Epithelial Cell Adhesion Molecule Gene Research Articles

Polymorphisms of EpCAM gene and prognosis for non‐small‐cell lung cancer in Han Chinese

The epithelial cell adhesion molecule (EpCAM) is overexpressed in a wide variety of human cancers and is associated with patient prognosis, including those with lung cancer. However, the association of single nucleotide polymorphisms (SNPs) in the EpCAM gene with the prognosis for non-small-cell lung cancer (NSCLC) patients has never been investigated. We evaluated the association between two SNPs, rs1126497 and rs1421, in the EpCAM gene and clinical outcomes in a Chinese cohort of 506 NSCLC patients. The SNPs were genotyped using the Sequenom iPLEX genotyping system. Multivariate Cox proportional hazards model and Kaplan–Meier curves were used to assess the association of EpCAM gene genotypes with the prognosis of NSCLC. We found that the non-synonymous SNP rs1126497 was significantly associated with survival. Compared with the CC genotype, the CT+TT genotype was a risk factor for both death (hazard ratio, 1.40; 95% confidence interval [CI], 1.02–1.94; P = 0.040) and recurrence (hazard ratio, 1.34; 95% CI, 1.02–1.77; P = 0.039). However, the SNP rs1421 did not show any significant effect on patient prognosis. Instead, the AG+GG genotype in rs1421 was significantly associated with early T stages (T1/T2) when compared with the AA genotype (odds ratio for late stage = 0.65; 95% CI, 0.44–0.96, P = 0.029). Further stratified analysis showed notable modulating effects of clinical characteristics on the associations between variant genotypes of rs1126497 and NSCLC outcomes. In conclusion, our study indicated that the non-synonymous SNP rs1126497 may be a potential prognostic marker for NSCLC patients.

EPCAM germline and somatic rearrangements in lynch syndrome: identification of a novel 3′EPCAM deletion

3'EPCAM (Epithelial Cell Adhesion Molecule) genomic rearrangements can be a cause of mismatch repair deficiency in rare Lynch syndrome families. 3'EPCAM deletions include the polyadenylation signal and might result in promoter hypermethylation of the centromeric MSH2 gene in cis. A somatic rearrangement in trans affecting MSH2 is responsible for the final mismatch repair deficiency in the corresponding tumors but the mechanisms are not well documented. In this report two germline 3'EPCAM deletions are described together with the corresponding somatic mutations in the patient's colorectal tumors. Mutation and breakpoint analysis resulted in the identification of one novel (c.556-531_*872del) and one known EPCAM deletion (c.859-689_*14697del). Both deletions resulted from Alu mediated homologous recombination causing aberrant EPCAM-MSH2 fusion transcripts. The colorectal tumors of the deletion carriers were MSI-high. Strong hypermethylation of the MSH2 promoter was measured. Analysis of somatic genomic rearrangements showed a 4 Mb deletion including the EPCAM, MSH2 and MSH6 genes in one tumor and copy neutral loss of heterozygosity in the EPCAM-MSH2 region in the other tumor. This indicates that hemi- and homozygous hypermethylation of the MSH2 promoter and hence complete silencing of MSH2 expression was responsible for the mismatch repair deficiency in both colorectal tumors.

Abstract 40: Mutation of EPCAM gene is less frequent in Korean and EPCAM immunohistochemistry can identify Lynch syndrome in patients who do not meet Bethesda criteria.

