Int. J . Cancer: 46, 712-718 (1990) 0 1990 Wiley-Liss, Inc.

Publication of the International Union Against Cancer Publication de I'Union lnternationale Contre 18 Cancer

OVER-EXPRESSION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR IN HUMAN BREAST CANCER CELLS FAILS TO INDUCE AN ESTROGEN-INDEPENDENT PHENOTYPE Eva M. VALVERIUS',~, Thieny VELU*,Vidya SHANKAR', Fortunato CIARDIELLO', Nancy KIM' and David S. SALOMON' 'Laboratory of Tumor Immunology and Biology, and 2Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. An association exists in primary human breast tumors between high epidermal growth factor receptor (EGFR) expression and a reduced number or even absence of estrogen receptors (ER). To determine whether an increase in EGFR expression might alter the estrogen responsiveness of an ERpositive human breast cancer cell line, ZR 75-1 cells were cotransfected with a plasmid containing the full-length cDNA for the human EGFR under the transcriptional control of the Harvey murine sarcoma virus (HaMSV) long terminal repeat (LTR) and with a pSV2neo plasmid. Two of the isolated G4 18resistant clones were found to constitutively express EGFR levels 15- to 60-fold higher than those found on nontransfectedZR 75- I cells. The EGFR in these clones were functionally normal since EGF could increase their autophosphorylation and since EGF could enhance the transphosphorylation of p185erbe-2. No change was seen in either the number or affinity of ER in these clones. In addition, the ability of estrogen to stimulate the anchorage-dependent and anchorageindependent growth of these clones was not significantly modified. These results suggest that an increase in EGFR expression alone i s not sufficient to induce a hormoneindependent phenotype in vitro in human breast cancer cells.

Epidermal growth factor (EGF) and transforming growth factor ci (TGFa), a peptide that structurally and functionally resembles EGF and that binds to the epidermal growth factor receptor (EGFR), are potent mitogens for normal and malignant mammary epithelial cells (Salomon and Kidwell, 1988). The EGFR is a 170-kDa transmembrane-associated glycoprotein that possesses an extracellular EGF/TGFci binding domain and an intracellular tyrosine kinase activity (Krupp et al., 1982). Since the EGFR is the product of the c-erbB protooncogene, and since TGFa is produced by a number of different human tumors and tumor cell lines expressing EGFR and therefore may function as an autocrine growth factor (Bates et al., 1986; Derynck et al., 1987; Salomon and Kidwell, 1988), then changes in the level of expression of the EGFR may be important in the pathogenesis of a number of different types of tumor. In this respect, amplification and/or over-expression of the EGFR has been detected in ovarian, bladder, brain, esophageal, gastric, head and neck, and lung tumors (Gullick et al., 1986; Berger et al., 1987; Wong et al., 1987; Lu et al., 1988; Yasui et al., 1988; Ishitoya et al., 1989). In breast cancer, approximately 35 to 50% of primary and metastatic human breast tumors express to varying degrees the EGFR (Macias et al., 1987; Rios et al., 1988; Harris and Nicholson, 1988). Increased levels of EGFR expression observed in primary breast tumors or in breast cancer cell lines are not generally due to gene amplification but rather due to an increased level of EGFR mRNA expression (Harris and Nicholson, 1988; Ro et al., 1988). A number of prognostic indicators are important for predicting the frequency of breast cancer relapse and the potential for response to endocrine therapy. These include estrogen receptor (ER) and progesterone receptor (PgR) status, number of positive axillary lymph nodes, tumor size and nuclear grade (Tandon et al., 1990). EGFR status may also be an independent prognostic marker in breast cancer. For example, high levels of EGFR expression are generally associated with tumors that

