Ets-1 Knockdown Research Articles

Disruption of blood-brain barrier and endothelial-to-mesenchymal transition are attenuated by Astragalus polysaccharides mediated through upregulation of ETS1 expression in experimental autoimmune encephalomyelitis

Blood-brain barrier (BBB) breakdown, an early hallmark of multiple sclerosis (MS), remains crucial for MS progression. Our previous works have confirmed that Astragalus polysaccharides (APS) can significantly ameliorate demyelination and disease progression in experimental autoimmune encephalomyelitis (EAE) mice. However, it remains unclear whether APS protects BBB and the potential mechanism. In this study, we found that APS effectively reduced BBB leakage in EAE mice, which was accompanied by a decreased level of endothelial-to-mesenchymal transition (EndoMT) in the central nervous system (CNS). We further induced EndoMT in the mouse brain endothelial cells (bEnd.3) by interleukin-1β (IL-1β) in vitro. The results showed that APS treatment could inhibit IL-1β-induced EndoMT and endothelial cell dysfunction. In addition, the transcription factor ETS1 is a central regulator of EndoMT related to the compromise of BBB. We tested the regulation of APS on ETS1 and identified the expression of ETS1 was upregulated in both EAE mice and bEnd.3 cells by APS. ETS1 knockdown facilitated EndoMT and endothelial cell dysfunction, which completely abolished the regulatory effect of APS. Collectively, APS treatment could protect BBB integrity by inhibiting EndoMT, which might be associated with upregulating ETS1 expression. Our findings indicated that APS has potential value in the prevention of MS.

Stiff extracellular matrix drives the differentiation of mesenchymal stem cells toward osteogenesis by the multiscale 3D genome reorganization

Extracellular matrix (ECM) stiffness is a major driver of stem cell fate. However, the involvement of the three-dimensional (3D) genomic reorganization in response to ECM stiffness remains unclear. Here, we generated comprehensive 3D chromatin landscapes of mesenchymal stem cells (MSCs) exposed to various ECM stiffness. We found that there were more long-range chromatin interactions, but less compartment A in MSCs cultured on stiff ECM than those cultured on soft ECM. However, the switch from compartment B in MSCs cultured on soft ECM to compartment A in MSCs cultured on stiff ECM included genes encoding proteins primarily enriched in cytoskeleton organization. At the topologically associating domains (TADs) level, stiff ECM tends to have merged TADs on soft ECM. These merged TADs on stiff ECM include upregulated genes encoding proteins enriched in osteogenesis, such as SP1, ETS1, and DCHS1, which were validated by quantitative real-time polymerase chain reaction and found to be consistent with the increase of alkaline phosphatase staining. Knockdown of SP1 or ETS1 led to the downregulation of osteogenic marker genes, including COL1A1, RUNX2, ALP, and OCN in MSCs cultured on stiff ECM. Our study provides an important insight into the stiff ECM-mediated promotion of MSC differentiation towards osteogenesis, emphasizing the influence of mechanical cues on the reorganization of 3D genome architecture and stem cell fate.

ERK/MAPK Signalling Pathway Regulates MMP2 through ETS1 in Renal Clear Cell Carcinoma.

The c-ETS-1 (ETS1) expression is high in clear cell renal cell carcinoma (ccRCC) tissues; however, how it impacts ccRCC is currently unknown. The online STRING web source was used to construct a protein network interacting with ETS1. The Cell Counting Kit-8 was used to detect the cell viability. A clonogenic assay, a wound-healing assay, and a Transwell assay were used to detect cell proliferation, invasion and migration abilities. Western blot was used to detect the expression of proteins. The data showed the expression of ETS1 in ccRCC tissues to be significantly increased compared to adjacent tissues (p<0.05). The positive expression of ETS1 in ccRCC patients aged 20-100 was statistically significant compared to adjacent normal tissues (p<0.05). The grade of ETS1 positive expression (1-4) and lymph node metastasis (N1) in ccRCC were significantly higher than those in adjacent normal tissues (p<0.05). The tumour stage (stages 1-4) in ccRCC patients with positive ETS1 expression was significantly higher than that in adjacent normal tissues (p<0.05). Knockdown of ETS1 and PERK inhibitors significantly inhibited the proliferation, migration and invasion of ccRCC cells. Knockdown of ETS1 inhibited MMP-2 expression, and an extracellular signal-related kinase (ERK) inhibitor inhibited both ETS1 and MMP-2 expression. A high expression of ETS1 is associated with the progression of ccRCC. This study suggests that ETS1 promotes proliferation by increasing MMP2 expression in ccRCC, and combined knockdown of ETS1 and inhibition of ERK can significantly inhibit the proliferation, migration and invasion of ccRCC. ETS1 may be a therapeutic and prognostic target for renal cell carcinoma.

