Genomic characterization and risk stratification of esophageal squamous dysplasia.

Objectives: The majority of esophageal squamous dysplasia (ESD) patients progress slowly, while a subset of patients can undergo recurrence rapidly or progress to invasive cancer even after proper treatment. However, the molecular mechanisms underlying these clinical observations are still largely unknown. Methods: By sequencing the genomic data of 160 clinical samples from 49 tumor-free ESD patients and 88 esophageal squamous cell carcinoma (ESCC) patients, we demonstrated lower somatic mutation and copy number alteration (CNA) burden in ESD compared with ESCC. Results: Cross-species screening and functional assays identified ACSM5 as a novel driver gene for ESD progression. Furthermore, we revealed that miR-4292 promoted ESD progression and could serve as a non-invasive diagnostic marker for ESD. Conclusions: These findings largely expanded our understanding of ESD genetics and tumorigenesis, which possessed promising significance for improving early diagnosis, reducing overtreatment, and identifying high-risk ESD patients.

Antitumour effects of artesunate via cell cycle checkpoint controls in human oesophageal squamous carcinoma cells.

Artesunate (ART), an effective antimalarial semisynthetic derivative of artemisinin, exhibits antitumour properties, but the mechanism(s) involved remain elusive. In this study, we investigated the antitumour effects of ART on human oesophageal squamous cell carcinoma (ESCC) cell lines. Treatment of ESCC cell lines with ART resulted in the production of excessive reactive oxygen species (ROS) that induced DNA damage, reduced cell proliferation and inhibited clonogenicity via G1-S cell cycle arrest and/or apoptosis in vitro. The administration of ART to nude mice with ESCC cell xenografts inhibited tumour formation in vivo. However, the cytotoxicity of ART strongly differed among the ESCC cell lines tested. Transcriptomic profiling revealed that although the expression of large numbers of genes in ESCC cell lines was affected by ART treatment, these genes could be functionally clustered into pathways involved in regulating cell cycle progression, DNA metabolism and apoptosis. We revealed that p53 and Cdk4/6-p16-Rb cell cycle checkpoint controls were critical determinants required for mediating ART cytotoxicity in ESCC cell lines. Specifically, KYSE30 cells with p53Mut/p16Mut were the most sensitive to ART, KYSE150 and KYSE180 cells with p53Mut/p16Nor exhibited intermediate responses to ART, and Eca109 cells with p53Nor/p16Nor exhibited the most resistance to ATR. Consistently, perturbation of p53 expression using RNA interference (RNAi) and/or Cdk4/6 activity using the inhibitor palbociclib altered ART cytotoxicity in KYSE30 cells. Given that the p53 and Cdk4/6-cyclin D1-p16-Rb genes are commonly mutated in ESCC, our results potentially shed new light on neoadjuvant chemotherapy strategies for ESCC.

Mammalian esophageal stratified tissue homeostasis is maintained distinctively by the epithelial pluripotent p63+Sox2+ and p63-Sox2+ cell populations.

Self-renewing, damage-repair and differentiation of mammalian stratified squamous epithelia are subject to tissue homeostasis, but the regulation mechanisms remain elusive. Here, we investigate the esophageal squamous epithelial tissue homeostasis in vitro and in vivo. We establish a rat esophageal organoid (rEO) in vitro system and show that the landscapes of rEO formation, development and maturation trajectories can mimic those of rat esophageal epithelia in vivo. Single-cell RNA sequencing (scRNA-seq), snapshot immunostaining and functional analyses of stratified "matured" rEOs define that the epithelial pluripotent stem cell determinants, p63 and Sox2, play crucial but distinctive roles for regulating mammalian esophageal tissue homeostasis. We identify two cell populations, p63+Sox2+ and p63-Sox2+, of which the p63+Sox2+ population presented at the basal layer is the cells of origin required for esophageal epithelial stemness maintenance and proliferation, whereas the p63-Sox2+ population presented at the suprabasal layers is the cells of origin having a dual role for esophageal epithelial differentiation (differentiation-prone fate) and rapid tissue damage-repair responses (proliferation-prone fate). Given the fact that p63 and Sox2 are developmental lineage oncogenes and commonly overexpressed in ESCC tissues, p63-Sox2+ population could not be detected in organoids formed by esophageal squamous cell carcinoma (ESCC) cell lines. Taken together, these findings reveal that the tissue homeostasis is maintained distinctively by p63 and/or Sox2-dependent cell lineage populations required for the tissue renewing, damage-repair and protection of carcinogenesis in mammalian esophagi.