have higher proliferation rates (Skoog et al., 1986; Walker et al., 1986; Spitzer et al., 1988) and with patients that have axillary lymph-node involvement and early breast cancer recurrence (Macias et al., 1987; Sainsbury et al., 1987; Nicholson et al., 1988; Harris and Nicholson, 1988; Rios et al., 1988). Further, in primary human breast tumors, there is an inverse relation between the presence of ER and the level of EGFR expression (Sainsbury et al., 1987; Harris and Nicholson, 1988; Pekonen et al., 1988; Rios et al., 1988; Wrba et al., 1988). Such an inverse correlation between ER and EGFR expression has also been clearly demonstrated in human breast cancer cell lines (Davidson et al., 1987). Generally, ERnegative patients have a shorter overall survival than ERpositive patients. Patients with tumors that are both ERnegative and EGFR-negative appear to have overall survival rates that are actually superior to those in patients whose tumors are ER-negative but EGFR-positive (Nicholson et al., 1988; Pekonen et al., 1988; Harris and Nicholson, 1988). However, it is still unclear whether there is any causal relationship between high levels of EGFR expression and the acquisition of an estrogen-independent phenotype. To determine if induction of constitutive EGFR overexpression could alter the estrogen responsiveness of a wellcharacterized ER-positive human breast cancer cell line, ZR 75-1 cells were co-transfected with an expression vector plasmid containing a full-length cDNA for the human EGFR under the transcriptional control of the Harvey murine sarcoma virus LTR (Velu et al., 1987) and with the pSV2neo selectable marker plasmid. Several stably transfected (3418-resistant ZR 75-1 clones were examined for EGFR expression and for their ability to respond to EGF both in growth assays and through the stimulation of autophosphorylation of the EGFR and the transphosphorylation of p185erbB-2(King et al., 1988). Further, clones that were expressing high levels of EGFR were assessed for their ability to respond both to physiological concentrations of estrogen by induction of PgR and also mitogenically in anchorage-dependent and anchorage-independent assays. MATERIAL AND METHODS

Cell culture and transfection ZR 75-1 human breast cancer cells were initially obtained from the American Type Culture Collection (Rockville, MD) T o whom correspondence and reprint requests should be sent, at the Department of Pathology, University Hospital, S-751 85 Uppsala, Sweden. ~~

Abbreviations: EGFR, epidermal growth factor receptor; ER, estrogen

receptor; PgR, progesterone receptor; TGFa, transforming growth factor alpha; IMEM, Improved Modified Eagle's Medium; FCS, fetal calf serum; CCS, charcoal-stripped, sulfatase-treated calf serum; kDa, kilo Dalton; kb, kilo base pair; LTR, long terminal repeat; HaMSV, Harvey murine sarcoma virus. Received: June 7, 1990.

EGFR TRANSFECTION OF BREAST CANCER CELLS

and routinely maintained in Improved Modified Eagle’s Medium (IMEM) with 10% fetal calf serum (FCS) at 37°C in a 5% CO, humidified atmosphere. Cells were co-transfected by calcium phosphate precipitation (Velu et al., 1987; Lowy et al., 1978) with 4 p.g/ml of the pCO12-EGFR plasmid and 1 pg/ml of the pSV2-neo plasmid in the presence of 1 mg/ml carrier salmon sperm DNA. Control cells were transfected with the pSV2-neo plasmid with carrier DNA. After 16 hr, cells were exposed to 15% glycerol for 3 min, then rinsed with complete medium and subcultured 24 hr later. Subsequently, cells were maintained in medium containing geneticin (G418; Gibco, Grand Island, NY) for 21 days, when 18 individual colonies from the pCOl2-EGFR/pSV2neo transfection and 12 colonies from the pSV2neo transfection were recovered and expanded for further characterization as individual clonal cell lines.

713

Immunoprecipitations Cells were grown to approximately 80% confluence in IMEM with 10% FCS in 25-cm2 culture flasks. Cells were then washed and labelled for 16 hr in either methionine-free IMEM with 2% dialyzed FCS containing 250 p.Ci/ml 35S-methionine (1 100 Ci/mmol, Amersham) or in phosphate-free IMEM with 2% dialyzed FCS containing 500 pCi/ml 32P-orthophosphate (200 Ci/mM, Amersham). Parallel flasks of the 32P-labelled cells were treated with 10 ng/ml EGF for 10 min prior to harvest. Cells were harvested by scraping, centrifuged, solubilized, and immunoprecipitated (Beguinot et al., 1984). Aliquots of cell lysates containing 2 x lo7 cpm of TCAprecipitable protein were immunoprecipitated using either the EGFRl mouse anti-human EGFR monoclonal antibody (MAb) (Amersham), the 21N rabbit anti-human erbB-2 polyclonal antibody (Gullick et al., 1987), or the corresponding non-specific preimmune sera. Immunoprecipitated proteins were then subjected to 10% SDS-PAGE analysis (Beguinot et al., 1984).