Abstract Lynch syndrome is an inherited tumor predisposition syndrome caused by germline mutations of DNA mismatch repair (MMR) genes, mainly MLH1 and MSH2. Recently, germline deletions affecting the epithelial cell adhesion molecule (EPCAM) gene located upstream of MSH2 were identified as a novel mutational mechanism causing Lynch syndrome. However, most studies were conducted in western populations, where the incidence of CRC is high. To identify the incidence of germline deletions of EPCAM gene in Korea where the incidence of CRC is relatively low and socioeconomic condition is different, we conducted a comprehensive study in 4,993 patients diagnosed and treated with CRCs at a single institute between 2006 and 2011. In all tumor samples, microsatellite instability (MSI) test and immunohistochemistry (IHC) for MMR proteins were performed. In cases with loss of MLH1 protein, methylation of MLH1 by MethyLight was performed and cases with hypermethylation of MLH1 gene were excluded. Germline mutations of MLH1, MSH2 and MSH6 were performed in patients who fulfilled at least one of the revised Bethesda criteria. In 97 patients with loss of MSH2 protein, EPCAM IHC and multiplex ligation-dependent probe amplification for germline deletions of the EPCAM and MSH2 gene were performed. Loss of expression of MMR proteins and/or MSI-high was observed in 337 (6.7%) patients, 112 (2.2%) patients met revised Bethesda criteria, and 54 (1.1%) patients harbored germline mutations of MMR genes. Out of 97 MSH2-negative CRCs, 5 (5.2%) showed concomitant loss of EPCAM expression and EPCAM exon 9 deletionmutations. Out of 5 cases with EPCAM mutation and protein loss, four patients showed familial histories of CRCs. However, one patient showed no family history. In this patient, although multiple liver metastases were the first manifestation, he lived 6 more years after diagnosis. In this large single study cohort, we found that 5.2% of MSH2-negative CRCs were caused by EPCAM deletion mutations, which is lower than previously reported incidences in western populations. Moreover, we could find one candidate patient with de novo mutation of EPCAM gene, which has not been reported yet. Our results strongly support that in routine screening of patients with CRC for Lynch syndrome, a simple and sensitive EPCAM IHC should be included even in cases do not meet Bethesda criteria. Citation Format: So Young Kang, Shin Woo Kang, In-Gu Do, Dong Kyung Chang, Kyoung M. Kim. Mutation of EPCAM gene is less frequent in Korean and EPCAM immunohistochemistry can identify Lynch syndrome in patients who do not meet Bethesda criteria. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 40. doi:10.1158/1538-7445.AM2013-40

Abstract 3992: The role of EpCAM expression in esophageal squamous cell carcinoma.

Abstract Background: The epithelial cell adhesion molecule (EpCAM) has been reported its various roles in variety of cancers, particularly in adenocarcinoma. EpCAM broad expression on human carcinomas and its involvement in cell proliferation have brought EpCAM into focus as an immunotherapeutic target for therapeutic strategies. However, its expression level and role in esophageal squamous cell carcinoma (ESCC) is remained unclear. This study is to investigate the clinicopathologic significance of EpCAM expression in ESCC. Methods: The EpCAM expression was immunohistochemically investigated in 74 primary ESCCs with R0 resection without any preoperative treatment using the monoclonal antibody. We evaluated the intensity of the stain in 4 grades. We considered 3 (strong) and 2 (medium) as “positive” and 1 (weak) and 0 (negative) as “negative”. Down regulation of EpCAM gene expression was performed using the shRNA in EpCAM high ESCC cell line (TE4-shRNA), TE4. The cell proliferation assay was performed using cell counting kit to assess cell proliferation in vitro. To evaluate cell proliferation and tumorgenesis in vivo, we inoculated TE4 and TE4-shRNA into nude mice subcutaneously. The size and incidence of subcutaneous tumors were recorded. Results: Thirty-six (48.6%) patients showed positive for EpCAM immunostaining and 38 (51.4%) patients showed negative. EpCAM expression was not significantly correlated with histology, tumor depth, regional lymph node metastasis, disease stage, lymphatic invasion, or venous invasion. However, high expression of EpCAM was significantly associated with poor survival in ESCC patients (P = 0.026). Multivariate Cox regression analysis showed overexpression of EpCAM was a significant and independent prognostic factor for surgically treated ESCC (P = 0.004). The cell proliferation rate of TE4-shRNA was lower than TE4 in vitro. TE4 cells could initiate tumors in 7 of 10 injected nude mice, whereas TE4-shRNA cells produced only 2 of 10 injected mice at 6 weeks after transplantation. The tumor sizes were larger in the TE4 cells than in the TE4-shRNA cells. Conclusion: High expression of EpCAM was independent prognostic factor for surgically treated ESCC. EpCAM expression level could change cell proliferation and tumorgenesis in vitro and in vivo. These results suggested that EpCAM might be a potential molecular target for esophageal cancer therapy. Citation Format: Tatsuo Matsuda, Hiroya Takeuchi, Sachiko Matsuda, Kunihiko Hiraiwa, Hirofumi Kawakubo, Rieko Nakamura, Tsunehiro Takahashi, Norihito Wada, Yoshiro Saikawa, Tai Ohmori, Yuko Kitagawa. The role of EpCAM expression in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3992. doi:10.1158/1538-7445.AM2013-3992

