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To study the mechanisms behind treatment resistance, we have previously established a large panel of antiestrogen resistant breast cancer cell lines based on the estrogen receptor (ER)-positive breast cancer cell line, MCF-7. It seems to be a general phenomenon that MCF-7 cells switch from ER driven to human epidermal growth factor receptor (HER) driven cell growth upon development of fulvestrant resistance. To obtain a broader knowledge of the resistance mechanisms, we have established four fulvestrant resistant subclones from the ER-positive breast cancer cell line, T47D. The parental T47D cells were confirmed to be estrogen responsive and short-term treatment of the parental T47D cells with fulvestrant severely reduced cell growth as well as protein expression of ER and the estrogen regulated proteins, insulin-like growth factor 1 receptor alpha (IGF-1 Rα) and progesterone receptor (PR). All four fulvestrant resistant cell lines had reduced mRNA and protein expression of HER1 and HER4 compared to T47D, while HER2 was highly up regulated in the fulvestrant resistant cell lines and ER was lost. No difference in response to the HER2 inhibitor trastuzumab or the specific HER2 kinase inhibitor AG825 was observed between the fulvestrant resistant cell lines and T47D, indicating that HER2 is not the main driver of fulvestrant resistance in T47D cells. We found no indication of involvement of the two classical signaling pathways downstream of the HER receptors (Ras/Raf/Erk and PI3K/Akt). However, the JNK inhibitor SP600125 preferentially inhibited growth of two tested fulvestrant resistant cell lines. Withdrawal of fulvestrant for several weeks did not result in regain of ER expression, or in change of HER receptor expression, supporting a stable resistant phenotype. In conclusion, loss of ER expression explains the lack of effect of fulvestrant in the T47D fulvestrant resistant cell lines. Noteworthy, the observed over expression of HER2 in the fulvestrant resistant T47D cells does not seem to be the main driver of cell growth, as HER2 targeted therapies like trastuzumab and HER2 kinase inhibitor did not exert a preferential growth inhibition of resistant cells. These breast cancer cells with high endogenous HER2 expression will be used as a model mimicking HER2 positive breast cancer which does not respond to standard HER2 targeted therapies. [ABSTRACT FROM AUTHOR]

The estrogen receptor variant lacking exon 5 (ERdeltaE5) encodes a truncated protein that lacks the hormone-binding domain and has been suggested to be responsible for the estrogen-independent growth of human breast tumors and resistance to antiestrogen therapy. The biological function of the ERdeltaE5 in human breast epithelial cells has been studied by transient transfection of HMT-3522S1 cells with wild-type (wt) estrogen receptor (ER) and ERdeltaE5. A 10-fold higher expression of ERdeltaE5 mRNA compared to that of wt ER mRNA was found. However, the expression of ERdeltaE5 protein was significantly lower than the expression of wt ER protein. The ERdeltaE5 was unable to activate the transcription of an estrogen-responsive reporter gene in the absence as well as in the presence of estrogen. The ERdeltaE5 disclosed a dominant negative activity when expressed together with wt ER. These data indicate that the biological significance of ERdeltaE5 in human breast cancer is dubious.

Development of resistance to tamoxifen is a serious problem in treatment of breast cancer patients. Although the mechanisms for development of resistance are unclear, an altered expression of alternatively spliced estrogen receptor (ER) mRNA has been suggested to be involved. We have looked for differential expression of ER splice variants lacking exon 2 (ERdeltaE2), exon 3 (ERdeltaE3), exon 4 (ERdeltaE4), exon 5 (ERdeltaE5), exon 7 (ERdeltaE7), and exons 4 and 7 (ERdeltaE4, 7) in the human breast cancer cell line MCF-7 and 10 ER-positive MCF-7 sublines resistant to the antiestrogens tamoxifen, ICI 164,384 or ICI 182,780. No major differences in the expression were demonstrated between MCF-7 cells and resistant cells, indicating that ER splice variants are not involved in antiestrogen resistance in this model system. Furthermore, despite a high mRNA level of some of the ER splice variants, no corresponding proteins could be detected using Western blot analysis.

A stable, tamoxifen-resistant subline, MCF-7/TAMR-1, of the human breast cancer cell line MCF-7 has been established in tissue culture after long-term treatment with 10(-6) M tamoxifen. The MCF-7/TAMR-1 cell line grows equally well in the presence and absence of tamoxifen, whereas the steroidal antiestrogens ICI 164,384 and ICI 182,780 exert profound inhibitory activity on cell proliferation, although higher concentrations are required to inhibit these cells compared to the parent cells. The MCF-7/TAMR-1 cells grown in tissue culture deviate from parent characteristics by the complete lack of expression of progesterone receptors even when grown with estradiol, by an altered tamoxifen regulation of M(r) 52,000 cathepsin D synthesis and secretion, and by lack of tamoxifen stimulation of an estradiol down-regulated M(r) 42,000 protein with presumed growth inhibitory function. MCF-7/TAMR-1 cells are estrogen receptor positive. The estrogen receptors have wild-type characteristics with respect to (a) binding of estradiol, tamoxifen, and ICI 164,384; (b) estrogen and antiestrogen regulation of the estradiol-regulated proteins pS2, M(r) 61,000 alpha 1-antitrypsin-like protein, M(r) 66,000 alpha 1-antichymotrypsin-like protein, and corresponding mRNAs; and (c) estrogen and antiestrogen regulation of a transiently transfected estrogen responsive reporter gene. We suggest that the lack of tamoxifen up-regulation of the M(r) 42,000 protein synthesis in MCF-7/TAMR-1 cells may at least partly explain the resistance to tamoxifen treatment. The sensitivity to the growth inhibitory activity of ICI 164,384 and ICI 182,780 may be ascribed to the maintenance of the pure antagonistic effect of these steroidal antiestrogens on MCF-7/TAMR-1 cells. Our results indicate that treatment with pure antiestrogens may be effective when patients become refractory to tamoxifen therapy.

