Proximal events in signaling by plasma membrane estrogen receptors

Estradiol (E2) rapidly stimulates signal transduction from plasma membrane estrogen receptors (ER) that are G protein-coupled. This is reported to occur through the transactivation of the epidermal growth factor receptor (EGFR) or insulin-like growth factor-1 receptor, similar to other G protein-coupled receptors. Here, we define the signaling events that result in EGFR and ERK activation. E2-stimulated ERK required ER in breast cancer and endothelial cells and was substantially prevented by expression of a dominant negative EGFR or by tyrphostin AG1478, a specific inhibitor for EGFR tyrosine kinase activity. Transactivation/phosphorylation of EGFR by E2 was dependent on the rapid liberation of heparin-binding EGF (HB-EGF) from cultured MCF-7 cells and was blocked by antibodies to this ligand for EGFR. Expression of dominant negative mini-genes for Galpha(q) and Galpha(i) blocked E2-induced, EGFR-dependent ERK activation, and Gbetagamma also contributed. G protein activation led to activation of matrix metalloproteinases (MMP)-2 and -9. This resulted from Src-induced MMP activation, implicated using PP2 (Src family kinase inhibitor) or the expression of a dominant negative Src protein. Antisense oligonucleotides to MMP-2 and MMP-9 or ICI 182780 (ER antagonist) each prevented E2-induced HB-EGF liberation and ERK activation. E2 also induced AKT up-regulation in MCF-7 cells and p38beta MAP kinase activity in endothelial cells, blocked by an MMP inhibitor, GM6001, and tyrphostin AG1478. Targeting of only the E domain of ERalpha to the plasma membrane resulted in MMP activation and EGFR transactivation. Thus, specific G proteins mediate the ability of E2 to activate MMP-2 and MMP-9 via Src. This leads to HB-EGF transactivation of EGFR and signaling to multiple kinase cascades in several target cells for E2. The E domain is sufficient to enact these events, defining additional details of the important cross-talk between membrane ER and EGFR in breast cancer.     

NDF induces expression of a novel 46 kD protein in estrogen receptor positive breast cancer cells

Most human breast tumors start as estrogen-dependent, but during the course of the disease become refractory to hormone therapy. The transition of breast tumors from estrogen dependent to independent behavior may be regulated by autocrine and/or paracrine growth factor(s) that are independent of the estrogen receptor (ER). We have investigated the role(s) of NDF (neu-differentiation factor) in the biology of estrogen positive breast cancer cells by using MCF-7 cells as a model system. Treatment of MCF-7 cells with human recombinant NDF-β2 (NDF) inhibited the ER expression by 70% and this was associated with growth stimulation in an estrogen-independent manner. To explore the mechanism(s) of action of NDF in MCF-7 cells, we examined the expression of NDF-inducible gene products. We report here that NDF stimulated the levels of expression of a 46 kD protein (p46) (in addition to few minor proteins) in ER positive breast cancer cells including MCF-7, T-47-D, and ZR-75-R cells but not in ER negative breast cancer cells including MDA-231, SK-BP-3, and MDA-468 cells. This effect of NDF was due to induction in the rate of synthesis of new p46. The observed NDF-mediated induction of p46 expression was specific as there was no such effect by epidermal growth factor or 17-β-estradiol, and inclusion of actinomycin D partially inhibited the p46 induction elicited by NDF. NDF-inducible stimulation of p46 expression was an early event (2–6 h) which preceded the period of down-regulation of ER expression by NDF. These results support the existence of NDF-responsive specific cellular pathway(s) that may regulate ER, and these interactions could play a role(s) in hormone-independence of ER positive breast cancer cells. © 1996 Wiley-Liss, Inc.     

Estrogen-induced apoptosis in a breast cancer model resistant to long-term estrogen withdrawal

Estrogen suppression through the use of an aromatase inhibitor is an effective endocrine treatment option for postmenopausal breast cancer patients with estrogen receptor (ER)-positive disease, however, there are concerns that long-term estrogen deprivation will inevitably lead to resistance. To address the issue of acquired resistance to long-term estrogen deprivation our laboratory has developed an ER+/PR- hormone-independent breast cancer cell line, MCF-7:5C which is a variant clone of wild-type MCF-7 cells. Originally, these cells were cultured in estrogen-free MEM containing 5% charcoal-stripped calf serum and were found to be resistant to both estradiol (E(2)) and antiestrogens. Interestingly, a completely different phenomenon was observed when MCF-7:5C cells were cultured in phenol red-free RPMI 1640 medium containing 10% charcoal-stripped fetal bovine serum (SFS). Using DNA quantitation assays, we examined the effect of E(2) on the growth of MCF-7:5C cells under different media conditions. Our results showed that 10(-9)M E(2) caused a dramatic 90% reduction in the growth of MCF-7:5C cells cultured in RPMI medium containing 10% SFS but did not have any significant inhibitory effects on cells cultured in MEM media. Additional experiments were performed to determine whether the medium or the serum facilitated the inhibitory effects of E(2) and the results indicated that it was the serum. Annexin V and DAPI staining confirmed that the E(2)-induced growth inhibition of MCF-7:5C cells was due to apoptosis. We also examined the tumorigenic potential of MCF-7:5C cells by injecting 1x10(7)cells/site into ovariectomized athymic mice and found that these cells, previously cultured in RPMI media, spontaneously grew into tumors in the absence of E(2). Overall, these results show that low concentrations (>10(-11)M) of E(2) are capable of inducing apoptosis in an aromatase resistant breast cancer cell model and that this effect is highly influenced by the medium in which the cells are grown.     