Phenotype-dependent effects of EpCAM expression on growth and invasion of human breast cancer cell lines

BackgroundThe epithelial cell adhesion molecule (EpCAM) has been shown to be overexpressed in breast cancer and stem cells and has emerged as an attractive target for immunotherapy of breast cancer patients. This study analyzes the effects of EpCAM on breast cancer cell lines with epithelial or mesenchymal phenotype.MethodsFor this purpose, shRNA-mediated knockdown of EpCAM gene expression was performed in EpCAMhigh breast cancer cell lines with epithelial phenotype (MCF-7, T47D and SkBR3). Moreover, EpCAMlow breast carcinoma cell lines with mesenchymal phenotype (MDA-MB-231, Hs578t) and inducible overexpression of EpCAM were used to study effects on proliferation, migration and in vivo growth.ResultsIn comparison to non-specific silencing controls (n/s-crtl) knockdown of EpCAM (E#2) in EpCAMhigh cell lines resulted in reduced cell proliferation under serum-reduced culture conditions. Moreover, DNA synthesis under 3D culture conditions in collagen was significantly reduced. Xenografts of MCF-7 and T47D cells with knockdown of EpCAM formed smaller tumors that were less invasive. EpCAMlow cell lines with tetracycline-inducible overexpression of EpCAM showed no increased cell proliferation or migration under serum-reduced growth conditions. MDA-MB-231 xenografts with EpCAM overexpression showed reduced invasion into host tissue and more infiltrates of chicken granulocytes.ConclusionsThe role of EpCAM in breast cancer strongly depends on the epithelial or mesenchymal phenotype of tumor cells. Cancer cells with epithelial phenotype need EpCAM as a growth- and invasion-promoting factor, whereas tumor cells with a mesenchymal phenotype are independent of EpCAM in invasion processes and tumor progression. These findings might have clinical implications for EpCAM-based targeting strategies in patients with invasive breast cancer.

Usefulness of epithelial cell adhesion molecule expression in the algorithmic approach to Lynch syndrome identification

Lynch syndrome (LS), the most frequent form of hereditary colorectal cancer syndrome, is caused by germ-line mutations in the mismatch repair system genes. Recently, a new mechanism involving the epithelial cell adhesion molecule (EPCAM)/TACSTD1 gene has been shown to be responsible in cases with abnormal MSH2 expression. Of interest, 3' exons deletions of the EPCAM gene, which is located upstream of MSH2 in chromosome 2, are associated with MSH2 promoter hypermethylation. EPCAM protein, expressed in epithelial tissues, is encoded by the EPCAM/TACSTD1 gene. Our study's aim was to explore EPCAM expression in colorectal carcinomas of MSH2-associated LS cases to evaluate the usefulness of EPCAM protein expression in the algorithm approach to LS population screening. We included a total of 19 MSH2-negative colorectal carcinomas from 14 different patients in whom we were able to perform a complete germ-line analysis. Nine patients showed a deleterious germ-line mutation that involved the MSH2 gene in 3 instances and the EPCAM gene exon 9 in 6 instances. All patients harboring the EPCAM mutation belonged to the same family. Of the 19 colorectal carcinomas, EPCAM expression loss was seen in only 5 tumors, all of them from patients showing a germ-line EPCAM deletion. Of interest, 6 tumors from 3 different patients carrying the same germ-line EPCAM deletion showed normal EPCAM expression. In conclusion, owing to the high specificity of EPCAM protein expression to identify LS patients carrying an EPCAM deletion, we recommend adding EPCAM immunohistochemistry to the LS diagnostic algorithm in MSH2-negative colorectal carcinoma.

