Susan M. Dougherty

Estradiol downregulates miR-21 expression and increases miR-21 target gene expression in MCF-7 breast cancer cells

Select changes in microRNA (miRNA) expression correlate with estrogen receptor α (ERα) expression in breast tumors. miR-21 is higher in ERα positive than negative tumors, but no one has examined how estradiol (E2) regulates miR-21 in breast cancer cells. Here we report that E2 inhibits miR-21 expression in MCF-7 human breast cancer cells. The E2-induced reduction in miR-21 was inhibited by 4-hydroxytamoxifen (4-OHT), ICI 182 780 (Faslodex), and siRNA ERα indicating that the suppression is ERα-mediated. ERα and ERβ agonists PPT and DPN inhibited and 4-OHT increased miR-21 expression. E2 increased luciferase activity from reporters containing the miR-21 recognition elements from the 3′-UTRs of miR-21 target genes, corroborating that E2 represses miR-21 expression resulting in a loss of target gene suppression. The E2-mediated decrease in miR-21 correlated with increased protein expression of endogenous miR-21-targets Pdcd4, PTEN and Bcl-2. siRNA knockdown of ERα blocked the E2-induced increase in Pdcd4, PTEN and Bcl-2. Transfection of MCF-7 cells with antisense (AS) to miR-21 mimicked the E2-induced increase in Pdcd4, PTEN and Bcl-2. These results are the first to demonstrate that E2 represses the expression of an oncogenic miRNA, miR-21, by activating estrogen receptor in MCF-7 cells.

Resveratrol stimulates nitric oxide production by increasing estrogen receptor α-Src-caveolin-1 interaction and phosphorylation in human umbilical vein endothelial cells

Epidemiological studies correlate moderate red wine consumption to reduced incidence of cardiovascular disease. Resveratrol is a polyphenolic compound in red wine that has cardioprotective effects in rodents. Although endothelial cell (EC) studies indicate that micromolar resveratrol has diverse biological activities, these concentrations are not physiologically relevant because human oral ingestion provides only brief exposure to nanomolar plasma levels. Previously, we reported that nanomolar resveratrol activated ERK1/2 signaling in bovine aortic ECs (BAECs). The goal of this study was to determine the mechanisms by which nanomolar resveratrol rapidly activates endothelial nitric oxide synthase (eNOS) in human umbilical vein ECs (HUVECs). We report for the first time that resveratrol increased interaction between estrogen receptor α ( ERα ), caveolin‐1 (Cav‐1) and c‐Src, and increased phosphorylation of Cav‐1, c‐Src, and eNOS. Pretreatment with the lipid raft disruptor beta‐methyl cyclodextrin or Gα inhibitor pertussis toxin blocked resveratrol‐ and E2‐induced eNOS activation and NO production. Depletion of endogenous ERα, not ERβ, by siRNA attenuated resveratrol‐ and E2‐induced ERK1/2, Src, and eNOS phosphorylation. Our data demonstrate that nanomolar resveratrol induces ERα‐Cav‐1‐c‐SRC interaction, resulting in NO production through a Gα‐protein‐coupled mechanism. This study provides important new insights into mechanisms for the beneficial effects of resveratrol in ECs.—Klinge, C. M., Wickramasinghe, N. S., Ivanova, M. M., Dougherty, S. M. Resveratrol stimulates nitric oxide produc tion by increasing estrogen receptor α‐Src‐caveolin‐1 interaction and phosphorylation in human umbilical vein endothelial cells. FASEB J. 22, 2185–2197 (2008)

Lipid Peroxidation Product 4-Hydroxy-trans-2-nonenal Causes Endothelial Activation by Inducing Endoplasmic Reticulum Stress

Background: Oxidized lipids cause endothelial activation. Results: Endothelial activation by the lipid peroxidation product, 4-hydroxy-trans-2-nonenal, was associated with ER stress and was prevented by chaperones of protein folding. Conclusion: ER stress regulates endothelial activation by oxidized lipids. Significance: Vascular inflammation caused by oxidized lipids could be attenuated by decreasing ER stress. Lipid peroxidation products, such as 4-hydroxy-trans-2-nonenal (HNE), cause endothelial activation, and they increase the adhesion of the endothelium to circulating leukocytes. Nevertheless, the mechanisms underlying these effects remain unclear. We observed that in HNE-treated human umbilical vein endothelial cells, some of the protein-HNE adducts colocalize with the endoplasmic reticulum (ER) and that HNE forms covalent adducts with several ER chaperones that assist in protein folding. We also found that at concentrations that did not induce apoptosis or necrosis, HNE activated the unfolded protein response, leading to an increase in XBP-1 splicing, phosphorylation of protein kinase-like ER kinase and eukaryotic translation initiation factor 2α, and the induction of ATF3 and ATF4. This increase in eukaryotic translation initiation factor 2α phosphorylation was prevented by transfection with protein kinase-like ER kinase siRNA. Treatment with HNE increased the expression of the ER chaperones, GRP78 and HERP. Exposure to HNE led to a depletion of reduced glutathione and an increase in the production of reactive oxygen species (ROS); however, glutathione depletion and ROS production by tert-butyl-hydroperoxide did not trigger the unfolded protein response. Pretreatment with a chemical chaperone, phenylbutyric acid, or adenoviral transfection with ATF6 attenuated HNE-induced monocyte adhesion and IL-8 induction. Moreover, phenylbutyric acid and taurine-conjugated ursodeoxycholic acid attenuated HNE-induced leukocyte rolling and their firm adhesion to the endothelium in rat cremaster muscle. These data suggest that endothelial activation by HNE is mediated in part by ER stress, induced by mechanisms independent of ROS production or glutathione depletion. The induction of ER stress may be a significant cause of vascular inflammation induced by products of oxidized lipids.