Identification and characterization of stem cells in mammalian esophageal stratified squamous epithelia.

Somatic stem cells are essential for the maintenance of tissue homeostasis. Despite its importance, how the esophageal stratified squamous epithelium executes its self-renewal and maintenance remains elusive. In this study, using 5-bromo-2'-deoxyuridine label-chase in rats in vivo and rat esophageal organoids in vitro together with genome-wide DNA methylation and single-cell RNA sequencing, we identified a slow-cycling/quiescent stem cell population that contained high levels of hemidesmosomes (HDs) and low levels of Wnt signaling localized spatially and randomly at the basal layer of the esophageal epithelium. Pseudotime cell trajectory analysis indicated that tissue cells originated from quiescent basal stem cells in the basal layer. Perturbations of HD component expression and/or Wnt signaling reduced the stem cell population in the basal layer of esophageal keratinocyte organoids, resulting in alterations in the organoid formation rate, size, morphogenesis, and proliferation-differentiation homeostasis. Furthermore, not only high levels of HDs and low levels of Wnt signaling but also an interplay between HD and Wnt signaling defined the stem cells of the basal layer. Hence, HDs and Wnt signaling are critical determinants for defining the stem cells of the basal layer required for tissue homeostasis in mammalian esophagi.

Aspirin enhances the therapeutic efficacy of cisplatin in oesophageal squamous cell carcinoma by inhibition of putative cancer stem cells.

BACKGROUND: Cancer stem cells (CSCs) are related to the patient's prognosis, recurrence and therapy resistance in oesophageal squamous cell carcinoma (ESCC). Although increasing evidence suggests that aspirin (acetylsalicylic acid, ASA) could lower the incidence and improve the prognosis of ESCC, the mechanism(s) remains to be fully understood. METHODS: We investigated the role of ASA in chemotherapy/chemoprevention in human ESCC cell lines and an N-nitrosomethylbenzylamine-induced rat ESCC carcinogenesis model. The effects of combined treatment with ASA/cisplatin on ESCC cell lines were examined in vitro and in vivo. Sphere-forming cells enriched with putative CSCs (pCSCs) were used to investigate the effect of ASA in CSCs. Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) was performed to determine the alterations in chromatin accessibility caused by ASA in ESCC cells. RESULTS: ASA inhibits the CSC properties and enhances cisplatin treatment in human ESCC cells. ATAC-seq indicates that ASA treatment results in remarkable epigenetic alterations on chromatin in ESCC cells, especially their pCSCs, through the modification of histone acetylation levels. The epigenetic changes activate Bim expression and promote cell death in CSCs of ESCC. Furthermore, ASA prevents the carcinogenesis of NMBzA-induced ESCC in the rat model. CONCLUSIONS: ASA could be a potential chemotherapeutic adjuvant and chemopreventive drug for ESCC treatment.

Metformin suppresses the esophageal carcinogenesis in rats treated with NMBzA through inhibiting AMPK/mTOR signaling pathway.