Mechanoresponsive ETS1 causes endothelial dysfunction and arterialization in varicose veins via NOTCH4/DLL4 signaling

Varicose veins are the most common venous disorder in humans and are characterized by hemodynamic instability due to valvular insufficiency and orthostatic lifestyle factors. It is unclear how changes in biomechanical signals cause aberrant remodeling of the vein wall. Our previous studies suggest that Notch signaling is implicated in varicose vein arterialization. In the arterial system, mechanoresponsive ETS1 is a transcriptional activator of the endothelial Notch, but its involvement in sensing disrupted venous flow and varicose vein formation has not been investigated. Here, we use human varicose veins and cultured human venous endothelial cells to show that disturbed venous shear stress activates ETS1-NOTCH4/DLL4 signaling. Notch components were highly expressed in the neointima, whereas ETS1 was upregulated in all histological layers of varicose veins. In vitro microfluidic flow-based studies demonstrate that even minute changes in venous flow patterns enhance ETS1-NOTCH4/DLL4 signaling. Uniform venous shear stress, albeit an inherently low-flow system, does not induce ETS1 and Notch proteins. ETS1 activation under altered flow was mediated primarily by MEK1/2 and, to a lesser extent, by MEK5 but was independent of p38 MAP kinase. Endothelial cell-specific ETS1 knockdown prevented disturbed flow-induced NOTCH4/DLL4 expression. TK216, an inhibitor of ETS-family, prevented the acquisition of arterial molecular identity and loss of endothelial integrity in cells exposed to the ensuing altered shear stress. We conclude that ETS1 senses blood flow disturbances and may promote venous remodeling by inducing endothelial dysfunction. Targeting ETS1 rather than downstream Notch proteins could be an effective and safe strategy to develop varicose vein therapies.

ETS1-mediated Regulation of SOAT1 Enhances the Malignant Phenotype of Oral Squamous Cell Carcinoma and Induces Tumor-associated Macrophages M2-like Polarization.

Oral squamous cell carcinoma (OSCC) is an aggressive cancer that poses a substantial threat to human life and quality of life globally. Lipid metabolism reprogramming significantly influences tumor development, affecting not only tumor cells but also tumor-associated macrophages (TAMs) infiltration. SOAT1, a critical enzyme in lipid metabolism, holds high prognostic value in various cancers. This study revealed that SOAT1 is highly expressed in OSCC tissues and positively correlated with M2 TAMs infiltration. Increased SOAT1 expression enhanced the capabilities of cell proliferation, tumor sphere formation, migration, and invasion in OSCC cells, upregulated the SREBP1-regulated adipogenic pathway, activated the PI3K/AKT/mTOR pathway and promoted M2-like polarization of TAMs, thereby contributing to OSCC growth both in vitro and in vivo. Additionally, we explored the upstream transcription factors that regulate SOAT1 and discovered that ETS1 positively regulates SOAT1 expression levels. Knockdown of ETS1 effectively inhibited the malignant phenotype of OSCC cells, whereas restoring SOAT1 expression significantly mitigated this suppression. Based on these findings, we suggest that SOAT1 is regulated by ETS1 and plays a pivotal role in the development of OSCC by facilitating lipid metabolism and M2-like polarization of TAMs. We propose that SOAT1 is a promising target for OSCC therapy with tremendous potential.

The Transcription Factor Ets1 Influences Axonal Growth via Regulation of Lcn2

Transcription factors are essential for the development and regeneration of the nervous system. The current study investigated key regulatory transcription factors in rat spinal cord development via RNA sequencing. The hub gene Ets1 was highly expressed in the spinal cord during the embryonic period, and then its expression decreased during spinal cord development. Knockdown of Ets1 significantly increased the axonal growth of cultured spinal cord neurons. Luciferase reporter assays and chromatin immunoprecipitation assays indicated that Ets1 could directly bind to the Lcn2 promoter and positively regulate Lcn2 transcription. In conclusion, these findings provide the first direct evidence that Ets1 regulates axon growth by controlling Lcn2 expression, and Ets1 may be a novel therapeutic target for axon regeneration in the central nervous system.

