ID2-ETS2 axis regulates the transcriptional acquisition of pro-tumoral microglia phenotype in glioma.

Glioblastoma is a highly aggressive brain tumour that creates an immunosuppressive microenvironment. Microglia, the brain's resident immune cells, play a crucial role in this environment. Glioblastoma cells can reprogramme microglia to create a supportive niche that promotes tumour growth. However, the mechanisms controlling the acquisition of a transcriptome associated with a tumour-supportive microglial reactive state are not fully understood. In this study, we investigated changes in the transcriptional profile of BV2 microglia exposed to C6 glioma cells. RNA-sequencing analysis revealed a significant upregulation of microglial inhibitor of DNA binding 1 (Id1) and Id2, helix-loop-helix negative transcription regulatory factors. The concomitant regulation of microglial ETS proto-oncogene 2, transcription factor (ETS2)-target genes, i.e., Dusp6, Fli1, Jun, Hmox1, and Stab1, led us to hypothesize that ETS2 could be regulated by ID proteins. In fact, ID2-ETS2 protein interactions increased in microglia exposed to glioma cells. In addition, perturbation of the ID2-ETS2 transcriptional axis influenced the acquisition of a microglial tumour-supportive phenotype. ID2 and ETS2 genes were found to be expressed by the tumour-associated microglia isolated from human glioblastoma tumour biopsies. Furthermore, ID2 and ETS2 gene expressions exhibited inverse prognostic values in patients with glioma in cohorts from The Cancer Genome Atlas. Collectively, our findings indicate that the regulation of ETS2 by ID2 plays a role in the transcriptional regulation of microglia in response to stimuli originating from glioblastoma cells, information that could lead to developing therapeutic strategies to manipulate microglial tumour-trophic functions.

A disease-associated gene desert directs macrophage inflammation through ETS2.

Increasing rates of autoimmune and inflammatory disease present a burgeoning threat to human health1. This is compounded by the limited efficacy of available treatments1 and high failure rates during drug development2, highlighting an urgent need to better understand disease mechanisms. Here we show how functional genomics could address this challenge. By investigating an intergenic haplotype on chr21q22-which has been independently linked to inflammatory bowel disease, ankylosing spondylitis, primary sclerosing cholangitis and Takayasu's arteritis3-6-we identify that the causal gene, ETS2, is a central regulator of human inflammatory macrophages and delineate the shared disease mechanism that amplifies ETS2 expression. Genes regulated by ETS2 were prominently expressed in diseased tissues and more enriched for inflammatory bowel disease GWAS hits than most previously described pathways. Overexpressing ETS2 in resting macrophages reproduced the inflammatory state observed in chr21q22-associated diseases, with upregulation of multiple drug targets, including TNF and IL-23. Using a database of cellular signatures7, we identified drugs that might modulate this pathway and validated the potent anti-inflammatory activity of one class of small molecules in vitro and ex vivo. Together, this illustrates the power of functional genomics, applied directly in primary human cells, to identify immune-mediated disease mechanisms and potential therapeutic opportunities.

USP39-Mediated Non-Proteolytic Control of ETS2 Suppresses Nuclear Localization and Activity.

ETS2 is a member of the ETS family of transcription factors and has been implicated in the regulation of cell proliferation, differentiation, apoptosis, and tumorigenesis. The aberrant activation of ETS2 is associated with various human cancers, highlighting its importance as a therapeutic target. Understanding the regulatory mechanisms and interacting partners of ETS2 is crucial for elucidating its precise role in cellular processes and developing novel strategies to modulate its activity. In this study, we conducted binding assays using a human deubiquitinase (DUB) library and identified USP39 as a novel ETS2-binding DUB. USP39 interacts with ETS2 through their respective amino-terminal regions, and the zinc finger and PNT domains are not required for this binding. USP39 deubiquitinates ETS2 without affecting its protein stability. Interestingly, however, USP39 significantly suppresses the transcriptional activity of ETS2. Furthermore, we demonstrated that USP39 leads to a reduction in the nuclear localization of ETS2. Our findings provide valuable insights into the intricate regulatory mechanisms governing ETS2 function. Understanding the interplay between USP39 and ETS2 may have implications for therapeutic interventions targeting ETS2-related diseases, including cancer, where the dysregulation of ETS2 is frequently observed.