Metformin is a widely used antidiabetic drug for the management of type 2 diabetes mellitus. Recently, epidemiological studies demonstrate that metformin has anticancer effects on esophageal squamous cell carcinoma (ESCC) and other cancers. However, the effects and potential mechanisms of metformin on ESCC remain elusive. In this study, we used N-nitroso-N-methylbenzylamine (NMBzA), a special carcinogen for esophagi, to develop a rat ESCC model, in which the carcinogenesis progression of ESCC in rat was induced and promoted. We investigated the effects of metformin on carcinogenesis of ESCC in this model. Our results revealed that metformin significantly decreased the incidence and precancerous lesions of ESCC and inhibited proliferation and promoted apoptosis of esophageal epithelial cells in rat treated with NMBzA. Moreover, metformin also increased apoptosis and inhibited migration, colony formation and tumor sphere formation of human ESCC cells in vitro. Immunohistochemistry and western blotting showed that without interfering the metabolism of NMBzA, metformin inhibited the inflammation of esophagi via reducing the expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6). Treatment of metformin led to activation of AMP-activated protein kinase (AMPK) and attenuated signaling of the downstream molecules such as p-mTOR, p-p70S6K and cyclin D1 expression both in vivo and in vitro. Taken together, our study demonstrated that metformin suppressed the carcinogenesis of ESCC through inhibiting AMPK/mammalian target of the rapamycin (mTOR) signaling pathway, resulting in its chemopreventive effects on the carcinogenesis of ESCC.

DNMT1 ablation suppresses tumorigenesis by inhibiting the self-renewal of esophageal cancer stem cells.

Cancer stem cells (CSCs) have been isolated from many tumors and considered as the main reason of cancer recurrence and metastasis. DNA methyltransferase 1 (DNMT1) mediates DNA methylation and plays an important role in CSCs maintenance. However, the function of DNMT1 in CSCs of esophageal squamous cell carcinoma (ESCC) remains unclear. In this study, we examined the role of DNMT1 in regulating self-renewal in CSCs of ESCC. We found a high expression of DNMT1 in both side population (SP) cells and sphere formation cells that represented as substitutes for CSCs in KYSE150 and EC109 ESCC cell lines. We performed the knockdown of DNMT1 using lentivirus-mediated RNA interference (RNAi) methods. We revealed that ablation of DNMT1 resulted in the numbers and self-renewal abilities of CSCs refrained significantly in ESCC cells. As a result of the CSCs inhibition, the malignant phenotypes such as cell proliferation, colony formation, migration and drug resistance abilities were dramatically inhibited in ESCC cells. Treatment of 5-aza-2'-deoxycytidine (5-aza-dC), a DNMT inhibitor, also resulted in the inhibition of CSCs and malignant profiles in ESCC cells. Our findings also provided the first evidence that 5-aza-dC inhibited the colony and sphere formation of CSCs. Thus, our results indicated that DNMT1 was important for the self-renewal maintenance of CSCs in ESCC, and 5-aza-dC could be a potential therapy for the CSCs of ESCC.

TM4SF1 promotes the self-renewal of esophageal cancer stem-like cells and is regulated by miR-141.

Cancer stem-like cells have been identified in primary human tumors and cancer cell lines. Previously we found TM4SF1 gene was highly expressed in side population (SP) cells from esophageal squamous cell carcinoma (ESCC) cell lines, but the role and underlying mechanism of TM4SF1 in ESCC remain unclear. In this study, we observed TM4SF1 was up-regulated but miR-141 was down-regulated in SP cells isolated from ESCC cell lines. TM4SF1 could stimulate the self-renewal ability and carcinogenicity of esophageal cancer stem-like cells, and promote cell invasion and migration. In miR-141 overexpression cells, the expression of TM4SF1 was significantly reduced. We also found that overexpression of miR-141 could abolish the self-renewal ability and carcinogenicity of esophageal cancer stem-like cells and decrease cell invasion and migration by suppressing TM4SF1. Consequently, TM4SF1 is a direct target gene of miR-141. The regulation of TM4SF1 by miR-141 may play an important role in controlling self-renewals of esophageal cancer stem-like cells. It may also promote the development of new therapeutic strategies and efficient drugs to target ESCC stem-like cells.

Prognostic impact of mutation profiling in patients with stage II and III colon cancer.

