Epidermal growth factor induces G protein-coupled receptor 30 expression in estrogen receptor-negative breast cancer cells

Different cellular receptors mediate the biological effects induced by estrogens. In addition to the classical nuclear estrogen receptors (ERs)-alpha and -beta, estrogen also signals through the seven-transmembrane G-protein-coupled receptor (GPR)-30. Using as a model system SkBr3 and BT20 breast cancer cells lacking the classical ER, the regulation of GPR30 expression by 17beta-estradiol, the selective GPR30 ligand G-1, IGF-I, and epidermal growth factor (EGF) was evaluated. Transient transfections with an expression plasmid encoding a short 5'-flanking sequence of the GPR30 gene revealed that an activator protein-1 site located within this region is required for the activating potential exhibited only by EGF. Accordingly, EGF up-regulated GPR30 protein levels, which accumulated predominantly in the intracellular compartment. The stimulatory role elicited by EGF on GPR30 expression was triggered through rapid ERK phosphorylation and c-fos induction, which was strongly recruited to the activator protein-1 site found in the short 5'-flanking sequence of the GPR30 gene. Of note, EGF activating the EGF receptor-MAPK transduction pathway stimulated a regulatory loop that subsequently engaged estrogen through GPR30 to boost the proliferation of SkBr3 and BT20 breast tumor cells. The up-regulation of GPR30 by ligand-activated EGF receptor-MAPK signaling provides new insight into the well-known estrogen and EGF cross talk, which, as largely reported, contributes to breast cancer progression. On the basis of our results, the action of EGF may include the up-regulation of GPR30 in facilitating a stimulatory role of estrogen, even in ER-negative breast tumor cells.     

Inducible expression of dominant negative insulin-like growth factor I receptor in MCF-7 breast cancer cells

The insulin-like growth factor I receptor (IGF-IR) is expressed in many cell types and is critical for normal growth and development. In the healthy mammary gland, the role of IGF-IR is not fully elucidated. However, IGF-IR, which is primarily expressed in the mammary epithelial cells, is known to play an obligatory role in cellular transformation, facilitating the progression to breast cancer. We have utilized the tetracycline regulatory (tet-on) system to generate an in vitro model system to allow us to further investigate IGF-I/IGF-IR function in mammary epithelial cells. A plasmid construct containing a mutant IGF-I receptor (IGF-IR-DN) fused to the tetracycline operator (tetOPh_CMV-IGF-IR-DN) was stably transfected into MCF-7 human breast cancer cells. The conditional regulation of the IGF-IR-DN gene expression was studied in four independent clonal lines. The translated IGF-IR-DN protein was detected only in the stably transfected doxycycline-induced cells, and its expression was up-regulated (three- to sixfold) following induction. IGF-I stimulated cell proliferation diminished (twofold) in doxycycline-induced cells compared to uninduced cells, demonstrating that the transgene construct was functional and ruling out any pleiotropic effect that may be attributed to doxycycline. Interestingly, autophosphorylation of the IGF-IR and phosphorylation of the downstream substrate, insulin receptor substrate-1 (IRS-1), was not inhibited in doxycycline/IGF-I treated cells, suggesting the possibility that activation of downstream substrates other than the IRS-1 may be critical for optimal cell proliferation. This novel in vitro model should allow us to more directly examine the role of IGF-I/IGF-IR signaling and function in mammary epithelial cells.     

Elevated expression of proprotein convertases alters breast cancer cell growth in response to estrogen and tamoxifen

Two proprotein convertase cDNAs, PC1 and furin, were stably transfected into the human breast cancer cell line MCF-7. The PC1 or furin over-expressing cells possessed an altered morphology. When grown in vitro in a serum-free medium, the population doubling time of the convertase-transfected cells was twice that of wild-type (WT) cells. High concentrations of estradiol stimulated the growth of all three cell types to a similar extent; however, at low concentrations of estradiol, the convertase-transfected cells grew more slowly than WT cells. In athymic nude mice implanted with 5 mg estradiol pellets, the growth of tumors of convertase-transfected MCF-7 cells was stimulated to a degree similar to that of WT MCF-7 tumors. However, in mice implanted with lower-dose (1.5 mg) estradiol pellets, the tumors of PC1- or furin-transfected MCF-7 cells grew approximately five times slower than those of WT MCF-7 cells. In mice implanted with tamoxifen pellets, tumors of PC1- or furin-transfected MCF-7 cells regressed approximately five times slower than the WT tumors. This study shows that the over-expression of proprotein convertases confers a greater estrogen dependency and anti-estrogen resistance on human breast cancer cells.     

