TMB Signature-Related RCAN2 Promotes Apoptosis by Upregulating EHF/DR5 Pathway in Hepatocellular Carcinoma.

BACKGROUND: The tumour mutation burden (TMB) is a valuable indicator of the accumulation of somatic mutations, and is thought to be associated with the biological behaviour and prognosis of tumours. However, the related genetic mechanism for these association is still unclear. The aim of the present study was to identify the key gene(s) associated with TMB in hepatocellular carcinoma (HCC) and to investigate its biological functions, downstream transcription factors, and mechanism of action. METHODS: Patients in The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) database were classified according to TMB signature-related genes. Key genes related to the TMB signature and tumour prognosis were identified. Immunohistochemistry and Quantitative Real-Time Polymerase Chain Reaction (qPCR) were then used to assess gene expression in clinical HCC tissues and HCC cells. Cells with altered gene expression were evaluated for the effect on cell proliferation and apoptosis, both in vitro and in vivo. Three independent databases and cell sequencing data were used to identify the mechanisms involved and the downstream transcription factors. The mechanism was also studied by altering the expression of downstream transcription factors in vitro. RESULT: The integrated cluster (IC) 2 group, characterized by 99 TMB signature-related genes, showed a significant different TMB score compared to the IC1 group (p < 0.001), as well as more favourable tumour prognosis (p = 0.031). We identified five key prognostic genes that were differentially expressed between IC2 and IC1 and were associated with overall survival. The expression of one of these key prognostic genes, RCAN2, was negatively correlated with TMB in 18 out of 33 tumour types examined. A high level of RCAN2 was correlated with better overall survival in HCC (p = 0.0009). Overexpression of RCAN2 enhanced apoptosis in vitro and in vivo, whereas knockdown of RCAN2 attenuated apoptosis. The mechanism by which RCAN2 promotes apoptosis may involve upregulation of the expression of ETS homologous factor (EHF) and of death receptor 5 (DR5). CONCLUSIONS: Downregulation of RCAN2 expression was found to correlate with elevated TMB in multiple cancer types. RCAN2 was also found to be a biomarker of HCC prognosis, and to promote the apoptosis of HCC cells through the EHF/DR5 pathway. These findings provide a new perspective on systemic treatment for advanced HCC with a high TMB.

ETS homologous factor, controlled by lysine-specific demethylase 5B, suppresses clear cell renal cell carcinoma by inducing Filamin-B.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) remains a deadly disease with a poor prognosis. Here, we identified the ETS homologous factor (EHF) and its target Filamin-B (FLNB) as molecules related to immune evasion in ccRCC. We also explored the upstream modifier that manipulates EHF in ccRCC. DESIGN: Cell proliferation and apoptosis assay, wound healing assay, and Transwell assay were designed to analyze the effects of EHF or FLNB knockdown on the biological activity of ccRCC cells. The growth of differently treated ccRCC cells was assessed by orthotopic tumors. ccRCC cells with different treatments were co-cultured with macrophages, and the role of the lysine-specific demethylase 5B (KDM5B)/EHF/FLNB axis on macrophage polarization or ccRCC progression was characterized by detecting the expression of M2 macrophage markers in the co-culture system or tumor tissues of tumor-bearing mice. RESULTS: The expression of EHF and FLNB was higher, while KDM5B was lower in HK2 cells than in ccRCC cells. EHF overexpression inhibited the biological behavior of ccRCC cells and tumor growth in mice. EHF activated FLNB transcription. Knockdown of FLNB supported the biological activity of ccRCC cells and tumor growth and reversed M2 macrophage polarization in tumor tissues of mice in the presence of EHF. KDM5B inhibited EHF expression by H3K4me3 demethylation, and EHF knockdown potentiated M2 macrophage polarization and tumor growth in vivo repressed by KDM5B knockdown. CONCLUSIONS: KDM5B inhibited the expression of EHF by repressing H3K4me3 modification and the transcription of FLNB by EHF to promote immune evasion and progression of ccRCC.