EGFR binding assay Initial screening of all ZR 75-1 clonal cell lines was performed using a single, saturating concentration Iz5I-EGF RNA preparation and hybridization (10nM; human; ICN, Irvine, CA) in the presence or absence of RNA was extracted from subconfluent cells by the guanidia 200-fold excess of unlabelled EGF (mouse; Collaborative Research, Bedford, MA). For binding isotherms, increasing nium isothiocyanate/cesium chloride centrifugation method concentrations (6 PM to 10 nM) 1251-EGF(80-110 p.Ci/pg) (Maniatis et al., 1982). Approximately 10 pg/lane poly(A)+were used. Assays were performed essentially as previously RNA were electrophoretically separated in a 1.2% agarose-2.2 described (Valverius et al., 1989) on whole-cell monolayers in M formaldehyde gel. Gels were stained with ethidium bromide 24-well cluster dishes at 4°C. Binding parameters were calcu- and examined to ensure that equivalent amounts of undegraded lated by the LIGAND program (Munson and Rodbard, 1980), RNA had been loaded for each sample. The RNA was subseand numbers of binding sites were normalized to cell numbers quently transferred from the gels to nitrocellulose by capillary blotting (Thomas, 1980) and hybridized with a 32P-labelled, determined from comparably treated parallel wells. nick-translated 2.4-kb human cDNA EGFR probe, pE7 (Merlino et al., 1984). Steroid hormone receptor determinations Determinations of ER and PgR binding parameters were DNA preparation and Southern blot analysis performed as described by Clarke et al. (1989) on whole-cell High-molecular-weight DNA was extracted from the cells monolayers in 24-well cluster dishes at 37°C using increasing using SDS-proteinase K (Maniatis et al., 1982). DNA samples concentrations of either 3H-estradiol (95 Ci/mmol) or 3H-ORG were then digested by the restriction enzymes Hind11 or 2058 (50.6 Ci/mmol) (Amersham, Arlington Heights, IL) in BamH-1, fractionated in 0.8% agarose gels, transferred to nithe presence or absence of a 100-fold excess of unlabelled trocellulose and hybridized to the labelled 2.4-kb EGFR insert, competitor. Prior to PgR determinations, cells were grown for pE7 (Merlin0 et al., 1984). a minimum of 7 days in phenol-red-free IMEM with 5% charcoal-stripped, sulfatase-treated calf serum (CCS). PgR inducRESULTS tion was assayed on cells pretreated with 1 n M estradiol for 48 to 96 hr. To reduce non-specific binding of the progesterone EGFR expression in the trangected ZR 75-1 clones compound to glucocorticoid receptors, PgR assays were initiTo ascertain whether the constitutive over-expression of the ated after pre-incubation of the cells with 100 nM hydrocortiEGFR might alter the estrogen responsiveness of human breast sone at 37°C for 30 min. cancer cells, the ZR 75-1 human breast cancer cell line was transfected with an expression vector plasmid, pCOl2-EGFR, Growth assays which contains the human EGFR cDNA under transcriptional Anchorage-dependent growth assays were performed on control by the HaMSV LTR (Velu et al., 1987). ZR 75-1 cells cells grown in 12-well cluster dishes either in IMEM with 10% were selected because they are a well-characterized human FCS and varying concentrations of EGF or in phenol-red-free breast cancer cell line that is ER-positive and that responds IMEM with 5% CCS in the presence or absence of 1 n M mitogenically to physiological concentrations of estrogen (Al17P-estradiol. For determinations of estrogen responsiveness, legra and Lippman, 1980; Bates et al., 1986). In addition, cells were pretreated in phenol-red-free IMEM with 5% CCS these cells express relatively low levels of EGFR and exogefor 1 to 3 weeks prior to assay. Routinely, lo3 to 3 X lo3 cells nous EGF or TGFa can produce an increase of up to 2-fold in were seeded per well and incubated overnight before the ad- their growth (Davidson el al., 1987; Fabbro et al., 1986; Nodition of either EGF or estradiol. Media with appropriate treat- vak-Hofer et al., 1987). ments were changed every 2 days, and after 5 to 8 days, cells Following selection in G418-containing medium, 19 G418were trypsinized and counted in a Coulter Counter. resistant EGFRheo co-transfected clones and clones that Anchorage-independent growth assays were performed with contained just the pSV2neo plasmid were initially screened for the same media, treatments, and pretreatments as for anchor- EGF binding using a single concentration of 12-9-EGF. Eleage-dependent assays (Valverius et al., 1989). Briefly, lo4 vated binding was detected in 3 of the EGFRJneo cocells were seeded with or without EGF or 17P-estradiol in the transfected clones, namely 4, 11 and 13, whereas the neoappropriate serum-containing medium with 0.36% Bactoagar transfected cells exhibited levels of EGF binding that were over a hardened 0.6% agar base layer in 35-mm dishes. After within the same range as the parental non-transfected ZR 75-1 10 to 12 days’ incubation, colonies were stained with nitroblue cells (data not shown). For determination of EGFR binding tetrazolium and counted on an Artek (Chantilly, VA) 880 col- parameters, saturation binding experiments were conducted ony counter. (Fig. 1). All of the clones and the parental cells bound