Physiological coupling of growth factor and steroid receptor signaling pathways: estrogen receptor knockout mice lack estrogen-like response to epidermal growth factor.

Past studies have shown that epidermal growth factor (EGF) is able to mimic the uterotropic effects of estrogen in the rodent. These studies have suggested a "cross-talk" model in which EGF receptor (EGF-R) signaling results in activation of nuclear estrogen receptor (ER) and its target genes in an estrogen-independent manner. Furthermore, in vitro studies have indicated the requirement for ER in this mechanism. To verify the requirement for ER in an in vivo system, EGF effects were studied in the uteri of ER knockout (ERKO) mice, which lack functional ER. The EGF-R levels, autophosphorylation, and c-fos induction were observed at equivalent levels in both genotypes indicating that removal of ER did not disrupt the EGF responses. Induction of DNA synthesis and the progesterone receptor gene in the uterus were measured after EGF treatment of both ERKO and wild-type animals. Wild-type mice showed increases of 4.3-fold in DNA synthesis, as well as an increase in PR mRNA after EGF treatment. However, these responses were absent in ERKO mice, confirming that the estrogen-like effects of EGF in the mouse uterus do indeed require the ER. These data conclusively demonstrate the coupling of EGF and ER signaling pathways in the rodent reproductive tract.     

ABCC11 expression is regulated by estrogen in MCF7 cells, correlated with estrogen receptor alpha expression in postmenopausal breast tumors and overexpressed in tamoxifen-resistant breast cancer cells

ABCC11 (Multidrug resistance protein 8; MRP8), a plasma membrane ATP-binding cassette transporter, has been implicated in drug resistance of breast cancer by virtue of its ability to confer resistance to fluoropyrimidines and to efflux methotrexate, and by its expression in this tumor. Expression of ABCC11 in breast, a hormonally regulated tissue, as well as the pump's ability to transport estrogen conjugates, suggest the possibility that expression of ABCC11 may be susceptible to regulation by estrogen. However, nothing is currently known about regulation of this gene. In this study, estradiol (E(2)) treatment reduced expression of ABCC11 mRNA in estrogen receptor (ER)-alpha-positive MCF7 cells, and E(2) antagonists such as ICI 182 780 and tamoxifen (TAM) abrogated E(2)-mediated downregulation. ABCC11 expression was positively correlated with ER-alpha expression in both breast cell lines, and two independent series of tumors from postmenopausal patients. In addition, expression of ABCC11 was upregulated in MCF7 cells exposed to TAM for 72 h, and was overexpressed in TAM-resistant cell lines. Drug sensitivity analysis of the TAM-resistant cells indicated that they were also resistant to 5-fluorouracil (5-FU), consistent with the reported ability of ABCC11 to confer resistance to this agent. These studies indicate that ABCC11 expression is negatively regulated by E(2), but that ABCC11 expression is high in high-expressing ER-alpha breast cancers. Our findings support the notion that expression of ABCC11 in ER-alpha-positive breast cancers may contribute to decreased sensitivity to chemotherapy combinations that include 5-FU. ABCC11 may be a potential predictive tool in the choice of anticancer therapies in ER-positive breast cancers resistant to TAM.     

Gefitinib and the modulation of the signaling pathways downstream of epidermal growth factor receptor in human liver cancer cells

Background The transforming growth factor-α (TGF-α)/epidermal growth factor receptor (EGFR) signaling pathway has been demonstrated to have a pivotal role in hepatocarcinogenesis. We examined whether abrogation of the TGF-α/EGFR signaling pathway with a selective EGFR tyrosine kinase inhibitor, gefitinib, could inhibit the proliferation of human hepatocellular carcinoma (HCC) cells. Methods Cellular growth was monitored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Cell-cycle distribution was analyzed by flow cytometric analysis. Activities of signaling molecules were evaluated by Western blot analysis. Results HCC cells expressed EGFR at variable levels; however, extracellular signal-regulated kinase (ERK)1/2 and AKT, key signaling molecules downstream of EGFR, were not constitutively active in the cells. When HCC cells were treated with TGF-α, cellular growth was accelerated in a manner dependent on activation of ERK1/2 and AKT. When the cells were co-treated with gefitinib and TGF-α, enhanced proliferation and activation of ERK1/2 and AKT were canceled, and the cell-cycle promotion by TGF-α was inhibited by co-treatment with gefitinib and TGF-α, independently of expression levels of EGFR. In contrast, gefitinib did not show an antiproliferative effect on HCC cells cultivated under the 10% serum condition. Conclusions The present data demonstrated that gefitinib exerted an antiproliferative action on HCC cells under a limited condition when signaling pathways downstream of EGFR were activated by TGF-α.     