Ehf controls mammary alveolar lineage differentiation and is a putative suppressor of breast tumorigenesis.

The transcription factor EHF is highly expressed in the lactating mammary gland, but its role in mammary development and tumorigenesis is not fully understood. Utilizing a mouse model of Ehf deletion, herein, we demonstrate that loss of Ehf impairs mammary lobuloalveolar differentiation at late pregnancy, indicated by significantly reduced levels of milk genes and milk lipids, fewer differentiated alveolar cells, and an accumulation of alveolar progenitor cells. Further, deletion of Ehf increased proliferative capacity and attenuated prolactin-induced alveolar differentiation in mammary organoids. Ehf deletion also increased tumor incidence in the MMTV-PyMT mammary tumor model and increased the proliferative capacity of mammary tumor organoids, while low EHF expression was associated with higher tumor grade and poorer outcome in luminal A and basal human breast cancers. Collectively, these findings establish EHF as a non-redundant regulator of mammary alveolar differentiation and a putative suppressor of mammary tumorigenesis.

Transcription factor EHF interacting with coactivator AJUBA aggravates malignancy and acts as a therapeutic target for gastroesophageal adenocarcinoma.

Transcriptional dysregulation of genes is a hallmark of tumors and can serve as targets for cancer drug development. However, it is extremely challenging to develop small-molecule inhibitors to target abnormally expressed transcription factors (TFs) except for the nuclear receptor family of TFs. Little is known about the interaction between TFs and transcription cofactors in gastroesophageal adenocarcinoma (GEA) or the therapeutic effects of targeting TF and transcription cofactor complexes. In this study, we found that ETS homologous factor (EHF) expression is promoted by a core transcriptional regulatory circuitry (CRC), specifically ELF3-KLF5-GATA6, and interference with its expression suppressed the malignant biological behavior of GEA cells. Importantly, we identified Ajuba LIM protein (AJUBA) as a new coactivator of EHF that cooperatively orchestrates transcriptional network activity in GEA. Furthermore, we identified KRAS signaling as a common pathway downstream of EHF and AJUBA. Applicably, dual targeting of EHF and AJUBA by lipid nanoparticles cooperatively attenuated the malignant biological behaviors of GEA in vitro and in vivo. In conclusion, EHF is upregulated by the CRC and promotes GEA malignancy by interacting with AJUBA through the KRAS pathway. Targeting of both EHF and its coactivator AJUBA through lipid nanoparticles is a novel potential therapeutic strategy.

Reproductive Ability Disparity in the Pacific Whiteleg Shrimp (Penaeus vannamei): Insights from Ovarian Cellular and Molecular Levels.

The Pacific whiteleg shrimp (Penaeus vannamei) is a highly significant species in shrimp aquaculture. In the production of shrimp larvae, noticeable variations in the reproductive capacity among female individuals have been observed. Some females experience slow gonadal development, resulting in the inability to spawn, while others undergo multiple maturations and contribute to the majority of larval supply. Despite numerous studies that have been conducted on the regulatory mechanisms of ovarian development in shrimp, the factors contributing to the differences in reproductive capacity among females remain unclear. To elucidate the underlying mechanisms, this study examined the differences in the ovarian characteristics between high and low reproductive bulks at different maturity stages, focusing on the cellular and molecular levels. Transmission electron microscopy analysis revealed that the abundance of the endoplasmic reticulum, ribosomes, mitochondria, and mitochondrial cristae in oocytes of high reproductive bulk was significantly higher than that of the low reproductive bulk in the early stages of ovarian maturation (stages I and II). As the ovaries progressed to late-stage maturation (stages III and IV), differences in the internal structures of oocytes between females with different reproductive capacities gradually diminished. Transcriptome analysis identified differentially expressed genes (DEGs) related to the mitochondria between two groups, suggesting that energy production processes might play a crucial role in the observed variations in ovary development. The expression levels of the ETS homology factor (EHF) and PRDI-BF1 and RIZ homology domain containing 9 (PRDM9), which were significantly different between the two groups, were compared using qRT-PCR in individuals at different stages of ovarian maturation. The results showed a significantly higher expression of the EHF gene in the ovaries of high reproductive bulk at the II and IV maturity stages compared to the low reproductive bulk, while almost no expression was detected in the eyestalk tissue of the high reproductive bulk. The PRDM9 gene was exclusively expressed in ovarian tissue, with significantly higher expression in the ovaries of the high reproductive bulk at the four maturity stages compared to the low reproductive bulk. Fluorescence in situ hybridization further compared the expression patterns of EHF and PRDM9 in the ovaries of individuals with different fertility levels, with both genes showing stronger positive signals in the high reproductive bulk at the four ovarian stages. These findings not only contribute to our understanding of the regulatory mechanisms involved in shrimp ovarian development, but also provide valuable insights for the cultivation of new varieties aimed at improving shrimp fecundity.

