary epithelial cells derived from specimens 184, 172, 161, and 102 were propagated in a 1% CO2 atmosphere, as previously described, in MCDB170 medium obtained from the University of California-San Francisco Tissue Culture Unit with serum-free supplements, including 5 ng/ml EGF, 70 ng/ ml bovine pituitary extract (BPE); Collaborative Research, Waltham, MA and Hammond Cell Technologies, Alameda, CA), 5 ng/ml insulin, and 0.5 fig/m\ hydrocortisone (14). Experiments with the cells were typically carried out at passages 10-13. Senescence occurred at passages 13-22. The benzo-apyrene-immortalized A1N4 subline was propagated in Improved Modified Eagle's Medium (IMEM) (Gibco) with 0.5% FBS (Gibco), 10 Mg/ml insulin (Sigma, St. Louis, MO), 0.1 (xg/ ml hydrocortisone (Sigma), and 10 ng/ml EGF (Collaborative Research) in a 5% CO2 atmosphere. Two additional populations of proliferating normal breast cells obtained from Kieran Horgan (Cardiff, Wales) were propagated in IMEM in 10% FBS, 100 ng/ml EGF, insulin, and hydrocortisone before RNA harvest. These cells were analyzed for TGFa mRNA expression only. Growth inhibition studies For evaluation of the effects of growth inhibition on TGFa and EGF receptor, cells were plated in their usual medium and incubated for a minimum of 24 h before the addition of growth inhibitors. Subsequently, inhibitors were added to final concentrations of 100 nM 12-0-tetradecanoylphorbol-13-acetate (TPA), 5 ng/ml TGF/3, or 2 mM sodium butyrate in the supplemented MCDB170 medium. Both the growth curves and analysis of mRNA after addition of the growth inhibitors were performed on at least three separate preparations of cells. The 24-h EGF withdrawal experiments in the 184 cells were carried out in the absence of both BPE, which contains TGFa (19), and EGF. The 7-day growth curve was performed in the presence of BPE. EGF withdrawal experiments in the A1N4 cells were performed in their usual medium, as described, without the addition of EGF. Blocking EGF receptor antibodies included the 96 (IgM) monoclonal antibody purified from ascites obtained after immunizing mice with hybridoma cells prepared using membranes isolated from Chinese hamster ovarian cells transfected with EGF receptor (the ascitic fluid was a gift of J. Schlessinger) (20). The monoclonal antibody binds to and blocks human, but not murine, EGF receptor (Schlessinger, J., personal communication). Monoclonal antibody 225 IgG was raised against EGF receptor from A431 cells as previously described (21, 22).

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The doses chosen for the EGF withdrawal experiments (20-40 nM), inhibited EGF-dependent growth of A1N4 cells in medium containing 1 ng/ml EGF by 75% relative to the nonspecific IgG or IgM control. Thymidine labeling index Proliferating 184 cells for simultaneous RNA analysis and [3H]thymidine labeling were plated at 8 X 104 cells/slide in 6 ml in each well of a four-well Quadriperm dish (Rupp and Bowman). Slides were prepared as for RNA in situ hybridization, described below. Thymidine labeling was carried out for 2 h at 37 C in 1 jj.Ci/m\ [3H]thymidine and 10"7 M cold thymidine. Subsequently, the cells were washed and fixed in 90% ethanol10% acetic acid, air dried, and dipped in a 1:2 dilution of NTB emulsion (Eastman Kodak, Rochester, NY) and developed after 1 week. The thymidine labeling index (TLI) was calculated as the number of labeled cells per 100 cells. EGF receptor binding assay Cells were plated in poly-D-lysine-coated 24-well cluster dishes (Falcon, Oxnard, CA) in their regular medium; after overnight incubation, cells were treated with TGF0, TPA, or sodium butyrate, at the concentrations given above, for 24 h. Before binding assays, cells were washed and incubated overnight in medium without EGF or BPE, maintaining treatments. Binding was carried out as previously described (9), using varying concentrations of [125I]EGF (human; ICN Radiochemicals, Irvine, CA; SA, 80-160 fiCi/ng). EGF receptor parameters were calculated using the Ligand program (23). RNA extraction, electrophoresis, and filter preparation Total cellular RNA was extracted from cells by homogenizing in guanidine isothiocyanate, followed by centrifugation over a cesium chloride cushion (24). RNA was electrophoresed in 1% agarose-6% formaldehyde gels. Gels were stained with 2 ng/m\ ethidium bromide to allow inspection of the quantity and quality of RNA. RNA gels were subjected to partial alkaline hydrolysis before transfer of RNA to nitrocellulose by capillary blot (24). Hybridization probes A 1.3-kilobase cDNA encoding the precursor for mature TGFa (25) ligated into the plasmid SP65 was used to make a synthetic riboprobe. 36B4, a cDNA constitutively expressed in MCF-7 cells, was subcloned into pGEM3 and used here to make a control probe (26). In the 184 cells 36B4 demonstrated less variability and reflected the ethidium bromide-stained RNA pattern better than the more commonly used /3-actin. A 137basepair HmdIII fragment from pE7 (a gift of Glen Merlino, NCI) was subcloned into pGEM 4 by Francis G. Kern (Lombardi Cancer Research Center) and generously donated to make a riboprobe vector for detection of EGF receptor mRNA (27). A 603-basepair Bg\l-EcoRl fragment of TGFa cDNA subcloned in pGEM 3 was used to generate radiolabeled antisense riboprobes and unlabeled antisense RNA for RNA in situ hybridizations.

