Edward M. Curran

Loss of Estrogen Receptor Signaling Triggers Epigenetic Silencing of Downstream Targets in Breast Cancer

Alterations in histones, chromatin-related proteins, and DNA methylation contribute to transcriptional silencing in cancer, but the sequence of these molecular events is not well understood. Here we demonstrate that on disruption of estrogen receptor (ER) α signaling by small interfering RNA, polycomb repressors and histone deacetylases are recruited to initiate stable repression of the progesterone receptor (PR) gene, a known ERα target, in breast cancer cells. The event is accompanied by acquired DNA methylation of the PR promoter, leaving a stable mark that can be inherited by cancer cell progeny. Reestablishing ERα signaling alone was not sufficient to reactivate the PR gene; reactivation of the PR gene also requires DNA demethylation. Methylation microarray analysis further showed that progressive DNA methylation occurs in multiple ERα targets in breast cancer genomes. The results imply, for the first time, the significance of epigenetic regulation on ERα target genes, providing new direction for research in this classical signaling pathway.

Phosphorylation of Estrogen Receptor α Blocks Its Acetylation and Regulates Estrogen Sensitivity

Estrogen receptor (ER) α is mutated (lysine 303 to arginine, K303R) in approximately one third of premalignant breast hyperplasias, which renders breast cancer cells expressing the mutant receptor hypersensitive for proliferation in response to low doses of estrogen. It is known that ERα is posttranslationally modified by protein acetylation and phosphorylation by a number of secondary messenger signaling cascades. The K303R ERα mutation resides at a major protein acetylation site adjacent to a potential protein kinase A (PKA) phosphorylation site at residue 305 within the hinge domain of the receptor. Mutation of this phosphorylation site to aspartic acid to mimic constitutive phosphorylation blocks acetylation of the K303 ERα site and generates an enhanced transcriptional response similar to that seen with the naturally occurring K303R mutant receptor. Activation of PKA signaling by the cell-permeable cyclic AMP (cAMP) analog 8-bromo-cAMP further enhances estrogen sensitivity of the mutant receptor, whereas a specific PKA inhibitor antagonizes this increase. We propose that the hypersensitive ERα mutant breast cancer phenotype involves an integration of coupled acetylation and phosphorylation events by upstream signaling molecules.

Estrogen Receptor α (ERα) Deficiency in Macrophages Results in Increased Stimulation of CD4+ T Cells while 17β-Estradiol Acts through ERα to Increase IL-4 and GATA-3 Expression in CD4+ T Cells Independent of Antigen Presentation

The effects of 17β-estradiol (E2) on immune function have been extensively reported. The effects are dependent on concentration and duration of exposure and potential differences in signaling between the known E2 receptors, estrogen receptors (ER) α and ERβ. Through the use of ER-deficient mice, we and others have begun to demonstrate the role of the two known receptors in modulating immune functional activities. Previous studies have shown that cells of the innate immune system have altered function (bactericidal capacity) and patterns of cytokine expression (increased proinflammatory cytokine expression) through amelioration of ERα signaling. In this study, we extend these studies to analysis of T cell differentiation and proliferation in APC-dependent and APC-independent in vitro assay systems. Our results demonstrate that ERα deficiency in splenic macrophages, but not CD11c+ splenic dendritic cells pulsed with OVA significantly enhances proliferative responses and IFN-γ production by transgenic OVA peptide-specific (OT-II) CD4+ T cells when compared with Ag-pulsed APC from wild-type littermates. The addition of E2 in this culture system did not significantly affect the production of IFN-γ. In addition, when purified CD4+ T cells from ERα-deficient and wild-type littermates were stimulated with anti-CD3/CD28 Ab in the absence of E2, there were no significant differences in IFN-γ or IL-4 production. However, the addition of E2 significantly increased IL-4 secretion, as well as increased GATA-3 mRNA levels from ERα-replete CD4+ T cells, while this effect was abrogated in ERα-deficient CD4+ T cells.

Estrogen receptors in membrane lipid rafts and signal transduction in breast cancer.

Regulation of breast cancer growth by estrogen is mediated by estrogen receptors (ER) in nuclear and extranuclear compartments. We assessed the structure and functions of extranuclear ER that initiate downstream signaling to the nucleus. ER, including full-length 66-kDa ER and a 46-kDa ER splice variant, are enriched in lipid rafts from MCF-7 cells with (MCF-7/HER-2) or without (MCF-7/PAR) HER-2 gene overexpression and co-localize with HER-1 and HER-2 growth factor receptors, as well as with lipid raft marker flotillin-2. In contrast, ER-negative MCF-7 cells do not express nuclear or lipid raft ER. ER knockdown with siRNA also elicits a marked loss of ER in MCF-7 cell rafts. In MCF-7/PAR cells, estrogen enhances ER association with membrane rafts and induces rapid phosphorylation of nuclear receptor coactivator AIB1, actions not detected in ER-negative cells. Thus, nuclear and lipid raft ER derive from the same transcript, and extranuclear ER co-localizes with HER receptors in membrane signaling domains that modulate downstream nuclear events leading to cell growth.

