Growth inhibition and growth stimulation by estradiol of estrogen receptor transfected human breast epithelial cell lines involve different pathways.

Epidermal growth factor (EGF) and estradiol (E2) are important mitogens in breast epithelial cells, and expression of epidermal growth factor receptor (EGFR) and estrogen receptor (ER) is often inversely correlated in human breast cancer cells. Stable transfection of ER-negative cells with ER cDNA is not sufficient to restore E2-mediated growth stimulation, on the contrary, E2 often inhibits growth of ER-transfected cell lines. In this study we used the ER-transfected human breast epithelial cell lines HMT-3522F9, growth inhibited by E2 in the presence of EGF, and HMT-3522F9/S3B, growth stimulated by E2 in the absence of EGF. In S3B cells, no active MAP kinase could be detected in response to E2, suggesting that signalling through the MAP kinase is not the major pathway in the E2-mediated growth stimulation. Interestingly, a decreased level of active MAP kinase was observed in HMT-3522F9 cells in response to E2, indicating that in these cells cross-talk between the ER and the MAP kinase signalling pathway could be due to the E2-mediated growth inhibition. Moreover, we found that EGF-induced signalling also could be reduced by E2 in S3B cells, suggesting a general mechanism of action by E2 in cells concomitantly expressing ER and EGFR.

Identification of two estrogen regulated genes associated with growth regulation of human breast cancer.

We have identified two estrogen regulated gene products in the E(2) growth inhibited human breast cancer xenograft, T61; one showing 100% homology to the human BAC clone RP11-112E16, the other 100% homology to the human CPR3/DNJ3 gene. Verification by Northern blot analyses showed an up-regulation of the BAC clone RP11-112E16 and the CPR3/DNJ3 mRNAs upon E(2) treatment. Treatment of T61 tumors with tamoxifen, leading to static tumor growth, also increased the expression of the BAC clone RP11-112E16 and the CPR3/DNJ3 mRNAs. A similar association between growth inhibition and BAC clone RP11-112E16 and CPR3/DNJ3 mRNA induction was observed in MCF-7 cells treated with ICI 182.780. In MCF-7 cells, treatment with E(2) resulted in growth stimulation concomitant with a decrease in the BAC clone RP11-112E16 and CPR3/DNJ3 mRNA expression. Treatment with a combination of E(2) and ICI 182.780 abolished the anti-estrogen induced increase in BAC clone RP11-112E16 and CPR3/DNJ3 mRNA expression, indicating that regulation of the gene products is mediated through the ER. The association between growth inhibition and BAC clone RP11-112E16 or CPR3/DNJ3 mRNA expression was supported by high expression of both gene products in brain tissue. Further investigations are ongoing to clarify the biological function of these two gene products.

Growth response of breast epithelial cells to estrogen is influenced by EGF.

Estrogen-induced growth stimulation has not previously been demonstrated in estrogen receptor (ER) cDNA transfected human cell lines in contrast to breast cancer cell lines expressing endogenous ER. On the contrary, estrogen usually inhibits cell growth of ER transfected cell lines. Growth inhibition by estrogen has also been demonstrated in our cell line, F9, which is an ER transfected subline of HMT-3522 breast epithelial cells derived from fibrocystic disease and propagated in chemically defined medium. By omitting EGF in the medium, we have demonstrated not only an increased transcriptional activity of the ER but also--after an adaptation period--estrogen-dependent growth of the cells, and we have succeeded in establishing a new subline, S3B, that requires 17beta-estradiol (E2) for growth. This is the first example of a nonmalignant, human breast epithelial cell line which is dependent on estrogen for continued growth. The S3B cells express functional ER as measured by transcriptional activity. ER-E2 induced transcription was not inhibited by EGF as in F9 cells. We propose that a growth-stimulatory response of breast epithelial cells in vitro to E2 is dependent on an inactive or down-regulated EGF receptor signaling pathway and it is possible that the effect of estrogen on normal breast epithelium in vivo also is modulated by the EGFR.

Differential regulation of specific genes in MCF-7 and the ICI 182780-resistant cell line MCF-7/182R-6.

To elucidate the mechanisms involved in anti-oestrogen resistance, two human breast cancer cell lines MCF-7 and the ICI 182780-resistant cell line, MCF-7/182R-6, have been compared with regard to oestrogen receptor (ER) expression, ER function, ER regulation, growth requirements and differentially expressed gene products. MCF-7/182R-6 cells express a reduced level of ER protein. The ER protein is functional with respect to binding of oestradiol and the anti-oestrogens tamoxifen, 4-hydroxy-tamoxifen and ICI 182780, whereas expression and oestrogen induction of the progesterone receptor is lost in MCF-7/182R-6 cells. The ER protein and the ER mRNA are regulated similarly in the two cell lines when subjected to treatment with oestradiol or ICI 182780. Oestradiol down-regulates ER mRNA and ER protein expression. ICI 182780 has no initial effect on ER mRNA expression whereas the ER protein level decreases rapidly in cells treated with ICI 182780, indicating a severely decreased stability of the ER protein when bound to ICI 182780. In vitro growth experiments revealed that the ICI 182780-resistant cell line had evolved to an oestradiol-independent phenotype, able to grow with close to maximal growth rate both in the absence of oestradiol and in the presence of ICI 182780. Comparison of gene expression between the two cell lines revealed relatively few differences, indicating that a limited number of changes is involved in the development of anti-oestrogen resistance. Identification of the differentially expressed gene products are currently in progress.

Characterization of a nontumorigenic human breast epithelial cell line stably transfected with the human estrogen receptor (ER) cDNA.

Estrogens play an important role in breast cancer and the effect of estrogen on growth of breast cancer cells has been extensively studied. However, only little information is available about the response of normal breast epithelial cells to estrogen, mainly due to the difficulties in establishing estrogen receptor (ER)-positive human breast epithelial cells in culture. We have stably transfected the human estrogen receptor (hER) wt cDNA into the ER-negative, spontaneously immortalized human breast epithelial cell line, HMT-3522S1, in order to develop a model for studying the effect of estrogen on nonmalignant human breast epithelial cells. Characterization of the transfected clone F9 confirmed incorporation of the estrogen receptor gene in the genome, expression of hER mRNA and hER protein. However, proliferation of F9 cells was inhibited by both estradiol (E2) and tamoxifen, whereas the pure antiestrogen ICI 182,780 had no effect on cell proliferation. This seems paradoxical since E2 stimulated the expression of the endogenous genes, TGF-alpha, cathepsin D, and alpha1-antitrypsin. In breast cancer cell lines, high expression of these genes is correlated to estrogen-stimulated cell proliferation. The spontaneously immortalized HMT-3522S1 cells transfected with wt ER cDNA behave similarly to cell lines from nonmalignant breast tissue immortalized by carcinogens and transfected with mutated ER cDNA as described by others. The discrepancy between growth inhibition and induction of positive growth factors by E2 indicates that either ER-positive nonmalignant breast epithelial cells are growth-inhibited by E2 in contrast to malignant cells or that introduction of the ER into ER-negative cells is not sufficient for restoring "normal' estrogen responsiveness.