Two distinct pathways of p16 gene inactivation in gallbladder cancer

Abstract

AIM:To examine the mechanism of inactivation of the p16 gene in gallbladder cancer, and to investigate p16 alterations and their correlation with clinicopathological features. METHODS:Specimens were collected surgically from 51 patients with gallbladder cancer. We evaluated the status of protein expression, loss of heterozygosity (LOH), homozygous deletion and promoter hypermethylation using immunohistochemistry, microsatellite analysis, quantitative real-time polymerase chain reaction (PCR) and methylation-specific PCR, respectively. In addition, mutations were examined by direct DNA sequencing. RESULTS:Homozygous deletions of the p16 gene exon2, LOH at 9p21-22, p16 promoter hypermethylation, and loss of p16 protein expression were detected in 26.0% (13/50), 56.9% (29/51), 72.5% (37/51) and 62.7% (32/51), respectively. No mutations were found. LOH at 9p21 correlated with the loss of p16 protein expression (P < 0.05). Homozygous deletion of the p16 gene, a combination LOH and promoter hypermethylation, and multiple LOH at 9p21 were significantly correlated with the loss of p16 protein expression (P < 0.05). LOH at 9p21 and promoter hypermethylation of the p16 gene were detected in 15.4% (2/13) and 92.3% (12/13) of the tumors with homozygous deletion of the p16 gene, respectively. P16 alterations were not associated with clinicopathological features. CONCLUSION:Our results suggest that LOH and homozygous deletion may be two distinct pathways in the inactivation of the p16 gene. Homozygous deletion, a combination of LOH and promoter hypermethylation, and multiple LOH are major mechanisms of p16 inactivation in gallbladder cancer.

DNA promoter methylation as a diagnostic and therapeutic biomarker in gallbladder cancer

“…The use of methylation in the search for new biomarkers in GBC is a promising alternative since this epigenetic modification is an early, progressive and cumulative event in GBC. The methylation frequency of promoter regions of some important tumor suppressor genes, such as p16 CDH1 REPRIMO DAPK-1 and SEMA3B , is high in GBC [29,40,48,50,51,88], and has also been well documented in other cancers.…”

“…This variation could be directly related to ethnic differences. Tadokoro et al [50] found a frequency of 73% of methylation in p16 in samples from Japan, and Takahashi et al [32] a frequency of 24% in samples from Chile. The age of the patients could also introduce variations in results.…”

“…Inactivation of p16 through methylation of the promoter region has been frequently identified in breast, prostate, head and neck, liver, lung, brain, colon and esophageal cancers and cell lines of bladder cancer [66-69] . This is a tendency that is also observed in GBC with a loss of expression of up to 62.5% [48,50]. Takahashi et al [32] reported that a considerable number of genes (8 out of 24 genes) are frequently methylated in GBC in comparison with chronic cholecystitis, varying in methylation frequency between 4% and 80%, with p16 having the lowest rate [32].…”

“…A lower expression of p16 has been reported to increase the activity of D-type cyclin-dependent kinase activity, which translates into aberrant phosphorylation of the retinoblastoma gene, producing uncontrolled cell proliferation [31,46]. Loss of heterozygosity and homozygote deletion are two different pathways of p16 inactivation and have been shown to be combined with hypermethylation of the promoter in GBC [50]. In hepatocellular carcinoma and intrahepatic cholangiocarcinoma, p16 is frequently inactivated by methylation of the promoter and rarely by deletion or mutation [31].…”

“…Furthermore, Ueki et al studied different alterations of p16 in 68 tumors in Chinese patients, finding that only 14.8% presented aberrant hypermethylation [31]. Other investigations have identified a methylation percentage of 72.5% at different stages of progression; nevertheless, a significant relationship to the loss of expression of this protein was not established [50]. When the methylation state of this gene was evaluated in samples from the US and Chile, methylation frequencies of 56% were recorded, with similar methylation patterns in both populations [28].…”