GPx3 promoter hypermethylation is a frequent event in human cancer and is associated with tumorigenesis and chemotherapy response

Glutathione peroxidase 3 (GPx3), a plasma antioxidant enzyme, maintains genomic integrity by inactivating reactive oxygen species (ROS), known DNA-damaging agents and mediators of cancer chemotherapy response. In this study, we demonstrate that loss of GPx3 expression by promoter hypermethylation is frequently observed in a wide spectrum of human malignancies. Furthermore, GPx3 methylation correlates with head and neck cancer (HNC) chemoresistance and may serve as a potential prognostic indicator for HNC patients treated with cisplatin-based chemotherapy. Our findings support the hypothesis that defects in the antioxidant system may contribute to tumorigenesis of a wide spectrum of human malignancies. GPx3 methylation may have implications in chemotherapy response and clinical outcome of HNC patients.     

Hypermethylation of the CDKN2A gene in colorectal cancer is associated with shorter survival

Promoter hypermethylation of the CDKN2A gene has been suspected to be involved in the carcinogenesis of colorectal cancers. However, the relations between CDKN2A hypermethylation and clinicopathological findings, and patient prognosis, remain inconclusive. We analyzed the CDKN2A methylation status in primary colorectal cancers using real-time methylation specific PCR (MethyLight) which enables quantitative measuring of the methylation level. CDKN2A promoter methylation was detectable in 55 (61.1%) of 90 tumor samples and the median level of the methylation was 0.187 (range 0.00-197.9). The methylation value of CDKN2A was correlated with certain clinicopathological findings, including tumor differentiation (p=0.018), and Dukes stage (p=0.016). In survival analysis, the CDKN2A methylation level was correlated with a poor prognosis of the patient with colorectal cancer for either all cases (p=0.014) or Dukes C (p=0.004) cases that were analyzed. These results suggest that CDKN2A methylation status is a prognostic factor in colorectal cancer and may be of use for the patient specific design of therapy.     

Silencing of estrogen receptor alpha (ERalpha) gene by promoter hypermethylation is a frequent event in Chinese women with sporadic breast cancer

Usually, the function of estrogen receptor alpha (ERα) could be silenced by ERα gene promoter hypermethylation. However, frequency of ERα promoter methylation and the clinicopathological characteristics of ERα methylation in Chinese women with sporadic breast cancer are unknown. The aim of this study was to determine the methylation status of ERα promoter and its possible correlation with clinicopathological features in a series of 138 sporadic breast cancers in Chinese women. ER1, ER3, ER4, and ER5 primers were used for methylation-specific polymerase chain reaction (MSP) to analyze the CpG methylation of promoter region of ERα gene. In general, we found that ERα was methylated in 60.1% (83/138) tumors, including 57 of 69 ERα negative tumors (82.6%, P < 0.00001). Specifically within each region the methylation percentage of ER1, ER3, ER4 and ER5 were 34.8%, 35.5%, 39.1%, and 36.9% respectively. The degree of methylation at four CpG sites was higher in breast cancer compared with benign breast hyperplasia (P < 0.00001). In addition, the levels of ERα protein expression diminished with the frequency of ERα methylation (P < 0.0001, r = −0.469), the probability of methylation was increased for cases with ERα and PgR negativity (P < 0.00001). Our preliminary findings demonstrate, for what we believe to be the first time, that ERα methylation occurs in high frequency and is one of the mechanisms of ERα expression silence in a subset of sporadic breast cancers from Chinese women. Epigenetic alteration of the ERα gene may play an important role in the pathogenesis of breast cancer. L. Zhao and L. Wang have contributed equally to this work.     