Endo • 1990 Voll26«Nol

Hybridizations Synthetic riboprobe was prepared by SP6 or T7 polymerase transcription (Promega Biotech, Madison, WI), as appropriate for each probe, using [a-32P]Uridine 5'-triphosphate, tetra (triethylammonium) salt (SA, 3000 Ci/mmol; New England Nuclear, Boston, MA) (28). Filters were prehybridized for 1-4 h at 55 C in 50% formamide (Fluka), 5 X Denhardt's, 5 X SSC, 0.1% sodium dodecyl sulfate (SDS), and 200 Mg/ml salmon sperm DNA (Sigma) and hybridized for 16-18 h in the same buffer with 2 x 106 cpm/ml labeled riboprobe. After hybridization, filters were washed twice for 30 min each with 1 x SSC0.1% SDS heated to 68 C and once for 30-45 min with 0.1 X SSC-0.1% SDS in a 68 C water bath. After hybridization, autoradiography was performed using Kodak XAR-5 film exposed for 2-4 days at -70 C. RNA in situ hybridizations RNA in situ hybridizations were performed according to the method of Han et al. with modifications (29). Solutions were generally prepared using double distilled water containing 0.1% diethylpyrocarbonate. Microscope slides were prepared in 1% aminopropyltriethoxysilane (Sigma). Cells grown on prepared slides were fixed for 1-2 min in 4% paraformaldehyde. Slides were then stored in 70% ethanol at 5 C until use. Pretreatment for in situ hybridization included washing in 2 X SSC, followed by acetylation in 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8) for 10 min, followed by rinsing with PBS and immersion in 0.1 M Tris-0.1 M glycine for 30 min. After a brief rinse in 2 X SSC, slides were prehybridized for 5 min at 52 C in 2 X SSC-50% formamide-10 mM dithiothreitol (DTT). Synthetically labeled riboprobes for in situ hybridization were labeled to a high specific activity using [35S] uridine 5'(athio)triphosphate (Amersham, Arlington Heights, IL), or [35S]cytidine 5'-(a thio)triphosphate (New England Nuclear). Each slide received 40 fA hybridization mixture containing 50% formamide (Fluka), 10% dextran sulfate (Sigma), 2 x SSC, 2 mg/ml BSA (Boehringer-Mannheim, Indianapolis, IN), 1 mg/ml E. coli tRNA (Sigma), 1 mg/ml herring sperm DNA (Sigma), 50 mM DTT (Sigma), and 3.75 x 107 cpm/ml radioactive probe. After a 5-h incubation at 50 C, coverslips were removed in 2 X SSC containing 10 mM DTT, and the slides were washed sequentially in 50 C 50% formamide-2 X SSC with and without 10 mM DTT for 10 and 20 min, respectively. After rinsing in 2 X SSC, the slides were treated with 100 Mg/ml RNAse-A for 30 min at 37 C. A subsequent 5-min 50% formamide-2 x SSC-10 mM DTT wash at 50 C was followed by rinses at room temperature in 2 X SSC and dehydration in an ascending ethanol series. Slides were dipped in Kodak NTB emulsion, diluted 1:2, and stored in the dark at 5 C for 2 weeks before development.

