Expression Of Esophageal Cancer-related Gene 4 Research Articles

A potential link between aberrant expression of ECRG4 and atrial fibrillation.

Esophageal cancer-related gene-4 (ECRG4), a 148-amino acid propertied and new tumor suppressor, is initially cloned from the normal esophageal epithelium. ECRG4 was found to be expressed not only in esophageal tissues but also in cardiomyocytes. Previous studies demonstrated that ECRG4 is constitutively expressed in esophageal epithelial cells, and its degree of downregulation is directly proportional to prognosis in patients with esophageal cancer. In the heart, ECRG4 shows greater expression in the atria than in the ventricles, which accounts for its heterogeneity. Downregulation of ECRG4 expression level correlates with esophageal cancer, as well as myocardial injuries and arrhythmias. As a result, this review summarizes the possible susceptibility gene, ECRG4 and its associated molecular mechanisms in cancer patients with atrial fibrillation and myocardial injury. The review begins by describing ECRG4's biological background, discusses its expression in the cardiovascular system, lists the clinical and animal research related to the downregulation of ECRG4 in atrial fibrillation, and focuses on its potential role in atrial fibrillation. Downregulation of ECRG4 may increase the risk of atrial fibrillation by affecting ion channels, MMPs expression and inflammatory response. We will then discuss how ECRG4 can be used in the treatment of tumors and arrhythmias, and provide a novel possible strategy to reduce the occurrence of perioperative cardiovascular adverse events in patients with tumors such as esophageal cancer and gastric cancer.

Esophageal cancer-related gene 4 inhibits gastric cancer growth and metastasis by upregulating Krüppel-like factor 2 expression.

There are a large number of people suffering from gastric cancer (GC) worldwide, so the study of biomarkers for GC is urgently needed. This study aimed to investigate the role of esophageal cancer-related gene 4 (ECRG4) in the growth, metastasis, and prognosis of GC and the possible underlying mechanism. The expression of ECRG4 was detected in GC tissues by quantitative polymerase chain reaction (PCR), Western blot, and immunohistochemistry. The relationships between ECRG4 expression and clinicopathological parameters of patients with GC were statistically analyzed, and Kaplan-Meier prognosis and survival curves of the patients were plotted. ECRG4 was overexpressed in the human gastric adenocarcinoma cell line (AGS) and human GC cell line 27 (HGC27), and the in vivo effects of ECRG4 overexpression on the growth, invasion, and metastasis of GC were analyzed and verified in nude mice. To identify the downstream transcription factors potentially regulated by ECRG4, ribonucleic acid (RNA) sequencing and differential gene expression analysis were performed on ECRG4-overexpressing cells. Quantitative PCR, Western blot, and immunohistochemistry were used to detect the expression of the downstream transcription factors targeted by ECRG4 in GC. The ECRG4 mRNA and protein expression levels were low in GC tissues and were associated with a poor prognosis. Least absolute shrinkage and selection operator (LASSO) Cox regression and Kaplan-Meier survival analyses showed that patients with low ECRG4 expression had worse prognosis and survival. Overexpression of ECRG4 inhibited the proliferation, metastasis, and invasion of GC cells. RNA sequencing analysis showed that overexpression of ECRG4 induced the upregulation of Krüppel-like factor 2. Our findings show that ECRG4 promotes GC progression via Krüppel-like factor 2 signaling and highlight ECRG4 as a potential GC biomarker and therapeutic target.

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.

MicroRNA-196b promotes gastric cancer progression by targeting ECRG4.