ETS2 promotes cardiomyocyte apoptosis and autophagy in heart failure by regulating lncRNA TUG1/miR-129-5p/ATG7 axis.

Heart failure (HF) is a chronic disease in which the heart is unable to provide enough blood and oxygen to the peripheral tissues. Cardiomyocyte apoptosis and autophagy have been linked to HF progression. However, the underlying mechanism of HF is unknown. In this study, H2 O2 -treated AC16 cells were used as a cell model of HF. The mRNA and protein levels of related genes were examined using RT-qPCR and western blot. Cell viability and apoptosis were assessed using CCK-8 and flow cytometry, respectively. The interactions between ETS2, TUG1, miR-129-5p, and ATG7 were validated by luciferase activity, ChIP, and RNA-Binding protein Immunoprecipitation assays. According to our findings, H2 O2 stimulation increased the expression of ETS2, TUG1, and ATG7 while decreasing the expression of miR-129-5p in AC16 cells. Furthermore, H2 O2 stimulation induced cardiomyocyte apoptosis and autophagy, which were reversed by ETS2 depletion, TUG1 silencing, or miR-129-5p upregulation. Mechanistically, ETS2 promoted TUG1 expression by binding to the TUG1 promoter, and TUG1 sponged miR-129-5p to increase ATG7 expression. Furthermore, TUG1 overexpression reversed ETS2 knockdown-mediated inhibition of cardiomyocyte apoptosis and autophagy and miR-129-5p inhibition abolished TUG1 depletion-mediated suppression of cardiomyocyte apoptosis and autophagy in H2 O2 -induced AC16 cells. As presumed, ATG7 overexpression reversed miR-129-5p mimics-mediated repression of cardiomyocyte apoptosis and autophagy in H2 O2 -induced AC16 cells. Finally, ETS2 silencing reduced cardiomyocyte apoptosis and autophagy to slow HF progression by targeting the ETS2/TUG1/miR-129-5p/ATG7 axis, which may provide new therapeutic targets for HF treatment.

Suppression of USP7 negatively regulates the stability of ETS proto-oncogene 2 protein.

Ubiquitin-specific protease 7 (USP7) is one of the deubiquitinating enzymes (DUBs) that remove mono or polyubiquitin chains from target proteins. Depending on cancer types, USP7 has two opposing roles: oncogene or tumor suppressor. Moreover, it also known that USP7 functions in the cell cycle, apoptosis, DNA repair, chromatin remodeling, and epigenetic regulation through deubiquitination of several substrates including p53, mouse double minute 2 homolog (MDM2), Myc, and phosphatase and tensin homolog (PTEN). The [P/A/E]-X-X-S and K-X-X-X-K motifs of target proteins are necessary elements for the binding of USP7. In a previous study, we identified a novel substrate of USP7 through bioinformatics analysis using the binding motifs for USP7, and suggested that it can be an effective tool for finding new substrates for USP7. In the current study, gene ontology (GO) analysis revealed that putative target proteins having the [P/A/E]-X-X-S and K-X-X-K motifs are involved in transcriptional regulation. Moreover, through protein-protein interaction (PPI) analysis, we discovered that USP7 binds to the AVMS motif of ETS proto-oncogene 2 (ETS2) and deubiquitinates M1-, K11-, K27-, and K29-linked polyubiquitination of ETS2. Furthermore, we determined that suppression of USP7 decreases the protein stability of ETS2 and inhibits the transcriptional activity of ETS2 by disrupting the binding between the GGAA/T core motif and ETS2. Therefore, we propose that USP7 can be a novel target in cancers related to the dysregulation of ETS2.

Distinct Prognostic and Immunological Roles of ETS1 and ETS2: A Pan-Cancer Analysis.

