Effects of Estrogen, Epidermal Growth Factor, and Transforming Growth Factor-a on the Growth of Human Breast Epithelial Cells in Primary Culture BRETT M. GABELMAN AND JOANNE T. EMERMAN Department
growth factors and their receptors . This information is important as it is hypothesized that disorders in the mechanisms of E, growth control may be critical steps in the initiation and/or the progression of breast cancer. It has been observed that many estrogen receptorpositive (ER+) breast cancer cell lines are growth stimulated by physiological concentrations of E,, whereas receptor-negative (ER-) lines are not [l]. Accompanying the increases in cell growth with E, treatment of ER+ cell lines are changes in the production and secretion of both autocrine growth factors and their receptors . In ER- cell lines the production of these growth factors is constitutively increased. The epidermal growth factor and transforming growth factor-a (EGF/TGF-LX!) pathway has received considerable attention as a mediator of BEC growth. EGF and TGF-a have been identified in milk, breast cyst fluid, and breast tumor tissue [7-g]. Exposure of ER+ breast cancer cell lines to E, leads to increases in mRNA and protein levels for both EGF and TGF-a and an increase in EGF receptor (EGFR) levels [6, lo]. The consistent observation that EGFR and ER levels are inversely related further supports a possible interrelationship between these two systems [ll, 121. The presence of the high levels of EGFR observed in many hormonally unresponsive ER- tumors may represent a critical step in the progression of the tumors to a hormonally independant state. Whether or not E, can act as a direct mitogen on human BEC (HBEC) has been investigated with mixed results on cells in primary culture. We and others have shown that E, can stimulate the proliferation of both normal- and tumor-derived HBEC in the presence of normal human serum [13-161. However, it has been reported that E, has no effect on the growth of normal HBEC in serum-free primary culture . In contrast, EGF has been shown to stimulate the growth of numerous cell lines [ 181 and primary cultures of HBEC in medium with [19, 201 and without serum [21, 221. We utilized the basic serum-free medium described by Yang et al.  to study the effects of E,, EGF, or TGF-(Y on the proliferation of HBEC from normal, benign, and malignant tissues. In addition, the combined effects of EGF
Since 17&estradiol (E&stimulated growth in human breast cancer cell lines has been shown to be accompanied by increased production of epidermal growth factor (EGF) and transforming growth factor-a (TGF-(w) and their receptor, we investigated the effects of E, and these growth factors on the growth of human breast epithelial cells (HBEC) in primary culture. HBEC from normal, benign, and malignant tissues were cultured in serum-free medium [DME:F12(1:1), 5 mg/ml BSA, 10 rig/ml cholera toxin, 0.5 fig/ml cortisol, 10 rglml insulin] in the presence and absence of E,, EGF, and TGF-a. Tritiated-thymidine (13H]TdR) incorporation into DNA was used as a measure of cell growth. E, did not stimulate growth of any of the cultures at all concentrations examined (lo-’ to lo-’ M). In contrast, EGF ranging the growth from 1 to 100 rig/ml consistently increased of cells of all three breast tissue types in a dose-dependent manner. The EGF stimulation was inhibited by MAb 528, a monoclonal antibody against the EGF receptor. TGF-(r was equally or more effective in stimulating proliferation, although its dose-response range was different than that of EGF. E, and EGF together acted in a synergistic manner in 50% of the samples examined. These studies suggest that E, can exert effects on HBEC growth via modulation of the cells’ response to 0 1992 Academic Press, Inc. EGF.
INTRODUCTION Data from in viva studies with rodents and epidemiological evidence clearly indicate that the ovarian steroid 17-P-estradiol (E,) is critically involved in the growth of both normal and malignant breast epithelial cells (BEC) [l, 21. Unfortunately, differences in the growth responses to E, in BEC from different species make it difficult to extrapolate data from experiments on mice to the situation in rats, let alone to humans. Furthermore, it is not clear whether E, exerts its effects on BEC indirectly, by stimulating the production of “estromedins” (growth factors) in organs other than the breast , by direct stimulation of DNA replication , or via the production of autocrineand paracrine-acting 113
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and E, were examined to determine if there are any interactive effects of the two on the growth of HBEC in primary culture.