Hearing in categories aids speech streaming at the "cocktail party".

Our perceptual system bins elements of the speech signal into categories to make speech perception manageable. Here, we aimed to test whether hearing speech in categories (as opposed to a continuous/gradient fashion) affords yet another benefit to speech recognition: parsing noisy speech at the "cocktail party." We measured speech recognition in a simulated 3D cocktail party environment. We manipulated task difficulty by varying the number of additional maskers presented at other spatial locations in the horizontal soundfield (1-4 talkers) and via forward vs. time-reversed maskers, promoting more and less informational masking (IM), respectively. In separate tasks, we measured isolated phoneme categorization using two-alternative forced choice (2AFC) and visual analog scaling (VAS) tasks designed to promote more/less categorical hearing and thus test putative links between categorization and real-world speech-in-noise skills. We first show that listeners can only monitor up to ~3 talkers despite up to 5 in the soundscape and streaming is not related to extended high-frequency hearing thresholds (though QuickSIN scores are). We then confirm speech streaming accuracy and speed decline with additional competing talkers and amidst forward compared to reverse maskers with added IM. Dividing listeners into "discrete" vs. "continuous" categorizers based on their VAS labeling (i.e., whether responses were binary or continuous judgments), we then show the degree of IM experienced at the cocktail party is predicted by their degree of categoricity in phoneme labeling; more discrete listeners are less susceptible to IM than their gradient responding peers. Our results establish a link between speech categorization skills and cocktail party processing, with a categorical (rather than gradient) listening strategy benefiting degraded speech perception. These findings imply figure-ground deficits common in many disorders might arise through a surprisingly simple mechanism: a failure to properly bin sounds into categories.

Targeting ESE3/EHF With Nifurtimox Inhibits CXCR2+ Neutrophil Infiltration and Overcomes Pancreatic Cancer Resistance to Chemotherapy and Immunotherapy.

