Elf5 Expression Research Articles

Modulation of angiopoietin-2 and Tie2: Organ specific effects of microvascular leakage and edema in mice

IntroductionCritical illness is associated with organ failure, in which endothelial hyperpermeability and tissue edema play a major role. The endothelial angiopoietin/Tie2 system, a regulator of endothelial permeability, is dysbalanced during critical illness. Elevated circulating angiopoietin-2 and decreased Tie2 receptor levels are reported, but it remains unclear whether they cause edema independent of other critical illness-associated alterations. Therefore, we have studied the effect of angiopoietin-2 administration and/or reduced Tie2 expression on microvascular leakage and edema under normal conditions. MethodsTransgenic male mice with partial deletion of Tie2 (heterozygous exon 9 deletion, Tie2+/−) and wild-type controls (Tie2+/+) received 24 or 72 pg/g angiopoietin-2 or PBS as control (n = 12 per group) intravenously. Microvascular leakage and edema were determined by Evans blue dye (EBD) extravasation and wet-to-dry weight ratio, respectively, in lungs and kidneys. Expression of molecules related to endothelial angiopoietin/Tie2 signaling were determined by ELISA and RT-qPCR. ResultsIn Tie2+/+ mice, angiopoietin-2 administration increased EBD extravasation (154 %, p < 0.05) and wet-to-dry weight ratio (133 %, p < 0.01) in lungs, but not in the kidney compared to PBS.Tie2+/− mice had higher pulmonary (143 %, p < 0.001), but not renal EBD extravasation, compared to wild-type control mice, whereas a more pronounced wet-to-dry weight ratio was observed in lungs (155 %, p < 0.0001), in contrast to a minor higher wet-to-dry weight ratio in kidneys (106 %, p < 0.05).Angiopoietin-2 administration to Tie2+/− mice did not further increase pulmonary EBD extravasation, pulmonary wet-to-dry weight ratio, or renal wet-to-dry weight ratio. Interestingly, angiopoietin-2 administration resulted in an increased renal EBD extravasation in Tie2+/− mice compared to Tie2+/− mice receiving PBS. Both angiopoietin-2 administration and partial deletion of Tie2 did not affect circulating angiopoietin-1, soluble Tie2, VEGF and NGAL as well as gene expression of angiopoietin-1, −2, Tie1, VE-PTP, ELF-1, Ets-1, KLF2, GATA3, MMP14, Runx1, VE-cadherin, VEGFα and NGAL, except for gene and protein expression of Tie2, which was decreased in Tie2+/− mice compared to Tie2+/+ mice. ConclusionsIn mice, the microvasculature of the lungs is more vulnerable to angiopoietin-2 and partial deletion of Tie2 compared to those in the kidneys with respect to microvascular leakage and edema.

HMGB1 regulates autophagy of placental trophoblast through ERK signaling pathway†.

The purpose of this study is to investigate the role of high mobility group protein B1 (HMGB1) in placental development and fetal growth. We employed the Cre-loxP recombination system to establish a placenta-specific HMGB1 knockout mouse model. Breeding HMGB1flox/flox mice with Elf5-Cre mice facilitated the knockout, leveraging Elf5 expression in extra-embryonic ectoderm, ectoplacental cone, and trophoblast giant cells at 12.5days of embryonic development. The primary goal of this model was to elucidate the molecular mechanism of HMGB1 in placental development, assessing parameters such as placental weight, fetal weight, and bone development. Additionally, we utilized lentiviral interference and overexpression of HMGB1 in human trophoblast cells to further investigate HMGB1's functional role. Our findings indicate that the HMGB1flox/floxElf5cre/+ mouse displays fetal growth restriction, characterized by decreased placental and fetal weight and impaired bone development. The absence of HMGB1 inhibits autophagosome formation, impairs lysosomal degradation, and disrupts autophagic flux. Depletion of HMGB1 in human trophoblast cells also suppresses cell viability, proliferation, migration, and invasion by inhibiting the ERK signaling pathway. Overexpression of HMGB1 observed the opposite phenotypes. HMGB1 participates in the regulation of autophagy through the ERK signaling pathway and affects placental development.

ELF5: A Molecular Clock for Breast Aging and Cancer Susceptibility.

Breast cancer is predominantly an age-related disease, with aging serving as the most significant risk factor, compounded by germline mutations in high-risk genes like BRCA1/2. Aging induces architectural changes in breast tissue, particularly affecting luminal epithelial cells by diminishing lineage-specific molecular profiles and adopting myoepithelial-like characteristics. ELF5 is an important transcription factor for both normal breast and breast cancer development. This review focuses on the role of ELF5 in normal breast development, its altered expression throughout aging, and its implications in cancer. It discusses the lineage-specific expression of ELF5, its regulatory mechanisms, and its potential as a biomarker for breast-specific biological age and cancer risk.