(E,) on Proliferation of Human Cells in Primary Culture Concentration
Sample procurement and assessment. Normal tissue was obtained from reduction mammoplasties; fibroadenoma and carcinoma samples were obtained from biopsies and mastectomies, all of which were performed by surgeons at local hospitals. All carcinoma samples chosen for this study were ER’ in situ or infiltrating ductal carcinomas. ER levels and the pathology reports were provided by the pathology departments of the hospitals. Cell culture. Our culture procedure has been described previously [13, 231. Briefly, breast tissue from reduction mammoplasties, biopsies, or mastectomies were minced and dissociated in a 1:l mixture of phenol red-free Ham’s FlZ:Dulbecco’s modified Eagle’s medium (F12:DMEM; Sigma Chemical Co., St. Louis, MO) containing 10 mM Hepes buffer, 2% bovine serum albumin, 10 @g/ml insulin, 300 U/ml collagenase, and 100 U/ml hyaluronidase (Sigma) at 37°C for approximately 18 h. The epithelial cell pellet was collected by centrifuging the cell suspension at 8Og for 4 min. The pellet was washed twice in F12:DMEM and the number of viable cells, determined by trypan blue exclusion, was counted on a hemacytometer. The cells were resuspended in culture medium (discussed below) and seeded at 3 X lo5 cells/cm’ onto collagen-coated tissue culture plates. Cultures were incubated at 37°C in 95% air:5% CO*. For the first 24 h of culture the culture medium consisted of Ham’s F12:DMEM (1:l) supplemented with 10 m&f Hepes buffer, 5 pg/ml insulin, and 5% pooled human serum from normal donors . The medium containing serum allowed attachment of the cells. After 24 h the medium was removed and the cultures were washed twice in F12:DMEM to remove any adhering serum, then a serum-free medium was added. This consisted of F12:DMEM (1:l) supplemented with 10 mM Hepes buffer, 5 mg/ml bovine serum albumin, 10 rig/ml cholera toxin, 0.5 pg/ml hydrocortisone, and 10 pg/ml insulin. The cells were then grown for a further 24 h in the serum-free medium to remove any remaining serum. Following this, media containing varying amounts of E, (10m9 to 10-a M) and/or EGF (l-100 rig/ml) or TGF-a (l-100 rig/ml; Sigma) were added to the cultures. It has been observed that the phenol red used as a pH indicator in tissue culture medium possesses estrogenic activity that might mask the growth effects of exogenously added E, . In order to avoid this, all experiments were carried out in phenol red-free medium. The insulin concentration was varied from 0.1 to 10 rg/ml in some experiments to determine if high concentrations of insulin were masking an E, effect on growth, as has been reported [25,26]. In some studies a monoclonal antibody to the EGF receptor, MAb 528 (Oncogene Science), was added to the medium at 1.5 rg/ml to block EGF binding. This MAb binds to the EGF receptor, blocking EGF binding, as well as blocking EGF stimulation of the receptor tyrosine-kinase activity . Growth studies. Media were changed every 3 days and cultures were observed daily. When the fastest growing cultures were 70-80% confluent, a final media change was done. Fourteen hours after the last media change, tritiated thymidine ([3H]TdR) at 1 &i/ml was added to each well. After 6 h the media were removed, the cultures were fixed in 10% trichloroacetic acid (TCA) at 4°C for 15 min and then washed 3~ in 5% TCA at 4°C for 5 min each. The acid-insoluble material was dissolved in 2 N NaOH at room temperature for 24 h. Aliquots from each well were measured for [3H]TdR incorporation into DNA by scintillation counting. Values were converted from absolute counts to the percentage of controls to allow for comparison between experiments. In all cases, the controls were cells grown in the absence of E,, EGF, and TGF-cr.
Redn” FA Ca TOTAL
of E, (M)
100 ri 136 n=8 100 + 18 n = 19 100 + 24 n = 11 100 k 18 n = 38
94 t 12 n=7 95 + 26 n=3 N.D.
