N-MYC Downstream Regulated Gene 4 (NDRG4), a Frequent Downregulated Gene through DNA Hypermethylation, plays a Tumor Suppressive Role in Esophageal Adenocarcinoma

SIMPLE SUMMARY: Esophageal adenocarcinoma has become a major clinical challenge in the western world due to its rapid increasing incidence and poor overall prognosis. Understanding the molecular events of its tumorigenesis is the key to better diagnosis and development of better therapeutic strategies. In the current study we aimed to identify epigenetic alteration targets in esophageal adenocarcinoma. We focused on a candidate gene, NDRG4 (N-myc downregulated gene 4). We found that NDRG4 was frequent downregulated in esophageal adenocarcinoma through DNA hypermethylation of its promoter region. Re-expression of NRDG4 in cancer cells significantly suppressed tumor growth via inhibition of cell proliferation. These results will improve our understanding on how dysfunction of NDRG4 contributes to esophageal adenocarcinoma. DNA hypermethylation of NDRG4 may be a useful biomarker in clinical monitoring of esophageal adenocarcinoma patients. ABSTRACT: The incidence of esophageal adenocarcinoma (EAC) has been rising dramatically in the past few decades in the United States and Western world. The N-myc downregulated gene 4 (NDRG4) belongs to the human NDRG family. In this study, we aimed to identify the expression levels, regulation, and functions of NDRG4 in EAC. Using an integrative epigenetic approach, we identified genes showing significant downregulation in EAC and displaying upregulation after 5-Aza-deoxycitidine. Among these genes, likely to be regulated by DNA methylation, NDRG4 was among the top 10 candidate genes. Analyses of TCGA (The Cancer Genome Atlas) and GEO (Gene Expression Omnibus) data sets and EAC tissue samples demonstrated that NDRG4 was significantly downregulated in EAC (p < 0.05). Using Pyrosequencing technology for quantification of DNA methylation, we detected that NDRG4 promoter methylation level was significantly higher in EAC tissue samples, as compared to normal esophagus samples (p < 0.01). A strong inverse correlation between NDRG4 methylation and its gene expression levels (r = −0.4, p < 0.01) was observed. Treatment with 5-Aza restored the NDRG4 expression, confirming that hypermethylation is a driving force for NDRG4 silencing in EAC. Pathway and gene set enrichment analyses of TCGA data suggested that NDRG4 is strongly associated with genes related to cell cycle regulation. Western blotting analysis showed significant downregulation of Cyclin D1, CDK4 and CDK6 in EAC cells after overexpression of NDRG4. Functionally, we found that the reconstitution of NDRG4 resulted in a significant reduction in tumor cell growth in two-dimensional (2D) and three-dimensional (3D) organotypic culture models and inhibited tumor cell proliferation as indicated by the EdU (5-ethynyl-2′-deoxyuridine) proliferation assay.