sodium-bisulfite and Breast-Neoplasms

Over the last two decades, survival rates from women's cancers (breast, ovarian, endometrial and cervical cancer) have all but modestly improved despite huge efforts from both research and clinical communities. In parallel with this, the field of epigenetics has grown from its infancy into a promising scientific discipline. In particular, DNA methylation analysis has been adopted by oncologists in an attempt to better understand and manage cancer. Now that the epigenetic technological base has caught up, the potential of methylation markers in cancer research is finally being realized. In this review, we present the current status of epigenetic research into women's cancers with a main focus on DNA methylation analysis. We provide an overview of technological development, current markers of risk prediction, early detection, diagnosis, prognosis and response to treatment, and highlight the progression of epigenetic therapies. Finally, we comment on the potential impact of epigenetic analyses on the future of women's health.

Topics: Biomarkers, Tumor; Breast Neoplasms; DNA Methylation; Epigenesis, Genetic; Female; Genital Neoplasms, Female; Gonadal Steroid Hormones; Histone Deacetylase Inhibitors; Humans; Immunoprecipitation; Models, Biological; Mullerian Ducts; Restriction Mapping; Sulfites

TREX (transcription/export) is a multiprotein complex that plays a key role in the transcriptional elongation and transport of mRNA from the nucleus to the cytoplasm. We previously reported the purification of the human TREX protein and found that expression of a member of this complex, p84N5 (referred to as hTREX84 or hHPR1), a RB binding protein, correlated with breast tumor size and metastasis. Here we examine the mechanisms of aberrant expression of hTREX84 in breast and ovarian cancer cells and evaluate its role in tumorigenesis. We show that ovarian tumor cells over-express hTREX84 4-fold and 10-fold compared to immortal, non-tumorigenic and primary ovarian surface epithelial cells, respectively. Reduction of hTREX84 levels by small interfering RNA result in inhibition of cellular proliferation and G(2/M) arrest. Even though we observed that hTREX84 expression was induced by treatment with a demethylation agent, 5-aza-2'-deoxycytidine (5-aza-dC), sodium bisulfite DNA sequencing and methylation specific PCR found no evidence of changes in DNA methylation in the CpG islands in the regulator region of hTREX84. We subsequently identify several transcriptional factors, including NF-κB binding sites in the hTREX84 gene promoter and demonstrate by chromatin immunoprecipation (ChIP) and site directed mutagenesis that RelA/p65 binds the NF-kB binding sites and induces hTREX84 expression. Finally, we show by immunohistochemistry (IHC) that RelA/p65 is abundantly expressed in malignant cells that aberrantly express hTREX84 indicating that RelA/p65 might play a pivotal role in regulating hTREX84 expression in cancer. Our results indicate that overexpression of hTREX84 is associated with cancer cell transformation, proliferation and may be regulated by RelA/p65.

Topics: Azacitidine; Base Sequence; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Transformation, Neoplastic; Decitabine; DNA-Binding Proteins; Exons; Female; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Nuclear Proteins; Ovarian Neoplasms; Promoter Regions, Genetic; RNA-Binding Proteins; Sequence Analysis, DNA; Sulfites; Transcription Factor RelA; Transcription, Genetic

Epigenetic alterations have emerged as a key mechanism involved in tumorigenesis. These disruptions are partly due to environmental factors that change normal DNA methylation patterns necessary for transcriptional regulation and chromatin compaction. Microarray technologies are allowing environmentally susceptible epigenetic patterns to be mapped and the precise targets of environmentally induced alterations to be identified. Previously, we observed BaP-induced epigenetic events and cell cycle disruptions in breast cancer cell lines that included time- and concentration-dependent loss of proliferation as well as sequence-specific hypo- and hypermethylation events. In this present report, we further characterized epigenetic changes in BaP-exposed MCF-7 cells. We analyzed DNA methylation on a CpG island microarray platform with over 5400 unique genomic regions. Depleted and enriched microarray targets, representative of putative DNA methylation changes, were identified across the genome; however, subsequent sodium bisulfite analyses revealed no changes in DNA methylation at a number of these loci. Instead, we found that the identification of DNA methylation changes using this restriction enzyme-based microarray approach corresponded with the regions of DNA bound by the BaP derived DNA adducts. This DNA adduct formation occurs at both methylated and unmethylated CpG dinucleotides and affects PCR amplification during sample preparation. Our data suggest that caution should be exercised when interpreting data from comparative microarray experiments that rely on enzymatic reactions. These results are relevant to genome screening approaches involving environmental exposures in which DNA adduct formation at specific nucleotide sites may bias target acquisition and compromise the correct identification of epigenetically responsive genes.

