Aberrant DNA Methylation Changes Research Articles

Abstract 2213: Aberrant expression of TRIM24 in breast cancer progression is linked to loss of repressive histone marks at the promoter

Abstract We recently discovered that TRIM24 is an E3-ubiquitin ligase that targets p53 for degradation in embryonic stem cells and breast cancer cell lines. TRIM24 functions in human cells as a reader of dual histone marks and binds chromatin and estrogen receptor (ER) to activate estrogen-dependent genes associated with cellular proliferation and tumor development. Intriguingly, we and others have shown that over-expression of TRIM24 is associated with poor prognosis and worse survival in breast cancer patients. Whether TRIM24 is causal in malignant transformation of breast epithelial cells during breast tumor development and progression is not known. Furthermore the mechanism that drives deregulated expression of TRIM24 in breast cancer is not understood. To develop a timeline of TRIM24 deregulation during malignant transformation, we used an isogenic human mammary epithelial cell (HMEC) model system that allows for assessment of molecular changes that occur at the earliest stages of multistep human breast carcinogenesis. Our analysis of TRIM24 expression in HMECs transitioning from finite lifespan, to immortality and to malignantly transformed HMECs showed gradual up-regulation of TRIM24 at both RNA and protein levels early in the transformation process. Consistent with these findings, Immunohistochemical (IHC) staining of TRIM24 in clinical breast tumor samples from a breast cancer progression array showed increased TRIM24 expression in hyperplasia, ductal carcinoma in situ (DCIS) and invasive carcinoma, compared to normal breast tissues. To understand the mechanism of TRIM24 deregulation in breast tumor progression, we studied the TRIM24 promoter region for aberrant DNA methylation changes by bisulfite genomic sequencing. There were no aberrant DNA methylation changes in HMEC transitioning from finite lifespan to malignant transformation suggesting that TRIM24 gene expression is regulated by mechanisms other than DNA methylation. Interestingly, Chromatin Immunoprecipitation (ChIP) studies in finite lifespan HMECs at the TRIM24 promoter showed an enrichment of repressive histone marks H3K9me2 and H3K9me3 which gradually decreased in immortal and malignantly transformed mammary epithelial cells. Further studies to see if TRIM24 is involved in transformation of normal mammary epithelial cells by stably overexpressing TRIM24 in HMECs are currently ongoing. Also more studies are ongoing to define additional levels of regulation of TRIM24, and which transcription factors are involved in its deregulated expression in breast cancer. Our studies suggest a unique role of TRIM24 in early steps of mammary carcinogenesis. Therefore, our study may provide valuable insights into the rational development of TRIM24-targeted therapeutic strategies against breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2213. doi:1538-7445.AM2012-2213

A Novel Pit Pattern Identifies the Precursor of Colorectal Cancer Derived From Sessile Serrated Adenoma

Sessile serrated adenomas (SSAs) are known to be precursors of sporadic colorectal cancers (CRCs) with microsatellite instability (MSI), and to be tightly associated with BRAF mutation and the CpG island methylator phenotype (CIMP). Consequently, colonoscopic identification of SSAs has important implications for preventing CRCs, but accurate endoscopic diagnosis is often difficult. Our aim was to clarify which endoscopic findings are specific to SSAs. The morphological, histological and molecular features of 261 specimens from 226 colorectal tumors were analyzed. Surface microstructures were analyzed using magnifying endoscopy. Mutation in BRAF and KRAS was examined by pyrosequencing. Methylation of p16, IGFBP7, MLH1 and MINT1, -2, -12 and -31 was analyzed using bisulfite pyrosequencing. Through retrospective analysis of a training set (n=145), we identified a novel surface microstructure, the Type II open-shape pit pattern (Type II-O), which was specific to SSAs with BRAF mutation and CIMP. Subsequent prospective analysis of an independent validation set (n=116) confirmed that the Type II-O pattern is highly predictive of SSAs (sensitivity, 65.5%; specificity, 97.3%). BRAF mutation and CIMP occurred with significant frequency in Type II-O-positive serrated lesions. Progression of SSAs to more advanced lesions was associated with further accumulation of aberrant DNA methylation and additional morphological changes, including the Type III, IV and V pit patterns. Our results suggest the Type II-O pit pattern is a useful hallmark of the premalignant stage of CRCs with MSI and CIMP, which could serve to improve the efficacy of colonoscopic surveillance.