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VALVERIUS ET AL.

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FIGURE1 - IZ5I-EGFsaturation binding curves. Assays were performed at 4"C, and values shown are the means of duplicate determinations. EGFR receptor parameters were calculated using the LIGAND program and are shown in Table I.

IZ5I-EGFin a concentration-dependent manner, and saturation of specific binding occurred between 10 and 30 nglml(l.5 and 5 nM) lZ51-EGF.Binding parameters were determined by the LIGAND program, and the total numbers of binding sites per cell are listed in Table I. All of the cell lines had a population of high-affinity EGFR (Kd 0.03-0.17 nM) while clones 4, 11 and 13 exhibited an additional class of low-affinity binding sites (Kd 1.9-2.2 nM) (data not shown). These last 3 clones possessed from 1.87 X lo5 to 1.2 X lo6 EGFR siteskell, which is a 6- to 60-fold increase above the parental ZR 75-1 or the neo-transfected clone 2 cells which ranged from 2 to 4 X lo4 EGFR siteslcell. The 2 highest EGFR-expressing clones, 11 and 13, were phenotypically stable over a period of 5 months with respect to this parameter. To determine if the increased level of lZ5I-EGFbinding in these different EGFR-transfected clones was also reflected by an increase in the amount of EGFR protein, clones 2 (neotransfected cells), 4 , 11 and 13 were labelled with 35S-methionine and subsequently lysed and immunoprecipitated with an anti-EGFR MAb (Fig. 2a). Increasing levels of immunoprecipitable 170- and 150-kDa species could be detected in the clones, in relative proportion to the previously determined increasing levels of T - E G F binding. The 170kDa band represents undegraded mature EGFR (Krupp et af., 1982) while the 150-kDa species probably represents a major receptor protease cleavage product. Cells were also labelled with 32P-orthophosphateto determine the relative activity of the EGFR tyrosine kinase with respect to its ability to autophosphorylate the EGFR before and after stimulation by EGF (10 ng/ml) for 10 min (Fig. 2b). The level of EGF-induced autophosphorylation of the 170-kDa band correlated reasonably well with the increased level of EGFR expressed on these different clones. In contrast, no significant difference could be

