Methylation status of oestrogen receptor-α gene promoter sequences in human ovarian epithelial cell lines

We have determined the methylation status of the CpG island of the oestrogen receptor α gene in seven human ovarian cell lines. Cell lines expressing oestrogen receptor α showed no evidence of hypermethylation. In three of four cell lines that produced no detectable oestrogen receptor α protein, hypermethylation was observed at the NotI site of the CpG island. These results indicate that aberrant hypermethylation may be responsible for a significant proportion of epithelial ovarian tumours in which oestrogen receptor α expression is lost

Reverting estrogen-receptor-negative phenotype in HER-2-overexpressing advanced breast cancer patients exposed to trastuzumab plus chemotherapy

INTRODUCTION: The amounts of estrogen receptor (ER) and progesterone receptor (PgR) in a primary tumor are predictive of the response to endocrine therapies of breast cancer. Several patients with ER-positive primary tumors relapse after adjuvant endocrine therapy with no ER expression in the recurrent tissue; much fewer with a recurrent disease after an ER-negative primary tumor may become endocrine responsive. These sequences of events indicate that a phenotype based on ER expression may not be a permanent feature of breast cancer. METHODS: Ten patients with advanced breast cancer whose tumors overexpressed HER-2, but not ER or PgR, were treated with weekly trastuzumab at standard doses with or without chemotherapy. RESULTS: Three out of 10 patients showed overexpression of ERs first appearing after 9, 12 and 37 weeks, respectively, from the initiation of trastuzumab. Two of these patients were subsequently treated with endocrine therapy alone: one of them received letrozole for 3 years without evidence of progression. CONCLUSION: Therapeutic targets enabling the appearance of an endocrine responsive disease may increase treatment options for patients with breast cancer. Furthermore, these clinical data suggest that an ER-negative phenotype is a multi-step process with a reversible repression modality, and that some ER-negative tumors may either revert to an ER-positive phenotype, allowing an endocrine treatment to be effective

Ribavirin restores ESR1 gene expression and tamoxifen sensitivity in ESR1 negative breast cancer cell lines

Tumor growth is estrogen independent in approximately one-third of all breast cancers, which makes these patients unresponsive to hormonal treatment. This unresponsiveness to hormonal treatment may be explained through the absence of the estrogen receptor alpha (ESR1). The ESR1 gene re-expression through epigenetic modulators such as DNA methyltransferase inhibitors and/or histone deacetylase inhibitors restores tamoxifen sensitivity in ESR1 negative breast cancer cell lines and opens new treatment horizons in patients who were previously associated with a poor prognosis

An empirical Bayes model for gene expression and methylation profiles in antiestrogen resistant breast cancer

<p>Abstract</p> <p>Background</p> <p>The nuclear transcription factor estrogen receptor alpha (ER-alpha) is the target of several antiestrogen therapeutic agents for breast cancer. However, many ER-alpha positive patients do not respond to these treatments from the beginning, or stop responding after being treated for a period of time. Because of the association of gene transcription alteration and drug resistance and the emerging evidence on the role of DNA methylation on transcription regulation, understanding of these relationships can facilitate development of approaches to re-sensitize breast cancer cells to treatment by restoring DNA methylation patterns.</p> <p>Methods</p> <p>We constructed a hierarchical empirical Bayes model to investigate the simultaneous change of gene expression and promoter DNA methylation profiles among wild type (WT) and OHT/ICI resistant MCF7 breast cancer cell lines.</p> <p>Results</p> <p>We found that compared with the WT cell lines, almost all of the genes in OHT or ICI resistant cell lines either do not show methylation change or hypomethylated. Moreover, the correlations between gene expression and methylation are quite heterogeneous across genes, suggesting the involvement of other factors in regulating transcription. Analysis of our results in combination with H3K4me2 data on OHT resistant cell lines suggests a clear interplay between DNA methylation and H3K4me2 in the regulation of gene expression. For hypomethylated genes with alteration of gene expression, most (~80%) are up-regulated, consistent with current view on the relationship between promoter methylation and gene expression.</p> <p>Conclusions</p> <p>We developed an empirical Bayes model to study the association between DNA methylation in the promoter region and gene expression. Our approach generates both global (across all genes) and local (individual gene) views of the interplay. It provides important insight on future effort to develop therapeutic agent to re-sensitize breast cancer cells to treatment.</p

