DNA methylation-mediated extracellular matrix gene silencing in colorectal cancer.

Simple SummaryThe role of DNA methylation in the carcinogenesis of colorectal cancer (CRC) diagnosed <50 years of age (early-onset CRC or EOCRC) is currently unknown. In this study, we investigated the genome-wide DNA methylation of 97 tumour and 54 normal colonic mucosa samples from people with EOCRC with the aim of identifying unique DNA methylation signatures and determining the role of ageing-related DNA methylation drift and age-acceleration in EOCRC aetiology. We found extensive DNA methylation alterations associated with EOCRC carcinogenesis, including a unique signature comprising 234 loci compared with CRCs from people >50 years of age. CpGs that undergo ageing-related methylation drift were significantly altered in EOCRC, and accelerated ageing was also evident in normal mucosa from people with EOCRC. Our study is the first study to identify unique DNA methylation changes in EOCRC, contributing novel information that may aid future efforts towards EOCRC prevention.We investigated aberrant DNA methylation (DNAm) changes and the contribution of ageing-associated methylomic drift and age acceleration to early-onset colorectal cancer (EOCRC) carcinogenesis. Genome-wide DNAm profiling using the Infinium HM450K on 97 EOCRC tumour and 54 normal colonic mucosa samples was compared with: (1) intermediate-onset CRC (IOCRC; diagnosed between 50–70 years; 343 tumour and 35 normal); and (2) late-onset CRC (LOCRC; >70 years; 318 tumour and 40 normal). CpGs associated with age-related methylation drift were identified using a public dataset of 231 normal mucosa samples from people without CRC. DNAm-age was estimated using epiTOC2. Common to all three age-of-onset groups, 88,385 (20% of all CpGs) CpGs were differentially methylated between tumour and normal mucosa. We identified 234 differentially methylated genes that were unique to the EOCRC group; 13 of these DMRs/genes were replicated in EOCRC compared with LOCRCs from TCGA. In normal mucosa from people without CRC, we identified 28,154 CpGs that undergo ageing-related DNAm drift, and of those, 65% were aberrantly methylated in EOCRC tumours. Based on the mitotic-based DNAm clock epiTOC2, we identified age acceleration in normal mucosa of people with EOCRC compared with normal mucosa from the IOCRC, LOCRC groups (p = 3.7 × 10−16) and young people without CRC (p = 5.8 × 10−6). EOCRC acquires unique DNAm alterations at 234 loci. CpGs associated with ageing-associated drift were widely affected in EOCRC without needing the decades-long accrual of DNAm drift as commonly seen in intermediate- and late-onset CRCs. Accelerated ageing in normal mucosa from people with EOCRC potentially underlies the earlier age of diagnosis in CRC carcinogenesis.