FIGURE2 - EGFR expression in transfected clones. (a) EGFR immunoprecipitation. Cells were labelled with 35S-methionineand lysates precipitated using an anti-EGFR MAb. Molecular weight markers are in lane s. (b) EGFR autophosphorylation. Transfected clones were labelled with 32P-orthophosphateand treated with EGF for 10 min ( + lanes) prior to lysis and precipitation with the anti-EGFR antibody. Untreated cell lysates ( - lanes) were labelled and immunoprecipitated identically. (c)EGFR mRNA expression. Poly(A)+ RNA (10 pgllane) from the parental ZR 75-1 cells (lane p) and clones neo-2, 4, 13 and 11 was hybridized to the human cDNA EGFR probe, pE7.

discerned in the basal levels of EGFR autophosphorylation among these clones. Another important functional characteristic of the EGFR tyrosine kinase is its ability to phosphorylate other cellular proteins that might be important in the signal transduction pathway for EGF (Krupp et al., 1982). The product of the c-erbB-2 proto-oncogene is an EGFR-related 185-kDa cell-surface glycoprotein, p185erbB-2,which possesses an intrinsic tyrosine kinase activity (Gullick et al., 1987). Since the p185erbB-2protein can serve as a substrate for the EGFR tyrosine kinase (King et al., 1988) and since ZR 75-1 cells express low to moderate levels of p185erbB-2protein (Kraus et al., 1987), we investigated whether there were any differences in the level of EGF-induced tsansphosphorylation of p 185erbB-2between the different EGFR-transfected ZR 75-1 clones. Cells which had been labelled with 32P-orthophosphate and subsequently stimulated with EGF (10 ng/ml) for 10 min were immunoprecipitated with the rabbit polyclonal, p185erbB-2-spe~ifi~ 21N antibody (Gullick el al., 1987). As illustrated in Figure 3, there

TABLE I - TOTAL NUMBERS OF EGF, ESTROGEN AND PROGESTERONE RECE€TOR SITESICELL ON ZR 1.5- I CELLS AND CLONES Cell

EGFR

ER

ZR 75-1 Clone 2-neo Clone 4

43,180 187,320

20,030

11,700

Clone 13 Clone 11

316,330

10,180

1.204.300

6,970 ND

16.470

PgR

(

PgR

- estroeen)

( +estrogen)

3,530 9,710

9,760 48,570

ND

ND

ND 3.190

ND 8.130

ND = not determined. Saturation binding experiments were performed on whole-cell monolayers in 24-well cluster dishes, for EGFR at 4"C,and for ER and PgR at 37°C. For determinationsof PgR parameters, cells were grown in estrogen-freemedium (phenol-red-free IMEM with sulfatase-treated. dextran-charcoal-snippedcalf serum) for 5-9 days prior to seeding for the assay. Parallel dishes were given short-term 17~-estradioltreatment prior to assay and binding parameters were compensated for differences in cell numbers between treated and non-treated dishes. Data analysis was performed using the LIGAND program.

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EGFR TRANSFECTION OF BREAST CANCER CELLS

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FIGURE3 - EGF-induced transphosphorylation of p185erbB-2. Aliquots of the 32P-orthophosphate-labelledcellular lysates with ( +lanes) or without ( - lanes) final EGF stimulation were precipitated with the 21N c-erbB-2-specific antibody.

