Ets-1 Protein Expression Research Articles

Potential influence of MIR-204-3P on ETS-1 protein expression in papillary thyroid carcinoma

Potential influence of MIR-204-3P on ETS-1 protein expression in papillary thyroid carcinoma

In Vivo Inhibition of MicroRNA-326 in a NOD.H-2h4 Mouse Model of Autoimmune Thyroiditis.

BackgroundPrevious studies reported that various miRNAs participate in autoimmune diseases, but the potential regulatory mechanism of miRNAs in autoimmune thyroiditis (AIT) needs further exploration.ObjectiveThis study aimed to further verify that miR-326 contributes to AIT by regulating Th17/Treg balance through Ets-1 using lentiviral gene delivery through tail vein and thyroid injection in NOD.H-2h4 mice.Materials and MethodsFive-week-old NOD.H-2h4 mice were divided randomly into tail vein and thyroid injection groups, and each received either mmu-miR-326 sponge (LV-sponge) or lentiviral vector control. Mice were divided for tail vein injection: the therapeutic LV-ctrl, therapeutic LV-sponge, prophylactic LV-ctrl, and prophylactic LV-sponge groups. The control group was fed high-iodine water without vein injection. The thyroid infiltration of lymphocytes and serum TgAb value were investigated by thyroid hematoxylin and eosin (HE) staining and ELISA, respectively. Ets-1 and lymphocyte counts were measured by RT-PCR, western blotting, and flow cytometry. The thyroid CD4+IL-17a+ cells and CD4+Ets-1+ cells were detected by immunofluorescence, and the serum cytokines were tested by ELISA.ResultsIn the tail vein injection groups, the thyroid inflammatory score and serum TgAb titer were significantly lower in the LV-sponge groups than in the control and LV-ctrl groups while Ets-1 protein expression in mouse spleens was increased in the LV-sponge groups. Moreover, Th17/Treg ratio declined in the LV-sponge group and decreased significantly in the prophylactic LV-sponge group (P = 0.036) tested by flow cytometry. Immunofluorescence showed that, in LV-sponge groups, CD4+IL-17a+ cells were decreased significantly (P = 0.001), while CD4+Ets-1+ cells were increased significantly in the LV-sponge group (P = 0.029). The serum IL-17/IL-10 was decreased significantly in the LV-sponge group (P < 0.05). In the thyroid injection groups, the thyroid inflammatory score and serum TgAb titer in the LV-sponge group decreased significantly compared with those in the LV-ctrl group (P < 0.05). In addition, in LV-sponge groups, CD4+IL-17a+ cells were decreased, while CD4+Ets-1+ cells were increased significantly in the inhibition group evaluated by immunofluorescence. Moreover, tail vein injection of LV-sponge resulted in much lower TgAb levels in thyroiditis compared with thyroid injection.ConclusionMiR-326 targeted therapy may be a promising approach for AIT. In addition, tail vein injection may achieve a better intervention effect than thyroid injection.

Regulation of cisplatin-resistant head and neck squamous cell carcinoma by the SRC/ETS-1 signaling pathway

BackgroundWe investigated the role of the ETS-1 transcription factor in Head and Neck Squamous Cell Carcinoma (HNSCC) in multiple cisplatin-resistant HNSCC cell lines.MethodsWe examined its molecular link with SRC and MEK/ERK pathways and determined the efficacy of either MEK/ERK inhibitor PD0325901 or SRC inhibitor Dasatinib on cisplatin-resistant HNSCC inhibition.ResultsWe found that ETS-1 protein expression levels in a majority of cisplatin-resistant HNSCC cell types were higher than those in their parental cisplatin sensitive partners. High ETS-1 expression was also found in patient-derived, cisplatin-resistant HNSCC cells. While ETS-1 knockdown inhibited cell proliferation, migration, and invasion, it could still re-sensitize cells to cisplatin treatment. Interestingly, previous studies have shown that MER/ERK pathways could regulate ETS-1 through its phosphorylation at threonine 38 (T38). Although almost all cisplatin-resistant HNSCC cells we tested showed higher ETS-1 phosphorylation levels at T38, we found that inhibition of MEK/ERK pathways with the MEK inhibitor PD0325901 did not block this phosphorylation. In addition, treatment of cisplatin-resistant HNSCC cells with the MEK inhibitor completely blocked ERK phosphorylation but did not re-sensitize cells to cisplatin treatment. Furthermore, we found that, consistent with ETS-1 increase, SRC phosphorylation dramatically increased in cisplatin-resistant HNSCC, and treatment of cells with the SRC inhibitor, Dasatinib, blocked SRC phosphorylation and decreased ETS-1 expression. Importantly, we showed that Dasatinib, as a single agent, significantly suppressed cell proliferation, migration, and invasion, in addition to survival.ConclusionsOur results demonstrate that the SRC/ETS-1 pathway plays a crucial role and could be a key therapeutic target in cisplatin-resistant HNSCC treatment.

