Promoter methylation of the PTEN gene is a common molecular change in breast cancer

About 25-50% of women with Cowden disease, a syndrome associated with germ-line mutations of the PTEN gene (at 10q23), develop breast cancer (BC), but PTEN mutations have been found in only 5% of sporadic BCs. However, 29-48% of BCs display loss of heterozygosity in 10q23, and about 40% of BCs show a decrease or absence of PTEN protein levels at the time of diagnosis. Promoter hypermethylation has been identified as an alternative mechanism of tumor-suppressor gene inactivation, but its importance in PTEN silencing in sporadic BC is unknown. We investigated PTEN promoter hypermethylation in 90 sporadic BCs and its correlations with 11 molecular and pathologic parameters, including mRNA levels of PTEN. The study, a methylation-specific PCR assay, was carried out with methylated specific primers designed in a region with scarce homology with the psiPTEN pseudogene. Expression was analyzed by real-time PCR. We found that the PTEN promoter was hypermethylated in 43 BCs (48%). PTEN hypermethylation was associated with ERBB2 overexpression, larger size, and higher histologic grade (P=0.012, 0.03, and 0.009, respectively). We concluded that PTEN promoter hypermethylation is a common event in sporadic BC, correlating with other well-established prognostic factors of this malignancy. Additionally, PTEN mRNA expression was lower in tumors with aberrant methylation.     

Promoter methylation status of the DNA repair genes hMLH1 and MGMT in gastric carcinoma and metaplastic mucosa.

Hypermethylation of CpG islands in the promoter region is associated with the silencing of a variety of tumor suppressor genes. DNA repair genes human Mut L homologue 1 (hMLH1) and O(6)-methylguanine-DNA methyltransferase (MGMT) have been shown to be hypermethylated in certain carcinomas. We studied DNA methylation of CpG islands in hMLH1 and MGMT in 50 gastric carcinomas and 10 intestinal metaplastic mucosa samples. We analyzed the methylation status of hMLH1 and MGMT using methylation-specific polymerase chain reaction and DNA sequencing analysis. We measured protein levels of hMLH1 using Western blot and immunohistochemical analysis. CpG island hypermethylation of hMLH1 and MGMT was detected in 11 (22%) and 8 (16%) of the 50 gastric tumors, respectively. Hypermethylation of the promoter was more common in intestinal-type gastric carcinomas than in poorly diffuse-type gastric carcinomas (p = 0.016 and 0.021, respectively; Fisher's exact test). However, hMLH1 promoter hypermethylation did not coincide with MGMT promoter hypermethylation except in 1 patient. Hypermethylation of the hMLH1 promoter but not the MGMT promoter occurred in intestinal metaplastic mucosae. Immunohistochemical analysis revealed a corresponding reduction in hMLH1 protein expression in some of the intestinal metaplastic mucosae. Our results suggest that at least two types of promoter methylation participate in the development of gastric carcinoma. Tumor-specific promoter hypermethylation of hMLH1 may be an early event in carcinogenesis in the stomach.     

Epigenetic PTEN silencing in malignant melanomas without PTEN mutation

A tumor suppressor gene at 10q 23.3, designated PTEN, encoding a dual specificity phosphatase with lipid and protein phosphatase activity, has been shown to play an important role in the pathogenesis of a variety of human cancers. Germline mutations in PTEN cause Cowden syndrome (CS), which is characterized by multiple hamartomas and a high risk of breast and thyroid cancers. Frequent loss of heterozygosity at 10q is found in both early and advanced-stage sporadic melanomas; however, mutations or deletions in PTEN are detected mainly in melanoma cell lines. In this study, we examined PTEN expression in 34 unselected sporadic melanomas (4 primary melanomas, 30 metastases) using immunohistochemistry and correlated this with the results of structural studies of this gene. Immunostaining of 34 melanoma samples revealed no PTEN expression in 5 (15%) and low PTEN expression in 17 (50%), whereas the rest of the tumors (35%) had high levels of expression. Hemizygous deletion was found in 32% of the tumors but neither intragenic PTEN mutation nor biallelic deletion was found in any of the samples. Of the 5 melanomas showing no PTEN expression, 4 had no mutation or deletion of PTEN. Of the 13 tumors having weak PTEN immunoreactivity and informative loss of heterozygosity results, 6 had evidence of hemizygous allelic loss of PTEN while the remaining 7 had intact PTEN. These results strongly support PTEN as a major tumor suppressor on 10q involved in melanoma tumorigenesis and suggest an epigenetic mechanism of biallelic functional inactivation not previously observed in other cancers where PTEN might be involved.     