Hypermethylation of epithelial-cadherin gene promoter is associated with Epstein-Barr virus in nasopharyngeal carcinoma

Background: Epstein-Barr virus (EBV) is documented as the important etiologic agent of nasopharyngeal carcinoma (NPC) but the mechanism of development and pathogenesis induced by EBV is presently unclear. Hypermethylation of epithelial-cadherin (E-cadherin) promoter has been shown to be induced in NPC cell line by EBV LMP1 via DNA methyltransferase activation. EBV genomes and hypermethylation of E-cadherin promoter were investigated in NPC tissues to evaluate the role of EBV in the hypermethylation and pathogenesis of NPC. Methods: Methylation-specific polymerase chain reaction (MSP) was performed to detect E-cadherin promoter hypermethylation in paraffin embedded tissues from patients with NPC and normal nasopharyngeal tissues. EBV genomes were detected by PCR in the tissue samples. Results: Hypermethylation of E-cadherin promoter and EBV were predominantly detected in undifferentiated and non-keratinizing NPC compared to those in squamous cell NPC. Hypermethylation of E-cadherin was found in 28 of 38 (73.7%) patient samples. EBV was detected in 22 of the 28 (78.6%) NPC samples demonstrating E-cadherin hypermethylation. EBV genomes and hypermethylation were not detected in normal nasopharyngeal tissues. Significant association was found between E-cadherin hypermethylation and EBV genomes (p < 0.001; Fisher's exact test). Hypermethylation of E-cadherin was more frequently detected in advanced stages compared to those in early stages of NPC (p = 0.036; Fisher's exact test). Conclusions: The high incidence of EBV with the consistency of E-cadherin hypermethylation, particularly in undifferentiated and non-keratinizing NPC suggests the role of EBV in the hypermethylation. EBV exists at early stage of NPC that induces the hypermethylation and contributes to progression of the disease to the advanced stage of NPC.     

Hypermethylation of epithelial-cadherin gene promoter is associated with Epstein-Barr virus in nasopharyngeal carcinoma

Background: Epstein-Barr virus (EBV) is documented as the important etiologic agent of nasopharyngeal carcinoma (NPC) but the mechanism of development and pathogenesis induced by EBV is presently unclear. Hypermethylation of epithelial-cadherin (E-cadherin) promoter has been shown to be induced in NPC cell line by EBV LMP1 via DNA methyltransferase activation. EBV genomes and hypermethylation of E-cadherin promoter were investigated in NPC tissues to evaluate the role of EBV in the hypermethylation and pathogenesis of NPC. Methods: Methylation-specific polymerase chain reaction (MSP) was performed to detect E-cadherin promoter hypermethylation in paraffin embedded tissues from patients with NPC and normal nasopharyngeal tissues. EBV genomes were detected by PCR in the tissue samples. Results: Hypermethylation of E-cadherin promoter and EBV were predominantly detected in undifferentiated and non-keratinizing NPC compared to those in squamous cell NPC. Hypermethylation of E-cadherin was found in 28 of 38 (73.7%) patient samples. EBV was detected in 22 of the 28 (78.6%) NPC samples demonstrating E-cadherin hypermethylation. EBV genomes and hypermethylation were not detected in normal nasopharyngeal tissues. Significant association was found between E-cadherin hypermethylation and EBV genomes (p < 0.001; Fisher's exact test). Hypermethylation of E-cadherin was more frequently detected in advanced stages compared to those in early stages of NPC (p = 0.036; Fisher's exact test). Conclusions: The high incidence of EBV with the consistency of E-cadherin hypermethylation, particularly in undifferentiated and non-keratinizing NPC suggests the role of EBV in the hypermethylation. EBV exists at early stage of NPC that induces the hypermethylation and contributes to progression of the disease to the advanced stage of NPC.     

