Silencing Of E-cadherin Research Articles

Activation of DNA Methyltransferase 1 by EBV LMP1 Involves c-Jun NH2-Terminal Kinase Signaling

EBV latent membrane protein 1 (LMP1) activates cellular DNA methyltransferases, resulting in hypermethylation and silencing of E-cadherin. However, the underlying mechanism remains to be elucidated. In this study, we show that LMP1 directly induces the dnmt1 promoter activity through its COOH-terminal activation region-2 YYD domain. Using (i) LMP1 mutants, (ii) dominant negative mutants c-jun NH(2)-terminal kinase (JNK)-DN, p38-DN, and constitutive active mutant IkappaB, as well as (iii) dsRNAs targeting c-Jun, JNK, and tumor necrosis factor receptor-associated death domain protein, and (iv) signal transduction inhibitors, we show that LMP1-mediated DNA methyltransferase-1 (DNMT1) activation involves JNK but not nuclear factor kappaB and p38/mitogen-activated protein kinase signaling. In addition, LMP1 is unable to activate dnmt1-P1 promoter with activator protein-1 (AP-1) site mutation. Chromatin immunoprecipitation assay results also confirm that LMP1 activates P1 promoter via the JNK-AP-1 pathway. Furthermore, chromatin immunoprecipitation assay data in LMP1-inducible cells disclose that LMP1 induces formation of a transcriptional repression complex, composed of DNMT1 and histone deacetylase, which locates on E-cadherin gene promoter. Treatment with JNK inhibitor, SP600125, prevents the formation of this repression complex. Statistical analyses of the immunohistochemical staining of 32 nasopharyngeal carcinoma (NPC) biopsies show LMP1 expression (18 of 32, 56.25%), DNMT1 expression (31 of 32, 97%), and phospho-c-Jun (27 of 32, 84.38%), suggesting that overexpression of these proteins is observed in NPC tumor. Overall, these results support a mechanistic link between JNK-AP-1 signaling and DNA methylation induced by the EBV oncogene product LMP1.

Sequential Molecular and Cellular Events during Neoplastic Progression: A Mouse Syngeneic Ovarian Cancer Model

Studies performed to identify early events of ovarian cancer and to establish molecular markers to support early detection and development of chemopreventive regimens have been hindered by a lack of adequate cell models. Taking advantage of the spontaneous transformation of mouse ovarian surface epithelial (MDSE) cells in culture, we isolated and characterized distinct transitional stages of ovarian cancer as the cells progressed from a premalignant nontumorigenic phenotype to a highly aggressive malignant phenotype. Transitional stages were concurrent with progressive increases in proliferation, anchorage-independent growth capacity, in vivo tumor formation, and aneuploidy. During neoplastic progression, our ovarian cancer model underwent distinct remodeling of the actin cytoskeleton and focal adhesion complexes, concomitant with downregulation and/or aberrant subcellular localization of two tumor-suppressor proteins Ecadherin and connexin-43. In addition, we demonstrate that epigenetic silencing of E-cadherin through promoter methylation is associated with neoplastic progression of our ovarian cancer model. These results establish critical interactions between cellular cytoskeletal remodeling and epigenetic silencing events in the progression of ovarian cancer. Thus, our MDSE model provides an excellent tool to identify both cellularand molecular changes in the early and late stages of ovarian cancer, to evaluate their regulation, and to determine their significance in an immunocompetent in vivo environment.

E-cadherin mutation and Snail overexpression as alternative mechanisms of E-cadherin inactivation in synovial sarcoma.

