Breast Neoplasms: Stoica A

Martin MB, Reiter R, Johnson M, Shah MS, Iann MC, Singh B, Richards JK, Wang A, Stoica A. · Department of Human Science, School of Nursing and Health Studies, Lombardi Comprehensive Cancer Center, Georgetown University, 3700 Reservoir Road Northwest, Washington, D.C. 20057-1107, USA. · Endocrinology. · Pubmed #17640996 links to  free full text

Abstract: Metallo-estrogens are a new class of potent environmental estrogens. This study investigates whether tobacco smoke condensate (TSC), which contains metals and metalloids, elicits estrogen-like effects at environmentally relevant doses. Treatment of human breast cancer cells, MCF-7, with 40 microg/ml TSC resulted in a 2.5-fold stimulation of cell growth. TSC decreased the concentration of estrogen receptor (ER)-alpha protein and mRNA (63 and 62%, respectively), and increased the expression of the estrogen-regulated genes, progesterone receptor and pS2 (5- and 2-fold, respectively). In addition, TSC activated ER-alpha in COS-1 or CHO cells transiently transfected with wild-type ER-alpha and an ERE-CAT or an ERE-luciferase reporter gene (11- and 6-fold, respectively). TSC also activated a chimeric receptor (GAL-ER) containing the hormone binding domain of ER-alpha (3.5-fold). It blocked the binding of estradiol to the receptor without altering the affinity of estradiol (K(d) = 2.2-6.8 x 10(-10) m). Transfection assays with ER-alpha mutants identified C381, C447, H524, N532, E523, and D538 in the hormone binding domain as important for activation by TSC. In ovariectomized rats, low doses of TSC [10 or 20 mg/kg body weight (bw)] increased uterine wet weight (1.7- and 2.1-fold), and induced the expression of progesterone receptor and complement C3 in the uterus (2- and 26-fold) and mammary gland (4.4- and 15-fold). Both the in vitro and in vivo TSC effects were blocked by the antiestrogen ICI 182,780, suggesting the involvement of ER. Collectively, these results provide strong evidence that low doses of TSC, acting through the hormone binding domain, exert estrogen-like effects in cell culture and animals.

Stoica GE, Franke TF, Moroni M, Mueller S, Morgan E, Iann MC, Winder AD, Reiter R, Wellstein A, Martin MB, Stoica A. · Department of Oncology, Lombardi Cancer Center, Georgetown University, Washington DC 20057, USA. · Oncogene. · Pubmed #12970748 No free full text.

Abstract: Epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and heregulin-beta1 (HRG-beta1), can modulate the expression and activity of the estrogen receptor-alpha (ER-alpha) via the phosphatidylinositol 3-kinase (PI 3-K)/Akt pathway in the ER-alpha-positive breast cancer cell line, MCF-7. Estradiol can also rapidly activate PI 3-K/Akt in these cells (nongenomic effect). The recent study examines whether Akt is involved in the ER-alpha regulation by estradiol (genomic effect). Stable transfection of parental MCF-7 cells with a dominant-negative Akt mutant, as well as the PI 3-K inhibitors wortmannin and LY 294,002, blocked the effect of estradiol on ER-alpha expression and activity by 70-80 and 55-63%, respectively. Stable transfection of MCF-7 cells with a constitutively active Akt mimicked the effect of estradiol. The changes in ER-alpha expression and activity were abrogated in response to estradiol by an arginine to cysteine mutation in the pleckstrin homology (PH) domain of Akt (R25C), suggesting the involvement of this amino acid in the interaction between Akt and ER-alpha. Experiments employing selective ErbB inhibitors demonstrate that the effect of estradiol on ER-alpha expression and activity is mediated by ErbB2 and not by EGFR. Moreover, anchorage-dependent and -independent growth assays, cell cycle and membrane ruffling analyses showed that Akt exerts estrogen-like activity on cell growth and membrane ruffling and that a selective ErbB2 inhibitor, but not anti-ErbB2 antibodies directed to the extracellular domain, can block these effects. In the presence of constitutively active Akt, tamoxifen only partially inhibits cell growth. In contrast, in cells stably transfected with either a dominant-negative Akt or with R25C-Akt, as well as in parental cells in the presence of a selective ErbB2 inhibitor, the effect of estradiol on anchorage-dependent and -independent cell growth was inhibited by 50-75 and 100%, respectively. Dominant-negative Akt inhibited membrane ruffling by 54%; however, R25C-Akt did not have any effect, suggesting that kinase activity plays an important role in this process. Scatchard analysis demonstrated a 67% reduction in estrogen-binding capacity in cells transfected with constitutively active Akt. No change in binding affinity of estradiol to the receptor was observed upon transfection with either Akt mutant. Taken together, our results suggest that estradiol treatment results in binding to membrane ER-alpha and interaction with a heterodimer containing ErbB2, leading to tyrosine phosphorylation. This results in the activation of PI 3-K and Akt. Akt, in turn, may interact with nuclear ER-alpha, altering its expression and activity.