Chromosomal catastrophe is a frequent event in clinically insignificant prostate cancer

Massive genomic rearrangements, a result of single catastrophic event termed chromothrispsis or chromosomal catastrophe, have been identified in a variety of human cancers. In a few cancer types, chromothripsis was found to be associated with poor prognosis. We performed mate-pair sequencing and analysis of structural rearrangements in 132 prostate cancer cases which included clinically insignificant Gleason score 6 tumors, clinically significant tumors of higher grade (7+) and high grade prostatic intraepithelial neoplasia. Chromothripsis was observed at least 30 per cent of the samples across different grades. Surprisingly, it was frequently observed in clinically insignificant Gleason score 6 tumors, indicating that chromothripsis does not define more aggressive phenotype. The degree of chromothripsis did not increase significantly in tumors of advanced grades and did not appear to contribute to tumor progression. Our data showed that distribution of chromothriptic rearrangements differed from that of fragile sites but correlated with the size of chromosomes. We also provided evidence that rearrangements resulting from chromothripsis were present in the cells of neighboring Gleason patterns of the same tumor. Our data suggest that that chromothripsis plays role in prostate cancer initiation.     

Hypermethylation of the nel-like 1 gene is a common and early event and is associated with poor prognosis in early-stage esophageal adenocarcinoma

The nel-like1 (NELL1) gene maps to chromosome 11p15, which frequently undergoes loss of heterozygosity in esophageal adenocarcinoma (EAC). NELL1 promoter hypermethylation was examined by real-time methylation-specific polymerase chain reaction in 259 human esophageal tissues. Hypermethylation of this promoter showed highly discriminative receiver-operator characteristic curve profiles, clearly distinguishing esophageal squamous cell carcinoma (ESCC) and EAC from normal esophagus (NE) (P<0.001). NELL1 normalized methylation values were significantly higher in Barrett's metaplasia (BE), dysplastic Barrett's (D) and EAC than in NE (P<0.0000001). NELL1 hypermethylation frequency was zero in NE but increased early during neoplastic progression, to 41.7% in BE from patients with Barrett's alone, 52.5% in D and 47.8% in EAC. There was a significant correlation between NELL1 hypermethylation and BE segment length. Three (11.5%) of 26 ESCCs exhibited NELL1 hypermethylation. Survival correlated inversely with NELL1 hypermethylation in patients with stages I-II (P=0.0264) but not in stages III-IV (P=0.68) EAC. Treatment of KYSE220 ESCC and BIC EAC cells with 5-aza-2'-deoxycytidine reduced NELL1 methylation and increased NELL1 mRNA expression. NELL1 mRNA levels in EACs with an unmethylated NELL1 promoter were significantly higher than those in EACs with a methylated promoter (P=0.02). Promoter hypermethylation of NELL1 is a common, tissue-specific event in human EAC, occurs early during Barrett's-associated esophageal neoplastic progression, and is a potential biomarker of poor prognosis in early-stage EAC.     

Hypermethylation of the APC (adenomatous Polyposis Coli) gene promoter region in human colorectal carcinoma

Germline mutations of the putative tumor suppressor gene APC are associated in high frequency with the familial adenomatous polyposis, predisposing the patients to colorectal neoplasia. Similarly, sequence analyses have revealed that in more than half of patients with sporadic colorectal carcinoma or adenoma, the APC gene was mutated. By employing genomic sequencing, i.e., base-specific analysis of methylated cytosines, we show here that the promoter region of the APC gene is heavily methylated at CpG sites in patients with colorectal carcinoma in comparison with normal colonic mucosa and premalignant adenomas. Our results suggest that cytosine methylation of the regulatory sequences of the APC gene could be involved in the progression of human colorectal cancer. Int. J. Cancer 70:644–648, 1997. © 1997 Wiley-Liss, Inc.     