Mechanism of decorin protein inhibiting invasion and metastasis of non-small cell lung cancer.

To detect the protein level of Decorin in non-small cell lung cancer (NSCLC) patients, and to study the mechanism of Decorin inhibiting invasion and metastasis of non-small cell lung cancer from the perspective of in vitro cells, and provide some theoretical support for the treatment of non-small cell lung cancer. Immunohistochemical staining was used to detect the expression of Decorin protein in 332 cases of stage I-IIIA clinical NSCLC and 70 cases of adjacent tissues. Then, in vitro cell experiments (cell scratch assay and transwell assay) were used to further study the effects of Decorin on migration and invasion of human lung cancer cell line A549; the effect of transforming growth factor-β on the expression of Decorin and Ets-1 protein was verified by Western blotting. The binding sites of Ets-1 and Decorin promoter were analyzed by bioinformatics. Exogenous Decorin inhibited invasion and metastasis of lung adenocarcinoma cells in vitro. Immunohistochemical staining showed that Decorin was lowly expressed in non-small cell lung cancer and Decorin had a certain effect on lung fibroblast activation. Western blotting results showed that TGF-β affects the expression of Decorin and Ets-1. Bioinformatics results showed that Ets-1 and Decorin gene DNA promoter regions have 18 binding sites. Decorin inhibits invasion and metastasis of non-small cell lung cancer through the TGF-β signaling pathway.

Expression of ETS1 and LEF1 in salivary glands of Sjögren syndrome patients.

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease affecting exocrine glands, thereby causing dry mouth and eyes (sicca). Our objective was to determine the expression of pSS pathogenic biomarker MMP9 and its putative transcription factors ETS1 and LEF1, in labial salivary glands of pSS patients. Sicca patients were assigned to three groups based on focus score (FS): non-pSS sicca (i.e., GR1 [FS=0] and GR2 [0<FS<1]) and pSS (i.e., GR3 [FS≥1]). We determined the mRNA and protein expression of MMP9, ETS1, and LEF1 in salivary gland biopsies. Also, ETS1-CD4 and LEF1-CD4 co-expression analyses were performed. The mRNA expression of MMP9, ETS1, and LEF1 was upregulated in GR3 compared to GR1 (p<0.01). Most GR3 salivary gland areas had moderate to high MMP9, ETS1, and LEF1 protein expression compared to GR1 and GR2. Further, ETS1-CD4 and LEF1-CD4 dual staining demonstrated that both salivary gland epithelial cells and lymphocytic infiltrates had increased levels of ETS1 and LEF1. Moreover, there was a strong correlation between ETS1(+)-CD4(-) and LEF1(+)-CD4(-) cells. These results suggest, for the first time, a concerted increase in ETS1 and LEF1 expression in salivary gland epithelial cells of pSS patients that is reflective of the etiopathogenesis of pSS.

Experimental and integrative analyses identify an ETS1 network downstream of BCR-ABL in chronic myeloid leukemia (CML)

The BCR-ABL oncogene, the hallmark of chronic myeloid leukemia (CML), has been shown to activate several signaling pathways in leukemic cells. The natural history of this disease has been radically modified by tyrosine kinase inhibitors (TKIs). However, resistance to several lines of TKI therapies and progression to blast crisis (BC) remain significant concerns. To identify novel signaling pathways induced by BCR-ABL, we performed a transcriptome analysis in a BCR-ABL-expressing UT-7 cell line. More than 2000 genes differentially expressed between BCR-ABL-expressing and parental UT-7 cells were identified and ETS1 was found to be the most upregulated. ETS1 protein expression was also shown to be highly increased in UT-7 cells expressing BCR-ABL either constitutively or under the control of TET-inducible promoters. ETS1 expression is tyrosine-kinase dependent because it was reduced by TKIs. A significant increase of ETS1 messenger RNA (mRNA) expression was observed in blood cells from CML patients at diagnosis compared with healthy controls. Integration of publicly available chromatin immunoprecipitation sequencing and transcriptomic data with our results allowed us to identify potential ETS1 targets, some of which are involved in the progression of CML. The messenger RNA expression of two of these genes (DNM3 and LIMS1) was found to be associated with the absence of major cytogenetic response after 1 year of imatinib therapy. The present work demonstrates for the first time the involvement of the ETS1 transcriptional program in the experimental UT-7 model and a large cohort of CML patients.