was an increase in the EGF-induced transphosphorylation of p185erbB-2 protein in all of the clones, with the highest levels in clones 11 and 13 as compared to the pSV2neo transfected clone 2. The increase in EGFR in these different clones is also reflected by an increase in the level of expression of the exogenous EGFR mRNA generated by the Hah4SV EGFR expression vector plasmid following transfection. Northern blot analysis of poly(A)+ RNA demonstrated an enhanced expression of a 4.5-kb mRNA transcript in clones 11 and 13, and lower levels of this transcript in clone 4 following hybridization with the nick-translated human pE7 EGFR cDNA insert (Fig. 2c). The size of this transcript is consistent with the expected size of the expression vector plasmid RNA (Velu et al., 1987). No endogenous EGFR mRNA transcripts of 10.0 or 5.6 kb could be detected by this method in either the parental or neo clone 2 cells, or in any of the EGFR-transfected clones. Southern blot analysis of DNA, extracted from clones 2, 4, 11 and 13 and digested with either HindIII or BarnHl , demonstrated the presence of unique restriction fragments at 2.2 and 2.6, or 9.0 kb, respectively, in the EGFR-transfected clones (data not shown). These restriction fragments were not present in the parental or neo clone 2 cells and suggest that integration and random rearrangements of the plasmid had occurred in the transfected clones. Response of EGFR-transfected ZR 75-1 clones to EGF and estrogen ZR 75-1 cells are normally responsive to exogenous EGF or to TGFa in that both peptides are able to elicit an approximately 2-fold increase in the anchorage-dependent growth of these cells (Davidson et al., 1987; Fabbro etal., 1986; NovakHofer et al., 1987). To determine if the EGFR-transfected clones possessed biologically functional receptors that could mediate a mitogenic response to exogenous EGF, and to ascertain if over-expression of the EGFR might in some manner modify the magnitude or the direction of response of these cells to EGF, parental, neo clone 2, clone 11 and clone 13 cells were grown in serum-containing medium for up to 8 days in the absence or presence of different concentrations of EGF (Fig. 4a). EGF (2.5-100 ng/ml) or TGFa (data not shown) generally produced a I .2- to 2.0-fold increase in the growth of the parental and neo clone 2 cells. In contrast, EGFR-transfected clone-13 cells were unresponsive to EGF whereas EGFRtransfected clone 11 cells, which expressed the highest level of EGFR, were growth inhibited by EGF. The basal growth rate of the EGFR-transfected clones did not differ significantly from that of the parental or neo-transfected clone 2 cells. Qualitatively comparable results were obtained when the soft-agar

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FIGURE4 - Growth responses to EGF in the presence of 10% FCS. (a)Monolayer growth responses. Cells (3-5 X 103/well)were seeded in 12-well dishes. The next day, EGF was added and, after 5-7 days, cells were bypsinized and counted. Values are means ? SD. Solid bars: no EGF treatment. Blank bars: low-dose EGF, as indicated. Cross-hatched bars: high-dose EGF, as indicated. (b) Anchorageindependent cellular response to EGF. Cells (lo4) were plated in 0.36% agar with varying concentrations of EGF. After 10 to 12 days, colonies were stained and those larger than 0.1 m were counted. Values shown are means 2 SD. Solid bars: no EGF. Blank bars: EGF 1 ng/ml. Wide cross-hatched bars: EGF 10 ng/ml. Narrow crosshatched bars: EGF 100 ng/ml.

growth of these different clones was tested in the absence or presence of EGF (Fig. 46). EGF was capable of stimulating colony formation in the parental and neo-transfected clone 2 cells. EGF had no significant effect upon the colony-forming capacity of clone 13 cells while the anchorage-independent growth of clone 11 cells was inhibited in a dose-dependent manner. ZR 75-1 cells possess functional estrogen receptors (ER) and are estrogen-responsive when maintained under estrogendepleted conditions in phenol-red-free medium containing CCS (Allegra and Lippman, 1980; Bates et al., 1986). An inverse correlation between EGFR expression and ER status has been noted in several human breast cancer cell lines and in a majority of primary human breast tumors (Davidson et ul., 1987; Pekonen et al., 1988; Wrba et al., 1988). Thus, it is conceivable that an increase in the level of EGFR expression might alter the number or affinity of the ER in the different ZR 75-1 clones andlor affect the subsequent estrogen responsiveness of the cells. However, no significant differences in either the number of ER binding sitedcell or the affinity of the ER for estrogen (Kd range 0.02-0.10 nM) were observed between the parental ZR 75-1 cells, neo clone 2 cells and the EGFRtransfected clone 11 and clone 13 cells (Table I). Another indication of ER functionality is the ability of estrogen to induce progesterone receptors (PgR) through an ER-mediated pathway (Horwitz et al., 1978). When previously estrogenstarved cells were maintained in the presence of 17P-estradiol (1 nM) for 2 to 4 days, there was a 3- to 5-fold increase in the

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VALVERIUS ET AL.

