Ecrg4 Expression Research Articles

ECRG4 mediates host response to cutaneous infection by regulating neutrophil recruitment and adhesion receptor expression.

Rapid neutrophil recruitment is critical for controlling infection, with dysfunctional neutrophil responses in diseases like diabetes associated with greater morbidity and mortality. We have shown that the leukocyte protein ECRG4 enhances early neutrophil recruitment to cutaneous wounds and hypothesized that ECRG4 regulates the early host response to infection. Using a cutaneous infection model, we found that ECRG4 KO mice had decreased early neutrophil recruitment with persistent larger lesions, increased bacterial proliferation and systemic dissemination. Although previous work identified ECRG4 as a negative regulator of CD44 on neutrophils, the mechanism regulating neutrophil recruitment remained unknown. We demonstrated that pro-inflammatory responses were intact in ECRG4 KO mice, but found decreased neutrophil mobilization from bone marrow and decreased migration to chemokines. ECRG4 KO mouse neutrophils demonstrated an increase in adhesion molecules that regulate recruitment, including enhanced induction of integrin CD11b and increased L-selectin and CD44 on bone marrow neutrophils. Analysis of gene expression in leukocytes from diabetic patients found decreased ECRG4 expression with similar increased L-selectin and CD44. We propose a previously unrecognized mechanism governing neutrophil recruitment, whereby ECRG4 mediates neutrophil surface adhesion molecules that determine both recruitment and outside-in signaling that modulates neutrophil response to pro-inflammatory stimuli.

Loss of Ecrg4 improves calcium oxalate nephropathy.

Kidney stone is one of the most frequent urinary tract diseases, affecting 10% of the population and displaying a high recurrence rate. Kidney stones are the result of salt supersaturation, including calcium and oxalate. We have previously identified Esophageal cancer-related gene 4 (Ecrg4) as being modulated by hypercalciuria. Ecrg4 was initially described as a tumor suppressor gene in the esophagus. Lately, it was shown to be involved as well in apoptosis, cell senescence, cell migration, inflammation and cell responsiveness to chemotherapy. To the best of our knowledge, nothing is known about ECRG4's function in the renal tissue and its relationship with calciuria. We hypothesized that the increased expression of Ecrg4 mRNA is triggered by hypercalciuria and might modulate intratubular calcium-oxalate precipitation. In this study, we have first (i) validated the increased Ecrg4 mRNA in several types of hypercalciuric mouse models, then (ii) described the Ecrg4 mRNA expression along the nephron and (iii) assessed ECRG4's putative role in calcium oxalate nephropathy. For this, Ecrg4 KO mice were challenged with a kidney stone-inducing diet, rich in calcium and oxalate precursor. Taken together, our study demonstrates that Ecrg4's expression is restricted mainly to the distal part of the nephron and that the Ecrg4 KO mice develop less signs of tubular obstruction and less calcium-oxalate deposits. This promotes Ecrg4 as a modulator of renal crystallization and may open the way to new therapeutic possibilities against calcium oxalate nephropathy.

Aberrant expression of the esophageal carcinoma related gene 4 as a prognostic signature for hepatocellular carcinoma

Background and objectiveHepatocellular carcinoma (HCC) is a lethal cancer with increasing incidence, yet the molecular biomarkers that have strong prognostic impact and also hold great therapeutic promise remain elusive. MethodsData mining approaches with a set of publicly accessible databases and immunohistochemistry were used to provide a novel insight into the expression pattern and prognostic significance of the esophageal cancer-related gene (ECRG) family members in HCC. ResultsWe found that elevated mRNA expression levels of ECRG factors were correlated with better overall survival, relapse-free survival and progression-free survival rates in patients with HCC. Subgroup analyses showed significant associations between ECRG expression and survival outcome in select HCC patients. In addition, immunohistochemical and multivariate analysis confirmed increased ECRG4 expression as an independent prognostic indicator for survival. ConclusionsOur data suggest that ECRG factors have significant impacts on the survival of HCC patients. The expression of ECRG factors may be involved in HCC progression and could serve as novel biomarkers for predicting more accurate prognosis.

