JOURNAL OF CELLULAR PHYSIOLOGY 145:207-216 (1990)

Stromal Influences on Transformation of Human Mammary Epithelial Cells Overexpressing c-myc and SV40T EVA M. VALVERIUS,* FORTUNATO CIARDIELLO, NILS-ERIK HELDIN, BRUNO BLONDEL, ClORGlO MERLO, GILBERT SMITH, MARTHA R. STAMPFER, MARC E. LIPPMAN, ROBERT B. DICKSON, AND DAVID S. SALOMON laboratory of Tumor Immunology and Biology, National Cancer Institute, Bethesda, Maryland 20892 (E.M.V.,F.C.,B.B.,C.M.,C.S.,D.S.S.I;Department of Pathology, University Hospital, 75 1 85 Uppsala, Sweden (N.-E.H.); Vincent Lombardi Cancer Research Center, Georgetown University Hospital, Washington, D.C. 20007 (M.E.L.,R.B.D.); Lawrence Berkeley Laboratory, Berkeley, California 94720 (M.R.S.) The proto-oncogene c-myc and the oncogene SV40T, both of which have been implicated in the process of cellular immortalization in vitro, have been introduced via amphotropic retroviral expression vectors into the human mammary epithelial cell (HMEC) line 184AlN4 (AlN4). Two stable cell lines were established by growth in selective medium and were found to overexpress either c-myc (A1N4-myc) or SV40T antigen (A1N4-T). Neither the A1 N4, A1 N4-myc, or AlN4-T cells will grow in soft agar or form tumors in nude mice. However, A1 N4-T or A1 N4-myc cells, but not the parental A1 N4 cells, form colonies in soft agar in response to either epidermal growth factor (EGF), transforming growth factor a (TGFa), or basic fibroblast growth factor (bFGF). Like EGF and TGFa, bFGF is moderately mitogenic for the anchorage-dependent growth (ADG) of all three cell lines. Further, co-cultivation of A1 N4-T or A1 N4-myc cells with primary diploid mammary fibroblasts can also induce the anchorage-independent growth (AIG) and stimulate the ADG of A1 N4-T or A1 N4-myc. In addition, conditioned medium obtained from these mammary fibroblasts also stimulated the AIG of the A1 N4-T and A1 N4-myc cells and was found to contain immunoreactive TGFa and bioactive FGF. The mammary fibroblasts express specific mRNA transcripts for bFGF and acidic FGF (aFGF). These results suggest that growth factors such as TFGa or FGF, which may be derived from the adjacent mammary stroma, might influence in a paracrine manner the phenotypic characteristics of a population of human mammary epithelial cells toward transformation.

FGF-like growth factors may have a particular significance in understanding the progression of human breast cancer. Acidic and basic FGF have primarily been considered potent mitogens for mesoderm- and neuroectoderm-derived cells (reviewed by Gospodarowicz, 1989). More recently, it has been realized that bFGF is also able to regulate the growth of epithelial cells. Specifically, bFGF is capable of stimulating the anchorage-dependent growth of normal and neoplastic human mammary epithelial cells (Karey and Sirbasku, 1988; Smith et al., 1984; Riss and Sirbasku, 1987; Levay-Young et al., 1989; Takahashi et al., 1989). In addition, human breast cancer cell lines have been reported to produce FGF-like proteins (Kern et al., 1990; Dickson and Lippman, 1988) which thus may function in an autocrine manner to regulate cellular proliferation. Furthermore, two FGF-related genes, int-2 and hstlK-FGF, have been implicated in the pathogenesis of human breast cancer since both genes are amplified in approximately 10-25% of primary human breast tumors (Theillet et al., 1989; Zhou et al., 1988; Varley et al., 1988; Lidereau et al., 1988). 0 1990 WILEY-LISS, INC.

Fibroblast feeder layers have served an important function for facilitating the establishment of primary cultures of rodent and human mammary epithelial cells (Smith et al., 1981). Further, the hormonal responsiveness and differentiation of rodent and human mammary epithelial cells is significantly enhanced in Received May 4, 1990; accepted July 16, 1990. *Eva M. Valverius is now at Department of Pathology, University Hospital, 751 85 Uppsala, Sweden. Address reprint requests/ correspondence there. Abbreviations used: aFGF, acidic fibroblast growth factor; AIG, anchorage-independent growth; bFGF, basic fibroblast growth factor; BPE, bovine pituitary extract; BSA, bovine serume albumin; CM, conditioned medium; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FCS, fetal calf serum; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; HMEC, human mammary epithelial cells; IGF, insulinlike growth factor; IMEM, Improved Modified Eagle’s Medium; kb, kilobase pair; PDGF, platelet-derived growth factor; RRA, radio-receptor assay; SD, standard deviation; TGFcYtransforming growth factor alpha; TGFp transforming growth factor beta.