Objective: ETS1 and ETS2, the main ETS family of transcription factors, have been found to act as downstream effectors of the RAS/MAPK pathway. This study explores the expression and prognostic values of ETS1 and ETS2 across cancers. We also aimed to explore the significance of ETS1 and ETS2 expression in normal immune cells with relation to tumorigenesis. Methods: The expression of ETS1 and ETS2 was examined in the HPA and GEPIA2 databases. The KM plotter was applied to examine prognostic value of ETS1 and ETS2. Correlation between ETS1/ETS2 and infiltrating immune cells and immune checkpoints was assessed using TIMER2.0. The mutation landscape of ETS1/ETS2 was explored using the cBioPortal. STRING and GEPIA2 were used to screen ETS1/ETS2 binding and correlated genes. Enrichr was applied to perform GO and KEGG enrichment analyses. Results: ETS1 showed enhanced expression in lymphoid tissue, while ETS2 showed low tissue specificity. ETS1 was increased in 12 and decreased in 6 cancers, while ETS2 was increased in 4 and decreased in 13 cancers. Both ETS1 and ETS2 were favorable prognostic markers in LIHC and KIRC, while they showed different prognostic roles in more cancers. ETS1 showed stronger correlation with several infiltrating immune cells and immune checkpoints compared with ETS2. Both ETS1 and ETS2 harbored low mutation ratio. ETS1 interacting and correlated genes were enriched in GO terms in response to cadmium ion and response to oxidative stress, while those of ETS2 were enriched in transcription regulation. Conclusion: ETS1 and ETS2 showed different patterns in expression, prognostic values, correlation with immune infiltrating, and immune checkpoints. ETS1 and ETS2 play distinct roles across cancer.

A distal super-enhancer activates oncogenic ETS2 via recruiting MECOM in inflammatory bowel disease and colorectal cancer.

Abnormal activities of distal cis-regulatory elements (CREs) contribute to the initiation and progression of cancer. Gain of super-enhancer (SE), a highly active distal CRE, is essential for the activation of key oncogenes in various cancers. However, the mechanism of action for most tumor-specific SEs still largely remains elusive. Here, we report that a candidate oncogene ETS2 was activated by a distal SE in inflammatory bowel disease (IBD) and colorectal cancer (CRC). The SE physically interacted with the ETS2 promoter and was required for the transcription activation of ETS2. Strikingly, the ETS2-SE activity was dramatically upregulated in both IBD and CRC tissues when compared to normal colon controls and was strongly correlated with the level of ETS2 expression. The tumor-specific activation of ETS2-SE was further validated by increased enhancer RNA transcription from this region in CRC. Intriguingly, a known IBD-risk SNP resides in the ETS2-SE and the genetic variant modulated the level of ETS2 expression through affecting the binding of an oncogenic transcription factor MECOM. Silencing of MECOM induced significant downregulation of ETS2 in CRC cells, and the level of MECOM and ETS2 correlated well with each other in CRC and IBD samples. Functionally, MECOM and ETS2 were both required for maintaining the colony-formation and sphere-formation capacities of CRC cells and MECOM was crucial for promoting migration. Taken together, we uncovered a novel disease-specific SE that distantly drives oncogenic ETS2 expression in IBD and CRC and delineated a mechanistic link between non-coding genetic variation and epigenetic regulation of gene transcription.

Ets-2 Propagates IL-6 Trans-Signaling Mediated Osteoclast-Like Changes in Human Rheumatoid Arthritis Synovial Fibroblast.