Cardiac Expression of Esophageal Cancer-Related Gene-4 is Regulated by Sp1 and is a Potential Early Target of Doxorubicin-Induced Cardiotoxicity.

Esophageal Cancer-Related Gene 4 (Ecrg4) expressed in cardiomyocytes and the cardiac conduction system is downregulated during cardiac ischemia and atrial fibrillation. To explore whether Ecrg4 plays any role in doxorubicin (DOX)-induced cardiotoxicity. Rats and neonatal rat cardiomyocytes (NRCMs) were employed to study the effect of DOX on Ecrg4 transcription. Bioinformatics combined with promoter analysis were used to map the rat Ecrg4 promoter. ChIP assay was used to evaluate the binding of Sp1 to the Ecrg4 promoter. Transient transfection was used to study the effect of Sp1 on the expression of endogenous Ecrg4. DOX decreased endogenous Ecrg4 gene expression in the heart and cultured NRCMs. In silico analysis showed that the 5'UTR immediately upstream of the start codon ATG, harbors a putative promoter that is GC-rich, and contains CpG islands, multiple overlapping Sp1sites. Transcription is initiated mainly on the 'C' at - 15. Serial 5'-deletion combined with dual-luciferase assays showed that the rat Ecrg4 core promoter resides at - 1/- 800. Sp1 transactivated Ecrg4 gene, which was almost abolished by DOX. Furthermore, ChIP assay showed that Sp1 specifically bound to the Ecrg4 promoter was interrupted by DOX. Finally, DOX suppressed Sp1 protein expression, and restoration of Sp1 increased Ecrg4 expression that was resistant to DOX-induced Ecrg4 downregulation. Importantly, cardiomyocyte-specific loss of Ecrg4 significantly enriched the differentially expressed proteins in the signaling pathways commonly involved in DOX-induced cardiotoxicity. Our results indicate that Sp1 mediates DOX-induced suppression of Ecrg4, which may contribute indirectly to its cardiotoxicity.

Potential functions of esophageal cancer-related gene-4 in the cardiovascular system

Esophageal cancer-related gene-4 (Ecrg4) is cloned from the normal epithelium of the esophagus. It is constitutively expressed in quiescent epithelial cells and downregulated during tumorigenesis, and Ecrg4 expression levels are inversely correlated with the malignant phenotype of tumor cells, validating that Ecrg4 is a real tumor suppressor gene. Unlike other tumor suppressor genes that usually encode membrane or intracellular proteins, Ecrg4 encodes a 148-amino acid pre-pro-peptide that is tethered on the cell surface in epithelial cells, specialized epithelial cells, and human leukocytes, where it can be processed tissue dependently into several small peptides upon cell activation. Ecrg4 is expressed in a wide variety of other cells/tissues, including cardiomyocytes and conduction system of the heart, the glomus cells of the carotid body, adrenal glands, choroid plexus, and leukocytes among others, where it exerts distinct functions, such as promoting/suppressing inflammation, inducing neuron senescence, stimulating the hypothalamus-pituitary-adrenal axis, maintaining the stemness of stem cells, participating in the rhythm and rate control of the heart, and possibly gauging the responsiveness of the cardiovascular system (CVS) to hypoxia, in addition to tumor suppression. Here, we briefly review the latest discoveries on Ecrg4 and its underlying molecular mechanisms as a tumor suppressor and focus on the emerging roles of Ecrg4 in the CVS.

