Epigenetic alteration of the Wnt inhibitory factor-1 promoter occurs early in the carcinogenesis of Barrett's esophagus

Abstract

The role of Wnt antagonists in the carcinogenesis of esophageal adenocarcinoma (EAC) remains unclear. We hypothesized that downregulation of the Wnt inhibitory factor-1 (WIF-1) might be involved in the neoplastic progression of Barrett's esophagus (BE). We analyzed the DNA methylation status of the WIF-1 promoter in normal, preneoplastic, and neoplastic samples from BE patients and in EAC cell lines. We investigated the role of WIF-1 on EAC cell growth and the chemosensitization of the cells to cisplatin. We found that silencing of WIF-1 correlated with promoter hypermethylation. EAC tissue samples showed higher levels of WIF-1 methylation compared to the matched normal epithelium. In addition, we found that WIF-1 hypermethylation was more frequent in BE samples from patients with EAC than in BE samples from patients who had not progressed to EAC. Restoration of WIF-1 in cell lines where WIF-1 was methylation-silenced resulted in growth suppression. Restoration of WIF-1 could sensitize the EAC cells to the chemotherapy drug cisplatin. Our results suggest that silencing of WIF-1 through promoter hypermethylation is an early and common event in the carcinogenesis of BE. Restoring functional WIF-1 might be used as a new targeted therapy for the treatment of this malignancy.

Figure 1

Correlation of methylation in the promoter region with silencing of the Wnt inhibitory factor‐1 (WIF‐1) gene in esophageal adenocarcinoma (EAC) cell lines. (a) Reverse transcription–polymerase chain reaction (RT‐PCR) analysis of the WIF‐1 gene in five EAC cell lines. The amplified fragment was 451 bp. A 180‐bp fragment of the glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) gene was used as a positive control for RNA quality and loading. C+ and C– corresponded to normal esophagus RNA (BD Biosciences Clontech, Palo Alto, CA) and H₂O, respectively, used as a control during the RT‐PCR. (b) Methylation‐specific polymerase chain reaction analysis of WIF‐1 methylation in EAC cell lines. Bands (198 bp) in lanes labeled “U” are unmethylated DNA product amplified with unmethylation‐specific primers. Bands in lanes labeled “M” are methylated DNA product amplified with methylation‐specific primers. (c) Bisulfite‐sequencing analysis of 64 CpG sites in the region –554 to +118 of the WIF‐1 promoter in EAC cell lines. White squares represent unmethylated CpG sites and black squares indicate methylated CpG sites. Black and gray arrows represent the primers used to amplify the promoter regions –554 to –141 and –161 to +118, respectively. Ten individual clones were analyzed for each cell line. Transcription (TSS) and translation (ATG) start sites are represented (RefSeq NM_007191). (d) Reactivation of WIF‐1 expression by 5‐aza‐2′‐deoxycytidine (5‐azadC) treatment in cell lines. RT‐PCR analysis of the WIF‐1 gene in EAC cell lines after treatment (2 µM for 6 days) or without treatment with the demethylating agent 5‐azadC. The GAPDH gene was used as a positive control for RNA quality and loading. C+ and C– corresponded to normal esophagus RNA and H₂O, respectively, used as a control during the RT‐PCR.

Figure 2

Methylation analysis of the Wnt inhibitory factor‐1 (WIF‐1) promoter in esophageal adenocarcinoma (EAC) tissue samples. (a) Methylation‐specific polymerase chain reaction analysis of 17 matched pairs of frozen normal (N) and EAC (T) tissue samples and of one unpaired EAC sample (T13). Bands (198 bp) in lanes labeled “U” are unmethylated DNA product amplified with unmethylation‐specific primers. Bands in lanes labeled “M” are methylated DNA product amplified with methylation‐specific primers. (b) WIF‐1 promoter methylation by methylation‐sensitive single‐strand conformation analysis in four microdissected formalin‐fixed paraffin‐embedded normal squamous epithelia and four EAC samples. Square brackets indicate the migration of either the umethylated DNA (unmethyl. CpGs) or the methylated DNA (methyl. CpGs). + and – indicate the methylation status of each sample.

Figure 3

Methylation analysis of the Wnt inhibitory factor‐1 (WIF‐1) promoter in Barrett's esophagus (BE) tissue samples. (a) WIF‐1 promoter methylation by methylation‐sensitive single‐strand conformation analysis in four BE patients without esophageal adenocarcinoma (EAC) and in four BE patients with EAC. Square brackets indicate the migration of either the umethylated DNA (unmethyl. CpGs) or the methylated DNA (methyl. CpGs). + and – indicate the methylation status of each sample. (b) Graphical representation of the methylated and unmethylated samples of BE without and BE with EAC.

Figure 4

Effect of the restoration of Wnt inhibitory factor‐1 (WIF‐1) expression on the Wnt‐dependent transcription activity. OE19 and BIC‐1 cells were co‐transfected with 1.5 µg of WIF‐1 expression vector or empty vector (pcDNA3.1) and 0.5 µg of TOPflash or FOPflash reporter. As a positive control, OE19 cells were cotransfected with 1.5 µg of SFPR4 expression vector or empty vector 2 (pcDNA3) and 0.5 µg of TOPflash or FOPflash reporter. Firefly luciferase activity of the reporters is represented after normalization of each sample with the Renilla luciferase activity. The experiment was carried out in triplicate. The expression level of WIF‐1 in OE19 and BIC‐1 cells was detected by reverse transcription–polymerase chain reaction (RT‐PCR) after transfection of the empty vector (e.v.) or WIF‐1 expression vector. The amplified fragment was 451 bp. A 180‐bp fragment of the glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) gene was used as a positive control for RNA quality and loading. C+, normal esophagus RNA; C–, H₂O, used as a control during RT‐PCR.

Figure 5

Growth suppression of OE19 cells by restoration of Wnt inhibitory factor‐1 (WIF‐1) expression. (a) Cell proliferation of OE19 cells after transfection of WIF‐1 expression vector or empty vector. Measurements were carried out 2 days after transfection (day 0) and repeated during the following five consecutive days. Results are means of sixplicates with error bars (standard deviation [SD]). Expression level of introduced WIF‐1 was detected by reverse transcription–polymerase chain reaction as for Fig. 4. (b) Colony formation assay using OE19 cells. The cells were transfected with empty vector or WIF‐1 expression vector, selected with G418 for 10 days and 2000 cells were plated in triplicate in 10‐cm dishes. Selection was maintained for four additional weeks. The bar graph represents the average of colony numbers of the triplicated experiments. Error bars are SD.

Figure 6

Growth suppression of OE19 cells by restoration of Wnt inhibitory factor‐1 (WIF‐1) expression and treatment with the chemotherapy drug cisplatin. (a) Cell proliferation of OE19 cells after transfection of WIF‐1 expression vector or empty vector. Cells were subjected to cisplatin 2 days after transfection (day 0) and measurements of the cell proliferation were repeated during the following five consecutive days. Cisplatin was added fresh every day. Results are means of sixplicates with error bars (standard deviation). (b) Graphical representation of the cell proliferation decrease at day 5 for each treatment.