Novel biomarkers to predict treatment response and prognosis in locally advanced rectal cancer undergoing neoadjuvant chemoradiotherapy

PurposeTo identify genes associated with treatment response and prognosis for locally advanced rectal cancer (LARC) patients receiving neoadjuvant chemoradiotherapy (NCRT).MethodsIn our cohort, gene expression profiles of 64 tumor biopsy samples before NCRT were examined and generated. Weighted gene co-expression network analysis was performed to identify gene modules. External validation datasets included GSE3493, GSE119409, and GSE133057. The expression of candidate genes was evaluated using immunohistochemistry (IHC). TIMER was used to assess immune infiltration.ResultsWe identified and validated the capability to predict the treatment response of CCT5 and ELF1 using our data and external validation datasets. The trends of survival differences of candidate genes in the GSE133057 dataset were similar to our cohort. High levels of CCT5 and ELF1 expression were associated with NCRT resistance and poor prognosis. Furthermore, the expression of CCT5 and ELF1 were also assessed in 117 LARC patients’ samples by the IHC method. Based on IHC results and Cox analysis, the risk score model with CCT5 and ELF1 was constructed and performed well. The risk score was an independent prognostic factor for progression-free survival and overall survival in LARC patients and was then used to build nomogram models. The underlying mechanisms of CCT5 and ELF1 were explored using gene set enrichment analysis. The underlying pathway including apoptosis, cell cycle, and other processes. CCT5 and ELF1 expressions were significantly correlated with immune cell infiltration.ConclusionCCT5 and ELF1 were determined as biomarkers for treatment response and prognosis in LARC patients. The risk score model and nomograms helped predict treatment response and survival outcomes for LARC patients undergoing NCRT.

Targeting G3BP2 Sensitizes Acute Myeloid Leukemia to Venetoclax Treatment Via ELF1-MCL1 Axis

Venetoclax-based combination therapies have emerged as promising treatments for elderly acute myeloid leukemia (AML) patients. However, venetoclax resistance renders the treatment ineffective, mainly due to the upregulation of MCL1. Thus, new venetoclax-based combination therapy is under development to circumvent venetoclax resistance. The Ras-GTPase-activating protein SH3-domain binding protein 2 (G3BP2) is upgraded in several solid tumors. Targeting G3BP2 re-sensitized tumor cells to chemotherapy. To date, whether targeting G3BP2 sensitizes venetoclax treatment in AML and its role in leukemogenesis has not been defined. To this end, the primary AML cells and AML cells were collected and cultured with compound C108 (G3BP2 inhibitor) and venetoclax alone or both. The result showed that co-culture with compound C108 and venetoclax decreased the cell viability in primary AML and AML cells, indicating the potent synergy of G3BP2 inhibitor and venetoclax. The Quantitative real-time PCR (RT-qPCR) showed that G3BP2 was overexpressed in AML patients and was associated with poor prognosis. Knockdown (KD) of G3BP2 decreased proliferation and induced apoptosis in AML cells. In addition, G3BP2 inhibition impeded the tumorigenicity in the subcutaneous AML xenograft tumor model. These data demonstrated that G3BP2 sensitized AML to venetoclax treatment and is critical in AML development. The RNA-seq analysis was performed to investigate the role of G3BP2 in venetoclax resistance and leukemogenesis. The results showed that differentially expressed genes (DEG) were enriched in apoptotic processes when G3BP2 were downregulated. The immunoblotting results indicated that G3BP2 knockdown decreased the expression of MCL1 and BCL2, and vice versa. Moreover, we found that the transcription factor ELF1 was positively correlated with G3BP2 by the immunoblotting and RNA-seq analysis. The result was further validated by the Pearson correlation analysis based on the online database. The expression of ELF1 was decreased when G3BP2 were downregulated. Thus, we concluded that G3BP2 is an upstream gene of ELF1 and MCL1. To further investigate the relationship among G3BP2, ELF1, and MCL1, the RNA binding protein immunoprecipitation assay (RIP) and mRNA stability analysis was performed. The results suggested that G3BP2 interacted with the ELF1 mRNA to enhance its stability. We employed bioinformatics analysis via the JASPAR database and RPISeq tools and found ELF1 may facilitate the transcription of MCL1, which was validated in the following study. The expression of MCL1 was restored in the G3BP2 KD cells when ELF1 was overexpressed. We subsequently performed CHIP-qPCR, dual-luciferase reporter gene assays with truncated MCL1 promoter fragments. A 576bp of the binding region upstream of MCL1 promoter was defined, indicating that ELF1 regulated MCL1 expression by modulating its transcription activity. Taken together, our data suggested that targeting G3BP2 decreases MCL1 expression by disturbing ELF1 mRNA stability, thus increasing the sensitivity to venetoclax treatment. We finally established the intra-venous tumorigenesis xenograft model to verify the role of G3BP2 downregulation in venetoclax-treated leukemia mice. The NCG immunodeficient mice were transplanted with U937 cells and administrated with venetoclax daily from 7-21 days post transplantation (Figure 1A). The data showed that G3BP2 KD in combination with venetoclax reduced clinical scores and prolonged the overall survival of AML mice (combination vs. venetoclax: p&amp;lt;0.01; combination vs. G3BP2 KD: p&amp;lt;0.05, Figure 1B). The reduced leukemic burden was observed when targeting G3BP2 in combination with venetoclax. Meanwhile, we verified the decreased expression of ELF1 and MCL1 in mice targeting G3BP2 in combination with venetoclax by immunofluorescence (IF) and flow cytometry analysis. These data supported the synergic effect of G3BP2 inhibition and venetoclax treatment in AML. Our findings emphasize the critical role of G3BP2 in conferring resistance to venetoclax in AML by stabilizing ELF1 mRNA to regulate MCL1 expression. Targeting G3BP2 represents an appealing therapeutic strategy for patients with venetoclax-resistant AML.