Increasing evidence suggests that gut microbiota plays a critical role in colorectal cancer (CRC) development; however, the underlying mechanism is largely unknown. We investigated the relationship of commensal microbiota with host DNA methylation (DNAm) and gene expression in colorectal adenoma, the major precursor of CRC. This study included 72 participants from the Tennessee Colorectal Polyp Study. Microbiome, DNA methylome, and transcriptome data of fresh frozen conventional adenoma samples were generated using 16S rRNA gene sequencing, HumanMethylation450 BeadChip, and RNA-seq, respectively. Among 35 participants with microbiome and matched DNA methylome data, microbial features were evaluated for their associations with DNAm at CpG sites (CpGs) via linear regression. False-discovery rate (FDR) correction was conducted separately for each microbial feature and significant associations were identified at FDR&amp;lt;0.1. DNAm at microbiome-associated CpGs were then tested for their correlations with expression of flanking genes (500Kb) among 45 subjects with DNA methylome and matched transcriptome data. For genes with expression significantly (FDR&amp;lt;0.1) correlated with DNAm at microbiome-associated CpGs, their differential expression between colorectal adenomas and adenocarcinomas were examined using microarray data from Gene Expression Omnibus (GEO, 363 conventional adenoma samples) and CRC Subtyping Consortium (CRCSC, 2,760 adenocarcinoma samples). The R package “limma” was used to identify significantly differentially expressed genes at FDR&amp;lt;0.1. Four alpha diversity indexes, three beta diversity matrices, and 194 taxa were investigated for their associations with DNAm at 28,081 variable CpGs (variance&amp;gt;0.02 across samples). Faith’s phylogenetic diversity index and abundance of 11 taxa were significantly associated with DNAm at 68 CpGs. Among them, the most significant association was observed between Lactococcus and cg03292388 (β=-0.67, P=4.39×10-9). DNAm at 11 of the 68 CpGs were significantly correlated with expression of 12 genes (551 genes tested). Six of these 12 genes were found in data from GEO and CRCSC, and three showed a significant differential expression between adenomas and adenocarcinomas. Integrating these results revealed potential bacteria-DNAm-gene-adenocarcinoma pathways in which Bacteroides ovatus, three CpGs, and three genes were involved. Specifically, increased abundance of B. ovatus was associated with decreased DNAm at cg19003815 (β=-0.28, P=7.64×10-5), which was correlated with increased expression of RARB (rho=-0.65, P=2.18×10-6). These findings are in line with the higher expression of RARB in adenocarcinomas compared to adenomas (fold-change=1.51, P=8.58×10-10). In conventional adenomas, bacterial-related host DNAm changes may affect expression of nearby genes, which might be implicated in adenoma-carcinoma development. Citation Format: Yaohua Yang, Jirong Long, Martha J. Shrubsole, Qiuyin Cai, Zhiguo Zhao, Fei Ye, Zhigang Li, Xingyi Guo, Bingshan Li, Seth R. Bordenstein, Ken S. Lau, Harvey J. Murff, Reid M. Ness, Robert J. Coffey, Wei Zheng. Commensal microbiota, host DNA methylation and gene expression: A pilot study in colorectal adenomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3051.

Retinal degeneration is a major cause of irreversible blindness. Stimulation with controlled low-level electrical fields, such as transcorneal electrical stimulation (TES), has recently been postulated as a therapeutic strategy. With promising results, there is a need for detailed molecular characterization of the therapeutic effects of TES. Controlled, non-invasive TES was delivered using a custom contact lens electrode to the retinas of Royal College of Surgeons (RCS) rats, a model of retinal degeneration. DNA methylation in the retina, brain and cell-free DNA in plasma was assessed by reduced representation bisulfite sequencing (RRBS) and gene expression by RNA sequencing. TES induced DNA methylation and gene expression changes implicated in neuroprotection in the retina of RCS rats. We devised an epigenomic-based retinal health score, derived from DNA methylation changes observed with disease progression in RCS rats, and showed that TES improved the epigenomic health of the retina. TES also induced DNA methylation changes in the superior colliculus: the brain which is involved in integrating visual signaling. Lastly, we demonstrated that TES-induced retinal DNA methylation changes were detectable in cell-free DNA derived from plasma. TES induced DNA methylation changes with therapeutic effects, which can be measured in circulation. Based on these changes, we were able to devise a liquid biopsy biomarker for retinal health. These findings shed light on the therapeutic potential and molecular underpinnings of TES, and provide a foundation for the further development of TES to improve the retinal health of patients with degenerative eye diseases.

