Krista A. Riggs

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.

Regulation of integrin endocytic recycling and chemotactic cell migration by syntaxin 6 and VAMP3 interaction

Summary Integrins are the primary receptors of cells adhering to the extracellular matrix, and play key roles in various cellular processes including migration, proliferation and survival. The expression and distribution of integrins at the cell surface is controlled by endocytosis and recycling. The present study examines the function of syntaxin 6 (STX6), a t-SNARE located in the trans-Golgi network, in integrin trafficking. STX6 is overexpressed in many types of human cancer. We show that depletion of STX6 inhibits chemotactic cell migration and the delivery of the laminin receptor &agr;3&bgr;1 integrin to the cell surface, whereas STX6 overexpression stimulates chemotactic cell migration, integrin delivery, and integrin-initiated activation of focal adhesion kinase. These data indicate that STX6 plays a rate-limiting role in cell migration and integrin trafficking. In STX6-depleted cells, &agr;3&bgr;1 integrin is accumulated in recycling endosomes that contain the v-SNARE VAMP3. Importantly, we show that STX6 and VAMP3 form a v-/t-SNARE complex, VAMP3 is required in &agr;3&bgr;1 integrin delivery to the cell surface, and endocytosed &agr;3&bgr;1 integrin traffics to both VAMP3 and STX6 compartments. Collectively, our data suggest a new integrin trafficking pathway in which endocytosed integrins are transported from VAMP3-containing recycling endosomes to STX6-containing trans-Golgi network before being recycled to the plasma membrane.

Estrogen receptor alpha 46 is reduced in tamoxifen resistant breast cancer cells and re-expression inhibits cell proliferation and estrogen receptor alpha 66-regulated target gene transcription

Resistance to endocrine therapy is a major clinical problem in breast cancer. The role of ERalpha splice variants in endocrine resistance is largely unknown. We observed reduced protein expression of an N-terminally truncated ERalpha46 in endocrine-resistant LCC2, LCC9, and LY2 compared to MCF-7 breast cancer cells. Transfection of LCC9 and LY2 cells with hERalpha46 partially restored growth inhibition by TAM. Overexpression of hERalpha46 in MCF-7 cells reduced estradiol (E(2))-stimulated endogenous pS2, cyclin D1, nuclear respiratory factor-1 (NRF-1), and progesterone receptor transcription. Expression of oncomiR miR-21 was lower in TAM-resistant LCC9 and LY2 cells compared to MCF-7 cells. Transfection with ERalpha46 altered the pharmacology of E(2) regulation of miR-21 expression from inhibition to stimulation, consistent with the hypothesis that hERalpha46 inhibits ERalpha activity. Established miR-21 targets PTEN and PDCD4 were reduced in ERalpha46-transfected, E(2)-treated MCF-7 cells. In conclusion, ERalpha46 appears to enhance endocrine responses by inhibiting selected ERalpha66 responses.

Decreased Chicken Ovalbumin Upstream Promoter Transcription Factor II Expression in Tamoxifen-Resistant Breast Cancer Cells

Tamoxifen (TAM) is successfully used for the treatment and prevention of breast cancer. However, many patients that are initially TAM responsive develop tumors that are antiestrogen/TAM resistant (TAM-R). The mechanism behind TAM resistance in estrogen receptor alpha (ERalpha)-positive tumors is not understood. The orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor (COUP-TF)-I interacts directly with 4-hydroxytamoxifen (4-OHT)- and estradiol (E(2))-occupied ERalpha, corepressors NCoR and SMRT, and inhibit E(2)-induced gene transcription in breast cancer cells. Here we tested the hypothesis that reduced COUP-TFI and COUP-TFII correlate with TAM resistance. We report for the first time that COUP-TFII, but not COUP-TFI, is reduced in three antiestrogen/TAM-R cell lines derived from TAM-sensitive (TAM-S) MCF-7 human breast cancer cells and in MDA-MB-231 cells compared with MCF-7. ERalpha and ERbeta protein expression was not different between TAM-S and TAM-R cells, but progesterone receptor (PR) was decreased in TAM-R cells. Further, E(2) increased COUP-TFII transcription in MCF-7, but not TAM-R, cells. Importantly, reexpression of COUP-TFII in TAM-S cells to levels comparable to those in MCF-7 was shown to increase 4-OHT-mediated growth inhibition and increased apoptosis. Conversely, knockdown of COUP-TFII in TAM-S MCF-7 cells blocked growth inhibitory activity and increased 4-OHT agonist activity. 4-OHT increased COUP-TFII-ERalpha interaction approximately 2-fold in MCF-7 cells. COUP-TFII expression in TAM-R cells also inhibited 4-OHT-induced endogenous PR and pS2 mRNA expression. These data indicate that reduced COUP-TFII expression correlates with acquired TAM resistance in human breast cancer cell lines and that COUP-TFII plays a role in regulating the growth inhibitory activity of TAM in breast cancer cells.