208

VALVERIUS ET AL.

the presence of nonproliferating fibroblasts (Haslam, 1986;McGrath, 1983).In part, this may be attributed to the production by the fibroblasts of extracellular matrix material, such as collagen and fibronectin (Ignotz and Massague, 1986). It is conceivable that an additional function of fibroblast feeder cells is to act as a source of growth factors for the adjacent mammary epithelial cells. For example, human foreskin and dermal fibroblasts have been shown to synthesize and secrete different members of the FGF family of peptides which are mitogenic specifically for keratinocytes (Kurokawa et al., 1987; Finch et al., 1989). Since bFGF can induce neovascularization and local fibroplasia in vivo, this growth factor assumes an even more important role as a possible paracrine growth regulator (Gospodarowicz, 1989). The present study was undertaken to investigate whether treatment with FGF or cocultivation with normal diploid mammary fibroblasts could induce the AIG of immortal HMEC overexpressing either the SV40T or c-myc genes. We have earlier reported that EGF or TGFa could facilitate the AIG of the immortal HMEC line expressing SV40T, AlN4-T (Valverius et al., 1989).Although the SV40T oncogene has not yet been shown to perform a role in human mammary carcinogenesis, expression of this gene can immortalize primary rodent and rabbit mammary epithelial cells (Chang, 1986). Another gene that can immortalize primary rodent fibroblasts and is capable of enhancing fibroblast responsiveness to various growth factors is c-myc or v-myc (Kelekar and Cole, 1987; Balk et al., 1985; Stern et al., 1986; Leof et al., 1987). In addition, a role for c-myc has been implicated in human mammary carcinogenesis in that amplification of the c-myc gene has been found in 1530% of primary breast tumors (Escot et al., 1986; Cline et al., 1987). We now report the establishment of an immortal HMEC line overexpressing c-myc, AlN4-myc. We also studied the soft agar responsiveness of AlN4-T and AlN4-myc to bFGF, EGF, and TGFa, and increased soft agar growth of AlN4-T and AlN4-myc cells by cocultivation with normal diploid fibroblasts derived from the same original mammary tissue. The production by these fibroblasts of FGF-like and TGFa-like activities was also assessed. MATERIALS AND METHODS Cell lines and growth factors The A1N4 and AlN4-T HMEC lines were isolated and maintained as previously described (Stampfer and Bartley, 1985; Clark et al., 1988). A1N4 cells were pro agated in IMEM containing 0.5% FCS (Gibco), hy rocortisone (0.5 pg/ml) (Sigma), insulin (5 pg/ml) (Sigma), and EGF (Collaborative Research) (10 ng/ml). AlN4-T cells were cultured in IMEM with 10% FCS. The AlN4-myc cell line was isolated followingexposure of the A1N4 cells to an amphotropic retrovirus (Blonde1 et al., 1990) that contained the neomycin resistance gene and the mouse c-myc gene under the transcriptional control of a Moloney leukemia virus LTR (Dean et al., 1987). AlN4-myc cells were obtained following growth in G418 (400 pg/ml) containing medium for 4 weeks. AlN4-myc cells were subsequently grown in the same medium as AlN4-T cells, IMEM with 10% FCS. Tumorigenicity of AlN4-myc cells was tested by sub-

cf

cutaneous injection of 5 x lo6 cells into the scapular region or cleared mammary fat pad of nude mice (Balbnulnu). The mice were observed for 3 months. Primary cultures of human diploid mammary 184FB fibroblasts were established from the same reduction mammoplasty that was utilized to obtain the original 184 mammary epithelial cells from which the A1N4 cell line was derived (Stampfer and Bartley, 1985). 184FB cells were maintained in IMEM containing 10% FCS and insulin (5 pg/ml). Experiments utilizing the 184FB cells, which senesce at passage 12 to 16, were performed while the cells were at passage 7 to 9. Growth factors used were mouse EGF and BPE (Collaborative Research, Bedford, MA), human synthetic TGFa (Bachem, Torrance, CAI, human recombinant bFGF (Boehringer-Mannheim), bovine brain-derived aFGF (Biomedical Technologies, Inc., Stoughton, MA), recombinant human IGF-I and IGF-I1 (KabiGen, Stockholm, Sweden), and human PDGF (R & D Systems, Inc., Minneapolis, MN). Conditioned medium collection 184FB and AlN4-T cells were grown to subconfluency in 175 cm2 tissue culture flasks. After two brief washes in PBS, the 184FB cells were incubated for 96 hours in DMEM containing ITS+ mixture (insulin 6.25 pg/ml, transferrin 6.25 pg/ml, selenious acid 6.25 ng/ ml, BSA 1.25 mglml, linoleic acid 5.35 pg/ml; Collaborative Research), and HEPES buffer (20 mM). AlN4-T cells were further incubated in PC-1 medium (Ventrex, Bangor, ME) with or without bFGF (10 ng/ml) for 48 hours. Upon collection, the CM samples were filtered and protease inhibitors were added (aprotinin 0.2%; leupeptin 2 pg/l). Samples were stored at -20°C until analysis. Conditioned medium analysis For assay of TGFa immunoreactivity, CM samples were thawed and lyophilized. TGFa-like material in the CM samples was then concentrated over a C18 Sep-Pak column and quantified using a TGFa-radioimmunoassay (RIA) kit (Biotope Inc., Redmond, WA) as previously described (Salomon et al., 1987). For determination of FGF receptor-competing activity, CM samples were dialyzed against distilled water through a 10,000 m.w. cut-off membrane, lyophilized, resuspended in PBS, and assayed in an FGF-RRA essentially as described (Neufeldt and Gospodarowicz, 1985). Briefly, monolayers of BHK cells in 24-well dishes were incubated with lZ5I-bFGF(bovine recombinant; Biomedical Technologies, Inc.) and dilutions of CM or standard curves of increasing concentrations of bFGF (human recombinant; Boehringer-Mannheim) in binding buffer (DMEM with 0.15% gelatin and 20 mM HEPES) at room temperature for 2 hours. The cells were then washed 3 times with ice-cold PBS with 0.15% gelatin, once with 2 M NaC1, and finally lysed with 0.1 M Na2P04and 0.5% Triton X-100. Cell surface receptor determinations EGFR binding assays were performed as described (Valverius et al., 1989) on cell monolayers in 24-well cluster dishes for 2.5 hours at 4"C, using increasing concentrations of 1251-EGF(80-120 pCi/p,g, ICN Radiochemicals, Irvine, CAI with or without 200-fold excess

