Abstract
BackgroundIt has been suggested that inactivation of p14ARF, 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 p14ARF alterations could be a mechanism for disabling the p53 pathway in this subgroup of CRCs.ResultsDetailed maps of the alterations in the p14ARF 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 p14ARF methylation. p14ARF alterations were then correlated with MSI status, TP53 mutations, and immunohistochemical expression of p53 and MDM2. The frequency of p14ARF 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 p14ARF 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 p14ARF 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 p14ARF 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).Conclusionp14ARF epigenetic silencing may represent an important deregulating mechanism of the p53-MDM2-p14ARF pathway in CRCs exhibiting a restricted p53 overexpression pattern.
Highlights
It has been suggested that inactivation of p14ARF, 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
Inactivation of the p14ARF gene has been proposed as a mechanism that is functionally equivalent to an inactivating p53 mutation, in that it disrupts p53 activity in tumors retaining the wild-type TP53 gene [4], and more in sporadic microsatellite instability (MSI)-H colorectal cancer (CRC) [8,9]
Evaluation of density of p14ARF promoter methylation in tumors and normal colon mucosa from patients with colorectal cancer we evaluated the degree of p14ARF promoter methylation, limiting the analysis to tumors and corresponding adjacent colon mucosa from 35 randomly selected patients (Table 1), including one of the seven CRCs that was identified as having heavy methylation by Methylation-specific polymerase chain reaction (PCR) (MSP)
Summary
It has been suggested that inactivation of p14ARF, 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. A significant proportion of CRCs, which include mainly microsatellite instability-high (MSI-H) CRCs, and a subset of microsatellite-stable (MSS) sporadic CRCs, display a particular immunohistochemical p53 expression pattern characterized by an accumulation of p53 protein restricted to a limited number of tumor cells, a profile that we previously termed ‘restricted p53 overexpression’ [7]. This CRC subgroup has an extremely low frequency of TP53 mutation, and displays overexpression of MDM2 and normal expression of p21, suggesting that deregulation of p53 pathway in this CRC subgroup may be due to other alternative mechanisms than TP53 mutation. We conducted detailed genetics and epigenetics analysis of the p14ARF gene in CRC tumors for which we had complete data on MSI status and DNA mismatch repair deficiency or sufficiency, and we investigated the relationships between p14ARF alterations and MSI phenotype, between p14ARF alterations and the p53 protein expression pattern and its mutational status, as well as with MDM2 protein expression
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