The molecular basis of EPCAM expression loss in Lynch syndrome-associated tumors

AbstractGermline deletions affecting the Epithelial cell adhesion molecule (EPCAM) gene lead to silencing of MSH2 and cause Lynch syndrome. We have recently reported that lack of EPCAM expression occurs in many, but not all tumors from Lynch syndrome patients with EPCAM germline deletions. The differences in EPCAM expression were not related to the localization of EPCAM germline deletions. We therefore hypothesized that the type of the second somatic hit, which leads to MSH2 inactivation during tumor development, determines EPCAM expression in the tumor cells. To test this hypothesis and to evaluate whether lack of EPCAM expression can already be detected in Lynch syndrome-associated adenomas, we analyzed four carcinomas and two adenomas from EPCAM germline deletion carriers for EPCAM protein expression and allelic deletion status of the EPCAM gene region by multiplex ligation-dependent probe amplification. In four out of six tumors we observed lack of EPCAM expression accompanied by biallelic deletions affecting the EPCAM gene. In contrast, monoallelic retention of the EPCAM gene was observed in the remaining two tumors with retained EPCAM protein expression. These results demonstrate that EPCAM expression in tumors from EPCAM deletion carriers depends on the localization of the second somatic hit that inactivates MSH2. Moreover, we report lack of EPCAM protein expression in a colorectal adenoma, suggesting that EPCAM immunohistochemistry may detect EPCAM germline deletions already at a precancerous stage.

Liver progenitor cell markers correlate with liver damage and predict short-term mortality in patients with alcoholic hepatitis

Alcoholic hepatitis (AH) is a severe condition developed in patients with underlying alcoholic liver disease. Ductular reaction has been associated with chronic alcohol consumption but there is no information regarding the extent of liver progenitor cell (LPC) proliferation in AH. The aim of this study was to investigate LPC markers in AH and its correlation with disease severity. Fifty-nine patients with clinical and histological diagnosis of AH were included in the study. LPC markers were assessed by real-time polymerase chain reaction (PCR) and immunohistochemistry. Standard logistic regression analysis and classification and regression trees (CART) analysis were used for statistical analysis. A microarray analysis showed an up-regulation of LPC markers in patients with AH. Real-time PCR demonstrated that epithelial cell adhesion molecule (EpCAM), Prominin-1, and Keratin7 were significantly increased in patients with AH compared with normal livers (P ≤ 0.01), chronic hepatitis C (P ≤ 0.01), and HCV-induced cirrhosis (P ≤ 0.01). Immunohistochemistry scores generated for Keratin7 and EpCAM demonstrated a good correlation with gene expression. Keratin7 gene expression correlated with liver failure as assessed by model for endstage liver disease score (r = 0.41, P = 0.006) and Maddrey's discriminant function (r = 0.43, P = 0.004). Moreover, Keratin7 (OR1.14, P = 0.004) and Prominin-1 (OR1.14, P = 0.002), but not EpCAM (OR1.16, P = 0.06), were identified as independent predictors of 90-day mortality. CART analysis generated an algorithm based on the combination of Keratin7 and EpCAM gene expression that stratified three groups of patients with high, intermediate, and low short-term mortality (89%, 33%, and 6%, respectively; area under the receiver operating curve 0.73, 95% confidence interval 0.60-0.87). Keratin7 expression provided additional discrimination potential to the age, bilirubin, international normalization ratio, creatinine (ABIC) score. LPC markers correlate positively with severity of liver disease and short-term mortality in AH patients. This study suggests that LPC proliferation may be an important feature of AH pathophysiology.

Significance of EpCAM and TROP2 expression in non-small cell lung cancer

BackgroundThe tumor-associated calcium signal transducer (TACSTD) genes, originally designated epithelial cell adhesion molecule (EpCAM) and TROP2, represent true oncogenes. Little is known about EpCAM and TROP2 gene expression in non-small cell lung carcinoma (NSCLC). This study evaluated EpCAM and TROP2 protein expression and clinicopathologic significance in cases of NSCLC.MethodsTissue microarray blocks acquired from 164 cases of NSCLC, including 100 cases of adenocarcinoma (AdC) and 64 of squamous cell carcinoma (SCC), were examined by immunohistochemical staining for EpCAM, and TROP2. The results were correlated with clinicopathologic data.ResultsEpCAM and TROP2 were significantly overexpressed in SCC than in AdC (P < 0.01). In AdC, EpCAM overexpression was closely related to sex, histologic grade, pathologic T stage, pathologic N stage, and TNM stage, and TROP2 overexpression was only related to histologic grade (P < 0.05, respectively). In SCC, correlations were evident between EpCAM overexpression and TNM stage (P = 0.01), and between TROP2 overexpression and pathologic T stage (P = 0.02). EpCAM overexpression showed no significance with overall survival in AdC and SCC patients. However, TROP2 overexpression in AdC had a positive influence on overall survival (P = 0.02) and disease-free survival (P = 0.03). In particular, AdC patients with stage II or III showed better overall survival (P = 0.05) and disease-free survival (P = 0.04).ConclusionsWhile EpCAM and TROP2 show weak and non-complete membranous staining in normal bronchial epithelium and pneumocyte, their complete membranous expression in carcinoma suggests their role in carcinogenesis. EpCAM and TROP2 were more frequently overexpressed in SCC. EpCAM overexpression had no prognostic value in this study, but TROP2 overexpression showed better survival in AdC patients and might be a better prognostic marker in advanced stage AdC.