Activity and intracellular location of estrogen receptors α and β in human bronchial epithelial cells

Gender differences in lung disease and cancer are well-established. We reported estrogenic transcriptional responses in lung adenocarcinoma cells from females but not males despite similar estrogen receptor (ER) expression. Here we tested the hypothesis that normal human bronchial epithelial cells (HBECs) show gender-independent estrogenic responses. We report that a small sample of HBECs express approximately twice as much ERbeta as ERalpha. ERalpha and ERbeta were located in the cytoplasm, nucleus, and mitochondria. In contrast to lung adenocarcinoma cells, estradiol (E2) induced estrogen response element (ERE)-mediated luciferase reporter activity in transiently transfected HBECs regardless of donor gender. Overexpression of ERalpha-VP16 increased ERE-mediated transcriptional activity in all HBECs. E2 increased and 4-hydroxytamoxifen and ICI 182,780 inhibited HBEC proliferation and cyclin D1 expression in a cell line-specific manner. In conclusion, the response of HBECs to ER ligands is gender-independent suggesting that estrogenic sensitivity may be acquired during lung carcinogenesis.

Sex Differences in Estrogen Receptor Subcellular Location and Activity in Lung Adenocarcinoma Cells

The role of estrogens in the increased risk of lung adenocarcinoma in women remains uncertain. We reported that lung adenocarcinoma cell lines from female, but not male, patients with non-small cell lung cancer respond proliferatively and transcriptionally to estradiol (E(2)), despite equal protein expression of estrogen receptors (ER) alpha and beta. To test the hypothesis that nuclear localization of ER alpha corresponds to genomic E(2) activity in lung adenocarcinoma cells from females, cell fractionation, immunoblot, and confocal immunohistochemical microscopy were performed. We report for the first time that E(2) increases phospho-serine-118-ER alpha (P-ser118-ER alpha) and cyclin D1 (CCND1) nuclear colocalization in H1793, but not A549 lung adenocarcinoma cells, derived from a female and male patient, respectively. ER beta was primarily in the cytoplasm and mitochondria, independent of E(2) treatment, and showed no difference between H1793 and A549 cells. E(2) induced higher transcription of endogenous ER alpha-regulated CCND1 in H1793 than in A549 cells. Likewise, higher rapid, non-genomic E(2)-induced extracellular signal-regulated kinase 1/2 activation was detected in H1793 compared with A549 cells, linking extracellular signal-regulated kinase activation to increased P-ser118-ER alpha. Furthermore, E(2) increased cyclin D1 and P-ser118-ER alpha nuclear localization in H1793, but not A549 cells. Together, our results indicate that nuclear localization of P-ser118-ER alpha provides one explanation for sex-dependent differences in E(2)-genomic responses in lung adenocarcinoma cell lines.

Anacardic acid inhibits estrogen receptor alpha-DNA binding and reduces target gene transcription and breast cancer cell proliferation

Anacardic acid (AnAc; 2-hydroxy-6-alkylbenzoic acid) is a dietary and medicinal phytochemical with established anticancer activity in cell and animal models. The mechanisms by which AnAc inhibits cancer cell proliferation remain undefined. AnAc 24:1ω5 was purified from geranium (Pelargonium × hortorum) and shown to inhibit the proliferation of estrogen receptor α (ERα)–positive MCF-7 and endocrine-resistant LCC9 and LY2 breast cancer cells with greater efficacy than ERα-negative primary human breast epithelial cells, MCF-10A normal breast epithelial cells, and MDA-MB-231 basal-like breast cancer cells. AnAc 24:1ω5 inhibited cell cycle progression and induced apoptosis in a cell-specific manner. AnAc 24:1ω5 inhibited estradiol (E2)–induced estrogen response element (ERE) reporter activity and transcription of the endogenous E2 target genes pS2, cyclin D1, and cathepsin D in MCF-7 cells. AnAc 24:1ω5 did not compete with E2 for ERα or ERβ binding, nor did AnAc 24:1ω5 reduce ERα or ERβ steady-state protein levels in MCF-7 cells; rather, AnAc 24:1ω5 inhibited ER-ERE binding in vitro. Virtual screening with the molecular docking software Surflex evaluated AnAc 24:1ω5 interaction with ERα ligand binding (LBD) and DNA binding (DBD) domains in conjunction with experimental validation. Molecular modeling revealed AnAc 24:1ω5 interaction with the ERα DBD but not the LBD. Chromatin immunoprecipitation experiments revealed that AnAc 24:1ω5 inhibited E2-ERα interaction with the endogenous pS2 gene promoter region containing an ERE. These data indicate that AnAc 24:1ω5 inhibits cell proliferation, cell cycle progression, and apoptosis in an ER-dependent manner by reducing ER-DNA interaction and inhibiting ER-mediated transcriptional responses. Mol Cancer Ther; 9(3); 594–605

