Involvement of TLRs/NF-κB/ESE-1 signaling pathway in T-2 toxin-induced cartilage matrix degradation.

T-2 toxin, a highly toxic type A monotrichothecene mycotoxin, has been found in many different types of cereals and is considered to be one of the most dangerous naturally occurring forms of food contamination. Globally, consuming grain-based food tainted with T-2 toxin poses significant risks to animal and human health. Prior research has indicated that the presence of T-2 toxin may lead to the demise of chondrocytes and the deterioration of the extracellular matrix of cartilage in degenerative bone and joint conditions, such as Kashin-Beck disease. However, the mechanisms by which T-2 toxin exerts its biological toxicity on the degradation of the extracellular matrix in cartilage are not well understood. In the current study, we found original results that demonstrate an upregulation of Toll-Like Receptors (TLR-2, TLR-4) and ESE-1 expression levels in the articular cartilage of a rat model subjected to T-2 toxin exposure. Furthermore, it was revealed that the exposure to T-2 toxin resulted in an increase in the expression of TLR-2, TLR-4, and ESE-1 in human C28/I2 chondrocytes. The findings of this study indicate that the increased expression of TLR-2, TLR-4, and ESE-1 may contribute to the development of degenerative osteoarthritic disease caused by T-2 toxin. Consistent with our hypotheses, we discovered that T-2 toxin increased the expression of MMP-1 and MMP-13 in human C28/I2 chondrocytes. We used a luciferase reporter gene assay to measure the activity of the ESE-1 promoter and transfected cells with plasmids encoding TLR-2 and TLR-4 to investigate their effects on this activity. TLR-2 and TLR-4 can activate ESE-1 transcriptional gene expression, and this expression is mediated through the NF-κB pathway, additional evidence is provided for the participation of the TLRs/NF-κB/ESE-1 signaling pathway in T-2 toxin-induced cartilage matrix degradation. Together, the findings indicated that the TLRs/NF-κB/ESE-1 signaling pathway played an essential part in T-2 toxin-induced cartilage matrix degradation.

ESE1/AGR2 axis antagonizes TGF-ß-induced epithelial-mesenchymal transition in low-grade pancreatic cancer.

Epithelium-specific ETS transcription factor 1 (ESE1) has been implicated in epithelial homeostasis, inflammation, as well as tumorigenesis, and cancer progression. However, numerous studies have reported contradictory roles-as an oncogene or a tumor suppressor of ESE1 in different cancers, and its function in the development and progression of pancreatic ductal adenocarcinoma (PDAC) has remained largely unexplored. Herein, we report that ESE1 was found upregulated in primary PDAC compared to normal pancreatic tissue, but high expression of ESE1 correlated to better relapse-free survival in patients with PDAC. Interestingly, ESE1 was found to exhibit dual roles in regulation of malignant properties of PDAC cells in that its overexpression promoted cell proliferation, whereas its downregulation enhanced epithelial-mesenchymal transition (EMT) phenotype. In the context of TGF-ß-induced EMT, ESE1 is markedly downregulated at post-transcriptional level, and reconstituted ESE1 expression partially reversed TGF-ß-induced EMT marker expression. Furthermore, we identify AGR2 as a novel transcriptional target of ESE1 that participates in TGF-ß-induced EMT in PDAC. Collectively, our findings reveal an ESE1/AGR2 axis that interacts with TGF-ß signaling to modulate EMT phenotype in PDAC.

Epithelium-specific ETS transcription factor-1 regulates NANOG expression and inhibits NANOG-induced proliferation of human embryonic carcinoma cells.

The epithelium-specific ETS transcription factor-1 (ESE-1) plays multiple roles in pathogenesis and normal development of epithelial tissues. NANOG, a key mediator of stem cell self-renewal and pluripotency, is also expressed in various cancers and pluripotent cells. In this study, we investigated how ESE-1 influences NANOG expression and NANOG-induced proliferation in human germ cell-derived embryonic carcinoma NCCIT cells. Endogenous ESE-1 expression in NCCIT cells significantly increased during differentiation, whereas NANOG expression decreased. In addition, NANOG expression was downregulated by exogenous overexpression of ESE-1, and increased by shRNA-mediated knockdown of ESE-1. NANOG transcriptional activity was reduced by dose-dependent ESE-1 overexpression and a putative ESE-1 binding site (EBS) was mapped within conserved region 2. Site-directed mutagenesis of the putative EBS abrogated the repressive effect of ESE-1 on NANOG promoter activity. ESE-1 directly interacted with the putative EBS to regulate transcriptional activity of NANOG. Furthermore, NANOG-induced proliferation and colony formation of NCCIT cells were inhibited by ESE-1 overexpression and stimulated by ESE-1 shRNA-mediated knockdown. Altogether, our results suggest that ESE-1 exerts an anti-proliferative effect on NCCIT cells by acting as a novel transcriptional repressor of NANOG.