Promoter methylation and clinical significance of GPX3 in esophageal squamous cell carcinoma

BackgroundGlutathione peroxidase 3 (GPX3) provides critical protection against reactive oxygen species (ROS) in cells. Downregulation of GPX3 may contribute to carcinogenesis of esophageal squamous cell carcinoma (ESCC), but the mechanisms are not clear.Materials and methodsWe examined the differences in gene expression between ESCC and normal esophageal epithelial, by using GEO datasets, and collected 136 ESCC tumors and adjacent normal tissues to confirm our findings. GPX3 expression was measured, at the mRNA and protein levels, by qRT-PCR, Western Blot and immunohistochemistry (IHC).ResultsGPX3 mRNA and protein levels were 3.3-fold higher in normal epithelium compared with case-matched ESCC tissues. There was no significant correlation between clinical parameters and expression levels of GPX3 in ESCC. Promoter methylation of the GPX3 gene correlated with decreased expression.ConclusionDownregulation of GPX3 might be a key factor in the process of ESCC carcinogenesis.     

Gene silencing of TSPYL5 mediated by aberrant promoter methylation in gastric cancers

DNA methylation is crucial for normal development, but gene expression altered by DNA hypermethylation is often associated with human diseases, especially cancers. The gene TSPYL5, encoding testis-specific Y-like protein, was previously identified in microarray screens for genes induced by the inhibition of DNA methylation and histone deacetylation in glioma cell lines. The TSPYL5 showed a high frequency of DNA methylation-mediated silencing in both glioma cell lines and primary glial tumors. We now report that TSPYL5 is also inactivated by DNA methylation and could be a putative epigenetic target gene in gastric cancers. We found that the expression of TSPYL5 mRNA was frequently downregulated and inversely correlated with DNA methylation in seven out of nine gastric cancer cell lines. TSPYL5 mRNA expression was also restored after treating with a DNA methyltransferase inhibitor. In primary gastric tumors, methylation-specific PCR results in 23 of the 36 (63.9%) cases revealed that the hypermethylation at CpG islands of the TSPYL5 was detectable at a high frequency. Furthermore, TSPYL5 suppressed the growth of gastric cancer cells as demonstrated by a colony formation assay. Thus, strong associations between TSPYL5 expression and hypermethylation were observed, and aberrant methylation at a CpG island of TSPYL5 may play an important role in development of gastric cancers.     

Epigenetic silencing of WNT5A in Epstein-Barr virus-associated gastric carcinoma

Epstein-Barr virus (EBV) is responsible for the development of multiple tumors, including EBV-associated gastric carcinoma (EBVaGC), but little is known about its mechanisms in EBVaGC. WNT5A expression and promoter methylation were measured in 5 EBV-positive and 15 EBV-negative GC cell lines. The methylation status of 23 EBV-positive and 25 EBV-negative paired tumor/normal tissue samples was also examined. EBV-positive GC had no or very low expression of WNT5A but a high level of methylation in the promoter region. In contrast, EBV-negative GC had higher WNT5A expression and a lower level of promoter methylation. The reduced WNT5A expression could be restored by treatment with Aza, a methyltransferase inhibitor. Increased expression of WNT5A in vitro inhibited β-catentin expression in EBVaGC cells (SNU719). These results suggest that hypermethylation of WNT5A induced by EBV may contribute to the development of EBVaGC. Ectopic introduction of WNT5A may have preventive/therapeutic potential for tumors with silenced WNT5A.     