Hypermethylation of RASSF1A promoter is associated with the age at starting smoking and a poor prognosis in primary non-small cell lung cancer

Cigarette smoking is the most common cause of lung cancer. The greatest risk of lung cancer is among those who started smoking early in life and continued throughout their lives. However, the molecular mechanisms responsible for the susceptibility to lung cancer in the young smoker are not clear. Recently, several groups have reported that the hypermethylation of CpG islands is associated with exposure to tobacco smoke. We studied the association between the age at starting smoking and hypermethylation of p14, p16, and RASSF1A promoters in 204 primary non-small cell lung cancer patients. We also examined whether hypermethylation of the RASSF1A promoter is an independent prognostic factor. Methylation rates in the 204 samples were detected in 9% for p14, 27% for p16, and 32% for RASSF1A. There was no relationship between the hypermethylation of p14 and p16 and the age at starting smoking. However, hypermethylation of the RASSF1A promoter was found to be significantly associated with the age at starting smoking (P = 0.001). No relationship was found between the methylation status of the RASSF1A promoter and other smoking variables, such as pack-years, smoking status, and the duration of smoking. The age at starting smoking in patients with hypermethylation of RASSF1A was earlier than that of patients without hypermethylation of the RASSF1A promoter (19 +/- 8 versus 25 +/- 7; P = 0.001). Young smokers who started smoking before age 19 were 4.23 times [95% confidence interval (CI) = 1.03-9.67; P = 0.001] more likely to have hypermethylation of the RASSF1A promoter than smokers who started smoking after the age of 19. Furthermore, hypermethylation of the RASSF1A promoter was found to be associated with a poor prognosis in non-small cell lung cancer patients at stages 1 and 2 (P = 0.02 and 0.01, respectively; Log-rank test). The hazard of failure was approximately 3.27 times higher for patients with hypermethylation of the RASSF1A promoter than for those without hypermethylation of the RASSF1A promoter (95% CI = 1.42-8.71; P = 0.01). Young smokers who started the habit before the age of 19 also had a poorer prognosis than those who started after the age of 19 (hazard ratio = 2.14, 95% CI = 1.22-9.11; P = 0.02). Our results suggest that starting cigarette smoking at an early age is associated with hypermethylation of the RASSF1A promoter and that hypermethylation of the RASSF1A promoter may be an independent prognostic factor in primary non-small cell lung cancer.     

Thymidylate synthase gene promoter polymorphisms are associated with TSmRNA expressions but not with microsatellite instability in colorectal cancer

BACKGROUND: Microsatellite instability (MSI) is a biological characteristic of most tumours, being involved in 85% of hereditary non-polyposis colorectal cancer (HNPCC). It also occurs in 10-15% of sporadic colorectal cancers (CRC). HNPCC appears to be caused by germline mutations in mismatch repair (MMR) genes, which are responsible for repairing single base-pair mismatches. MSI is also associated with a better response of CRC to adjuvant chemotherapy with fluoropyrimidines. We investigated any relationship between the MSI status and the TSmRNA expression, the polymorphisms of 5-Fluorouracil (5-FU cellular target, the enzyme thymidylate synthase (TS) and TS expression evaluated by means of immunohistochemistry. MATERIALS AND METHODS: A series of 80 colorectal cancers was evaluated for MSI and polymorphisms in the 3'UTR and the 5'UTR of the TS gene by a PCR assay. TSmRNA was quantified by real-time PCR and the TS expression by immunohistochemical assay. RESULTS: There was no significant association between the polymorphisms in the TS gene and the MSI or between the TSmRNA expression and the MSI status. CRC with a 3R/3R or 2R/3R genotype showed a significantly higher TSmRNA expression than those with the 2R/2R genotype (p = 0.001 and p = 0.028, respectively). Another significant association was found between the TSmRNA expression and the TS immunohistochemical determination (p = < 0.05). No association was found between the polymorphism of the 3'UTR and the TSmRNA expression. CONCLUSION: Our data show that there is no association between MSI status and the polymorphisms in the 3' and 5' UTRs and the TS expression. Tumour samples displaying the 3R/3R or 2R/3R genotype of TS have higher TSmRNA levels than the 2R/2R genotype. Polymorphic variant of the 3'UTR does not influence the TSmRNA level. We found a relationship between the TSmRNA expression, evaluated by real-time PCR, and with the TS level determined by immunohistochemical assay. Thus, genotyping of the 5'UTR and quantification of the TSmRNA expression in human CRC could be considered as predictors for response to SFU-based chemotherapy. The evaluation of the TS expression by means of immunohistochemistry assay remains a safe and reliable assay in CRC.     