209

STROMAL INFLUENCES ON HMEC TRANSFORMATION

unlabeled EGF. After incubation, cells were washed and then solubilized in 0.5% SDS, 1mM EDTA, 10 mM Tris-HC1. FGFR characteristics were determined essentially as described (Moscatelli, 1987) on cell monolayers in 24-well dishes for 2 hours at 4°C using increasing concentrations of 1251-bFGF(40-80 pCi/kg, Biomedical Technologies) in the absence or presence of 100-fold excess unlabeled bFGF (Boehringer-Mannheim). Previous kinetic binding experiments showed that steady state was attained (data not shown). EGFR and FGFR binding parameters were calculated using the LIGAND computer program (Munson and Rodbard, 1980) and normalized to cell numbers determined from comparably treated parallel wells. Anchorage-dependent growth assays AlN4, AlN4-T, and AlN4-myc cells were seeded at 200 to 500 cells/cm2 into triplicate wells in 12-well cluster dishes. 184FB cells were seeded at a density of 2,000 cells/cm2due to their slower growth rate. After 18 hours, the medium was changed and various growth factors were added at the indicated concentrations. Medium with growth factors was replaced every 48 hours. After 6 days, cells were trypsinized and counted in a Coulter Counter. For cocultivation ex eriments, indicator cells and feeder cells were cu tured in Transwell'" dishes (Costar). Indicator cells were plated in duplicate in 6-well cluster dishes and the feeder cells were seeded into the corresponding Transwell '"inserts at an equal density. AlN4-T and AlN4-myc were seeded at 50 cells/cm2 while the 184FB cells were plated at 4,000 cells/cm2.Medium was changed every 48 hours. After 8 days of growth, the cells in the bottom 6-well dishes were trypsinized and counted in a Coulter Counter. Anchorage-independent growth assays The cells (1 x lo4)were suspended in 0.36% agar and seeded into 35 mm dishes over a 0.6% agar base layer in IMEM containing 10% FCS and various growth factors at the indicated concentrations or containing dilutions of CM. After 14 days, cells were stained with nitroblue tetrazolium and colonies larger than 50 km diameter were counted on an Artek Colony Counter. For cocultivation experiments, the feeder cells were plated either directly onto the plastic surface of 35 mm dishes beneath the 0.6% agar base layer (2,000 184FB cells/cm2; 50 cells/cm2 for the two HMEC lines), or incorporated directly into the 0.6% agar base layer

r

(4 x lo4 184FB cells; 400 AlN4-T or AlN4-myc cells) before overlaying with 1 x lo4 indicator cells in 0.36% agar. After 14 days of growth, colonies were counted either by direct microscopic examination or following nitroblue tetrazolium staining. RNA preparation and analysis Total cellular RNA was isolated from cells by homogenizing in guanidine isothiocyanate, followed by centrifugation over a cesium chloride cushion (Maniatis et al., 1982). RNA was electrophoresed in 1%agarose6% formaldehyde gels. Gels were stained with 2 kg/ml ethidium bromide to allow inspection of the quantity and quality of RNA. RNA gels were subjected t o partial alkaline hydrolysis before transfer of RNA to nitrocellulose by capillary blot (Maniatis et al., 1982). The filters were hybridized to the following human 32Plabeled nick-translated cDNA probes as previously described (Valverius et al., 1989): a 1.4 kb EcoRl fragment covering the entire bFGF sequence (pJJ11-1; gift of Dr. J. Abraham, California Biotechnology Inc, Mountain View, CA; [Abraham et al., 19861; a 2.1 kb insert coding for aFGF (pDH15; gift of Dr. J. Winkles, Meloy Laboratories Inc., Rockville, MD); a 2.2 kb FGFR cDNA covering the intracellular region of the receptor (gift of Dr. L. Claesson-Welsh, Ludwig Institute for Cancer Research, Uppsala, Sweden [Wennstrom et al., 19901); and a 925 bp EcoRl fragment of the human TGFa cDNA containing the entire TGFa coding region (gift of Dr. J. Scott, London). Autoradiography was performed with Kodak XAR-5 film at -70°C.

