Ets-1 Knockdown Research Articles

Abstract A50: The identification of an Ets1-driven gene signature in head and neck squamous cell carcinoma

Abstract Head and Neck Squamous Cell Carcinoma (HNSCC) is a heterogeneous disease of significant mortality and morbidity and with limited treatment options. This highlights the urgent need for a better understanding of the underlying biology of HNSCC. Recent genomic analysis of HNSCC tumors has identified four distinct molecular classes, of which the mesenchymal subtype is particularly aggressive in nature and associated with poor survival. We have discovered that Ets1, a prototypic oncogenic transcription factor, is selectively enriched in the mesenchymal HNSCC tumors along with other key drivers of Epithelial to Mesenchymal transition (EMT). Further analysis of gene expression datasets reveals that Ets1 expression, while low in normal epithelium, increases in pre-malignant and malignant tissues, suggesting a possible role for Ets1 during early tumorigenesis. Strikingly, Kaplan Meier analysis shows that Ets1 expression is associated with both poor survival in HNSCC and high incidence of tumor recurrence. We have performed hierarchical clustering of 33 HNSCC cell lines to show that these cell lines exhibit gene expression profiles that are representative of the four tumor subtypes. To better understand the Ets1-driven molecular processes in EMT high tumors, we have utilized SCC25, a mesenchymal HNSCC cell line to perform global transcriptomic analysis of effects of Ets1 knockdown. Additionally, we have also performed ChIP-seq experiments to identify genome wide transcriptional targets of Ets1 in SCC25. Taken together, these studies have uncovered an Ets1 intrinsic core gene signature that can serve as a valuable tool for further tumor classification and cancer-specific pathway discovery. Interestingly, by cross-referencing with the Cancer Genome Atlas HNSCC RNA-Seq dataset, we demonstrate that Ets1 expression is highly correlated with the majority of the Ets1 signature target genes thus reinforcing a functional relationship. Collectively, our findings not only identify several Ets1 dependent genes that are key to initiating and maintaining EMT in HNSCC, but also unearth novel targets that can be exploited for future therapeutics against this deadly cancer. Citation Format: Christian Gluck, Isha Sethi, Maria Tsompana, Satrajit Sinha. The identification of an Ets1-driven gene signature in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A50.

Luteolin Inhibits Ischemia/Reperfusion-Induced Myocardial Injury in Rats via Downregulation of microRNA-208b-3p.

BackgroundLuteolin (LUT), a kind of flavonoid which is extracted from a variety of diets, has been reported to convey protective effects of various diseases. Recent researches have suggested that LUT can carry out cardioprotective effects during ischemia/reperfusion (I/R). However, there have no reports on whether LUT can exert protective effects against myocardial I/R injury through the actions of specific microRNAs (miRs). The purpose of this study was to determine which miRs and target genes LUT exerted such function through.MethodsExpression of various miRs in perfused rat hearts was detected using a gene chip. Target genes were predicted with TargetScan, MiRDB and MiRanda. Anoxia/reoxygenation was used to simulate I/R. Cells were transfected by miR-208b-3p mimic, inhibitor and small interfering RNA of Ets1 (avian erythroblastosis virus E26 (v ets) oncogene homolog 1). MiR-208b-3p and Ets1 mRNA were quantified by real-time quantitative polymerase chain reaction. The percentage of apoptotic cells was detected by annexin V-fluorescein isothiocyanate/propidium iodide dyeing and flow cytometry. The protein expression levels of cleaved caspase-3, Bcl-2, Bax, and Ets1 were examined by western blot analysis. A luciferase reporter assay was used to verify the combination between miR-208b-3p and the 3’-untranslated region of Ets1.ResultsLUT pretreatment reduced miR-208b-3p expression in myocardial tissue, as compared to the I/R group. And LUT decreased miR-208b-3p expression and apoptosis caused by I/R. However, overexpression of miR-208b-3p further aggravated the changes caused by I/R and blocked all the effects of LUT. Knockdown of miR-208b-3p expression also attenuated apoptosis, while knockdown of Ets1 promoted apoptosis. Further, the luciferase reporter assay showed that miR-208b-3p could inhibit Ets1 expression.ConclusionLUT pretreatment conveys anti-apoptotic effects after myocardial I/R injury by decreasing miR-208b-3p and increasing Ets1 expression levels.

Transcription factor Ets-1 links glucotoxicity to pancreatic beta cell dysfunction through inhibiting PDX-1 expression in rodent models.