Rheumatoid arthritis synovial fibroblasts (RASFs) contribute to synovial inflammation and bone destruction by producing a pleiotropic cytokine interleukin-6 (IL-6). However, the molecular mechanisms through which IL-6 propels RASFs to contribute to bone loss are not fully understood. In the present study, we investigated the effect of IL-6 and IL-6 receptor (IL-6/IL-6R)-induced trans-signaling in human RASFs. IL-6 trans-signaling caused a significant increase in tartrate-resistant acid phosphatase (TRAP)-positive staining in RASFs and enhanced pit formation by ~3-fold in the osteogenic surface in vitro. IL-6/IL-6R caused dose-dependent increase in expression and nuclear translocation of transcription factor Ets2, which correlated with the expression of osteoclast-specific signature proteins RANKL, cathepsin B (CTSB), and cathepsin K (CTSK) in RASFs. Chromatin immunoprecipitation (ChIP) analysis of CTSB and CTSK promoters showed direct Ets2 binding and transcriptional activation upon IL-6/IL-6R stimulation. Knockdown of Ets2 significantly inhibited IL-6/IL-6R-induced RANKL, CTSB, and CTSK expression and TRAP staining in RASFs and suppressed markers of RASF invasive phenotype such as Thy1 and podoplanin (PDPN). Mass spectrometry analysis of the secretome identified 113 proteins produced by RASFs uniquely in response to IL-6/IL-6R that bioinformatically predicted its impact on metabolic reprogramming towards an osteoclast-like phenotype. These findings identified the role of Ets2 in IL-6 trans-signaling induced molecular reprogramming of RASFs to osteoclast-like cells and may contribute to RASF heterogeneity.

Cooperative Binding of ETS2 and NFAT Links Erk1/2 and Calcineurin Signaling in the Pathogenesis of Cardiac Hypertrophy.

BACKGROUND: Cardiac hypertrophy is an independent risk factor for heart failure, a leading cause of morbidity and mortality globally. The calcineurin/NFAT (nuclear factor of activated T cells) pathway and the MAPK (mitogen-activated protein kinase)/Erk (extracellular signal-regulated kinase) pathway contribute to the pathogenesis of cardiac hypertrophy as an interdependent network of signaling cascades. How these pathways interact remains unclear and few direct targets responsible for the prohypertrophic role of NFAT have been described. METHODS: By engineering cardiomyocyte-specific ETS2 (a member of the E26 transformation-specific sequence [ETS] domain family) knockout mice, we investigated the role of ETS2 in cardiac hypertrophy. Primary cardiomyocytes were used to evaluate ETS2 function in cell growth. RESULTS: ETS2 is phosphorylated and activated by Erk1/2 on hypertrophic stimulation in both mouse (n=3) and human heart samples (n=8 to 19). Conditional deletion of ETS2 in mouse cardiomyocytes protects against pressure overload-induced cardiac hypertrophy (n=6 to 11). Silencing of ETS2 in the hearts of calcineurin transgenic mice significantly attenuates hypertrophic growth and contractile dysfunction (n=8). As a transcription factor, ETS2 is capable of binding to the promoters of hypertrophic marker genes, such as ANP, BNP, and Rcan1.4 (n=4). We report that ETS2 forms a complex with NFAT to stimulate transcriptional activity through increased NFAT binding to the promoters of at least 2 hypertrophy-stimulated genes: Rcan1.4 and microRNA-223 (=n4 to 6). Suppression of microRNA-223 in cardiomyocytes inhibits calcineurin-mediated cardiac hypertrophy (n=6), revealing microRNA-223 as a novel prohypertrophic target of the calcineurin/NFAT and Erk1/2-ETS2 pathways. CONCLUSIONS: Our findings point to a critical role for ETS2 in calcineurin/NFAT pathway-driven cardiac hypertrophy and unveil a previously unknown molecular connection between the Erk1/2 activation of ETS2 and expression of NFAT/ETS2 target genes.

Linarin Protects the Kidney against Ischemia/Reperfusion Injury via the Inhibition of Bioactive ETS2/IL-12.

