Anti-epidermal growth factor receptor antibodies inhibit the autocrine-stimulated growth of MDA-468 human breast cancer cells

The response of malignant and nonmalignant human breast cell lines to the growth inhibitory effects of monoclonal antibodies against the epidermal growth factor (EGF) receptor was studied. A series of human breast cell lines, which express EGF receptor, were used: MDA-468, MDA-231, and Hs578T human breast cancer cells and the transformed human mammary epithelial cell lines 184A1N4 and 184A1N4-T that have been benzo[a]pyrene immortalized and further transformed with SV40T, respectively. Four antibodies of two different classes were tested: 225 immunoglobulin G (IgG), 108.4 IgG, 96 immunoglobulin M (IgM), and 42 IgM. All four antibodies inhibited the anchorage-dependent and -independent, EGF-stimulated growth of 184A1N4 and 184A1N4-T cells, respectively, and this growth inhibition could be reversed by the addition of increasing concentrations of EGF. In contrast, the antibodies inhibited the anchorage-dependent and -independent growth of MDA-468 cells in the absence of exogenous EGF suggesting that the antibodies were acting to block access of an endogenously produced ligand to the EGF receptor. In the presence of antibody and increasing concentrations of EGF, MDA-468 cell growth was first stimulated then inhibited as the EGF concentration increased, thus, uncovering the growth stimulatory potential of low concentrations of EGF in these cells. Data is presented that indicates MDA-468 cells secrete a transforming growth factor with autocrine growth stimulatory capabilities. The growth of MDA-231 and Hs578T cells, which contain activated ras oncogenes, was not inhibited by the antibodies and the growth of these cell lines was not stimulated by EGF. Of the cell lines studied only MDA-468 cells appear to possess an autocrine growth stimulatory capacity.     

Modulation of transforming growth factor alpha-dependent expression of epidermal growth factor receptor gene by transforming growth factor beta, triiodothyronine, and retinoic acid

We have investigated the actions of transforming growth factor (TGF) type alpha on epidermal growth factor (EGF) receptor mRNA expression in MDA-468 human mammary carcinoma cells in serum-free media. We found that exposure of MDA-468 cells to TGF alpha results in elevated levels of EGF receptor mRNA. This increase in mRNA accumulation showed time and dose dependence. Addition of TGF beta 1 enhanced the accumulation of EGF receptor mRNA induced by TGF alpha in a time- and dose-dependent manner. We also found that triiodothyronine at physiological concentrations exerts synergistic control on the action of TGF alpha alone, or in association with TGF beta 1, on EGF receptor mRNA expression. Similarly, retinoic acid treatment also enhanced in a time- and dose-dependent manner the TGF alpha-dependent response of EGF receptor mRNA and acted synergistically with TGF beta 1. The results described here suggest that optimum regulation of EGF receptor gene expression by TGF alpha is a complex process involving synergistic interactions with heterologous growth factors and hormones.     

G-protein-mediated epidermal growth factor signal transduction in a human breast cancer cell line. Evidence for two intracellular pathways distinguishable by pertussis toxin

The MDA-468 human breast cancer cell line has an amplified epidermal growth factor (EGF) receptor gene (20 x) and correspondingly overexpresses the EGF receptor. Since this cell line is growth inhibited by supra-physiological levels of EGF in tissue culture, it has been possible to select variant cells which have lost the chromosome bearing the amplified EGF receptor domain and which are capable of growing in high levels of EGF. One such cell line (MDA-468-S4) shows an absolute requirement for EGF for growth in anchorage-independent tissue culture conditions. We have utilized MDA-468 and MDA-468-S4 to examine the intracellular transduction of EGF signals leading to growth inhibition and proliferation, respectively. We report that in anchorage-independent conditions, pertussis toxin can abrogate both the EGF-dependent growth inhibition in MDA-468 cells and the EGF-dependent cell proliferation in MDA-468-S4 cells. This inhibition is paralleled by the ADP-ribosylation of an endogenous 41,000-dalton membrane protein in both MDA-468 and MDA-468-S4 cells. In contrast, the toxin does not prevent the transient, augmented expression of c-myc and c-fos mRNA seen in response to EGF in both cell types. These data suggest 1) the notion of more than one simultaneous, parallel, intracellular EGF-dependent signal transduction pathway and 2) G-protein involvement in at least one pathway mandatory for the growth modulating responses to EGF in anchorage-independent conditions, but distinct from that inducing c-myc and c-fos mRNA expression.     