82 f 12 n=8 105 + 17 n = 19 95 f 20 n = 10 97 f 17 n = 37
94 t 16 n = 10
10m7 88 f n=5 902 n=3 75 f n=3 85 f n =
12 11 21 14 11
1O-8 8Ort n=8 74 2 n = 66 2 n=8 73 f n =
11 17 10 16 15 25
a Cells obtained from reduction mammoplasties (Redn), fibroadenomas (FA), and carcinomas (Ca). * Values shown are the percentage of control values (control = no E2) ?SEM.
Effects of E, on HBEC Proliferation In a total of 37 cultures examined, 8 reductions (Redn), 19 fibroadenomas (FA), and 10 carcinomas (Ca), E, did not stimulate a significant increase in [3H]TdR incorporation into DNA at any of the concentrations tested. Table 1 summarizes the responses of cells from the three types of breast tissue to the different concentrations of E,. There was a trend toward inhibition of proliferation at the higher concentrations of E, (lop7 and 10e6 M); however, this trend was not significant in the grouped data. Figure 1 illustrates the absence of an E, effect at a physiological concentration (lo-’ M) and significant inhibition of [3H]TdR incorporation at a supraphysiological concentration (lop6 M) in representative single experiments with each of the three tissue types. The significant inhibition at high doses of E, was seen in 5112 Redn, 5110 FA, and 316 Ca. Effects of Insulin
on HBEC Proliferation
High concentrations of insulin have been shown to mask E, stimulation of growth in the MCF-7 human breast cancer cell line [25, 261. Therefore, we reduced the concentration of insulin from 10.0 to 0.1 pg/ml in a number
fered with the effect of E, on HBEC growth in primary culture. In 9 samples examined (7 FA and 2 Ca), reduction of insulin to 0.1 pg/ml decreased proliferation significantly (Fig. 2). The magnitude of this decreased growth ranged from 3 to 67% of control values. The E, effect on growth was the same in all cultures containing either low or high concentrations of insulin; there was no E, stimulation of growth (Fig. 2).
FA 47 Ca1Ol
FIG. 1. Effect of E, on cultures of cells from a reduction mammoplasty (R ll), a fibroadenoma (FA 47), and a mammary carcinoma (Ca 101). Cells were cultured in serum-free medium [DME/FlS(l:l), 10 mM Hepes buffer, 5 mg/ml bovine serum albumin, 10 rig/ml cholera toxin, 5.0 ag/ml hydrocortisone, 10 pg/ml insulin] and different concentrations of E, (10m9 to 10-e M). When cultures were 80% confluent they were incubated in [aH]TdR for 6 h, then assayed for [3H]TdR incorporation into DNA. Results are the mean values -+ SEM from triplicate wells and are expressed as a percentage of control, where the control condition was serum-free medium with no E,. E, at 0.01 aM (10-s M) had no effect, whereas at 1 r&f (10-a M), E, caused a dramatic inhibition of growth.
Effects of EGF and TGF-a on HBEC Proliferation EGF was added to cultures to examine its effect on the proliferation of HBEC in serum-free primary cul-
Ins 10 pg/ml Ins 0.1 pg/ml
FIG. 3. Summary of the effect of EGF on the growth of HBEC from normal, benign, and malignant breast tissue (Redn, reduction mammoplasties, n = 3; FA, fibroadenomas, n = 13; Ca, carcinomas, n = 9). EGF stimulated the growth of cells from all breast tissues. The effect was maximal at 5-10 rig/ml (data not shown). Results are the mean + SEM.
