Temporal evolution in caveolin 1 methylation levels during human esophageal carcinogenesis

Abstract

Background: Esophageal cancer ranks eighth among frequent cancers worldwide. Our aim was to investigate whether and at which neoplastic stage promoter hypermethylation of CAV1 is involved in human esophageal carcinogenesis.

Methods: Using real-time quantitative methylation-specific PCR (qMSP), we examined CAV1 promoter hypermethylation in 260 human esophageal tissue specimens. Real-time RT-PCR and qMSP were also performed on OE33 esophageal cancer cells before and after treatment with the demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC).

Results: CAV1 hypermethylation showed highly discriminative ROC curve profiles, clearly distinguishing esophageal adenocarcinomas (EAC) and esophageal squamous cell carcinomas (ESCC) from normal esophagus (NE) (EAC vs. NE, AUROC = 0.839 and p < 0.0001; ESCC vs. NE, AUROC = 0.920 and p < 0.0001). Both CAV1 methylation frequency and normalized methylation value (NMV) were significantly higher in Barrett's metaplasia (BE), low-grade and high-grade dysplasia occurring in BE (D), EAC, and ESCC than in NE (all p < 0.01, respectively). Meanwhile, among 41 cases with matched NE and EAC or ESCC, CAV1 NMVs in EAC and ESCC (mean = 0.273) were significantly higher than in corresponding NE (mean = 0.146; p < 0.01, Student's paired t-test). Treatment of OE33 EAC cells with 5-Aza-dC reduced CAV1 methylation and increased CAV1 mRNA expression.

Conclusions: CAV1 promoter hypermethylation is a frequent event in human esophageal carcinomas and is associated with early neoplastic progression in Barrett's esophagus.

Figure 1

Receiver-operator characteristic (ROC) curve analysis of normalized methylation value (NMV). ROC curve analysis of CAV1 NMVs of normal esophagus (NE) vs. esophageal adenocarcinoma (EAC) (A), and NE vs. esophageal squamous cell carcinoma (ESCC) (B). The area under the ROC curve (AUROC) conveys this biomarker’s accuracy in distinguishing NE from EAC and from ESCC in terms of its sensitivity and specificity.

Figure 2

Methylation status of CAV1 in matched esophageal tissue samples. A, NMVs of CAV1 in 41 patients with matched NE and esophageal cancer (T, EAC or ESCC). B, methylation status of CAV1 in 41 cases with corresponding NE and T. C, methylation status of CAV1 in 15 cases with corresponding NE, BE and EAC.

Figure 3

Methylation profiles of CAV1 in different esophageal tissue samples. A, The mean NMV of CAV1 was significantly higher in ESCC, EAC, D, and BE than in NE, and in BE than in D and EAC. B. The frequency of CAV1 hypermethylation was significantly higher in BE, D, EAC and ESCC than in NE, and in BE than in D and EAC. NE: normal esophagus; BE: Barrett’s metaplasia; D: Dysplasia in BE; EAC: esophageal adenocarcinoma; ESCC: esophageal squamous cell carcinoma; *: Student’s t test, p < 0.01 (comparisons made to NE); $: Student’s t test, p < 0.01 (comparisons made to BE); †: Chi-square for independence test, p < 0.01 (comparisons made to NE); ‡: Fisher’s exact test, p < 0.01 (comparisons made to NE); §: Chi-square for independence test, p < 0.05 (comparisons made to BE).

Figure 4

CAV1 methylation and mRNA expression in OE33 cells after treatment with 5-aza-2’-deoxycytidine (5-Aza-dC). After 5-Aza-dC treatment, the NMV of CAV1 was diminished, while the normalized mRNA value (NRV) of CAV1 was increased.