Ischemia/reperfusion injury (IRI), a participant in acute kidney injury (AKI), can occur as a series of pathological processes such as inflammation. Linarin (LIN) has been widely used for different diseases. To confirm the anti-inflammatory value and relevant mechanism of LIN during IRI, in vivo and vitro models were established. LIN or dissolvent was given, and histologic analysis, quantitative (q)RT-PCR, serum creatinine and blood urea nitrogen testing were used to evaluate kidney injury. Microarray analysis, protein-protein interaction (PPI) analysis and molecular docking were used to identify the target protein of LIN, and small interfering RNA (siRNA) transfection was applied to explore the crucial role of identified protein. First, we found that LIN inhibited kidney injury in an in vivo IRI model and decreased the expression of interleukin-12 (IL-12) p40 in vivo and in vitro IRI models. To explore the mechanism of LIN, we collected raw data from a public microarray database and identified E26 oncogene homolog 2 (ETS2) as a crucial protein of LIN according to microarray analysis and PPI. Meanwhile, qRT-PCR indicated that IL-12 p40 showed no significant difference between ETS2 knock down group and LIN treated ETS2 knock down group after hypoxia reoxygenation treatment. In addition, according to molecular docking the contact area is highly conserved and located on a PPI domain of ETS2 which indicates that LIN may alter the interaction with synergistic proteins in the regulation of IL-12 p40 expression. Our study demonstrated the anti-inflammatory effect of LIN during IRI-AKI, broadening the medicinal value of LIN and the therapeutic options for IRI-AKI.

Transcription factor Ets-2 regulates the expression of key lymphotropic factors.

Transcription factor Ets-2 downregulates the expression of cytokine genes and HIV-1 in resting T-cells. Herein, we studied whether Ets-2 regulates the expression of lymphotropic factors (LFs) NFAT2, NF-κΒ/p65, c-Jun, c-Fos, which regulate the activation/differentiation of T-cells, and kinase CDK10, which controls Ets-2 degradation and repression activity. In silico analysis revealed Ets-2 binding sites on the promoters of NFAT2, c-Jun, c-Fos. The T-cell lines Jurkat (models T-cell signaling/activation) and H938 (contains the HIV-1-LTR) were transfected with an Ets-2 overexpressing vector, in the presence/absence of mitogens. mRNA and protein levels were assessed by qPCR and Western immunoblotting, respectively. Ets-2 overexpression in unstimulated Jurkat increased NFAT2 and c-Jun mRNA/protein, c-Fos mRNA and NF-κΒ/p65 protein, and decreased CDK10 protein. In unstimulated H938, Ets-2 upregulated NFAT2, c-Jun and CDK10 mRNA/protein and NF-κΒ/p65 protein. In stimulated Jurkat, Ets-2 increased NFAT2, c-Jun and c-Fos mRNA/protein and decreased CDK10 mRNA/protein. In stimulated H938 Ets-2 increased NFAT2, c-Jun and c-Fos protein and reduced CDK10 protein levels. Furthermore, Ets-2 overexpression modulated the expression of pro- and anti-apoptotic genes in both cell lines. Ets-2 upregulates the expression of key LFs involved in the activation of cytokine genes or HIV-1 in T-cells, either through its physical interaction with gene promoters or through its involvement in signaling pathways that directly impact their expression. The effect of Ets-2 on CDK10 expression in H938 vs Jurkat cells dictates that, additionally to Ets-2 degradation, CDK10 may facilitate Ets-2 repression activity in cells carrying the HIV-1-LTR, contributing thus to the regulation of HIV latency in virus-infected T-cells.

RNA-binding protein CELF1 enhances cell migration, invasion, and chemoresistance by targeting ETS2 in colorectal cancer.