Fascin is essential for mammary gland lactogenesis

Fascin expression has commonly been observed in certain subtypes of breast cancer, where its expression is associated with poor clinical outcome. However, its role in normal mammary gland development has not been elucidated. Here, we used a fascin knockout mouse model to assess its role in normal mammary gland morphogenesis and lactation. Fascin knockout was not embryonically lethal, and its effect on the litter size or condition at birth was minimal. However, litter survival until the weaning stage significantly depended on fascin expression solely in the nursing dams. Accordingly, pups that nursed from fascin−/− dams had smaller milk spots in their abdomen, suggesting a lactation defect in the nursing dams. Mammary gland whole-mounts of pregnant and lactating fascin−/− mice showed significantly reduced side branching and alveologenesis. Despite a typical composition of basal, luminal, and stromal subsets of mammary cells and normal ductal architecture of myoepithelial and luminal layers, the percentage of alveolar progenitors (ALDH+) in fascin−/− epithelial fraction was significantly reduced. Further in-depth analyses of fascin−/− mammary glands showed a significant reduction in the expression of Elf5, the master regulator of alveologenesis, and a decrease in the activity of its downstream target p-STAT5. In agreement, there was a significant reduction in the expression of the milk proteins, whey acidic protein (WAP), and β-casein in fascin−/− mammary glands. Collectively, our data demonstrate, for the first time, the physiological role of fascin in normal mammary gland lactogenesis, an addition that could reveal its contribution to breast cancer initiation and progression.

Abstract 11: Prophylactic use of tamoxifen could reduce the risk of breast cancer in women who do not breast feed postpartum