ture. In all 23 samples examined (3 Redn, 11 FA, 9 Ca), EGF at 10 rig/ml stimulated proliferation significantly. The stimulation ranged from 167 to 3455% of control values. The means of the responses of cells from the different types of tissue is shown in Fig. 3. MAb 528, a monoclonal antibody against the EGFR added to the medium at 1.5 pg/ml, reduced EGF stimulation by 5060%, which is comparable to results for cell lines . The effect of TGF-a (10 rig/ml) on cell growth was also examined in 6 FA samples and 2 Ca samples. In all cases, TGF-a was stimulatory. The degree of stimulation was equal to or greater than that of EGF in all experiments. Figure 4 shows two representative samples demonstrating this result. It is interesting to note that TGF-(Y was active over a shorter dose range than EGF. TGF-(Y at 100 rig/ml caused only 30% of the growth stimulation seen at 10 rig/ml. In contrast, EGF at 100 rig/ml elicited growth responses only slightly less (80-90%) than the peak response seen at 10 rig/ml (data not shown). Effects of EGF Plus E, on HBEC Proliferation
FIG. 2. Effect of insulin concentration on the growth of HBEC from fibroadenomas (n = 7). Insulin at 0.1 pg/ml reduced [3H]TdR incorporation into DNA by 60% on average (range 33-97% reduction), compared to insulin at 10 aglml. E, failed to stimulate any of the cultures examined regardless of insulin concentration. Results are the mean + SEM.
Hormonal stimulation of growth by E, may be mediated by the secretion of autocrine and paracrine growth factors or altered expression of their receptors . If this occurs in our cultures, E, and EGF together might produce a greater than additive effect on cell growth. We examined the growth effects of E, ( 10e8 M) and EGF (10 rig/ml) alone and in combination in 6 FA samples and 2 Ca samples. In 216 FA samples and 2/2 Ca samples, E, plus EGF was synergistic. In the 4/6 FAs
FA6S FA54 l-
FIG. 4. Comparison of the effects of EGF and TGF-(Y on the growth of HBEC from 2 representative cultures of cells from fibroadenomas (FA 85 and FA 54). TGF-a was either equal to or more effective than EGF in stimulating growth at 10 rig/ml; however, this difference was reversed at 100 rig/ml (data not shown). Results are expressed as in Fig. 1. Control condition refers to cells grown in the serum-free medium described in Fig. 1, without EGF or TGF-(Y.
in which no synergism was observed, the effect of E, and EGF together was the same as EGF alone. Representative samples are illustrated in Fig. 5.
Although we have shown that E, at physiological concentrations can stimulate the growth of HBEC in primary cultures in the presence of serum , we are unable to demonstrate a growth effect on cells from normal, benign, and malignant breast tissues in serum-free medium. This is consistent with a previously published report showing no effect of E, on the growth of normal HBEC in primary culture [ 171. The absence of an effect in serum-free medium suggests that factors present in the medium either block or mask an estrogenic stimulation of growth or, alternatively, that factors absent from the medium are required for an E, effect. In the carcinoma samples, all of which were ER+, there was no correlation between the ER levels and the growth response to estrogen. Unfortunately, ER levels were not determined for the normal and fibroadenoma samples; however, both of these tissue types have been shown to contain significant numbers of ER+ cells in a majority of specimens [ 28,291. Furthermore, it has been shown that cells from ER+ normal samples and fibroadenomas cultured under conditions similar to ours remained ER+ throughout the culture period [30, 311. Therefore, it is unlikely that the absence of ER+ cells in
our samples is a factor related to the absence of estrogenie growth stimulation. We examined the possibility that the pharmacological concentration of insulin was masking an estrogenic effect on cell growth, as has been demonstrated for MCF-7 cells [25, 261. A loo-fold reduction of the initial insulin concentration leads to a significant reduction in cell growth but still does not result in a stimulatory effect of E,. Using this serum-free medium, but simultaneously deleting hydrocortisone with the addition of E,, Yang and co-workers fail to show growth stimulation by E, . These experiments suggest that hydrocortisone is not blocking or masking a growth effect of E,. The finding that addition of EGF to the medium is consistently able to stimulate the proliferation of HBEC from normal, benign, and malignant tissues demonstrates that the cells are able to respond to growth stimulatory factors added to the basic serum-free medium used in this culture system. Therefore, the absence of stimulation by E, is not likely due to the fact that the cells have already reached a maximal growth rate. Also, there does not appear to be factors in the medium that render the cells incapable of growing at increased rates, at least in response to EGF. Taken together, these findings support the absence of a factor required for estrogenic stimulation of proliferation, rather than the presence of a blocking or masking agent.