Gastric cancer is one of the most common malignant tumors. MicroRNA-196b (miR-196b) has been demonstrated to play important roles in human cancers. However, its functions in gastric cancer progression were still largely unknown. In this study, the expression of miR-196b was determined by quantitative real-time PCR. Esophageal cancer-related gene 4 (ECRG4) level was examined by western blot assay and immunohistochemistry staining assay. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) assay and colony formation assay. Cell migration and invasion were analyzed by transwell assay. The association between miR-196b and ECRG4 was analyzed by dual-luciferase reporter assay. The functional role of miR-196b in vivo was analyzed by murine xenograft assay. As a result, we found the expression of miR-196b was elevated and the protein expression of ECRG4 was reduced in gastric cancer tissues and cells. MiR-196b inhibition suppressed gastric cancer cell proliferation, migration and invasion. ECRG4 was a target of miR-196b and its protein expression was negatively regulated by miR-196b. Moreover, ECRG4 overexpression showed similar effects with miR-196b inhibition on the malignant behaviors of GC cells and ECRG4 knockdown reversed the effects of miR-196b inhibition on gastric cancer cell proliferation, migration and invasion. In addition, miR-196b inhibition suppressed tumor volume and weight in vivo. In conclusion, downregulation of miR-196b inhibited gastric cancer progression by modulating ECRG4 expression, indicating that miR-196b might be a potential therapeutic target for gastric cancer.

Esophageal cancer related gene-4 inhibits the migration and proliferation of oral squamous cell carcinoma through BC200 lncRNA/MMP-9 and -13 signaling pathway

Esophageal cancer related gene-4 (ECRG4) inhibits the malignant phenotype of oral squamous cell carcinoma. However, the molecular mechanisms remain to be explored. Using the tongue carcinoma cell line, TCA8113 as a cell model, we showed that forced expression of ECRG4 down-regulated the expression of the BC200 long non-coding RNA (lncRNA) and matrix metalloproteinases (MMP-9 and MMP-13). Restoration of BC200 lncRNA rescued ECRG4-mediated down-regulation of MMP-9 and -13. Furthermore, over-expression of Ecrg4 inhibited cell proliferation and migration, which was abolished by forced expression of BC200 lncRNA in TCA8113 cells. Our results indicate that ECRG4 inhibits the malignant phenotype of TCA8113 cells most likely through suppression of BC200 lncRNA/MMPs signaling pathway, rationalizing that BC200 lncRNA may be a potential target for oral squamous cell carcinoma (OSCC) therapy.

Downregulation and DNA methylation of ECRG4 in gastric cancer.

BackgroundEsophageal cancer-related gene 4 (ECRG4) is a novel candidate tumor suppressor gene. Our study investigated the expression and function of ECRG4 in gastric cancer and highlighted the role of DNA hypermethylation at the promoter in silencing the ECRG4 expression.MethodsThe GSE63089 data set was obtained from the Gene Expression Omnibus and analyzed for differentially expressed genes. Carcinoma and para-carcinoma tissues of 102 patients with gastric cancer were collected from January 2010 to July 2011. Immunohistochemistry, real-time polymerase chain reaction (PCR), and western blot analyses were performed to evaluate the expression of ECRG4. After measuring the change in the level of ECRG4 expression, CCK-8, Transwell, and flow cytometric cell cycle assays were performed. In addition, methylation-specific PCR was performed to detect the methylation state of ECRG4, and 5-aza-2′-deoxycytidine was used for demethylation of ECRG4. All statistical analyses were performed using the SPSS 17.0 software.ResultsWe found that ECRG4 expression was downregulated in gastric cancer, and this was closely related to lymph node metastasis. After ECRG4 was silenced using a specific small interfering RNA, the BGC-823 cell line became highly aggressive and proliferative. In addition, we verified whether downregulation of ECRG4 was highly correlated with DNA methylation of the ECRG4 promoter and found that the demethylating agent 5-aza-2′-deoxycytidine could effectively enhance ECRG4 expression.ConclusionThe aberrant expression of ECRG4 is associated with hypermethylation in the promoter region and plays an important role in the malignancy of gastric cancer. Therefore, ECRG4 may be a potential biomarker for molecular diagnosis of gastric cancer, and the use of 5-Aza-dC to reverse the hypermethylation of ECRG4 may be a new approach to the treatment of gastric cancer.

Downregulation of esophageal cancer-related gene 4 promotes proliferation and migration of hepatocellular carcinoma.