Counter regulation of ECRG4 gene expression by hypermethylation-dependent inhibition and the Sp1 transcription factor-dependent stimulation of the c2orf40 promoter

The human cytokine precursor ECRG4 has been associated with multiple physiological, developmental and pathophysiological processes involving cell proliferation, cell migration, innate immunity, inflammation, cancer progression and metastases. Although down-regulation of ECRG4 gene expression has been largely attributed to hypermethylation of CpG islands in the 5’untranslated region of the ECRG4 promoter, the mechanisms that underlie the dynamics of its regulation have never been systematically described. Here we show that the ECRG4 gene is widely expressed in human tissues and report that its core promoter lies between the −780 to +420 base pairs relative to the ATG start codon of the ECRG4 open reading frame. This sequence, which contains several CpG islands, also includes multiple overlapping Sp1 consensus binding sequences and a putative binding site for NF-kB activation. 5′RACE of mRNA derived from human leukocytes shows that ECRG4 transcription initiates from the guanidine at −11 from the initiation ATG of the ECRG4 open reading frame. While there is no canonical TATA- or CAAT-boxes proximal to this translational initiation site, there is a distal TATA-sequence in the 5′UTR. This region was identified as the sequence targeted by hypermethylation because in vitro methylation of plasmids encoding the ECRG4 promoter abolish promoter activity and the treatment of Jurkat cells (which naturally express ECRG4) with the methylation inhibitor 5-AzaC, increases endogenous ECRG4 expression. Because ChIP assays show that Sp1 binds the ECRG4 promoter, that forced Sp1 expression trans-activates the ECRG4 promoter and Sp1 inhibition with mithramycin inhibits ECRG4 expression, we conclude that the dynamic positive and negative regulatory elements controlling ECRG4 expression include a counter regulation between promoter methylation and Sp1 activation.

A Potential Role of Esophageal Cancer Related Gene-4 for Atrial Fibrillation

Epidemiological studies have shown a strong correlation between tumor and AF. However, the molecular link between tumor and AF remains unknown. ECRG4, a tumor suppressor gene that is expressed in the A-V node and in sporadic ventricular myocytes, inhibits tumorigenesis and monitors tissue homeostasis by functioning as a ‘sentinel’ molecule gauging inflammatory and cell proliferative responses. To explore the potential physiological function of Ecrg4 in heart, we evaluated its distribution in heart, analyzed its expression in patients with persistent AF and in a canine AF model, and dissected the molecular events downstream of Ecrg4. The results showed that the level of Ecrg4 expression is homogenously high in atria and the conduction systems and in sporadic ventricular myocytes. Importantly, the expression of Ecrg4 was significantly decreased in atrial appendages of AF patients than patients with SR. Moreover, in rapid pacing canine AF models, the expression of ECRG4 in atria was significantly decreased compared to that of the controls. Mechanistically, knockdown ECRG4 in atrial myocytes significantly shortened the APDs, inhibited the expression of Gja1, and activated pro-inflammatory cascades and genes involved in cardiac remodeling. These results suggest that Ecrg4 may play a critical role in the pathogenesis of AF.

Abstract 11439: A Role of Esophageal Cancer Related Gene-4 for Atrial Fibrillation

Introduction: Epidemiological studies have shown that the incidence of atrial fibrillation(AF), the most common type of arrthymia, is more than doubled in cancer patients than that in general population, and reciprocally that the incidence of tumor is ten times higher in AF patients than in non-AF patients. However, the molecular link between tumor and AF remain obscure. Esophageal Cancer Related Gene-4 (Ecrg4), a newly-discovered tumor suppressor gene that is highly expressed in atria and conduction systems of heart, has been shown to function as a ‘sentinel’ molecule in tissue homeostasis, meaning that the presence of Ecrg4 calls for no actions, whereas the loss of Ecrg4 upon injury causes the activation of pro-inflammatory cascade and proliferative responses including fibrosis. Hypothesis: Since injury-induced loss of Ecrg4 in tumor leads to systemic inflammation and increased fibrosis that are well-known inducing factors to AF, we hypothesize that Ecrg4 in heart plays a critical role in the pathogenesis of AF. Methods: The expression of Ecrg4 was evaluated by IHC, IF, and RT-PCR. Canine AF model was established by rapid right atrial pacing at 600 beats/min. for 6 hours. Results: The expression of Ecrg4 was higher in atria and conduction systems than that in ventricles of rat heart by IHC, which was confirmed by RT-PCR. In neonatal rat cardiomyocytes, the expression of Ecrg4, localized in perinucler region and in the ER, is higher in atria than in ventricles. Knock-down Ecrg4 expression by siRNA in atrial cardiomyocytes significantly shortened the duration of action potential. Moreover, in a canine AF model, the expression of Ecrg4 in both Sinus node and left atria was significantly down-regulated compared to that in control animals. Conclusions: Expression of Ecrg4 is high in atria and heart conduction systems and down-regulated in AF, suggesting that Ecrg4 may play a critical role in the pathogenesis of AF.