Chemically induced DNA hypomethylation in breast carcinoma cells detected by the amplification of intermethylated sites

INTRODUCTION: Compromised patterns of gene expression result in genomic instability, altered patterns of gene expression and tumour formation. Specifically, aberrant DNA hypermethylation in gene promoter regions leads to gene silencing, whereas global hypomethylation events can result in chromosomal instability and oncogene activation. Potential links exist between environmental agents and DNA methylation, but the destabilizing effects of environmental exposures on the DNA methylation machinery are not understood within the context of breast cancer aetiology. METHODS: We assessed genome-wide changes in methylation patterns using a unique methylation profiling technique called amplification of intermethylated sites (AIMS). This method generates easily readable fingerprints that represent the investigated cell line's methylation profile, based on the differential cleavage of DNA with methylation-specific isoschisomeric restriction endonucleases. RESULTS: We validated this approach by demonstrating both unique and reoccurring sites of genomic hypomethylation in four breast carcinoma cell lines treated with the cytosine analogue 5-azacytidine. Comparison of treated with control samples revealed individual bands that exhibited methylation changes, and these bands were excized and cloned, and the precise genomic location individually identified. In most cases, these regions of hypomethylation coincided with susceptible target regions previously associated with chromosome breakage, rearrangement and gene amplification. Similarly, we observed that acute benzopyrene exposure is associated with altered methylation patterns in these cell lines. CONCLUSION: These results reinforce the link between environmental exposures, DNA methylation and breast cancer, and support a role for AIMS as a rapid, affordable screening method to identify environmentally induced DNA methylation changes that occur in tumourigenesis

Genotypes and haplotypes of the methyl-CpG-binding domain 2 modify breast cancer risk dependent upon menopausal status

INTRODUCTION: MBD2, the gene encoding methyl-CpG-binding domain (MBD)2, is a major methylation related gene and functions as a transcriptional repressor that can specifically bind to the methylated regions of other genes. MBD2 may also mediate gene activation because of its potential DNA demethylase activity. The present case-control study investigated associations between two single nucleotide polymorphisms (SNPs) in the MBD2 gene and breast cancer risk. METHODS: DNA samples from 393 Caucasian patients with breast cancer (cases) and 436 matched control individuals, collected in a recently completed breast cancer case–control study conducted in Connecticut, were included in the study. Because no coding SNPs were found in the MBD2 gene, one SNP in the noncoding exon (rs1259938) and another in the intron 3 (rs609791) were genotyped. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to estimate cancer risk associated with the variant genotypes and the reconstructed haplotypes. RESULTS: The variant genotypes at both SNP loci were significantly associated with reduced risk among premenopausal women (OR = 0.41 for rs1259938; OR = 0.54 for rs609791). Further haplotype analyses showed that the two rare haplotypes (A-C and A-G) were significantly associated with reduced breast cancer risk (OR = 0.40, 95% CI = 0.20–0.83 for A-C; OR = 0.47, 95% CI = 0.26–0.84 for A-G) in premenopausal women. No significant associations were detected in the postmenopausal women and the whole population. CONCLUSION: Our results demonstrate a role for the MBD2 gene in breast carcinogenesis in premenopausal women. These findings suggest that genetic variations in methylation related genes may potentially serve as a biomarker in risk estimates for breast cancer

The promoter and the enhancer region of the KLK 3 (prostate specific antigen) gene is frequently mutated in breast tumours and in breast carcinoma cell lines

KLK3 or prostate specific antigen (PSA) is a serine protease, which is an established tumour marker of prostatic adenocarcinoma. PSA is now used widely for the diagnosis and monitoring of patients with prostate cancer. Recent studies have demonstrated that about 70% of breast cancers produce PSA. In this study, we examined the molecular mechanism underlying the expression of the PSA gene in breast cancer and breast cancer cell lines. We analysed nine breast tumours categorized on the basis of high- or low-PSA expression in tumour cytosols and four breast cancer cell lines. To determine abnormalities associated with PSA expression in breast tumours, genomic DNA was extracted and all five exons of the PSA gene were polymerase chain reaction (PCR) amplified and sequenced on both strands. PCR amplification was also performed for the promoter and enhancer elements of the PSA gene. No mutations were observed in the coding portion of the gene. A polymorphism was observed in exon 2 from three breast tumours. However, sequencing of the promoter and the enhancer elements of the PSA gene reveals several point mutations. Within a 5.8-kb promoter/enhancer region of the PSA gene, we detected 16 different mutational hotspots (appearing more than once in the nine tumours). Among these hotspots, two appeared in seven out of nine tumours. Most importantly, the androgen response element (ARE I) in the proximal promoter was found mutated in four tumours and in the breast carcinoma cell line MCF-7. Mutations associated with the ARE I have been shown previously to result in an 80% decrease in PSA gene expression. The mutations in the core enhancer and promoter region probably contribute to the aberrant expression of the PSA gene in breast tumours, possibly by altering the regulation of the gene by steroid hormones. © 1999 Cancer Research Campaig