Recent evidence has indicated an important role for DNA methylation in the control of gene expression, silencing of transposons and maintenance of genome stability. The current study utilised the Amplified Methylation Polymorphism (AMP) technique to investigate the extent of DNA methylation polymorphisms and their biological importance in several plant species. DNA methylation patterns were examined in response to different environments and propagation through tissue culture, and in different tissue types of one plant species. The technique was also used to investigate the extent of DNA loss in transgenic sugarcane as a result of genetic engineering procedures. Widespread DNA methylation changes were detected in response to the environment in both species investigated, namely sugarcane (a complex monocot polyploid) and Arabidopsis thaliana (the simple dicot diploid). Sugarcane cultivar Q155 grown at three diverse locations in Queensland, Australia, showed location-specific DNA methylation patterns, and when clones were moved to common glasshouses they showed new glasshouse-specific DNA methylation patterns whilst generally retaining the older location-specific DNA methylation signatures. In addition, prolonged cold treatment (vernalisation) in Arabidopsis induced DNA methylation changes. These findings indicate that DNA methylation may be a general control mechanism for mediating phenotypic responses to environmental change. Plant genome instability with passage through tissue culture was shown to vary greatly between plant species, with wheat cultivar Hartog being particularly stable in comparison to sugarcane cultivars. It was also demonstrated that the process of producing double haploids of Hartog resulted in few, if any DNA methylation or sequence changes, thereby validating this approach for producing mapping populations for mapping important traits in wheat. In sugarcane, on the other hand, genome instability in conventionally tissue culture-propagated plants was demonstrated to be a major problem. Furthermore, gene transfer and/or selection processes increased the extent of genome instability in the form of DNA loss compared to that resulting from the conventional tissue culture-propagation process alone. While genome instability in conventionally tissue culture-propagated and genetically engineered sugarcane is currently a major problem, AMP analysis of non-transgenic sugarcane plants propagated by a new direct regeneration method indicated that the prospects of overcoming this problem of genome instability in sugarcane are encouraging. AMP profiles were shown to be distinguishable between leaf and pollen DNA in lily (Lilium longiforum), however, the extent of tissue-specific DNA methylation changes was shown to be less than that reported for mammals. Throughout the present study, the AMP technique has been proven to be a robust and efficient tool for analysing plant genome instability and assessing the DNA methylation status of specific loci throughout the genome. This study has also demonstrated the potential importance of DNA methylation in the plant response mechanism to changing environments, including responses to stresses imposed by the tissue culture process. However, more studies are necessary to elucidate the environmentally-induced DNA methylation changes. In particular, the priority would be the cloning, sequencing and mapping of the environmentally-induced DNA methylation changes. In view of the fact that the Arabidopsis genome is completely sequenced, mapping of the vernalisation-induced DNA methylation markers is currently achievable in this species and could greatly enhance our understanding of vernalisation and the control of flowering time in plants. Future applications of the AMP protocol would include efficient screening for genome integrity in tissue culture-derived and genetically engineered plants, assessing the effect of tissue culture conditions and gene transfer and selection procedures on genome instability, and further identification and characterisation of genomic regions displaying environment-specific DNA methylation.

S-adenosylmethionine Levels Regulate the Schwann Cell DNA Methylome

Proliferation-Dependent Alterations of the DNA Methylation Landscape Underlie Hematopoietic Stem Cell Aging

SummaryCircular RNAs (circRNAs) have been identified as naturally occurring RNAs that are highly represented in the eukaryotic transcriptome. Although a large number of circRNAs have been reported, the underlying regulatory mechanism of circRNAs biogenesis remains largely unknown. Here, we integrated in-depth multi-omics data including epigenome, transcriptome, and non-coding RNA and identified candidate circRNAs in six cellular contexts. Next, circRNAs were divided into two classes (high versus low) with different expression levels. Machine learning models were constructed that predicted circRNA expression levels based on 11 different histone modifications and host gene expression. We found that the models achieve great accuracy in predicting high versus low expressed circRNAs. Furthermore, the expression levels of host genes of circRNAs, H3k36me3, H3k79me2, and H4k20me1 contributed greatly to the classification models in six cellular contexts. In summary, all these results suggest that epigenetic modifications, particularly histone modifications, can effectively predict expression levels of circRNAs.