Abstract A21: Thrombin-processed Ecrg4 recruits myeloid cells and induces anti-tumorigenic inflammation

Abstract Myeloid cells regulate tumor angiogenesis and promote tumor invasion, yet few strategies exist to exploit their presence to reverse the overall immunosuppressive tumor microenvironment. Based on bioinformatic mining of sentinel genes encoding biologically active peptides, we have recently identified Ecrg4 (Esophageal Cancer-Related Gene 4), as a novel secreted pro-inflammatory mediator of macrophage activation. While higher Ecrg4 expression levels correlate with improved survival in cancers of the esophagus, prostate and breast, indicating a role as a candidate tumor suppressor gene, we have demonstrated that the biological activity of Ecrg4 in cancer functions through a paracrine mechanism, in which Ecrg4 regulates myeloid cells in brain tumors. Moreover, mutational analysis reveals that the biological activity of Ecrg4 is dependent on a thrombin-processing site at the C-terminus, inducing monocyte invasion in vivo and in vitro, demonstrating its role as a novel chemoattractant. Here we establish Ecrg4 as a thrombin-processed chemoattractant using subcutaneous tumor models. Ecrg4 gene delivery or Ecrg4 (133-148) peptide administration to tumor reduced melanoma progression through mobilization and activation of myeloid cells to tumor site. Furthermore, in Ecrg4-knockout mice, myeloid cell recruitment is impaired, leading to increased tumor burden. Together, these results identify Ecrg4 as a paracrine factor that is chemotactic for monocytes, with translational potential as an anti-tumor therapeutic. Citation Format: Jisook Lee, Alexandra Borboa, Andrew Baird, Brian Eliceiri. Thrombin-processed Ecrg4 recruits myeloid cells and induces anti-tumorigenic inflammation. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A21. doi:10.1158/1538-7445.CHTME14-A21

Expression of ECRG4 is associated with lower proliferative potential of esophageal cancer cells

We have shown that ECRG4 suppressed Fas-induced apoptosis in Jurkat cells. ECRG4 mRNA expression was ubiquitously detected in normal adult human tissues, suggesting that ECRG4 plays a major role in human tissues. ECRG4 mRNA expression was down-regulated in tumor cells. Expression of ECRG4 suppressed cell growth. We established an anti-ECRG4 monoclonal antibody. Our immunohistochemical analysis demonstrated that ECRG4-positive cells tended to be distributed in the region that was negative for Ki-67 in esophageal squamous cell carcinoma tissues. There was a significant inverse correlation between ECRG4 expression and Ki-67 labeling index in esophageal squamous cell carcinoma. This study provides the first functional evidence for an association of endogenous expression of ECRG4 with cell proliferation. ECRG4 is a candidate tumor suppressor gene that might be involved in the proliferation of esophageal squamous cell carcinoma.

ECRG4 inhibits growth and invasiveness of squamous cell carcinoma of the head and neck in vitro and in vivo