Results TGFa gene expression in nonproliferating and proliferating normal human mammary epithelial cells Our initial observation of high levels of expression of TGFa mRNA in the 184 proliferating normal mammary

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epithelial cells (9) was extended to other populations of rapidly proliferating mammary epithelial cells. As shown in Fig. 1, marked levels of TGFa expression could be detected in three proliferating normal epithelial cell populations, 119, 99, and 184, with lower levels in 102. Shown in Fig. 4, below, are two additional samples (161 and 172) which demonstrate levels of TGFa mRNA expression comparable to that of 184. To determine whether TGFa mRNA was expressed in the nonproliferating organoids, we examined expression in total RNA extracted from four organoid samples stored over liquid nitrogen after isolation from mammary tissue. Because of the RNA quantities required for Northern analysis, the number of samples analyzed in this way was limited. In three of four different organoid preparations, one which had given rise to the normal culture 102, levels of TGFa were at or below the level of detection in total RNA. In one of the organoid samples (123) TGFa mRNA was clearly detectable. The organoid 186 was derived in the same way as the organoids from reduction mammoplasty, but from nontumor tissue in a patient with breast cancer. To confirm these observations on a cellular basis, RNA in situ hybridization was carried out on four additional organoid samples. In Fig. 2, the level of TGFa in four organoids was beneath the level of detection (A-D), including that observed in the 184 organoid (D), from which the proliferating normal 184 cells were derived. These levels contrast to the mRNA in the 184 organoid hybridizing to the 36B4 control probe (Fig. 2E) and to CO CM CD O>

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36B4 FlG. 1. Expression of TGFa mRNA in normal mammary cells. Eight micrograms of total RNA were analyzed for each sample. RNA samples from the human breast cancer cell lines MDA-MB-231 and Hs578T were used for positive and negative controls, respectively, in this figure and in the Northern blots shown in subsequent figures. Samples 102, 184,119, and 99 are mRNA harvested from rapidly proliferating normal mammary epithelial cell cultures. The organoid samples in the remaining four lanes were obtained by thawing frozen stored organoids, centrifuging the pellet, and resuspending in guanidinium isothiocyanate. Hybridization to the 36B4 probe is shown.

FlG. 2. Demonstration of TGFa mRNA by RNA in situ hybridization. Where sample size limited isolation of total RNA, cells were placed on prepared slides by cytocentrifugation. Shown in A, B, and C are organoid samples 190, 195, and 186, respectively. D is organoid 184, from which the rapidly proliferating 184 cells used in these studies were derived. Levels of TGFa mRNA hybridizing in the organoid samples in A-D can be compared to levels of TGFa mRNA hybridizing in the rapidly proliferating 184 cells in F. E demonstrates that control 36B4 mRNA is detectable in the cytocentrifuged organoid samples.

the level of expression of TGFa mRNA in the proliferating 184 cells (Fig. 2). Thus, increased expression of TGFa mRNA appears to be consistently associated with the conversion of normal human breast epithelial cells from nonproliferating organoids to a proliferative state in vitro. Regulation of TGFa and EGF receptor gene expression in the normal cells We hypothesized that a TGFa/EGF receptor autocrine loop could be a principal pathway mediating growth control in normal human mammary epithelial cells. To determine whether expression of this loop was directly

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linked to the growth state of the cells, we studied the cells during growth inhibition achieved by diverse agents. Examination of TGFa and EGF receptor gene expression was performed after addition of the phorbol ester, TPA; sodium butyrate, a differentiating agent (30); or TGF/?, a growth inhibitor and differentiating agent widely produced in cell lines and tissues (5, 31). As demonstrated in Fig. 3, each of these growth inhibitors resulted in a decrease in [3H]thymidine incorporation to 5-10% of control values 24 h after treatment (see Fig. 3, inset) and a decrease in cell number to 15-30% of control values 47 days after treatment. We initially examined the effects of TGF/3 on TGFa mRNA expression in the rapidly proliferating 184 cells. No alteration in level of expression was found, and we sought to learn whether this was unique to the 184 cells. As shown in Fig. 4, RNA harvested after 48 h of TGF0 treatment of proliferating cells from samples 184, 172, and 161 demonstrated no regulation of TGFa mRNA. Subsequently, we examined mRNA harvested from 184 cells treated with the other inhibitors, TPA and sodium butyrate. RNA harvest for these experiments was carried out 3 and 24 h after the addition of TPA and 24 or 48 h after the addition of TGF/3 and sodium butyrate. Actively 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0