Loss of MSH3 protein expression is frequent in MLH1-deficient colorectal cancer and is associated with disease progression

Mononucleotide repeat sequences are particularly prone to frameshift mutations in tumors with biallelic inactivation of the mismatch repair (MMR) genes MLH1 or MSH2. In these tumors, several genes harboring mononucleotide repeats in their coding region have been proposed as targets involved in tumor progression, among which are also the MMR genes MSH3 and MSH6. We have analyzed the expression of the MSH3 and MSH6 proteins by immunohistochemistry in 31 colorectal carcinomas in which MLH1 was inactivated. Loss of MSH3 expression was identified in 15 tumors (48.5%), whereas all tumors expressed MSH6. Frameshift mutations at coding microsatellites were more frequent in MSH3 (16 of 31) than in MSH6 (3 of 31; Fisher's exact test, P < 0.001). Frameshift mutations and allelic losses of MSH3 were more frequent in MSH3-negative tumors compared with those with normal expression (22 mutations in 30 alleles versus 8 mutations in 28 alleles; chi(2), P = 0.001). Biallelic inactivation was evident or inferred for 60% of MSH3-negative tumors but none of the tumors with normal MSH3 expression. In contrast, we did not identify frameshift mutations in the (A)8 tract of MSH3 in a control group of 18 colorectal carcinomas in which the MMR deficiency was based on the inactivation of MSH2. As it has been suggested that mutations of MSH3 might play a role in tumor progression, we studied the association between MSH3 expression and disease stage assessed by lymph node and distant metastases status. Dukes stages C and D were more frequent in primary tumors with loss of MSH3 expression (9 of 13), compared with tumors with retained expression (1 of 14; Fisher's exact test, P = 0.001), suggesting that MSH3 abrogation may be a predictor of metastatic disease or even favor tumor cell spread in MLH1-deficient colorectal cancers.     

Methylation of homeobox genes is a frequent and early epigenetic event in breast cancer

Introduction  

Aberrant methylation of CpG islands is a hallmark of cancer and occurs at an early stage in breast tumorigenesis. However, its impact on tumor development is not fully determined, and its potential as a diagnostic biomarker remains to be validated. Methylation profiling of invasive breast carcinoma has been largely explored. Conversely, very little and sparse information is available on early-stage breast cancer. To gain insight into the epigenetic switches that may promote and/or contribute to the initial neoplastic events during breast carcinogenesis, we have analyzed the DNA methylation profile of ductal carcinoma in situ, a premalignant breast lesion with a great potential to progress toward invasive carcinoma.     

Hypermethylation of 14-3-3 sigma (stratifin) is an early event in breast cancer.

We have identified 14-3-3 sigma (sigma) as a gene whose expression is lost in breast carcinomas, primarily by methylation-mediated silencing. In this report, we investigated the timing of loss of sigma gene expression during breast tumorigenesis in vivo. We analysed the methylation status of sigma in breast cancer precursor lesions using microdissection for selective tissue sampling. We found hypermethylation of sigma in 24 of 25 carcinomas (96%), 15 of 18 (83%) of ductal carcinoma in situ, and three of eight (38%) of atypical hyperplasias. None of the five hyperplasias without atypia showed sigma-hypermethylation. Unexpectedly, patients with breast cancer showed sigma hypermethylation in adjacent histologically normal breast epithelium, while this was never observed in individuals without evidence of breast cancer. Also, samples of periductal stromal breast tissue were consistently hypermethylated, underscoring the importance of selective tissue sampling for accurate assessment of 14-3-3-sigma methylation in breast epithelium. These results suggest that hypermethylation of 14-3-3-sigma occurs at an early stage in the progression to invasive breast cancer, and may occur in apparently normal epithelium adjacent to breast cancer. These results provide evidence that loss of expression of sigma is an early event in neoplastic transformation.