BACKGROUND & AIMS: Because pancreatic cancer responds poorly to chemotherapy and immunotherapy, it is necessary to identify novel targets and compounds to overcome resistance to treatment. METHODS: This study analyzed genomic single nucleotide polymorphism sequencing, single-cell RNA sequencing, and spatial transcriptomics. Ehf-knockout mice, KPC (LSL-KrasG12D/+, LSL-Trp53R172H/+ and Pdx1-Cre) mice, CD45.1+ BALB/C nude mice, and CD34+ humanized mice were also used as subjects. Multiplexed immunohistochemistry and flow cytometry were performed to investigate the proportion of tumor-infiltrated C-X-C motif chemokine receptor 2 (CXCR2)+ neutrophils. In addition, multiplexed cytokines assays and chromatin immunoprecipitation assays were used to examine the mechanism. RESULTS: The TP53 mutation-mediated loss of tumoral EHF increased the recruitment of CXCR2+ neutrophils, modulated their spatial distribution, and further induced chemo- and immunotherapy resistance in clinical cohorts and preclinical syngeneic mice models. Mechanistically, EHF deficiency induced C-X-C motif chemokine ligand 1 (CXCL1) transcription to enhance in vitro and in vivo CXCR2+ neutrophils migration. Moreover, CXCL1 or CXCR2 blockade completely abolished the effect, indicating that EHF regulated CXCR2+ neutrophils migration in a CXCL1-CXCR2-dependent manner. The depletion of CXCR2+ neutrophils also blocked the in vivo effects of EHF deficiency on chemotherapy and immunotherapy resistance. The single-cell RNA-sequencing results of PDAC treated with Nifurtimox highlighted the therapeutic significance of Nifurtimox by elevating the expression of tumoral EHF and decreasing the weightage of CXCL1-CXCR2 pathway within the microenvironment. Importantly, by simultaneously inhibiting the JAK1/STAT1 pathway, it could significantly suppress the recruitment and function of CXCR2+ neutrophils, further sensitizing PDAC to chemotherapy and immunotherapies. CONCLUSIONS: The study demonstrated the role of EHF in the recruitment of CXCR2+ neutrophils and the promising role of Nifurtimox in sensitizing pancreatic cancer to chemotherapy and immunotherapy.

Corrigendum: Navigating formula shortages: associations of parental perspectives on transitioning to alternative infant formulas for cow's milk protein allergy during the 2022 national formula shortage.

[This corrects the article DOI: 10.3389/falgy.2023.1333570.].

Anticancer Effect of E26 Transformation-Specific Homologous Factor through the Induction of Senescence and the Inhibition of Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells.

The aim of the present study was to evaluate the effect of ETS homologous factor (EHF) in malignant breast cancer cells. The overexpression and knockdown of the EHF gene in human and mouse breast cancer cells were performed, and the TCGA dataset and Q-omics were analyzed. We found that the tumor suppressor NDRG2 is correlated with EHF gene expression in triple-negative breast cancer cells, that EHF overexpression results in reduced cell proliferation and that apoptosis is promoted by the chemotherapeutic reagent treatment of EHF-overexpressing cells. By EHF overexpression, senescence-associated ß-galactosidase activity and p21WAF1/CIP1 expression were increased, suggesting that EHF may induce cellular senescence. In addition, the overexpression of EHF reduced the migratory ability and inhibited epithelial-mesenchymal transition (EMT). Furthermore, EHF inhibited the phosphorylation of STAT3. The overexpression of EHF also reduced the tumor size, and lung metastasis in vivo. At the tumor site, ß-galactosidase activity was increased by EHF. Finally, the Kaplan-Meier-plotter analysis showed that TNBC patients with a high expression of EHF had a longer relapse-free survival rate. Our findings demonstrated that EHF inhibits breast tumor progression by inducing senescence and regulating EMT in TNBC cells.

[Retracted] miR­206 inhibits cancer initiating cells by targeting EHF in gastric cancer.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that the colony formation assay data shown in Figs. 2, 4 and 8 were strikingly similar to data that had already appeared in another article written by different authors at different research institutes [Chen W, Wang J, Liu S, Wang S, Cheng Y, Zhou W, Duan C and Zhang C: MicroRNA­361­3p suppresses tumor cell proliferation and metastasis by directly targeting SH2B1 in NSCLC. J Exp Clin Cancer Res 35: 76, 732516, 2016]. Owing to the fact that the contentious data in the above article had already been published prior to its submission to Oncology Reports, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [Oncology Reports 38: 1688­1694, 2017; DOI: 10.3892/or.2017.5794].

Extensively Hydrolyzed Formula Improves Allergic Symptoms in the Short Term in Infants with Suspected Cow's Milk Protein Allergy.