We have recently reported frequent E-cadherin gene mutations in synovial sarcoma (SS), suggesting mutational inactivation of E-cadherin as a potential mechanism of spindle cell morphology in SS, a spindle cell sarcoma that shows areas of glandular epithelial differentiaton in some cases (biphasic SS) and only pure spindle cell morphology in most cases (monophasic SS). However, the mechanism of downregulation of E-cadherin in SS remains unknown. To further address this issue, we analysed the mechanisms of E-cadherin silencing in 40 SS. Genetic and epigenetic changes in the E-cadherin gene, and the expression level of its transcriptional repressor Snail were examined by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), methylation-specific PCR, and real-time quantitative PCR, respectively. Expression of E-cadherin was examined by RT-PCR and immunohistochemistry. We also examined ELF3, a transcription factor associated with epithelial differentiation in SS in a previous cDNA microarray, by RT-PCR. E-cadherin and ELF3 transcripts were detected, respectively, in 27/40 (67.5%) and in 25/40 (62.5%) of SS, and these epithelial-related genes were almost always coexpressed. Hypermethylation of the promoter of the E-cadherin gene was detected in five cases (12.5%) in SS; however, E-cadherin was silenced at mRNA level in only one of the five cases. E-cadherin missense mutations were observed in five cases (12.5%) of SS. In SS, all five cases with E-cadherin missense mutations had the SYT-SSX1 fusion and were monophasic tumors, suggesting a relationship between the SYT-SSX fusion type and E-cadherin missense mutation (P=0.07). E-cadherin mRNA expression in SS was associated with reduced Snail expression level (P=0.03). E-cadherin membranous expression was observed in 14/40 (35.0%) of SS, and was also correlated with SYT-SSX1 fusion type and biphasic histology. ELF3 was confirmed to be more highly expressed in biphasic than monophasic SS by real-time quantitative PCR. These results suggest that in SS the loss of E-cadherin expression occurs either by Snail trans-repression or by inactivating mutations. Thus, E-cadherin downregulation is associated with the loss or absence of glandular epithelial differentiation in certain SS.

Hypermethylation-associated transcriptional silencing of E-cadherin in primary sporadic colorectal carcinomas.

Loss of E (epithelial)-cadherin expression has been previously documented in sporadic colorectal carcinomas (SCRCs), but not as a consequence of mutations or allelic loss. In this study, the methylation status of the E-cadherin promoter was examined by utilizing the methylation-specific polymerase chain reaction (MSP) assay in 63 primary SCRCs and paired adjacent normal tissues. This was correlated with E-cadherin expression at both the RNA and the protein levels using multiplex reverse transcription (RT)-PCR and immunohistochemistry (IHC), respectively. Data were associated with the patients' clinicopathological features. Methylated alleles were present in 34/61 (56%) of the samples examined. Decreased E-cadherin mRNA expression was demonstrated in 29/61 carcinomas (47.5%) and was significantly associated with lymph node (LN) metastases (p = 0.03, Kruskal-Wallis) and tumour stages Astler-Coller B1 and B2 (p = 0.01, chi(2)). E-cadherin IHC expression was significantly associated with the absence of LN metastases (p = 0.01, chi(2)) and tumour stages Astler-Coller B1 and B2 (p = 0.002, Kruskal-Wallis) in 28/63 (44.4%) of the samples examined. Twenty-three out of 29 (79.3%) samples with decreased mRNA expression and 20/33 (60.6%) with detected protein expression revealed methylated (p = 0.03, Kruskal-Wallis) and unmethylated (p = 0.01, Kruskal-Wallis) alleles, respectively. In agreement with previous work demonstrating that somatic mutations and loss of heterozygosity of the E-cadherin gene are rare or absent in the majority of SCRCs studied so far, this study reports a consistent and uniform decrease or absence of E-cadherin expression, associated with aberrant methylation, in the majority of carcinomas examined, suggesting an epigenetically mediated loss of E-cadherin function in these carcinomas.

E-cadherin expression is silenced by DNA methylation in cervical cancer cell lines and tumours

A previous study showed E-cadherin expression was lost in some cervical cancer cell lines and tumours. This study was designed to clarify the significance of DNA methylation in silencing E-cadherin expression. We examined promoter methylation of E-cadherin in five cervical cancer cell lines and 20 cervical cancer tissues using methylation-specific PCR (MSP) and bisulphite DNA sequencing. The correlation of E-cadherin methylation and expression together with methyltransferase ( DNMT1) were further studied. We found that hypermethylation of E-cadherin was involved in five cervical cancer cell lines and 40% (8/20) of cervical cancer tissues. E-cadherin protein was lost in 6/8 (75%) samples and 3/5 (60%) cell lines with promoter methylation. E-cadherin methylation was significantly correlated with increased DNMT1. Using an antisense DNMT1 oligo to transfect into SiHa HeLa C33A cell line, E-cadherin protein was re-expressed. We concluded that loss of E-cadherin expression was in part correlated with DNA methylation and DNMT1 expression in cervical cancer.

Multiple ways of silencing E-cadherin gene expression in lobular carcinoma of the breast.