ETS transcription factor ESE-1/Elf3 is an independent prognostic factor of survival in HR+HER2+ breast cancer patients.

PURPOSE: The ETS transcription factor ESE-1 has been shown to be important in HER2+ breast cancer and ESE-1 mRNA expression has been shown to associate with prognostic outcomes in the HER2+ subtype, as well as in ER+, HER2+ luminal B patients. However, the clinical significance of ESE-1 protein expression remains unknown. The purpose of the current exploratory study is to evaluate the prognostic value of ESE-1 protein expression in molecular breast cancer subtypes with special emphasis on hormone receptor positive HER2+(HR+ HER2+) and the HER2 positive (HER2+-only) breast cancer patients. METHODS: We developed a mouse monoclonal anti-ESE-1 antibody, verified its specificity, epitope, and used immunohistochemical staining to assess ESE-1 expression in an IBC approved archive of 957 breast tumor samples. Using Pearson product correlation, contingency analysis, and long rank P value testing, we analyzed the association of ESE-1 expression with clinicopathological features and survival outcomes in HR+HER2-; HR+HER2+; HR- HER2- (Triple negative) and HR-HER2+ (HER2 subtype) patients. RESULTS: ESE-1, nuclear or cytoplasmic, was not significantly associated with survival outcomes in HR+HER2-, triple-negative, or HER2+-only breast cancer patients. However, high nuclear ESE-1 was associated with poor survival outcomes in hormone receptor positive (ERα+, PR+) HER2+ patients and was an independent prognostic marker for that group. CONCLUSIONS: This study provides evidence for prognostic significance of nuclear ESE-1 in ERalpha positive breast cancers patients also positive for HER2 indicating that crosstalk between ERalpha and ESE-1 in HER2+ tumors could be important for prognostic outcomes. Further studies regarding the nature of interaction between ESE-1 and ERalpha in these tumors are warranted.

Ultrasound-mediated delivery of siESE complexed with microbubbles attenuates HER2+/- cell line proliferation and tumor growth in rodent models of breast cancer.

The highly tunable, noninvasive and spatially targeted nature of microbubble-enhanced, ultrasound-guided (MB+US) drug delivery makes it desirable for a wide variety of therapies. In breast cancer, both HER2+ and HER2- type neoplasms pose significant challenges to conventional therapeutics in greater than 40% of breast cancer patients, even with the widespread application of biologics such as trastuzumab. To address this therapeutic challenge, we examined the novel combination of tumor-injected microbubble-bound siRNA complexes and monodisperse size-isolated microbubbles (4-µm diameter) to attenuate tumor growth in vivo, as well as MB+US-facilitated shRNA and siRNA knockdown of ESE-1, an effector linked to dysregulated HER2 expression in HER2+/- cell line propagation. We first screened six variants of siESE and shESE for efficient knockdown of ESE in breast cancer cell lines. We demonstrated efficient reduction of BT-474 (PR+, ER+, HER2+; luminal B) and MDA-MB-468 (PR-, ER-, HER2-; triple-negative) clonogenicity and non-adherent growth after knockdown of ESE-1. A significant reduction in proliferative potential was seen for both cell lines using MB+US to deliver shESE and siESE. We then demonstrated significant attenuation of BT-474 xenograft tumor growth in Nod/SCID female mice using direct injection of microbubble-adsorbed siESE to the tumor and subsequent sonication. Our results suggest a positive effect on drug delivery from MB+US, and highlights the feasibility of using RNAi and MB+US for breast cancer pathologies. RNAi coupled with MB+US may also be an effective theranostic approach to treat other acoustically accessible tumors, such as melanoma, thyroid, parotid and skin cancer.

Identification of epithelial-specific ETS-1 (ESE-1) as a tumor suppressor and molecular target of green tea compound, EGCG.