ETS1 regulates NDRG1 to promote the proliferation, migration, and invasion of OSCC.

The aim of this study was to investigate the molecular mechanism by which the transcription factor ETS1 regulates N-myc downstream regulatory gene 1 (NDRG1) to provide a new theoretical basis for the study of oral squamous cell carcinoma (OSCC). In this study, eight human OSCC and paraneoplastic samples were collected. The expressions of NDRG1, ETS1, and Ki67 were detected by immunohistochemistry; apoptosis was detected by tdt-mediated dUTP notched end labeling; cell migration and invasion were detected by Transwell; quantitative real-time PCR was performed to detect the expression of NDRG1; RNA-binding protein immunoprecipitation (RIP) assays detected NDRG1 expression; immunofluorescence assays detected ETS1 expression. NDRG1 and ETS1 expression was significantly upregulated in cancer tissues and CAL-27 and SCC-6 cells. Knockdown of NDRG1 and ETS1 inhibited cell proliferation, migration, invasion, cloning, and EMT while promoting apoptosis and inhibited tumor development; ETS1 positively regulated NDRG1 expression; Finally, overexpression of NDRG1 in vivo and in vitro reversed the effect of ETS1 knockdown on CAL-27 and SCC-6 cells. ETS1 positively regulates the expression of NDRG1 and promotes OSCC. Therefore, ETS1 may serve as a new target for the clinical diagnosis and treatment of OSCC.

ETS1-HMGA2 Axis Promotes Human Limbal Epithelial Stem Cell Proliferation.

This study aimed to investigate the role and molecular mechanism of ETS1 in the proliferation and differentiation of human limbal epithelial stem cells (LESCs). RNA-seq and quantitative real-time PCR were used to determine gene expression changes when ETS1 and HMGA2 was knocked down using short-hairpin RNAs or overexpressed by lentivirus. Immunofluorescence and flow cytometry experiments were performed to assess the roles of ETS1 and HMGA2 in LESC proliferation. ETS1-bound cis-regulatory elements and target genes in LESCs were identified using chromatin immunoprecipitation sequencing. The epigenetic features of ETS1-binding sites were assessed by the published histone modification and chromatin accessibility profiles. ETS1 was robustly expressed in LESCs but dramatically reduced on differentiation into corneal epithelial cells (CECs). ETS1 knockdown in LESCs inhibited cellular proliferation and activated CEC markers (KRT3, KRT12, CLU, and ALDH3A1). When ETS1 was overexpressed during CEC differentiation, LESC-associated genes were upregulated while CEC-associated genes were downregulated. The genome-wide binding profile of ETS1 was identified in LESCs. ETS1 occupied H3K4me3-marked promoters and H3K27ac/H3K4me1-marked enhancers. ETS1-binding sites were also enriched for chromatin accessibility signal. HMGA2 showed a consistent expression pattern with ETS1. ETS1 activates HMAG2 by binding to its promoter. Knockdown and overexpression experiments suggested that HMGA2 can promote LESC proliferation and inhibits its differentiation. ETS1 promotes LESC proliferation and inhibits its differentiation via activating HMGA2.

Regulation of MMP9 transcription by ETS1 in immortalized salivary gland epithelial cells of patients with salivary hypofunction and primary Sjögren’s syndrome

Primary Sjögren’s syndrome (pSS) patients exhibit enhanced degradation of the salivary epithelium initially through MMP9 overexpression. We assessed the expression of MMP9 and an associated transcription factor, ETS1, in primary salivary gland epithelial cells (SGECs) and investigated potential regulatory mechanism(s) in immortalized SGECs. SGECs and iSGECs were derived from pSS and/or xerostomic “sicca” patients. siRNA knockdown of ETS1 in iSGECs was performed to determine MMP9 mRNA (qRT-PCR) and protein expression (ELISA). ETS1 binding to MMP9 promoter was assessed by luciferase activity and binding confirmed by mutagenesis and ChIP. Effects of ETS1 overexpression on progenitor and Epithelial-Mesenchymal transition (EMT) associated markers were determined by Western blot. Expression of ETS1 and its phosphorylated form in iSGECs was determined by immunofluorescence microscopy. ETS1 and MMP9 were overexpressed in SGECs of pSS and non-pSS sicca patients with salivary gland lymphocytic infiltration compared to non-pSS sicca patients without infiltration. ETS1 siRNA knockdown reduced both MMP9 mRNA and protein levels. ETS1 overexpression affected the expression of EMT and progenitor cell markers. Lastly, ETS1 bound the MMP9 promoter within the DNA region of −296 bp to −339 bp. ETS1 may impair salivary function through direct transcriptional control of the MMP9 promoter. ETS1 upregulation may also affect other factors involved in repair of the dysfunctional pSS salivary epithelium.