DNA甲基化与基因转录抑制

DNA甲基化作为一种重要的表观遗传修饰,调节着机体许多生物学过程。DNA甲基化由DNA甲基转移酶催化 ,在多种调节因子的参与下,与组蛋白修饰相互作用,抑制基因转录,导致基因沉默。探讨DNA甲基化与基因转录抑制之间的关系,对许多人类疾病的研究具有重要意义。     

Promoter methylation status of E-cadherin,hMLH1, and p16 genes in nonneoplastic gastric epithelia

Silencing of tumor suppressor and tumor-related genes by hypermethylation at promoter CpG islands is one of the major events in human tumorigenesis. Promoter methylation is also present in nonneoplastic cells as an age-related tissue-specific phenomenon that precedes the development of neoplasia. To clarify the significance of promoter methylation in nonneoplastic gastric epithelia as a precancerous signal, we investigated promoter methylation status of E-cadherin, hMLH1, and p16 genes in nonneoplastic cells of various organs obtained at autopsy, and compared the results with those of nonneoplastic epithelia of a cancerous stomach. Methylation of these genes was not seen in nonneoplastic cells of organs from people who were 22 years and younger (0%, 0 of 6). In contrast, E-cadherin and p16 were methylated in nonneoplastic gastric epithelia of persons who were 45 years or older. The numbers were 86% (12 of 14) and 29% (4 of 14), respectively. E-cadherin methylation occurred preferentially in the intestines, whereas p16 methylation was almost restricted to the stomach. For samples obtained from patients with stomach cancer, methylation was frequently observed in both neoplastic and corresponding nonneoplastic gastric epithelia: 47% (44 of 94) and 67% (63 of 94) for E-cadherin, 32% (30 of 94) and 24% (23 of 94) for hMLH1, and 22% (21 of 94) and 44% (41 of 94) for p16, respectively. hMLH1 methylation was not seen in nonneoplastic gastric epithelia from autopsy samples but occurred significantly in samples from nonneoplastic tissues of individuals with stomach cancer. Therefore, detection of hMLH1 methylation in nonneoplastic gastric epithelia may be useful for screening patients who may be at risk of developing gastric cancer.     

Epstein-barr virus-positive gastric carcinoma demonstrates frequent aberrant methylation of multiple genes and constitutes CpG island methylator phenotype-positive gastric carcinoma

CpG island methylation is an important mechanism for inactivating the genes involved in tumorigenesis. Gastric carcinoma (GC) is one of the tumors that exhibits a high frequency of aberrant CpG island methylation. There have been many reports suggesting a close link between Epstein-Barr virus (EBV) and the development of GC. However, little is known about the oncogenic mechanism of EBV in gastric carcinogenesis. Twenty-one cases of EBV-positive GC and 56 cases of EBV-negative GC were examined for aberrant DNA methylation of the CpG islands of 19 genes or loci and the differences in the methylation frequency between EBV-positive and -negative GCs were investigated to determine a role of aberrant methylation in EBV-related gastric carcinogenesis. The average number of methylated genes or loci was higher in EBV-positive GCs than in EBV-negative GCs (13.4 versus 7.8, respectively, P < 0.001). EBV-positive GCs showed methylation in at least 10 CpG islands (52.6% of the tested genes), whereas 62.5% of EBV-negative GCs showed methylation in <10 CpG islands. THBS1, APC, p16, 14-3-3 sigma, MINT1, and MINT25 were methylated at a frequency >90% in EBV-positive GCs. The methylation frequency difference in the respective CpG islands between EBV-positive and -negative GCs was statistically significant (P < 0.05). Among these genes or loci, the methylation frequency of p16 in the EBV-positive GCs was more than three times higher than in the EBV-negative GCs. The PTEN, RASSF1A, GSTP1, MGMT, and MINT2 were methylated in EBV-positive GCs at a frequency of more than three times that of the EBV-negative GCs. These results demonstrate a relationship between EBV and aberrant methylation in GC and suggest that aberrant methylation may be an important mechanism of EBV-related gastric carcinogenesis.     