Endo • 1990 Voll26«Nol

growing cells from passages 12- to 13 were used; senescence of the cells occurred after passage 15. As demonstrated in Fig. 5, there was no decrease in TGFa or EGF receptor gene expression by the addition of any of the growth inhibitors. Cells examined after confluence maintained for 3 days or at senescence (no observable growth for 3 weeks despite medium changes) also failed to show significant alteration of the high level of expression of TGFa and EGF receptor mRNA. Also shown in Fig. 5 is the level of TGFa mRNA detectable in 184 cells in mass culture after withdrawal of EGF and BPE from the medium. This experiment and several similar ones failed to show a significant decrease in TGFa expression; at most, a 20% decrease in TGFa mRNA expression was observed. Regulation of TGFa protein and EGF receptor binding in human mammary epithelial cells Although the levels of TGFa and EGF receptor mRNA in the 184 cells and the A1N4 subline are unchanged by the addition of growth inhibitors, effects on the pathway could be mediated postranscriptionally. Therefore, conditioned medium was examined for TGFa expression. In experiments not shown here, production of TGFa in the medium continued unabated after addition of the growth inhibitors. Similarly, EGF receptor-binding characteristics in the 184 cells were unaltered by the addition of TPA, TGFjS, or butyrate. In each case receptor binding approximated 2 X 105 binding sites/cell, with a Kd in the 1- to 3-nM range for the lower affinity binding component (Table 1). In contrast to observations in the control cells, a small fraction (

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EGF receptor determinations were performed on confluent cell monolayers in 24-well dishes (Costar). Cells were pretreated in the complete MCDB170 medium with butyrate, TPA, or TGF/3 for 24 h, then withdrawn from EGF and BPE, while maintaining the treatments, for 14-18 h before assay. Assays were incubated for 2.5 h at 4 C with 0.001-10 nM [126I]EGF. Binding parameters were calculated using the Ligand program (18). Results shown are the average ± SD of duplicate determinations from two separate experiments for each treatment.

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FIG. 5. Effects of growth inhibition on expression of TGFa and EGF receptor mRNA expression in rapidly proliferating 184 cells. One-half microgram of total RNA from MDA-MB-468 cells was included as a positive control for EGF receptor mRNA hybridization, while 8 ng total RNA were used in all other lanes. MDA-MB-231 and Hs578T mRNAs are included as positive and negative controls for TGFa hybridization, respectively. In the first panel, levels of mRNA in control 184 cells can be compared to levels in cells harvested 72 h after reaching confluence and 24 h after treatment with butyrate and TGF/3 at the concentrations given above. In the second panel, mRNA expression in control 184 cells can be compared to cells that were noted to be senescent 3 weeks before mRNA harvest. Effects of 48-h butyrate and 3-h TPA addition are shown in this panel as well. In the last panel, mRNA harvested 24 h after withdrawal of EGF and BPE from the medium is shown. Cells were seeded at as light a density as possible for mRNA harvest. Hybridization to 36B4 riboprobe is shown as a control. Kb, Kilobases.

the A1N4 cells (Fig. 6). Unlike the 184 cells, growth inhibition could be easily achieved in mass culture by EGF withdrawal in the A1N4 cells. The decrease in TGFa mRNA level was noted as early as 4 h after EGF withdrawal. Twenty-four hours after EGF withdrawal, the expression of TGF« mRNA had fallen 2.5-fold, as measured by scanning densitometry. There was also a decrease in EGF receptor mRNA observed at the 24 h time point. Effective EGF withdrawal in 184 normal mammary epithelial cells We had previously observed growth inhibition after EGF withdrawal in the 184 line only at clonal density (9). This would imply that sufficient cross-feeding by secreted TGFa occurred in normal density 184 cells to negate the effect of EGF withdrawal, shown in the experiment in Fig. 5. We postulated that a decrease in TGFa mRNA after EGF withdrawal similar to that observed in the A1N4 cells would be seen if the 184 cells were plated sparsely. To test this hypothesis we performed RNA in situ hybridization of 184 cells plated on