Colorectal cancer (CRC) is often diagnosed at later stages after it has metastasized to other organs. The development of chemoresistance also contributes to a poor prognosis. Therefore, an increased understanding of the metastatic properties of CRC and chemoresistance could improve patient survival. CUGBP elav-like family member 1 (CELF1) is an RNA-binding protein, which is overexpressed in many human malignant tumors. However, the influence of CELF1 in CRC is unclear. V-ets erythroblastosis virus E26 oncogene homologue 2 (ETS2) is an evolutionarily conserved proto-oncogene known to be overexpressed in a variety of human cancers including CRC. In thespresent tudy, we investigated the association between CELF1 and ETS2 in CRC tumorigenesis and oxaliplatin (L-OHP) resistance. We found a positive correlation between the elevated expression of CELF1 and ETS2 in human CRC tissues. Overexpression of CELF1 increased CRC cell proliferation, migration, and invasion in vitro and in a xenograft tumor growth model in vivo, and induced resistance to L-OHP. In contrast, CELF1 knockdown improved the response of CRC cells to L-OHP. Overexpression of ETS2 increased the malignant behavior of CRC cells (growth, migration, and invasion) and L-OHP resistance in vitro. Moreover, L-OHP resistance induced by CELF1 overexpression was reversed by ETS2 knockdown. The results of luciferase reporter and ribonucleoprotein immunoprecipitation assays indicated that CELF1 up-regulates ETS2 by binding to its 3'-UTR. Taken together, our findings have identified that CELF1 regulates ETS2 in a mechanism that results in CRC tumorigenesis and L-OHP resistance, and CELF1 may be a promising target for overcoming chemoresistance in CRC.

Direct Reprogramming of Mouse Embryonic Fibroblasts to Induced Trophoblast Stem Cells.

Trophoblast cells are the first committed lineage to emerge during mammalian preimplantation embryo development. Trophoblast stem (TS) cells can be derived from the trophectoderm (TE) of blastocyst-stage embryos and differentiate into extraembryonic trophoblast cells of the placenta. While mouse TS cells are an indispensable tool to study placental development, and reproductive diseases such as implantation failure and recurrent miscarriage, human TS cells have not been isolated. To model human trophoblast development and to investigate trophoblast-specific causes of reproductive diseases, it will be important to derive human induced trophoblast stem (iTS) cells. Recent studies have shown that fibroblasts can be reprogrammed to iTS cells by overexpressing four transcription factors (TFs) including TFAP2C, GATA3, EOMES, and ETS2. Here, we describe a protocol to directly convert mouse embryonic fibroblasts (MEFs) to iTS cells following overexpression of 10 TFs. iTS cells are capable of self-renewing using conventional TS cell culture media supplemented with the external signal FGF4 and heparin. iTS cells are also able to differentiate into trophoblast lineages.

Protein C-ets-2 epigenetically suppresses TLRs-induced interleukin 6 production in macrophages.

Interleukin 6 (IL-6) is a major proinflammatory cytokine involved in several aspects of the immune response. Excessive IL-6 production and dysregulated IL-6 receptor signaling lead to multiple inflammatory and autoimmune diseases, such as asthma, even cancer. Thus, its precise regulatory mechanisms need to be fully addressed. Here we found that knockdown of protein C-ets-2 (Ets2) resulted in higher IL-6 production after TLRs activation in macrophages. Mechanistically, Ets2 associated with an epigenetic modifier histone deacetylase 1 (HDAC1) and promoted its recruitment to the Il6 promoter after TLRs activation. Subsequentially, it enhanced histone deacetylation and inhibited Il6 mRNA transcription. Thus, Ets2 epigenetically suppresses TLRs-induced IL-6 production in both human and murine macrophages via promoting histone deacetylation of the Il6 promoter, serving as a new potential therapeutic target in inflammatory diseases therapy.

ETS2 promotes epithelial-to-mesenchymal transition in renal fibrosis by targeting JUNB transcription.