The cell-cell adhesion receptor gene E-cadherin (CDH1) is expressed by epithelial cells, in which it mediates adhesion and morphogenesis. Invasive lobular carcinoma (ILC) characteristically infiltrates diffusely as single cells; by immunohistochemistry, many of these tumours lack E-cadherin expression. In the present study we investigated various ways in which loss of function of the E-cadherin gene could occur in ILCs, namely, promoter methylation, mutation and allelic loss. We analysed 22 ILCs and found 12 (55%) E-cadherin-negative samples by immunohistochemical analysis. Methylation-specific polymerase chain reaction (PCR) showed that 17/22 (77%) of these tumours had methylation of the CDH1 promoter, including 11/12 (91%) of the E-cadherin-negative tumours. All 16 exons of E-cadherin (including intron-exon boundaries) were amplified from chromosomal DNA and screened for mutations by conformation-sensitive gel electrophoresis (CSGE). Bands with altered mobility were analysed by direct sequencing. We identified five frameshift mutations, which resulted in downstream stop codons and one splice site mutation in six different tumours (29%). Loss of heterozygosity (LOH) was assessed using microsatellite markers, and 9/18 (50%) informative tumours showed LOH. We conclude that most ILCs show genetic or epigenetic changes affecting the E-cadherin gene and that many of these tumours lack E-cadherin expression. In all cases in which there was loss of expression, this was consistent with biallelic inactivation of CDH1 by promoter methylation, mutation or allelic loss in any combination.

Molecular characteristics of eight gastric cancer cell lines established in Japan.

Molecular characterization of eight gastric cancer cell lines established in Japan are summarized according to the genetic and epigenetic alterations and growth factor status. TMK-1 poorly differentiated adenocarcinoma cell line harbors mutant p53 tumor suppressor gene and rearrangement of p15MTS2. MKN-1 adenosquamous carcinoma line with mutant p53 reveals silencing of E-cadherin by promoter CpG hypermethylation. MKN-7 well-differentiated adenocarcinoma cell line has amplification of c-erbB2 oncogene and cyclin E gene. MKN-28 well-differentiated adenocarcinoma cell line reveals mutations in p53 and APC tumor suppressor genes and silencing of CD44. The MKN-45 poorly differentiated adenocarcinoma cell line with wild-type p53 is characterized by homozygous deletion of p16CDKN2/MTS1/INK4A and p15MTS2, amplification of c-met oncogene and promoter mutation of E-cadherin. MKN-74 derived from moderately differentiated tubular adenocarcinoma has wild-type p53. KATO-III signet ring cell carcinoma line has genomic deletion of p53, amplification of K-sam and c-met oncogene and mutation of E-cadherin. HSC-39 signet ring cell carcinoma cell line harboring p53 missense mutation has homozygous deletion of p16CDKN2/MTS1/INK4A and p15MTS2, amplifications of c-myc, c-met, K-sam and CD44 gene and mutation in beta-catenin gene.

E-Cadherin gene promoter hypermethylation in primary human gastric carcinomas.

E (epithelial)-cadherin, the cell adhesion molecule also considered a potential invasion/metastasis suppressor, is mutationally inactivated in nearly half of all undifferentiated-scattered (diffuse-type) gastric carcinomas. In addition, silencing of E-cadherin by CpG methylation within its promoter region has been reported in several gastric carcinoma cell lines. We investigated the methylation status of the E-cadherin promoter region in 53 primary human gastric carcinomas. Hypermethylation of the E-cadherin promoter was determined by utilizing methylation-specific polymerase chain reaction (PCR)-single-strand conformation polymorphism (MSP-SSCP) analysis followed by direct sequencing of PCR products. Expression of E-cadherin was studied by western blot analysis. All statistical tests were two-sided. Hypermethylation of the E-cadherin promoter was evident in 27 (51%) of 53 primary gastric carcinomas examined by MSP-SSCP. It occurred more frequently in carcinomas of the undifferentiated-scattered type (in 15 [83%] of 18) than in other histologic subtypes (in 12 [34%] of 35) (P =.0011, Fisher's exact test), and it was present at similar rates in early (in six [60%] of 10) versus advanced (in 21 [49%] of 43) carcinomas (P =.73, Fisher's exact test). Methylation occurring at all cytosine-guanosine sequences (CpGs) near the transcriptional start site was confirmed in six of six tumors examined by bisulfite-DNA sequencing, including two early gastric carcinomas. In addition, loss or diminished expression of E-cadherin was confirmed by western blotting in four of the six tumor tissues demonstrating hypermethylation. The E-cadherin promoter frequently undergoes hypermethylation in human gastric cancers, particularly those of the undifferentiated-scattered histologic subtype. E-cadherin promoter hypermethylation is associated with decreased expression and may occur early in gastric carcinogenesis.