Epithelial specific ETS-1 (ESE-1) belongs to the E26 transformation-specific transcription factor superfamily and is of great interest as a potential target for managing several types of cancer. Despite its clinical significance, the documented effects of ESE-1 on cancer development and progression are contradictory and its underlying biological mechanism of action remains elusive. The objectives of this study are to investigate whether ESE-1 is a tumor suppressor and to identify dietary anti-cancer compound to activate ESE-1 expression in human colon cancer model. ESE-1 knockout and xenograft mouse models were used to examine the effect of ESE-1 in colon tumorigenesis. Stable human colon cancer cell lines were used for in vitro mechanistic studies. ESE-1 knockout in mice increased azoxymethane (AOM)-induced and dextran sulfate sodium (DSS)-promoted formation of aberrant crypt foci (ACF). Conversely, overexpression of ESE-1 suppressed tumorigenicity in a xenograft mouse study, and repressed anchorage-independent growth and migration/invasion in human colon cancer cells. Full length ESE-1 localized abundantly in the nucleus, and internal deletion of nuclear localization sequence 2 (NLS2) reduced nuclear ESE-1. Three lysine residues (318 KKK320 ) in the NLS2 determine its nuclear localization. We identified epigallocatechin-3-gallate (EGCG) that acts as a transcriptional activator of ESE-1 in human colon cancer cells. These findings propose a novel and promising molecular target of dietary anti-cancer compounds for prevention of colon cancer.

Ets1 and ESE1 reciprocally regulate expression of ZEB1/ZEB2, dependent on ERK1/2 activity, in breast cancer cells.

The epithelial-mesenchymal transition (EMT) is a crucial morphological event that occurs during progression of epithelial tumors. We reported previously that levels of the δ-crystallin/E2-box factor 1 (δEF1) family proteins (Zinc finger E-box binding homeobox 1 [ZEB1]/δEF1 and ZEB2/ Smad-interacting protein 1), key regulators of the EMT, are positively correlated with EMT phenotypes and aggressiveness of breast cancer. Here, we show that Ets1 induces ZEB expression and activates the ZEB1 promoter, independently of its threonine 38 phosphorylation status. In the basal-like subtype of breast cancer cells, siRNAs targeting Ets1 repressed expression of ZEBs and partially restored their epithelial phenotypes and sensitivity to antitumor drugs. Epithelium-specific ETS transcription factor 1 (ESE1), a member of the Ets transcription factor family, was originally characterized as being expressed in an epithelial-restricted pattern, placing it within the epithelium-specific ETS subfamily. ESE1, highly expressed in the luminal subtype of breast cancer cells, was repressed by activation of the MEK-ERK pathway, resulting in induction of ZEBs through Ets1 upregulation. Conversely, Ets1, highly expressed in the basal-like subtype, was repressed by inactivation of MEK-ERK pathway, resulting in reduction of ZEBs through ESE1 upregulation. These findings suggest that ESE1 and Ets1, whose expressions are reciprocally regulated by the MEK-ERK pathway, define the EMT phenotype through controlling expression of ZEBs in each subtype of breast cancer cells.

ESE-1 in Early Development: Approaches for the Future.

E26 transformation-specific (Ets) family of transcription factors are characterized by the presence of Ets-DNA binding domain and have been found to be highly involved in hematopoiesis and various tissue differentiation. ESE-1, or Elf3 in mice, is a member of epithelium-specific Ets sub-family which is most prominently expressed in epithelial tissues such as the gut, mammary gland, and lung. The role of ESE-1 during embryogenesis had long been alluded from 30% fetal lethality in homozygous knockout mice and its high expression in preimplantation mouse embryos, but there has been no in-depth of analysis of ESE-1 function in early development. With improved proteomics, gene editing tools and increasing knowledge of ESE-1 function in adult tissues, we hereby propose future research directions for the study of ESE-1 in embryogenesis, including studying its regulation at the protein level and at the protein family level, as well as better defining the developmental phase under investigation. Understanding the role of ESE-1 in early development will provide new insights into its involvement in tissue regeneration and cancer, as well as how it functions with other Ets factors as a protein family.

ESE1 is Associated with Neuronal Apoptosis in Lipopolysaccharide Induced Neuroinflammation.

Neuronal apoptosis induced by the over-activation of microglia during neuroinflammation contributes to the pathology of central nervous system (CNS) degenerative diseases. ESE1 regulates apoptosis of intestinal epithelial cells in ulcerative colitis via accelerating NF-κB activation. NF-κB activation participates in neuronal apoptosis. However, the expression and functions of ESE1 in neuronal apoptosis during CNS inflammatory response remain unclear. In present study, ESE1 expression significantly increased in cerebral cortex after lipopolysaccharide (LPS) intracerebroventricular injection. Immunofluorescence staining indicated that ESE1 was located in neurons. Furthermore, there was a concomitant up-regulation of apoptotic markers including active caspase-3, BAX and decreased expression of anti-apoptosis protein Bcl-2. In vitro, ESE1 depletion in cortical primary neurons inhibited active caspase-3 and BAX expression as well as lactate dehydrogenase (LDH) release with up-regulation of Bcl-2, while ESE1 overexpression can exert opposite effects, indicating that ESE1 promoted neuronal apoptosis induced by LPS or LPS exposed microglia conditioned media (CM). ESE1 accelerated NF-κB activation in neurons with CM treatment. Collectively, all these data suggested that ESE1 might boost neuronal apoptosis during neuroinflammation via up-regulating NF-κB activation. These findings have implications on the potential target of ESE1 in CNS inflammation treatment.