Epithelial-to-mesenchymal transition (EMT) plays an important role in the progression of renal tubulointerstitial fibrosis, a common mechanism leading to end-stage renal failure. V-ets erythroblastosis virus E26 oncogene homolog 2 (ETS2), a transcription factor, exhibits diverse roles in pathogenesis; however, its role in renal fibrosis is not yet fully understood. In this study, we detected the expression of ETS2 in an animal model of renal fibrosis and evaluated the potential role of ETS2 in tubular EMT induced by TGF-ß1. We found that ETS2 and profibrogenic factors, alpha-smooth muscle actin (α-SMA) and fibronectin (FN), were significantly increased in the unilateral ureteral obstruction (UUO)-induced renal fibrosis model in mice. In vitro, TGF-ß1 induced a high expression of ETS2 dependent on Smad3 and ERK signaling pathway in human proximal tubular epithelial cells (HK2). Knockdown of ETS2 abrogated TGF-ß1-mediated expression of profibrogenic factors vimentin, α-SMA, collagen I, and FN in HK2 cells. Mechanistically, ETS2 promoted JUNB expression in HK2 cells after TGF-ß1 stimulation. Furthermore, luciferase and Chromatin Immunoprecipitation (ChIP) assays revealed that the binding of ETS2 to three EBS motifs on the promoter of JUNB triggered its transcription. Notably, silencing JUNB reversed the ETS2-induced upregulation of the profibrogenic factors in HK2 cells after TGF-ß1 stimulation. These findings suggest that ETS2 mediates TGF-ß1-induced EMT in renal tubular cells through JUNB, a novel pathway for preventing renal fibrosis.

ΔNp73/ETS2 complex drives glioblastoma pathogenesis- targeting downstream mediators by rebastinib prolongs survival in preclinical models of glioblastoma.

BACKGROUND: Glioblastoma (GBM) remains one of the least successfully treated cancers. It is essential to understand the basic biology of this lethal disease and investigate novel pharmacological targets to treat GBM. The aims of this study were to determine the biological consequences of elevated expression of ΔNp73, an N-terminal truncated isoform of TP73, and to evaluate targeting of its downstream mediators, the angiopoietin 1 (ANGPT1)/tunica interna endothelial cell kinase 2 (Tie2) axis, by using a highly potent, orally available small-molecule inhibitor (rebastinib) in GBM. METHODS: ΔNp73 expression was assessed in glioma sphere cultures, xenograft glioblastoma tumors, and glioblastoma patients by western blot, quantitative reverse transcription PCR, and immunohistochemistry. Immunoprecipitation, chromatin immunoprecipitation (ChiP) and sequential ChIP were performed to determine the interaction between ΔNp73 and E26 transformation-specific (ETS) proto-oncogene 2 (ETS2) proteins. The oncogenic consequences of ΔNp73 expression in glioblastomas were examined by in vitro and in vivo experiments, including orthotopic zebrafish and mouse intracranial-injection models. Effects of rebastinib on growth of established tumors and survival were examined in an intracranial-injection mouse model. RESULTS: ΔNp73 upregulates both ANGPT1 and Tie2 transcriptionally through ETS conserved binding sites on the promoters by interacting with ETS2. Elevated expression of ΔNp73 promotes tumor progression by mediating angiogenesis and survival. Therapeutic targeting of downstream ΔNp73 signaling pathways by rebastinib inhibits growth of established tumors and extends survival in preclinical models of glioblastoma. CONCLUSION: Aberrant expression of ΔNp73 in GBM promotes tumor progression through autocrine and paracrine signaling dependent on Tie2 activation by ANGPT1. Disruption of this signaling by rebastinib improves tumor response to treatment in glioblastoma.

Exosomal miR-663b targets Ets2-repressor factor to promote proliferation and the epithelial-mesenchymal transition of bladder cancer cells.

Exosomes circulating in biological fluids have the potential to be utilized as cancer biomarkers and are associated with cancer progression and metastasis. MicroRNA (miR)-663b has been found to be elevated in plasma from patients with bladder cancer (BC). However, the functional role of exosomal miR-663b in BC processes remains unknown. Here, we isolated exosomes from plasma and found that the miR-663b level was elevated in exosomes from plasma of patients with BC compared with healthy controls. Exosomal miR-663b from BC cells promoted cell proliferation and epithelial-mesenchymal transition. Moreover, exosomal miR-663b targeted Ets2-repressor factor and acted as a tumor promoter in BC cells. Taken together, our findings suggested that exosomal miR-663b is a promising potential biomarker and target for clinical detection and therapy in BC.

EGF suppresses the expression of miR-124a in pancreatic ß cell lines via ETS2 activation through the MEK and PI3K signaling pathways.