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TGFa/EGF RECEPTOR IN NORMAL BREAST CELLS

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FIG. 6. Effects of growth inhibition on the immortalized AlN4subline. Cells were plated in the usual EGF-containing medium. After 24 h, fresh medium was applied, and RNA was harvested after 3-h TPA and 24-h butyrate treatment. Medium lacking EGF was added in separate dishes to determine the effects of EGF withdrawal. Cells were harvested both 4 and 24 h after EGF withdrawal. Two separate EGF-containing controls are shown. Kb, Kilobases.

prepared slides at very low density. Shown in Fig. 7A are the levels of TGFa mRNA in the 184 proliferating parental cells. The signal demonstrated is specific, as shown by competition with unlabeled antisense TGFa probe (Fig. 7B). As with the observations made in the A1N4 cells, levels of TGFa mRNA decreased after EGF withdrawal (Fig. 7C). Effects of EGF withdrawal were also demonstrated by the addition of 20 nM 96 IgM, an EGF receptor antibody known to block binding of EGF or TGFa to its receptor (Fig. 7D). The level of TGFa mRNA detected by in situ hybridization was again markedly decreased. Thus, the results in both the A1N4 cells and the rapidly proliferating 184 cells are consistent, with a decrease in TGF« mRNA upon EGF withdrawal from the medium. The experiment was then repeated with the 225 IgG monoclonal antibody, and both DNA synthesis, determined by [3H]thymidine labeling, and TGFa expression were studied. Cells were plated at comparable densities and on the same pretreated slides for both TGF« RNA in situ hybridization and determination of the TLI. After 24-h exposure to 225 IgG antibody, the TLI had fallen from 22% in the control to 2% in the treated cells (Table 2). A nonspecific IgG antibody reduced labeling insignif-

Endo • 1990 Vol 126-No 1

icantly to 17%. In this experiment, EGF withdrawal had no effect, with labeling at 21%. Figure 8E demonstrates control cells with thymidine incorporated in the nuclei, and antibody-treated cells are shown in Fig. 8F. Companion slides were analyzed for TGFa RNA expression. Levels of TGFa expression were seen to fall 80%, estimated by counting grains in representative areas of the slide. Taken together, these data demonstrate that inhibition of binding of ligand to the EGF receptor by monoclonal blocking antibodies results in decreased DNA synthesis as well as decreased TGFa expression. Withdrawal of EGF from the medium in this experiment, with cells plated at a higher density than in Fig. 7, fails to result in a significant decrease in the TLI or in TGFa mRNA expression. In Fig. 7, EGF withdrawal did result in decreased TGFa expression. These cells were plated more sparsely and presumably did not have sufficient levels secreted to replace that contributed by EGF supplemented normally present in the medium. The variations seen with EGF withdrawal here were mirrored in total RNA preparations, where a somewhat decreased TGFa expression was observed in some experiments but not in others. Addition of antibodies that block binding to the EGF receptor provide confirmation that EGF withdrawal in the proliferating normal cells results in decreased growth and TGFa mRNA expression. Growth inhibition by EGF receptor antibodies To confirm the inhibition seen in DNA synthesis after 24 h, growth effects of a longer durations of antibody treatment were examined. As shown in Fig. 9, growth of the cells is completely inhibited by treatment with EGF receptor-blocking antibody 225 IgG. Effects of the antibody could be observed in both the usual growth medium, which contains EGF and BPE, and EGF-free medium. EGF in the medium competed with the antibody to result in a decreased effect compared to that seen in the EGFfree medium. Development of TGFa mRNA expression in organoids in tissue culture Thus, the experimental data demonstrate TGF« expression in the proliferating cells and its regulation by levels of ligand binding to the receptor. The low level of expression of TGFa mRNA seen in the organoids could indicate low levels of expression in vivo or, if such autoregulation were present in vivo, could result from decreased exposure to TGFa/EGF in the EGF-free isolation conditions (14). Figure 10 demonstrates that placement of the organoids in tissue culture results in early homogeneous expression of TGFa mRNA, preceding the time in passage 3 when the majority of cells die before selection of expanded rapidly proliferating cultures.