Interaction between transcriptional factor E26 transformation specific 1 and peroxiredoxin 1 in nicotine-induced oral precancerous lesion cells

Objective: To investigate the interaction between nuclear transcriptional factor E26 transformation specific 1 (Ets1) and peroxiredoxin 1 (Prx1) in nicotine-induced oral precancerous lesion cells. Methods: Human oral precancerous lesion dysplastic oral keratinocyte (DOK) cells were cultured and divided into nicotine group, control group, knockdown group and knockdown control group. The nicotine group, knockdown group and knockdown control group were treated with 1 μmol/L nicotine for 7 days while the control group was untreated. Western blotting, co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) were performed to detect Prx1 and Ets1 protein expression, Prx1 and Ets1 protein interaction, combined activity of Ets1 with PRDX1 gene promoter region in nicotine group and control group DOK cells. In nicotine group, DOK cells were transfected with siRNA or lentivirus to knockdown Ets1 and Prx1 expression. Prx1 and Ets1 protein expression was examined by Western blotting. Results: Nicotine increased the expression of Prx1 and Ets1 protein in DOK cells. The relative expression of Prx1 and Ets1 was 0.71±0.02, 0.12±0.01 in nicotine group and 0.53±0.06, 0.01±0.01 in control group (P=0.009, P=0.000). Co-IP showed that Prx1 could form protein complex with Ets1. The expression of Prx1 and Ets1 complex protein was increased in nicotine group. ChIP revealed that nicotine upregulated the combination of transcriptional factor Ets1 with PRDX1 gene promoter region, and the enrichment fold was 80.9±19.7 in nicotine group and 13.8±1.2 in control group (P=0.004). Ets1 and Prx1 protein expression was knocked down. The relative expression of Ets1 and Prx1 was 0.60±0.06, 0.48±0.03 in knockdown group and 0.83±0.08, 0.80±0.06 in knockdown control group (P=0.016, P=0.002). Ets1 knockdown suppressed the expression of Prx1 (P=0.002). Conversely, Prx1 knockdown also inhibited the expression of Ets1 significantly (P=0.000). Conclusions: In oral precancerous lesion cells, Ets1 directly regulates Prx1 expression and nicotine might promote the development of oral precancerous lesion by magnifying the positive feedback signal pathway between Ets1 and Prx1.

MicroRNA-326 contributes to autoimmune thyroiditis by targeting the Ets-1 protein.

MicroRNA-326 (miR-326), as a member of the microRNA (miRNA) family, which includes endogenous single-stranded, conserved, noncoding small RNAs, has been reported to play important roles in autoimmune diseases such as multiple sclerosis and systemic lupus erythematosus. However, few studies of the role of miR-326 in autoimmune thyroiditis (AIT) have been published. Here, we explored the roles of miR-326 and the involved pathway in iodine-induced AIT. NOD.H-2h4 mice, which are a model of human AIT, were randomly divided into a normal water control group and a high-iodine group. Mice in the high-iodine group were administered 0.05% NaI (~1000 times the normal daily iodine intake), and mice in the control group received sterile water. Furthermore, we evaluated small interfering RNA (siRNA) interference in spleen mononuclear cell experiments in vitro. In this study, we found that Th17 cells were significantly increased with a high expression of miR-326 in an iodine-induced thyroiditis NOD.H-2h4 mouse model. In addition, the expression of Ets-1 protein, a negative regulator of Th17 differentiation, was significantly decreased. Intriguingly, our analysis showed that Ets-1 protein expression was negatively correlated with miR-326 levels in AIT mice (r = -0.814, p < 0.01). Our study indicated that miR-326 inhibited Ets-1 protein expression and promoted the differentiation of Th17 cells during the onset and development of AIT. The addition of a miR-326 inhibitor reversed Th17 cell production and Ets-1 protein expression, supporting this hypothesis. The results of our study suggest that miR-326 may target the Ets-1 protein to contribute to iodide-induced thyroiditis, providing a new theoretical basis for the use of miRNA targeting therapy for the treatment of autoimmune diseases.