Functional polymorphism in the EpCAM gene is associated with occurrence and advanced disease status of cervical cancer in Chinese population

The epithelial cell adhesion molecule (EpCAM) was originally identified as a tumor associated antigen, attributable to its high expression on rapidly proliferating tumors of epithelial origin. EpCAM plays vital roles in carcinogenesis, tumor progression and metastasis in most tumors. A non-synonymous polymorphism (rs1126497 C/T) was found in exon 3 of EpCAM, which cause a transition from 115 Met to 115 Thr. Another polymorphism (rs1421 A/G) in the 3'UTR causes loss of has-miR-1183 binding. We performed a multiple independent case-control analysis to assess the association between EpCAM genotypes and cervical cancer risk. Genotyping a total of 518 patients with cervical cancer and 723 control subjects in a Chinese population, we observed that the variant EpCAM genotypes (rs1126497 CT, and TT) were associated with substantially increased risk of cervical cancer. Compared with the rs1126497 CC genotype, CT genotype had a significantly increased risk of cervical cancer (Crude OR = 1.70; 95% CI = 1.33-2.20; adjusted OR = 1.72; 95% CI = 1.33-2.22), the TT carriers had a further increased risk of cervical cancer (Crude OR = 1.94; 95% CI = 1.01-3.72; adjusted OR = 1.96; 95%CI = 1.01-3.81), and there was a trend for an allele dose effect on risk of cervical cancer (P < 0.001). Moreover, the allele T increases the risk for invasive disease or metastatic disease, compared with C allele. However, there exists no significant difference in genotype frequencies of rs1421 A/G site between cases and controls (P = 0.798). These findings suggest that rs1126497 C/T polymorphism in EpCAM may be a genetic modifier for developing cervical cancer.

Expression of prospero-related homeobox 1 (prox–1) transcription factor and epithelial adhesion molecule (EpCAM) in a rat model of intrahepatic cholangiocarcinogenesis

Deregulation of homeobox genes may give rise to tumorigenesis in target organs (1). Immuhistochemical studies have shown that the Prospero-related homeobox 1 (Prox–1) gene is expressed in mature hepatocytes but is not detectable in non-parenchymal liver cells (2). EpCAM is a surface marker on human hepatic stem/progenitor cells that is reported as absent on mature hepatocytes.However, hepatocytes (that have recently derived from hepatobiliary progenitors) are reported to have EPCAM surface expression in different cirrhotic livers (3). In the current study we investigated the expression pattern of Prox–1 in a rat model of damage, fibrosis, cirrhosis and intrahepatic cholangiocarcinoma (CC). Sprague dawley rats were given thioacetamide in drinking water (500mg/l) until they developed tumors (for 18 weeks). Control rats were given normal tap water. Animals were sacrificed, liver was removed and RNA was isolated for real time PCR. Cryostat and Paraffin sections were used for histopathology. The mRNA expression levels of Prox–1, Cytokeratin–19, and Epithelial Cell Adhesion Molecule genes were examined by RT-PCR.Immunohistochemical analysis of the normal liver revealed that Prox–1 is expressed in hepatocytes, while it could not be detected in EpCAM and CK–19 positive biliary epithelial cells. A co-expression of Prox–1 with CK–19 and EpCAM was found in proliferating ductular cells, which were located within the fibrotic tissue of the cirrhotic liver after 12 and 16 weeks of TAA administration. Immunohistochemical analysis of the CC tissue demonstrated Prox–1 positivity in CK–19 and EpCAM positive tumoral cells as well. Compared to the normal liver tissue, no significant up-regulation of the Prox–1 gene-mRNA expression could be found during the development of CC whereas an up-regulation of CK–19 and EpCAM could be detected. These data suggest that EpCAM and Prox–1 positive cells might be tumor initiating cells and Prox–1 might be involved in neoplastic changes of the biliary epithelium during cholangiocarcinogenesis.