Implication of Reprimo and hMLH1 gene methylation in early diagnosis of gastric carcinoma

DNA methylation has been recently recognized as a novel tumor marker. This study investigated the methylation status of Reprimo and hMLH1 gene in both plasma and tissue samples from gastric cancer patients, in an attempt to investigate their diagnostic implications in gastric cancer. A total of 180 tissue and plasma samples (including 50 cases of gastric cancer, 50 dysplasia, 50 chronic atrophic gastritis with intestinal metaplasia and 30 normal controls) were collected for detecting DNA methylation status of Reprimo and hMLH1 genes using MSP method. Tissue protein expression levels were further tested by immunohistochemical (IHC) staining. The positive rate of DNA methylation rate was, in ascending sequence, gastritis tissue, dysplasia tissue and gastric carcinoma tissue. All those tissues had significantly elevated DNA methylation level compared to normal group (P < 0.05). Expression level of Reprimo and hMLH1 proteins were, however, decreased in pathological tissues compared to normal ones (P < 0.05). A significantly negative relationship existed between protein level and promoter region methylation level. The DNA methylation occurred in promoter regions of both Reprimo and hMLH1 genes depressed the protein expression, and may participate in the occurrence and progression and gastric cancer. The combined assay of serum Reprimo and hMLH1 DNA methylation levels thus had critical importance in the early diagnosis and gastric cancer.     

Promoter methylation and protein expression of the E-cadherin gene in the clinicopathologic assessment of adenoid cystic carcinoma.

Adenoid cystic carcinoma, a relatively uncommon tumor of salivary glands, is characterized by a prolonged clinical course and a fatal outcome. The molecular events underlying their progression are unknown. In this study, we examined the methylation status of E-cadherin gene and its protein expression in 23 cases of adenoid cystic carcinoma and correlated the results with the clinicopathologic factors to determine its role in these tumors. We also analyzed the effect of 5-azacytidine on the re-expression in a methylated cell line of adenoid cystic carcinoma for this gene. In our study, E-cadherin immunoreactivity, although heterogeneous, showed a progressive reduction with high histological grade and in metastatic and recurrent lesions. Promoter methylation was detected in 16 of 23 cases (70%), but there was no correlation with the histological grade or patient prognosis. Microdissection of immuno-negative cells in heterogeneous tumors showed positive methlyation. In the cell line from salivary adenoid cystic carcinoma with methylated E-cadherin, 5-azacytidine restored the E-cadherin expression. Our results indicate that: (1) E-cadherin gene promoter is frequently methylated in adenoid cystic carcinoma, leading to reduced E-cadherin expression, (2) variable E-cadherin expression might result from the intratumoral heterogeneity, and (3) increased extent of methylated areas may be associated with progression and advancement of the disease.     

Promoter methylation of E-cadherin in hepatocellular carcinomas and dysplastic nodules

In order to clarify the significance of E-cadherin methylation in multistep hepatocarcinogenesis, we examined the methylation status of the E-cadherin promoter region, using methylation-specific polymerase chain reaction in 64 hepatocellular carcinomas (HCCs) and 13 dysplastic nodules (DNs), and correlated these results with E-cadherin protein expression and clinicopathologic factors of HCCs. Promoter methylation was detected in 1 of 13 (7.7%) DNs, in 5 of 13 (38.5%) Edmondson and Steiner grade I HCCs, and in 27 of 51 (52.9%) grade II or III HCCs, and a significant correlation was observed between the methylation status and the stepwise progression of hepatocarcinogenesis (p=0.004). Reduced E-cadherin immunoreactivity was found in 18 of 64 (28%) HCCs, but in none of DNs. E-cadherin methylation status in HCCs was significantly correlated with microvascular invasion (p=0.02) and tumor recurrence (p=0.04), but not with reduced E-cadherin immunoreactivity. The Kaplan-Meier method showed that methylation status did not have a significant influence on the recurrence-free survival of HCC patients (p=0.15). Our results indicate that methylation of the E-cadherin promoter region is a frequent event in HCC, which may play an important role in the stepwise progression of hepatocarcinogenesis. And the promoter methylation of E-cadherin in HCC was found to be significantly correlated with microvascular invasion and recurrence.     

