Esophageal Barrett's adenocarcinoma (BA) develops through a multistage process, which is associated with the transcriptional silencing of tumor-suppressor genes by promoter CpG island hypermethylation. In this study, we explored the promoter hypermethylation and protein expression of proapoptotic death-associated protein kinase (DAPK) during the multistep Barrett's carcinogenesis cascade. Early BA and paired samples of premalignant lesions of 61 patients were analyzed by methylation-specific polymerase chain reaction and immunohistochemistry. For the association of clinicopathological markers and protein expression, an immunohistochemical tissue microarray analysis of 66 additional BAs of advanced tumor stages was performed. Hypermethylation of DAPK promoter was detected in 20% of normal mucosa, 50% of Barrett's metaplasia, 53% of dysplasia, and 60% of adenocarcinomas, and resulted in a marked decrease in DAPK protein expression (P < .01). The loss of DAPK protein was significantly associated with advanced depth of tumor invasion and advanced tumor stages (P < .001). Moreover, the severity of reflux esophagitis correlated significantly with the hypermethylation rate of the DAPK promoter (P < .003). Thus, we consider DAPK inactivation by promoter hypermethylation as an early event in Barrett's carcinogenesis and suggest that a decreased protein expression of DAPK likely plays a role in the development and progression of BA.

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BACKGROUND and PURPOSE: Salivary gland adenoid cystic carcinoma (ACC) is the second most common malignancy of the salivary glands. It is characterized by a heterogeneous morphology, perineural invasion, and a variable clinical course. The molecular mechanisms underlying ACC are still unknown. We previously developed a high-throughput integrative epigenomic screening method to discover novel proto-oncogene candidates in ACC. With this screening method, we found that cat eye syndrome chromosome region, candidate 1 (CECR1) was a promising oncogene candidate for ACC. CECR1 encodes a member of a subfamily of the adenosine deaminase protein family and acts as a growth factor through its adenosine deaminase activity. No previous studies have correlated CECR1 in human cancers. In the current study, we validated several features of CECR1 in primary ACC samples, including promoter methylation status and expression levels. METHODS and RESULTS: Bisulfite genomic sequencing was first employed to analyze the DNA methylation levels in the promoter of CECR1 in four ACC samples and four normal salivary gland tissues. Decreased methylation in CECR1 was found in three out of four ACC samples compared to zero out of four normal tissues. Secondly, quantitative methylation specific PCR (qMSP) was performed in a larger ACC cohort including 15 ACCs and 18 normal tissues. Consistent with the bisulfite genomic sequencing results, significant demethylation of CECR1 was detected in ACC samples (methylation levels ranged from 0.1 to 300.1; median, 19.8) compared to normal tissues (range from 10.2 to 600.0; median, 235.6), p &lt; 0.05. Thirdly, we induced global demethylation in an ACC cell line, ACC83, by 5-aza-2′-deoxycytidine/ trichostatin A (5 Aza dC/TSA) treatment. Quantitative reverse transcription PCR (qRT-PCR) showed that CECR1 was significantly re-expressed in 5 Aza dC/TSA treated cell lines. Lastly, we analyzed the expression levels of CECR1 by qRT-PCR in the same ACC cohort. We found significant overexpression of CECR1 in ACCs compared to normal salivary tissue, p &lt; 0.05. CONCLUSIONS: This is the first study to associate elevated CECR1 levels with salivary gland ACC. The transcription of CECR1 appears to be upregulated by promoter demethylation. Both overexpression and promoter hypomethylation of CECR1 are hallmarks of ACC. CECR1 might play an important role in salivary gland ACC. FUTURE DIRECTIONS: Functional studies of CECR1's role(s) in the carcinogenesis of salivary gland ACC are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4802. doi:10.1158/1538-7445.AM2011-4802