Esophageal cancer-related gene 4 (ECRG4) is a candidate tumor suppressor gene, which is involved in cell apoptosis, migration, infection and inflammation responsiveness; however, its expression level and clinical significance in hepatocellular carcinoma (HCC) remains unclear. In the present study, the authors aim to evaluate the clinical significance and potential role of ECRG4 in HCC. Level of ECRG4 protein expression in HCC and peripheral tissues was investigated in tissue specimens obtained from 56 consecutive HCC patients by immunohistochemistry. Cell proliferation, cell migration and invasion regulations were examined by MTT curves, flow cytometry, Transwell assays and western blotting. ECRG4 expression was weak positive in normal liver cells but was downregulated in HCC cells in vivo or in vitro. A decreased expression of ECRG4 was associated with the age of the patients, metastasis and Ki-67 proliferation index. However, decreased ECRG4 expression was not associated with differentiation, tumor size, the presence of portal vein tumor thrombosis, satellite lesions, tumor relapse or mortality. Further investigations revealed that ectopic expression of ECRG4 inhibited cell proliferation, migration and invasion and promoted cell apoptosis in SMMC-7721 cells, which was mediated by the regulation of BAX and B cell lymphoma-2, in addition to the upregulation of epithelial-mesenchymal transition markers. In conclusion, the results of the present study indicated that ECRG4 was downregulated in HCC and served important roles in promoting cell proliferation and migration.

Beclin 1 promotes apoptosis and decreases invasion by upregulating the expression of ECRG4 in A549 human lung adenocarcinoma cells.

Although Beclin1 has been demonstrated to exert an important role in cell autophagy during carcinogenesis, its biological function in lung cancer has yet to be fully elucidated. A previous study by our laboratory identified that knockdown of Beclin1 promoted cell growth and inhibited apoptosis in the A549lung cancer cell line. In the present study, a Beclin1 lentiviral expression vector was constructed, and an A549cell line was established with a steady expression of Beclin1. Furthermore, the effect of Beclin1 overexpression on cell invasion and apoptosis, changes in the activities of the apoptosis‑associated caspases‑3 and ‑9, and the overexpression of esophageal cancer‑related gene4 (ECRG4) were examined. The results demonstrated that the overexpression of Beclin1 in A549cells reduced cell invasion by Matrigel invasion assay and promoted apoptosis by flow cytometric analysis (P<0.01) compared with Lenex‑packaged lentiviral particles and non‑transfected control groups. Furthermore, the overexpression of Beclin1 in A549cells increased the activities of caspases‑3 and ‑9 and the expression of ECRG4 (P<0.01) compared with Lenex‑packaged lentiviral particles and non‑transfected control groups. In conclusion, the overexpression of Beclin1 promoted apoptosis and decreased invasion by upregulating the expression of ECRG4 in A549lung adenocarcinoma cells. Therefore, the selection of Beclinl as a target for gene therapy represents a more effective method for the treatment of lung cancer.

ECRG4 as a novel tumor suppressor gene inhibits colorectal cancer cell growth in vitro and in vivo.

Esophageal cancer related gene 4 (ECRG4) as a tumor suppressor gene inhibits the growth and development of various tumors. Colorectal cancer (CRC), a common malignant tumor in the digestive tract worldwide, is a leading cause of death. The aim of our study was to assess the tumor-suppressing effect of ECRG4 on CRC and explore its related mechanisms in vitro and in vivo. The expression levels of ECRG4 were measured in colorectal cancer tissues and para-carcinoma tissues. ECRG4 gene was transfected into CRC cells to investigate its effect on cell proliferation by MTT, colony formation assay, and cell cycle analysis. Cell apoptosis was assessed by annexin-V/PI, Hoechst 33342 staining, and analysis of apoptosis-related protein expressions in vitro. The in vivo tumorigenesis assays were performed in nude mice. According to the results, there was a lower expression of ECRG4 in CRC tissues compared with normal tissues, which was strongly associated with histology differentiation and lymph node metastasis. Additionally, overexpression of ECRG4 had a significant inhibitory effect on proliferation and promoted apoptosis in Caco-2 and SW480 cells. Moreover, we found that the overexpression of ECRG4 inhibited tumorigenesis in vivo by diminishing the volume and weight of the tumors and inducing apoptosis of tumor cells. Our study indicates that ECRG4 may be a new potential target and prognostic factor for patients with CRC.