Abstract Background: Triple negative breast cancer (TNBC) is associated with poor survival, particularly affecting African American women (AAW). Epidemiological studies indicate prolonged breast feeding reduces breast cancer (BC) risk, including TNBC. AAW have significantly lower rates of breast feeding compared to Caucasian women. To understand this link we developed a mouse model mimicking abrupt (AI) and gradual involution (GI). AI led to increased estrogen signaling, cell proliferation and chronic inflammation, which was followed by hyperplasia and squamous metaplasia in mammary glands1. There was an increase in the luminal progenitor (LP) cell population, the cells of origin of TNBC, and a decrease in mature luminal (ML) cells in AI glands. In this study, we sought to determine if blocking estrogen signaling with tamoxifen (TAM) could revert the negative effects of AI, and if so, could be a prophylactic option to reduce BC risk in women who do not breast feed. Methods: Uniparous FVB/N mice (~8 weeks) were allowed to nurse six pups per dam at partum. To induce AI, all pups were removed on postpartum (PP) day 7 (d7). For TAM treatment, 5mg sustained release TAM citrate pellet or placebo was implanted in the subscapular region on PP d8. Mammary glands were harvested on PP d28 and d120. FFPE sections were used for histology and immunohistochemistry. Single cell suspensions were analyzed for mammary epithelial subpopulations using Fluorescence Activated Cell Sorting. Affymetrix and qPCR were used for gene expression analysis. Mass cytometry was performed on mammary glands harvested at PP d120. Results: TAM treatment for 21 days completely abrogated hyperplastic and metaplastic changes in AI glands harvested on d120. Treatment initiation on PP d8, d15 and d35 had the same effect. TAM treatment reduced the cell proliferation and collagen deposition in AI glands. De-enrichment of estrogen signaling pathways and decrease in Elf5 expression, a luminal progenitor marker, were observed upon TAM treatment in d28 glands. Mass cytometry revealed a marked reduction in LP population and a significant increase in ML population in TAM treated AI glands on d120, restoring to the levels in age matched virgin mice. Significant increases in progenitor-like markers TSPAN8, Ly6D, CD200 and decreases in CD49f and CD47 expression in LP cells were observed, indicating return to a normal uniparous LP state. Expression of Ly-6D in ML cells, a ML cell marker, was also rescued upon TAM treatment. Conclusion: Using our mouse model of AI and GI, we show that suppression of estrogen signaling after initiation of AI offered marked protection against precancerous changes. TAM restored the balance of epithelial lineages and normalized the LP and basal cells in AI glands to the post-involution phenotype. Our data provide a rationale for considering short-term TAM treatment for women who do not breastfeed to reduce risk of BC. 1. Basree et.al. PMID 31315645 Citation Format: Bhuvaneswari Ramaswamy, Neelam Shinde, Gary K. Gray, Resham Mawalkar, Allen Zhang, Mustafa Basree, Xiaoli Zhang, Ramesh Ganju, Gina M. Sizemore, Joan S. Brugge, Sarmila Majumder. Prophylactic use of tamoxifen could reduce the risk of breast cancer in women who do not breast feed postpartum [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 11.

PTHrP induces STAT5 activation, secretory differentiation and accelerates mammary tumor development

BackgroundParathyroid hormone-related protein (PTHrP) is required for embryonic breast development and has important functions during lactation, when it is produced by alveolar epithelial cells and secreted into the maternal circulation to mobilize skeletal calcium used for milk production. PTHrP is also produced by breast cancers, and GWAS studies suggest that it influences breast cancer risk. However, the exact functions of PTHrP in breast cancer biology remain unsettled.MethodsWe developed a tetracycline-regulated, MMTV (mouse mammary tumor virus)-driven model of PTHrP overexpression in mammary epithelial cells (Tet-PTHrP mice) and bred these mice with the MMTV-PyMT (polyoma middle tumor-antigen) breast cancer model to analyze the impact of PTHrP overexpression on normal mammary gland biology and in breast cancer progression.ResultsOverexpression of PTHrP in luminal epithelial cells caused alveolar hyperplasia and secretory differentiation of the mammary epithelium with milk production. This was accompanied by activation of Stat5 and increased expression of E74-like factor-5 (Elf5) as well as a delay in post-lactation involution. In MMTV-PyMT mice, overexpression of PTHrP (Tet-PTHrP;PyMT mice) shortened tumor latency and accelerated tumor growth, ultimately reducing overall survival. Tumors overproducing PTHrP also displayed increased expression of nuclear pSTAT5 and Elf5, increased expression of markers of secretory differentiation and milk constituents, and histologically resembled secretory carcinomas of the breast. Overexpression of PTHrP within cells isolated from tumors, but not PTHrP exogenously added to cell culture media, led to activation of STAT5 and milk protein gene expression. In addition, neither ablating the Type 1 PTH/PTHrP receptor (PTH1R) in epithelial cells nor treating Tet-PTHrP;PyMT mice with an anti-PTH1R antibody prevented secretory differentiation or altered tumor latency. These data suggest that PTHrP acts in a cell-autonomous, intracrine manner. Finally, expression of PTHrP in human breast cancers is associated with expression of genes involved in milk production and STAT5 signaling.ConclusionsOur study suggests that PTHrP promotes pathways leading to secretory differentiation and proliferation in both normal mammary epithelial cells and in breast tumor cells.

Effect of WWOX on epithelial-mesenchymal transition in ovarian cancer cells in vivo, and its association with Elf5/Snail-1 signal pathway

