Colorectal cancer progression to metastasis is associated with dynamic genome-wide biphasic 5-hydroxymethylcytosine accumulation.

Colorectal cancer (CRC) progression from adenoma to adenocarcinoma is associated with global reduction in 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). DNA hypomethylation continues upon liver metastasis. Here we show that 5hmC is increased in metastatic liver tissue relative to the primary colon tumour and that expression of TET2 and TET3 is negatively correlated with risk for metastasis in patients with CRC. Genes associated with increased 5-hydroxymethylcytosine show KEGG enrichment for adherens junctions, cytoskeleton and cell migration around a core cadherin (CDH2) network. Overall, the 5-hydroxymethylcyosine profile in the liver metastasis is similar to normal colon appearing to recover at many loci where it was originally present in normal colon and then spreading to adjacent sites. The underlying sequences at the recover and spread regions are enriched for SALL4, ZNF770, ZNF121 and PAX5 transcription factor binding sites. SALL4 and PAX5 are not expressed during CRC progression, but ZNF770 and ZNF121 transcripts are dynamically regulated by TET2 during CRC progression. Finally, we show in a zebrafish migration assay using SW480 CRISPR-engineered TET knockout and rescue cells that reduced TET expression leads to a reduced migration frequency. Together these results suggest a biphasic trajectory for 5-hydroxymethyation dynamics that has bearing on potential therapeutic interventions aimed at manipulating 5-hydroxymethylcytosine levels. Research using patient tumour samples was conducted under the principles of the World Medical Association Helsinki agreement with ethical approval obtained from the Cambridgeshire Local Research Ethics Committee (LREC references 04/Q0108/ 125 and 06/Q0108/307). From a previously reported cohort of 119 CRC patients, available DNA samples included normal colonic mucosa (n=22), taken from tissue some distance away from the tumour, invasive primary carcinoma (n=65), liver metastatic deposits (n=32) and normal liver (5), taken from a site adjacent to the tumour. Genomic DNA was sonicated (Bioruptor) and pulled down using anti-5hmC (Active Motif, 39769). hMeDIP-seq libraries were prepared using the TruSeq DNA sample preparation kit (Illumina) following manufacturer’s instructions. Adaptor modified genomic DNA was then immunoprecipitated, whole genome amplified, and sequenced on an Illumina Genome Analyzer IIx. Sequence reads were aligned to the human reference genome build 38, filtered to remove greylist regions, and analysed for changes in 5mC/5hmC enrichment according to tissue/disease state.