Monitoring Neutrophil-Expressed Cell Surface Esophageal Cancer Related Gene-4 after Severe Burn Injury.

We identified recently esophageal cancer related gene-4 (ECRG4) as a candidate cytokine that is expressed on the surface of quiescent polymorphonuclear leukocytes (PMNs) and shed in response to ex vivo treatment with lipopolysaccharide. To investigate the potential biologic relevance of changes in cell surface ECRG4 in human samples, we performed a pilot study to examine a population of burn patients in whom blood could be analyzed prospectively. We hypothesized that cutaneous burn injury would alter cell surface expression of ECRG4 on PMNs. Patients admitted with more than 20% total burn surface area (TBSA) (n = 10) had blood collected at the time of admission and weekly thereafter. For comparison, blood was obtained from a control group of healthy human volunteers (n = 4). We used flow cytometry to measure changes in ECRG4(+) PMNs from patients during recovery from injury. Esophageal cancer related gene-4 expression at each time point was compared with the patient's clinical status based on a Multiple Organ Dysfunction (MOD) score. Esophageal cancer related gene-4 was detected on the PMN surface of cells collected from healthy volunteers, however, within 48 h of admission after burn injury (n = 10 patients), the number of PMNs with cell surface ECRG4 was decreased. Esophageal cancer related gene-4 expression in PMNs was re-established over the course of patient recovery, unless complications occurred. In this case, the decrease in cell surface ECRG4(+) PMNs preceded the clinical diagnosis of infectious complications and was reflected by increased organ injury scores. From a small sample set, we were able to determine that PMN cell surface ECRG4 expression was decreased after burn injury and returned to baseline during recovery from injury. Although larger studies are needed to define the role of ECRG4 in human PMNs further, this report is the first assessment of cell surface ECRG4 protein in a patient population to support analogous findings in animal studies.

Downregulated ECRG4 is associated with poor prognosis in renal cell cancer and is regulated by promoter DNA methylation.

Esophageal cancer-related gene 4 (ECRG4) has been proposed as a putative tumor suppressor gene in several tumors. However, the role and regulation of ECRG4 in the pathogenesis of human renal cancer remain largely unknown. Our current study revealed that expression of ECRG4 is downregulated in renal cell lines and renal cancer tissues. ECRG4 expression was significantly associated with histological grade of tumors (p < 0.001), primary tumor stage (p = 0.017), and distant metastasis (p = 0.017). Low expression of ECRG4 was an independent prognostic indicator for survival of renal cancer patients. Silencing of ECRG4 expression in renal cell lines was associated with its promoter methylation. Moreover, ectopic expression of ECRG4 markedly inhibited cell proliferation and invasion in renal cancer cell lines. These results indicated that ECRG4 is frequently silenced by the methylation of promoter in renal cell cancers. ECRG4 may be a tumor suppressor in renal cancer and serve as a prognostic marker.

A preliminary study of the effect of ECRG4 overexpression on the proliferation and apoptosis of human laryngeal cancer cells and the underlying mechanisms.

Human esophageal cancer-related gene 4 (ECRG4) is a potential tumor suppressor gene isolated from human esophageal epithelial cells. Studies have shown that ECRG4 effectively inhibits the proliferation of tumor cells and induces apoptosis. However, the role of ECRG4 in laryngeal cancer has not yet been clearly defined. In this study, a human laryngeal cancer cell line stably overexpressing ECRG4 was established. The effect of ECRG4 on the proliferation and apoptosis of laryngeal cancer cells and the associated mechanisms were investigated. The Hep-2 human laryngeal carcinoma cell line exhibited a low basal level of ECRG4 expression and was selected for the present study. The eukaryotic expression plasmid pcDNA3.1-ECRG4 was constructed and introduced into Hep-2 cells by transfection reagents. Western blot analysis, reverse transcription-quantitative polymerase chain reaction and immunofluorescence staining confirmed high-level expression of ECRG4. The 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and colony formation assay showed that ECRG4 over-expression suppressed the proliferative capacity of laryngeal cancer cells in vitro. Cell cycle analysis showed that ECRG4 induced cell cycle arrest at the G0/G1 phase. Flow cytometric analysis and Hoechst staining demonstrated that overexpres-sion of ECRG4 significantly induced apoptosis. Western blot analysis confirmed that Bcl-2-associated X protein, cleaved-caspase-3 and cleaved-poly (ADP-ribose) polymerase were upregulated in the apoptotic process, whereas B-cell lymphoma 2 was downregulated. In conclusion, overexpression of ECRG4 inhibited laryngeal cancer cell proliferation and induced cancer cell apoptosis. Therefore, ECRG4 exhibits potential as an effective target in gene therapy for laryngeal cancer.