ECRG4 has been shown to be a candidate tumor suppressor in several tumors, but its role in head and neck cancer remains poorly understood. In the present study, the effect of ECRG4 on head and neck cancer was investigated in vitro and in vivo. pFLAG-CMV-2-ECRG4 was stably transfected into squamous cell carcinoma of the head and neck (SCCHN) M2 cell lines to overexpress the ECRG4 gene. Real-time PCR and western blot analysis were performed to detect gene and protein expression, respectively. An MTT assay and flow cytometric analysis were used to detect the growth of M2 cells. Matrigel™ invasion and scratch assays were applied to observe the invasion and migration of the cells. A tumorigenicity assay was applied to test the tumor growth and cervical lymph node metastasis in vivo. Based on the data, pFLAG-CMV-2-ECRG4 significantly increased the expression of ECRG4 in the M2 cells. The constructed plasmid inhibited cell proliferation and promoted cell cycle arrest and apoptosis in the M2 cells. The growth rate and metastasis of the tumor cells in xenografts were suppressed following the overexpression of ECRG4 in nude mice. These data suggest that ECRG4 plays a significant role in the regulation of growth and metastasis in SCCHN, providing new clues for the diagnosis and therapy of SCCHN.

Abstract A19: Ecrg4 downregulation in glioma enables transformed cells to escape immunosurveillance by tumor-associated macrophages/microglia

Abstract Tumor-associated microglia generally promote tumor invasion, adopting an immunosuppressive phenotype that is distinct from their pro-inflammatory functions. Based on bioinformatic mining of novel sentinel genes encoding biologically active peptides, we have recently identified Ecrg4, as a novel secreted pro-inflammatory mediator of macrophage activation. While downregulation of Ecrg4 expression occurs in various tumor cells, in this study, we demonstrate that the biological activity of Ecrg4 in cancer functions through a paracrine mechanism, where tumor cell-exposed Ecrg4 acts upon tumor-associated microglia. In orthotopic glioma models, tumor cells expressing Ecrg4 induce proliferation and morphological changes in tumor-associated microglia. These Ecrg4-mediated effects on microglia correlate with a reduction in tumor burden and prolonged survival. Together, these results identify Ecrg4 as a novel mediator of glioma progression functioning through a paracrine mechanism that regulates the inflammatory state of tumor-associated microglia/macrophages. Citation Format: Jisook Lee, Xitong Dang, Alexandra Borboa, Raul Coimbra, Andrew Baird, Brian Eliceiri. Ecrg4 downregulation in glioma enables transformed cells to escape immunosurveillance by tumor-associated macrophages/microglia. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A19.

The candidate tumor suppressor gene Ecrg4 as a wound terminating factor in cutaneous injury

The Esophageal cancer-related gene-4 (Ecrg4) is a candidate tumor suppressor gene whose secreted protein product has been implicated in the development and progression of epithelial cancers, neuroprogenitor cell activation after central nervous system injury, cell senescence in neurodegeneration, and the survival of hematopoietic stem cells. Here, we investigated the temporal and spatial localization of Ecrg4 expression in healthy and injured mouse skin, and evaluated the biological activity of Ecrg4 using viral-mediated gene delivery in cutaneous wound healing models. Using in situ hybridization and immunohistochemistry, we found both Ecrg4 mRNA and its protein product localized to the epidermis, dermis, and hair follicles of healthy mouse skin. Upon cutaneous injury, Ecrg4 redistributed to the wound margins where gene microarray and quantitative RT-PCR showed an increased gene expression5-10days post-injury as a late phase injury response gene. Ecrg4 over-expression inhibited the directional migration of fibroblasts in modified Boyden chambers in vitro, but had no effect on rates of fibroblast proliferation. Ecrg4 over-expression in vivo at the wound margins delayed the rate of wound closure at 1 and 2days after full-thickness punch injury. These findings point to the candidate tumor suppressor gene Ecrg4 as a novel, biologically active, constituent of skin and skin injury. The possibility that Ecrg4 serves as a wound termination factor during wound resolution is discussed.