'Glucotoxicity' is a term used to convey the negative effect of hyperglycaemia on beta cell function; however, the underlying molecular mechanisms that impair insulin secretion and gene expression are poorly defined. Our objective was to define the role of transcription factor v-ets avian erythroblastosis virus E26 oncogene homologue 1 (Ets-1) in beta cell glucotoxicity. Primary islets and Min6 cells were exposed to high glucose and Ets-1 expression was measured. Recombinant adenovirus and transgenic mice were used to upregulate Ets-1 expression in beta cells in vitro and in vivo, and insulin secretion was assessed. The binding activity of H3/H4 histone on the Ets-1 promoter, and that of forkhead box (FOX)A2, FOXO1 and Ets-1 on the Pdx-1 promoter was measured by chromatin immunoprecipitation and quantitative real-time PCR assay. High glucose induced upregulation of Ets-1 expression and hyperacetylation of histone H3 and H4 at the Ets-1 gene promoter in beta cells. Ets-1 overexpression dramatically suppressed insulin secretion and biosynthesis both in vivo and in vitro. Besides, Ets-1 overexpression increased the activity of FOXO1 but decreased that of FOXA2 binding to the pancreatic and duodenal homeobox 1 (PDX-1) homology region 2 (PH2), resulting in inhibition of Pdx-1 promoter activity and downregulation of PDX-1 expression and activity. In addition, high glucose promoted the interaction of Ets-1 and FOXO1, and the activity of Ets-1 binding to the Pdx-1 promoter. Importantly, PDX-1 overexpression reversed the defect in pancreatic beta cells induced by Ets-1 excess, while knockdown of Ets-1 prevented hyperglycaemia-induced dysfunction of pancreatic beta cells. Our observations suggest that Ets-1 links glucotoxicity to pancreatic beta cell dysfunction through inhibiting PDX-1 expression in type 2 diabetes.

MicroRNA-129-5p inhibits hepatocellular carcinoma cell metastasis and invasion via targeting ETS1

MiR-129-5p is deregulated in various human cancers and has been associated with hepatocellular carcinoma (HCC) progression. However, the underlying mechanisms of miR-129-5p involvement in the development and progression of HCC and the effects of miR-129-5p deregulation on the clinical characteristics observed in HCC patients remain poorly understood. We therefore investigated the correlation between low miR-129-5p expression and vascular invasion, intrahepatic metastasis, and poor patient survival. Ectopic restoration of miR-129-5p expression in HCC cells suppressed cellular migration and invasion and the expression of v-ets erythroblastosis virus E26 oncogene homolog 1 (ETS1), while inhibition of endogenous miR-129-5p caused an increase in these parameters. We identified the ETS1 gene as a novel direct target of miR-129-5p. SiRNA-mediated ETS1 knockdown rescued the effects of anti-miR-129-5p inhibitor in HCC cell lines, while the effects of miR-129-5p overexpression were partially phenocopied in the knockdown model. In addition, miR-129-5p levels inversely correlated with those of ETS1 in HCC cells and tissues. Taken together, our findings indicate an important role for miR-129-5p in the molecular etiology of invasive HCC and suggest that miR-129-5p could have potential therapeutic applications in HCC.

β6 integrin induces the expression of metalloproteinase-3 and metalloproteinase-9 in colon cancer cells via ERK-ETS1 pathway

We previously reported that β6 integrin played an important role in the progression of colon cancer. In this study, we demonstrated that β6 integrin induced the expression of MMP-3/MMP-9 and the invasion of colon cancer cells. Moreover, that function was abolished by the inhibition of ERK/MAPK pathways or knockdown of ETS1, an important transcription factor of MMP genes. Here, we showed that β6 induced phosphorylation of ETS1 via the ERK/MAPK pathways, through which the MMP-3/MMP-9 promoters were stimulated, thereby leading to the up-regulation of MMP-3/MMP-9, and subsequent the invasion of colon cancer cells.

Transcription factor Ets1 regulates expression of thioredoxin-interacting protein and inhibits insulin secretion in pancreatic β-cells.