Pulmonary preconditioning, injury, and inflammation modulate expression of the candidate tumor suppressor gene ECRG4 in lung

ABSTRACTPurpose: The human c2orf40 gene encodes a candidate tumor suppressor called Esophageal Cancer-Related Gene-4 (ECRG4) that is a cytokine-like epigenetically-regulated protein that is characteristically downregulated in cancer, injury, inflammation, and infection. Here, we asked whether ECRG4 gene expression is detectable in lung epithelial cells and if its expression changes with inflammation, infection, and/or protective preconditioning. Materials and Methods: We used immunoblotting, PCR, and quantitative PCR to measure ECRG4 and either inhalation anesthesia preconditioning, lipopolysaccharide injection, or laparotomy to modulate lung inflammation. Results: Immunoblotting establishes the presence of the full-length 14 kDa ECRG4 peptide in mouse lung. Immunohistochemistry localizes ECRG4 to type l alveolar epithelial cells. Basal ECRG4 mRNA is greater than TNF-α, IL-1β, and IL-6 but following inflammatory lung injury, TNF-α, IL-1β, IL-6, and IL-10 are upregulated while ECRG4 gene expression is decreased. Similar findings are observed after an intravenous administration of lipopolysaccharide. In contrast, lung preconditioning with isoflurane anesthesia increases lung ECRG4 gene expression. Over-expression of ECRG4 in human lung epithelial cells in vitro decreases cell proliferation implying that a loss of ECRG4 in vivo would be permissive to cell growth. Conclusions: This study supports the hypothesis that ECRG4 acts as a sentinel growth inhibitor in lung alveolar epithelial cells. Its downregulation by injury, infection, and inflammation and upregulation by preconditioning supports a role for ECRG4 in regulating the alveolar epithelium response to injury and inflammation. By extension, the findings support a functional consequence to its inhibition by promoter hypermethylation (i.e. lung cancer) and suggest potential benefits to its upregulation.

The tumor-promoting function of ECRG4 in papillary thyroid carcinoma and its related mechanism.

This study aimed to explore the tumor-promoting function of esophageal cancer-related gene 4 (ECRG4) in the papillary thyroid cancer and its related mechanism. ECRG4 Messenger RNA (mRNA) and protein expression analysis in papillary thyroid cancer tissues was performed by quantitative real-time PCR (Q-RT-PCR), Western blot, and immunohistochemistry methods. Ten pairs of fresh samples from the papillary thyroid carcinoma patients were analyzed for ECRG4 promoter CpG island methylation status by bisulfite sequencing analysis. We also transfected ECRG4 into papillary thyroid cancer cell lines W3 and K1 with lentivirus and analyzed ECRG4 functions through evaluating the changes of the proliferation activity, the cell cycle, and the cell apoptosis rate of these transformed cells. We found that ECRG4 expression was upregulated in most papillary thyroid cancer samples (70.0%, 28 out of 40 papillary thyroid cancer samples) on the protein level, and the ECRG4 mRNA level was also enhanced in tumor tissues compared to their matched nontumor tissues. CpG islands around the ECRG4 promoter region were demethylated in the papillary thyroid cancer samples. At the same time, the upregulated expression of ECRG4 in papillary thyroid cancer cell lines W3 and K1 could promote both the proliferation activity and the cell cycle transition from the G1 phase into the G2 but could not affect the cell apoptosis rate. The expression of ECRG4 is frequently upregulated in a papillary thyroid carcinoma through the demethylation mechanism of CpG islands in the gene promoter region, and the ECRG4 has a tumor-promoting function through inducing the cell cycle transition from the G1 phase to the G2 in papillary thyroid carcinoma cells.