Toxicity and effects of epidermal growth factor on glucose metabolism of MDA-468 human breast cancer cells

Epidermal growth factor (EGF) has an in vitro inhibitory effect on tumor cells which exhibit a high number of EGF receptors (EGFR). Studies were performed in order to delineate the effects of EGF on glucose metabolism of MDA-468 human breast cancer cells, which have a large number of EGFR. Glucose consumption and lactate production were found to be substantially increased in MDA-468 cells following EGF exposure, while no such effects were detected in MCF-7 breast cancer cells, which have a very low number of EGFR. When glucose levels in the growth medium were increased, the toxicity of EGF was diminished. The energetic status of MDA-468 cells perfused with growth medium containing EGF was monitored by 31P magnetic resonance spectroscopy, and no signs of compromised metabolic state or viability were noted for up to 36 h. The rate of glucose transport and phosphorylation was quantitated by 13C magnetic resonance spectroscopy, utilizing [6-13C]2-deoxyglucose, and a 97% increase was found in MDA-468 cells following EGF administration. The profound effects of EGF on glucose metabolism in cells with very high numbers of EGFR and the lack of toxicity in the perfused system may indicate that the growth-inhibitory effect is confined to the in vitro cultured cells.     

MDA-468, a human breast cancer cell line with a high number of epidermal growth factor (EGF) receptors, has an amplified EGF receptor gene and is growth inhibited by EGF

Epidermal growth factor (EGF) has been noted to stimulate proliferation of a variety of normal and malignant cells including those of human breast epithelium. We report here that MDA-468, a human breast cancer cell line with a very high number of EGF receptors, is growth-inhibited at EGF concentrations that stimulate most other cells. The basis for the elevated receptor level is EGF receptor gene amplification and over-expression. An MDA-468 clone selected for resistance to EGF-induced growth inhibition shows a number of receptors within the normal range. The results are discussed in relation to a threshold model for EGF-induced growth inhibition.     

Epidermal growth factor receptor gene-amplified MDA-468 breast cancer cell line and its nonamplified variants.

We have recently reported (J. Filmus, M. N. Pollak, R. Cailleau, and R. N. Buick, Biochem. Biophys. Res. Commun. 128:898-905, 1985) that MDA-468, a human breast cancer cell line with a high number of epidermal growth factor (EGF) receptors, has an amplified EGF receptor gene and is growth inhibited in vitro pharmacological doses of EGF. We have derived several MDA-468 clonal variants which are resistant to EGF-induced growth inhibition. These clones had a number of EGF receptors, similar to normal human fibroblasts, and had lost the EGF receptor gene amplification. Karyotype analysis showed that MDA-468 cells had an abnormally banded region (ABR) in chromosome 7p which was not present in the variants. It was shown by in situ hybridization that the amplified EGF receptor sequences were located in that chromosome, 7pABR. Five of the six variants studied were able to generate tumors in nude mice, but their growth rate was significantly lower than that of tumors derived from the parental cell line. The variant that was unable to produce tumors was found to be uniquely dependent on EGF for growth in soft agar.     

