The DNA hypermethylation phenotype of colorectal cancer liver metastases resembles that of the primary colorectal cancers

Abstract

Abstract Background Identifying molecular differences between primary and metastatic colorectal cancers—now possible with the aid of omics technologies—can improve our understanding of the biological mechanisms of cancer progression and facilitate the discovery of novel treatments for late-stage cancer. We compared the DNA methylomes of primary colorectal cancers (CRCs) and CRC metastases to the liver. Laser microdissection was used to obtain epithelial tissue (10 to 25 × 106 μm2) from sections of fresh-frozen samples of primary CRCs (n = 6), CRC liver metastases (n = 12), and normal colon mucosa (n = 3). DNA extracted from tissues was enriched for methylated sequences with a methylCpG binding domain (MBD) polypeptide-based protocol and subjected to deep sequencing. The performance of this protocol was compared with that of targeted enrichment for bisulfite sequencing used in a previous study of ours. Results MBD enrichment captured a total of 322,551 genomic regions (249.5 Mb or ~ 7.8% of the human genome), which included over seven million CpG sites. A few of these regions were differentially methylated at an expected false discovery rate (FDR) of 5% in neoplastic tissues (primaries: 0.67%, i.e., 2155 regions containing 279,441 CpG sites; liver metastases: 1%, i.e., 3223 regions containing 312,723 CpG sites) as compared with normal mucosa samples. Most of the differentially methylated regions (DMRs; 94% in primaries; 70% in metastases) were hypermethylated, and almost 80% of these (1882 of 2396) were present in both lesion types. At 5% FDR, no DMRs were detected in liver metastases vs. primary CRC. However, short regions of low-magnitude hypomethylation were frequent in metastases but rare in primaries. Hypermethylated DMRs were far more abundant in sequences classified as intragenic, gene-regulatory, or CpG shelves-shores-island segments, whereas hypomethylated DMRs were equally represented in extragenic (mainly, open-sea) and intragenic (mainly, gene bodies) sequences of the genome. Compared with targeted enrichment, MBD capture provided a better picture of the extension of CRC-associated DNA hypermethylation but was less powerful for identifying hypomethylation. Conclusions Our findings demonstrate that the hypermethylation phenotype in CRC liver metastases remains similar to that of the primary tumor, whereas CRC-associated DNA hypomethylation probably undergoes further progression after the cancer cells have migrated to the liver.

Keywords

• Normal colorectal mucosa
• Colorectal cancer
• Liver metastasis
• DNA methylation
• CpG sites
• CpG islands