Ets1 mediates sorafenib resistance by regulating mitochondrial ROS pathway in hepatocellular carcinoma

The incidence and mortality of hepatocellular carcinoma (HCC) are on a rise in the Western countries including US, attributed mostly to late detection. Sorafenib has been the first-line FDA-approved drug for advanced unresectable HCC for almost a decade, but with limited efficacy due to the development of resistance. More recently, several other multi-kinase inhibitors (lenvatinib, cabozantinib, regorafenib), human monoclonal antibody (ramucirumab), and immune checkpoint inhibitors (nivolumab, pembrolizumab) have been approved as systemic therapies. Despite this, the median survival of patients is not significantly increased. Understanding of the molecular mechanism(s) that govern HCC resistance is critically needed to increase efficacy of current drugs and to develop more efficacious ones in the future. Our studies with sorafenib-resistant (soraR) HCC cells using transcription factor RT2 Profiler PCR Arrays revealed an increase in E26 transformation–specific-1 (Ets-1) transcription factor in all soraR cells. HCC TMA studies showed an increase in Ets-1 expression in advanced HCC compared to the normal livers. Overexpression or knocking down Ets-1 modulated sorafenib resistance-related epithelial–mesenchymal transition (EMT), migration, and cell survival. In addition, the soraR cells showed a significant reduction of mitochondrial damage and mitochondrial reactive oxygen species (mROS) generation, which were antagonized by knocking down Ets-1 expression. More in-depth analysis identified GPX-2 as a downstream mediator of Ets-1-induced sorafenib resistance, which was down-regulated by Ets-1 knockdown while other antioxidant pathway genes were not affected. Interestingly, knocking down GPX2 expression significantly increased sorafenib sensitivity in the soraR cells. Our studies indicate the activation of a novel Ets-1–GPX2 signaling axis in soraR cells, targeting which might successfully antagonize resistance and increase efficacy.

Function of AXL and molecular mechanisms in regulation of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignant tumor with unique geographical and ethnic distribution characteristics. NPC is mostly found in south China and Southeast Asia, and its treatment mainly depends on radiotherapy and chemotherapy. However, NPC is usually found in the late stage, and local recurrence and distant metastasis are common, leading to poor prognosis. The receptor tyrosine kinase AXL is up-regulated in various tumors and it is involved in tumor proliferation, migration, invasion, and other processes, which are associated with poor prognosis of tumors. This study aims to detect the expression of AXL in NPC cell lines and tissues, and to investigate its biological function of AXL and the underlying molecular mechanisms in regulation of NPC. The expression levels of AXL in normal nasopharyngeal epithelial tissues and NPC tissues were analyzed by GSE68799, GSE12452, and GSE53819 data sets based on Gene Expression Omnibus (GEO) database. The Cancer Genome Atlas (TCGA) database was used to analyze the relationship between AXL and prognosis of head and neck squamous cell carcinoma (HNSC). The indicators of prognosis included overall survival (OS), disease-free interval (DFI), disease-specific survival (DSS), and progression-free interval (PFI). Western blotting assay was used to detect the AXL protein expression levels in normal nasopharyngeal epithelial cell line and NPC cell lines. Immunohistochemical method was used to detect AXL expression levels in normal nasopharyngeal epithelial tissues and NPC tissues. Cell lines with stable AXL knockdown were established by infecting 5-8F and Fadu cells with lentivirus interference vector, and cell lines with stable AXL overexpression were established by infecting C666-1 and HK-1 cells with lentivirus expression vector. Real-time PCR and Western blotting were used to detect the efficiency of knockdown and overexpression in stable cell lines. The effects of AXL knockdown or overexpression on proliferation, migration, and invasion of NPC cells were detected by CCK-8, plate colony formation, and Transwell assays, and the effect of AXL knockdown on tumor growth in nude mice was detected by subcutaneous tumor formation assay. The sequence of AXL upstream 2.0 kb promoter region was obtained by UCSC online database. The PROMO online database was used to predict AXL transcription factors with 0% fault tolerance, and the JASPAR online database was used to predict the binding sites of ETS1 to AXL. Real-time PCR and Western blotting were used to detect the effect of ETS1 on AXL protein and mRNA expression. The AXL upstream 2.0 kb promoter region was divided into 8 fragments, each of which was 250 bp in length. Primers were designed for 8 fragments. The binding of ETS1 to AXL promoter region was detected by chromatin immuno-precipitation (ChIP) assay to determine the direct regulatory relationship between ETS1 and AXL. Rescue assay was used to determine whether ETS1 affected the proliferation, migration, and invasion of NPC cells through AXL. Bioinformatics analysis showed that AXL was highly expressed in NPC tissues (P<0.05), and AXL expression was positively correlated with OS, DFI, DSS, and PFI in HNSC patients. Western blotting and immunohistochemical results showed that AXL was highly expressed in NPC cell lines and tissues compared with the normal nasopharyngeal epithelial cell line and tissues. Real-time PCR and Western blotting results showed that knockdown and overexpression efficiency in the stable cell lines met the requirements of subsequent experiments. The results of CCK-8, plate colony formation, Transwell assays and subcutaneous tumor formation in nude mice showed that down-regulation of AXL significantly inhibited the proliferation, migration, invasion of NPC cells and tumor growth (all P<0.05), and the up-regulation of AXL significantly promoted the proliferation, migration, and invasion of NPC cells (all P<0.05).As predicted by PROMO and JASPAR online databases, ETS1 was a transcription factor of AXL and had multiple binding sites in the AXL promoter region. Real-time PCR and Western blotting results showed that knockdown or overexpression of ETS1 down-regulated or up-regulated AXL protein and mRNA expression levels. ChIP assay result showed that ETS1 bound to AXL promoter region and directly regulate AXL expression. Rescue assay showed that AXL rescued the effects of ETS1 on proliferation, migration and invasion of NPC cells (P<0.05). AXL is highly expressed in NPC cell lines and tissues, which can promote the malignant progression of NPC, and its expression is regulated by transcription factor ETS1.