Transforming growth factors type beta 1 and beta 2 are equipotent growth inhibitors of human breast cancer cell lines

At least one member of the TGF-beta family, TGF-beta 1, has been previously shown to inhibit the anchorage-independent growth of some human breast cancer cell lines (Knabbe et al., 1987; Arteaga et al., 1988). Members of the TGF-beta family might, therefore, provide new strategies for breast cancer therapy. We have studied the inhibitory effects of TGF-beta 1 and TGF-beta 2 on the anchorage-independent growth of the oestrogen receptor-negative cell lines MDA-MB-231, SK-BR-3, Hs578T, MDA-MB-468, and MDA-MB-468-S4 (an MDA-MB-468 clone not growth inhibited by EGF) and the estrogen receptor-positive cell lines MCF7, ZR-75-1, T-47D. TGF-beta 1 and TGF-beta 2 caused a 75-90% growth inhibition of MDA-MB-231, SK-BR-3, Hs578T, and MDA-MB-468 cells and a 50% growth inhibition of ZR-75-1 and early passage (less than 100) MCF7 cells. T-47D cells responded to TGF-beta only in serum-free conditions in the presence of IGF-1 or EGF. The growth of MDA-MB-468-S4 cells and late passage (greater than 500) MCF7 cells was not inhibited by TGF-beta 1 or TGF-beta 2. TGF-beta-sensitive MCF7 and MDA-MB-231 cells did not respond to Muellerian inhibiting substance (MIS), a TGF-beta-related polypeptide. TGF-beta 1 or TGF-beta 2 were mutually competitive for receptor binding with a similar affinity (Kd 25-130 pM, 1,000-13,000 sites per cell). To determine the time course of the TGF-beta effect, an anchorage-dependent growth assay was carried out using MDA-MB-231 cells. Growth inhibition occurred at 6 days, and cell-cycle changes were seen 12 hr after the addition of TGF-beta. Cells accumulated in the G1 phase and were thus inhibited from entering the S-phase. These data indicate that TGF-beta is a potent growth inhibitor in most breast cancer cell lines and provide a basis for studying TGF-beta effects in vivo.     

Transforming growth factor alpha production and epidermal growth factor receptor expression in normal and oncogene transformed human mammary epithelial cells

We have characterized the expression of transforming growth factor alpha (TGF alpha) and its receptor, the epidermal growth factor receptor (EGF-R), in normal and malignantly transformed human mammary epithelial cells. Human mammary epithelial cells were derived from a reduction mammoplasty (184), immortalized by benzo-a-pyrene (184A 1N4), and further transformed by the oncogenes simian virus 40 T (SV40 T), v-Ha-ras, and v-mos alone or in combination using retroviral vectors. 184 and 184A 1N4 cells require EGF for anchorage-dependent clonal growth. In mass culture, they secrete TGF alpha at high concentrations and exhibit an attenuated requirement for exogenous EGF/TGF alpha. SV40 T transformed cells have 4-fold increased EGF-R, have acquired the ability to clone in soft agar with EGF/TGF alpha supplementation, but are not tumorigenic. Cells transformed by v-mos or v-Ha-ras are weakly tumorigenic and capable of both anchorage dependent and independent growth in the absence of EGF/TGF alpha. Cells transformed by both SV40 T and v-Ha-ras are highly tumorigenic, are refractory to EGF/TGF alpha, and clone with high efficiency in soft agar. The expression of v-Ha-ras is associated with a loss of the high (but not low) affinity binding component of the EGF-R. Malignant transformation and loss of TGF alpha/EGF responsiveness did not correlate with an increase in TGF alpha production. Thus, TGF alpha production does not appear to be a tumor specific marker for human mammary epithelial cells. Differential growth responses to EGF/TGF alpha, rather than enhanced production of TGF alpha, may determine the transition from normal to malignant human breast epithelium.     

