Epigenetic Instability via Genomic-Wide DNA Methylation and Histone Modification Profile Predicts Colorectal Adenoma and Cancer

Abstract

Background: Epigenetic silencing including chromatin modification likely precedes genetic alterations in early colorectal cancer (CRC) events. We aimed to investigate global histone acetylation, HDAC expression and genome wide DNA methylation (GWM) of all CpG island in normal, adenoma and CRC from African Americans (AA). Materials and methods: Tissue microarrays (TMA) with colon adenoma (134), cancer (58) and matched normal tissue from AA patients were stained for H3K18, H4K12 and HDAC2 by immunohistochemistry. Trends in intensity of staining were tested by analysis of variance. The correlation between expression of these proteins and various clinicopathological features were analyzed. In addition, DNA from 22 (two normal, eight adenoma, 12 cancer) fresh frozen samples was analyzed for GWM on an Illumina Infinium methylation array (HumanMethylation27). Data was normalized using the methylumi package for the R statistical analysis software. For each type of sample (normal, adenoma or cancer), the top 100-200 hyperand hypo-methylated genes were selected by comparing the confidence intervals for the methylation status to 0 (unmethylated) or 1 (methylated). We also compared the cancer and adenoma samples. From this comparison, 24 genes were found (by inspection) to have high methylation in CRC and adenoma compared to normal or vice versa which we did an Ingenuity Pathway Analysis (IPA). Methylation specific PCR (MSP) was used to validate few candidate genes. Comparative analysis was also done between our data and data from The Cancer Genome Atlas (TCGA) study. Results: HDAC2 nuclear expressionwas 81.9%, 62.1%, and 53.1% in cancer, adenoma, and normal tissue, respectively (P<0.002). H4K12 acetylation was 71.7%, 61%, and 43.6%, respectively (P<0.002). H4K12 acetylation and HDAC2 expression correlated with progression of adenoma to carcinoma (P<0.002). The GWM and IPA, revealed that Wnt/b-catenin signaling was one of the top canonical pathways implicated in the 24 genes differentially methylated between cancer and adenoma compared to normal. Hypermethylated genes were 307 in AA adenoma (including CBFA, ATP1A, ANKDD1, FLI, GDPD3. DRD5, TCF4 and SerpinB3) vs. normal, while there was 185 genes in Whites using TCGA. A near perfect [adenoma + normal] cluster and cancer cluster was observed indicating the significance and importance of DNA methylation in CRC progression. Methylation for TCF was 92%, in CRC and 70% of adenoma cases. Conclusion: These results suggest that global H4 acetylation and HDAC2 expression might be associated with colorectal tumor progression. Our data indicated a high methylation profile of SerpinB3 and TCF4 as potential markers in adenoma as part of the early events of colon carcinogenesis. DNA methylation profile and histone modification can predict colorectal adenoma and cancer.