Long-term activation of extracellular-regulated kinase (ERK1/2) pathway has been shown to cause glucotoxicity and inhibit insulin gene expression in β-cells. Transcription factor Ets1 is activated by ERK1/2-mediated phosphorylation at the Thr38 residue. We hypothesize that Ets1 plays an important role in mediating ERK1/2 induced glucotoxicity in β-cells. We determined the role of Ets1 in Min6 cells and isolated mouse islets using overexpression and siRNA mediated knockdown of Ets1. The results show that Ets1 was localized in insulin-staining positive cells but not in glucagon-staining positive cells. Overexpression of Ets1 reduced glucose-stimulated insulin secretion in primary mouse islets. Overexpression of Ets1 in Min6 β-cells and mouse islets increased expression of thioredoxin-interacting protein (TXNIP). Conversely, knockdown of Ets1 by siRNA reduced expression of TXNIP in Min6 cells. Ets1 was associated with the txnip promoter in min6 cells and transfection of 293 cells with Ets1 and p300 synergistically increased txnip promoter reporter activity. Moreover, overexpression of Ets1 inhibited Min6 cell proliferation. Our results suggest that Ets1, by promoting TXNIP expression, negatively regulates β-cell function. Thus, over-activation of Ets1 may contribute to diet-induced β-cell dysfunction.

Ets‐1 controls breast cancer cell balance between invasion and growth

Ets-1 overexpression in human breast cancers is associated with invasiveness and poor prognosis. By overexpressing Ets-1 or a dominant negative mutant in MMT breast cancer cells, we previously highlighted the key role of Ets-1 in coordinating multiple invasive features of these cells. Interestingly, we also noticed that Ets-1 decreased the density of breast cancer cells cultured in three-dimensional extracellular matrix gels. The 3D context was instrumental to this phenomenon, as such downregulation was not observed in cells grown on two-dimensional plastic or matrix-coated dishes. Ets-1 overexpression was deleterious to anchorage-independent growth of MMT cells in soft agar, a standard model for in vitro tumorigenicity. The relevance of this mechanism was confirmed in vivo, during primary tumor growth and in a metastatic assay of lung colonization. In these models, Ets-1 was associated with epithelial-to-mesenchymal transition features and modulated the ratio of Ki67-positive cells, while hardly affecting in vivo apoptotic cell death. Finally, siRNA-mediated knockdown of Ets-1 in human breast cancer cell lines also decreased colony growth, both in anchorage-independent assays and 3D extracellular matrix cultures. These in vitro and in vivo observations shed light on an unsuspected facet of Ets-1 in breast tumorigenesis. They show that while promoting malignancy through the acquisition of invasive features, Ets-1 also attenuates breast tumor cell growth and could therefore repress the growth of primary tumors and metastases. This work also demonstrates that 3D models may reveal mechanisms of tumor biology that are cryptic in standard 2D models.

P082 Divergent effects of gut bacteria altered in dysbiosis of inflammatory bowel disease on monocytes and macrophage polarisation

Background: Fistulae are a frequent complication in Crohn’s disease patients. Epithelial to mesenchymal transition (EMT) has been identified as an important driving force of their formation. On the other hand, the importance of ER stress and related pathways in intestinal inflammation has been demonstrated by recent evidence from animal models, the phenotype of human IBD, as well as genetic data. We showed previously, that ER stress leads to the onset of EMT in intestinal epithelial cells. Here, we investigated candidate signaling pathways and therapeutic substances. Methods: HT-29 cells were stimulated with the ER stress inducer tunicamycin (TM). In order to identify candidate pathways, we used specific inhibitors targeting JNK, Src, Smad or siRNA targeting Ets1 transcription factor prior to stimulation with TM. Polyamines, butyrates and bile acids were tested for their potential therapeutic effect by pre-treating the cells with the substances followed by addition of TM. The extent of ER stress and EMT was determined by measuring typical markers by Western blot and quantitative real time PCR. Results: Upon treatment with TM induction of the transcription factor Ets1 was found, which is known to be induced upon ER stress and to play a role in induction of EMT. However, knockdown of Ets1 by siRNA did not influence mRNA levels of the typical fibroblast markers vimentin, beta-6-integrin or the ER stress marker gp96. Similar, inhibiting Smad only lead to slightly reduced levels of beta-6-integrin. On the other hand, stimulation with JNK inhibitor II revealed that induction of Ets, beta-6-integrin and gp96 was JNK-dependent. In addition, the Src kinase inhibitor PP2 reduced the expression of Ets1 and gp96, while induction of beta-6-integrin was found to be Src-independent. Analysing potential therapeutic substances, neither spermidine nor sodium phenylbutyrate could counteract the effect of TM. However, pre-incubation of the cells with 10 mM of the bile acid tauroursodeoxycholic acid decreased significantly the levels of vimentin, beta-6-integrin and gp96. Conclusions: Our data suggest that the JNK and Src pathway are involved in the regulation of ER stress-mediated EMT during fistula formation in Crohn’s disease. The bile acid tauroursodeoxycholic acid represents a therapeutic treatment option.