Abstract ED03-01: Validation and quantitation of DNA methylation changes

Abstract Advances in the molecular mechanisms underlying prostate cancer demonstrates that prostate cancer cells carry a myriad of genomic defects including both genetic and epigenetic alterations such as DNA methylation. Underlying this aberrant DNA methylation is the accumulating body of data hinting that normal prostate cells may be subjected to a relentless barrage of genome-damaging stresses due to both exogenous and endogenous carcinogens, with damage accumulating over time and age. There are increasing numbers of genes that show aberrant DNA methylation changes in human prostate cancer. In addition, recent reports indicate widespread methylation changes that correlate with a broad range of clinical behavior in prostate cancer patients. Thus aberrant DNA methylation changes may represent the integration of environmental or lifestyle exposures and genetic predisposition to prostate cancer. Such events may differ between individuals belonging the same ethnic group or individuals belonging to different ethnic groups. We hypothesize that differential methylation patterns maybe associated with ethnicity and could potentially contribute to the incidence and mortality of prostate cancer. Our research studies validate and quantify selected regions of genes from a genome-wide methylation analysis in a large sample number from African American and Caucasian male patients with prostate cancer. Our preferred method for studying DNA methylation is pyrosequencing over other methods of methylation analysis in that it is processive and quantitative. The principle of pyrosequencing is the real-time detection of pyro-phosphate (PPi), released after each incorporation of a specific nucleotide, initiated by simple primer extension on a PCR product template. Pyrosequencing has both qualitative and quantitative applications, and is optimal for allele quantification, DNA methylation estimation, and for regions that contain multiple polymorphisms (SNPs, IN/DELs) adjacent or in close proximity. Using this approach, we have observed significant differences in the methylation pattern for several genes in prostate tissue samples from African American and Caucasian men. Such alterations can contribute to the biological differences underlying prostate cancer disparity. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):ED03-01.

Epigenetics, development, and cancer: Zebrafish make their mark

Zebrafish embryos are an exceptional system for studying vertebrate development. Historically, studies using zebrafish to uncover key players in developmentally regulated gene expression have entailed detailed analysis of transcription factors. It is now apparent that epigenetic modifications of both DNA and histone tails are equally important in the regulation of gene expression during development. As such, blocking the function of key epigenetic modifiers impairs development, albeit with surprising tissue specificity. For instance, DNA methylation is an important epigenetic mark that is depleted in embryos lacking dnmt1 and uhrf1. These embryos display developmental defects in the eye, liver, pancreas, and larval lethality. Interestingly, human tumors derived from these same organs have aberrant changes in DNA methylation and altered expression of genes that are thought to contribute to formation of these cancers. These observations have provided a mechanistic basis for treating cancer with drugs that block the enzymes that facilitate DNA and histone modifications. Thus, it is important to understand the consequences of targeting these factors in a whole animal. We review the use of zebrafish for probing the genetic, cellular, and physiological response to alterations in the epigenome and highlight exciting data illustrating that epigenetic studies using zebrafish can inform and impact cancer biology.

Abstract 4791: Genome-wide DNA methylation maps in chronic lymphocytic leukemia cells determined by next-generation sequencing

Abstract Chronic lymphocytic leukemia (CLL) is the most common leukemia of adults in the western world. The clinical course of CLL is highly variable. Some patients are able to live years with no need for treatment; whereas others progress rapidly requiring therapy within a short time after diagnosis. The identification and validation of prognostic molecular markers (including surface markers, cytogenetic abnormalities, and IGHV mutational status) have resulted in refinements in the approach to the management of these patients. Further discovery of biologically relevant factors that influence the heterogeneity and progression of CLL will not only promote our understanding of the disease process, but also allow us to identify rational therapeutic approaches. In this study, we conducted genome-wide DNA methylation analyses in purified CD19+ B-cells from 11 CLL patients with a range of CD38 expression using reduced representation bisulfite sequencing (RRBS). Using one lane of the Illumina sequencing data, we were able to consistently determine the methylation status of approximately 1.8 million CpGs; 45% of these CpGs are located in more than 23,000 CpG islands (CGIs), accounting for more than 80% of all annotated CGIs across the genome. We have identified a large number of CGIs that were differentially methylated between CLL cells and normal CD19+ B-cells. The promoter hypermethylation patterns of many genes previously reported in CLL, such as FOXD3, GRM7, DLEU7 etc, were determined in the 11 CLL samples. In addition, we have observed aberrant DNA methylation changes in all 4 HOX gene clusters in CLL. For instance, HOXD8, HOXD9, and HOXD11 were hypermethylated in 11 out of 11 CLL samples. In addition, we also identified a significant number of DNA methylation alterations in the WNT signaling pathway genes. One-way ANOVA analysis of DNA methylation profiles of 764,984 CpGs shared by all CLL samples identified 570 CpGs that were differentially methylated (p<0.001) between CD38high and CD38low group (with >20% CLL cells expressing CD38 as CD38high group). The cluster analysis of the 570 CpGs reveals that it is possible to separate these CLL samples into two distinct groups based on the DNA methylation profiles. The study further confirms our early findings that CLL is affected by CpG island methylation in some genes that segregate with CD38 expression levels, while most others show similar methylation patterns across all levels. The aberrant promoter hypermethylation in certain functional gene groups and pathway-associated genes that are known to be deregulated in CLL provides additional insights into the CLL methylome and epigenetic contribution to cellular dysfunction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4791. doi:10.1158/1538-7445.AM2011-4791

