Metallothionein expression is suppressed in primary human hepatocellular carcinomas and is mediated through inactivation of CCAAT/enhancer binding protein α by phosphatidylinositol 3-kinase signaling cascade

Reactive oxygen species (ROS) resulting from chronic inflammation cause liver injury leading to transformation of regenerating hepatocytes. Metallothioneins (NIT), induced at high levels by oxidative stress, are potent scavengers of ROS. Here, we report that the levels of MT-1 and MT-2A are drastically reduced in primary human hepatocellular carcinomas (HCCs) and in diethylnitrosamine-induced liver tumors in mice, which is primarily due to transcriptional repression. Expression of the transcription factor, MTF-1, essential for NIT expression, and its target gene Zn-T1 that encodes the zinc transporter-1 was not significantly altered in HCCs. Inhibitors of both phosphatidylinositol 3-kinase (PI3K) and its downstream target AKT increased expression of MT genes in HCC cells but not in liver epithelial cells. Suppression of MT-1 and MT-2A by ectopic expression of the constitutively active PI3K or AKT and their up-regulation by dominant-negative PI3K or AKT mutant confirmed negative regulation of NIT expression by PI3K/AKT signaling pathway. Further, treatment of cells with a specific inhibitor of glycogen synthase kinase-3 (GSK-3), a downstream effector of PI3K/AKT, inhibited NIT expression specifically in HCC cells. Short interfering RNA-mediated depletion of CCAAT/enhancer binding protein alpha (C/ERP alpha), a target of GSK-3, impeded NIT expression, which could not be reversed by PI3K inhibitors. DNA binding activity of C/EBP alpha and its phosphorylation at T222 and T226 by GSK-3 are required for NIT expression. MTF-1 and C/EBP alpha act in concert to increase MT-2A expression, which probably explains the high level of NIT expression in the liver. This study shows the role of PI3K/AKT signaling pathway and C/EBP alpha in regulation of NIT expression in hepatocarcinogenesis.