RESULTS Growth factor responsiveness of AlN4-T cells in soft agar A1N4 cells will not form colonies in soft agar either in 0.5% FCS or in 10% FCS despite the addition of a variety of growth factors (Clark et al., 1988; Valverius et al., 1989).However, AlN4-T cells, which overexpress the SV40T antigen, will grow in soft agar in response to either EGF or TGFa (Valverius et al., 1989). In addition, AlN4-T cells express five- to ten-fold higher levels of EGFR than the parental A1N4 cells (Valverius et al., 1989; Table 1).To determine if growth in soft agar of AlN4-T cells was a response specific for EGF and TGFa or the result of a generalized hypersensitivity of these cells to other growth stimulatory factors, BPE or various growth factors either alone or in combination were tested for their ability to stimulate the AIG of AlN4-T

TABLE 1. Binding parameters for bFGF and EGF receptors'

EGFR bFGFR Cell line

KdbM)

Sitedcell

* 710

A1N4

28.2 k 6.5

4,650

AlN4-T

20.0 k 4.4

5,540 f 1,600

68.0

1,810

AIN4-myc

high low

Sites/cell, high low

0.03 0.55 0.10 1.75 0.03 1.07

15,500 128,800 28,800 791.600 161400 219,200

KdbM),

Total 144,300

820.400 235,600

'Binding experiments for the FGFR and EGFR were performed on cell monolayer in 24-well cluster dishes at 4 ° C using increasing concentrations of lZ5I-bFGF or 1251-EGF. respectively, as described in "Materials and Methods." For the FGFR, values shown are means f SE from up to five separate experiments per cell line.

VALVERIUS ET AL.

't

I

-

K

I

111 10

,

L

0 0.001

1 .o

0.1

10

100

CONCENTRATION, NG/ML

Fig. 1. Soft agar cloning specificity of AlN4-T cells to various growth factors; lo4 cells were seeded in the upper of two layers containing semi-solid agar in regular medium with 10%FCS in 35 mm dishes, in the presence of the following growth factors: EGF 5 ngiml, TGFa 10 ngiml, BPE 70 pglml, bFGF 10 ng/ml, aFGF 10 ngiml, PDGF 10 ngiml, insulin 5 pg/ml, IGF I 100 n /ml, and IGF I1 100 ngiml. After 11days incubation cells were stainecfovernight and colonies 350 pm diameter were counted. Bars represent means ? SD of triplicate determinations, and are expressed as cloning efficiency, i.e., number of colonies/ number of cells seeded.

cells. As shown in Figure 1, in the absence of any growth factors, AlN4-T cells exhibit a cloning efficiency of approximately 1%in soft agar. However, addition of BPE, at the same concentration that is used in the MCDB-170 medium to support the growth of the normal mammary epithelial 184 cell strain from which the A1N4 and AlN4-T cells lines were originally established (Stampfer and Barley, 1989, was able to stimulate the AIG of the AlN4-T to the same extent as either EGF or TGFa (Fig. 1).The ability of BPE to stimulate the AIG of AlN4-T cells may in part be due to the presence in the pituitary of TGFa (Kudlow et al., 1989)and bFGF (Gospodarowicz,1989). Both aFGF and bFGF stimulated a three- to five-fold increase in the soft agar growth of the AlN4-T cells, although aFGF was less potent than bFGF. Other growth factors that were screened either alone or in combination, including PDGF, IGF-1, IGF-11, or insulin, were ineffective in stimulating the AIG of AlN4-T cells. The soft agar cloning response of AlN4-T in response to EGF, TGFa, or bFGF, was dose-dependent (Fig. 2), with the optimal concentration for both EGF and TGFa found at approximately 1nglml, and for bFGF between 5 and 10 ng/ml. Suboptimal or optimal concentrations of EGF or TGFa were tested in combination with similar concentrations of bFGF, and were found to have additive, rather than synergistic, effects on the AIG of the AlN4-T cells (data not shown). Establishment and characterization of the AlNCmyc cell line Since amplification and possible overexpression of the c-myc gene has been demonstrated in a subset of primary human breast tumors (Escot et al., 1986; Lidereau et al., 1988), it seemed worthwhile t o attempt to overexpress this ene in the A1N4 cells and to analyze its effect on t e growth behavior of these cells. Total cellular RNA from A1N4 and AlN4-myc cells was

f

Fig. 2. Titration of AlN4-T cells soft agar response to EGF, TGFa, and bFGF. Dose-response curves were performed as in Figure 1 on AlN4-T cells in soft agar for the three growth factors found to be most potent, EGF (open circles), TGFa (open triangles), and bFGF (solid squares). Values are means ? SD of triplicate determinations, and are expressed as cloning efficiency.