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FIG. 7. Demonstration of effect of EGF withdrawal on 184 parent cells. Rapidly proliferating cells were placed on prepared slides at very low density in supplemented medium. The cells received fresh medium with or without 5 ng/ml EGF 24 h after plating. Some of the cells were incubated with both EGF and 20 nM anti-EGF receptor antibody. Twenty-four hours later, slides were fixed in 4% paraformaldehyde before RNA in situ hybridization. Each slide was hybridized to the TGFa riboprobe. In A and B are 184 cells grown as controls in EGF; the slide shown in B also received competing unlabeled antisense RNA in the in situ hybridization to demonstrate nonspecific background binding. C demonstrates TGFa mRNA expression in cells grown in the absence of EGF. D demonstrates expression in cells cultured in the presence of both EGF and anti-EGF receptor antibody.

TABLE 2. TLI in 184 cells treated with 225 IgG antibody Treatment

TLI ± SD

Control IgG 225 IgG Without EGF

22.3 ± 1.9 17.3 ± 5.6 2.3 ± 1.6 21.0 ± 4.4 Cells were incubated at 37 C in medium containing 1 ng/ml EGF for 24 h, then washed, and the media with the respective treatments were applied for another 24 h. Thymidine labeling was carried out for 2 h at 37 C in 1 fiCi/ml [3H]thymidine and 10"7 M cold thymidine. The TLI was the number of labeled nuclei per 100 cells counted. Slides were made in duplicate, and triplicate counts were obtained on each slide to obtain the SD given.

Discussion In these studies we have observed that conversion of normal human breast epithelial cells to a proliferative state in vitro is accompanied by increased TGFa mRNA expression. The levels of TGFa: mRNA expression in nonproliferating organoid samples were far below levels seen in proliferating samples of human breast epithelial cells. High levels of EGF receptor mRNA and binding are observed in the proliferating cells, with lower levels in the organoids as well (data not shown). Addition of TGF/3, TPA, or sodium butyrate failed to alter TGFa or EGF receptor gene or protein expression. While exogeneous inhibitors failed to inhibit the putative TGFa/ EGF receptor loop, withdrawal of either TGFa or EGF

in both the proliferating (at low cell density) and immortalized normal breast epithelial cells resulted in a decrease in the level of TGFa mRNA expression. In the 184 proliferating normal cells, addition of antibody to

the EGF receptor resulted in decreases in DNA synthesis, growth, and TGFa mRNA expression, suggesting the presence of an autoregulated autocrine loop. TGFa was originally identified by its ability to confer the transformed phenotype on NRK cells (2). It was hypothesized that TGFa might act in an autocrine fashion, that is stimulating the growth of the cell that secreted it after interaction with the cell surface EGF receptor (32). For a time it appeared that production of TGFa was specific to malignant cells, since expression was found in both spontaneously and virally or chemically induced malignancies (33). It was thought that the autocrine loop accounted for the relative growth deregulation of malignant cells compared to their normal counterparts (34). However, the increasing number of normal cell types described as expressing the TGFa gene suggests that this growth system may be operant in normal as well as malignant cells (6-13). For regulation of normal cellular growth, the existence of an inhibitory pathway would be required to provide opposing stimuli to the TGFa/EGF receptor-positive growth stimuli. Growth inhibition could be mediated by decreasing expression of some component of the TGFa/ EGF receptor autocrine loop or could occur by a separate

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FIG. 8. Effects of 225 EGF receptor antibody on TGFa mRNA expression and [3H]thymidine incorporation in 184 cells. The experiment in Fig. 7 was repeated using the IgG antibody. Cells were plated on slides and prepared as for the experiment in Fig. 7. In this experiment 1 ng/ ml EGF was used in the MCDB 170 complete control medium, since other experiments had demonstrated no difference in TGFa mRNA expression with the lower dose of EGF. Anti-EGF receptor IgG antibody (40 nM) was used. The 603-basepair antisense TGFa riboprobe was hybridized to the slides in A, C, and D. Shown in the figure are control cells (A), control cells hybridized to a sense TGFa riboprobe to demonstrate the background level of hybridization (B), cells treated with the 225 IgG antibody (C), and cells treated with control IgG antibody (D). [3H]Thymidine labeling of control (E) and 225 IgGtreated cells (F) are shown in the lower panels. Five densely labeled nuclei are seen in E.

pathway. In the 184 cells, addition of diverse agents to achieve growth inhibition did not return expression of the putative TGFa/EGF receptor autocrine loop to levels in the organoid. The duration of treatment in the studies was chosen to coincide with documented physiological changes in the cells after the addition of growth inhibitors. Thus, RNA was harvested at a time when clear alterations in cell morphology and [3H]thymidine incorporation were present.