ETS1 is a novel transcriptional regulator of adult T-cell leukemia/lymphoma of North American descent

AbstractAdult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell lymphoma associated with the human T-cell lymphotropic virus type 1 virus endemic in regions including Japan, the Caribbean islands, and Latin America. Although progress has been made to understand the disease, survival outcomes with current standard therapy remain extremely poor particularly in acute ATLL, underlying the need for better understanding of its biology and identification of novel therapeutic targets. Recently, it was demonstrated that ATLL of North American–descendent patients (NA-ATLL) is both clinically and molecularly distinct from Japanese-descendent (J-ATLL), with inferior prognosis and higher incidence of epigenetic-targeting mutations compared with J-ATLL. In this study, combined chromatin accessibility and transcriptomic profiling were used to further understand the key transcriptional regulators of NA-ATLL compared with J-ATLL. The ETS1 motif was found to be enriched in chromatin regions that were differentially open in NA-ATLL, whereas the AP1/IRF4 motifs were enriched in chromatin regions more open in J-ATLL. ETS1 expression was markedly elevated in NA-ATLL in both cell line and primary tumor samples, and knockdown of ETS1 in NA-ATLL cells resulted in inhibition of cell growth. CCR4, a previously identified oncogenic factor in ATLL, was found to be a direct ETS1 transcriptional target in NA-ATLL. As such, ETS1 provides an alternate mechanism to enhance CCR4 expression/activity in NA-ATLL, even in the absence of activating CCR4 mutations (CCR4 mutations were identified in 4 of 9 NA-ATLL cases). Taken together, this study identifies ETS1 as a novel dominant oncogenic transcriptional regulator in NA-ATLL.

Uncovering a Key Role of ETS1 on Vascular Abnormality in Glioblastoma.

Glioblastoma (GBM) is the most aggressive type of brain tumor. Microvascular proliferation and abnormal vasculature are the hallmarks of the GBM, aggravating disease progression and increasing patient morbidity. Here, we uncovered a key role of ETS1 on vascular abnormality in glioblastoma. ETS1 was upregulated in endothelial cells from human tumors compared to endothelial cells from paired control brain tissue. Knockdown of Ets1 in mouse brain endothelial cells inhibited cell migration and proliferation, and suppressed expression of genes associated with vascular abnormality in GBM. ETS1 upregulation in tumor ECs was dependent on TGFβ signaling, and targeting TGFβ signaling by inhibitor decreased tumor angiogenesis and vascular abnormality in CT-2A glioma model. Our results identified ETS1 as a key factor regulating tumor angiogenesis, and suggested that TGFβ inhibition may suppress the vascular abnormality driven by ETS1.