Transcriptional Read-Through Induction Treatment Trial in Intestinal Failure Induced by anEpCAMNonsense Mutation

Congenital tufting enteropathy (CTE) is a rare autosomal recessive diarrheal disorder where epithelial tufts can be present from the duodenum to the large intestine. CTE has been linked to mutations in the epithelial cell adhesion molecule gene (EpCAM) Sivagnanam et al. (2008). We recently reported the first case with a nonsense mutation in EpCAM Sivagnanam et al. (2010). Here, we explored the clinical and molecular effects of enterally administered gentamicin in this CTE patient. Altogether, our findings indicate that the therapy employed was insufficient to produce notable read-through induction of the EpCAM premature termination codon. This report highlights the utility of genetic testing not only in respect of diagnostics, prognostics, and family planning, but potential mutation-specific therapeutic considerations as well.

Expression and role of epithelial cell adhesion molecule in dysplastic nodule and hepatocellular carcinoma

Epithelial cell adhesion molecule (EpCAM) has been proposed as a marker for cancer stem cells in human hepatocellular carcinoma (HCC). However, the function and clinical significance of EpCAM in HCC is largely unknown. We examined EpCAM expression and localization in 28dysplastic nodules (DNs) and their corresponding cirrhotic nodules, 79HCC tissue sections and 132HCC tissue microarray cores by immunohistochemistry and determined the relationship to clinicopathologic findings. We also examined the role of EpCAM in HCC using synthetic small interfering RNA to silence EpCAM gene expression in Huh-7 cells. EpCAM expression was very rare in DNs but dominantly appeared in a distinctly nodular type of small HCC. Expression of EpCAM was observed in 39% (31/79) of HCC tissue sections and in 34.1% (45/132) of tissue microarray sections. EpCAM expression in HCC was significantly associated with high tumor grade and serum α-fetoprotein level. Silencing EpCAM gene expression significantly decreased the proliferative activity and invasiveness of HCC cells. EpCAM expression was an independent prognostic factor for survival in patients with T1 HCC. The data indicate that EpCAM expression occurs at distinct nodular stage of HCC and could play an important role in HCC progression.

Positive Darwinian Selection Drives the Evolution of the Morphology-Related Gene, EPCAM, in Particularly Species-Rich Lineages of African Cichlid Fishes

The study of genetic evolution within the context of adaptive radiations offers insights to genes and selection pressures that result in rapid morphological change. Cichlid fishes are very species-rich and variable in coloration, behavior, and morphology, and so provide a classical model system for studying the genetics of adaptive radiation. In this study, we researched the evolution of the epithelial cell adhesion molecule (EPCAM), a candidate gene for the adaptive evolution of morphology broadly, and skin development specifically, in fishes. We compared EPCAM gene sequences from a rapidly speciating African cichlid lineage (the haplochromines), a species-poor African lineage (Nile tilapia Oreochromis niloticus), and a very young adaptive radiation in the Neotropics (sympatric crater lake Midas cichlids, Amphilophus sp.). Our results, based on a hierarchy of evolutionary analyses of nucleotide substitution, demonstrate that there are different selection pressures on the EPCAM gene among the cichlid lineages. Several waves of positive natural selection were identified not only on the terminal branches, but also on ancestral branches. Interestingly, significant positive or directional selection was found in the haplochromine cichlids only but not the comparatively species-poor tilapia lineage. We hypothesize that the strong signal of selection in the ancestral African cichlid lineage coincided with the transition from riverine to lacustrine habitat. The two neotropical species for which we collected new sequence data were invariant in the EPCAM locus. Our results suggest that functional changes promoted by positive Darwinian selection are widespread in the EPCAM gene during African cichlid evolution.