Endo • 1990 Voll26«Nol

Effects of TPA and EGF on TGFa and the EGF receptor have been previously described. EGF, after binding to the EGF receptor and inducing EGF receptor internalization and degradation with loss of cell surface binding, has been shown to simultaneously stimulate EGF receptor synthesis (both mRNA and protein) in the KB human epidermoid cancer cell line and in the WB rat hepatic epithelial cell line (35, 36). Time-course studies demonstrated a peak in EGF receptor mRNA 4 h after EGF addition. Studies were not carried out to 24 h, but at 10 h levels were still above baseline in KB cells (35). In MDA-MB-468 human breast cancer cells, which overexpress EGF receptor and are growth inhibited by EGF, both TPA and EGF stimulate EGF receptor synthesis while inhibiting cell growth (37, 38). Likewise, stimulation of TGF« mRNA has been observed after TPA and/or EGF treatment of human keratinocytes (39). In these studies the peak stimulation occurred at 5 h, but levels remained elevated after 24 h. We also observed stimulation by TPA at 3 h in A1N4 cells, but in 184 cells the effects of TPA were less consistent. In some of our experiments TPA addition resulted in an increase in TGFa or EGF receptor mRNA at 4 or 24 h in 184 normal mammary epithelial cells. However, the increase was inconsistently observed, and levels were often unchanged. Other studies have demonstrated effects of TGF/3 on the EGF receptor. Assoian et al. (40) reported an increase in total EGF binding in NRK cells 6-8 h after treatment with TGF/3. Massague and Like (41, 42) reported a decrease in high affinity binding of EGF to the receptor in NRK cells, but not in mink lung epithelial cells after 2-h treatment with TGF/3. In contrast, at 24 h other investigators have found an increase in NRK cell EGF receptor high affinity binding and a decrease in high affinity binding in rat heart endothelial cells (43). In mouse keratinocytes, Coffey et al. (44) demonstrated inhibition of DNA synthesis after treatment with TGF0 without effects on EGF receptor binding or internalization. In MDA-MB-468 cells TGF0 increases EGF receptor mRNA, reaching maximal stimulation from 6-8 h (45). In the present study, TGF/3 failed to regulate expression of TGFa or EGF receptor mRNA. EGF receptor binding after 24 (or 48)-h treatment with TGF/3 demonstrated no effect on affinity or total number of binding sites. In contrast to the results obtained with the growth inhibitors, effective withdrawal of EGF from the medium of both 184 and A1N4 cells resulted in down-regulation of TGFa mRNA expression. In A1N4 cells, decreased expression of EGF receptor mRNA was obtained as well. The autoregulation observed in the normal mammary epithelial cells would suggest that secretion of TGFa, its binding to cell surface receptor, and stimulation of eel-

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TGFa/EGF RECEPTOR IN NORMAL BREAST CELLS

605

10.0

FIG. 9. Growth of 184 cells after EGF withdrawal or EGF receptor blockade. To confirm the results seen in inhibition of DNA synthesis, growth curves were performed in the presence of the EGF receptor 225 antibody. 184 cells were plated at 0.25 x 105 cells in each well of a 24-well dish. To obtain adequate growth over the 7 days, BPE was included in the medium in all cells. Four conditions were used: MCDB 170 complete plus IgG control antibody (O), MCDB 170 without EGF plus IgG control antibody (•), MCDB 170 complete plus 225 IgG (•), and MCDB 170 without EGF plus 225 IgG (•). Cells were fed every 48 h and counted on the days they were not fed. Results on the y-axis show mean cell counts x 106 per well, with SD.