Expression of the lncRNA Maternally Expressed Gene 3 (MEG3) Contributes to the Control of Lung Cancer Cell Proliferation by the Rb Pathway

Maternally expressed gene 3 (MEG3, mouse homolog Gtl2) encodes a long noncoding RNA (lncRNA) that is expressed in many normal tissues, but is suppressed in various cancer cell lines and tumors, suggesting it plays a functional role as a tumor suppressor. Hypermethylation has been shown to contribute to this loss of expression. We now demonstrate that MEG3 expression is regulated by the retinoblastoma protein (Rb) pathway and correlates with a change in cell proliferation. Microarray analysis of mouse embryonic fibroblasts (MEFs) isolated from mice with genetic deletion of all three Rb family members (TKO) revealed a significant silencing of Gtl2/MEG3 expression compared to WT MEFs, and re-expression of Gtl2/MEG3 caused decrease in cell proliferation and increased apoptosis. MEG3 levels also were suppressed in A549 lung cancer cells compared with normal human bronchial epithelial (NHBE) cells, and, similar to the TKO cells, re-constitution of MEG3 led to a decrease in cell proliferation and elevated apoptosis. Activation of pRb by treatment of A549 and SK-MES-1 cells with palbociclib, a CDK4/6 inhibitor, increased the expression of MEG3 in a dose-dependent manner, while knockdown of pRb/p107 attenuated this effect. In addition, expression of phosphorylation-deficient mutant of pRb increased MEG3 levels in both lung cancer cell types. Treatment of these cells with palbociclib also decreased the expression of pRb-regulated DNA methyltransferase 1 (DNMT1), while conversely, knockdown of DNMT1 resulted in increased expression of MEG3. As gene methylation has been suggested for MEG3 regulation, we found that palbociclib resulted in decreased methylation of the MEG3 locus similar to that observed with 5-aza-deoxycytidine. Anti-sense oligonucleotide silencing of drug-induced MEG3 expression in A549 and SK-MES-1 cells partially rescued the palbociclib-mediated decrease in cell proliferation, while analysis of the TCGA database revealed decreased MEG3 expression in human lung tumors harboring a disrupted RB pathway. Together, these data suggest that disruption of the pRb-DNMT1 pathway leads to a decrease in MEG3 expression, thereby contributing to the pro-proliferative state of certain cancer cells.

Inhibition of 6-phosphofructo-2-kinase (PFKFB3) suppresses glucose metabolism and the growth of HER2+ breast cancer

PurposeHuman epidermal growth factor receptor-2 (HER2) has been implicated in the progression of multiple tumor types, including breast cancer, and many downstream effectors of HER2 signaling are primary regulators of cellular metabolism, including Ras and Akt. A key downstream metabolic target of Ras and Akt is the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 isozyme (PFKFB3), whose product, fructose-2,6-bisphosphate (F26BP), is a potent allosteric activator of a rate-limiting enzyme in glycolysis, 6-phosphofructo-1-kinase (PFK-1). We postulate that PFKFB3 may be regulated by HER2 and contribute to HER2-driven tumorigenicity.MethodsImmunohistochemistry and Kaplan–Meier analysis of HER2+ patient samples investigated the relevance of PFKFB3 in HER2+ breast cancer. In vitro genetic and pharmacological inhibition of PFKFB3 was utilized to determine effects on HER2+ breast cancer cells, while HER2 antagonist treatment assessed the mechanistic regulation on PFKFB3 expression and glucose metabolism. Administration of a PFKFB3 inhibitor in a HER2-driven transgenic breast cancer model evaluated this potential therapeutic approach in vivo.ResultsPFKFB3 is elevated in human HER2+ breast cancer and high PFKFB3 transcript correlated with poorer progression-free (PFS) and distant metastatic-free (DFMS) survival. Constitutive HER2 expression led to elevated PFKFB3 expression and increased glucose metabolism, while inhibition of PFKFB3 suppressed glucose uptake, F26BP, glycolysis, and selectively decreased the growth of HER2-expressing breast cancer cells. In addition, treatment with lapatinib, an FDA-approved HER2 inhibitor, decreased PFKFB3 expression and glucose metabolism in HER2+ cells. In vivo administration of a PFKFB3 antagonist significantly suppressed the growth of HER2-driven breast tumors and decreased 18F-2-deoxy-glucose uptake.ConclusionsTaken together, these data support the potential clinical utility of PFKFB3 inhibitors as chemotherapeutic agents against HER2+ breast cancer.