isolated and analyzed by Northern blot hybridization with a mouse c-myc probe. Both the A1N4 and AlN4myc cells express the endogenous human c-myc 2.4 kb mRNA transcript (Fig. 3). The AlN4-myc cells expressed the 4.3 kb splice product mRNA species, encoding a functional myc protein product, as well as an additional transcript at approximately 7.5 kb (Dean et al., 1987). Several other mRNA species cross-hybridizing with the myc probe are also seen. Expression of the mouse c-myc transcripts in the AlN4-myc cells was stable over 5 months and after adaptation of these cells for growth in the presence of 10% FCS (data not shown). Like the AlN4-T cells (Clark et al., 1988), the AlN4myc cells were not tumorigenic in nude mice after either subcutaneous injection or after injection into cleared mammary fat pads. AlN4-myc growth factor responsiveness A1N4 cells grow poorly in medium containing concentrations of FCS in excess of 0.5% and cannot be induced to clone in soft agar despite the addition of 10 ng/ml EGF or bFGF (Clark et al., 1988, and data not shown). In contrast, AlN4-T cells can be maintained indefinitely in medium containing 10% FCS without EGF (Clark et al., 1988).Likewise, the AlN4-myc cells were also capable of growin in medium containing 10%FCS although at a slight y reduced growth rate as compared to their growth in medium with 0.5% FCS and supplemented with insulin, EGF, and hydrocortisone (data not shown). In soft agar assays, like the AlN4-T cells, AlN4-myc cells were unable to form colonies in the absence of exogenous growth factors (Fig. 4). However, when AlN4-myc cells were plated in the presence of either EGF or bFGF, these cells exhibited AIG cloning efficiencies that were comparable to similarly treated AlN4-T cells. While the cellular growth factor responses were qualitatively consistent, a certain inter-assay variability in the magnitude of cellular responsiveness was noted. The monolayer mitogenic response of AlN4-myc and AlN4-T cells to TGFa and bFGF were tested in the presence of 10% FCS. The AlN4-myc cells exhibited a

ff

STROMAL INFLUENCES ON HMEC TRANSFORMATION

u

211

20 r

L

F

d-

z

a

F

a

A1 N4

7.5 kb

A1 N4-T

A1 N4-myc

Fig. 4. Soft agar growth responses of AlN4-T and AlN4-myc cells. bFGF and EGF were tested on the AlN4-T and AlN4-myc cells, respectively, in soft agar as described in Figure 1. Bars represent means 2 SD of colony counts expressed as cloning efficiency. Solid bars, no additions; narrow crosshatch, bFGF 1ng/ml; wide crosshatch, bFGF 10 ng/ml; blank bars. EGF 5 ng/ml.

144000

i20000 LL

100000

-

W

m I 3

BQOOO-

z

-1

w 1

6MOO-

40000 -

nMoo0

2.4 kb

h

A1 N4-myc

A1 N4-T

184FB

Fig. 5 . Monolayer TGFa and bFGF growth responses. AlN4-myc and AlN4-T were seeded in medium with 10% FCS at 200-500 cellsicm' and 184FB at 2000 cellsicm' in 12-well cluster dishes, treated the following day, and counted after 5-6 days. Bars represent means t SD of cell counts. Solid bars, no additions; blank bars, TGFcr 10 ngiml; crosshatch, bFGF 10 ngiml.

EGFR sites per cell, as compared to the A1N4 cells (Valverius et al., 1989; Table 1).The enhanced sensitivity of the AlN4-T cells to EGF or TGFa in soft agar could, therefore, in part be related to the increased EGFR expression. In contrast, although the AlN4-myc cells exhibited a similar increase in AIG in response to either EGF or TGFa, there was no significant change in either total EGFR numbers or in the proportion of highpronounced sensitivity to TGFa (Fig. 5). This may in and low-affinity population of EGFR binding sites as part be a reflection of the recent adaptation of these compared to the parental A1N4 cells (Table 1). cells to growth in the absence of EGF. The monolayer Since bFGF can also induce the AIG of AlN4-T and responsiveness of AlN4-T to TGFa was less impressive, AlN4-myc cells, it is possible that this response may be as previously reported (Valverius et al., 1989). AlN4-T due to an indirect effect of bFGF on enhancing EGFR cells showed a moderate increase in monolayer growth expression on these cells and thereby hypersensitizing in response to bFGF while the response of AlN4-myc the cells to the effects of EGF or TGFa. Therefore, cells to bFGF was not significant (Fig. 5). AlN4-T cells were pretreated for 48 hours with bFGF (10 ng/ml) and examined for EGFR expression. No Expression of cell surface EGFR and FGFR significant difference in EGFR binding parameters We have earlier reported that the AlN4-T cells could be detected on AlN4-T cells either before or after exhibit a nearly six-fold increase in the number of treatment with bFGF (data not shown). Fig. 3. Expression of c-myc mRNA in the A1N4 and AlNCmyc cells: 20 kg of total RNA from AlN4myc and the parental A1N4 cells was separated electrophoretically ,transferred to nitrocellulose filters, and hybridized to the mouse c-myc probe. The functional 4.3 kb c-myc splice product mRNA species is indicated by the arrow.

212

VALVERIUS ET AL.