Identification of a novel AU-Rich element in the 3' untranslated region of epidermal growth factor receptor mRNA that is the target for regulated RNA-binding proteins

The epidermal growth factor receptor (EGF-R) plays an important role in the growth and progression of estrogen receptor-negative human breast cancers. EGF binds with high affinity to the EGF-R and activates a variety of second messenger pathways that affect cellular proliferation. However, the underlying mechanisms involved in the regulation of EGF-R expression in breast cancer cells are yet to be described. Here we show that the EGF-induced upregulation of EGF-R mRNA in two human breast cancer cell lines that overexpress EGF-R (MDA-MB-468 and BT-20) is accompanied by stabilization (>2-fold) of EGF-R mRNA. Transient transfections using a luciferase reporter identified a novel EGF-regulated approximately 260-nucleotide (nt) cis-acting element in the 3' untranslated region (3'-UTR) of EGF-R mRNA. This cis element contains two distinct AU-rich sequences (~75 nt), EGF-R1A with two AUUUA pentamers and EGF-R2A with two AUUUUUA extended pentamers. Each independently regulated the mRNA stability of the heterologous reporter. Analysis of mutants of the EGF-R2A AU-rich sequence demonstrated a role for the 3' extended pentamer in regulating basal turnover. RNA gel shift analysis identified cytoplasmic proteins (~55 to 80 kDa) from breast cancer cells that bound specifically to the EGF-R1A and EGF-R2A cis-acting elements and whose binding activity was rapidly downregulated by EGF and phorbol esters. RNA gel shift analysis of EGF-R2A mutants identified a role for the 3' extended AU pentamer, but not the 5' extended pentamer, in binding proteins. These EGF-R mRNA-binding proteins were present in multiple human breast and prostate cancer cell lines. In summary, these data demonstrate a central role for mRNA stabilization in the control of EGF-R gene expression in breast cancer cells. EGF-R mRNA contains a novel complex AU-rich 260-nt cis-acting destabilizing element in the 3'-UTR that is bound by specific and EGF-regulated trans-acting factors. Furthermore, the 3' extended AU pentamer of EGF-R2A plays a central role in regulating EGF-R mRNA stability and the binding of specific RNA-binding proteins. These findings suggest that regulated RNA-protein interactions involving this novel cis-acting element will be a major determinant of EGF-R mRNA stability.     

A transforming growth factor-beta like growth factor in mouse embryonal carcinoma cells

Our previous study indicated that polypeptides isolated from acid/ethanol extracts of solid tumors of a cloned F9-3 embryonal carcinoma cells by Bio-Gel P60 column chromatography were found to be able to stimulate anchorage independent growth of either NIH 3T3 cells or NRK 49 F cells in soft agar. The major peak of active elute had a molecular weight of about 15 kDa as determined by SDS-polyacrylamide gel electrophoresis. In the present report we further isolated and purified the active compound corresponding to molecular weight of 15 kDa by gel filteration on Bio-Gel P10 column (Fig. 1) and then by high pressure liquid chromatography (Fig. 2). It was found that the purified 15 kDa molecules showed some properties similar to transforming growth factor-beta (TGF-beta): 1. Colony-stimulating activity in soft agar can be induced in NRK 49 F cells only in the presence of mouse epidermal growth factor (EGF) (Plate I); 2. Increase in relative uptake of 3H-thymidine in NRK 49 F cells occurred in the presence of EGF, but with the same amount of EGF, not much change in 3H-thymidine incorporation could be found with further increasing amounts of purified 15 kDa molecules (Fig. 3); 3. Like human blood platelets derived TGF-beta, inhibition effect on the growth of mink lung epithelial cells (CCL/64) can also be exhibited by purified 15 kDa molecules (Fig. 4). In addition, using ELISA procedure, we have also demonstrated that the 15 kDa molecules had immunological reactivity with the antibody raised against a synthetic oligopeptide identical to the N-terminal residues 1-29 of TGF-beta 1 from human blood platelets (Fig.5). Thus, the 15 kDa molecules isolated from mouse F9-3 embryonal carcinoma cells appeared to share some common antigenic determinants with human TGF-beta 1 molecule. These results taken together provide strong support for the existence of TGF-beta like growth factor in mouse embryonal carcinoma cells.     

Epidermal growth factor induces tyrosine phosphorylation of epidermal growth factor receptors not occupied with ligand in intact A431 cells.