Estrogen receptor‐α deficiency promotes increased TNF‐α secretion and bacterial killing by murine macrophages in response to microbial stimuli in vitro

In this series of studies, we determined the potential role of intracellular estrogen receptors (ER), ERα and ERβ, on macrophage function in response to bacterial stimuli. The sex hormone 17β‐estradiol (E2) and ER have been shown to modulate inflammatory responses as well as T helper cell type 1 (TH1)/TH2 responses. The mechanisms E2 and its receptors use to alter these immune functions remain largely unknown. ERα and ERβ possess complex actions in tissues where they are expressed. We have characterized the receptor repertoire of murine dendritic cells and thioglycollate‐elicited peritoneal macrophages (PM). Both cell types express mRNA for ERα. Neither cell type expressed detectable amounts of ERβ mRNA, as determined by reverse transcriptase‐polymerase chain reaction using exon‐specific primers spanning each of the seven intron/exon junctions. Primary macrophages from ERα‐ and ERβ‐deficient severe combined immunodeficiency mice [ERα knockout (KO) and ERßKO, respectively] were used to delineate the effects and potential mechanisms via which steroid receptors modulate macrophage function. ERα‐deficient PM exposed ex vivo to lipopolysaccharide or Mycobacterium avium exhibited significant increases in tumor necrosis factor α (TNF‐α) secretion as well as reduction in bacterial load when compared with wild‐type (WT) PM. In contrast, ERβ‐deficient PM possessed no significant difference in TNF‐α secretion or in bacterial load when compared with WT littermates. These studies suggest that ERα, but not ERβ, modulates murine PM function.

Estrogen receptor expression and sensitivity to paclitaxel in breast cancer.

A retrospective analysis of CALGB trial 9344 suggested paclitaxel administration following cyclophosphamide and doxorubicin adjuvant chemotherapy is most beneficial for patients with ER? negative (ER?-) breast cancer. Since the cytotoxic effects of paclitaxel are cell cycle dependent, we postulated that the relationship between ER? and the effectiveness of adjuvant paclitaxel reflects the observation that ER? positive (ER?+) breast cancers proliferate more slowly than ER?- breast cancers. Three in vitro models (MCF-7, T47D and ZR-75) were examined to compare growth rates and paclitaxel-induced apoptosis in ER?+ and ER?- clones of the same, originally ER?+ cell line. For the T47D and ZR-75 cell lines, loss of ER? was associated with a decrease in doubling time and an increase in paclitaxel sensitivity. However, when cell culture conditions were altered to achieve equivalent cell proliferation rates, no difference in paclitaxel sensitivity was observed. Similarly, an ER?- clone of MCF-7 cells that did not exhibit an enhanced growth rate compared to its ER?+ counterpart also did not show increased paclitaxel sensitivity. The combined apoptotic effects of tamoxifen and paclitaxel on MCF-7 cells were not synergistic or even clearly additive. In these in vitro models, the effectiveness of paclitaxel correlated more closely with growth rate than ER? expression. These data suggest that measurements of tumor proliferation may provide more accurate predictive markers for the benefits of adjuvant paclitaxel than ER? analysis.

Estrogenic regulation of host immunity against an estrogen receptor-negative human breast cancer.

Purpose: The risk of developing breast cancer is positively correlated with exposure to increased levels of estrogen and/or an increased duration of estrogen exposure. Many different mechanisms have been proposed to explain the association of estrogens with breast cancer risk; however, the well-documented immune modulatory properties of estrogen have received little attention. In part, this is due to a lack of suitable models for studying this relationship. Experimental Design: We have developed an animal model using estrogen receptor (ER)-negative human breast cancer cell line, MDA-MB-468, xenografted into severe combined immunodeficient (SCID) mice. We also generated the ER-α knockout (ER-αKO) mice on the SCID background and then tested the ability of 17β-estradiol to stimulate growth of xenografted ER-negative human breast cancer tumors in wild-type and ER-αKO SCID mice. We quantified vascularization of tumors, macrophage recruitment to the tumor site by immunocytochemistry, and inflammatory cytokine production. Results: We show that estrogen treatment of C57BL/6/SCID mice promotes the growth of xenografted ER-negative tumors in wild-type mice and this estrogen-induced tumor growth is abrogated in ER-αKO mice. Tumor neovascularization of estrogen-treated mice was unchanged versus control; however, estrogen treatment of the C57BL/6/SCID host suppressed macrophage recruitment to and inflammatory cytokine production at the tumor site. Conclusions: These data are consistent with estrogen modulation of the inflammatory response as a contributing factor in estrogen-stimulated growth of an ER-negative tumor. This effect on the host innate immune response was mediated by ER-α.

Immunolocalization of NuMA and phosphorylated proteins during the cell cycle in human breast and prostate cancer cells as analyzed by immunofluorescence and postembedding immunoelectron microscopy

Abstract. The formation of mitotic centrosomes is a complex process in which a number of cellular proteins translocate to mitotic poles and play a critical role in the organization of the mitotic apparatus. The 238-kDa nuclear mitotic apparatus protein NuMA is one of the important proteins that plays a significant role in this process. NuMA resides in the nucleus during interphase and becomes transiently associated with mitotic centrosomes after multiple steps of phosphorylations. The role of NuMA in the interphase nucleus is not well known but it is clear that NuMA responds to external signals (such as hormones) that induce cell division, or heat shock that induces apoptosis. In order to determine the function of NuMA it is important to study its localization. Here we report on nuclear organization of NuMA during the cell cycle in estrogen responsive MCF-7 breast cancer cells and in androgen responsive LNCaP prostate cancer cells using immunoelectron microscopy, and on correlation to MPM-2 monoclonal phosphoprotein antibody. These results show that NuMA is present in speckled and punctate form associated with distinct material corresponding to a speckled or punctate immunofluorescence appearance in the nucleus while MPM-2 is uniformly dispersed in the nucleus. At prophase NuMA disperses in the cytoplasm and associates with microtubules while MPM-2 is uniformly distributed in the cytoplasm. During metaphase or anaphase anti-NuMA labeling is associated with spindle fibers. During telophase NuMA relocates to electron-dense areas around chromatin and finally to the reconstituted nuclei. These results demonstrate NuMA organization in MCF-7 and LNCaP cells in the log phase of cell culture growth.