BackgroundVitamin D is a fat-soluble vitamin that has a protective role in colorectal cancer. Several studies have identified the association between vitamin D and changes in DNA methylation in different types of tumours. Dickkopf-1 (DKK1) inhibits the Wnt/β-catenin signalling pathway, and 1,25(OH)2D3 can induce DKK1 expression in colorectal cancer. However, whether 1,25(OH)2D3 can affect DKK1 expression by regulating DNA methylation in colorectal cancer is not known.MethodsFifty-seven colorectal cancer (CRC) patients and fifty-five healthy controls were included in this study. Serum DKK1 and 25(OH)D levels were measured via ELISA and liquid chromatography‒tandem mass spectrometry, respectively, and the associations of DKK1 with clinicopathological characteristics and 25(OH)D were analysed. A DKK1 expression plasmid was transfected into cells to assess the functional significance of DKK1 in CRC progression via CCK8, wound healing and migration assays. BiSulphite Amplicon Sequencing (BSAS) and methylation-specific PCR were used to detect the DKK1 methylation status of colorectal cancer cells and tissues. The effect of 1,25(OH)2D3 on DKK1 methylation was investigated by pyrosequencing. A dual-luciferase reporter assay was performed to investigate the influence of CpG island methylation on DKK1 transcriptional activity.ResultsA decreased serum DKK1 level was closely associated with nerve infiltration and 25(OH)D status in patients with colorectal cancer. Overexpression of DKK1 reduced the proliferative and migratory capabilities of colorectal cancer cells. The methylation patterns of DKK1 (− 195 to + 231), including 31 CpG sites, were assayed via BSAS in CRC cells and tissues. Compared with those in adjacent normal tissues, the methylation levels of multiple CpG sites located in the promoter, 5’UTR and exon 1 were increased in tumour tissues. DKK1 hypermethylation was associated with decreased DKK1 expression in colorectal cancer cells and tissues. 1,25(OH)2D3 induced DKK1 expression in colorectal cancer cells, and pyrosequencing revealed that 1,25(OH)2D3 treatment induced demethylation of CpG sites located in the promoter (− 97 to − 32) and 5’UTR (+ 39 to + 97). The dual-luciferase reporter assay further confirmed that CpG island methylation (-120 to + 225) directly represses DKK1 transcription.ConclusionDKK1 functions as a tumour suppressor in colorectal cancer, and 1,25(OH)2D3 upregulates DKK1 expression by inducing demethylation of the DKK1 promoter and 5’UTR in specific colorectal cancer cell lines.

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Background: Long non-coding RNAs may play role in colorectal cancer (CRC) development, however, lncRNA expression profile in the colorectal adenoma-carcinoma sequence (C-ACS) and its relation to the complex epigenetic regulation system still remain incomplete. Aims: We aimed the whole genomic lncRNA expression profiling with up- and downstream epigenetic analyses of the C-ACS in order to explore the underlying mechanisms and consequences of aberrantly expressed lncRNAs. Materials&amp;methods: lncRNA expression levels were analyzed on 60 colonic biopsy samples (20 CRCs, 20 adenomas, 20 normals) by Human Transcriptome Array (HTA) 2.0 (Affymetrix). Data analysis was perfomed using Expression Console and Transcriptome Analysis Console. Expression of certain candidates was verified in silico on HGU133 Plus 2.0 array data and also by qRT-PCR. DNA methylation status of lncRNA promoter regions was studied by methyl-capture sequencing. miRNA targets of lncRNAs were predicted with miRCODE algorithm and miRNA expression was analysed using GeneChip miRNA 3.0 Array. In the respective regulatory networks mRNA expression changes were also evaluated on the basis of the above-mentioned whole genome expression arrays. Results: According to HTA results analyzing 40.914 non-coding transcripts on whole genome level, in adenomas 12 lncRNAs (e.g. CCAT1, LINC00278) were significantly upregulated and 6 lncRNAs (e.g.FLJ22763, RP11.747D18.1) were downregulated compared to the healthy controls, while in CRC samples 1 lncRNA (UCA1) was overexpressed and 8 lncRNAs (e.g. LINC00350, LINC00261) were underexpressed compared to adenomas (p&amp;lt;0.05; -2≥Fold change≥2). In CRC samples 8 lncRNAs (e.g. MACC1, AC123023.1) were upregulated and 11 lncRNAs (e.g. RP13-497K6.1) were downregulated compared to normal controls. Furthermore, 42% of lncRNAs upregulated in CRC samples showed significantly elevated expression (p&amp;lt;0.05) already in adenoma samples (e.g. LINC350, CCAT1 were upregulated and LINC01133, FLJ22763 were downregulated compared to healthy controls). Promoter DNA methylation showed inverse relation with lncRNA expression along the C-ACS (e.g. CCAT1). In line with aberrant lncRNA expression in tumors, miRNA and mRNA targets’ expression showed systematic alterations, e.g. UCA1 upregulation in CRC samples in parallel with miR-1 downregulation accompanied by MET proto-oncogene target mRNA overexpression (p&amp;lt;0.05). Conclusion: The defined lncRNA sets (including MACC1, CCAT1, UCA1) have a central regulatory role in colorectal adenoma development and in tumor cell growth pathways. The underlying DNA methylation changes and miRNA and mRNA target expression alterations were proven using whole genomic array technologies. The identified lncRNA candidate sets can be further investigated as early diagnostic biomarkers and as potential therapeutic molecular targets for CRC. Citation Format: Alexandra Kalmar, Zsofia B. Nagy, Orsolya Galamb, Barnabas Wichmann, Barbara K. Bartak, Zsolt Tulassay, Bela Molnar. Key tumor growth controlling long non-coding RNA (lncRNA) expression alterations in the colorectal adenoma-carcinoma sequence. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 990.