MiR-129 inhibits cell proliferation and metastasis by targeting ETS1 via PI3K/AKT/mTOR pathway in prostate cancer

New evidence suggests that microRNAs (miRNAs) play an important role in regulating the development and progression of prostate cancer. However, their specific functions and mechanisms remained to be further explored. MiR-129 has been reported in gastric cancers, colon cancer and lung cancer. In this study, we disclosed a new tumor suppresser function of miR-129 in prostate cancer. The purpose of our study is to clarify the effects of miR-129 in cellular processes correlated with cancer development and progression of prostate cancer cell by regulating ETS1. MiR-129 and ETS1 expression in prostate cancer tissues, tumor adjacent tissues and cells were tested by quantitative real-time PCR. We validated the target relationship between miR-129 and ETS1 by dual luciferase reporter gene system. MTT, colony formation, tumorigenesis assays, flow cytometry, wound healing and transwell assays were used to analyze cell viability, proliferation, migration, and invasiveness in vivo and in vitro. The level of ETS1 protein expression was detected by western blot. Here we demonstrate that miR-129 have a relatively reduced expression in prostate cancer cell lines and tissues. Morever, the miR-129 inhibits the expression of ETS1 by binding its 3′-UTR. The overexpression of miR-129 can inhibit PC-3 cell viability, proliferation, migration and invasion through targeting ETS1 by PI3K/AKT/mTOR signaling pathway. These findings suggested that miR-129 could directly suppress ETS1, which might be one of potential mechanisms in inhibiting cell processes including viability, proliferation, migration and invasiveness of prostate cancercells and it provides new clues for us to understand the carcinogenesis of prostate cancer. In addition, it may help to develop a treatment approach for ETS1-activated prostate cancer.

MiR-204 is dysregulated in metastatic prostate cancer in vitro.

During cancer progression, the genome instability incurred rearrangement could possibly turn some of the tumor suppressor micro-RNAs into pro-oncogenic ones. We aimed to investigate miR-204 in the context of prostate cancer progression using a cell line model of different levels of genome instability (LNCaP, PC3, VCaP and NCI H660), as demonstrated by the availability of ERG fusion. We studied the effect of miR-204 modulation on master transcription factors important for lineage development, cell differentiation and prostate cancer bone marrow metastasis. We followed c-MYB, ETS1 and RUNX2 transcript and protein expression and the miR-204 affected global proteome. We further investigated if these transcription factors exert an effect on miR-204 expression (qPCR, luciferase reporter assay) by silencing them using esiRNA. We found dualistic miR-204 effects, either acting as a tumor suppressor on c-MYB, or as an oncomiR on ETS1. RUNX2 and ETS1 regulation by miR-204 was ERG fusion dependent, demonstrating regulatory circuitry disruption in advanced metastatic models. miR-204 also differentially affected mRNA splicing and protein stability. miR-204 levels were found dependent on cancer hypermethylation and supported by positive feedback induced by all three transcription factors. In this regulatory circuitry among miR-204, c-MYB, RUNX2 and ETS1, the c-MYB was found to induce all three other members, but its expression was differentially affected by the methylation status in lymph node vs. bone metastasis. We demonstrate that not only tumor suppressor micro-RNA loss, but also significant genome rearrangement-driven regulatory loop perturbations play a role in the advanced cancer progression, conferring better pro-survival and metastatic potential.

Silencing of ETS1 reverses adriamycin resistance in MCF-7/ADR cells via downregulation of MDR1

BackgroundClinical resistance to chemotherapeutic agents is one of the major hindrances in the treatment of human cancers. Erythroblastosis virus E26 oncogene homolog 1 (ETS1) is involved in the drug resistance of various cancer cells, and is overexpressed in drug-resistant human breast cancer cell lines. In this study, we investigated the effects of ETS1 on adriamycin resistance in MCF-7/ADR cells.MethodssiRNAs against ETS1 or negative control siRNAs was transfected to MCF-7/ADR breast cancer cells. Reverse transcription-PCR and Western blotting were used to determine the mRNA and protein expression of ETS1 and MDR1. The cytotoxicity of adriamycin was assessed using the MTT assay. Drug efflux was investigated by flow cytometry using the Rhodamine 123 intracellular accumulation assay.ResultsETS1 mRNA and protein was significantly overexpressed in MCF-7/ADR cells, compared to MCF-7 cells. ETS1 siRNA successfully silenced ETS1 mRNA and protein expression. Silencing of ETS1 also significantly reduced the mRNA and protein expression levels of MDR1 (multidrug resistance 1; also known as ABCB1, P-glycoprotein/P-gp), which is a major ATP-binding cassette (ABC) transporter linked to multi-drug resistance in cancer cells. Silencing of ETS1 significantly increased the sensitivity of MCF-7/ADR cells to adriamycin, compared to cells transfected with negative control siRNA. In addition, intracellular accumulation of Rhodamine 123 significantly increased in MCF-7/ADR cells transfected with ETS1 siRNA, indicating that silencing of ETS1 may reduce drug efflux.ConclusionsThis study demonstrates that drug resistance can be effectively reversed in adriamycin-resistant breast carcinoma cells through delivery of siRNAs targeting ETS1.