Martin MB, Reiter R, Pham T, Avellanet YR, Camara J, Lahm M, Pentecost E, Pratap K, Gilmore BA, Divekar S, Dagata RS, Bull JL, Stoica A. · Department of Biochemistry, Georgetown University, Washington, DC 20007, USA. · Endocrinology. · Pubmed #12746304 links to  free full text

Abstract: The ability of metals to activate estrogen receptor-alpha (ERalpha) was measured in the human breast cancer cell line, MCF-7. Similar to estradiol, treatment of cells with the divalent metals copper, cobalt, nickel, lead, mercury, tin, and chromium or with the metal anion vanadate stimulated cell proliferation; by d 6, there was a 2- to 5-fold increase in cell number. The metals also decreased the concentration of ERalpha protein and mRNA by 40-60% and induced expression of the estrogen-regulated genes progesterone receptor and pS2 by1.6- to 4-fold. Furthermore, there was a 2- to 4-fold increase in chloramphenicol acetyltransferase activity after treatment with the metals in COS-1 cells transiently cotransfected with the wild-type receptor and an estrogen-responsive chloramphenicol acetyltransferase reporter gene. The ability of the metals to alter gene expression was blocked by an antiestrogen, suggesting that the activity of these compounds is mediated by ERalpha. In binding assays the metals blocked the binding of estradiol to the receptor without altering the apparent binding affinity of the hormone (K(d) = 10(-10) M). Scatchard analysis employing either recombinant ERalpha or extracts from MCF-7 cells demonstrated that (57)Co and (63)Ni bind to ERalpha with equilibrium dissociation constants of 3 and 9.5 x 10(-9) and 2 and 7 x 10(-9) M, respectively. The ability of the metals to activate a chimeric receptor containing the hormone-binding domain of ERalpha suggests that their effects are mediated through the hormone-binding domain. Mutational analysis identified amino acids C381, C447, E523, H524, N532, and D538 as potential interaction sites, suggesting that divalent metals and metal anions activate ERalpha through the formation of a complex within the hormone-binding domain of the receptor.

Stoica A, Pentecost E, Martin MB. · Department of Biochemistry and Molecular Biology, Lombardi Cancer Center, Georgetown University, Washington, DC 20007, USA. · Endocrinology. · Pubmed #11014213 links to  free full text