Purpose: To investigate how WW domain-containing oxidoreductase (WWOX) gene affects epithelial-mesenchymal transition (EMT) of ovarian cancer (OC) cells, and the implication of Elf5/Snail-1 pathway in the process.&#x0D; Methods: This study employed female BALB/c nude mice aged 6 weeks. The expression levels of E-cadherin, β-Catenin, N-cadherin, Vimentin, Snail1 and Elf5 were assayed using qRT-PCR. The protein levels of WW1, WW2, E-cad, β-Cat, N-cad, vimentin, Snail-1 and Elf5 were determined using western blotting (WB). Invasion changes in cells in each group were analyzed by Transwell invasion test while WWOX and Elf5 were evaluated by GST pull-down test; furthermore, WWOX and ELF5 were evaluated by co-immunoprecipitation (COIP).&#x0D; Results: Relative to control group, tumor volume and weight of WWOX high-expression mice were significantly decreased, while those of WWOX low-expression group were increased (p &lt; 0.05). The levels of E-cad, Elf5 and β-catenin increased, but those of N-cad, Snail-1 and vimentin decreased in WWOX high expression group. In the WWOX low-expression group, the levels of E-cad, Elf5 and β-cat were down-regulated, while those of N-cad, Snail-1 and vimentin were up-regulated (p &lt; 0.05). The expression of E-cad, Elf5 and β-cat declined; on the other hand, those of N-Cad, Snail-1 and vimentin were elevated after mutation of WW1 domain in WWOX gene (p &lt; 0.05).&#x0D; Conclusion: Based on the in vivo results, the role of WWOX/Elf5/Snail1 signal route in the epithelial-mesenchymal transition of ovarian cancer, and its related mechanisms have been clarified. This provides a new target for gene therapy of ovarian cancer.

Breast-Specific Molecular Clocks Comprised of ELF5 Expression and Promoter Methylation Identify Individuals Susceptible to Cancer Initiation.

A robust breast cancer prevention strategy requires risk assessment biomarkers for early detection. We show that expression of ELF5, a transcription factor critical for normal mammary development, is downregulated in mammary luminal epithelia with age. DNA methylation of the ELF5 promoter is negatively correlated with expression in an age-dependent manner. Both ELF5 methylation and gene expression were used to build biological clocks to estimate chronological ages of mammary epithelia. ELF5 clock-based estimates of biological age in luminal epithelia from average-risk women were within three years of chronological age. Biological ages of breast epithelia from BRCA1 or BRCA2 mutation carriers, who were high risk for developing breast cancer, suggested they were accelerated by two decades relative to chronological age. The ELF5 DNA methylation clock had better performance at predicting biological age in luminal epithelial cells as compared with two other epigenetic clocks based on whole tissues. We propose that the changes in ELF5 expression or ELF5-proximal DNA methylation in luminal epithelia are emergent properties of at-risk breast tissue and constitute breast-specific biological clocks. PREVENTION RELEVANCE: ELF5 expression or DNA methylation level at the ELF5 promoter region can be used as breast-specific biological clocks to identify women at higher than average risk of breast cancer.

ELF5 inhibits the proliferation and invasion of breast cancer cells by regulating CD24.

E74-like factor five (ELF5) is a basic transcription factor that plays a key role in breast tissue and gland development. However, the molecular mechanism of ELF5 in breast cancer cells has not been elucidated. In this study, we examined the effect of ELF5 on the human breast cancer cell lines MCF-7 and T47D and confirmed that ELF5 can inhibit cell proliferation, migration and invasion. In further research, the relationship between ELF5 and CD24 was characterized in breast cancer cells. We found that CD24 was a target gene of ELF5 through chromatin immunoprecipitation (ChIP) -Sequence assays, and proved that ELF5 could bind to the ETS cis-element on the proximal promoter of the CD24 gene and regulate the expression of CD24. Moreover, overexpression of ELF5 in MCF-7 cells significantly increased both the mRNA and protein levels of CD24, while knockdown of CD24 expression restored cell proliferation, migration and invasion through adaptive ELF5 expression in MCF-7 cells. Therefore, these data suggest that ELF5 inhibits migration and invasion of breast cancer cells by regulating CD24 expression, which make provides a molecular mechanism for ELF5 to inhibit breast cancer from a new perspective and provides further theoretical support for the treatment and prevention of breast cancer.

EPC-Derived Exosomal miR-1246 and miR-1290 Regulate Phenotypic Changes of Fibroblasts to Endothelial Cells to Exert Protective Effects on Myocardial Infarction by Targeting ELF5 and SP1.