Prognostic values of ETS-1, MMP-2 and MMP-9 expression and co-expression in breast cancer patients

The aim of this study was to analyse expression of ETS-1 protein and two gelatinases (MMP-2 and MMP-9) and their possible prognostic value in breast carcinoma patients, as well as correlation of their expression with other known prognostic factors such as tumor size, grade, vascular invasion, steroid receptor values, HER2 values and proliferative index. The expression of MMP-2, MMP-9 and ETS-1 was immunohistochemicaly analysed in 121 consecutive primary breast carcinoma patients who underwent surgery at the Clinical Hospital Centre Zagreb during 2002. Three representative areas from each tumor paraffin blocks were taken and arranged on a recipient paraffin block with predefined coordinates for simultaneous analyses of multiple tissue samples (TMA). ETS-1, MMP-2 and MMP-9 expression and co-expression were correlated with other clinico-pathological parameters and based on the available clinical follow up data survival analysis was performed. The ETS-1 protein is found to be expressed in tumor cell nuclei and cytoplasm as well as in stromal lymphocytes, fibroblasts and endothelial cells. MMP-2 and MMP-9 were found to be expressed in cytoplasm of both, tumor and stromal cells. For our analysis only tumor cell expression was used for statistical analysis. We found 56,2% ETS-1 positive tumors, 77,7% were MMP-2 positive, and MMP-9 was expressed in 90% of primary breast carcinomas. There were no significant correlations between MMP-s expression and other patohistological prognostic factors, but expression of ETS-1 was significantly correlated with higher tumor size and grade, as well as with negative steroid receptors. Co-expression of MMP-2, MMP-9 and ETS-1 was found in 40,5 % of tumors, and more commonly was found in tumors larger than 2 cm, high grade tumors, and steroid receptor negative tumors. In univariate analysis, statistically significant negative impact on overall survival (OS) had tumor size, nuclear and tumor grade, ETS-1 expression in tumor cells, co-expression of ETS-1 either with MMP-2 or MMP-9, as well as co-expression of ETS-1, MMP-2 and MMP-3. Disease free survival (DFS) was significantly shorter in patients with tumors greater than 2 cm, ETS-1 positive tumors, ETS-1 and MMP-2 or MMP-9 co-expressed tumors, and additionally in tumors with ETS-1, MMP-2 and MMP-9 co-expression. These results suggest that expression of ETS-1 as well as MMP-2, MMP-9 and ETS-1 co-expression might be used as a poor prognostic factor in breast cancer patients.

MiR-222 contributes to sex-dimorphic cardiac eNOS expression via ets-1

It is well recognized that there is sex-dimorphic expression of mRNA and protein in the heart; however, the underlying mechanism is poorly understood. Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiac function, and the expression levels of eNOS differ between male and female hearts. The aim of this study was to examine whether expression of specific microRNA (miRNA, miR) in males and females contributes to changes in the expression of eNOS. miRNA was extracted from the myocardium of male and female C57BL/6 mice and subjected to an Affymetrix miRNA array. Decreased expression of miR-222 was discovered in females and confirmed by qRT-PCR from whole heart or isolated cardiomyocytes. The transcription factor V-ets erythroblastosis virus E26 oncogene homolog-1 (ets-1) was identified as a potential target of miR-222 using TargetScan, and fivefold increased ets-1 protein expression in females was confirmed by Western blot. Targeting of ets-1 by miR-222 was determined in HEK293 cells overexpressing luciferase under regulation of either the ets-1 3'-UTR, a null 3'-UTR control, or a scrambled ets-1 3'-UTR and treated with a small molecule miR-222 mimic or inhibitor. Additionally qRT-PCR confirmed that mRNA levels of the ets-1 transcriptional target, eNOS, were 25% higher in females. Compared with untreated myocyte controls, 50% inhibition of eNOS expression was achieved by treatment with a miR-222 mimic, compared with a 25% increase due to miR-222 inhibitor. Our findings indicate that sex-dependent miR-222 regulation alters the expression of the cardiac regulatory protein eNOS.