The mechanism of epidermal growth factor receptor (EGF-R) autophosphorylation in intact A431 cells was studied. We detected epidermal growth factor (EGF) induced tyrosine phosphorylation of EGF-R not occupied with ligand. Cell monolayers were subjected to irradiation after incubation with photoreactive derivative of EGF and uncoupled EGF was extracted by acidic treatment. Subsequent immunoprecipitation with antiphosphotyrosine antibodies resulted in precipitation of both EGF-R complexes with EGF and EGF-R with unoccupied ligand-binding site. The fact of precipitation of EGF-R with unoccupied ligand-binding site in conjunction with our finding of rapid dephosphorylation of EGF-R after EGF extraction by acidic treatment, strongly supports the interpretation that cross-phosphorylation of EGF-R may take place in intact cells.     

Transforming growth factor beta modulates phosphorylation of the epidermal growth factor receptor and proliferation of A431 cells.

Transforming growth factor beta (TGF-beta) increased the phosphorylation of the epidermal growth factor (EGF) receptor and inhibited the growth of A431 cells. Incubation with TGF-beta induced maximal EGF receptor phosphorylation to levels 1.5-fold higher than controls. Phosphorylation increased more prominently (4-5-fold) on tyrosine residues as determined by phosphoamino acid analysis and antiphosphotyrosine antibody immunoblotting. The kinase activity of EGF receptor was also elevated 2.5-fold when cells were cultured in the presence of TGF-beta. The antiproliferative effect of TGF-beta on A431 cells was accompanied by prolongation of G0-G1 phase and by morphological changes. TGF-beta augmented the growth inhibition of A431 cells which could be induced by EGF. In parallel, the specific EGF-induced increase in total phosphorylation of the EGF receptor was also augmented in the presence of TGF-beta. In cells cultured with TGF-beta, the phosphorylation of EGF receptor tyrosines induced by 20-min exposure to EGF was further increased 2-3-fold, suggesting additive effects upon receptor phosphorylation. EGF receptor activation by TGF-beta is characterized by kinetics quite distinct from that induced by EGF and therefore appears to take place through an independent mechanism. The TGF-beta-induced elevation in the phosphorylation of the EGF receptor may have a role in the augmented growth inhibition of A431 cells observed in the presence of EGF and TGF-beta.     

Induction of apoptosis in porcine thyroid follicles by transforming growth factor beta1 and epidermal growth factor.

For thyroid cells in culture DNA fragmentation and morphological changes related to apoptosis were first described in dog thyroid cells after deprivation of serum, epidermal growth factor or thyrotropin. With intact porcine thyroid follicles in three-dimensional culture, the effect of deprivation of growth factors and of incubation with transforming growth factor beta1 (TGF-beta1), epidermal growth factor (EGF), thyrotropin (TSH) or insulin-like growth factor I (IGF-I) on the incidence of apoptosis was studied. Thyroid follicles were embedded in growth factor-depleted Matrigel and cultured in serum-free medium with or without growth factors for 7 days followed by incubation for 4, 24 and 72 h with TGF-beta1 (2 or 5 ng/mL). The percentage of apoptotic cells was determined by direct counting in electron-microscopy. Approximately 1% of apoptotic bodies could be detected in unstimulated follicles. This was unchanged in the presence of TSH (1 mU/mL) or IGF (10 ng/mL) but significantly increased up to 3.99 +/- 1.24% with 2 ng/mL of EGF. After incubation with TGF-beta apoptosis increased dose-dependently to 4.05 +/- 0.67% with 2 ng/mL TGF-beta1 and 5.16 +/- 1.75% with 5 ng/mL TGF-beta1. The incidence of necrotic cells remained constant at about 1 to 2%. Preincubation of follicles with 2 ng/mL of EGF followed by incubation with 5 ng/mL TGF-beta1 increased the rate of apoptic bodies up to 13.19 +/- 1.9%. We conclude that growth factor depletion in thyroid follicles in three-dimensional culture does not lead to apoptosis. TGF-beta1, however, induces apoptosis even in quiescent thyroid follicular cells and is significantly more pronounced in growing thyroid cells. EGF, which is a dedifferentiating growth factor for thyroid cells, also induces apoptosis. As EGF enhances TGF-beta1 mRNA and protein in thyroid follicular cells, the induction of apoptosis by EGF might also be due to TGF-beta1.     