Overexpression of lncRNA SLC26A4-AS1 inhibits papillary thyroid carcinoma progression through recruiting ETS1 to promote ITPR1-mediated autophagy.

Papillary thyroid carcinoma (PTC), accounting for approximately 85% cases of thyroid cancer, is a common endocrine tumour with a relatively low mortality but an alarmingly high rate of recurrence or persistence. Long non‐coding RNAs (lncRNAs) is emerging as a critical player modulating diverse cellular mechanisms correlated with the progression of various cancers, including PTC. Herein, we aimed to investigate the role of lncRNA SLC26A4‐AS1 in regulating autophagy and tumour growth during PTC progression. Initially, ITPR1 was identified by bioinformatics analysis as a differentially expressed gene. Then, Western blot and RT‐qPCR were conducted to determine the expression of ITPR1 and SLC26A4‐AS1 in PTC tissues and cells, both of which were found to be poorly expressed in PTC tissues and cells. Then, we constructed ITPR1‐overexpressing cells and revealed that ITPR1 overexpression could trigger the autophagy of PTC cells. Further, we performed a series of gain‐ and loss‐of function experiments. The results suggested that silencing of SLC26A4‐AS1 led to declined ITPR1 level, up‐regulation of ETS1 promoted ITPR1 expression, and either ETS1 knockdown or autophagy inhibitor Bafilomycin A1 could mitigate the promoting effects of SLC26A4‐AS1 overexpression on PTC cell autophagy. In vivo experiments also revealed that SLC26A4‐AS1 overexpression suppressed PTC tumour growth. In conclusion, our study elucidated that SLC26A4‐AS1 overexpression promoted ITPR1 expression through recruiting ETS1 and thereby promotes autophagy, alleviating PTC progression. These finding provides insight into novel target therapy for the clinical treatment of PTC.

ETS1, ELK1, and ETV4 Transcription Factors Regulate Angiopoietin-1 Signaling and the Angiogenic Response in Endothelial Cells.

BackgroundAngiopoietin-1 (Ang-1) is the main ligand of Tie-2 receptors. It promotes endothelial cell (EC) survival, migration, and differentiation. Little is known about the transcription factors (TFs) in ECs that are downstream from Tie-2 receptors.ObjectiveThe main objective of this study is to identify the roles of the ETS family of TFs in Ang-1 signaling and the angiogenic response.MethodsIn silico enrichment analyses that were designed to predict TF binding sites of the promotors of eighty-six Ang-1-upregulated genes showed significant enrichment of ETS1, ELK1, and ETV4 binding sites in ECs. Human umbilical vein endothelial cells (HUVECs) were exposed for different time periods to recombinant Ang-1 protein and mRNA levels of ETS1, ELK1, and ETV4 were measured with qPCR and intracellular localization of these transcription factors was assessed with immunofluorescence. Electrophoretic mobility shift assays and reporter assays were used to assess activation of ETS1, ELK1, and ETV4 in response to Ang-1 exposure. The functional roles of these TFs in Ang-1-induced endothelial cell survival, migration, differentiation, and gene regulation were evaluated by using a loss-of-function approach (transfection with siRNA oligos).ResultsAng-1 exposure increased ETS1 mRNA levels but had no effect on ELK1 or ETV4 levels. Immunostaining revealed that in control ECs, ETS1 has nuclear localization whereas ELK1 and ETV4 are localized to the nucleus and the cytosol. Ang-1 exposure increased nuclear intensity of ETS1 protein and enhanced nuclear mobilization of ELK1 and ETV4. Selective siRNA knockdown of ETS1, ELK1, and ETV4 showed that these TFs are required for Ang-1-induced EC survival and differentiation of cells, while ETS1 and ETV4 are required for Ang-1-induced EC migration. Moreover, ETS1, ELK1, and ETV4 knockdown inhibited Ang-1-induced upregulation of thirteen, eight, and nine pro-angiogenesis genes, respectively.ConclusionWe conclude that ETS1, ELK1, and ETV4 transcription factors play significant angiogenic roles in Ang-1 signaling in ECs.