Although extensively hydrolyzed formula is widely accepted for managing cow's milk protein allergy (CMPA) long-term, there is a lack of evidence on its short-term efficacy. This study's objective was to investigate the short-term symptom changes (within 3-6 weeks) of infants diagnosed with CMPA and managed with extensively hydrolyzed formula containing Lactobacillus rhamnosus at their subsequent physician visit. Healthcare providers treating 202 patients diagnosed with CMPA under six months old completed de-identified surveys, which were then analyzed in this prospective study. After their first visit, the patients were started on extensively hydrolyzed formula, and their baseline symptoms were scored on a severity scale of 0-3. Patients were then reevaluated at their next follow-up visit to assess changes in symptom severity. The study found statistically significant improvements in gastrointestinal (93%), skin (83%), respiratory (73%), and uncategorized symptoms (90%). These symptom improvements were consistent across different follow-up visit durations. This study is the largest prospective analysis conducted in the United States evaluating short-term change in CMPA symptoms severity in infants under six months old using extensively hydrolyzed formula. These findings suggest that extensively hydrolyzed formula is associated with clinical symptom relief, which is often noticeable by the next follow-up visit. However, additional randomized control trials are needed to validate these results.

Extremely high frequency Schottky diodes based on single GaN nanowires.

Gallium nitride (GaN) is one of the most promising materials for high-frequency devices owing to its prominent material properties. We report on the fabrication and study of a series of Schottky diodes in the ground-signal-ground topology based on individual GaN nanowires. The electrical characterization ofI-Vcurves demonstrated relatively high ideality factor value (about 6-9) in comparison to the planar Au/GaN diodes that can be attributed to the nanowire geometry. The effective barrier height in the studied structures was defined in the range of 0.25-0.4 eV. The small-signal frequency analysis was employed to study the dependency of the scattering parameters in the broad range from 0.1 to 40 GHz. The approximation fitting of the experimental data indicated the record high cutoff frequency of about 165.8 GHz.

Spatio-temporal evolution of heat waves severity and expansion across the Iberian Peninsula and Balearic islands.

In the current climate change scenario, heat waves have become one of the most concerning extreme climatic events, both because of their implications for human health and the economy, and because of their increase in intensity and frequency in recent decades. This work presents for the first time a climatological analysis of heat waves in the Iberian Peninsula and Balearic Archipelago (IPB) using the Excess Heat Factor index (EHF). This index considers the factor of intensity and the acclimatization process of human body in the study of heat waves. We focused on the intensity (also called severity), duration, frequency and spatial extension of heat waves in the IPB in the 1950-2020 period. The exceptional heat wave of August 2018 was approached in a similar way to further explore the usefulness of the EHF index. We found that the EHF index identified heat wave conditions 2 days earlier than indices that used only maximum temperatures. Results showed a significant increase in intensity, duration, frequency and spatial extension of heat waves for the whole IPB for 1950-2020 period. The average extent of heat waves increased by 4.0% per decade and the maximum extent by 4.1% per decade. This trend suggested a significant increase in human exposure, droughts, fire risk and energy demand in this region in the last decades.

The roles of ETS transcription factors in liver fibrosis.

E26 transformation specific or E twenty-six (ETS) protein family consists of 28 transcription factors, five of which, named ETS1/2, PU.1, ERG and EHF, are known to involve in the development of liver fibrosis, and are expected to become diagnostic markers or therapeutic targets of liver fibrosis. In recent years, some small molecule inhibitors of ETS protein family have been discovered, which might open up a new path for the liver fibrosis therapy targeting ETS. This article reviews the research progress of ETS family members in the development liver fibrosis as well as their prospect of clinical application.

Ehf and Fezf2 regulate late medullary thymic epithelial cell and thymic tuft cell development.