number of PgR siteskell in the parental and neo clone 2 cells (Table I). Estrogen induction of PgR of an equivalent magnitude was also observed in the highest EGFR-expressing clone11 cells. Finally, the ultimate test of whether these different clones possess biologically functional ER is to determine if estrogen is capable of stimulating cellular growth. As illustrated in Figure 5a, after 8 days in monolayer culture with 17P-estradiol (1 nM), there is a 2- to 3-fold increase in the growth of all the different ZR 75-1 clones irrespective of the level of EGFR expression in the cells. Further, in the absence of estrogen, the parental and various transfected clones do not exhibit appreciable anchorage-independent growth (Fig. 5b). Addition of 17P-estradiol (1 nM) results in a 12- to 29-fold increase in the ability of the different cell lines to form colonies in soft agar, with no significant quantitative difference observed between the parental ZR 75-1 cells (29-fold increase) and the EGFR-over-expressing clone 11 cells (26-fold increase). Finally, when 5 x lo6 cells were injected subcutaneously into ovariectomized nude mice in the absence or presence of estrogen supplementation, no difference in tumorigenicity was seen between the neo transfected clone 2 cells and the EGFR-over-expressing clone 11 or 13 cells. DISCUSSION

A number of established prognostic factors have been described for breast cancer including axillary 1ymph-node involvement, nuclear and histologic grade, tumor size, and steroid hormone receptor status (Tandon et al., 1990). EGFR status and amplification and/or over-expression of c-erbB-2 have both been shown to be possible independent prognostic markers for specific subsets of human breast tumors (Sainsbury et al., 1987; Harris and Nicholson, 1988; Nicholson et a l . , 1988; Slamon et al., 1989; Harris et al., 1989). More importantly, there appears to be a unique negative association between ER and EGFR expression, suggesting that a subset of human breast tumors whose growth is not regulated by estrogens may in fact be regulated by a series of tumor-derived growth factors, such as TGFu, which could function in an autocrine fashion through the EGFR system (Salomon and Kidwell, 1988). These data also suggest that progression to an estrogen-independent phenotype in breast cancer cells might result in the over-expression of EGFR, or vice versa. Therefore, it appeared worthwhile to determine if over-expression of the EGFR in an ER-positive human breast cancer cell line might lead to a change in or a loss of estrogen responsiveness that might suggest a functional or causal relationship between these 2 phenotypes. 175 -

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The present study demonstrates that successful transfection of an EGFR expression vector plasmid into a human breast cancer cell line leads to the outgrowth of different EGFRexpressing clones that exhibit a spectrum of EGFR binding. In some of these clones, such as clones 11 and 13, the number of EGFR was equal to or greater than the level of EGFR expression observed in NIH 3T3 cells which had been transformed using a similar EGFR expression vector (Velu et al., 1987). The EGFR over-expressed on the transfected ZR 75-1 clones were found to be structurally normal as determined by immunoprecipitation with an anti-EGFR antibody. Furthermore, the EGFR in these clones possessed a functionally active tyrosine kinase since EGF was capable of inducing the autophosphorylation of the EGFR. In fact, the level of EGF-induced EGFR autophosphorylation was directly proportional to the amount of immunoprecipitable EGFR protein and to the level of EGF binding that were detected in these clones. Another index of a functional EGFR tyrosine kinase was the ability of EGF to induce transphosphorylation of p l 85erbB-2in these clones, also in proportion to the amount of EGFR protein present. In NIH 3T3 cells, EGFR over-expression results in a liganddependent transformation of the cells that can be directly correlated with the level of EGFR binding (Velu et al., 1987). Under these conditions, NIH 3T3 cells that expressed more than 4 X lo5 siteskell generally became hypersensitive to the mitogenic effects of low concentrations of EGF. Similarly, over-expression of EGFR in ZR 75- 1 breast cancer cells could be expected to lead to a biologically functional receptor capable of transducing a growth-regulatory signal(s) in response to EGF. The EGFR-transfected ZR 75-1 clones examined in this study possess varying levels of EGFR expression and exhibit a gradual transition in the nature of the growth response produced by EGF under both anchorage-dependent and anchorage-independent culture conditions. In clones that possess low numbers of EGFR, EGF was mitogenic, whereas in cells such as clone 13, which exhibited an intermediate level of EGFR expression, EGF had no effect on cell growth. In ZR 75-1 clone 11 cells, where the number of EGFR was more than lo6 binding siteskell, EGF became growth-inhibitory . A similar EGF-induced growth inhibition has been reported in the MDAMB-468 human breast cancer cell line that also exhibits a comparably high number of EGFR due to amplification of the EGFR gene (Filmus et al., 1985). In fact, clonal variants of MDA-MB-468 cells have been isolated that express lower levels of EGFR and which are stimulated rather than inhibited by EGF (Filmus et al., 1987). Among the ZR 75-1-transfected clones, there appears to be a threshold level of EGFR expression for alterations in EGF responsiveness. This was postulated