Activation of protooncogenes and constitutive secretion of autocrine growth factors are thought to be involved in the uncontrolled growth of cancer cells. We have attempted to elucidate the role of oncogenes and growth factors in the premalignant progression of human breast epithelial cells by using an immortalized, nontumorigenic, near-diploid human mammary epithelial cell line, HMT-3522, derived from a fibrocystic lesion and established in our laboratory. During propagation in tissue culture, the growth factor requirements of the HMT-3522 cells decreased simultaneously with an amplification and overexpression of the c-myc protooncogene. Other protooncogenes related to human breast cancer were unaltered with regard to gene copy number and expression. In passage 118, in which the most important growth factor still was epidermal growth factor (EGF), we were able to isolate an EGF-independent subline (S2). The EGF independence of S2 was accompanied by an overexpression of the mRNAs for epidermal growth factor receptor (EGF-R), transforming growth factor-alpha, and c-erb-B2 as compared to the EGF-dependent subline (S1). Moreover, by application of a blocking anti-EGF-R antibody, growth of S2 cells in EGF-free medium was inhibited significantly, indicating that EGF-R was involved in an autocrine loop probably with transforming growth factor-alpha as ligand. Neither the late passages of S1 cells nor S2 cells were tumorigenic after subcutaneous transplantation to athymic mice. Our results indicate that c-myc amplification and overexpression are correlated with a decreased requirement for growth factors. Even when these alterations are combined with immortalization and EGF independence, they are insufficient for malignant transformation of these human breast epithelial cells.

A rapid, convenient method for the quantitative determination of estrogen receptors (ER) under high salt (0.6 M KCl) conditions (such as in extracts of nuclear pellets from human breast cancer biopsies) using a commercially available kit [estrogen receptor enzyme immunoassay (ER-EIA) Monoclonal; Abbott] is described. This assay has been validated using breast tumor cytosol ER preparations. It determines total (both occupied and unoccupied) ER, it is insensitive to KCl at concentrations up to 0.8 M, and it can be used with ER preparations having very low protein concentrations. Results obtained using the ER-EIA method for breast tumor nuclear extracts have been compared to those obtained using the hydroxylapatite method, and higher values have been found using the ER-EIA method. The reasons for this discrepancy may be due to: the sensitivity of ER binding to hydroxylapatite in high concentrations of KCl; the temperature dependent degradation of the receptor complex at 30 degrees C, the temperature commonly used to achieve exchange between radioactive estradiol and the endogenously bound estradiol; and possible detection of immunoreactive, but non-ligand-binding forms of ER. The possibility that occurrence of "free" receptors in high salt extracts from nuclear pellets may be an artifact is discussed. The availability of this ER-EIA suitable for nuclear ER determinations opens the possibility of extending correlations between the clinical course of breast cancer and the levels of the ER form (nuclear) that are thought to be of greatest physiological significance.

The human breast cancer cell line MCF-7 has been adapted to long-term growth at 0.5 and 0.05% fetal bovine serum. Free cytoplasmic and filled nuclear estrogen receptors were found in cells grown at 5, 0.5, and 0.05% fetal bovine serum. Cells grown in medium with 0.05% dextran-charcoal-treated serum contained cytoplasmic receptors but no filled nuclear receptors, indicating that this medium did not contain biologically active concentrations of estrogen. Addition of estradiol to the medium translocated the cytoplasmic receptor to the nucleus and stimulated progesterone receptor synthesis but did not increase the growth rate. The antiestrogen tamoxifen (TAM) inhibited cell growth at all serum concentrations investigated, at least in part by a reduction of the growth fraction. The sensitivity to TAM was highest at low serum concentrations. The effect of TAM could be reversed by estradiol at TAM concentrations of 10(-6) M or lower. It is concluded that estradiol does not have a direct growth-stimulatory effect on our MCF-7 cells in monolayer cultures although the cells contain functional estrogen receptors and growth of the cells in athymic mice requires estrogens. TAM has an estrogen-competitive, inhibitory effect on the growth of MCF-7 cells at concentrations lower than 10(-6) M. At higher concentrations, the growth inhibition is unspecific and noncompetitive by estradiol.