Computational identification of conserved transcription factor binding sites upstream of genes induced in rat brain by transient focal ischemic stroke

Microarray analysis has been used to understand how gene regulation plays a critical role in neuronal injury, survival and repair following ischemic stroke. To identify the transcriptional regulatory elements responsible for ischemia-induced gene expression, we examined gene expression profiles of rat brains following focal ischemia and performed computational analysis of consensus transcription factor binding sites (TFBS) in the genes of the dataset. In this study, rats were sacrificed 24h after middle cerebral artery occlusion (MCAO) stroke and gene transcription in brain tissues following ischemia/reperfusion was examined using Affymetrix GeneChip technology. The CONserved transcription FACtor binding site (CONFAC) software package was used to identify over-represented TFBS in the upstream promoter regions of ischemia-induced genes compared to control datasets. CONFAC identified 12 TFBS that were statistically over-represented from our dataset of ischemia-induced genes, including three members of the Ets-1 family of transcription factors (TFs). Microarray results showed that mRNA for Ets-1 was increased following tMCAO but not pMCAO. Immunohistochemical analysis of Ets-1 protein in rat brains following MCAO showed that Ets-1 was highly expressed in neurons in the brain of sham control animals. Ets-1 protein expression was virtually abolished in injured neurons of the ischemic brain but was unchanged in peri-infarct brain areas. These data indicate that TFs, including Ets-1, may influence neuronal injury following ischemia. These findings could provide important insights into the mechanisms that lead to brain injury and could provide avenues for the development of novel therapies.

Ets-1 expression and gemcitabine chemoresistance in pancreatic cancer cells

Gemcitabine, a novel pyrimidine nucleoside analog, has become the standard chemotherapeutic agent for pancreatic cancer patients. The clinical impact of gemcitabine remains modest owing to the high degree of inherent and acquired resistance. There are various lines of evidence that confirm the role of Ets-1, a proto-oncoprotein, in tumor invasion, progression, and chemoresistance. This study examines a hypothesis that implicates Ets-1 in the development of gemcitabine-resistance in pancreatic cancer cells. Ets-1 protein expression was assessed in parental pancreatic cancer cells and their gemcitabine-resistant clones. Western blot analysis revealed elevated levels of Ets-1 protein expression in gemcitabine-resistant PANC1GemRes (4.8-fold increase; P < 0.05), MIA PaCa2GemRes (3.2-fold increase; P < 0.05), and Capan2GemRes (2.1-fold increase; P < 0.05) cells as compared to their parental counterparts. A time course analysis was conducted to determine the change in Ets-1 expression in the parental cells after incubation with gemcitabine. Reverse transcriptase quantitative real-time PCR (RT-qPCR) and Western blot analysis revealed a significant increase in Ets-1 expression. All the three parental cells incubated with gemcitabine showed elevated Ets-1 protein expression at 6 h. By 24 h, the expression level had decreased. Using small interfering RNA (siRNA) against Ets-1 in gemcitabine-resistant cells, we demonstrated a reversal in gemcitabine chemosensitivity and also detected a marked reduction in the expression of the Ets-1 target genes MMP1 and uPA. Our novel finding demonstrates the significance of Ets-1 in the development of gemcitabine chemoresistance in pancreatic cancer cells. Based on these results, a new siRNA-based therapeutic strategy targeting the Ets-1 genes can be designed to overcome chemoresistance.

Transcriptional silencing of the ETS1 oncogene contributes to human granulocytic differentiation