Epigenetic marks are likely to explain variability of response to antigen in granulomatous lung disease. The objective of this study was to identify DNA methylation and gene expression changes associated with chronic beryllium disease (CBD) and sarcoidosis in lung cells obtained by BAL. BAL cells from CBD (n = 8), beryllium-sensitized (n = 8), sarcoidosis (n = 8), and additional progressive sarcoidosis (n = 9) and remitting (n = 15) sarcoidosis were profiled on the Illumina 450k methylation and Affymetrix/Agilent gene expression microarrays. Statistical analyses were performed to identify DNA methylation and gene expression changes associated with CBD, sarcoidosis, and disease progression in sarcoidosis. DNA methylation array findings were validated by pyrosequencing. We identified 52,860 significant (P < 0.005 and q < 0.05) CpGs associated with CBD; 2,726 CpGs near 1,944 unique genes have greater than 25% methylation change. A total of 69% of differentially methylated genes are significantly (q < 0.05) differentially expressed in CBD, with many canonical inverse relationships of methylation and expression in genes critical to T-helper cell type 1 differentiation, chemokines and their receptors, and other genes involved in immunity. Testing of these CBD-associated CpGs in sarcoidosis reveals that methylation changes only approach significance, but are methylated in the same direction, suggesting similarities between the two diseases with more heterogeneity in sarcoidosis that limits power with the current sample size. Analysis of progressive versus remitting sarcoidosis identified 15,215 CpGs (P < 0.005 and q < 0.05), but only 801 of them have greater than 5% methylation change, demonstrating that DNA methylation marks of disease progression changes are more subtle. Our study highlights the significance of epigenetic marks in lung immune response in granulomatous lung disease.

BackgroundDNA methylation is likely a key mechanism regulating changes in gene transcription in traits that show temporal fluctuations in response to environmental conditions. To understand the transcriptional role of DNA methylation we need simultaneous within-individual assessment of methylation changes and gene expression changes over time. Within-individual repeated sampling of tissues, which are essential for trait expression is, however, unfeasible (e.g. specific brain regions, liver and ovary for reproductive timing). Here, we explore to what extend between-individual changes in DNA methylation in a tissue accessible for repeated sampling (red blood cells (RBCs)) reflect such patterns in a tissue unavailable for repeated sampling (liver) and how these DNA methylation patterns are associated with gene expression in such inaccessible tissues (hypothalamus, ovary and liver). For this, 18 great tit (Parus major) females were sacrificed at three time points (n = 6 per time point) throughout the pre-laying and egg-laying period and their blood, hypothalamus, ovary and liver were sampled.ResultsWe simultaneously assessed DNA methylation changes (via reduced representation bisulfite sequencing) and changes in gene expression (via RNA-seq and qPCR) over time. In general, we found a positive correlation between changes in CpG site methylation in RBCs and liver across timepoints. For CpG sites in close proximity to the transcription start site, an increase in RBC methylation over time was associated with a decrease in the expression of the associated gene in the ovary. In contrast, no such association with gene expression was found for CpG site methylation within the gene body or the 10 kb up- and downstream regions adjacent to the gene body.ConclusionTemporal changes in DNA methylation are largely tissue-general, indicating that changes in RBC methylation can reflect changes in DNA methylation in other, often less accessible, tissues such as the liver in our case. However, associations between temporal changes in DNA methylation with changes in gene expression are mostly tissue- and genomic location-dependent. The observation that temporal changes in DNA methylation within RBCs can relate to changes in gene expression in less accessible tissues is important for a better understanding of how environmental conditions shape traits that temporally change in expression in wild populations.

