Hypermethylation of the 5′ CpG island of the p14(ARF) flanking exon 1β in human colorectal cancer displaying a restricted pattern of p53 overexpression concomitant with increased MDM2 expression

BACKGROUND: It has been suggested that inactivation of p14(ARF), a tumor suppressor central to regulating p53 protein stability through interaction with the MDM2 oncoprotein, abrogates p53 activity in human tumors retaining the wild-type TP53 gene. Differences in expression of tumor suppressor genes are frequently associated with cancer. We previously reported on a pattern of restricted p53 immunohistochemical overexpression significantly associated with microsatellite instability (MSI), low TP53 mutation frequency, and MDM2 overexpression in colorectal cancers (CRCs). In this study, we investigated whether p14(ARF) alterations could be a mechanism for disabling the p53 pathway in this subgroup of CRCs. RESULTS: Detailed maps of the alterations in the p14(ARF) gene were determined in a cohort of 98 CRCs to detect both nucleotide and copy-number changes. Methylation-specific PCR combined with bisulfite sequencing was used to evaluate the prevalence and distribution of p14(ARF) methylation. p14(ARF) alterations were then correlated with MSI status, TP53 mutations, and immunohistochemical expression of p53 and MDM2. The frequency of p14(ARF) mutations was extremely low (1/98; 1%), whereas coexistence of methylated and unmethylated alleles in both tumors and normal colon mucosa was common (91/98; 93%). Only seven of ninety-eight tumors (7%) had a distinct pattern of methylation compared with normal colon mucosa. Evaluation of the prevalence and distribution of p14(ARF) promoter methylation in a region containing 27 CpG sites in 35 patients showed a range of methylated CpG sites in tumors (0 to 25 (95% CI 1 to 13) versus 0 to 17 (95% CI 0 to 2)) in adjacent colon mucosa (P = 0.004). Hypermethylation of the p14(ARF) promoter was significantly correlated with the restricted p53 overexpression pattern (P = 0.03), and MDM2 overexpression (P = 0.02), independently of MSI phenotype. Although no significant correlation between p14(ARF) methylation and TP53 mutational status was seen (P = 0.23), methylation involving the proximal CpG sites within the 5′ CpG flanking exon 1β was present more frequently in tumors with restricted p53 overexpression than in those with diffuse p53 overexpression (range of methylated clones 17 to 36% (95% CI 24 to 36%) versus range 0 to 3% (95% CI 0 to 3%), P = 0. 0003). CONCLUSION: p14(ARF) epigenetic silencing may represent an important deregulating mechanism of the p53-MDM2-p14(ARF) pathway in CRCs exhibiting a restricted p53 overexpression pattern.