Identification and characterization of nucleolin as a COUP-TFII coactivator of retinoic acid receptor β transcription in breast cancer cells.

Introduction The orphan nuclear receptor COUP-TFII plays an undefined role in breast cancer. Previously we reported lower COUP-TFII expression in tamoxifen/endocrine- resistant versus sensitive breast cancer cell lines. The identification of COUP-TFII-interacting proteins will help to elucidate its mechanism of action as a transcriptional regulator in breast cancer. Results FLAG-affinity purification and multidimensional protein identification technology (MudPIT) identified nucleolin among the proteins interacting with COUP-TFII in MCF-7 tamoxifen-sensitive breast cancer cells. Interaction of COUP-TFII and nucleolin was confirmed by coimmunoprecipitation of endogenous proteins in MCF-7 and T47D breast cancer cells. In vitro studies revealed that COUP-TFII interacts with the C-terminal arginine-glycine repeat (RGG) domain of nucleolin. Functional interaction between COUP-TFII and nucleolin was indicated by studies showing that siRNA knockdown of nucleolin and an oligonucleotide aptamer that targets nucleolin, AS1411, inhibited endogenous COUP-TFII-stimulated RARB2 expression in MCF-7 and T47D cells. Chromatin immunoprecipitation revealed COUP-TFII occupancy of the RARB2 promoter was increased by all-trans retinoic acid (atRA). RARβ2 regulated gene RRIG1 was increased by atRA and COUP-TFII transfection and inhibited by siCOUP-TFII. Immunohistochemical staining of breast tumor microarrays showed nuclear COUP-TFII and nucleolin staining was correlated in invasive ductal carcinomas. COUP-TFII staining correlated with ERα, SRC-1, AIB1, Pea3, MMP2, and phospho-Src and was reduced with increased tumor grade. Conclusions Our data indicate that nucleolin plays a coregulatory role in transcriptional regulation of the tumor suppressor RARB2 by COUP-TFII.

Atherogenesis and metabolic dysregulation in LDL receptor–knockout rats

Mechanisms of atherogenesis have been studied extensively in genetically engineered mice with disturbed cholesterol metabolism such as those lacking either the LDL receptor (Ldlr) or apolipoprotein E (apoe). Few other animal models of atherosclerosis are available. WT rabbits or rats, even on high-fat or high-cholesterol diets, develop sparse atherosclerotic lesions. We examined the effects of Ldlr deletion on lipoprotein metabolism and atherosclerotic lesion formation in Sprague-Dawley rats. Deletion of Ldlr resulted in the loss of the LDLR protein and caused a significant increase in plasma total cholesterol and triglycerides. On normal chow, Ldlr-KO rats gained more weight and were more glucose intolerant than WT rats. Plasma proprotein convertase subtilisin kexin 9 (PCSK9) and leptin levels were higher and adiponectin levels were lower in KO than WT rats. On the Western diet, the KO rats displayed exaggerated obesity and age-dependent increases in glucose intolerance. No appreciable aortic lesions were observed in KO rats fed normal chow for 64 weeks or Western diet for 16 weeks; however, after 34-52 weeks of Western diet, the KO rats developed exuberant atherosclerotic lesions in the aortic arch and throughout the abdominal aorta. The Ldlr-KO rat may be a useful model for studying obesity, insulin resistance, and early-stage atherosclerosis.

Analysis of SNARE-Mediated Exocytosis Using a Cell Fusion Assay

Exocytosis is the fusion of transport vesicles with the plasma membrane. By exocytosis, eukaryotic cells secrete soluble proteins and endogenous chemicals to the extracellular space, and deliver new membrane proteins and lipids to the plasma membrane. A large body of work has demonstrated that the interactions of SNARE (soluble N-ethylmaleimidesensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion events, including exocytosis (Bonifacino and Glick, 2004; Jahn et al., 2003; Rothman, 1994) (Fig. 1). The vesicle-associated membrane proteins (VAMPs), i.e., VAMPs 1, 2, 3, 4, 5, 7 and 8, are v-SNAREs that reside in various post-Golgi vesicular compartments, and have been implicated in exocytosis. In this chapter, we review recent progress of using a novel cell fusion assay to analyze the specificity and membrane fusion activities of VAMPs (Hasan et al., 2010).