Akt2- and ETS1-Dependent IP3 Receptor 2 Expression in Dendritic Cell Migration

Background/Aims: The protein kinase Akt2/PKBβ is a known regulator of macrophage and dendritic cell (DC) migration. The mechanisms linking Akt2 activity to migration remained, however, elusive. DC migration is governed by Ca²⁺ signaling. We thus explored whether Akt2 regulates DC Ca²⁺ signaling. Methods: DCs were derived from bone marrow of Akt2-deficient mice (akt2^-/-) and their wild type littermates (akt2^+/+). DC maturation was induced by lipopolysaccharides (LPS) and evaluated by flow cytometry. Cytosolic Ca²⁺ concentration was determined by Fura-2 fluorescence, channel activity by whole cell recording, transcript levels by RT-PCR, migration utilizing transwells. Results: Upon maturation, chemokine CCL21 stimulated migration of akt2^+/+ but not akt2^-/- DCs. CCL21-induced increase in cytosolic Ca²⁺ concentration, thapsigargin-induced release of Ca²⁺ from intracellular stores with subsequent store-operated Ca²⁺ entry (SOCE), ATP-induced inositol 1,4,5-trisphosphate (IP₃)-dependent Ca²⁺ release as well as Ca²⁺ release-activated Ca²⁺ (CRAC) channel activity were all significantly lower in mature akt2^-/- than in mature akt2^+/+ DCs. Transcript levels of IP₃ receptor IP₃R2 and of IP₃R2 regulating transcription factor ETS1 were significantly higher in akt2^+/+ than in akt2^-/- DCs prior to maturation and were upregulated by LPS stimulation (1h) in akt2^+/+ and to a lower extent in akt2^-/- DCs. Following maturation, protein abundance of IP₃R2 and ETS1 were similarly higher in akt2^+/+ than in akt2^-/- DCs. The IP₃R inhibitor Xestospongin C significantly decreased CCL21-induced migration of akt2^+/+DCs and abrogated the differences between genotypes. Finally, knock-down of ETS1 with siRNA decreased IP₃R2 mRNA abundance, thapsigargin- and ATP-induced Ca²⁺ release, SOCE and CRAC channel activation, as well as DC migration. Conclusion: Akt2 upregulates DC migration at least in part by ETS1-dependent stimulation of IP₃R2 transcription.

Resveratrol Inhibits Hydrogen Peroxide-Induced Apoptosis in Endothelial Cells via the Activation of PI3K/Akt by miR-126

Resveratrol(RSV) is an edible polyphenolic phytoalexin present in different plant species that plays an important role in improving endothelial dysfunction. However, the molecular mechanisms underlying these effects are unknown. In the present study, the mechanism underlying the protection of CRL-1730 cells by RSV against oxidative stress was examined. We first assessed the effects of RSV on the cell viability and apoptosis of CRL-1730 cells exposed to hydrogen peroxide(H2O2). Real-time PCR was used to determine the microRNA-126(miR-126) expression in cells treated with RSV and/or H2O2. We also evaluated the PI3K/Akt signaling pathway in CRL-1730 cells following upregulation of the miR-126 expression. Finally, we determined the effects of miR-126 on RSV against oxidative injury using an miR-126 inhibitor. Treatment with RSV resulted in a significant increase in survival and a decrease in the apoptosis of CRL-1730 cells exposed to H2O2. We also found that H2O2 significantly suppressed the expression of miR-126, which was reversed by RSV in a dose-dependent manner. The overexpression of miR-126 decreased PIK3R2(p85-β) and enhanced Akt phosphorylation, which resulted in an increase in the survival of CRL-1730 cells exposed to H2O2. More importantly, the downregulation of the miR-126 expression reversed the effects of RSV on the survival and apoptosis of CRL-1730 cells exposed to H2O2. In addition, the knockdown of Ets-1 reversed the effects of RSV on the miR-126 expression in CRL-1730 cells exposed to H2O2. In this study, we demonstrated that the protection of endothelial cells by RSV against oxidative injury is due to the activation of PI3K/Akt by miR-126.