Epithelium-specific Ets transcription factor-1 acts as a negative regulator of cyclooxygenase-2 in human rheumatoid arthritis synovial fibroblasts.

BACKGROUND: Rheumatoid arthritis (RA) is characterized by excessive synovial inflammation. Cyclooxygenase-2 (COX-2) is an enzyme that catalyzes the conversion of arachidonic acid (AA) into prostaglandins. Epithelium-specific Ets transcription factor-1 (ESE-1) was previously demonstrated to upregulate COX-2 in co-operation with nuclear factor kappa B (NFκB) in macrophages and chondrocytes. However, the role of ESE-1 in RA pathology has remained unclear. In this study, we aimed to elucidate the relationship between ESE-1 and COX-2 in RA synovial fibroblasts (RASFs) using a HD-Ad-mediated knockdown approach. RESULTS: ESE-1 and COX-2 were induced by IL-1ß in RASFs that corresponded with an increase in PGE2. Endogenous levels of ESE-1 and COX-2 in human RASFs were analyzed by RT-qPCR and Western blot, and PGE2 was quantified using competitive ELISA. Interestingly, knockdown of ESE-1 using helper-dependent adenovirus (HD-Ad) led to a significant upregulation of COX-2 at a later phase of IL-1ß stimulation. Examination of ESE-1 intracellular localization by nuclear fractionation revealed that ESE-1 was localized in the nucleus, occupying disparate cellular compartments to NFκB when COX-2 was increased. To confirm the ESE-1-COX-2 relationship in other cellular systems, COX-2 was also measured in SW982 synovial sarcoma cell line and ESE-1 knockout (KO) murine macrophages. Similarly, knockdown of ESE-1 transcriptionally upregulated COX-2 in SW982 and ESE-1 KO murine macrophages, suggesting that ESE-1 may be involved in the resolution of inflammation. CONCLUSION: ESE-1 acts as a negative regulator of COX-2 in human RASFs and its effect on COX-2 is NFκB-independent.

Identification of ESE1 as a ß-Catenin Binding Protein.

BACKGROUND/AIM: ß-Catenin regulates cell-cell adhesion and gene transcription and acts as a master switch that controls proliferation in several types of cancer. ESE1 is an epithelium-restricted transcription factor and its multiple domain structure predicts its interaction with other proteins with diverse cellular functions. Here, for the first time, we report that endogenous ß-catenin binds to and co-localizes with endogenous ESE1 in the cytoplasm. MATERIALS AND METHODS: The binding sites were mapped to E26 transformation-specific (ETS) domain at carboxyl terminus of ESE1 and N-terminus of ß-catenin. RESULTS: We found that C-terminus of ESE1 also binds to α-catenin and that ESE1/ß-catenin interaction was abrogated by knockdown of either ß-catenin or α-catenin. CONCLUSION: The data suggest that interactions between ESE1 and ß-/α-catenins might be a mechanism by which the ESE1 protein determines the ß-catenin function and tumorigenesis.

A transcriptional repressive role for epithelial-specific ETS factor ELF3 on oestrogen receptor alpha in breast cancer cells.

Oestrogen receptor-α (ERα) is a ligand-dependent transcription factor that primarily mediates oestrogen (E2)-dependent gene transcription required for mammary gland development. Coregulators critically regulate ERα transcription functions by directly interacting with it. In the present study, we report that ELF3, an epithelial-specific ETS transcription factor, acts as a transcriptional repressor of ERα. Co-immunoprecipitation (Co-IP) analysis demonstrated that ELF3 strongly binds to ERα in the absence of E2, but ELF3 dissociation occurs upon E2 treatment in a dose- and time-dependent manner suggesting that E2 negatively influences such interaction. Domain mapping studies further revealed that the ETS (E-twenty six) domain of ELF3 interacts with the DNA binding domain of ERα. Accordingly, ELF3 inhibited ERα's DNA binding activity by preventing receptor dimerization, partly explaining the mechanism by which ELF3 represses ERα transcriptional activity. Ectopic expression of ELF3 decreases ERα transcriptional activity as demonstrated by oestrogen response elements (ERE)-luciferase reporter assay or by endogenous ERα target genes. Conversely ELF3 knockdown increases ERα transcriptional activity. Consistent with these results, ELF3 ectopic expression decreases E2-dependent MCF7 cell proliferation whereas ELF3 knockdown increases it. We also found that E2 induces ELF3 expression in MCF7 cells suggesting a negative feedback regulation of ERα signalling in breast cancer cells. A small peptide sequence of ELF3 derived through functional interaction between ERα and ELF3 could inhibit DNA binding activity of ERα and breast cancer cell growth. These findings demonstrate that ELF3 is a novel transcriptional repressor of ERα in breast cancer cells. Peptide interaction studies further represent a novel therapeutic option in breast cancer therapy.