Development of colorectal cancer (CRC) associates with accumulation of genetic mutations include the epidermal growth factor receptor (EGFR) signaling pathway. However, whether mutations in KRAS together with downstream factors BRAF, PIK3CA and NRAS impact prognosis is still unclear for stage II-III colon cancer. In the present study a total of 228 stage II-III colon cancer samples were retrospectively collected, KRAS (codons 12, 13 and 61), BRAF (exon 11 and exon 15), PIK3CA (exon 9 and exon 20) and NRAS (codons 12, 13 and 61) status was detected by Sanger sequencing, 37.89% (86/227) tumors harbored a KRAS mutation, 7.02% (16/228) harbored a BRAF mutation, 13.18% (29/220) harbored a PIK3CA mutation and 0.89% (2/224) harbored a NRAS mutation. NRAS mutations existed only in stage II colon cancer. Older groups harbored a higher KRAS and BRAF mutation (P < 0.05), PIK3CA (exon9) mutations appeared more common in worse differentiation tumors (P = 0.032). Moreover, PIK3CA (E545K) mutation was significantly associated with tumor recurrence (P = 0.031) and acted independently prognostic for poor OS (P = 0.044), while only in stage III colon cancer. KRAS, BRAF and NRAS mutations do not have major prognostic value in stage II and III colon cancer, subtypes of gene mutations should be further investigated for a better understanding in CRC.

RFT2 is overexpressed in esophageal squamous cell carcinoma and promotes tumorigenesis by sustaining cell proliferation and protecting against cell death.

Human riboflavin transporter 2 (RFT2, also termed as SLC52A3) was recently identified as a susceptibility gene to esophageal squamous cell carcinoma (ESCC), however, its expression and biologic function has remained unclear in ESCC. In this study, we demonstrated that RFT2 was frequently overexpressed in tumor samples compared with normal adjacent tissue in ESCC patients. Knockdown of RFT2 in ESCC cells resulted in decreases of intracellular flavin status, mitochondrial membrane potential and cellular ATP levels, and inhibitions of cell proliferation, colony formation and anchorage-independent growth. Knockdown of RFT2 increased p21 and p27 protein levels, decreased their downstream targets cyclin E1 and Cdk2 protein levels and caused pRb hypophosphorylation, leading to cell cycle arrest at G1-G1/S. Knockdown of RFT2 also reduced anti-apoptotic proteins Bcl-2, Bcl-xl and survivin levels, caused activation of caspase-3 and apoptosis. In contrast, ectopic overexpression of RFT2 in ESCC cells promoted cell proliferation under restricted conditions (soft agar), conferred resistance to cisplatin, and enhanced tumorigenicity in nude mice. These results suggest that RFT2 contributes to ESCC tumorigenesis and may serve as a potential therapeutic target.

Macrophage inhibitory factor 1 acts as a potential biomarker in patients with esophageal squamous cell carcinoma and is a target for antibody-based therapy.

Macrophage inhibitory factor 1 (MIC1) is frequently altered in various cancers. The aim of this study was to investigate the clinical significance of MIC1 for esophageal squamous cell carcinoma (ESCC). Serum MIC1 of 286 ESCC and 250 healthy subjects was detected, the diagnostic performance was assessed and compared with SCC, CEA, CA199 and CA724, and the value as a prognostic indicator was also evaluated. The expression of MIC1 in ESCC cell lines, tissues were detected, and the inhibition of MIC1 antibody on ESCC was carried out in vitro and in vivo. The results showed that the serum MIC1 of ESCC was significantly higher than normal groups (P < 0.001), and was positively associated with tumor invasion (P = 0.030) as well as lymph node metastasis (P = 0.007). The sensitivity of MIC1 was significantly better than SCC, CEA, CA199 and CA724, especially for stage I ESCC. Patients with higher serum MIC1 also had a poorer prognosis in relapse-free (P = 0.050) and tumor-specific survival (P = 0.005). In vitro studies showed that the expression of MIC1 was upregulated in 37.5% (3/8) ESCC cell lines and 45% (18/40) tissues, and the transcription of MIC1 in tumor tissues was significantly higher than paired adjacent normal tissues (P = 0.001). The antibody of MIC1 inhibited the tumor growth (P < 0.001), and showing preference for tumor tissues in xenograft model. The decreased formation of neovascularization lumen may be involved in the mechanism. We conclude that MIC1 plays an important role in the progression of ESCC and can serve as a potential biomarker and therapeutic target for ESCC.

miR-203 inhibits the proliferation and self-renewal of esophageal cancer stem-like cells by suppressing stem renewal factor Bmi-1.