Ets-1 is a widely expressed transcription factor implicated in several biological processes including hematopoiesis, where it contributes to the regulation of cellular differentiation. The functions of Ets-1 are regulated by transcription factors as well as by phosphorylation events: phosphorylation of threonine 38 activates Ets-1, whereas phosphorylation of a cluster of serines within exon VII reduces DNA binding activity. This study focuses on the role of Ets-1 during granulocytic differentiation of NB4 promyelocytic and HL60 myeloblastic leukemia cell lines induced by all-trans retinoic acid. Ets-1 expression was measured by real-time reverse transcriptase polymerase chain reaction and western blotting. The role of Ets-1 during all-trans retinoic acid-induced differentiation was analyzed by using a transdominant negative molecule or small interfering RNA. NB4 and HL60 cell lines expressed high levels of p51 Ets-1, while the splice variant isoform that lacks exon VII (p42) was almost undetectable. The addition of all-trans retinoic acid reduced p51 Ets-1 levels and induced inhibitory phosphorylation of the remaining protein. Expression of Ets-1 was also reduced during dimethylsulfoxide-induced differentiation and during granulocytic differentiation of human CD34(+) hematopoietic progenitor cells but not in NB4.R2 and HL60R cells resistant to all-trans retinoic acid. In line with these observations, transduction of a transdominant negative molecule of Ets-1, which inhibited DNA binding and transcriptional activity of the wild-type Ets-1, significantly increased chemical-induced differentiation. Consistently, Ets-1 knockdown by small interfering RNA increased the number of mature neutrophils upon addition of all-trans retinoic acid. Interestingly, p51 Ets-1 over-expression was frequently observed in CD34(+) hematopoietic progenitor cells derived from patients with acute myeloid leukemia, as compared to its expression in normal CD34(+) cells. Our results indicated that a decreased expression of Ets-1 protein generalizes to granulocytic differentiation and may represent a crucial event for granulocytic maturation.

Abstract 2096: The role of microRNA-1 as a candidate tumor suppressor in cutaneous squamous cell carcinoma

Abstract Cutaneous squamous cell carcinoma (SCC) is the second most common form of skin cancer, affecting over 250,000 people in the United States annually. Using miRNA expression arrays, we identified lower levels of microRNA-1 (miR-1) expression in skin from FVB/NJ mice, which are susceptible to chemically-induced skin cancer, compared to skin from Spr/EiJ mice, which are resistant to chemically-induced skin cancer. Additionally, murine SCC cell lines showed greatly reduced levels of miR-1 compared to normal skin. We hypothesize that decreased expression of miR-1 in susceptible mice and SCC cell lines suggests a possible role of miR-1 as a tumor suppressor in skin. To test this hypothesis, we transfected a precursor for miR-1 into a murine SCC cell line, A5, and measured proliferation and cell viability via an MTT assay at 24, 48, and 72 hours post-transfection. The growth rate for cells transfected with miR-1 was significantly decreased at 48 to 72 hours post-transfection relative to those mock-transfected cells and corresponded to miR-1 expression levels. These data are consistent with our hypothesis that miR-1 may act as a tumor suppressor in the skin. Using in silico prediction programs, we identified a number of putative targets of miR-1. The transcription factor oncogene, Ets1, emerged as an interesting potential direct target of miR-1. Ets1 has three predicted target binding sites for miR-1. Quantitative PCR and Western analyses revealed decreased Ets1 mRNA and protein expression in A5 cells transfected with miR-1 compared to mock-transfected A5 control cells. Ets1 mRNA expression is also decreased in a normal keratinocyte cell line, C5N, compared to A5 cells. From these experiments we showed that Ets1 expression shows an inverse correlation with miR-1 levels. These results suggest Ets1 is regulated by miR-1. Future studies will determine whether Ets1 is a direct target of miR-1. We will also clarify the role of Ets1 in SCC cell proliferation and migration and identify other tumor-related phenotypes associated with miR-1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2096.

Transcription factor Ets-1 expression in human fetal membranes and its clinical significance