Novel DNA Binding Domain-Based Assays For Detection of Methylated And Nonmethylated DNA

Methylation of cytosine in the context of CpG dinucleotides is an epigenetic phenomenon in eukaryotes that plays important roles in genome function and transcription regulation. Aberrant changes in DNA methylation is an important feature of several human diseases such as cancer and neurological disorders. These discoveries have opened a new field of new therapies and diagnostics. During recent years, there has been a revolution in DNA methylation analysis technologies. This article focuses on methods with which to study DNA methylation that employ protein domains that specifically recognize either 5-methyl-cytosine in the CpG context or nonmethylated DNA, and methods developed for the detection of 5-hydroxymethylcytosine, the recently described epigenetic mark known as the sixth base of the epigenome.

Identification of Differentially Methylated Genes in Normal Prostate Tissues from African American and Caucasian Men

Aberrant DNA methylation changes are common somatic alterations in prostate carcinogenesis. We examined the methylation status of six genes in prostate tissue specimens from African American (AA) and Caucasian (Cau) males. We used pyrosequencing to quantitatively measure the methylation status of GSTP1, AR, RARbeta2, SPARC, TIMP3, and NKX2-5. Real-time PCR was used to determine gene expression, and gene reactivation was analyzed by 5-aza-2'-deoxycytidine and/or trichostatin A treatment. Statistical analysis showed significantly higher methylation in the prostate cancer tissue samples in comparison with matched normal samples for GSTP1 (P = 0.0001 for AA; P = 0.0008 for Cau), RARbeta2 (P < 0.001 for AA and Cau), SPARC (P < 0.0001 for AA and Cau), TIMP3 (P < 0.0001 for AA and Cau), and NKX2-5 (P < 0.0001 for AA; P = 0.003 for Cau). Overall, we observed significant differences (P < 0.05) in the methylation level for all genes, except GSTP1, in the AA samples in comparison with the Cau samples. Furthermore, regression analysis revealed significantly higher methylation for NKX2-5 (P = 0.008) and TIMP3 (P = 0.039) in normal prostate tissue samples from AA in comparison with Cau, and a statistically significant association of methylation with age for NKX2-5 (P = 0.03) after adjusting for race. Our findings show higher methylation of several genes in prostate tissue samples from AA in comparison with Cau and may potentially contribute to the racial differences that are observed in prostate cancer pathogenesis.

DNA Methylation: An Introduction to the Biology and the Disease-Associated Changes of a Promising Biomarker

DNA methylation occurring on the 5 position of the pyrimidine ring of cytosines in the context of the dinucleotide sequence CpG forms one of the multiple layers of epigenetic mechanisms controlling and modulating gene expression through chromatin structure. It closely interacts with histone modifications and chromatin remodeling complexes to form the genomic chromatin landscape. DNA methylation is essential for proper mammalian development, crucial for imprinting and plays a role in maintaining genomic stability as well as in dosage compensation. DNA methylation patterns are susceptible to change in response to environmental stimuli such as diet or toxins, whereby the epigenome seems to be most vulnerable during early in utero development. Aberrant DNA methylation changes have been detected in several diseases, particularly cancer where genome-wide hypomethylation coincides with genespecific hypermethylation. DNA methylation patterns can be used to detect cancer at very early stages, to classify tumors as well as predict and monitor the response to antineoplastic treatment. As a stable nucleic-acid-based modification with limited dynamic range that is technically easy to handle, DNA methylation is a promising biomarker for many applications.

Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization

The timing and progression of DNA methylation changes during carcinogenesis are not completely understood. To develop a timeline of aberrant DNA methylation events during malignant transformation, we analyzed genome-wide DNA methylation patterns in an isogenic human mammary epithelial cell (HMEC) culture model of transformation. To acquire immortality and malignancy, the cultured finite lifespan HMEC must overcome two distinct proliferation barriers. The first barrier, stasis, is mediated by the retinoblastoma protein and can be overcome by loss of p16(INK4A) expression. HMEC that escape stasis and continue to proliferate become genomically unstable before encountering a second more stringent proliferation barrier, telomere dysfunction due to telomere attrition. Rare cells that acquire telomerase expression may escape this barrier, become immortal, and develop further malignant properties. Our analysis of HMEC transitioning from finite lifespan to malignantly transformed showed that aberrant DNA methylation changes occur in a stepwise fashion early in the transformation process. The first aberrant DNA methylation step coincides with overcoming stasis, and results in few to hundreds of changes, depending on how stasis was overcome. A second step coincides with immortalization and results in hundreds of additional DNA methylation changes regardless of the immortalization pathway. A majority of these DNA methylation changes are also found in malignant breast cancer cells. These results show that large-scale epigenetic remodeling occurs in the earliest steps of mammary carcinogenesis, temporally links DNA methylation changes and overcoming cellular proliferation barriers, and provides a bank of potential epigenetic biomarkers that may prove useful in breast cancer risk assessment.

CHILDREN’S HEALTH: Methylation Links Prenatal PAH Exposure to Asthma

CHILDREN’S HEALTH: Methylation Links Prenatal PAH Exposure to Asthma

Global Epigenomic Profiling Demonstrates That Myelodysplasia Is Characterized by a Distinct Epigenetic Signature with Aberrant DNA Methylation Changes Involving Various Malignant and Hematopoietic Pathways.

Myelodysplasia (MDS) is a clonal hematopoietic disorder that leads to ineffective hematopoiesis and peripheral cytopenias. DNMT inhibitors such as azacytidine have led to clinical responses in patients, though the genes affected by epigenetic alterations are not well known. Whole genome DNA methylation was analyzed by a recently described novel method, The HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific restriction digestion by HpaII and MspI followed by amplification, two color labeling and cohybridization to quantitatively determine individual promoter island methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. Peripheral blood leucocytes from 13 patients with MDS were compared to 9 age matched normal and anemic controls. Gene expression analysis was performed using 37K oligo maskless arrays on cDNA obtained from the same samples. Analysis showed that whole genome methylation profiling has greater discriminatory power in separating clusters of MDS samples from normal and anemic controls when compared to gene expression analysis. Unsupervised clustering based on epigenetic profiling demonstrated that only two cases of early MDS clustered with normals as compared to absolutely no separation between MDS and normals with clustering based on gene expression patterns. A high correlation (r=0.88–0.96) was observed between global methylation profiles of matched sets of bone marrow and peripheral blood leucocyte samples from selected patients demonstrating that peripheral blood leucocytes can be a valid surrogate for epigenomic analysis. Further analysis showed that genes consistently aberrantly methylated in MDS included Syk kinase, HOXB3, several histone acetyltranferases and others. Functional analysis by Ingenuity showed that cancer and cell signaling pathways were the most affected by epigenetic silencing. Most interestingly, a large proportion of gene promoters were also aberrantly hypomethylated. These included genes from Ras oncogene family, the CDC42 GTPase, various methyl binding proteins and other proteins mainly encoding for cancer and hematopoiesis functional pathways, thus biologically validating our analysis. Therefore, our data demonstrates that MDS is characterized by distinct epigenetic aberrations that are preserved in peripheral blood leucocytes. These can be the basis of future studies on pathogenesis and diagnosis for this disease and can potentially uncover a new set of therapeutic gene targets.

Active demethylation of individual genes in intracytoplasmic sperm injection rabbit embryos

Intracytoplasmic sperm injection (ICSI), as an assisted reproduction technique, has been widely used in animal and human. However, its possible effect on epigenetic changes has not been well studied. To investigate whether ICSI can induce aberrant DNA methylation changes in rabbit preimplantation embryos, we examined the methylation status of the SP-A promoter region and the satellite sequence Rsat IIE by bisulfite-sequencing technology. The SP-A promoter region was extensively demethylated before the first round of DNA replication commences, and the unmethylated status was maintained until morula when dynamic remethylation occurred. A similar but more moderate demethylation process was observed in satellite sequence Rsat IIE. These results are in contrast with the previous reports of no active demethylation in normal rabbit embryos, suggesting that the active demethylation we observed may be induced by ICSI.