Thymic epithelial cells are indispensable for T cell maturation and selection and the induction of central immune tolerance. The self-peptide repertoire expressed by medullary thymic epithelial cells is in part regulated by the transcriptional regulator Aire (Autoimmune regulator) and the transcription factor Fezf2. Due to the high complexity of mTEC maturation stages (i.e., post-Aire, Krt10+ mTECs, and Dclk1+ Tuft mTECs) and the heterogeneity in their gene expression profiles (i.e., mosaic expression patterns), it has been challenging to identify the additional factors complementing the transcriptional regulation. We aimed to identify the transcriptional regulators involved in the regulation of mTEC development and self-peptide expression in an unbiased and genome-wide manner. We used ATAC footprinting analysis as an indirect approach to identify transcription factors involved in the gene expression regulation in mTECs, which we validated by ChIP sequencing. This study identifies Fezf2 as a regulator of the recently described thymic Tuft cells (i.e., Tuft mTECs). Furthermore, we identify that transcriptional regulators of the ELF, ESE, ERF, and PEA3 subfamily of the ETS transcription factor family and members of the Krüppel-like family of transcription factors play a role in the transcriptional regulation of genes involved in late mTEC development and promiscuous gene expression.

Navigating formula shortages: associations of parental perspectives on transitioning to alternative infant formulas for cow's milk protein allergy during the 2022 national formula shortage.

The COVID-19 pandemic led to supply chain disruptions causing a severe shortage of infant formula. The shortage impacted parents of infants with cow's milk protein allergy (CMPA) who rely on specialized formulas. However, research on parent perspectives during formula shortages is limited. We aimed to understand the factors guiding parents' decisions when transitioning to alternative amino acid formula (AAF) or extensively hydrolyzed formula (eHF) during the national formula shortage. We conducted a survey using the ZSMoments platform and found that before the shortage, parents valued safety (83%), tolerability (78%), and reputability (78%) as primary factors in selecting eHFs and AAFs. Post-shortage, formula tolerability (86%), assurance (84%), and safety (80%) gained more importance. Among those switching eHF (n = 54), health care provider recommendations (81%), reputability (78%), taste (78%), and tolerability (78%) were rated as "extremely important." Among those switching AAF (n = 26), top factors included tolerability (77%), assurance (73%), safety (73%), cost-effectiveness (73%), and formula trustworthiness (73%). These data suggest that parents carefully weigh various factors when managing their child's CMPA and transitioning to different AAF or eHF options.

Robust adaptive active disturbance rejection control of an electric furnace using additional continuous sliding mode component.

The temperature control process of electric heating furnace (EHF) systems is a quite difficult and changeable task owing to non-linearity, time delay, time-varying parameters, and the harsh environment of the furnace. In this paper, a robust temperature control scheme for an EHF system is developed using an adaptive active disturbance rejection control (AADRC) technique with a continuous sliding-mode based component. First, a comprehensive dynamic model is established by using convection laws, in which the EHF systems can be characterized as an uncertain second order system. Second, an adaptive extended state observer (AESO) is utilized to estimate the states of the EHF system and total disturbances, in which the observer gains are updated online by a non-linear observer bandwidth, that is as a function of the observation errors. Moreover, with the help of disturbance estimation, a novel sliding manifold is constructed with parameters adaptively adjusted by a dynamic nonlinear bandwidth function to reduce the impact of high gain problems, especially noise-sensitivity. A continuous sliding-mode (CSM) based component is also designed to handle disturbance estimation errors. Third, the stability of the closed loop system, including the proposed controller and estimator, is mathematically proved using the Lyapunov theorem. Finally, the comparative simulation results show that the proposed method has superior robustness and temperature tracking performance.

Pancreatic cancer cell-derived microRNA-155-5p-containing extracellular vesicles promote immune evasion by triggering EHF-dependent activation of Akt/NF-κB signaling pathway.

