Center for Human Genetics, Université Catholique de Louvain, Cliniques universitaires Saint-Luc, Avenue Hippocrate 10, Brussels, B-1200, Belgium.
Clin Epigenetics. 2012 Jun 15;4(1):9. doi: 10.1186/1868-7083-4-9.
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. 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.
Detailed 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).
p14ARF epigenetic silencing may represent an important deregulating mechanism of the p53-MDM2-p14ARF pathway in CRCs exhibiting a restricted p53 overexpression pattern.
已经有人提出，p14ARF 的失活，作为一种通过与 MDM2 癌蛋白相互作用来调节 p53 蛋白稳定性的肿瘤抑制因子，会破坏保留野生型 TP53 基因的人类肿瘤中的 p53 活性。肿瘤抑制基因的表达差异通常与癌症有关。我们之前报道过一种模式，即微卫星不稳定（MSI）、TP53 突变频率低和结直肠癌（CRC）中 MDM2 过表达与明显的 p53 免疫组化过表达显著相关。在这项研究中，我们研究了 p14ARF 改变是否可能是这种 CRC 亚群中 p53 途径失活的机制。
我们在 98 例 CRC 队列中确定了 p14ARF 基因改变的详细图谱，以检测核苷酸和拷贝数的变化。甲基化特异性 PCR 结合亚硫酸氢盐测序用于评估 p14ARF 甲基化的流行率和分布。然后将 p14ARF 改变与 MSI 状态、TP53 突变以及 p53 和 MDM2 的免疫组化表达相关联。p14ARF 突变的频率极低（1/98；1%），而肿瘤和正常结肠黏膜中同时存在甲基化和未甲基化等位基因是常见的（91/98；93%）。只有 98 例肿瘤中的 7 例（7%）与正常结肠黏膜有明显不同的甲基化模式。在 35 例患者中，对包含 27 个 CpG 位点的 p14ARF 启动子区域的甲基化流行率和分布进行评估，显示肿瘤中存在甲基化 CpG 位点的范围为 0 至 25（95%CI 1 至 13），而相邻结肠黏膜中的范围为 0 至 17（95%CI 0 至 2）（P=0.004）。p14ARF 启动子的高甲基化与限制型 p53 过表达模式显著相关（P=0.03），并且与 MDM2 过表达相关（P=0.02），独立于 MSI 表型。尽管未观察到 p14ARF 甲基化与 TP53 突变状态之间有显著相关性（P=0.23），但在限制型 p53 过表达的肿瘤中，5'CpG 侧翼外显子 1β内的近端 CpG 位点的甲基化比弥漫性 p53 过表达的肿瘤更常见（甲基化克隆的范围为 17%至 36%（95%CI 24%至 36%），而范围为 0%至 3%（95%CI 0%至 3%），P=0.0003）。
p14ARF 的表观遗传沉默可能代表了在表现出限制型 p53 过表达模式的 CRC 中 p53-MDM2-p14ARF 途径的一个重要调节机制。