Abstract: To determine whether arsenite has estrogen-like activities, the effects of this compound on estrogen receptor-alpha (ERalpha) and other estrogen-regulated genes were measured in the human breast cancer cell line MCF-7. Treatment of cells with 1 microM arsenite resulted in a 60% decrease in the amount of ERalpha and in a parallel decrease of 40% in ERalpha messenger RNA. Progesterone receptor concentration increased 22-fold after arsenite treatment. pS2 messenger RNA also increased 2. 1-fold after treatment. The induction of progesterone receptor and pS2 was blocked by the antiestrogen ICI-182,780. In transient cotransfection experiments of wild-type ERalpha and an estrogen response element-reporter construct, arsenite stimulated chloramphenicol acetyltransferase (CAT) activity. In growth assays, arsenite significantly stimulated the proliferation of MCF-7 cells compared with cells grown in estrogen-depleted medium. Addition of an antiestrogen blocked growth stimulation by arsenite. In binding assays, arsenite blocked the binding of estradiol to ERalpha (Ki = 5 +/- 0.5 nM; n = 3), suggesting that the compound interacts with the hormone-binding domain of the receptor. To determine whether interaction of arsenite with the hormone-binding domain results in receptor activation, COS-1 cells were transiently cotransfected with the chimeric receptors GAL-ER, which contains the hormone-binding domain of ERalpha and the DNA-binding domain of the transcription factor GAL4, and a GAL4-responsive CAT reporter gene. Treatment of cells with estradiol or arsenite resulted in a 4-fold increase in CAT activity. The effects of arsenite on the chimeric receptor were blocked by the antiestrogen, suggesting that arsenite activates ERalpha through an interaction with the hormone-binding domain of the receptor. Transfection assays with ERalpha mutants identified C381, C447, H524, and N532 as interaction sites of arsenite with the hormone-binding domain.

Stoica A, Pentecost E, Martin MB. · Department of Biochemistry and Molecular Biology, Lombardi Cancer Center, Georgetown University, Washington DC 20007, USA. · J Cell Biochem. · Pubmed #10967555 No free full text.

Abstract: To determine whether selenite has estrogen-like activities, the effects of this compound on estrogen receptor-alpha (ER-alpha) and other estrogen-regulated genes were measured in the human breast cancer cell line MCF-7. Treatment of cells with 1 uM of sodium selenite resulted in a 40% decrease in the amount of estrogen receptor-alpha and in a parallel decrease of 40% in ER-alpha mRNA. Progesterone receptor concentration increased 2.6-fold and pS2 mRNA increased 2.4-fold after selenite treatment. The induction of progesterone receptor and pS2 was blocked by the anti-estrogen ICI-182,780. In transient co-transfection experiments of Wild-type ER-alpha and an estrogen response element-reporter construct, selenite stimulated CAT activity. In binding assays, selenite blocked the binding of estradiol to ER-alpha (K(i) = 23 +/- 17 nM, n = 3) suggesting that this compound interacts with the hormone binding domain of the receptor. To determine whether interaction of selenite with the hormone binding domain results in receptor activation, COS-1 cells were transiently co-transfected with the chimeric receptors GAL-ER, which contains the hormone binding domain of ER-alpha and the DNA binding domain of the transcription factor GAL4, and a GAL4-responsive CAT reporter gene. Treatment of cells with estradiol or selenite resulted in a three- to five-fold increase in CAT activity. The effects of selenite on the chimeric receptor were blocked by the antiestrogen, suggesting that selenite activates ER-alpha through an interaction with the hormone binding domain of the receptor. Transfection assays with ER-alpha mutants identified C381, C447, H524, and N532 as interaction sites of selenite with the hormone binding domain.

Stoica A, Saceda M, Doraiswamy VL, Coleman C, Martin MB. · Lombardi Cancer Center, Department of Biochemistry and Molecular Biology, Georgetown University, Washington, DC 20007-2197, USA. · J Endocrinol. · Pubmed #10810301 links to  free full text