Cell specific processing and release of the pro‐hormone candidate tumor suppressor, Ecrg4, from the epithelial cell surface

ObjectiveTo study the processing of human Ecrg4 in epithelial cells.MethodsPlasmids encoding human Ecrg4, Ecrg4 mutants and Ecrg4‐derived peptides fused with green fluorescent protein were transiently expressed in the prostate cancer (PC‐3) or human embryonic kidney (HEK) epithelial cells. The expression of Ecrg4 was analyzed by immunoblotting, immunohistochemistry and fluorescence and confocal microscopy. Cell surface‐bound Ecrg4 was detected by cell surface fractionation.ResultsEcrg4 can be detected in the conditioned media of PC‐3 but not HEK cells 30 hours after transient transfection of pcDNA3‐hEcrg4. In both HEK and PC3 cells however, a “secreted” 14 kDa Ecrg4 product was found tethered to the outer cell surface and could not be released by washing cells with neutral (PBS), high salt (2M NaCl), acidic (Glycine, pH 2.8) or basic (Na2CO3, pH 11) buffers. Fusion of leader sequences with GFP established that Ecrg4 retention was mediated by amino acids 1–15 of the 30 amino acid leader sequence while Ecrg4(16–30) promoted retention. Mutagenesis studies further established that processing occurred at predicted furin and thrombin consensus sites at the cell surface.ConclusionEcrg4 is cell membrane protein that can be shed into media by cell stimulation. This surface bound ECRG4 can be further processed at the furin site to release the ligand‐like peptides in conditioned media.

The Candidate Tumor Suppressor Gene Ecrg4 Inhibits Proliferation of the Inflammed Mucosal Epithelium

Mucosal membranes play an important role in innate and adaptive immunity acting as physical and biological barriers that prevent and regulate the response to infections. Using whole‐genome gene arrays, we identified a small number of regulatory genes that are differentially expressed during otitis media (OM), a mucosal epithelial proliferative response to infection in the middle ear (ME). One such gene is Esophageal cancer related gene 4 (Ecrg4), a candidate tumor suppressor that we found is normally expressed in the quiescent ME. We found that Ecrg4 expression is strongly down‐regulated during the proliferative phase of infection but recovers as mucosal hyperplasia recedes. When tested in vitro, mucosal epithelial proliferation and migration was reduced by Ecrg4 over‐expression when hyper‐proliferating mucosal epithelial cells were harvested from infected ME. When the analogous experiment was performed in vivo, we observed a decrease in the thickness of the mucosal epithelial layer and a reduction in inflammatory infiltrate entering the ME cavity 48 hrs after infection. Together these data suggest that the down‐regulation of Ecrg4 gene expression after infection plays a determinant function in mucosal epithelial hyperplasia and are the first that point to the value of a therapeutic strategy targeting Ecrg4 in the inflammation‐immunity cascade that precipitates mucosal epithelial proliferation.

Cell Surface Localization and Shedding of the Candidate Tumor Suppressor Ligand Ecrg4 after Neutrophil Activation and Polarization

We identified fresh human leukocytes as an abundant source of the candidate epithelial tumor suppressor gene, Ecrg4; an epigenetically regulated gene that, unlike other tumor suppressor genes, encodes an orphan secreted ligand‐like protein. In cultured human cell lines, Ecrg4 gene expression was low, the Ecrg4‐ encoded protein undetectable and Ecrg4 promoter hypermethylation was high (45–90%). Low gene expression was also reversible by incubation with the methylation inhibitor 5‐Aza‐ Cytidine. In contrast, Ecrg4 gene expression in fresh normal human peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) was 600–800 times higher than cultured cells, the protein was readily detected in cell lysates and Ecrg4 promoter hyper‐methylation in normal leukocytes was low (<3%). Immunoblotting, cell surface biotinylation, immunofluorescent staining and cell flow analyses also established that the Ecrg4‐encoded protein was not constitutively secreted but instead exists as an extracellular, membrane‐anchored protein on the surface leukocytes. Its presence on the surface however is dynamic and upon leukocyte activation by 5 min incubations with fMLP, cell surface Ecrg4 protein is shed in concordance with fMLP‐activation of neutrophils and polarization. In light of these findings, we propose that the pathological sequelae of losing Ecrg4 expression in cancer may reflect a more global physiological response that links Ecrg4 to the biology of leukocytes in cancer surveillance, innate immunity, injury and inflammation.