Transforming growth factor beta regulates the inhibitory actions of epidermal growth factor during granulosa cell differentiation

The effects of transforming growth factor beta (TGF-beta) on epidermal growth factor (EGF) receptor content and EGF action were studied in cultured granulosa cells from immature diethylstilbestrol-implanted rats. During follicle-stimulating hormone (FSH)-induced differentiation in vitro, EGF receptors increased by 20-fold as measured by the binding of 125I-EGF to the intact cells. Addition of TGF-beta during the 48-h culture period amplified the stimulatory effects of FSH on EGF receptors up to 2-fold, with ED50 and maximal concentrations of 2.5 and 8 pM, respectively. Also TGF-beta alone in amounts from 1.6 to 16 pM increased EGF receptor content 4-fold. The stimulatory effects of TGF-beta were due to increased numbers of EGF receptors/cell, since the growth factor had no effect on the Kd (3-5 X 10(-11) M) of the high-affinity EGF binding site. TGF-beta action was observed within 20 h of granulosa cell culture and was maximal by 48 h of a 96-h culture. The stimulatory actions of TGF-beta in gonadotropin-induced cells were exerted through the cAMP effector system of the granulosa cell, since the growth factor also amplified the induction of EGF receptors by cholera toxin, forskolin, and 8-bromo-cAMP. The augmentation of EGF receptors by TGF-beta resulted in a parallel 2-fold increase in the inhibitory effects of EGF on FSH-induced cAMP production and luteinizing hormone receptor expression during granulosa cell development. TGF-beta did not increase granulosa cell numbers during culture although it elevated [3H]thymidine incorporation into DNA by 2-fold over that of FSH-treated cells. These results indicate that TGF-beta regulates the effects of both FSH and EGF during granulosa cell differentiation and provides evidence that ovarian function may be controlled by the combined actions of gonadotropins and multiple growth factors.     

Mouse Balb/c3T3 cell mutant with low epidermal growth factor receptor activity: induction of stable anchorage-independent growth by transforming growth factor beta

A mutant clone (MO-5) was originally isolated as a clone resistant to Na+/K+ ionophoric antibiotic monensin from mouse Balb/c3T3 cells. MO-5 was found to show low receptor-endocytosis activity for epidermal growth factor (EGF): binding activity for EGF in MO-5 was less than one tenth of that in Balb/c3T3. Anchorage-independent growth of MO-5 was compared to that of Balb/c3T3 when assayed by colony formation capacity in soft agar. Coadministration of EGF and TGF-beta efficiently enhanced anchorage-independent growth of normal rat kidney (NRK) cells, but neither factor alone was competent to promote the anchorage-independent growth. The frequency of colonies appearing in soft agar of MO-5 or Balb/c3T3 was significantly enhanced by TGF-beta while EGF did not further enhance that of MO-5 or Balb/c3T3. Colonies of Balb/c3T3 formed in soft agar in the presence of TGF-beta showed low colony formation capacity in soft agar in the absence of TGF-beta. Colonies of MO-5 formed by TGF-beta in soft agar, however, showed high colony formation capacity in soft agar in the absence of TGF-beta. Pretreatment of MO-5 with TGF-beta induced secretion of TGF-beta-like activity from the cells, while the treatment of Balb/c3T3 did not induce the secretion of a significant amount of TGF-beta-like activity. The loss of EGF-receptor activity in the stable expression and maintenance of the "transformed" phenotype in MO-5 is discussed.