Topics: Benzopyrenes; Breast Neoplasms; Cell Line, Tumor; CpG Islands; DNA Adducts; DNA Methylation; Epigenesis, Genetic; Female; Genome, Human; Humans; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Sulfites

Aberrant methylation of promoter CpG islands is causally linked with a number of inherited syndromes and most sporadic cancers, and may provide valuable diagnostic and prognostic biomarkers. In this report, we describe an approach to simultaneous analysis of multiple CpG islands, where methylation-specific oligonucleotide probes are joined by ligation and subsequently amplified by polymerase chain reaction (PCR) when hybridized in juxtaposition on bisulfite-treated DNA. Specificity of the ligation reaction is achieved by (i) using probes containing CpGpCpG (for methylated sequences) or CpApCpA (for unmethylated sequences) at the 3' ends, (ii) including three or more probes for each target, and (iii) using a thermostable DNA ligase. The external probes carry universal tails to allow amplification of multiple ligation products using a common primer pair. As proof-of-principle applications, we established duplex assays to examine the FMR1 promoter in individuals with fragile-X syndrome and the SNRPN promoter in individuals with Prader-Willi syndrome or Angelman syndrome, and a multiplex assay to simultaneously detect hypermethylation of seven genes (ID4, APC, RASSF1A, CDH1, ESR1, HIN1 and TWIST1) in breast cancer cell lines and tissues. These data show that ligation of oligonucleotide probes hybridized to bisulfite-treated DNA is a simple and cost-effective approach to analysis of CpG methylation.

Topics: Angelman Syndrome; Autoantigens; Breast Neoplasms; Cell Line, Tumor; CpG Islands; DNA; DNA Ligases; DNA Methylation; Female; Fragile X Mental Retardation Protein; Fragile X Syndrome; Humans; Male; Oligonucleotide Probes; Polymerase Chain Reaction; Prader-Willi Syndrome; Promoter Regions, Genetic; Ribonucleoproteins, Small Nuclear; snRNP Core Proteins; Sulfites

Aberrant methylation of promoter CpG islands is known to be responsible for the alteration and silencing of genes in cancer. The data presented here describes that the CpG methylations of ERalpha and ERbeta are found in breast cancer tissues, and methylation exerts a considerable effect on gene silencing, investigated by bisulfite genomic sequencing and reverse transcriptase polymerase chain reaction. Consequently, hypermethylation of the ERalpha and ERbeta genes was found in 66.0% and 50.0% of 50 breast cancers, respectively. Eleven of 50 samples (22.0%) did not show any methylation of either ERalpha or ERbeta, whereas 19 samples (38.0%) showed methylation of both ERalpha and ERbeta. The tumors that showed aberrant methylation of ERalpha and ERbeta did not express mRNA, compared with unmethylated cases (p < 0.01). The methylated case was negatively correlated with the expression of the ERalpha protein (p < 0.01). In addition, ERbeta methylation demonstrated significant associations with the lower level of Ki67 (9.36 +/- 2.43 versus 19.68 +/- 3.42, p = 0.02). Although the number of samples was relatively small, our results suggest that DNA methylation in ERalpha and ERbeta are present in breast cancer tissue, and that the methylation causes a significant effect on transcriptional silencing. Furthermore, the CpG methylation of the ERbeta gene seems to play a role in cell proliferation of breast cancer tissue.

Topics: Aged; Base Sequence; Breast Neoplasms; Cell Line, Tumor; CpG Islands; DNA Methylation; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Gene Silencing; Humans; Immunohistochemistry; Ki-67 Antigen; Middle Aged; Molecular Sequence Data; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulfites

A number of different genes are known to be inactivated by aberrant hypermethylation in breast cancer, but it is still unknown to what extent these epigenetic alterations differ according to specific breast cancer phenotypes. We sought to determine whether the extent of hypermethylation or defined profiles of gene hypermethylation are associated with biological characteristics of breast cancers.. We evaluated methylation status of 12 different genes in a series of 109 invasive breast tumors, representing the ductal, lobular, and mucinous histologic subtypes using methylation-specific PCR. Frequencies of methylation were compared across the recognized histologic classes, and multivariate techniques (latent class analysis, factor analysis, recursive partitioning, and hierarchical clustering) were used to seek patterns of methylation for individual genes that distinguish recognized histologic types of breast cancer or define breast cancer phenotypes on a molecular level.. All 109 cases studied have aberrant methylation of multiple genes (3 to 10 genes per case), demonstrating that gene hypermethylation is pervasive in breast cancer. Lobular cancers and mucinous cancers, which often have relatively low levels of chromosomal changes, have higher overall frequencies of hypermethylation than ductal cancers (49% in lobular and mucinous versus 40% in ductal), but there is a relatively unimodal distribution of methylation frequency for all three histologic types. Only one of the individual genes studied, BRCA1, has a variable frequency of methylation that is significantly dependent on histologic pattern of tumor growth, with a higher frequency of methylation in mucinous cancers than ductal or lobular cancers. Although some trends of histology-specific gene methylation were seen, methylation patterns could not definitively classify breast cancers according to histologic type.. Although a more comprehensive hypermethylation profile could potentially be useful for breast cancer classification and understanding the biology of this disease, it appears that the hypermethylation patterns across various forms of breast cancer are less distinct than those between breast cancer and cancers of different tissue origins. Furthermore, the relatively unimodal distribution of methylation frequency for all three histologic types does not support there being a distinct CpG island methylator phenotype for breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; CpG Islands; DNA; DNA Methylation; Female; Genes, BRCA1; Humans; Lymphatic Metastasis; Male; Middle Aged; Multivariate Analysis; Odds Ratio; Phenotype; Phylogeny; Polymerase Chain Reaction; Sulfites