Abstract: The role of epidermal growth factor (EGF) in the regulation of estrogen receptor-alpha (ER-alpha) gene expression in the human breast cancer cell line MCF-7 was investigated. Treatment of cells with 0.4 ng/ml EGF resulted in an approximately 60% decrease in ER-alpha protein concentration by 6 h and the amount of receptor remained suppressed for 24 h. Ligand binding assays demonstrated that the decrease in ER-alpha protein corresponded to a similar decrease (approximately 50%) in estradiol binding sites. Although EGF treatment resulted in a decrease in the number of binding sites, it had no effect on the binding affinity of ER-alpha. The dissociation constant of the estradiol-ER-alpha complex in the presence or absence of EGF was the same (K(d)=2.3x10(-)(10) M in control cells versus K(d)=1.98x10(-)(10) M in EGF-treated cells). The decrease in ER-alpha protein concentration paralleled a decrease in the steady-state amount of ER-alpha mRNA. By 9 h there was an approximately 60% decrease in ER-alpha mRNA. The amount of ER-alpha mRNA remained suppressed for 48 h. Transcription run-on experiments demonstrated that there was a decrease of approximately 70% in ER-alpha gene transcription upon EGF treatment, suggesting that the mechanism by which EGF regulates ER-alpha gene expression is transcriptional. In addition to regulating the amount of ER-alpha, EGF affected the activity of the receptor. At high concentrations, EGF induced progesterone receptor. Estradiol and high concentrations of EGF had an additive effect on progesterone receptor. In contrast to high concentrations, low concentrations of EGF had no effect on progesterone receptor and blocked estradiol induction. The effects of EGF on ER-alpha expression were inhibited by tyrophostins and wortmannin, suggesting that the effects of the growth factor are mediated by the EGF receptor and protein kinase B. When the cells were placed in serum-free medium and then treated with EGF, there was no effect on ER-alpha protein concentration or activity. However, increasing concentrations of serum restored the effects of EGF on ER-alpha, suggesting that an additional serum factor was required for the EGF-mediated effect on the decrease in ER-alpha protein concentration.

Stoica A, Katzenellenbogen BS, Martin MB. · Department of Biochemistry and Molecular Biology, Vincent T. Lombardi Cancer Center, Georgetown University, Washington, DC 20007, USA. · Mol Endocrinol. · Pubmed #10770491 links to  free full text

Abstract: Previous studies from this laboratory have shown that the heavy metal cadmium (Cd) mimics the effects of estradiol in estrogen-responsive breast cancer cell lines. To understand the mechanism by which cadmium activates estrogen receptor-alpha (ER-alpha), the ability of cadmium to bind to and activate wild-type and various mutants of ER-alpha was examined. When tested in transient cotransfection assays in COS-1 cells, cadmium concentrations as low as 10(-11) M activated ER-alpha. Scatchard analysis employing either purified human recombinant ER-alpha or extracts from ER-containing MCF-7 cells demonstrated that l09Cd binds to the ER with an equilibrium dissociation constant of approximately 4 to 5 x 10(-10) M. Cadmium also blocks the binding of estradiol to ER-alpha in a noncompetitive manner (K(i) = 2.96 x 10(-10) M), suggesting that the heavy metal interacts with the hormone-binding domain of the receptor. To study the role of the hormone-binding domain in cadmium activation, COS-1 cells were transiently cotransfected with GAL-ER, a chimeric receptor containing the DNA-binding domain of the transcription factor GAL4 and the hormone-binding domain of ER-alpha, and a GAL4-responsive reporter gene. Treatment of the transfected cells with either 10(-6) M cadmium or 10(-9) M estradiol resulted in a 4-fold increase in reporter gene activity. The effect of cadmium on the chimeric receptor was blocked by the antiestrogen, ICI-164,384, suggesting that cadmium activates ER-alpha through an interaction with the hormone-binding domain of the receptor. Transfection and binding assays with ER-alpha mutants identified C381, C447, E523, H524, and D538 as possible interaction sites of cadmium with the hormone-binding domain of ER-alpha.

Stoica A, Saceda M, Fakhro A, Solomon HB, Fenster BD, Martin MB. · Department of Biochemistry and Molecular Biology, Vincent T. Lombardi Cancer Center, Georgetown University, Washington D.C. 20007, USA. · J Cell Biochem. · Pubmed #10572247 No free full text.

