Widespread Hypomethylation Occurs Early and Synergizes with Gene Amplification during Esophageal Carcinogenesis

Abstract

Although a combination of genomic and epigenetic alterations are implicated in the multistep transformation of normal squamous esophageal epithelium to Barrett esophagus, dysplasia, and adenocarcinoma, the combinatorial effect of these changes is unknown. By integrating genome-wide DNA methylation, copy number, and transcriptomic datasets obtained from endoscopic biopsies of neoplastic progression within the same individual, we are uniquely able to define the molecular events associated progression of Barrett esophagus. We find that the previously reported global hypomethylation phenomenon in cancer has its origins at the earliest stages of epithelial carcinogenesis. Promoter hypomethylation synergizes with gene amplification and leads to significant upregulation of a chr4q21 chemokine cluster and other transcripts during Barrett neoplasia. In contrast, gene-specific hypermethylation is observed at a restricted number of loci and, in combination with hemi-allelic deletions, leads to downregulatation of selected transcripts during multistep progression. We also observe that epigenetic regulation during epithelial carcinogenesis is not restricted to traditionally defined “CpG islands,” but may also occur through a mechanism of differential methylation outside of these regions. Finally, validation of novel upregulated targets (CXCL1 and 3, GATA6, and DMBT1) in a larger independent panel of samples confirms the utility of integrative analysis in cancer biomarker discovery. The incidence of esophageal adenocarcinoma (EA) is increasing at an alarming pace in the United States. Distinct pathological stages of Barrett's metaplasia and low- and high-grade dysplasia can be seen preceding malignant transformation. These precursor lesions provide a unique in vivo model for deepening our understanding the early steps in human neoplasia. By integrating genome-wide DNA methylation, copy number, and transcriptomic datasets obtained from endoscopic biopsies of neoplastic progression within the same individual, we are uniquely able to define the molecular events associated progression of Barrett esophagus. We show that the predominant change during this process is loss of DNA methylation. We show that this global hypomethylation occurs very early during the process and is seen even in preinvasive lesions. This loss of DNA methylation drives carcinogenesis by cooperating with gene amplifications in upregulating proteins during this process. Finally we uncovered proteins that upregulated by loss of methylation or gene amplification (CXCL1 and 3, GATA6, and DMBT1) and show their relevance by validating their levels in larger independent panel of samples, thus confirming the utility of integrative analysis in cancer biomarker discovery.