Fine needle aspiration (FNA) is used widely in diagnostic assessment of breast lesions. However, cytomorphological evaluation depends heavily on the proficiency of cytopathologists. Because epigenetic alterations are frequent and specific enough to potentially augment the accuracy of malignant disease detection, we tested whether hypermethylation analysis of a panel of genes would distinguish benign from malignant breast FNA washings.. FNA washings were collected from 123 female patients harboring suspicious mammary lesions. Sodium bisulfite-modified DNA was amplified by methyl-specific PCR (MSP) for CDH1, GSTP1, BRCA1, and RARbeta to detect gene promoter CpG island methylation. Paired samples of 27 breast cancer tissue and 7 fibroadenomas and 12 samples of normal breast tissue, collected postoperatively, were also analyzed. MSP results were compared with conventional cytomorphological diagnosis.. FNAs were cytomorphologically diagnosed as benign (25 cases), malignant (76 cases), suspicious for malignancy (6 cases), and unsatisfactory (16 cases). Percentages of methylated CDH1, GSTP1, BRCA1, and RARbeta in FNA washings were 60, 52, 32, and 16%, and 65.8, 57.9, 39.5, and 34.2% for benign and malignant lesions, respectively. These differences did not reach statistical significance. In all of the paired benign lesions tested, there was absolute concordance. Sixty-seven percent (18 of 27) of FNA washings displayed hypermethylation patterns identical to malignant paired tissue. No methylation was found in the normal breast samples for any of the genes.. Detection of gene hypermethylation in FNA washings by MSP analysis is feasible, but the selected gene panel does not discriminate between benign and malignant breast lesions.

Topics: Biopsy, Fine-Needle; Breast; Breast Neoplasms; CpG Islands; DNA Methylation; Female; Gene Silencing; Genes, BRCA1; Humans; Promoter Regions, Genetic; Receptors, Retinoic Acid; Sulfites

We have previously shown that the distal promoter (promoter B) of the estrogen receptor alpha (ER alpha) gene is responsible for the enhanced expression of the ER alpha gene seen in human breast cancer and that a novel trans-acting factor, estrogen receptor promoter B associated factor 1 (ERBF-1), is required for transcription from promoter B in breast cancer cells. In development of breast cancer, loss of ER alpha gene expression is one of the most important steps in acquiring hormone resistance, though the mechanisms are poorly understood. Recent studies have reported that methylation of the ER alpha gene promoter A and exon 1 was inversely associated with ER alpha gene expression in human breast cancer and cell lines. The methylation status of the promoter B region, which is responsible for overexpression of ER alpha protein in cancer tissue, has not been investigated. In this report, we found that the methylation status of promoter B, as well as that of promoter A, was inversely associated with ER alpha gene expression in human breast cancer and cell lines. Specific methylation of ER alpha gene promoters in vitro directly decreased transcription of the ER alpha gene in a reporter assay. Demethylating treatment induced transcription of ER alpha mRNA from promoter B in ZR-75-1 cells, which showed no transcription from promoter B, despite weak ERBF-1 expression, but not in ER alpha-negative MDA-MB-231 and BT-20 cells, which lack ERBF-1. ZR-75-1 cells showed promoter activity equal to that of MCF-7 cells in a reporter assay. Our results indicate that methylation of promoter B of the ER alpha gene is important for loss of ER alpha gene expression in human breast cancer, and methylation of the promoters can directly modulate ER alpha gene expression. However, loss of critical transcriptional factors such as ERBF-1 may also be involved in some ER alpha-negative cases.

Topics: Base Sequence; Breast Neoplasms; DNA Methylation; Estrogen Receptor alpha; Exons; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Introns; Luciferases; Molecular Sequence Data; Promoter Regions, Genetic; Receptors, Estrogen; Restriction Mapping; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulfites; Trans-Activators; Transcription, Genetic; Tumor Cells, Cultured