Background: Colorectal cancer (CRC) is the third most common type of cancer. The Wnt signaling pathway is known to play an important role in initiation and progression of CRC. Suppression of Wnt antagonists by promoter hypermethylation has been proposed to increase Wnt activity. DNA methylation changes of Wnt antagonists during CRC progression through the preinvasive adenoma stage have not been previously characterized. We analyzed promoter methylation changes of selected Wnt antagonists in a set of synchronous normal, hyperplastic or adenomatous polyps and adenocarcinoma tissue samples obtained from colectomy specimens. Method: We profiled methylation changes in CpG islands associated with the Wnt signaling antagonists DKK1, DKK2, DKK3, WIF1, SFRP1, SFRP2, SFRP4, SFRP5, and SOX17 using pyrosequencing assays. We also examined methylation changes of two members of the β-catenin destruction complex, APC and AXIN2, and two non-antagonist genes (CDH1 and DVL2) involved in Wnt signaling. The sample set included 48 cases comprising matched normal (n=73), hyperplastic polyp (n=9), adenoma (n=42) and adenocarcinoma (n=48) tissue samples. Results: Our samples showed significant widespread hypermethylation changes of a subset of the analyzed Wnt antagonists in the transition from normal to hyperplastic polyp to carcinoma and from normal to adenoma to carcinoma, mostly in an increasing stepwise pattern (p&amp;lt;0.05, Wilcoxon rank sum test). DKK1 showed no significant change during CRC progression, DKK3 did not show significant methylation changes during the hyperplastic polyp to carcinoma stage and SFRP4 was not significantly hypermethylated during adenoma to adenocarcinoma progression. APC was hypermethylated in a small subset of carcinomas, however, these changes were not statistically significant. AXIN2 and the non-antagonist Wnt genes, CDH1 and DVL2 showed consistent normal levels of methylation throughout CRC progression. Conclusion: Wnt antagonists are progressively hypermethylation during CRC neoplastic development. We propose that CpG island hypermethylation of Wnt antagonists could be used as biomarkers for early detection of CRC. 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 94. doi:10.1158/1538-7445.AM2011-94

Colorectal cancer (CRC) is characterized by genome-wide alterations to DNA methylation that influence gene expression and genomic stability. Less is known about the extent to which methylation is disrupted in the earliest stages of CRC development. In this study we have combined laser-capture microdissection (LCM) with reduced representation bisulfite sequencing (RRBS) to identify cancer-associated DNA methylation changes in human aberrant crypt foci (ACF), the earliest putative precursor to CRC. Using this approach, methylation profiles have been generated for 10 KRAS-mutant ACF and 10 CRCs harboring a KRAS mutation, as well as matched samples of normal mucosa. Of 811 differentially methylated regions (DMRs) identified in ACF, 537 (66%) were hypermethylated and 274 (34%) were hypomethylated. DMRs located within intergenic regions were heavily enriched for AP-1 transcription factor binding sites and were frequently hypomethylated. Furthermore, gene ontology (GO) analysis demonstrated that DMRs associated with promoters were enriched for genes involved in intestinal development, including homeobox genes and targets of the Polycomb repressive complex 2 (PRC2). Consistent with their role in the earliest stages of colonic neoplasia, 75% of the loci harboring methylation changes in ACF were also altered in CRC samples, though the magnitude of change at these sites was lesser in ACF. While aberrant promoter methylation was associated with altered gene expression in CRC, this was not the case in ACF, suggesting the insufficiency of methylation changes to modulate gene expression in early colonic neoplasia. Together, these data demonstrate that DNA methylation changes, including significant hypermethylation, occur more frequently in early colonic neoplasia than previously believed, and identify epigenomic features of ACF that may provide new targets for cancer chemoprevention or lead to the development of new biomarkers for CRC risk.