Down-Regulation of ECRG4, a Candidate Tumor Suppressor Gene, in Human Breast Cancer

Introduction ECRG4/C2ORF40 is a potential tumor suppressor gene (TSG) recently identified in esophageal carcinoma. Its expression, gene copy number and prognostic value have never been explored in breast cancer.MethodsUsing DNA microarray and array-based comparative genomic hybridization (aCGH), we examined ECRG4 mRNA expression and copy number alterations in 353 invasive breast cancer samples and normal breast (NB) samples. A meta-analysis was done on a large public retrospective gene expression dataset (n = 1,387) in search of correlations between ECRG4 expression and histo-clinical features including survival.Results ECRG4 was underexpressed in 94.3% of cancers when compared to NB. aCGH data revealed ECRG4 loss in 18% of tumors, suggesting that DNA loss is not the main mechanism of underexpression. Meta-analysis showed that ECRG4 expression was significantly higher in tumors displaying earlier stage, smaller size, negative axillary lymph node status, lower grade, and normal-like subtype. Higher expression was also associated with disease-free survival (DFS; HR = 0.84 [0.76–0.92], p = 0.0002) and overall survival (OS; HR = 0.72 [0.63–0.83], p = 5.0E-06). In multivariate analysis including the other histo-clinical prognostic features, ECRG4 expression remained the only prognostic factor for DFS and OS.ConclusionsOur data suggest that ECRG4 is a candidate TSG in breast cancer, the expression of which may help improve the prognostication. If functional analyses confirm this TSG role, restoring ECRG4 expression in the tumor may represent a promising therapeutic approach.

Esophageal Cancer Related Gene-4 Is a Choroid Plexus-Derived Injury Response Gene: Evidence for a Biphasic Response in Early and Late Brain Injury

By virtue of its ability to regulate the composition of cerebrospinal fluid (CSF), the choroid plexus (CP) is ideally suited to instigate a rapid response to traumatic brain injury (TBI) by producing growth regulatory proteins. For example, Esophageal Cancer Related Gene-4 (Ecrg4) is a tumor suppressor gene that encodes a hormone-like peptide called augurin that is present in large concentrations in CP epithelia (CPe). Because augurin is thought to regulate senescence, neuroprogenitor cell growth and differentiation in the CNS, we evaluated the kinetics of Ecrg4 expression and augurin immunoreactivity in CPe after CNS injury. Adult rats were injured with a penetrating cortical lesion and alterations in augurin immunoreactivity were examined by immunohistochemistry. Ecrg4 gene expression was characterized by in situ hybridization. Cell surface augurin was identified histologically by confocal microscopy and biochemically by sub-cellular fractionation. Both Ecrg4 gene expression and augurin protein levels were decreased 24–72 hrs post-injury but restored to uninjured levels by day 7 post-injury. Protein staining in the supraoptic nucleus of the hypothalamus, used as a control brain region, did not show a decrease of auguin immunoreactivity. Ecrg4 gene expression localized to CPe cells, and augurin protein to the CPe ventricular face. Extracellular cell surface tethering of 14 kDa augurin was confirmed by cell surface fractionation of primary human CPe cells in vitro while a 6–8 kDa fragment of augurin was detected in conditioned media, indicating release from the cell surface by proteolytic processing. In rat CSF however, 14 kDa augurin was detected. We hypothesize the initial release and proteolytic processing of augurin participates in the activation phase of injury while sustained Ecrg4 down-regulation is dysinhibitory during the proliferative phase. Accordingly, augurin would play a constitutive inhibitory function in normal CNS while down regulation of Ecrg4 gene expression in injury, like in cancer, dysinhibits proliferation.

Ecrg4 expression and its product augurin in the choroid plexus: impact on fetal brain development, cerebrospinal fluid homeostasis and neuroprogenitor cell response to CNS injury.