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FIGURE5 - Growth responses to 17P-estradiol (1 IIM) in 5 % CCS. ( a ) Monolayer cellular growth response. Cells (3 to 5 X 103/well)were seeded in 12-welldishes. Estradiol was added the next day and cells were trypsinized and counted after 5-8 days. Values are means f SD.Solid bars: no E, treatment. Blank bars: E, 1 nM. (b)Anchorage-independent cellular responses. Cells ( lo4)were plated in 0.36%agar with or without estradiol(1 nM). After 10 to 12 days’ incubation, colonies were stained and counted. Values shown are means SD of number of colonies larger than 0.1 mm diameter. Solid bars: no E,. Blank bars: E, 1 nM.

*

EGFR TRANSFECTION OF BREAST CANCER CELLS

to be the case in the study of the MDA-MB-468-derived clones (Filmus et al., 1987) but in that study, clones with intermediate receptor levels were not available to test the hypothesis. Also in support of the idea of a threshold level of EGFR expression, when comparing several different breast cancer cell lines, Davidson reported a tendency for EGF responsiveness only in those lines with fewer than 7 X lo4 EGFR per cell, whereas cell lines with higher receptor numbers were generally unresponsive (Davidson et al., 1987). An inverse relationship between ER and EGFR expression has been described in several breast cancer cell lines and in a large cohort of breast cancer tissues, suggesting that EGFR over-expression may desensitize breast cancer cells to the growth-regulatory effects of estrogen (Davidson et al., 1987; Sainsbury et al., 1987; Pekonen et al., 1988; Rios et al., 1988). The results of this study using an EGFR expression vector plasmid in an estrogen-responsive human breast cancer cell line demonstrate that no direct causal relationship exists between these 2 properties, in that EGFR over-expression does not induce an estrogen-independent phenotype. A large increase in EGFR binding, as was observed in clone-11 cells, failed to alter either the level of ER expression or ER functionality. Physiological concentrations of 17P-estradiol were still capable of stimulating the monolayer and soft-agar growth of clone 11 cells and to induce the PgR to the same degree as was observed in the parental or neo-transfected ZR 75-1 cells. The mechanism behind the observed inverse relationship be-

717

tween ER and EGFR expression and its correlation to tumor progression is not known. It is conceivable that different clones of tumor cells in vivo during the early stages of tumor formation may possess varying levels of EGFR expression that could confer upon these cells a selective growth advantage irrespective of their ER status. A change in a growth factor receptor status late in the cellular life history, such as that occurring in the cells used in this study which were originally established from an ascites effusion (Engel and Young, 1978) and were thus metastatic by definition, might then not be sufficient to substantially alter the hormone-responsive phenotype. It is difficult to entirely exclude the possibility that an increase in EGFR expression in normal non-malignant mammary epithelial cells or in mammary tumor cells that are not yet metastatic would alter their response to estrogens. In any event, the results of this study clearly demonstrate that no immediate functional or causal relationship exists between over-expression of the EGFR and the acquisition of an estrogen-independent phenotype in a human breast cancer cell line.

ACKNOWLEDGEMENTS

The authors thank Dr. J . Bergh and A. Johnson, Department of Oncology, University Hospital, Uppsala, Sweden, and Dr. B.K. Vonderhaar, National Cancer Institute, Bethesda, Maryland, for expert assistance with the nude mouse experiments.

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