Promoter methylation of TGFbeta receptor I and mutation of TGFbeta receptor II are frequent events in MSI sporadic gastric carcinomas

Transforming growth factor beta (TGFbeta) is a potent inhibitor of cell growth, whose action is transduced through interaction between type I (RI) and type II (RII) receptors. Abnormal expression of these receptors has been identified in several human cancers and was found to be associated with resistance to TGFbeta. TGFbeta RII mutations occur in many types of malignancy. TGFbeta RI hypermethylation has been suggested as a cause of abnormal or absent expression of this receptor in cancer. This study has analysed the methylation status of the promoter region of the TGFbeta RI gene using a methylation-sensitive enzyme followed by polymerase chain reaction (PCR), and TGFbeta RII mutations (BAT-RII and a GT(3)) in order to determine the frequency of alteration of the TGFbeta receptors in a series of 40 sporadic gastric carcinomas (SGCs), 25 of which showed microsatellite instability (MSI) and 15 of which were microsatellite stable (MSS). Methylation in the promoter region of the TGFbeta RI gene was detected in 20 of the 40 (50%) SGCs (64% of the MSI cases and 26.7% of the MSS); 17 of the 40 (42.5%) cases had mutations in the BAT-RII region of the TGFbeta RII gene (68% in the MSI cases; 0% in the MSS). In total, 25 of the 40 (62.5%) SGCs had alterations in at least one of the TGFbeta receptors (84% of the cases in the MSI group, in contrast with 16% of the MSS cases) (p = 0.0003). The clinicopathological features of the cases were also studied and significant associations were found between the presence of alterations in TGFbeta receptors and the age of the patients (p = 0.046), size (p = 0.011), and proliferative rate of the tumours (p = 0.048). In conclusion, alterations in the receptors of TGFbeta (TGFbeta RI promoter hypermethylation and TGFbeta RII mutations) are frequent events in MSI SGC and are associated with large size and high proliferative activity of the tumours, in keeping with loss of the growth inhibitory effects of TGFbeta in this setting.     

Role of PTEN and MMP-7 expression in growth, invasion, metastasis and angiogenesis of gastric carcinoma

To investigate the role of PTEN and matrix metalloproteinase-7 (MMP-7) expression in tumorigenesis and progression of gastric carcinoma, their expression in 113 gastric carcinomas was studied by immunohistochemistry. Microvessel density (MVD) was counted using the anti-CD34 antibody. The expressions of PTEN and MMP-7, and MVD were compared with the clinicopathological parameters of tumors, and the relationship between PTEN and MMP-7 expression and MVD was analyzed. It was found that PTEN was expressed less frequently in primary gastric carcinoma cells than in adjacent epithelial cells (P < 0.05), whereas this was reversed for MMP-7 (P < 0.05). PTEN expression was negatively correlated with invasion, metastasis, growth pattern, Lauren's classification and histological classification (P < 0.05). Matrix metalloproteinase-7 expression was positively associated with tumor size, Borrmann's classification, invasive depth, metastasis and TNM staging (P < 0.05), but negative with PTEN expression (P < 0.05). A positive correlation of MVD with tumor size, invasive depth, metastasis and TNM staging was found (P < 0.05). Microvessel density depended on decreased PTEN expression and increased MMP-7 expression (P < 0.05). The results of the present study suggested that down-regulated PTEN expression and up-regulated MMP-7 expression were greatly implicated in tumorigenesis and progression of gastric carcinoma. Close correlation between PTEN on MMP-7 expression provided a novel insight into the regulatory effects of PTEN on MMP-7 expression in gastric carcinoma.     

Promoter methylation analysis of seven clock genes in Parkinson's disease

The expression of clock genes is altered in leukocytes from patients with Parkinson's disease (PD). However, the underlying mechanisms are unknown. To determine whether abnormal CpG methylation contributes to the dysregulated expression of these genes, the methylation status of the promoters of seven major human clock genes, PER1, PER2, CRY1, CRY2, Clock, NPAS2, and BMAL1, was examined using methylation-specific PCR (MSP) and sequencing in 206 PD patients and 181 healthy controls. This analysis revealed that most clock gene promoters were devoid of methylation. Methylation was only detectable in the CRY1 and NPAS2 promoters. Interestingly, the methylation frequency of the NPAS2 promoter was significantly decreased in PD patients. These results suggest that altered promoter methylation may contribute to the abnormal expression of clock genes in PD.