Activation of the Ig Iα1 promoter by the transcription factor Ets-1 triggers Ig Iα1–Cα1 germline transcription in epithelial cancer cells

Immunoglobulins (Igs) are known to be synthesized and secreted only by B lymphocytes. Class switch recombination (CSR) is a key event that enables B cells to express Igs, and one of the crucial steps for CSR initiation is the germline transcription of Ig genes. Surprisingly, recent studies have demonstrated that the Ig genes are also expressed in some epithelial cancer cells; however, the mechanisms underlying how cancer cells initiate CSR and express Igs are still unknown. In this study, we confirmed that the Ig Iα1 promoter in cancer cell lines was activated by the Ets-1 transcription factor, and the activity of the Ig Iα1 promoter and Ig Iα1-Cα1 germline transcription were attenuated after knockdown of Ets-1 by specific small interfering RNAs (siRNA). Furthermore, the expression of Ets-1 and Igα heavy chain in cancer cells was dose dependently upregulated by TGF-β1. These results indicate that activation of the Ig Iα1 promoter by the transcription factor Ets-1 is a critical pathway and provides a novel mechanism for Ig expression in non-B cell cancers.

ETS-1-mediated Transcriptional Up-regulation of CD44 Is Required for Sphingosine-1-phosphate Receptor Subtype 3-stimulated Chemotaxis

Sphingosine-1-phosphate (S1P)-regulated chemotaxis plays critical roles in various physiological and pathophysiological conditions. S1P-regulated chemotaxis is mediated by the S1P family of G-protein-coupled receptors. However, molecular details of the S1P-regulated chemotaxis are incompletely understood. Cultured human lung adenocarcinoma cell lines abundantly express S1P receptor subtype 3 (S1P3), thus providing a tractable in vitro system to characterize molecular mechanism(s) underlying the S1P3 receptor-regulated chemotactic response. S1P treatment enhances CD44 expression and induces membrane localization of CD44 polypeptides via the S1P3/Rho kinase (ROCK) signaling pathway. Knockdown of CD44 completely diminishes the S1P-stimulated chemotaxis. Promoter analysis suggests that the CD44 promoter contains binding sites of the ETS-1 (v-ets erythroblastosis virus E26 oncogene homolog 1) transcriptional factor. ChIP assay confirms that S1P treatment stimulates the binding of ETS-1 to the CD44 promoter region. Moreover, S1P induces the expression and nuclear translocation of ETS-1. Knockdown of S1P3 or inhibition of ROCK abrogates the S1P-induced ETS-1 expression. Furthermore, knockdown of ETS-1 inhibits the S1P-induced CD44 expression and cell migration. In addition, we showed that S1P3/ROCK signaling up-regulates ETS-1 via the activity of JNK. Collectively, we characterized a novel signaling axis, i.e., ROCK-JNK-ETS-1-CD44 pathway, which plays an essential role in the S1P3-regulated chemotactic response.

Ets-1 global gene expression profile reveals associations with metabolism and oxidative stress in ovarian and breast cancers

BackgroundThe Ets-1 proto-oncogene is frequently upregulated in cancer cells, with known involvement in cancer angiogenesis, metastasis, and more recently energy metabolism. In this study we have performed various bioinformatic analyses on existing microarray data to further clarify the role of Ets-1 in ovarian cancer, and validated these results with functional assays.MethodsFunctional pathway analyses were conducted on existing microarray data comparing 2008 and 2008-Ets1 ovarian cancer cells. Methods included over-representation analysis, functional class scoring and pathway topology, and network representations were visualized in Cytoscape. Oxidative stress regulation was examined in ovarian cancer cells by measuring protein expression and enzyme activity of glutathione peroxidases, as well as intracellular reactive oxygen species using dichlorofluorescin fluorescence. A stable Ets-1 knockdown MDA-MB-231 cell line was created using short hairpin RNA, and glycolytic dependence of these cells was measured following treatment with 2-deoxy-D-glucose and Hoechst nuclear staining to determine cell number. High-resolution respirometry was performed to measure changes in basal oxygen flux between MDA-MB-231 cells and MDA-Ets1KD variants.ResultsEnrichments in oxidoreductase activity and various metabolic pathways were observed upon integration of the different analyses, suggesting that Ets-1 is important in their regulation. As oxidative stress is closely associated with these pathways, we functionally validated our observations by showing that Ets-1 overexpression resulted in decreased reactive oxygen species with increased glutathione peroxidase expression and activity, thereby regulating cellular oxidative stress. To extend our findings to another cancer type, we developed an Ets-1 knockdown breast cancer cell model, which displayed decreased glycolytic dependence and increased oxygen consumption following Ets-1 knockdown confirming our earlier findings.ConclusionsCollectively, this study confirms the important role of Ets-1 in the regulation of cancer energy metabolism in ovarian and breast cancers. Furthermore, Ets-1 is a key regulator of oxidative stress in ovarian cancer cells by mediating alterations in glutathione antioxidant capacity.

Abstract 4286: Hepatocyte growth factor-mediated gastrin-releasing peptide induces IL-8 expression through Ets-1 in gastric cancer cells.