Conditioned medium from adipose-derived stem cells attenuates ischemia/reperfusion-induced cardiac injury through the microRNA-221/222/PUMA/ETS-1 pathway.

Rationale: Cardiovascular diseases, such as myocardial infarction (MI), are the leading causes of death worldwide. Reperfusion therapy is the common standard treatment for MI. However, myocardial ischemia/reperfusion (I/R) causes cardiomyocyte injury, including apoptosis and fibrosis. We aimed to investigate the effects of conditioned medium from adipose-derived stem cells (ADSC-CM) on apoptosis and fibrosis in I/R-treated hearts and hypoxia/reoxygenation (H/R)-treated cardiomyocytes and the underlying mechanisms.Methods: ADSC-CM was collected from ADSCs. The effects of intramuscular injection of ADSC-CM on cardiac function, cardiac apoptosis, and fibrosis examined by echocardiography, Evans blue/TTC staining, TUNEL assay, and Masson's trichrome staining in I/R-treated mice. We also examined the effects of ADSC-CM on apoptosis and fibrosis in H/R-treated H9c2 cells by annexin V/PI flow cytometry, TUNEL assay, and immunocytochemistry.Results: ADSC-CM treatment significantly reduced heart damage and fibrosis of I/R-treated mice and H/R-treated cardiomyocytes. In addition, the expression of apoptosis-related proteins, such as p53 upregulated modulator of apoptosis (PUMA), p-p53 and B-cell lymphoma 2 (BCL2), as well as the fibrosis-related proteins ETS-1, fibronectin and collagen 3, were significantly reduced by ADSC-CM treatment. Moreover, we demonstrated that ADSC-CM contains a large amount of miR-221/222, which can target and regulate PUMA or ETS-1 protein levels. Furthermore, the knockdown of PUMA and ETS-1 decreased the induction of apoptosis and fibrosis, respectively. MiR-221/222 overexpression achieved similar results. We also observed that cardiac I/R markedly increased apoptosis and fibrosis in miR-221/222 knockout (KO) mice, while ADSC-CM decreased these effects. The increased phosphorylation of p38 and NF‐κB not only mediated myocardial apoptosis through the PUMA/p53/BCL2 pathway but also regulated fibrosis through the ETS-1/fibronectin/collagen 3 pathway.Conclusions: Overall, our results show that ADSC-CM attenuates cardiac apoptosis and fibrosis by reducing PUMA and ETS-1 expression, respectively. The protective effect is mediated via the miR-221/222/p38/NF-κB pathway.

TAMI-58. A NOVEL ROLE FOR REST IN THE CONTROL OF THE MEDULLOBLASTOMA MICROENVIRONMENT

Abstract The REI Silensing Transcription Factor (REST) is a transcriptional repressor and a canonical regulator of neurogenesis. Its expression is elevated in human sonic hedgehog (SHH) subgroup of medulloblastomas (MBs), where functional studies shown its elevated expression to promote proliferation and block neuronal specification. A role for REST in the control of the MB tumor microenvironment (TME) has not been described previously. Here, we demonstrate that REST also controls the MB-TME, and specifically vascular remodeling. Using our unique RESTTG mouse model, we show that conditional expression of human REST transgene in cerebellar granule neuron progenitors (CGNPs), the cell of origin of a subset of SHH MBs, promoted increased vascular growth. In the context of constitutive activation of SHH signaling, a key driver of SHH-MB development, REST elevation drove tumor progression by altering the tumor vasculature. These findings were validated in mouse orthotopic models of human MB cell lines and through analyses of publicly available transcriptomic database of human MB samples. A strong positive correlation between REST and that of endothelial genes CD31/VEGFR1/ETS1 was seen in samples from patients with SHH-MBs subtypes that are associated with the worst prognosis. Proteomic analyses identified increased secretion of a number of pro-angiogenic factors in the context of upregulated REST expression in MB cells. Unexpectedly, in vitro and in vivo studies showed that MB cells expressed these endothelial markers and co-localized with endothelial cells suggesting that REST elevation may have altered the fate of cells that were destined to become neurons. Finally, ETS1 knockdown in MB cell lines not only downregulated VEGFR1 levels in these cells, and blocked tube formation in vitro, but also caused a reduction in tumor cell co-localization with endothelial cells. Collectively, these data suggest that REST elevation remodels MB vasculature through cell-intrinsic and cell-extrinsic mechanisms.