Pancreatic cancer (PC)-derived EVs have been extensively investigated due to their promising potential as disease biomarkers for diagnosis, monitoring, and treatment decisionmaking. Herein, we explored the mechanism underlying PC-derived EVs in immune evasion of PC. Initially, microRNA (miR)-155-5p level was quantified by RT-qPCR in tumor tissue samples from PC patients, EVs isolated from PC cell lines and PC cell lines. Then, the interaction between miR-155-5p and EHF was identified using dual-luciferase reporter assay. Ectopic expression and knockdown experiments were conducted in PC cells, PC cells-derived EVs, or mouse xenograft model of PC. Afterwards, cell invasion, proportion of macrophage and immune cell subsets, and expression of NF-κB signaling-related genes were assessed using Transwell assay, flow cytometry, RT-qPCR and western blot analysis, respectively. Accordingly, miR-155-5p was upregulated in clinical tissue samples, Pan02-derived EVs and PC cell lines. miR-155-5p knockdown in PC cells enhanced anti-tumor immunity. PC cell-derived EVs facilitated immunosuppressive microenvironment by promoting T cell depletion. In addition, PC cell-derived EVs transferred miR-155-5p to macrophages and then promoted polarization of macrophages to M2 phenotype. EHF was downregulated in PC and could be targeted by miR-155-5p, which resulted in the activation of the Akt/NF-κB signaling. Our findings revealed a previously unrecognized tumor immune evasion-promoting function of PC-derived EV miR-155-5p in PC development by  suppressing EHF and activating NF-κB signaling. This study suggested that the miR-155-5p/EHF/Akt/NF-κB axis can be exploited to prevent cancer immune evasion triggered by therapies.

EHF is essential for epidermal and colonic epithelial homeostasis, and suppresses Apc-initiated colonic tumorigenesis.

Ets homologous factor (EHF) is a member of the epithelial-specific Ets (ESE) family of transcription factors. To investigate its role in development and epithelial homeostasis, we generated a series of novel mouse strains in which the Ets DNA-binding domain of Ehf was deleted in all tissues (Ehf-/-) or specifically in the gut epithelium. Ehf-/- mice were born at the expected Mendelian ratio, but showed reduced body weight gain, and developed a series of pathologies requiring most Ehf-/- mice to reach an ethical endpoint before reaching 1 year of age. These included papillomas in the facial skin, abscesses in the preputial glands (males) or vulvae (females), and corneal ulcers. Ehf-/-mice also displayed increased susceptibility to experimentally induced colitis, which was confirmed in intestinal-specific Ehf knockout mice. Gut-specific Ehf deletion also impaired goblet cell differentiation, induced extensive transcriptional reprogramming in the colonic epithelium and enhanced Apc-initiated adenoma development. The Ets DNA-binding domain of EHF is therefore essential for postnatal homeostasis of the epidermis and colonic epithelium, and its loss promotes colonic tumour development.

Activation of EHF via STAT3 phosphorylation by LMP2A in Epstein-Barr virus-positive gastric cancer.

Epstein-Barr virus (EBV) is associated with approximately 10% of gastric cancers (GCs). We previously showed that EBV infection of gastric epithelial cells induces aberrant DNA methylation in promoter regions, which causes silencing of critical tumor suppressor genes. Here, we analyzed gene expressions and active histone modifications (H3K4me3, H3K4me1, and H3K27ac) genome-widely in EBV-positive GC cell lines and in vitro EBV-infected GC cell lines to elucidate the transcription factors contributing to tumorigenesis through enhancer activation. Genes associated with "signaling of WNT in cancer" were significantly enriched in EBV-positive GC, showing increased active ß-catenin staining. Genes neighboring activated enhancers were significantly upregulated, and EHF motif was significantly enriched in these active enhancers. Higher expression of EHF in clinical EBV-positive GC compared with normal tissue and EBV-negative GC was confirmed by RNA-seq using The Cancer Genome Atlas cohort, and by immunostaining using our cohort. EHF knockdown markedly inhibited cell proliferation. Moreover, there was significant enrichment of critical cancer pathway-related genes (eg, FZD5) in the downstream of EHF. EBV protein LMP2A caused upregulation of EHF via phosphorylation of STAT3. STAT3 knockdown was shown to inhibit cellular growth of EBV-positive GC cells, and the inhibition was rescued by EHF overexpression. Our data highlighted the important role of EBV infection in gastric tumorigenesis via enhancer activation.