Interactions between estrogen and insulin-like growth factor signaling pathways in human breast tumor cells

Estrogens and insulin-like growth factors (IGFs) act as mitogens promoting cell proliferation in normal breast tissue as well as in breast carcinomas. Both hormones have been shown to play a role in the development of breast cancer and were found to activate multiple signaling pathways leading to proliferation of human breast cancer cell lines in vitro. Originally, it was considered that these agents manifest their mitogenic actions through separate pathways, but a growing body of evidence suggests that the IGF- and estrogen-mediated signaling pathways are intertwined. 17beta-Estradiol (E2) has been shown to enhance IGF signaling at multiple levels. E2 treatment of breast cancer cells alters expression of nearly all of the IGF family members including IGF-I, IGF-II, IGF-binding proteins, IGF type I receptor (IGF-RI), and insulin receptor substrate 1. The ligand-bound estrogen receptor has been reported to bind to and to activate the IGF-RI directly. Vice versa, IGF signaling has been reported to enhance estrogen receptor activation in human breast cancer cells by inducing phosphorylation of the estrogen receptor. Finally, several groups have described synergistic effects of the combination of E2 and IGF-I on S phase entry in breast tumor cell lines. Here, we review recent, often contradictory, reports describing the effects of E2 and IGFs on the proliferation of breast tumor cells, with special emphasis on the synergistic effects of the two hormones.     

Endogenously expressed estrogen receptor and coactivator AIB1 interact in MCF-7 human breast cancer cells

Coactivators are believed to mediate estrogen-induced gene responses via interaction with estrogen receptors (ER). Currently, a major challenge is to determine the importance of each coactivator in a specific cell type and promoter context in response to a particular ligand. The potential of ER to interact with a growing list of coactivators has been shown in a variety of in vitro and gene transfer assays, yet very few data have demonstrated the interaction of endogenous coactivators with ER in intact cells. We report here a ligand-specific interaction of endogenous human ER (hER) and the AIB1 coactivator in MCF-7 human breast cancer cells by using immunoprecipitation analyses. Complexes between endogenously expressed hER and AIB1 were detected in estradiol-treated cells and to a much lesser extent in cells treated with the partial agonist, monohydroxytamoxifen. We were unable to detect an hER-SRC-1 complex in our immunoprecipitations from MCF-7 cells. The in vitro-binding affinity for mouse ER interaction with AIB1 was estimated to be 40-120 nM. We conclude that AIB1 is a major coactivator for hER in MCF-7 human breast cancer cells.     

Expression of c-erbB-2 in node negative breast cancer does not correlate with estrogen receptor status,predictors of hormone responsiveness,or PCNA expression

The aim of this study was to analyse the relationships between the expression of c-erbB-2, estrogen receptor (ER), progesterone receptor (PR), Bcl-2 and PCNA in node negative breast cancer. Expression of these markers was determined by HercepTest, by immunohistochemistry and quantified by morphometry in the group of 125 selected breast carcinoma patients with broad spectrum of histological types and grades. Multivariate statistical analysis revealed only relationships between ER/PR, ER/Bcl-2, ER/grade and ER/age. There was not found any significant relationship between c-erbB-2 expression and any other immunohistochemical marker, apocrine metaplasia, histological type or patient characteristics. The same result was found in complete group of tumors as well as in individual groups divided according to histological type. These results indicate that in node negative breast tumors, c-erbB-2 expression does not correlate inversely with hormone receptor status and hormone responsiveness like previously reported metastasising breast cancer and that the prognostic significance of c-erbB-2 expression in these tumors is not clear.     

Chimeric Fv-zeta or Fv-epsilon receptors are not sufficient to induce activation or cytokine production in peripheral T cells

In current clinical trials, chimeric antibody-like receptors fused to signaling domains derived from TCR-zeta or Fc(epsilon)RIgamma-chain are tested for their ability to lyse tumor cells in vivo. In this study, the function of primary T cells expressing such receptors has been investigated in transgenic mice. These receptors cannot induce proliferation of resting T cells or trigger the production of optimal amounts of cytokines. It is further demonstrated that an initial low presence of cytokine message and protein is disappearing rather fast, whereas the triggering of endogenous TCR/CD3 in the same cells leads to normal prolonged cytokine production. The direct clinical relevance of these findings is further underlined by the increased in vivo tumor rejection by T cells expressing chimeric receptors in presence of exogenous interleukin-2. Therefore, adoptive T-cell therapy using primary T cells transfected with single chain receptors might benefit substantially from the accompanying administration of cytokines. (Blood. 2000;96:1999-2001)