Abstract Gastric cancer cells secrete a variety of pro-angiogenic molecules, including IL-8 and VEGF. However, factors regulating the expression of pro-angiogenic genes for gastric cancer remain largely undefined. We investigated the role of HGF-induced activation of GRP and Ets-1 transcription factor in expression of the pro-angioigenic factor, IL-8. The genes associated with angiogenesis induced by HGF were screened using cDNA microarray technology in two gastric cancer cell lines (NUGC-3 and MKN-28). First, GRP RNA and protein were confirmed to be up-regulated. Then, expression of GRP, Ets-1, and IL-8 were further estimated by Western blot analysis. A role for Ets-1 in HGF-induced up-regulation of IL-8 was determined by knockdown of Ets-1 with Ets-1 sh-RNA and a chromatin immune precipitation assay. The levels of GRP, Ets-1, and IL-8 were up-regulated in cells treated with HGF in a dose-dependent manner. HGF-induced expression of Ets-1 and IL-8 was increased more by GRP treatment, and inhibited by pre-treatment with an ERK 1/2 inhibitor (PD098059). HGF-induced up-regulation of IL-8 was repressed by Ets-1 knockdown. HGF enhanced the binding activity of Ets-1 to the IL-8 promoter in control cells, but not in the Ets-1 shRNA cells. We confirmed the functional role of HGF-induced Ets-1 in activation of the IL-8 promoter by the reporter gene assay. Down-regulation of IL-8 also decreased in vitro cell invasion. In conclusion, HGF mediated the GRP induction of IL-8 expression through Ets-1, which thus might serve as a promising target for gastric cancer therapy. Key words: HGF, GRP, Ets-1, IL-8 Citation Format: Sung Ae Koh, Kyung Hee Lee, Eun Young Choi, Min Kyung Kim, Byung Ik Jang, Se Won Kim, Sang Woon Kim, Sun Kyo Song, Jae Ryong Kim. Hepatocyte growth factor-mediated gastrin-releasing peptide induces IL-8 expression through Ets-1 in gastric cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4286. doi:10.1158/1538-7445.AM2013-4286

Ets-1 Is Required for the Activation of VEGFR3 during Latent Kaposi's Sarcoma-Associated Herpesvirus Infection of Endothelial Cells

Kaposi's sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi's sarcoma (KS), is present in the predominant tumor cells of KS, the spindle cells. Spindle cells express markers of lymphatic endothelium and, interestingly, KSHV infection of blood endothelial cells reprograms them to a lymphatic endothelial cell phenotype. KSHV-induced reprogramming requires the activation of STAT3 and phosphatidylinositol 3 (PI3)/AKT through the activation of cellular receptor gp130. Importantly, KSHV-induced reprogramming is specific to endothelial cells, indicating that there are additional host genes that are differentially regulated during KSHV infection of endothelial cells that contribute to lymphatic reprogramming. We found that the transcription factor Ets-1 is highly expressed in KS spindle cells and is upregulated during KSHV infection of endothelial cells in culture. The KSHV latent vFLIP gene is sufficient to induce Ets-1 expression in an NF-κB-dependent fashion. Ets-1 is required for KSHV-induced expression of VEGFR3, a lymphatic endothelial-cell-specific receptor important for lymphangiogenesis, and Ets-1 activates the promoter of VEGFR3. Ets-1 knockdown does not alter the expression of another lymphatic-specific gene, the podoplanin gene, but does inhibit the expression of VEGFR3 in uninfected lymphatic endothelium, indicating that Ets-1 is a novel cellular regulator of VEGFR3 expression. Knockdown of Ets-1 affects the ability of KSHV-infected cells to display angiogenic phenotypes, indicating that Ets-1 plays a role in KSHV activation of endothelial cells during latent KSHV infection. Thus, Ets-1 is a novel regulator of VEGFR3 and is involved in the induction of angiogenic phenotypes by KSHV.