All of the cell lines were then examined for FGFR expression and were found to possess a comparable number of FGFR binding sites with a similar set of affinities within the 20-60 pM range (Table 1).These observations were confirmed by Northern blot analysis (Fig. 8).A specific 4.0 kb FGFR mRNA transcript could be detected in the AlN4-T cells and to a lesser extent in the AlN4-myc cells, as demonstrated in the binding experiments. Cocultivation of mammary epithelial cells and mammary fibroblasts Since fibroblast feeder layers have been employed to aid the establishment and growth of human mammary epithelial cell cultures (Smith et al., 1981), we examined the effect of cocultivation of AlN4-T or AlN4-myc cells with diploid human mammary stromal fibroblasts under ADG or AIG conditions. The 184FB fibroblasts were derived from the same donor from which the original 184 mammary epithelial cell strain had been obtained (Stampfer and Bartley, 1985). For ADG assays, AlN4-derived cell lines and 184FB cells were grown in Transwelll'' cluster dishes where the indicator cells and feeder cells are separated by a 0.4 pm pore filter. To observe whether autocrine or paracrine stimulation occurred, parallel wells were seeded with either no feeder cells, the same cell line as both feeder and indicator cells, or with the 184FB as feeder cells. Growth of both AlN4-myc and AlN4-T was more than doubled by 184FB as feeder cells (Fig. 6). AlN4-myc, but not AlN4-T, also showed evidence for autocrine growth stimulation. The ADG of 184FB cells was not significantly affected by either the AlN4-T or 184FB as feeder cells (Fig. 6). In parallel monolayer assays, the 184FB were less than two-fold growth stimulated by TGFa or bFGF (Fig. 5). To ascertain if the 184FB could also facilitate the AIG of either AlN4-T or AlN4-myc cells, the AlN4derived cells were grown in soft agar over an agar base layer containing the 184FB or in dishes where the 184FB had previously been plated directly on the plastic beneath the two agar layers. We had previously determined that the 184FB cells were unable to clone in soft agar in the absence or presence of EGF, TGFa, bFGF, or TGFp or when cocultivated with AlN4-T feeder layers (data not shown). Cocultivation of AlN4-T or AlN4-myc with 184FB resulted in the appearance of distinct colonies which were less numerous than when purified growth factors such as EGF or bFGF were used (Fig. 7). Nevertheless, there was a five- to ten-fold increase in the AIG of AlN4-T and AlN4-myc in the presence of fibroblast feeder layers (Fig. 7). Further, concentrated CM from 184FB could also induce the AIG of AlN4-T cells. Analysis of growth factor ex ression in normal mammary fibro lasts The results from the cocultivation experiments and soft agar assays using 184FB CM indicated that these mammary fibroblasts secreted growth factors which could stimulate the growth of the AlNCderived cells (Figs. 6. 7). To determine whether Dart of this nowth stiGulation was due to the secretionof immunoreactive TGFa, CM samples from the 184FB cells were analyzed

1

250 r

feedercell:

indicator cell:

FB T

-

T FB

- M FB

FB FB

T

T

M M M

T

Fig. 6. Monolayer cocultivation of AlN4-myc, AlN4-T, and 184FB in "Transwell" dishes. The indicator cell line was seeded in the bottom of a 6-well dish and the feeder cell was seeded into the "Transwell" insert. Due to the different rowth rates, 184FB were seeded a t 4,100 cells/cm2,and AlN4-myc an! AlN4-T at 50 cells/cm2.After 8 days, the indicator cells were trypsinized and counted. Bars represent means ? SD expressed as % increase above cell numbers without feeder cells. FB = 184FB; T = AlN4-T; M = AlN4-myc; - = no feeder cells.

300

250 200 150 100

'1

A1 N4-T

A1 N4-MYC

Fig. 7. Soft a ar cocultivation of AlN4-T or AlN4-myc cells with 184FB;. 4 x 10f 184FB cells (FB in Fig.) used as feeder cells were mixed in the bottom agar layer while 0.5-1 x lo4 AlN4-T or AlN4myc, respectively, were mixed into the top agar layer. Alternatively, CM from 184FB was mixed with AlN4-T cells into the top agar layer. Bars represent means ? SD of actual colony numbers.

in a TGFa-specific RIA. 184FB CM was found to contain high levels of TGFa, approximately 65 ng/108 cells (Table 2), comparable to the amounts previously reported in CM from human breast cancer cell lines (Valverius et al., 1989).In contrast, TGFa mRNA could not be detected either in the 184FB or in AG1523 human foreskin fibroblasts using conventional Northern blot analysis (Fig. 8). Both the AlN4-T and AlN4myc cells expressed the 4.8 kb TGFa mRNA transcript. In addition, upregulation of TGFa mRNA was evident in the AlN4-myc cells grown in medium containing EGF compared"t0 when grown at later passages in medium with 10% FCS, without EGF (Fig. 8).A similar

STROMAL INFLUENCES ON HMEC TRANSFORMATION

F

*ZI

h 7.0-

bFGF

3.7-

bFGF

4.1 -

aFGF

4.0-

TGFa

4.0-

FGFR

Fig. 8. Expression of mRNA transcripts for bFGF, aFGF, TGFa, and FGFR in the AlN4, AlNCT, AlN4-myc, and 184FB cells. RNA was harvested from the AlNCmyc cells both at early passages (grown in low serum, EGF-supplementedmedium) and late passages (adaptedto growth in 10%FCS without EGF). The human foreskin fibroblast cell line AG1523 is included for comparison. Twenty micrograms of total RNA was run on agarose-formaldehyde gels, transferred to nitrocellulose filters, and subjected to sequential hybridization with the indicated probes. The appropriatemRNA sizes are marked in kb to the left.