Cancer stem-like cells exist in many malignancies and several stem cell-related genes and microRNAs, such as Bmi-1 and miR-203, have been identified as cancer stem-like cell regulators using gene microarray or sequencing analysis. Previously, we used side population (SP) sorting to enrich cancer stem-like cells from esophageal squamous cell carcinoma (ESCC) cell line EC9706. Our results demonstrated that EC9706 SP cells shared common features of cancer stem-like cells. In this study, we examined the expression of Bmi-1 and miR-203 in ESCC SP and non-SP (NSP) cells. Our results showed that, when compared with NSP cells, Bmi-1 was up-regulated and miR-203 was down-regulated in SP cells. During the differentiation from SP to NSP cells, the expression levels of Bmi-1 were gradually decreased. Overexpression of miR-203 resulted in a significant reduction of endogenous Bmi-1 protein level in EC9706 cells. SP and NSP analyses revealed that the SP cell fraction was markedly decreased in miR-203 overexpressed cells. miR-203 overexpressed cells also showed a significant reduction in colony formation, which was resistant to chemotherapeutic drug treatment and tumorigenicity in nude mice. Rescue experiments demonstrated that ectopic expression of Bmi-1 in miR-203 overexpressed cells increased the SP fraction and restored cell proliferation. Taken together, these results indicated that stem renewal factor Bmi-1 was a direct target of miR-203. The regulation of Bmi-1 by miR-203 may play an important role in controlling cell proliferation and self-renewal of esophageal cancer stem-like cells. It may also promote the development of new therapeutic strategies and efficient drugs that target ESCC stem-like cells.

A novel tumor suppressor gene ECRG4 interacts directly with TMPRSS11A (ECRG1) to inhibit cancer cell growth in esophageal carcinoma.

BACKGROUND: The esophageal carcinoma related gene 4 (ECRG4) was initially identified and cloned from human normal esophageal epithelium in our laboratory (GenBank accession no.AF325503). ECRG4 has been described as a novel tumor suppressor gene associated with prognosis in esophageal squamous cell carcinoma (ESCC). METHODS: In this study, binding affinity assay in vitro and co-immunoprecipitation experiment in vivo were utilized to verify the physical interaction between ECRG4 and transmembrane protease, serine 11A (TMPRSS11A, also known as ECRG1, GenBank accession no. AF 071882). Then, p21 protein expression, cell cycle and cell proliferation regulations were examined after ECRG4 and ECRG1 co-transfection in ESCC cells. RESULTS: We revealed for the first time that ECRG4 interacted directly with ECRG1 to inhibit cancer cell proliferation and induce cell cycle G1 phase block in ESCC. Binding affinity and co-immunoprecipitation assays demonstrated that ECRG4 interacted directly with ECRG1 in ESCC cells. Furthermore, the ECRG4 and ECRG1 co-expression remarkably upregulatd p21 protein level by Western blot (P < 0.001), induced cell cycle G1 phase block by flow cytometric analysis (P < 0.001) and suppressed cell proliferation by MTT and BrdU assay (both P < 0.01) in ESCC cells. CONCLUSIONS: ECRG4 interacts directly with ECRG1 to upregulate p21 protein expression, induce cell cycle G1 phase block and inhibit cancer cells proliferation in ESCC.

Molecular cloning and characterization of a novel esophageal cancer related gene.