The E3 ubiquitin ligase HOS1 is involved in ethylene regulation of leaf expansion in Arabidopsis.

Ethylene regulates a variety of physiological processes, such as flowering, senescence, abscission, and fruit ripening. In particular, leaf expansion is also controlled by ethylene in Arabidopsis. Exogenous treatment with ethylene inhibits leaf expansion, and consistently, ethylene insensitive mutants show increased leaf area. Here, we report that the RING finger-containing E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1) regulates leaf expansion in an ethylene signaling pathway. The HOS1-deficient mutant showed reduced leaf area and was insensitive to ethylene perception inhibitor, silver thiosulfate (STS). Accordingly, genes encoding ethylene signaling components were significantly up-regulated in hos1-3. This study demonstrates that the HOS1 protein is involved in ethylene signal transduction for the proper regulation of leaf expansion possibly under environmentally stressful conditions.

Epithelium-Specific ETS (ESE)-1 upregulated GP73 expression in hepatocellular carcinoma cells.

BACKGROUND: Golgi protein-73 (GP73) is a Golgi transmembrane glycoprotein elevated in numerous liver diseases. Clinically, GP73 is strongly elevated in the serum of HCC patients and is thus regarded as a novel potential biomarker for HCC. However, the mechanism leading to GP73 dysregulation in liver diseases remains unknown. RESULTS: This study determined that epithelium-specific ETS (ESE)-1, an epithelium-specific transcription factor, and GP73 expressions were induced by IL-1ß stimulation in vitro, and both were triggered during liver inflammation in vivo. In hepatocellular carcinoma cells, the overexpression of ESE-1 induced GP73 expression, whereas its knock-down did the opposite. Mechanistically, ESE-1 activated GP73 expression by directly binding to its promoter. CONCLUSIONS: Our findings supported a novel paradigm for ESE-1 as a transcriptional mediator of GP73. This study provided a possible mechanism for GP73 upregulation in liver diseases.

Transcriptional regulation of OCT4 by the ETS transcription factor ESE-1 in NCCIT human embryonic carcinoma cells.

The epithelium-specific ETS transcription factor-1 (ESE-1) is physiologically important in the pathogenesis of various diseases. Recently, OCT4, a transcription factor involved in stem cell pluripotency, has been implicated in tumorigenesis. In this study, we invested the molecular mechanism by which ESE-1 regulates transcription of OCT4 in NCCIT human embryonic carcinoma cells. Real-time PCR analysis revealed that OCT4 levels were high in undifferentiated NCCIT cells but significantly decreased upon retinoic acid-mediated differentiation, concomitant with up-regulation of ESE-1 expression. OCT4 mRNA level rose following shRNA-mediated knockdown of ESE-1, but declined when ESE-1 was overexpressed, suggesting that the expression levels of OCT4 and ESE-1 may be coordinated in an opposite manner. Promoter-reporter assays revealed that induced OCT4 promoter activity in NCCIT cells was significantly down-regulated by ESE-1 overexpression in a dose-dependent manner. The inhibitory effect of ESE-1 on OCT4 promoter activity was relieved by co-expression of an ESE-1 mutant lacking the transactivation domain, but not by mutants lacking other domains. Serial deletion and site-directed mutagenesis of the OCT4 promoter revealed that a potential ETS binding site (EBS) is present in the conserved region 2 (CR2). ESE-1 interacted with the EBS element in CR2 and enrichment of CR2 significantly increased upon RA-mediated differentiation of NCCIT cells, suggesting that this binding is likely to be involved in ESE-1-mediated repression of OCT4 promoter activity upon differentiation. Taken together, the results of this study reveal the molecular details of the mechanism by which the oncogenic factor ESE-1 regulates expression of the stem cell transcription factor OCT4 in pluripotent NCCIT cells.