Diabetes mellitus is characterized by pancreatic ß cell dysfunction. Previous studies have indicated that epidermal growth factor (EGF) and microRNA-124a (miR-124a) play opposite roles in insulin biosynthesis and secretion by beta cells. However, the underlying mechanisms remain poorly understood. In the present study, we demonstrated that EGF could inhibit miR-124a expression in beta cell lines through downstream signaling pathways, including mitogen-activated protein kinase kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) cascades. Further, the transcription factor ETS2, a member of the ETS (E26 transformation-specific) family, was identified to be responsible for the EGF-mediated suppression of miR-124a expression, which was dependent on ETS2 phosphorylation at threonine 72. Activation of ETS2 decreased miR-124a promoter transcriptional activity through the putative conserved binding sites AGGAANA/TN in three miR-124a promoters located in different chromosomes. Of note, ETS2 played a positive role in regulating beta cell function-related genes, including miR-124a targets, Forkhead box a2 (FOXA2) and Neurogenic differentiation 1 (NEUROD1), which may have partly been through the inhibition of miR-124 expression. Knockdown and overexpression of ETS2 led to the prevention and promotion of insulin biosynthesis respectively, while barely affecting the secretion ability. These results suggest that EGF may induce the activation of ETS2 to inhibit miR-124a expression to maintain proper beta cell functions and that ETS2, as a novel regulator of insulin production, is a potential therapeutic target for diabetes mellitus treatment.

Association of Cyclin Dependent Kinase 10 and Transcription Factor 2 during Human Corneal Epithelial Wound Healing in vitro model.

Proper wound healing is dynamic in order to maintain the corneal integrity and transparency. Impaired or delayed corneal epithelial wound healing is one of the most frequently observed ocular defect and difficult to treat. Cyclin dependen kinase (cdk), a known cell cycle regulator, required for proper proliferating and migration of cell. We therefore investigated the role of cell cycle regulator cdk10, member of cdk family and its functional association with transcriptional factor (ETS2) at active phase of corneal epithelial cell migration. Our data showed that cdk10 was associated with ETS2, while its expression was upregulated at the active phase (18 hours) of cell migration and gradually decrease as the wound was completely closed. Topical treatment with anti-cdk10 and ETS2 antibodies delayed the wound closure time at higest concentration (10 µg/ml) compared to control. Further, our results also showed increased mRNA expression of cdk10 and ETS2 at active phase of migration at approximately 2 fold. Collectively, our data reveals that cdk10 and ETS2 efficiently involved during corneal wound healing. Further studies are warranted to better understand the mechanism and safety of topical cdk10 and ETS2 proteins in corneal epithelial wound-healing and its potential role for human disease treatment.

Integration of Wnt and FGF signaling in the Xenopus gastrula at TCF and Ets binding sites shows the importance of short-range repression by TCF in patterning the marginal zone.

During Xenopus gastrulation, Wnt and FGF signaling pathways cooperate to induce posterior structures. Wnt target expression around the blastopore falls into two main categories: a horseshoe shape with a dorsal gap, as in Wnt8 expression; or a ring, as in FGF8 expression. Using ChIP-seq, we show, surprisingly, that the FGF signaling mediator Ets2 binds near all Wnt target genes. However, ß-catenin preferentially binds at the promoters of genes with horseshoe patterns, but further from the promoters of genes with ring patterns. Manipulation of FGF or Wnt signaling demonstrated that 'ring' genes are responsive to FGF signaling at the dorsal midline, whereas 'horseshoe' genes are predominantly regulated by Wnt signaling. We suggest that, in the absence of active ß-catenin at the dorsal midline, the DNA-binding protein TCF binds and actively represses gene activity only when close to the promoter. In contrast, genes without functional TCF sites at the promoter may be predominantly regulated by Ets at the dorsal midline and are expressed in a ring. These results suggest recruitment of only short-range repressors to potential Wnt targets in the Xenopus gastrula.