9.0 L ") 7.0 6.0 5.0 4.0 3.0 2.0

c o O Q5

O

3

lular growth would be accompanied by further increases in TGFa levels. Such an amplification mechanism for TGFa was described by Coffey and co-workers in normal mouse keratinocytes (7, 44) and has been suggested in both normal bovine pituitary and regenerating liver (46, 47). Likewise, amplification mechanisms have been previously described in other growth factor systems (1). These include platelet-derived growth factor (48), interleukin-1 (49), and melanoma growth stimulatory activity (50). The data presented in this manuscript would suggest that a positive amplification system for TGFa/EGF receptor in vivo could be controlled by a protein or factor which blocked binding of TGFa to its receptor rather than directly controlling TGFa gene expression. Alternatively, increased secretion of a protease which acted specifically to degrade TGFa could result in removal of TGFa from the cellular milieu and thereby control gene expression. According to this hypothesis, endogenous growth inhibitors may exist which regulate an autocrine loop indirectly through controlling access to the ligand. Thus, the yin-yang hypothesis that growth is controlled by a balance of positive and negative regulatory influences may be extended to suggest that regulation in some cases occurs at the level of the ligand in the stimulatory loop itself (34, 51, 52), and that a separate pathway to achieve growth inhibition is not always required. The failure to see TGFa expression in the organoid samples may be interpreted to suggest that normal breast epithelium in vivo does not express TGFa mRNA unless it is proliferating. However, expression has been previously described at both mRNA and protein levels (53, 54). Alternatively, if the autoregulatory loop exists in

4

5 Day

6

7

8

9

vivo in normal breast epithelium, the EGF-free isolation conditions for the organoids may have resulted in decreased expression of TGFa mRNA. The presence of TGFa mRNA in normal breast epithelium in vivo raises the question of whether TGFa exists in normal breast tissue as a proliferative stimulus. Clearly, in tissues such as regenerating liver (46), TGFa may easily be seen as playing a role in proliferation. Likewise, during times of growth of the mammary epithelium, such as in puberty and pregnancy, the normally quiescent epithelium could rely upon TGFa to provide a stimulus for cellular growth. The correlation of expression of TGFa with conversion to proliferation in vitro suggests that such could occur in vivo as well. During quiescent times, a role for TGFa is more difficult to envision. Perhaps TGFa acts to maintain a basal or steady state level of epithelial renewal, but in the neurons of the brain, growth factor-induced proliferation seems out of place (55). Other potential mechanisms of action for TGFa remain to be defined in tissues such as brain and normal breast. The data presented in this manuscript suggest that in vivo proliferation of normal human breast epithelial cells could be dependent upon expression of both TGFa and EGF receptor in an autoregulatory autocrine loop. Addition of EGF receptor-blocking antibody results in the reversible cessation of mitosis in the cells in vitro. Such a finding does not preclude a role for the TGFa/EGF receptor system in malignancy. Rather than an association with specific levels of ligand or receptor, the system could be specifically associated with malignancy by alterations in signal transduction after the ligand-receptor interaction. As proposed several years ago, the cell's

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Endo • 1990 Vol 126 • No 1

and Francis G. Kern for subcloning the EGF receptor cDNA. We thank J. Schlessinger and Rorer Biotechnology, Inc. (King of Prussia, PA), for use of the 96 IgM monoclonal antibody directed against the EGF receptor. The authors also thank Antonio Fojo for critical reading of the manuscript.

References

FIG. 10. Expression of TGFa mRNA in normal breast organoids in tissue culture. The isolated breast epithelial cells (organoids) were gently placed on prepared slides and cultured in the compete MCDB 170 medium. After 10 days, outgrowth of proliferating cells was observed, and RNA in situ hybridization was performed for TGFa expression as described above. The figure demonstrates the original cluster of epithelial cells surrounded by cells proliferating outward from the center (A); B is a higher power view of the same, with the matching darkfield photomicrograph shown in C. The single cells outside the organoid are fibroblasts and do not show TGFa expression.

phenotype determines its response to a growth factor (56) more than the nature of the factor or its level of expression. For the normal human mammary epithelial cells in culture, the TGFa/EGF receptor system represents a critical permissive pathway for proliferation.

Acknowledgments The authors would like to thank K. Horgan and R. E. Mansel for their donation of flasks of normal human breast epithelial cells. We want to thank Glen Merlino for use of the pE7 EGF receptor cDNA

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