TABLE 2. TGFa-and FGF-like activities in conditioned media from 184FB cells' ~~~~~~~

TGFa-like(ng/108 cells) FGF-like (nn/106 cells)

65.0 (28-100) 77.0 f 27.4 ~

'For quantitation of TGFa-activity in the 184FB CM, a specific TGFa-RIA kit was used. Values are the mean of duplicate determinations from two separate experiments. The range is givenin parentheses. CM fromthe 184FB cells was analyzed for FGF-like activity in the FGF-RRA utilizing BHK cell monolayer and lZ5I-bFGF,in comparison with standard curves of human recombinant bFGF. Values shown are means f SD of duplicate determinations from three separate experiments.

213

EGF response has earlier been reported for the A1N4 and the original normal 184 cells (Bates et al., 1990). Analysis of 184FB CM in a n FGF-RRA in competition with 1251-bFGFfor binding to BHK cells (Neufeldt et al., 19851,indicated that the 184FB produced approximately 75 ng/106 cells FGF-like activity (Table 2). Northern blot analysis demonstrated high levels of expression in the 184FB of the characteristic bFGF 3.7 and 7.0 kb mRNA transcripts, at levels comparable to those of the human foreskin fibroblasts AG1523 (Kurokawa et al., 1987). Low levels of a 4.1 kb aFGF mRNA were also detected in the 184FB (Gospodarowicz, 1989) (Fig. 8). Moreover, the 184FB also expressed considerable amounts of FGFR mRNA. DISCUSSION The ability of cells to grow in soft agar has been used as one index to monitor cellular transformation in vitro (Kahn and Shin, 1979).With respect to HMEC, we have previously shown that the HMEC line AlN4-T, which expresses the SV40T antigen, can be induced to grow in soft agar following treatment with either EGF or TGFa and this enhanced responsiveness might in part be due to an enhanced EGFR expression in these cells (Valverius et al., 1989). The present study extends these observations and shows that AIG of AlN4-T cells can also be elicited by either bFGF or, to a lesser extent, aFGF. In addition, AlN4-myc cells, which overexpress the c-my gene, exhibit a nearly identical profile with respect to their growth factor sensitivity as the AlN4-T cells. However, in contrast to the AlN4-T cells, AlN4m y do not exhibit any change in EGFR binding characteristics. Likewise, although AlN4-T and AlN4myc cells also exhibit an increased responsiveness to bFGF with respect to AIG, there was no significant change in FGFR expression on either of these two cell lines as compared to the A1N4 cells. This contrasts to a study of cultured rat mammary epithelial cells, where the appearance of FGFR directly parallels the development of cellular responsiveness to FGF during differentiation (Fernig et al., 1990). Thus, the enhanced sensitivity to EGF/TGFa and bFGF and AlN4-T and AlN4-myc cells as compared to the A1N4 cells is apparently due to additional change(s) induced by the oncogenes distal to the receptors for these growth factors, possibly in the intracellular signal transduction pathways. Enhancement of AIG by either EGF, bFGF, PDGF, and TGFp following over-expression of either the c-myc or v-myc genes has previously been reported in immortalized Fischer rate 3T3 and mouse C3H/lOT1,, cells and in primary cultures of chicken heart mesenchymal cells (Balk et al., 1985; Stern et al., 1986; Sorrentino et al., 1986; Leof et al., 1987). Potential paracrine mechanisms in the regulation of breast cancer growth have earlier been proposed since it was found that several human mammary carcinoma cell lines can synthesize and secrete growth factors such as PDGF, bFGF-like peptides, or IGF-I1 that may act oh the surrounding stroma and contribute to desmoplasia and neovascularization (Yee et al., 1988; reviewed in Dickson and Lippman, 1988; Kern et al., 1990).Reciprocally, a supportive effect of mesenchymal host cells on adjacent tumor cells may be equally important. In this regard, the present study demon-

