Genetic alterations were previously identified in normal epithelia adjacent to invasive cancers. The aim of this study was to determine DNA methylation in histologically normal tissues from multiple geographic zones adjacent to primary breast tumors.First, methylation status of a 4-kb region of RASSF1A promoter was interrogated using oligonucleotide-based microarray in 144 samples (primary tumors, 47; adjacent normals, 69; reduction mammoplasty tissues, 28). Second, allelic imbalance (AI)/loss of heterozygosity (LOH) surrounding RASSF1A promoter were analyzed in 30 samples (tumors, 8; adjacent normals, 22). Third, global methylation screening of 49 samples (tumors, 12; adjacent normals, 25; reduction mammoplasty, 12) was done by differential methylation hybridization. Real-time quantitative methylation-specific PCR was used to validate the microarray findings.DNA methylation in the core RASSF1A promoter was low in reduction mammoplasty tissues (P=0.0001) when compared with primary tumors. The adjacent normals had an intermediate level of methylation. The regions surrounding the core were highly methylated in all sample types. Microsatellite markers showed AI/LOH in tumors and some of the adjacent normals. Concurrent AI/LOH and DNA methylation in RASSF1A promoter occurred in two of six tumors. Global methylation screening uncovered genes more methylated in adjacent normals than in reduction mammoplasty tissues. The methylation status of four genes was confirmed by quantitative methylation-specific PCR.Our findings suggest a field of methylation changes extending as far as 4 cm from primary tumors. These frequent alterations may explain why normal tissues are at risk for local recurrence and are useful in disease prognostication.
The current concept of epigenetic repression is based on one repressor unit corresponding to one silent gene. This notion, however, cannot adequately explain concurrent silencing of multiple loci observed in large chromosome regions. The long-range epigenetic silencing (LRES) can be a frequent occurrence throughout the human genome. To comprehensively characterize the influence of estrogen signaling on LRES, we analyzed transcriptome, methylome, and estrogen receptor alpha (ESR1)-binding datasets from normal breast epithelia and breast cancer cells. This “omics” approach uncovered 11 large repressive zones (range, 0.35∼5.98 megabases), including a 14-gene cluster located on 16p11.2. In normal cells, estrogen signaling induced transient formation of multiple DNA loops in the 16p11.2 region by bringing 14 distant loci to focal ESR1-docking sites for coordinate repression. However, the plasticity of this free DNA movement was reduced in breast cancer cells. Together with the acquisition of DNA methylation and repressive chromatin modifications at the 16p11.2 loci, an inflexible DNA scaffold may be a novel determinant used by breast cancer cells to reinforce estrogen-mediated repression.
Abstract Alterations in histones, chromatin-related proteins, and DNA methylation contribute to transcriptional silencing in cancer, but the sequence of these molecular events is not well understood. Here we demonstrate that on disruption of estrogen receptor (ER) α signaling by small interfering RNA, polycomb repressors and histone deacetylases are recruited to initiate stable repression of the progesterone receptor (PR) gene, a known ERα target, in breast cancer cells. The event is accompanied by acquired DNA methylation of the PR promoter, leaving a stable mark that can be inherited by cancer cell progeny. Reestablishing ERα signaling alone was not sufficient to reactivate the PR gene; reactivation of the PR gene also requires DNA demethylation. Methylation microarray analysis further showed that progressive DNA methylation occurs in multiple ERα targets in breast cancer genomes. The results imply, for the first time, the significance of epigenetic regulation on ERα target genes, providing new direction for research in this classical signaling pathway.
