Loss of disabled-2 expression is an early event in esophageal squamous tumorigenesis

Abstract

Aim: Disabled-2 (DAB2) is a candidate tumor-suppressor gene identified in ovarian cancer that negatively influences mitogenic signal transduction of growth factors and blocks ras activity. In a recent study, we observed down-regulation of DAB2 transcripts in ESCCs using cDNA microarrays. In the present study, we aimed to determine the clinical significance of loss of DAB2 protein in esophageal tumorigenesis, hypothesizing that DAB2 promoter hypermethylation-mediated gene silencing may account for loss of the protein.

Methods: DAB2 expression was analyzed by immunohistochemistry in 50 primary esophageal squamous cell carcinomas (ESCCs), 30 distinct hyperplasia, 15 dysplasia and 10 non-malignant esophageal tissues. To determine whether promoter hypermethylation contributes to loss of DAB2 expression in ESCCs, methylation status of DAB2 promoter was analyzed in DAB2 immuno-negative tumors using methylation-specific PCR.

Results: Loss of DAB2 protein was observed in 5/30 (17%) hyperplasia, 10/15 (67%) dysplasia and 34/50 (68%) ESCCs. Significant loss of DAB2 protein was observed from esophageal normal mucosa to hyperplasia, dysplasia and invasive cancer (P(trend) < 0.001). Promoter hypermethylation of DAB2 was observed in 2 of 10 (20%) DAB2 immuno-negative ESCCs.

Conclusion: Loss of DAB2 protein expression occurs in early pre-neoplastic stages of development of esophageal cancer and is sustained down the tumorigenic pathway. Infrequent DAB2 promoter methylation in ESCCs suggests that epigenetic gene silencing is only one of the mechanisms causing loss of DAB2 expression in ESCCs.

Figure 1

Immunohistochemical analysis of DAB2 in esophageal squamous cell carcinogenesis. A: Cytoplasmic expression of DAB2 in non-malignant normal esophageal mucosa; B: Hyperplastic esophageal mucosa showing no detectable expression of DAB2 protein; C: Dysplastic esophageal mucosa showing loss of DAB2 protein expression; D: ESCC section showing loss of DAB2 protein expression; and E: ESCC showing faint cytoplasmic DAB2 protein expression (A-D: Original magnification x 100; and E: Original magnification x 200).

Figure 2

DAB2 exon-1 promoter hypermethylation. Methylation-specific PCR was done to determine the possibility of promoter methylation-mediated gene silencing of DAB2. Eight of 10 ESCCs showed signal (PCR amplicon) for the unmethylated DAB2 alleles (samples 1, 4-10), while 2 cases showed the presence of both unmethylated and methylated PCR amplicon (samples 2 and 3). Burkitt’s lymphoma cell line Raji was used as a positive control for hypermethylation of DAB2 gene (sample 11). In each sample, lane U represents the unmethylated product, while M represents the methylated product, lane NTC refers to the no template control.