Esophageal cancer related gene 4 in tumor

Esophageal cancer related gene 4 (ECRG4) serves as a tumor suppressor gene through interacting with NF-κB and p53 pathways.Multiple studies have demonstrated that the down-regulated expression of ECRG4 occurs in a variety of cancer types,indicating a potential application of ECRG4 as a molecular diagnostic marker as well as a therapeutic target Key words: Genes, tumor suppressor; Neoplasms; Tumor markers, biological; Diagnosis

Esophageal cancer related gene 4 (ECRG4) is a marker of articular chondrocyte differentiation and cartilage destruction

With the aim of identifying novel genes regulating cartilage development and degeneration, we screened a cartilage-specific expressed sequence tag database. Esophageal cancer related gene 4 (ECRG4) was selected, based on the criteria of ‘chondrocyte-specific’ and ‘unknown function.’ ECRG4 expression was particularly abundant in chondrocytes and cartilage, compared to various other mouse tissues. ECRG4 is a secreted protein that undergoes cleavage after secretion. The protein is specifically expressed in chondrocytes in a manner dependent on differentiation status. The expression is very low in mesenchymal cells, and dramatically increased during chondrogenic differentiation. The ECRG4 level in differentiated chondrocytes is decreased during hypertrophic maturation, both in vitro and in vivo, and additionally in dedifferentiating chondrocytes induced by interleukin-1β or serial subculture, chondrocytes of human osteoarthritic cartilage and experimental mouse osteoarthritic cartilage. However, ectopic expression or exogenous ECRG4 treatment in a primary culture cell system does not affect chondrogenesis of mesenchymal cells, hypertrophic maturation of chondrocytes or dedifferentiation of differentiated chondrocytes. Additionally, cartilage development and organization of extracellular matrix are not affected in transgenic mice overexpressing ECRG4 in cartilage tissue. However, ectopic expression of ECRG4 reduced proliferation of primary culture chondrocytes. While the underlying mechanisms of ECRG4 expression and specific roles remain to be elucidated in more detail, our results support its function as a marker of differentiated articular chondrocytes and cartilage destruction.

Expression of esophageal cancer related gene 4 (ECRG4), a novel tumor suppressor gene, in esophageal cancer and its inhibitory effect on the tumor growth in vitro and in vivo

The ECRG4 gene was initially identified and cloned in our laboratory from human normal esophageal epithelium (GenBank accession no. AF325503). We revealed the expression of ECRG4 protein was downregulated in 68.5% (89/130) ESCC samples using tissue microarray. The low ECRG4 protein expression was significantly associated with regional lymph node metastasis, primary tumor size, and tumor stage in ESCC (p < 0.05). ECRG4 mRNA expression was downregulated in ESCC due to the hypermethylation in the gene promoter. The treatment with 5-aza-2'-deoxycytidine, which is a DNA methyltransferase inhibitor restored ECRG4 mRNA expression in ESCC cells. The result indicated that promoter hypermethylation may be 1 main mechanism leading to the silencing of ECRG4. The high expression of ECRG4 in patients with ESCC was associated with longer survival compared with those with low ECRG4 expression by Kaplan-Meier survival analysis (p < 0.05). ECRG4 protein was an independent prognostic factor for ESCC by multivariable Cox proportional hazards regression analysis (p < 0.05). The restoration of ECRG4 expression in ESCC cells inhibited cell proliferation, colony formation, anchorage-independent growth, cell cycle progression and tumor growth in vivo (p < 0.05). The transfection of ECRG4 gene in ESCC cells inhibited the expression of NF-kappaB and nuclear translocation, in addition to the expression of COX-2, a NF-kappaB target gene, was attenuated. Taken together, ECRG4 is a novel candidate tumor suppressor gene in ESCC, and ECRG4 protein is a candidate prognostic marker for ESCC.