Downregulation of SBF2-AS1 functions as a tumor suppressor in clear cell renal cell carcinoma by inhibiting miR-338-3p-targeted ETS1.

Clear cell renal cell carcinoma (ccRCC) is the most prevalent type of kidney cancer in adults, accompanied by an increasing incidence rate worldwide. We found that SBF2-AS1 was a differentially expressed long-noncoding RNA (lncRNA) in ccRCC through the microarray-based expression analyses. The aim of the present study was to explore the role of SBF2-AS1 in ccRCC development by assessing its effects on cellular processes and further investigate the underlying mechanism. SBF2-AS1 was found to be highly expressed in ccRCC tissues and cells. Ectopic expression and knockdown of SBF2-AS1 and miR-338-3p were performed in ccRCC 768-O cells to explore their effects on cell proliferation, migration, invasion, apoptosis and autophagy by EdU assay, scratch test, Transwell assay, calcein-AM/PI, and GFP-LC3 immunofluorescence assays, respectively. The interactions among SBF2-AS1, miR-338-3p and ETS1 were analyzed using dual-luciferase reporter, RIP and RNA pull-down assays. SBF2-AS1 specifically bound to miR-338-3p and inhibited its expression. Moreover, ETS1 was targeted by miR-338-3p. The knockdown of SBF2-AS1 or ETS1 or overexpression of miR-338-3p resulted in reduced cell proliferation, migration and invasion but elevated cell apoptosis and autophagy. In vivo experiments verified the tumor-suppressive role of silencing SBF2-AS1 in tumor growth of nude mice xenografted with ccRCC cells. Thus, silencing SBF2-AS1 exerted suppressive effects on ccRCC by elevating miR-338-3p and suppressing ETS1.

Endothelial-specific deletion of Ets-1 attenuates Angiotensin II-induced cardiac fibrosis via suppression of endothelial-to-mesenchymal transition

Cardiac fibrosis is a common feature in chronic hypertension patients with advanced heart failure, and endothelial-to-mesenchymal transition (EndMT) is known to promote Angiotensin II (Ang II)-mediated cardiac fibrosis. Previous studies have suggested a potential role for the transcription factor, ETS-1, in Ang II-mediated cardiac remodeling, however the mechanism are not well defined. In this study, we found that mice with endothelial Ets-1 deletion showed reduced cardiac fibrosis and hypertrophy following Ang II infusion. The reduced cardiac fibrosis was accompanied by decreased expression of fibrotic matrix genes, reduced EndMT with decreased Snail, Slug, Twist, and ZEB1 expression, as well as reduced cardiac hypertrophy and expression of hypertrophy-associated genes was observed. In vitro studies using cultured H5V cells further confirmed that ETS-1 knockdown inhibited TGF-β1-induced EndMT. This study revealed that deletion of endothelial Ets-1 attenuated Ang II-induced cardiac fibrosis via inhibition of EndMT, indicating an important ETS-1 function in mediating EndMT. Inhibition of ETS-1 could be a potential therapeutic strategy for treatment of heart failure secondary to chronic hypertension.

Ets-1 enhances tumor migration through regulation of CCR7 expression

Ets-1 is a prototype of the ETS protein family. Members of the ETS protein family contain a unique ETS domain. Ets-1 is associated with cancer progression and metastasis in many types of cancer. Many studies have shown a link between elevated expression of Ets-1 in cancer biopsies and poor survival. CCR7 is a chemokine that binds to specific ligand CCL21/CCL19. CCR7 expression is associated with tumor metastasis and infiltration into lymph nodes. The objective of this study was to test whether Ets-1 could regulate CCR7 expression and enhance tumor metastasis. Our data showed that CCR7 expression was downregulated in Ets-1-deficient T cells upon T-cell stimulation. Overexpression of Ets-1 increased CCR7 expression in breast cancer cell lines. In contrast, knockdown of Ets-1 reduced CCR7 expression. Ets-1 could directly bind to CCR7 promoter and mediate CCR7 expression in luciferase reporter assays and chromatin immunoprecipitation assays. Transactivation activity of Ets-1 was independent of the Pointed domain of Ets-1. Ets-1 could also enhance NF-κB and CBP transactivation of CCR7 promoter. Our results also showed that Ets-1 could modulate cancer cell transmigration by altering CCR7 expression in transwell assay and wound healing assay. Taken together, our data suggest that Ets-1 can enhance CCR7 expression and contribute to tumor cell migration.