BackgroundThe content and composition of cerebrospinal fluid (CSF) is determined in large part by the choroid plexus (CP) and specifically, a specialized epithelial cell (CPe) layer that responds to, synthesizes, and transports peptide hormones into and out of CSF. Together with ventricular ependymal cells, these CPe relay homeostatic signals throughout the central nervous system (CNS) and regulate CSF hydrodynamics. One new candidate signal is augurin, a newly recognized 14 kDa protein that is encoded by esophageal cancer related gene-4 (Ecrg4), a putative tumor suppressor gene whose presence and function in normal tissues remains unexplored and enigmatic. The aim of this study was to explore whether Ecrg4 and its product augurin, can be implicated in CNS development and the response to CNS injury.MethodsEcrg4 gene expression in CNS and peripheral tissues was studied by in situ hybridization and quantitative RT-PCR. Augurin, the protein encoded by Ecrg4, was detected by immunoblotting, immunohistochemistry and ELISA. The biological consequence of augurin over-expression was studied in a cortical stab model of rat CNS injury by intra-cerebro-ventricular injection of an adenovirus vector containing the Ecrg4 cDNA. The biological consequences of reduced augurin expression were evaluated by characterizing the CNS phenotype caused by Ecrg4 gene knockdown in developing zebrafish embryos.ResultsGene expression and immunohistochemical analyses revealed that, the CP is a major source of Ecrg4 in the CNS and that Ecrg4 mRNA is predominantly localized to choroid plexus epithelial (CPe), ventricular and central canal cells of the spinal cord. After a stab injury into the brain however, both augurin staining and Ecrg4 gene expression decreased precipitously. If the loss of augurin was circumvented by over-expressing Ecrg4 in vivo, BrdU incorporation by cells in the subependymal zone decreased. Inversely, gene knockdown of Ecrg4 in developing zebrafish embryos caused increased proliferation of GFAP-positive cells and induced a dose-dependent hydrocephalus-like phenotype that could be rescued by co-injection of antisense morpholinos with Ecrg4 mRNA.ConclusionAn unusually elevated expression of the Ecrg4 gene in the CP implies that its product, augurin, plays a role in CP-CSF-CNS function. The results are all consistent with a model whereby an injury-induced decrease in augurin dysinhibits target cells at the ependymal-subependymal interface. We speculate that the ability of CP and ependymal epithelium to alter the progenitor cell response to CNS injury may be mediated, in part by Ecrg4. If so, the canonic control of its promoter by DNA methylation may implicate epigenetic mechanisms in neuroprogenitor fate and function in the CNS.

ECRG-4 expression in normal and neoplastic choroid plexus

The choroid plexus is a major site of gene expression of esophageal cancer-related gene (ECRG)-4 during development, suggesting that its gene product may be involved in cerebrospinal fluid (CSF) homeostasis. Yet, ECRG-4 is also a novel candidate tumor suppressor gene whose expression is downregulated and is inversely associated with a worse prognosis in several different cancers. Reduced expression of ECRG-4 has been demonstrated in most tumors, including colorectal carcinoma and malignant glioma, to be mediated by hypermethylation of its promoter. Materials and methods In this study, samples of normal human choroid plexus (both fetal and adult) and choroid plexus neoplasms (WHO grade I papilloma, grade II atypical papilloma, and grade III carcinoma) were stained with antibodies that we generated to augurin, the gene product of ECRG4. DNA was then extracted from the tissue, treated with bisulfite, and subjected to PCR using a 217-base pair region encompassing the ECRG-4 promotor to detect methylation. Results Both fetal and adult human choroid plexus cells demonstrated a robust positive immunostaining at the apical surface that is consistent with our prior results in human, rat, and mouse brains. In contrast, there was a near-complete absence of immunostaining in all of the choroid plexus neoplasms examined. The choroid plexus carcinoma demonstrated significant methylation of the ECRG-4 promotor region. Conclusions Taken together, these data suggest that ECRG-4 is downregulated in neoplasms of the choroid plexus just as has been observed in other central nervous system (CNS) and non-CNS cancers. This is likely due to hypermethylation of the ECRG-4 promotor, as shown in the choroid plexus carcinoma. Further analysis is underway to determine the (1) physiologic and (2) pathophysiologic consequences of ECRG-4 over- and under- expression in the choroid plexus on CSF formation, function, and composition.