MiRNA-mediated gene silencing of epithelial cell adhesion molecule inhibits growth of human gallbladder cancer cells in vitro

Objective To observe the effect of epithelial cell adhesion molecule (EP-CAM) gene silencing by RNA interfering (RNAi) technology on the proliferation, apoptosis of GBC-SD cells. Methods Recombinant plasmid pcDNA? 6. 2-GW/EraGFPmiR-EPCAM-l, -2, -3 and -4 targeting human EP-CAM gene were respectively transfected into GBC-SD cells by lipofectamine? 2000 after DNA sequencing. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the mRNA and protein expression of EP-CAM and adopted to select the recombinant plasmid which showed the most optimal inhibition effct. After tranfection by pcDNA?6. 2-GW/EmGFPmiR-EPCAM-l with the highest inhibition rate, cell proliferation was analyzed by methyl thiazol tetrazolium (MTT) assay, and apoptosis was assayed by flow cytometry. Results Four EP-CAM-targeted microRNA (miRNA)-l, -2, -3 and -4 were successfully inserted into the plasmid vector pcDNA?6. 2-GW/EmGFPmiR, and the coding sequences of the obtained miRNA were consistent with the designed fragments. RT-PCR results showed that the mRNA expression of EP-CAM gene in GBC-SD cells transfected with recombinant plasmid-1 ,-2,-3 and -4 were 0. 26 ± 0. 02, 0. 79 ± 0. 03, 0. 37 ± 0. 02, 0.45 ± 0.04 respectively, which was significantly lower than that in blank and negative control groups (0. 94 ± 0. 03, 0. 92 ± 0. 03, respectively, P ＜ 0. 01). Western blotting revealed that the relative expression of EP-CAM protein in GBC-SD cells transfected with recombinant plasmid-1, -2, -3 and 4 was 0. 22 ±0. 03, 0.71 ±0.03, 0.32 ±0.02, and 0.41 ±0.04 respectively, which was significantly lower than that in blank and negative control groups (0. 85 ±0.03, 0. 84 ±0.04, respectively,P＜0. 01). After transfection with pcDNA?6. 2-GW/EmGFPmiR-EPCAM-l, the proliferation ability of the cells was significantly inhibited and the inhibition rate was 39% after 48 h. As compared with the control groups, the difference was significant (P ＜ 0. 05). However cell apoptosis had no significant change ( P ＞ 0. 05 ). Conclusion The recombinant plasmid pcDNA? 6. 2-GW/EmGFPmiR miRNA targeting human EP-CAM gene could effectively down-regulate EP-CAM expression and inhibit proliferation of GBC-SD cells in vitro. Key words: Gallbladder carcinoma; Epithelial cell; Adhesion molecule; RNA interference; Proliferation

Analysis of EPCAM Protein Expression in Diagnostics of Lynch Syndrome

Lynch syndrome is an inherited tumor predisposition syndrome caused by germline mutations of DNA mismatch repair (MMR) genes, mainly MLH1 and MSH2. Recently, germline deletions affecting the epithelial cell adhesion molecule (EPCAM) gene located upstream of MSH2 were identified as a novel mutational mechanism causing Lynch syndrome by epigenetic inactivation of the respective MSH2 allele. Immunohistochemical analysis of MMR protein expression is a hallmark of Lynch syndrome diagnostics, but it cannot distinguish between EPCAM deletion carriers and MSH2 mutation carriers. We hypothesized that EPCAM protein expression might be altered in tumors from patients with a germline EPCAM deletion. Immunohistochemistry was used to assess EPCAM expression in Lynch syndrome-associated MSH2-negative tumors (n = 26). Multiplex ligation-dependent probe amplification (MLPA) analysis was performed to detect germline deletions of the EPCAM and MSH2 gene loci. In four MSH2-negative tumors, a concomitant lack of EPCAM expression was detected. MLPA analysis revealed heterozygous EPCAM deletions in all patients with EPCAM-negative tumors. In contrast, EPCAM expression was positive in all cancers from patients with germline alterations affecting MSH2 but not EPCAM. Two EPCAM deletions were detected in patients with an EPCAM-positive tumor. These results indicate that loss of EPCAM protein expression is frequent in tumors from patients with EPCAM germline deletions. EPCAM immunohistochemistry therefore represents a promising novel tool for the identification of Lynch syndrome patients with EPCAM germline deletions.