Expression and epigenetic silencing of ECRG4gene in breast cancer

11046 Previous whole-genome expression array studies in our laboratory had shown the expression of ECRG4 (esophageal cancer related gene 4) was significantly down-regulated in primary breast cancer. The present study was undertaken to investigate the expression and epigenetic regulation ofECRG4in human breast cancer cell lines and primary carcinomas. Here we showed that the expression ofECRG4, as measured by quantitative reverse transcription-PCR, was noticeably increased in 3 methylated breast cancer cell lines (MDA-MB-435, Bcap-37, MCF-7), after treated with 5-aza-2’-deoxycytidine - a demethylation agent. Analysis from 16 paired tissue samples revealed expression of ECRG4 was significantly reverse-correlated with methylation (Spearman correlation coefficient r=-0.419, P=0.017). Analysis of 67 additional samples showed that the ECRG4 CpGs island was in high methylation level in 59.7% of cancer tissues compared with 14.9% in tumor adjacent tissues,with an odds ratio of 3.393 (95% CI, 1.912–6.021,P=8.39E-8). And patients with high methylation level had a higher tumor grade. 90% samples were high methylation levels in grade III vs. 50% in grade I-II (95% CI, 1.063–76.213, P=0.033). In conclusion, the inactivation of ECRG4 gene by hypermethylation appears to be a frequent molecular event in development of primary breast cancer and may be involved in the carcinogenesis of this cancer. No significant financial relationships to disclose.

Expression of ECRG4 is an independent prognostic factor for poor survival in patients with esophageal squamous cell carcinoma

In this study, we examined the expression of esophageal cancer-related gene 4 (ECRG4) mRNA and evaluated its clinical significance in esophageal squamous cell carcinoma (ESCC). ECRG4 mRNA expression was quantified by real-time RT-PCR in 63 ESCC and corresponding normal esophageal mucosal samples. ECRG4 mRNA expression levels were significantly lower in ESCC tissues compared with corresponding normal esophageal mucosa (P<0.0001), in patients with locally invasive T2-4 tumors compared with less invasive T1 tumors (P=0.0229) and in stage 4 tumors compared with stage 0-3 tumors (P=0.0120). Furthermore, low ECRG4 mRNA expression levels were associated with significantly shorter survival after surgery compared with high ECRG4 mRNA expression levels (P=0.0150) in ESCC patients. On the basis of multivariate analysis, we conclude that ECRG4 mRNA expression level could be a candidate for an independent prognostic factor for ESCC patients.

Expression of ECRG4, a novel esophageal cancer-related gene, downregulated by CpG island hypermethylation in human esophageal squamous cell carcinoma.

To study the mechanisms responsible for inactivation of a novel esophageal cancer related gene 4 (ECRG4) in esophageal squamous cell carcinoma (ESCC). A pair of primers was designed to amplify a 220 bp fragment, which contains 16 CpG sites in the core promoter region of the ECRG 4 gene. PCR products of bisulfite-modified CpG islands were analyzed by denaturing high-performance liquid chromatography (DHPLC), which were confirmed by DNA sequencing. The methylation status of ECRG 4 promoter in 20 cases of esophageal cancer and the adjacent normal tissues, 5 human tumor cell lines (esophageal cancer cell line-NEC, EC109, EC9706; gastric cancer cell line- GLC; human embryo kidney cell line-Hek293) and 2 normal esophagus tissues were detected. The expression level of the ECRG 4 gene in these samples was examined by RT-PCR. The expression level of ECRG 4 gene was varied. Of 20 esophageal cancer tissues, nine were unexpressed, six were lowly expressed and five were highly expressed compared with the adjacent tissues and the 2 normal esophageal epithelia. In addition, 4 out of the 5 human cell lines were also unexpressed. A high frequency of methylation was revealed in 12 (8 unexpressed and 4 lowly expressed) of the 15 (80 %) downregulated cancer tissues and 3 of the 4 unexpressed cell lines. No methylation peak was observed in the two highly expressed normal esophageal epithelia and the methylation frequency was low (3/20) among the 20 cases in the highly expressed adjacent tissues. The methylation status of the samples was consistent with the result of DNA sequencing. These results indicate that the inactivation of ECRG 4 gene by hypermethylation is a frequent molecular event in ESCC and may be involved in the carcinogenesis of this cancer.