Myocardial infarction (MI) remains a leading cause of morbidity and mortality worldwide. Endothelial progenitor cell (EPC)-derived exosomes have been found to be effective in alleviating MI, while the detailed mechanisms remain unclear. The present study aimed to determine the protective effects of EPC-derived exosomal miR-1246 and miR-1290 on MI-induced injury and to explore the underlying molecular mechanisms. The exosomes were extracted from EPCs; gene expression levels were determined by quantitative real-time PCR, and protein expression levels were determined by western blot and immunofluorescence staining, respectively. The angiogenesis and proliferation of human cardiac fibroblasts (HCFs) were determined by tube formation assay and immunofluorescence staining of PKH67, respectively. Luciferase reporter, CHIP, and EMSA assays determined the interaction between miR-1246/1290 and the targeted genes (EFL5 and SP1). The protective effects of miR-1246/1290 on MI were evaluated in a rat model of MI. EPC-derived exosomes significantly upregulated miR-1246 and miR-1290 expression and promoted phenotypic changes of fibroblasts to endothelial cells, angiogenesis, and proliferation in HCFs. Exosomes from EPCs with miR-1246 or miR-1290 mimics transfection promoted phenotypic changes of fibroblasts to endothelial cells and angiogenesis in HCFs, while exosomes from EPCs with miR-1246 or miR-1290 knockdown showed opposite effects in HCFs. Mechanistically, miR-1246 and miR-1290 from EPC-derived exosomes induced upregulation of ELF5 and SP1, respectively, by targeting the promoter regions of corresponding genes. Overexpression of both ELF5 and SP1 enhanced phenotypic changes of fibroblasts to endothelial cells and angiogenesis in HCFs pretreated with exosomes from EPCs with miR-1246 or miR-1290 mimics transfection, while knockdown of both EFL5 and SP1 exerted the opposite effects in HCFs. Both ELF5 and SP1 can bind to the promoter of CD31, leading to the upregulation of CD31 in HCFs. Furthermore, in vivo animal studies showed that exosomes from EPCs with miR-1246 or miR-1290 overexpression attenuated the MI-induced cardiac injury in the rats and caused an increase in ELF5, SP1, and CD31 expression, respectively, but suppressed α-SMA expression in the cardiac tissues. In conclusion, our study revealed that miR-1246 and miR-1290 in EPC-derived exosomes enhanced in vitro and in vivo angiogenesis in MI, and these improvements may be associated with amelioration of cardiac injury and cardiac fibrosis after MI.

An upstream open reading frame regulates vasculogenic mimicry of glioma via ZNRD1-AS1/miR-499a-5p/ELF1/EMI1 pathway.

Increasing evidence has suggested that gliomas can supply blood through vasculogenic mimicry. In this study, the expression and function of ZNRD1‐AS1‐144aa‐uORF (144aa‐uORF) and some non‐coding RNAs in gliomas were assessed. Real‐time quantitative PCR or Western blot was used to discover the expression of 144aa‐uORF, ZNRD1‐AS1, miR‐499a‐5p, ELF1 and EMI1 in gliomas. In addition, RIP and RNA pull‐down assays were applied to explore the interrelationship between 144aa‐uORF and ZNRD1‐AS1. The role of the 144aa‐uORF\\ZNRD1‐AS1\\miR‐499a‐5p\\ELF1\\EMI1 axis in vasculogenic mimicry formation of gliomas was analysed. This study illustrates the reduced expression of the 144aa‐uORF in glioma tissues and cells. Up‐regulation of 144aa‐uORF inhibits proliferation, migration, invasion and vasculogenic mimicry formation within glioma cells. The up‐regulated 144aa‐uORF can increase the degradation of ZNRD1‐AS1 through the nonsense‐mediated RNA decay (NMD) pathway. Knockdown of ZNRD1‐AS1 inhibits vasculogenic mimicry in glioma cells by modulating miR‐499a‐5p. At the same time, miR‐499a‐5p is down‐regulated and has a tumour‐suppressive effect in gliomas. In addition, ZNRD1‐AS1 serves as a competitive endogenous RNA (ceRNA) and regulates the expression of ELF1 by binding to miR‐499a‐5p. Notably, ELF1 binds to the promoter region of EMI1 and up‐regulates EMI1 expression, while simultaneously promoting vasculogenic mimicry in glioma cells. This study suggests that the 144aa‐uORF\\ZNRD1‐AS1\\miR‐499a‐5p\\ELF1\\EMI1 axis takes key part in regulating the formation of vasculogenic mimicry in gliomas and may provide a potential target for glioma treatment.

Loss of ELF5-FBXW7 stabilizes IFNGR1 to promote the growth and metastasis of triple-negative breast cancer through interferon-γ signalling.