Tufting Enteropathy with EpCAM Mutations in Two Siblings

Tufting enteropathy is a rare autosomal recessive disorder presenting with early-onset severe intractable diarrhea. The epithelial cell adhesion molecule gene (EpCAM) has recently been identified as the gene responsible for tufting enteropathy. Based on histology, a diagnosis of tufting enteropathy was made in two Korean siblings. They developed chronic diarrhea and failure to thrive. They had a broad nasal bridge and micrognathia. Duodenal and colonic biopsies showed villous atrophy, disorganization of surface enterocytes, and focal crowding resembling tufts. Protracted diarrhea continued and so cyclic parenteral nutrition was supplied. The sister had juvenile rheumatoid arthritis. Mutation analysis of EpCAM identified two compound heterozygous mutations in these siblings: 1) a donor splicing site mutation in intron 5 (c.491+1G>A) and 2) a novel nonsense mutation in exon 3 (c.316A>T, Lys106X). Analysis of EpCAM will be useful for genetic counseling and prenatal diagnosis of tufting enteropathy.

A non-synonymous polymorphism Thr115Met in the EpCAM gene is associated with an increased risk of breast cancer in Chinese population

As a tumor-associated antigen and a surface marker of breast cancer stem cells (BCSCs), epithelial cell adhesion molecule (EpCAM) plays an important role in not only cell adhesion, morphogenesis, metastases but also carcinogenesis. A non-synonymous C/T polymorphism (rs1126497) in exon3 of EpCAM causes a transition of 115 amino acid from Met to Thr. Another polymorphism (A/G, rs1421) in the 3'UTR causes loss of has-miR-1183 binding. A multiple independent case-control analysis was performed to assess the association between EpCAM genotypes and breast cancer risk. We observed that the variant EpCAM genotype (rs1126497 CT, and TT) was associated with substantially increased risk of breast cancer. Genotyping a total of 1643 individuals with breast cancer and 1818 control subjects in Eastern and Southern Chinese populations showed that rs1126497 CT + TT genotype had an odd ratio of 1.40 (95% confidence interval, 1.16-1.57) for developing breast cancer compared with CC genotype. The allele T increases the risk of breast cancer in a dose-dependent response manner (P (trend) < 0.001). Moreover, compared to breast cancer patients carrying the CC genotype, the EpCAM SNP rs1126497 CT or TT carrier was significantly associated with early breast cancer onset (P = 0.0023). However, no significant difference was found in genotype frequencies at the rs1421 A/G site between cases and controls. These findings suggest that M115T polymorphism in EpCAM may be a genetic modifier for developing breast cancer.

Targeted DNA methylation by a DNA methyltransferase coupled to a triple helix forming oligonucleotide to down-regulate the epithelial cell adhesion molecule.

The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein that has been identified as a marker of cancer-initiating cells. EpCAM is highly expressed on most carcinomas, and transient silencing of EpCAM expression leads to reduced oncogenic potential. To silence the EpCAM gene in a persistent manner via targeted DNA methylation, a low activity mutant (C141S) of the CpG-specific DNA methyltransferase M.SssI was coupled to a triple-helix-forming oligonucleotide (TFO−C141S) specifically designed for the EpCAM gene. Reporter plasmids encoding the green fluorescent protein under control of different EpCAM promoter fragments were treated with the TFO−C141S conjugate to determine the specificity of targeted DNA methylation in the context of a functional EpCAM promoter. Treatment of the plasmids with TFO−C141S resulted in efficient and specific methylation of the targeted CpG located directly downstream of the triple helix forming site (TFS). No background DNA methylation was observed neither in a 700 bp region of the EpCAM promoter nor in a 400 bp region of the reporter gene downstream of the TFS. Methylation of the target CpG did not have a detectable effect on promoter activity. This study shows that the combination of a specific TFO and a reduced activity methyltransferase variant can be used to target DNA methylation to predetermined sites with high specificity, allowing determination of crucial CpGs for promoter activity.

Hereditary and Familial Colon Cancer

Between 2% to 5% of all colon cancers arise in the setting of well-defined inherited syndromes, including Lynch syndrome, familial adenomatous polyposis, MUTYH-associated polyposis, and certain hamartomatous polyposis conditions. Each is associated with a high risk of colon cancer. In addition to the syndromes, up to one-third of colon cancers exhibit increased familial risk, likely related to inheritance. A number of less penetrant, but possibly more frequent susceptibility genes have been identified for this level of inheritance. Clarification of predisposing genes allows for accurate risk assessment and more precise screening approaches. This review examines the colon cancer syndromes, their genetics and management, and also the common familial colon cancers with current genetic advances and screening guidelines.