To investigate the role of transcription factor Ets-1 involved in premature rupture of membranes(PROM)by detecting its expression and location in human fetal membranes. Between Feb. and Nov. 2007, 100 pregnant women who delivered in the First Affiliated Hospital, Chongqing Medical University were enrolled in this study. According to gestational weeks, rupture of amniochorionic membranes and delivery mode, those women were classified into preterm labor group, preterm premature rupture of membranes (PPROM) group, term labor group and term premature rupture of membranes (TPROM) group, matched with elective term cesarean sections women as control group. There were 20 pregnant women in every group. The expression and distribution of Ets-1 protein in fetal membranes were detected by immunohistochemical streptravidin-biotin peroxidase (SP) method and histoscore. In the mean time, 6 cases were chosen from each group randomly and reverse transcription-PCR was used to measure the Ets-1 mRNA expression. (1) The expression of Ets-1 mRNA in fetal membranes were 0.342 ± 0.016 in preterm labor group, 0.603 ± 0.027 in PPROM group, 0.325 ± 0.013 in term labor group, 0.582 ± 0.075 in TPROM group, 0.139 ± 0.012 in control group, respectively. When compared with that in control group, Ets-1 mRNA expression were significantly increased in both PPROM and TPROM group (P < 0.05). However, it did not show remarkably difference between preterm group and term labor group, as well as between PPROM and TPROM group (P > 0.05). (2) Ets-1 was in stromal layers of amniochorionic membranes and in both cytoplasm and nuclei of trophoblast, which were shown with diffuse intracellular positive granularities (brown-yellow) clearly. No expression of Ets-1 was observed in amniotic epithelial cells. (3) The expression of Ets-1 protein was 0.552 ± 0.018 in preterm labor group, 2.853 ± 0.174 in PPROM group, 0.538 ± 0.042 in term labor group, 2.731 ± 0.090 in TPROM group and 0.214 ± 0.013 in control group, respectively. Ets-1 protein was significantly increased in the stroma of both PPROM and TPROM group than that in control group (P < 0.05). However, no remarkable different expression of Ets-1 was observed between preterm and term labor group, so was that between PPROM and TPROM group (P > 0.05). Transcription factor Ets-1 is expressed in human amniochorionic membranes and it could be up-regulated in PROM.

Relationship between Ets-1 expression and angiogenesis, clinicopathological features and survival of patients with gastric carcinoma

To investigate the expression of Ets-1 in gastric carcinoma, para-cancerous tissue and metastatic lymph nodes, and to determine the relationship between Ets-1 expression and clinicopathological features, angiogenesis and survival of patients with gastric carcinoma. Gastric carcinoma tissue microarray was used to determine Ets-1 protein expression by SP immunohistochemical staining in 189 advanced gastric cancer, 54 papacancerous tissues, 41 metastatic lymph nodes and 32 control tissues. The positive rates for Ets-1 expression of the carcinoma, paracancerous and control tissues were 71.4%, 29.6% and 18.8%, respectively, with a significant difference among the three groups (P < 0.01). In the cancer tissues, the positive rate of Ets-1 protein expression was significantly associated with depth of invasion and lymph node metastasis (P < 0.01), but not associated with degree of differentiation, Lauren's histological type, sex, age, and size of tumor (P > 0.05). The positive rates for Ets-1 expression of the 41 gastric cancer and 41 metastatic lymph nodes were significantly different (P < 0.05). In metastatic lymph nodes, the positive rate for Ets-1 expression was higher. The MVD in Ets-1 positive tumors was higher than that in the Ets-1 negative tumors, with a significant difference (P < 0.05). Kaplan-Meier survival analysis showed that the survival time of Ets-1-negative patients was longer than that of Ets-1-positive patients (P < 0.05). Cox regression analysis showed that Ets-1 expression was not an independent prognostic factor of gastric carcinoma. A higher expression of Ets-1 is involved in carcinogenesis, development, invasion, and metastasis of gastric cancer. Ets-1 plays an important role in angiogenesis in gastric cancer. Ets-1 is a useful marker for predicting the outcome for patients with gastric carcinoma, though it is not an independent prognostic indicator.

Expression and localization of transcription factor Ets-1 in the rat ovary during the estrous cycle and pregnancy

To examine the expression and localization of Ets-1 in the rat ovary during the estrous cycle and pregnancy, and to investigate its effects on ovarian function. Prospective, randomized study. Department of Physiology at Harbin Medical University. Pubertal female Wistar rats. Vaginal smears were taken daily from female rats to determine the stage of the estrous cycle. Pregnancies were achieved by caging female and male rats together overnight. Ovaries were collected from both cycling and pregnant rats for tissue sectioning and RNA and protein extractions. Real-time quantitative polymerase chain reaction, Western blot, in situ hybridization, and immunohistochemistry were performed to investigate the expression and localization of Ets-1 messenger RNA (mRNA) and protein in the rat ovary during the estrous cycle and pregnancy. During the estrous cycle, the levels of Ets-1 mRNA and protein expression increased during the follicular phase, achieving their highest measurements at proestrus and lowest at metestrus. The expression of Ets-1 mRNA and protein fluctuated during pregnancy, increasing during early pregnancy, then decreasing during mid-pregnancy, and again increasing until parturition. Ets-1 mRNA and protein were present throughout the estrous cycle and pregnancy, principally localized in follicles of various sizes and in the corpus luteum. Ets-1 may participate and play an important role in the regulation of follicular development, corpus luteum formation, maintenance, and regression.