Triple-negative breast cancer (TNBC) is characterized by a high degree of immune infiltrate in the tumour microenvironment, which may influence the fate of TNBC cells. We reveal that loss of the tumour suppressive transcription factor Elf5 in TNBC cells activates intrinsic interferon-γ (IFN-γ) signalling, promoting tumour progression and metastasis. Mechanistically, we find that loss of the Elf5-regulated ubiquitin ligase FBXW7 ensures stabilization of its putative protein substrate IFN-γ receptor 1 (IFNGR1) at the protein level in TNBC. Elf5low tumours show enhanced IFN-γ signalling accompanied by an increase of immunosuppressive neutrophils within the tumour microenvironment and increased programmed death ligand 1 expression. Inactivation of either programmed death ligand 1 or IFNGR1 elicited a robust anti-tumour and/or anti-metastatic effect. A positive correlation between ELF5 and FBXW7 expression and a negative correlation between ELF5, FBXW7 and IFNGR1 expression in the tumours of patients with TNBC strongly suggest that this signalling axis could be exploited for patient stratification and immunotherapeutic treatment strategies for Elf5low patients with TNBC.

ELF5 modulates the estrogen receptor cistrome in breast cancer.

Acquired resistance to endocrine therapy is responsible for half of the therapeutic failures in the treatment of breast cancer. Recent findings have implicated increased expression of the ETS transcription factor ELF5 as a potential modulator of estrogen action and driver of endocrine resistance, and here we provide the first insight into the mechanisms by which ELF5 modulates estrogen sensitivity. Using chromatin immunoprecipitation sequencing we found that ELF5 binding overlapped with FOXA1 and ER at super enhancers, enhancers and promoters, and when elevated, caused FOXA1 and ER to bind to new regions of the genome, in a pattern that replicated the alterations to the ER/FOXA1 cistrome caused by the acquisition of resistance to endocrine therapy. RNA sequencing demonstrated that these changes altered estrogen-driven patterns of gene expression, the expression of ER transcription-complex members, and 6 genes known to be involved in driving the acquisition of endocrine resistance. Using rapid immunoprecipitation mass spectrometry of endogenous proteins, and proximity ligation assays, we found that ELF5 interacted physically with members of the ER transcription complex, such as DNA-PKcs. We found 2 cases of endocrine-resistant brain metastases where ELF5 levels were greatly increased and ELF5 patterns of gene expression were enriched, compared to the matched primary tumour. Thus ELF5 alters ER-driven gene expression by modulating the ER/FOXA1 cistrome, by interacting with it, and by modulating the expression of members of the ER transcriptional complex, providing multiple mechanisms by which ELF5 can drive endocrine resistance.

Expression and clinical significance of WWOX, Elf5, Snail1 and EMT related factors in epithelial ovarian cancer.

Expression and clinical significance of WW domain-containing oxidoreductase (WWOX), Elf5, Snail1 and epithelial-mesenchymal transition (EMT) related factors in epithelial ovarian cancer were investigated. Ovarian cancer tissues of 300 epithelial ovarian cancer patients and the adjacent normal tissues were analyzed. Immunohistochemical method was used to detect the expressions of WWOX, Elf5, Snail1 and EMT marker molecules in the specimens. The relationship between the indicators and clinicopathological parameters, and prognosis of patients with ovarian cancer was analyzed. The relationship between WWOX, Elf5, Snail1 and EMT marker molecules E-cadherin, N-cadherin and vimentin in ovarian cancer tissues was analyzed. The expression levels of WWOX, Elf5, Snail1 and EMT marker molecules in epithelial ovarian cancer tissues were significantly different from those in adjacent normal tissues, and were related to surgical pathological stage, pathological grade and lymph node metastasis. High expressions of WWOX and Elf5 were related to the survival rate of patients. The survival rate of patients with positive expression was significantly higher than that of negative expression. FIGO stage, pathological grade, lymph node metastasis and expression of WWOX and Elf5 were all independent factors affecting postoperative prognosis in ovarian cancer patients. In conclusion, the expression levels of WWOX, Elf5, Snail1 and EMT related factors in epithelial ovarian cancer tissues are consistent and different. The expression levels of WWOX and Elf5 are related to the survival and prognosis of patients with epithelial ovarian cancer.

C1orf106, an innate immunity activator, is amplified in breast cancer and is required for basal-like/luminal progenitor fate decision.

Basal-like breast cancer with a luminal progenitor gene expression profile is an aggressive subtype of breast cancer with a poorer prognosis compared with other subtypes. However, genes that specifically promote basal-like breast cancer development remain largely unknown. Here, we report that a novel gene C1orf106 plays an important role in maintaining the feature of basal-like/luminal progenitors. C1orf106 is frequently amplified and overexpressed in basal-like breast cancer and is associated with a poor outcome in patients. In human TCGA database, C1orf106 expression was correlated with upregulation of ELF5 and downregulation of GATA3, two transcription factors that regulate mammary gland stem cell fate. Enhanced expression of C1orf106 promotes tumor progression and expression of basal-like/luminal progenitor marker ELF5; depletion of C1orf106 suppresses tumorigenesis and expression of basal-like/luminal progenitor marker GATA3. These findings suggest that C1orf106 maintains the basal-like/luminal progenitor character through balancing the expression of ELF5 and GATA3. Taken together, we demonstrated that C1orf106 is an important regulator for basal-like/luminal progenitors and targeting C1orf106 is of therapeutic value for breast cancer.

