Some colon cancer (CC) patients present synchronous cancers at diagnosis and others develop metachronous neoplasms, but the risk factors are unclear for non-hereditary CC. We showed previously that global DNA demethylation increased with aging and correlated with genomic damage in CC, and we show now that preferentially associates to CCs with wild-type p53. This study aimed to elucidate the extent of DNA hypomethylation in patients with single and multiple CC, its relationship with aging, and its potential as predictive tool. We compared by real-time methylation-specific PCR the relative demethylation level (RDL) of long interspersed nucleotide element-1 (LINE-1) sequences in matched cancer tissues and non-cancerous colonic mucosa (NCM) from patients with single and multiple right-sided CCs. Although no RDL difference was found in NCM from single CC patients and healthy volunteers (P =0.5), there was more demethylation (higher RDL) in NCM from synchronous cancer patients (P =1.1 × 10−5) multiple CCs also were more demethylated than single CCs (P =0.0014). High NCM demethylation was predictive for metachronous neoplasms (P =0.003). In multivariate logistic regression analyses RDL was the only independent predictor for metachronous (P =0.02) and multiple (P =4.9 × 10−5) tumors. The higher LINE-1 demethylation in NCM from patients with multiple (synchronous and metachronous) tumors (P =9.6 × 10−7) was also very significant in patients with tumors without (P =3.8 × 10−6), but not with (P =0.16) microsatellite instability. NCM demethylation increased with aging in patients with single tumors, but decreased in those with multiple tumors. Moreover, the demethylation difference between patients with single vs multiple tumors appeared higher in younger (P =3.6 × 10−4) than in older (P =0.0016) patients. These results predict that LINE-1 hypomethylation in NCM can be used as an epigenetic predictive biomarker for multiple CC risk. The stronger association of demethylation in NCM with multiple CC risk from younger patients also suggests an inherited predisposition for the apparent field cancerization effect of somatic demethylation.

DNA hypomethylation at repetitive elements accounts for the genome-wide DNA hypomethylation common in cancer, including colorectal cancer (CRC). We identified a pericentromeric repeat element called SST1 frequently hypomethylated (>5% demethylation compared with matched normal tissue) in several cancers, including 28 of 128 (22%) CRCs. SST1 somatic demethylation associated with genome damage, especially in tumors with wild-type TP53. Seven percent of the 128 CRCs exhibited a higher ("severe") level of demethylation (≥10%) that co-occurred with TP53 mutations. SST1 demethylation correlated with distinct histone marks in CRC cell lines and primary tumors: demethylated SST1 associated with high levels of the repressive histone 3 lysine 27 trimethylation (H3K27me3) mark and lower levels of histone 3 lysine 9 trimethylation (H3K9me3). Furthermore, induced demethylation of SST1 by 5-aza-dC led to increased H3K27me3 and reduced H3K9me3. Thus, in some CRCs, SST1 demethylation reflects an epigenetic reprogramming associated with changes in chromatin structure that may affect chromosomal integrity. The chromatin remodeler factor, the helicase lymphoid-specific (HELLS) enzyme, called the "epigenetic guardian of repetitive elements", interacted with SST1 as shown by chromatin immunoprecipitation, and down-regulation of HELLS by shRNA resulted in demethylation of SST1 in vitro. Altogether these results suggest that HELLS contributes to SST1 methylation maintenance. Alterations in HELLS recruitment and function could contribute to the somatic demethylation of SST1 repeat elements undergone before and/or during CRC pathogenesis.