Integrative array-based approach identifies MZB1 as a frequently methylated putative tumor suppressor in hepatocellular carcinoma

Purpose: The aim of this study was the identification of novel tumor suppressor genes (TSG) silenced by DNA hypermethylation in hepatocellular carcinoma (HCC). Experimental Design: We conducted integrative array-based approach for genome-wide screening of methylation targets using a methylated DNA immunoprecipitation-CpG island microarray and expression array in three universal hepatoma cell lines and normal liver tissue. Through detailed expression and functional analyses using hepatoma cell lines and primary HCC samples, we isolated novel TSGs for HCC. Results: A total of 642 genes were identified as methylated in three hepatoma cell lines but unmethylated in normal liver tissue, whereas 204 genes on autosomes were identified as genes unexpressed but restored after treatment with 5-aza-2′-deoxycytidine in these cell lines and expressed in normal tissue. Through the integration of results of the two-array analyses and further validation analyses of expression and methylation status in 17 cell lines and 30 primary tumors of hepatoma, we identified MZB1, marginal zone B and B1 cell-specific protein, encoding an endoplasmic reticulum protein, as a putative TSG frequently methylated within its CpG island in hepatoma. Among 162 patients with primary HCC, silencing of MZB1 protein was significantly and independently associated with a worse outcome. Restoration of MZB1 expression in hepatoma cells reduced cell proliferation in vitro and in vivo through G ₁-arrest. Conclusions: These results suggest that methylation-mediated silencing of MZB1 expression leads to loss of its tumor-suppressive activity, which may be a factor in the hepatocarcinogenesis, and is a useful prognosticator in HCC.