<div>Abstract<p><b>Purpose:</b> Genetic alterations were previously identified in normal epithelia adjacent to invasive cancers. The aim of this study was to determine DNA methylation in histologically normal tissues from multiple geographic zones adjacent to primary breast tumors.</p><p><b>Experimental Design:</b> First, methylation status of a 4-kb region of <i>RASSF1A</i> promoter was interrogated using oligonucleotide-based microarray in 144 samples (primary tumors, 47; adjacent normals, 69; reduction mammoplasty tissues, 28). Second, allelic imbalance (AI)/loss of heterozygosity (LOH) surrounding <i>RASSF1A</i> promoter were analyzed in 30 samples (tumors, 8; adjacent normals, 22). Third, global methylation screening of 49 samples (tumors, 12; adjacent normals, 25; reduction mammoplasty, 12) was done by differential methylation hybridization. Real-time quantitative methylation-specific PCR was used to validate the microarray findings.</p><p><b>Results:</b> DNA methylation in the core <i>RASSF1A</i> promoter was low in reduction mammoplasty tissues (<i>P</i> = 0.0001) when compared with primary tumors. The adjacent normals had an intermediate level of methylation. The regions surrounding the core were highly methylated in all sample types. Microsatellite markers showed AI/LOH in tumors and some of the adjacent normals. Concurrent AI/LOH and DNA methylation in <i>RASSF1A</i> promoter occurred in two of six tumors. Global methylation screening uncovered genes more methylated in adjacent normals than in reduction mammoplasty tissues. The methylation status of four genes was confirmed by quantitative methylation-specific PCR.</p><p><b>Conclusions:</b> Our findings suggest a field of methylation changes extending as far as 4 cm from primary tumors. These frequent alterations may explain why normal tissues are at risk for local recurrence and are useful in disease prognostication.</p></div>
<div>Abstract<p>Genetic amplification, mutation, and translocation are known to play a causal role in the upregulation of an oncogene in cancer cells. Here, we report an emerging role of microRNA, the epigenetic deregulation of which may also lead to this oncogenic activation. <i>SOX4</i>, an oncogene belonging to the SRY-related high mobility group box family, was found to be overexpressed (<i>P</i> < 0.005) in endometrial tumors (<i>n</i> = 74) compared with uninvolved controls (<i>n</i> = 20). This gene is computationally predicted to be the target of a microRNA, <i>miR-129-2</i>. When compared with the matched endometria, the expression of <i>miR-129-2</i> was lost in 27 of 31 primary endometrial tumors that also showed a concomitant gain of <i>SOX4</i> expression (<i>P</i> < 0.001). This inverse relationship is associated with hypermethylation of the <i>miR-129-2</i> CpG island, which was observed in endometrial cancer cell lines (<i>n</i> = 6) and 68% of 117 endometrioid endometrial tumors analyzed. Reactivation of <i>miR-129-2</i> in cancer cells by pharmacologic induction of histone acetylation and DNA demethylation resulted in decreased SOX4 expression. In addition, restoration of <i>miR-129-2</i> by cell transfection led to decreased SOX4 expression and reduced proliferation of cancer cells. Further analysis found a significant correlation of hypermethylated <i>miR-129-2</i> with microsatellite instability and <i>MLH1</i> methylation status (<i>P</i> < 0.001) and poor overall survival (<i>P</i> < 0.039) in patients. Therefore, these results imply that the aberrant expression of <i>SOX4</i> is, in part, caused by epigenetic repression of <i>miR-129-2</i> in endometrial cancer. Unlike the notion that promoter hypomethylation may upregulate an oncogene, we present a new paradigm in which hypermethylation-mediated silencing of a microRNA derepresses its oncogenic target in cancer cells. [Cancer Res 2009;69(23):9038–46]</p></div>
Aberrant DNA methylation of promoter CpG islands is associated with transcriptionally repressive heterochromatin in neoplasia. The dynamics of this epigenetic process in mediating the transition from an active to an inactive state of transcription remains to be elucidated, however. Here, we used the methylation-specific oligonucleotide microarray to map the methylation patterns of a CpG island, located within the promoter and the first exon regions of RASSF1A, in normal breast tissue controls, primary tumors, and breast cancer cell lines. Oligonucleotide pairs, spaced along the CpG island region, were designed to discriminate between methylated and unmethylated alleles of selected sites. The methylation-specific oligonucleotide data indicate that the majority of test samples show widespread methylation in the first exon of RASSF1A. In contrast, the promoter area was usually undermethylated in normal controls and in 32% of the primary tumors tested, whereas the rest of the primary tumors and breast cancer cell lines showed various degrees of methylation in the region. Methylation profiling of individual tumors further suggest that DNA methylation progressively spreads from the first exon into the promoter area of this gene. Functional analysis indicates that increased density of RASSF1A promoter methylation is associated with altered chromatin, marked by a depletion of acetylated histones and methylated histone 3-lysine 4 and an enrichment of methylated histone 3-lysine 9 in the studied area. The combination of these epigenetic modifications may engender a stable silencing of the gene in breast cancer cells. Thus, this study underscores the importance of detailed mapping of methylation patterns within a CpG island locus that may provide insights into the progressive nature of aberrant DNA methylation and its relationship with transcriptional silencing during the neoplastic process.