FIG. 5. Comparison of the effects of E, (10-e M) and EGF (10 rig/ml), added separately and together, on the growth of cells from a fibroadenoma (FA 54) and a carcinoma (Ca 111). The control condition consists of the serum-free medium described in Fig. 1, without E, or EGF. In the eight experiments in which E, and EGF were examined together, E, alone had no effect. In all experiments, the effect of EGF alone was stimulatory. In 4 out of 8 experiments, E, and EGF added together had a synergistic effect, similar to the two examples shown above. In the 4 experiments where E, and EGF together did not act synergistically, the 2 together had the same effect as EGF alone (data not shown). Results are expressed as the mean + SEM.
EGF has been shown to stimulate BEC growth, both in uiuo [32, 331 and in vitro [19, 201. In mice it appears that EGF plays a role in both the initiation and maintenance of the breast cancer process . Our results indicate EGF is indeed a potent and direct mitogen for normal, benign, and malignant HBEC in serum-free primary culture. Using the same medium, Yang et al.  failed to see any effect of EGF, unless the cells are cultured in three-dimensional collagen gels or hydrocortisone is deleted from the medium. The reason for this discrepancy is not clear. Similar to our results, Ethier et al.  have also shown that EGF greatly stimulates the growth of primary cultures of BEC from normal and cancerous breast tissues maintained on collagen-coated dishes, although they used an enriched serum-free medium. The variation in response to EGF among individual samples (167-3455s) is of considerable interest due to the large degree of variability of receptor levels observed in biopsy samples . We are currently investigating the relationship between EGFR levels in the original tissue and cultured cells and the in vitro growth response to EGFITGF-a. TGF-a also stimulates the growth of HBEC in primary culture. However, samples vary as to whether TGF-(U is equal to or more potent than EGF in stimulating HBEC growth. At present there is no explanation for the differences in the growth promoting activity between EGF and TGF-(u, considering they act via a common receptor pathway. One possibility is that interaction between exogenously added growth factors and endogenously produced factors may occur, with different interactions between EGF and TGF-a. Both TGF-cx and EGF are likely candidates for such endogenously produced growth factors [l]. Synergism between EGF and insulin-like growth factor-l (IGF-1) has been shown previously in cultures of bovine BEC [ 371. Another possibility is that novel forms of the EGFR, such as EGFR/ erbB-2 heterodimers, may interact differently with the different growth factors . Although our data suggest that in general cells from breast cancers are not different from cells obtained from normal tissue or fibroadenomas in their growth response, to EGF, it is possible that such differences may occur in normal and pathological samples from the same individual. By comparing growth responses of normal cells obtained distal to the tumor site and cells from the tumor from individual mastectomy samples, it would be possible to address this question directly. Alternatively, gross differences in response to individual factors may play a small role in malignancy as compared to multiple defects in growth response to a number of factors. Future experiments investigating the effects of multiple growth factors and hormones alone and in combination will address this issue. The synergistic effect on growth of E, and EGF on
cells from several samples is very exciting. Although E, alone is unable to elicit a mitogenic effect, it is capable of enhancing the growth-stimulating effect of EGF in some samples. This observation gives support to the idea that E, may act as a modulator of cellular responses to growth factors, rather than acting directly as a mitogen. E, has been shown to regulate production of IGF-1 in MCF-7 cells [ 391, and E, and insulin  or EGF and IGF-1  can interact in a synergistic fashion. However, our data suggest that there is likely no interaction between E, and I under the experimental conditions used in this study on primary cultures. Alternatively, an E,-induced increase in EGFR levels could explain the synergism between E, and EGF observed in our studies. Such increases in EGFR have been demonstrated in ER+ cell lines [6, lo]. In uterine tissues it has been shown that estrogen is capable of modulating functional EGF and EGFR levels in uiuo (32,401. We are currently investigating the effects of E, on EGFR levels in HBEC in primary culture. The authors thank Darcy Wilkinson for her excellent technical assistance. This work was funded by a grant from the National Cancer Institute of Canada. B.M.G. is a recipient ofthe Evelyn Martin Memorial Fellowship.
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