MiRNA-145 Targets v-ets Erythroblastosis Virus E26 Oncogene Homolog 1 to Suppress the Invasion, Metastasis, and Angiogenesis of Gastric Cancer Cells

Recent evidence shows that v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets1) is implicated in tumor development and progression. However, the clinical potentials and underlying mechanisms of Ets1 in gastric cancer progression and metastasis remain largely unknown. In this study, Ets1 immunostaining was identified in 56 of 84 (66.7%) gastric cancer tissues, which was correlated with tumor invasion and metastasis. In gastric cancer specimens and cell lines, miRNA-145 (miR-145) was downregulated and inversely correlated with Ets1 expression. Bioinformatics analysis and luciferase reporter assay revealed that miR-145 directly targeted the 3'-untranslated region (3'-UTR) of Ets1 mRNA. Overexpression or knockdown of miR-145 responsively altered both the mRNA and protein levels of Ets1 and its downstream genes, matrix metalloproteinase (MMP-1)-1 and -9, in gastric cancer cell lines SGC-7901 and MKN-45. Ectopic expression of miR-145 suppressed the invasion, metastasis, and angiogenesis of SGC-7901 and MKN-45 cells in vitro and in vivo. In addition, the effects of miR-145 on Ets1 expression, migration, invasion, and angiogenesis were rescued by restoration of Ets1 expression in these cells. Furthermore, anti-miR-145 inhibitor promoted the migration, invasion, and angiogenesis, whereas siRNA-mediated Ets1 knockdown phenocopied the effects of miR-145 overexpression in gastric cancer cells. These results show that miR-145 suppresses Ets1 expression via the binding site in the 3'-UTR, thus inhibiting the invasion, metastasis, and angiogenesis of gastric cancer cells.

Ets1 and Elk1 transcription factors regulate cancerous inhibitor of protein phosphatase 2A expression in cervical and endometrial carcinoma cells

Cancerous inhibitor of protein phosphatase 2A (CIP2A) has been identified as a proto-oncogene that is overexpressed in various types of human cancers. CIP2A acts by inhibiting protein phosphatase 2A-dependent destabilization of c-Myc, resulting in increased cell proliferation. Here, we have characterized the proximal promoter region of the human CIP2A gene in cervical, endometrial and liver carcinoma cells. The 5′ flanking minimal proximal promoter of the CIP2A gene consists of putative binding sites for Ets1 and Elk1 in forward and reverse orientations. Here, we show that Ets1 and Elk1 binding is essential for CIP2A basal expression in several urogenital cancer cell lines. Interestingly, both Ets1 and Elk1 are required together for CIP2A expression, as siRNA knockdown of Ets1 and Elk1 together decreased CIP2A gene transcription, whereas knockdown of Ets1 or Elk1 alone had no effect. Moreover, ectopic expression of Ets1 and Elk1 together increased CIP2A expression. To gain physiological significance of the Ets1 and Elk1 regulation we observed, a panel of matched human cervical carcinoma samples was analyzed for the expression of CIP2A and Ets1 and/or Elk1. We found a direct correlation between the levels of CIP2A and the levels of Ets1 and Elk1. Our results suggest that the binding of Ets1 and Elk1 together to the proximal CIP2A promoter is absolutely required for CIP2A expression in cervical, endometrial and liver carcinoma cell lines. Thus, different factors regulate CIP2A expression in a cell-type specific manner. As previous work has shown a requirement for only Ets1 in prostate and gastric carcinomas, our results now indicate that CIP2A regulation is more complex than previously determined.

PI3K Inhibition Impairs BRCA1/2 Expression and Sensitizes BRCA-Proficient Triple-Negative Breast Cancer to PARP Inhibition

PARP inhibitors are active in tumors with defects in DNA homologous recombination (HR) due to BRCA1/2 mutations. The phosphoinositide 3-kinase (PI3K) signaling pathway preserves HR steady state. We hypothesized that in BRCA-proficient triple-negative breast cancer (TNBC), PI3K inhibition would result in HR impairment and subsequent sensitization to PARP inhibitors. We show in TNBC cells that PI3K inhibition leads to DNA damage, downregulation of BRCA1/2, gain in poly-ADP-ribosylation, and subsequent sensitization to PARP inhibition. In TNBC patient-derived primary tumor xenografts, dual PI3K and PARP inhibition with BKM120 and olaparib reduced the growth of tumors displaying BRCA1/2 downregulation following PI3K inhibition. PI3K-mediated BRCA downregulation was accompanied by extracellular signal-regulated kinase (ERK) phosphorylation. Overexpression of an active form of MEK1 resulted in ERK activation and downregulation of BRCA1, whereas the MEK inhibitor AZD6244 increased BRCA1/2 expression and reversed the effects of MEK1. We subsequently identified that the ETS1 transcription factor was involved in the ERK-dependent BRCA1/2 downregulation and that knockdown of ETS1 led to increased BRCA1/2 expression, limiting the sensitivity to combined BKM120 and olaparib in 3-dimensional culture. Treatment options are limited for patients with TNBCs. PARP inhibitors have clinical activity restricted to a small subgroup of patients with BRCA mutations. Here, we show that PI3K blockade results in HR impairment and sensitization to PARP inhibition in TNBCs without BRCA mutations, providing a rationale to combine PI3K and PARP inhibitors in this indication. Our findings could greatly expand the number of patients with breast cancer that would benefit from therapy with PARP inhibitors. On the basis of our findings, a clinical trial with BKM120 and olaparib is being initiated in patients with TNBCs.