We previously identified four novel cDNA fragments related to human esophageal cancer. One of the fragments was named esophageal cancer related gene 2 (ECRG2). We report here the molecular cloning, sequencing, and expression of the ECRG2 gene. The ECRG2 cDNA comprises a 258 bp nucleotide sequence which encodes for 85 amino acids with a predicted molecular weight of 9.2 kDa. Analysis of the protein sequence reveals the presence at the N terminus of a signal peptide followed by 56 amino acids with a significant degree of sequence similarity with the conserved Kazal domain which characterizes the serine protease inhibitor family. Pulse-chase experiments showed that ECRG2 protein was detected in both cell lysates and culture medium, indicating that the ECRG2 protein was extracellularly secreted after the post-translational cleavage. In vitro uPA/plasmin activity analysis showed the secreted ECRG2 protein inhibited the uPA/plasmin activity, indicating that ECRG2 may be a novel serine protease inhibitor. Northern blot analysis revealed the presence of the major band corresponding to a size of 569 kb throughout the fetal skin, thymus, esophagus, brain, lung, heart, stomach, liver, spleen, colon, kidney, testis, muscle, cholecyst tissues and adult esophageal mucosa, brain, thyroid tissue and mouth epithelia. However, ECRG2 gene was significantly down-regulated in primary esophageal cancer tissues. Taken together, these results indicate that ECRG2 is a novel member of the Kazal-type serine protease inhibitor family and may function as a tumor suppressor gene regulating the protease cascades during carcinogenesis and migration/invasion of esophageal cancer.

The candidate tumor suppressor gene ECRG4 inhibits cancer cells migration and invasion in esophageal carcinoma.

BACKGROUND: The esophageal cancer related gene 4 (ECRG4) was initially identified and cloned in our laboratory from human normal esophageal epithelium (GenBank accession no.AF325503). ECRG4 was a new tumor suppressor gene in esophageal squamous cell carcinoma (ESCC) associated with prognosis. In this study, we investigated the novel tumor-suppressing function of ECRG4 in cancer cell migration, invasion, adhesion and cell cycle regulation in ESCC. METHODS: Transwell and Boyden chamber experiments were utilized to examined the effects of ECRG4 expression on ESCC cells migration, invasion and adhesion. And flow cytometric analysis was used to observe the impact of ECRG4 expression on cell cycle regulation. Finally, the expression levels of cell cycle regulating proteins p53 and p21 in human ESCC cells transfected with ECRG4 gene were evaluated by Western blotting. RESULTS: The restoration of ECRG4 expression in ESCC cells inhibited cancer cells migration and invasion (P < 0.05), which did not affect cell adhesion capacity (P > 0.05). Furthermore, ECRG4 could cause cell cycle G1 phase arrest in ESCC (P < 0.05), through inducing the increased expression of p53 and p21 proteins. CONCLUSION: ECRG4 is a candidate tumor suppressor gene which suppressed tumor cells migration and invasion without affecting cell adhesion ability in ESCC. Furthermore, ECRG4 might cause cell cycle G1 phase block possibly through inducing the increased expression of p53 and p21 proteins in ESCC.

ECRG2 regulates ECM degradation and uPAR/FPRL1 pathway contributing cell invasion/migration.

ECRG2 is a novel tumor suppressor gene that shows sequence similarity to KAZAL-type serine protease inhibitor. We have previously demonstrated ECRG2 inhibits migration/invasion of lung cancer PG cells. However, the mechanism by which ECRG2 performs these activities remains unknown. In this study, we found that ECRG2 inhibits proteolysis activity of uPA/plasmin and MMP2, and substantially reduces the ability of HT1080 and HCT-116 cells to invade ECM. Moreover, we demonstrated ECRG2 prevents the cleavage of uPAR, disrupts the association of sD2D3 with FPRL1, and that disruption impairs FPRL1 function. Conversely, depletion of ECRG2 not only markedly increased proteolysis activity of uPA/plasmin and MMP2 but also enhanced the association of uPAR with FPRL1, stimulated cell migration/invasion. Together, our results provide evidence that ECRG2 regulates invasion/migration partly through ECM degradation and uPA/uPAR/FPRL1 pathway, and may represent a novel therapeutic target for cancer.

ECRG2 regulates cell migration/invasion through urokinase-type plasmin activator receptor (uPAR)/beta1 integrin pathway.