Expression of ELF1, a lymphoid ETS domain-containing transcription factor, is recurrently lost in classical Hodgkin lymphoma.

Loss of B cell-specific transcription factors (TFs) and the resulting loss of B-cell phenotype of Hodgkin and Reed-Sternberg (HRS) cells is a hallmark of classical Hodgkin lymphoma (cHL). Here we have analysed two members of ETS domain containing TFs, ELF1 and ELF2, regarding (epi)genomic changes as well as gene and protein expression. We observed absence or lower levels of ELF1 protein in HRS cells of 31/35 (89%) cases compared to the bystander cells and significant (P<0·01) downregulation of the gene on mRNA as well as protein level in cHL compared to non-cHL cell lines. However, no recurrent loss of ELF2 protein was observed. Moreover, ELF1 was targeted by heterozygous deletions combined with hypermethylation of the remaining allele(s) in 4/7 (57%) cell lines. Indeed, DNA hypermethylation (range 95-99%, mean 98%) detected in the vicinity of the ELF1 transcription start site was found in all 7/7 (100%) cHL cell lines. Similarly, 5/18 (28%) analysed primary biopsies carried heterozygous deletions of the gene. We demonstrate that expression of ELF1 is impaired in cHL through genetic and epigenetic alterations, and thus, it may represent an additional member of a TF network whose downregulation contributes to the loss of B-cell phenotype of HRS cells.

Generation of Elf5-Cre knockin mouse strain for trophoblast-specific gene manipulation.

Placental development is a complex and highly controlled process during which trophoblast stem cells differentiate to various trophoblast subtypes. The early embryonic death of systemic gene knockout models hampers the investigation of these genes that might play important roles during placentation. A trophoblast specific Cre mouse model would be of great help for dissecting out the potential roles of these genes during placental development. For this purpose, we generate a transgenic mouse with the Cre recombinase inserted into the endogenous locus of Elf5 gene that is expressed specifically in placental trophoblast cells. To analyze the specificity and efficiency of Cre recombinase activity in Elf5-Cre mice, we mated Elf5-Cre mice with Rosa26mT/mG reporter mice, and found that Elf5-Cre transgene is expressed specifically in the trophoectoderm as early as embryonic day 4.5 (E4.5). By E12.5, the activity of Elf5-Cre transgene was detected exclusively in all derivatives of trophoblast lineages, including spongiotrophoblast, giant cells, and labyrinth trophoblasts. In addition, Elf5-Cre transgene was also active during spermatogenesis, from spermatids to mature sperms, which is consistent with the endogenous Elf5 expression in testis. Collectively, our results provide a unique tool to delete specific genes selectively and efficiently in trophoblast lineage during placentation.

Effect of the normal mammary differentiation regulator ELF5 upon clinical outcomes of triple negative breast cancers patients.

Elf5 is a transcription factor previously shown to be involved in regulating cell differentiation in both normal and pathological breast tissues. Pertinently, Elf5 was reported to interact with the FOXA1 transcription factor, a pivotal regulatory factor in a subset of AR overexpressing triple negative cancer (TNBC) cases. We examined the correlation among AR, FOXA1, and Elf5 expression in a series of TNBC cases. The cases were retrieved from surgical pathological files of Tohoku University Hospital Japan and consisted of 60 cases operated between the year 1999 and 2007. An additional cohort cases of 51 TNBC ductal carcinoma in situ was used to compare invasive and non-invasive TNBC. In our cohort, 47% of all carcinomas were positive for Elf5, with a significantly higher proportion of Elf5 positive cases occurring in the younger age groups (p=0.0061). Elf5 immunoreactivity was not associated with any other clinicopathological factors examined in this study. However, Elf5 expression was associated with decreased overall and disease-free survival of the patients (Peto-Peto modification of Gehan-Wilcoxon test, OS p=0.132, DFS p=0.1 (LI cutoff 10%); OS p=0.038, DFS p=0.021 (LI cutoff 50%)). Of particular interest, its effects on survival were more pronounced in the EGFR-/CK5/6- (non-basal surrogate) than the EGFR+and/or CK5/6+(basal-surrogate) subtype of TNBC. Elf5 is present in TNBC and its status was significantly correlated with overall survival of the patients. Further studies examining possible interactions between Elf5 and other factors in TNBC could contribute to disentangling TNBC biology.