The MicroRNA miR-199a-5p Down-regulation Switches on Wound Angiogenesis by Derepressing the v-ets Erythroblastosis Virus E26 Oncogene Homolog 1-Matrix Metalloproteinase-1 Pathway

miR-199a-5p plays a critical role in controlling cardiomyocyte survival. However, its significance in endothelial cell biology remains ambiguous. Here, we report the first evidence that miR-199a-5p negatively regulates angiogenic responses by directly targeting v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1). Induction of miR-199a-5p in human dermal microvascular endothelial cells (HMECs) blocked angiogenic response in Matrigel® culture, whereas miR-199a-5p-deprived cells exhibited enhanced angiogenesis in vitro. Bioinformatics prediction and miR target reporter assay recognized Ets-1 as a novel direct target of miR-199a-5p. Delivery of miR-199a-5p blocked Ets-1 expression in HMECs, whereas knockdown endogenous miR-199a-5p induced Ets-1 expression. Matrix metalloproteinase 1 (MMP-1), one of the Ets-1 downstream mediators, was negatively regulated by miR-199a-5p. Overexpression of Ets-1 not only rescued miR-199a-5p-dependent anti-angiogenic effects but also reversed miR-199a-5p-induced loss of MMP-1 expression. Similarly, Ets-1 knockdown blunted angiogenic response and induction of MMP-1 in miR-199a-5p-deprived HMECs. Examination of cutaneous wound dermal tissue revealed a significant down-regulation of miR-199a-5p expression, which was associated with induction of Ets-1 and MMP-1. Mice carrying homozygous deletions in the Ets-1 gene exhibited blunted wound blood flow and reduced abundance of endothelial cells. Impaired wound angiogenesis was associated with compromised wound closure, insufficient granulation tissue formation, and blunted induction of MMP-1. Thus, down-regulation of miR-199a-5p is involved in the induction of wound angiogenesis through derepressing of the Ets-1-MMP1 pathway.

Ets-1 is a transcriptional mediator of oncogenic nitric oxide signaling in estrogen receptor-negative breast cancer

IntroductionThe Ets-1 transcription factor is a candidate breast cancer oncogene that regulates the expression of genes involved in tumor progression and metastasis. Ets-1 signaling has also been linked to the development of a basal-like breast cancer phenotype. We recently described a nitric oxide (NO)-induced gene signature that is associated with poor disease outcome in estrogen receptor-negative (ER-) breast cancer and contains both stem cell-like and basal-like components. Thus, we examined the role of Ets-1 in NO signaling and NO-induced phenotypes in ER- human breast cancer cells.MethodsPromoter region analyses were performed on genes upregulated in inducible nitric oxide synthase (NOS2) high expressing tumors for Ets-binding sites. In vitro mechanisms were examined in human basal-like breast cancer cells lines. NO signaling effects were studied using either forced NOS2 expression or the use of a chemical NO-donor, diethlylenetriamine NONOate (DETANO).ResultsPromoter region analysis of genes that are up-regulated in human ER-negative breast tumors with high NOS2 expression revealed that the Ets-binding sequence is the only common promoter element present in all of these genes, indicating that Ets-1 is the key transcriptional factor down-stream of oncogenic NOS2-signaling. Accordingly, both forced NOS2 over-expression and exposure to NO-donors resulted in significant Ets-1 transcriptional activation in ER- breast cancer cells. Functional studies showed that NO activated Ets-1 transcriptional activity via a Ras/MEK/ERK signaling pathway by a mechanism that involved Ras S-nitrosylation. RNA knock-down of Ets-1 suppressed NO-induced expression of selected basal-like breast cancer markers such as P-cadherin, S100A8, IL-8 and αβ-crystallin. Additionally, Ets-1 knock-down reduced NO-mediated cellular proliferation, matrix metalloproteinase and cathepsin B activities, as well as matrigel invasion.ConclusionsThese data show that Ets-1 is a key transcriptional mediator of oncogenic NO signaling that promotes the development of an aggressive disease phenotype in ER- breast cancer in an Ets-1 and Ras-dependent manner, providing novel clues of how NOS2 expression in human breast tumors is functionally linked to poor patient survival.