Inactivation of CCAAT/Enhancer Binding Protein Human Hepatocellular Carcinomas and Is Mediated through Metallothionein Expression Is Suppressed in Primary

Reactive oxygen species (ROS) resulting from chronic inflammation cause liver injury leading to transformation of regenerating hepatocytes. Metallothioneins (MT), 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 MT 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 MT 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 MT expression specifically in HCC cells. Short interfering RNA– mediated depletion of CCAAT/enhancer binding protein A (C/EBPA), a target of GSK-3, impeded MT expression, which could not be reversed by PI3K inhibitors. DNA binding activity of C/EBPA and its phosphorylation at T222 and T226 by GSK-3 are required for MT expression. MTF-1 and C/EBPA act in concert to increase MT-2A expression, which probably explains the high level of MT expression in the liver. This study shows the role of PI3K/AKT signaling pathway and C/EBPA in regulation of MT expression in hepatocarcinogenesis. [Cancer Res 2007;67(6):2736–46] Introduction Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer in the world and is most common form of liver cancer with 5-year survival rate of only 7% (1–3). The high mortality is due to late-stage detection of this cancer when most of the therapies available are not effective (4, 5). Hepatitis B (HBV) or hepatitis C (HCV) viral infection that causes chronic liver disease and inflammation and cirrhosis plays an important role in etiology of HCC. In United States, alcoholism is the most common cause of HCC, whereas consumption of food contaminated with aflatoxin is the major cause of HCC in Africa and Asia. Alcohol or aflatoxin intake further increases the risk for HCC in individuals exposed to chronic HCV (6). Metastasis from other cancerous organs that include colorectal, pancreas, and breast and diabetes (3) also contribute to liver cancer. The disease is progressive and death usually occurs within 10 months of initial diagnosis from cachexia, gastrointestinal bleeding, liver failure, or rupture of the tumor with massive hemorrhage. Generation of excessive free radicals in tissues resulting from chronic inflammation damages cellular macromolecules, including DNA, which lead to mutation, apoptosis, hyperproliferation, and ultimately cancer (7). Metallothioneins (MT), a group of stress response proteins induced at a high level by oxidative stress, are efficient scavengers of reactive oxygen species (ROS) and reactive nitrogen species (8–10). These are evolutionarily conserved, ubiquitously expressed, and cysteine-rich, heavy metal binding proteins. Four isoforms of MT are arranged in tandem on mouse chromosome 8 and on human chromosome 16 (11). Human genome contains several MT-1 variants of which some are pseudogenes. MT-2A is the major MT isoform expressed in human. Expression of MT-1 and MT-2 are coordinately regulated in all tissues. An important role of MTs is to preserve homeostasis of biologically essential metals, such as zinc and copper, and to scavenge the toxic metals, such as cadmium and mercury (8, 9). The major function of MT is to scavenge free radicals by using its unique metal-thiolate clusters that act as redox sensor and are rapidly oxidized by ROS releasing apo-MT and the metal ions (12, 13). Overproduction of MT at a high level selectively in the heart can protect mice from the cardiotoxic effects of the potent anticancer drug Adriamycin (13). MTs are expressed at high levels in the liver and are dramatically induced by a variety of agents (8, 9), whereas these genes are not induced in rodent hepatomas following exposure to heavy metals Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). This work is dedicated to the memory of Dr. Robert Costa. Requests for reprints: Samson T. Jacob, Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, Columbus, OH 43210. Phone: 614-688-5494; Fax: 614-688-5600; E-mail: [email protected] and Kalpana Ghoshal, Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, Columbus, OH 43210. Phone: 614-292-8865; Fax: 614-2924118; E-mail: [email protected]. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-4433 Cancer Res 2007; 67: (6). March 15, 2007 2736 www.aacrjournals.org Research Article American Association for Cancer Research Copyright © 2007 on February 23, 2013 cancerres.aacrjournals.org Downloaded from DOI:10.1158/0008-5472.CAN-06-4433