Regulation of estrogen receptor messenger ribonucleic acid and protein levels in human breast cancer cell lines by sex steroid hormones, their antagonists, and growth factors

Since sex steroid hormones and growth factors are known to modulate the proliferation of breast tumors, we have studied the effects of estrogen and progestin, their antagonists, and growth factors on the regulation of estrogen receptor (ER) mRNA and protein levels in T47D breast cancer cells, which contain low levels of ER, and in two sublines of MCF-7 cells which contain high ER levels. The mRNA levels were measured by Northern blot analysis using lambda OR8, a cDNA probe for ER, and protein levels were measured by hormone binding or Western blot analysis. Treatment of T47D cells with estradiol (E2) caused a 2.5-fold increase in ER mRNA (6.6 kilobases) levels after 48 h. The progestin R5020 evoked a marked decrease in ER mRNA and protein levels to 20% of control values, while the antiprogestin RU38,486 caused no change in ER. In MCF-7 cells, the effect of E2 on ER levels was dependent on the prior growth history of the cells. In cells grown in low estrogen [5% charcoal-dextran-treated calf serum with phenol red for 8 yr (MCF-7-K2)], which are still E2 responsive, treatment with E2, the antiestrogen LY117018, or both produced little change in ER mRNA or protein; in contrast, ER mRNA and protein were reduced by E2 to 40% and 50% of control levels, respectively, in MCF-7 cells (denoted MCF-7-K1) which had been maintained routinely in medium containing 5% calf serum. This decrease in ER mRNA was dose dependent; 10(-11) E2 reduced levels to 60%, and 10(-10) M E2 evoked the maximal drop to 40% of the control level in 2 days. LY117018 alone did not alter ER mRNA levels in these cells, but it completely prevented the down-regulation of ER by E2. Administration of progestin, but not antiprogestin, along with E2 partially prevented the decrease in ER evoked by E2. Addition of epidermal growth factor or insulin-like growth factor-I to MCF-7-K1 cells, which increased cell proliferation, had no detectable effect on ER levels. Treatment with transforming growth factor-beta, which decreased cell proliferation, reduced ER by about 20%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Resveratrol, a natural product derived from grape, exhibits antiestrogenic activity and inhibits the growth of human breast cancer cells

Resveratrol is a natural phytoalexin compound found in grapes and other food products. In this study, the effect of resveratrol on the growth of human breast cancer cells was examined. Results show that resveratrol inhibits the growth of estrogen receptor(ER)-positive MCF-7 cells in a dose-dependent fashion. Detailed studies with MCF-7 cells demonstrate that resveratrol antagonized the growth-promoting effect of 17-beta-estradiol (E2) in a dose-dependent fashion at both the cellular (cell growth) and the molecular (gene activation) levels. At 5 x 10(-6) M, resveratrol abolished the growth-stimulatory effect mediated by concentrations of E2 up to 10(-9) M. The antiestrogenic effect of resveratrol could be observed at a concentration of 10(-6) M and above. The antiestrogenic effect of resveratrol was also demonstrated at the molecular level. Resveratrol in a dose-dependent fashion antagonized the stimulation by E2 of progesterone receptor gene expression in MCF-7 cells. Moreover, expression of transforming growth factor-alpha and insulin-like growth factor I receptor mRNA was inhibited while the expression of transforming growth factor beta2 mRNA was significantly elevated in MCF-7 cells cultivated in the presence of resveratrol (10(-5) M). In summary, our results show that resveratrol, a partial ER agonist itself, acts as an ER antagonist in the presence of estrogen leading to inhibition of human breast cancer cells.     

17beta-Estradiol responsiveness of MCF-7 laboratory strains is dependent on an autocrine signal activating the IGF type I receptor

BACKGROUND: Human MCF-7 cells have been studied extensively as a model for breast cancer cell growth. Many reports have established that serum-starved MCF-7 cells can be induced to proliferate upon the sole addition of 17beta-estradiol (E2). However, the extent of the mitogenic response to E2 varies in different MCF-7 strains and may even be absent. In this study we compared the E2-sensitivity of three MCF-7 laboratory strains. RESULTS: The MCF-7S line is non-responsive to E2, the MCF-7 ATCC has an intermediate response to E2, while the MCF-7 NKI is highly E2-sensitive, although the levels and activities of the estrogen receptor (ER) are not significantly different. Both suramin and IGF type I receptor blocking antibodies are able to inhibit the mitogenic response to E2-treatment in MCF-7 ATCC and MCF-7 NKI cells. From this we conclude that E2-induced proliferation is dependent on IGF type I receptor activation in all three MCF-7 strains. CONCLUSIONS: The results presented in this article suggest that E2-responsiveness of MCF-7 cells is dependent on the secretion of an autocrine factor activating the IGF-IR. All three strains of MCF-7 breast cancer cells investigated do not respond to E2 if the IGF-RI-pathway is blocked. Generally, breast cancer therapy is targeted at inhibiting estrogen action. This study suggests that inhibition of IGF-action in combination with anti-estrogen-treatment may provide a more effective way in treatment or even prevention of breast cancer.