ESR1 gene promoter region methylation in free circulating DNA and its correlation with estrogen receptor protein expression in tumor tissue in breast cancer patients

[Background] Tumor expression of estrogen receptor (ER) is an important marker of prognosis, and is predictive of response to endocrine therapy in breast cancer. Several studies have observed that epigenetic events, such methylation of cytosines and deacetylation of histones, are involved in the complex mechanisms that regulate promoter transcription. However, the exact interplay of these factors in transcription activity is not well understood. In this study, we explored the relationship between ER expression status in tumor tissue samples and the methylation of the 5′ CpG promoter region of the estrogen receptor gene (ESR1) isolated from free circulating DNA (fcDNA) in plasma samples from breast cancer patients. [Methods] Patients (n = 110) with non-metastatic breast cancer had analyses performed of ER expression (luminal phenotype in tumor tissue, by immunohistochemistry method), and the ESR1-DNA methylation status (fcDNA in plasma, by quantitative methylation specific PCR technique). [Results] Our results showed a significant association between presence of methylated ESR1 in patients with breast cancer and ER negative status in the tumor tissue (p = 0.0179). There was a trend towards a higher probability of ESR1-methylation in those phenotypes with poor prognosis i.e. 80% of triple negative patients, 60% of HER2 patients, compared to 28% and 5.9% of patients with better prognosis such as luminal A and luminal B, respectively. [Conclusion] Silencing, by methylation, of the promoter region of the ESR1 affects the expression of the estrogen receptor protein in tumors of breast cancer patients; high methylation of ESR1-DNA is associated with estrogen receptor negative status which, in turn, may be implicated in the patient’s resistance to hormonal treatment in breast cancer. As such, epigenetic markers in plasma may be of interest as new targets for anticancer therapy, especially with respect to endocrine treatment.The study was funded, in part, by a grant from the Ministerio de Educación y Ciencia (CICYT: SAF 2004–00889)

Gene promoter hypermethylation in ductal lavage fluid from healthy BRCA gene mutation carriers and mutation-negative controls

INTRODUCTION: Female germline BRCA gene mutation carriers are at increased risk for developing breast cancer. The purpose of our study was to establish whether healthy BRCA mutation carriers demonstrate an increased frequency of aberrant gene promoter hypermethylation in ductal lavage (DL) fluid, compared with predictive genetic test negative controls, that might serve as a surrogate marker of BRCA1/2 mutation status and/or breast cancer risk. METHODS: The pattern of CpG island hypermethylation within the promoter region of a panel of four genes (RAR-β, HIN-1, Twist and Cyclin D2) was assessed by methylation-specific polymerase chain reaction using free DNA extracted from DL fluid. RESULTS: Fifty-one DL samples from 24 healthy women of known BRCA mutation status (7 BRCA1 mutation carriers, 12 BRCA2 mutation carriers and 5 controls) were available for methylation analysis. Eight of 19 (42.1%) BRCA mutation carriers were found to have at least one hypermethylated gene in the four-gene panel. Two BRCA mutation carriers, in whom aberrant methylation was found, also had duct epithelial cell atypia identified. No hypermethylation was found in DL samples from 5 negative controls(p = 0.13). CONCLUSION: We found substantial levels of aberrant methylation, with the use of a four-gene panel, in the fluid from the breasts of healthy BRCA mutation carriers compared with controls. Methylation analysis of free DNA in DL fluid may offer a useful surrogate marker for BRCA1/2 mutation status and/or breast cancer risk. Further studies are required for the evaluation of the specificity and predictive value of aberrant methylation in DL fluid for future breast cancer development in BRCA1/2 mutation carriers