Abstract: This report describes an investigation of the role of 1, 25-dihydroxyvitamin D (VD(3)) in the regulation of estrogen receptor-alpha (ER) in the ER-positive breast cancer cell line, MCF-7. Treatment of cells with 10 nM VD(3) resulted in a 50% decline in the concentration of ER protein at 24 h. Scatchard analysis showed a corresponding decrease in the number of estradiol binding sites and no alteration in the binding affinity of estradiol for the ER (K(d) = 0.08 nM in VD(3)-treated cells compared with K(d) = 0.07 nM in control cells). Vitamin D treatment also caused a 50% decrease in the steady state amount of ER mRNA, which was maximal by 18 h. In vitro transcription run-on experiments demonstrated a decrease of approximately 60% in transcription of the estrogen receptor gene. Transient transfections using an ER promoter-CAT construct also demonstrated a 40% decrease in CAT activity after VD(3) treatment. Sequence analysis identified a potential vitamin D response element (nVDRE) within the ER promoter. When this element was mutated, the ability of VD(3) to block transcription from the ER promoter was lost. When the nVDRE was placed upstream of a heterologous promoter, nVDRE-SV40-CAT, treatment with VD(3) resulted in a 50% decrease in CAT activity. Interestingly, co-transfection of either the ER promoter-CAT or the nVDRE-SV40-CAT construct and a vitamin D receptor expression vector into COS-1 or CV-1 cells showed an approximately 4-fold increase in CAT activity after VD(3) treatment. Taken together these data suggest that VD(3) inhibition of ER gene transcription is mediated through a nVDRE in the ER promoter. Inhibition appears to be cell specific.

Martin MB, Stoica A. · Department of Oncology, Vincent T. Lombardi Cancer Center, Georgetown University, Washington, DC, USA. · J Nutr. · Pubmed #12468626 links to  free full text

Abstract: There is increasing evidence that estradiol and insulin-like growth factor-I (IGF-I) act through a complex cross-talk mechanism to stimulate the proliferation of normal mammary epithelium to increase the risk of breast cancer. The emerging model of cross-talk suggests that estradiol regulates the expression of IGF-I and the IGF receptor I. The subsequent binding of IGF-I to its receptor initiates an intracellular signal transduction pathway that activates transcription factors, including the estrogen receptor. Recent studies show that the effects of IGF-I on estrogen receptor activity are mediated in part by the protein kinase A and phosphatidylinositol-3-kinase/Akt pathways.

Stoica GE, Franke TF, Wellstein A, Czubayko F, List HJ, Reiter R, Morgan E, Martin MB, Stoica A. · Department of Oncology, Lombardi Cancer Center, Georgetown University, Washington, DC 20007, USA. · Mol Endocrinol. · Pubmed #12554767 links to  free full text

Abstract: Previously, we have demonstrated that the two mitogenic growth factors epidermal growth factor and IGF-I can activate Akt and estrogen receptor-alpha (ERalpha) in the hormone-dependent breast cancer cell line, MCF-7. In this report we now show that estradiol can also rapidly activate phosphatidylinositol 3-kinase (PI 3-K)/Akt and that this effect is mediated by the ErbB2 signaling pathway. Treatment of cells with estradiol resulted in phosphorylation of Akt and a 9-fold increase in Akt activity in 10 min. Akt activation was blocked by wortmannin and LY 294,002, two inhibitors of PI 3-K; by genistein, a protein tyrosine kinase inhibitor and an ER agonist; by AG825, a selective ErbB2 inhibitor; and by the antiestrogens ICI 182,780 and 4-hydroxy-tamoxifen; but not by rapamycin, an inhibitor of the ribosomal protein kinase p70S6K; nor by AG30, a selective epidermal growth factor receptor inhibitor. Akt activation by estradiol was abrogated by an arginine-to-cysteine mutation in the pleckstrin homology domain of Akt (R25C). Growth factors also activated Akt in the ER-negative variant of MCF-7, MCF-7/ADR, but estradiol did not induce Akt activity in these cells. Transient transfection of ERalpha into these cells restored Akt activation by estradiol, suggesting that estradiol activation of Akt requires the ERalpha. Estradiol did not activate Akt in MCF-7 cells stably transfected with an anti-ErbB2-targeted ribozyme, further confirming a role for ErbB2. In vitro kinase assays using immunoprecipitation and anti-Akt1, -Akt2, and -Akt3-specific antibodies demonstrated that Akt1 is activated by estradiol in MCF-7 cells whereas Akt3 is the activated isoform in ER-negative MDA-MB231 cells, implying that selective activation of Akt subtypes plays a role in the actions of estradiol. Taken together, our data suggest that estradiol, bound to membrane ERalpha, interacts with and activates an ErbB dimer containing ErbB2, inducing activation of PI 3-K/Akt.