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strates that the cocultivation of AlN4-T or AlN4-myc cells with normal diploid mammary fibroblasts can increase both the ADG and, perhaps more importantly, the AIG of these cells. This effect is at least in part due to the secretion of diffusible growth factors from the fibroblasts since the culture conditions precluded direct contact between the fibroblasts and the HMEC. In fact, concentrated 184FB CM could stimulate the AIG of the AlN4-T cells to a level comparable to that achieved in the cocultivation experiments. The paracrine influence of stromal cells on epithelial cell growth has been documented in several systems. For example, the human prostatic cancer cells PC-3 are stimulated t o grow in soft agar in the presence of normal human lung fibroblasts (Kirk et al., 1981). Mouse fibroblast CM can stimulate mouse mammary epithelial cell growth in monolayer culture (Enami et al., 1983), and human dermal fibroblasts can support the monolayer growth of human follicular keratinocytes (Limat et al., 1989). Further, monolayer stimulation of clonal growth of human prostatic epithelial cells by coculture with fibroblasts or CM from fibroblasts has been reported (Kabalin et al., 1989). Recently, another FGF-related growth factor, KGF, was purified from human embryonic lung fibroblasts and found to be mitogenic for keratinocytes and other epithelial cells in monolayer culture (Finch et al., 1989). The 184FB CM was found to contain high levels of TGFa-activity. The secretion of TGFa by these normal diploid human mammary fibroblasts is particularly noteworthy since in rodent fibroblasts, TGFa expression has generally been closely associated with transformation (reviewed in Sporn and Roberts, 1985). Recently, however, TGFa expression has been reported in a variety of normal human cell types and tissues (summarized in Bates et al., 1990). Although immunoreactive TGFa was found in the 184FB CM, no TGFa mRNA could be detected in 20 p,g of total RNA by Northern analysis. Such a discrepancy between immunoreactive TGFa and TGFa mRNA expression has also been reported for the human mammary carcinosarcoma cell line Hs578T (Clarke et al., 1989). In that report, Western blotting with the same antibody as used in the TGFa-RIA showed a TGFa-specific band for the Hs578T cells similar to that found in the other TGFaexpressing breast cancer cell lines. It is possible that a more sensitive method for detection of TGFa mRNA in the 184FB may have to be employed, such as the use of poly(A)+enriched RNA or RNase protection assays. Nevertheless, that some mammary fibroblasts may indeed express TGFa mRNA has recently been demonstrated by in situ hybridization in a small subpopulation of mammary stromal cells in human and rat mammary gland during pregnancy (Liscia et al., 1990). CM from 184FB cells also contained FGF-like activity as determined in a FGF-RRA, and total RNA isolated from the 184FB showed expression of transcripts for both basic and acidic FGF. Since bFGF and aFGF lack a classical hydrophobic signal sequence for their secretion by a conventional secretory pathway (Gospodarowicz, 19891, it is unclear how the FGF-like activity is released from the 184FB. A possibility is that the FGF might have been released into the CM follow-

ing cellular death and lysis of the 184FB during the serum-free CM collection period (Gospodarowicz,1989). However, other as-yet undefined secretory pathways have been proposed for FGF, as suggested by transfection experiments using mouse BALBlc 3T3 fibroblasts and baby hamster kidney cells transfected by the human bFGF cDNA without a leader sequence (Sasada et al., 1988; Neufeld et al., 1988). In both studies, the CM of the transfected cells was found to contain bFGF. Alternatively, it has been proposed that FGF is secreted together with and bound to extracellular matrix components (Folkman et al., 1988; Moscatelli, 1988; Gospodarowicz, 19891, and subsequently the FGF would be released from these matrix componentsby the action of heparanase or other secreted proteolytic enzymes. Thus, in the cocultivation experiments above, it is possible that proteolytic enzymes secreted by the HMEC could have facilitated the release of FGF secreted by the 184FB bound to basement membrane components. In the CM analysis, it is conceivable that even a limited amount of 184FB cell lysis during the CM collection could provide sufficient lysosomal enzymes for the release of FGF. Finally, it is possible that part of the FGF-like activity detected in the 184FB CM consisted of kFGF, which has a signal peptide sequence and is known to be secreted (reviewed in Rifkin and Moscatelli, 1989). Several strains of human mammary fibroblasts have been reported to express kFGF mRNA (Cullen et al., 1990). In conclusion, these results suggest that there are various interactions between the stroma and epithelium in the human mammary gland that may contribute to the multi-step process of tumorigenesis (Land et al., 1983). These heterotypic cellular interactions could be mediated by soluble growth factors and/or extracellular matrix components and might be enhanced by the expression of certain oncogenes. The present results demonstrate that human mammaryderived fibroblasts can accentuate the growth and induce an in vitro characteristic of transformation, e.g., AIG, in two nontumorigenic immortal human mammary epithelial cell lines that are overexpressing one of two functionally related oncogenes, SV40T or c-myc. These effects of the mammary fibroblasts may apparently in part be due to the secretion of growth factors such as TGFa or FGF. Fibroblasts have previously been shown to enhance the growth and differentiation of rodent and human mammary epithelial cells in vitro (McGrath, 1983; Haslam, 1986) and to stimulate the tumorigenicity of human breast cancer cells in vivo (Horgan et al., 1988). Thus, the in vitro model system described in this report could be useful for further identifying and defining the role of endogenous growth factors as potential mediators of paracrine mammary stromal-epithelial cell interactions.

ACKNOWLEDGMENTS This work was 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, and by American Cancer Society Grant BC 52754.

STROMAL INFLUENCES ON HMEC TRANSFORMATION

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