ECRG2 is a novel gene that shows sequence similarity to KAZAL-type serine protease inhibitor. We have previously demonstrated that ECRG2 inhibits migration/invasion of lung cancer PG cells. However, the mechanism by which ECRG2 performs these activities is a compelling question. Urokinase-type plasmin activator (uPA) binding to uPAR induces migration/invasion through multiple interactors including integrins. In this study, we found that ECRG2 binds specifically to the kringle domain of uPA. Moreover, we demonstrated that ECRG2 forms a complex with uPA.uPAR, that such a complex modifies the dynamical association of uPAR with beta1 integrins, and that disruption inhibits Src/MAP (mitogen-activated protein) kinase pathway, resulting in suppression of cell migration/invasion in an in vitro Matrigel migration/invasion assay. Conversely, depletion of ECRG2 markedly enhanced the association of uPAR with beta1 integrins, elevated basal Src/MAP kinase activation, and stimulated HT1080, MDA-MB-231, and MCF-7 cell migration/invasion. Together, our results provide evidence that ECRG2 is involved in the regulation of migration/invasion through uPA/uPAR/beta1 integrins/Src/MAP kinase pathway and may represent a novel therapeutic target for cancer.

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.

Mapping the putative binding site for uPA protein in Esophageal Cancer-Related Gene 2 by heteronuclear NMR method.

Esophageal Cancer-Related Gene 2 (ECRG2) is a novel member of the KAZAL-type serine proteinase inhibitor family and plays an important role in the inhibition of human esophageal cancer cell proliferation. The previous studies have shown that ECRG2 can bind the urokinase-type plasminogen activator (uPA)/plasmin system and inhibit its activity. In this study, the strategy of cloning, overexpression, and purification of ECRG2 for obtaining a properly folded ECRG2 with accurately formed disulfide bonds was established. The heteronuclear NMR experiments were performed with isotope labeled ECRG2 to investigate the binding interface of the protein with uPA. The sequence regions of ECRG2 for uPA binding were determined. Analysis indicates that the uPA-binding loops of ECRG2 are in correspondence with the reactive site loops for binding of serine proteinase in turkey ovomucoid third domain (OMTKY3). The structural similarity of ECRG2 to OMTKY3 was identified and a model for ECRG2 was proposed.

Genomic profiling of 766 cancer-related genes in archived esophageal normal and carcinoma tissues.

We employed the BeadArraytrade mark technology to perform a genetic analysis in 33 formalin-fixed, paraffin-embedded (FFPE) human esophageal carcinomas, mostly squamous-cell-carcinoma (ESCC), and their adjacent normal tissues. A total of 1,432 single nucleotide polymorphisms (SNPs) derived from 766 cancer-related genes were genotyped with partially degraded genomic DNAs isolated from these samples. This directly targeted genomic profiling identified not only previously reported somatic gene amplifications (e.g., CCND1) and deletions (e.g., CDKN2A and CDKN2B) but also novel genomic aberrations. Among these novel targets, the most frequently deleted genomic regions were chromosome 3p (including tumor suppressor genes FANCD2 and CTNNB1) and chromosome 5 (including tumor suppressor gene APC). The most frequently amplified genomic region was chromosome 3q (containing DVL3, MLF1, ABCC5, BCL6, AGTR1 and known oncogenes TNK2, TNFSF10, FGF12). The chromosome 3p deletion and 3q amplification occurred coincidently in nearly all of the affected cases, suggesting a molecular mechanism for the generation of somatic chromosomal aberrations. We also detected significant differences in germline allele frequency between the esophageal cohort of our study and normal control samples from the International HapMap Project for 10 genes (CSF1, KIAA1804, IL2, PMS2, IRF7, FLT3, NTRK2, MAP3K9, ERBB2 and PRKAR1A), suggesting that they might play roles in esophageal cancer susceptibility and/or development. Taken together, our results demonstrated the utility of the BeadArray technology for high-throughput genetic analysis in FFPE tumor tissues and provided a detailed genetic profiling of cancer-related genes in human esophageal cancer.