Overexpression of Jab 1 in Hepatocellular Carcinoma and Its Inhibitionby PeroxisomeProliferator-ActivatedReceptor ; Ligands In vitro and In vivo

Purpose: Jun activation domain-binding protein 1 (Jab1) is the fifth subunit of the COP9 signalosome and exhibits oncogenic activity.We investigated Jab1expression in hepatocellular carcinoma (HCC) tissues and cell lines and tested the effect of peroxisome proliferator-activated receptor g (PPARg) ligands onJab1expression. Experimental Design: Jab1expression in HCC tissues and cell lines was studied by real-time reverse transcription-PCR, immunohistochemical staining, and Western blotting. Promoter activity and chromatin immunoprecipitation assays were done to address the inhibition of Jab1 promoter by PPARg ligands. RNA interference was used to clarify PPARg ligand-induced inhibition of Jab1. Anticancer and Jab1-suppressing activity of PPARg ligands was tested in nude mice. Results: Jab1was detected in the nucleus and cytoplasm of HCC tissues and 37% (37 of 99) of tissues exhibited Jab1overexpression. Jab1 expression correlated with sex and hepatitis C virus infection, whereas it was negatively associated with hepatitis B virus infection. Additionally, Jab1was overexpressed in HCC cell lines. PPARg ligands troglitazone and rosiglitazone down-regulated Jab1 expression in HCC cells, and troglitazone directly suppressed Jab1 promoter activity by inhibiting Sp1and Tcf4-mediated transcription. This suppression was mediated via both PPARg-dependent and PPARg-independent mechanisms. Ectopic expression of Jab1 counteracted troglitazone-induced growth inhibition. Animal studies verified that intratumor or i.p. injection of troglitazone attenuated HCC growth and reducedJab1 expression in tumor tissues. Conclusions: Our results indicate that Jab1 is overexpressed in HCC and PPARg ligands may suppressJab1to inhibit the proliferation of HCC cells. Jun activation domain-binding protein 1 (Jab1) was originally identified as a coactivator of activator protein-1 transcription factor and was important for the specificity of activation of target genes by activator protein-1 (1). Subsequent studies indicated that Jab1 is also the fifth subunit of the 450K COP9/ signalosome complex (2, 3). Recent studies suggested that Jab1 is a potential oncogene and may play critical roles in tumorigenesis. First, Jab1 may induce p27 nuclear exportation and degradation to promote cell proliferation (4). Second, Jab1 may trigger the proteolysis of p53 to enhance oncogenic activity (5). Third, Jab1 interacts directly with Smad4 and induces its degradation via the ubiquitin/proteasome pathway, which leads to inhibition of transforming growth factor-h– induced gene transcription (6). Because transforming growth factor-h exhibits anticancer activity in the early stages of tumorigenesis, inhibition of transforming growth factor-h signaling pathway by Jab1 may promote tumor formation. Fourth, Jab1 binds hypoxia-inducible factor-1, prevents its degradation, and enhances its transcriptional activity (7). Upregulation of hypoxia-inducible factor-1 by Jab1 stimulates the expression of vascular endothelial growth factor to increase tumor angiogenesis. Pathologic investigations also showed that Jab1 was up-regulated in several types of human cancer including breast, colon, pancreatic, and lung carcinomas (8–11). These data suggest that Jab1 is a tumor promoter. However, the functional role of Jab1 in the development of hepatocellular carcinoma (HCC) is still unclear. Peroxisome proliferator-activated receptor g (PPARg) are ligand-activated nuclear receptors involved in the control of diverse biological processes via activation of downstream target Human Cancer Biology Authors’ Affiliations: Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University ; Department of Pathology, and Division of Gastroenterology, Department of Internal Medicine, Chang Gung Memorial Hospital-Kaohsiung Medical Center; Institute of Biomedical Sciences, National Sun Yat-Sen University; Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University, Tainan, Taiwan; and National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center, Taiwan, People’s Republic of China Received12/2/07; revised1/21/08; accepted 2/12/08. Grant support: Center for Gene Regulation and Signal Transduction Research of Cheng Kung University and National SunYat-Sen University-Kaohsiung Medical UniversityJoint Research Center (W-C. Hung). The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints:Wen-Chun Hung, Institute of Biomedical Sciences, National SunYat-Sen University, No. 70, Lien-Hai Road, Kaohsiung 804,Taiwan, People’s Republic of China. Phone: 886-7-5252000, ext. 5817; Fax: 886-72259573; E-mail: hung1228@ms10.hinet. net. F2008 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-07-5040 www.aacrjournals. org Clin Cancer Res 2008;14(13) July1, 2008 4045 Research. on August 14, 2017. © 2008 American Association for Cancer clincancerres.aacrjournals. org Downloaded from genes (12). Ligands for PPARg are clinically used for the treatment of non-insulin-dependent diabetes mellitus (13). Recent studies indicated that these ligands exhibited potent anticancer activity (reviewed in ref. 14). Two previous studies showed that a PPARg ligand troglitazone effectively inhibited proliferation of HCC cells in vitro and in vivo (15, 16). The authors also showed that the F-box protein p45 was a molecular target for troglitazone. In this study, we tested whether Jab1 may be another target for PPARg ligands. In addition, the expression of Jab1 in HCC tissues and its clinical significance were also addressed. Materials andMethods Patients and tissues. Surgically excised HCC tissues and surrounding nontumor liver tissues were collected from the archives of the Department of Pathology at Chang Gung Memorial Hospital-Kaohsiung Medical Center between January 1986 and December 1996. Nineteen females and 80 males were included in this study. The mean age of these patients is 55.2 years. Among 99 patients, 65 were infected hepatitis B virus (HBV) and 27 were infected with hepatitis C virus (HCV). Sixty-two HCC cases were concurrent with cirrhosis and 66 cases were with grade 3 to 4 fibrosis. Tumors with size larger than 5 cm were detected in 55 cases, and 26 HCC tissues were poorly differentially. a-Fetoprotein was assayed by MEIA method by using Abbot AXSYM autoanalyzer system, and the cutoff value is 15 ng/mL. Seventy-three HCC cases were a-fetoprotein positive (range, 16-42,350 ng/mL). Written consent forms were obtained before tissue collection, and the study was approved by the Medical Ethics and the Human Clinical Trial Committee of Chang Gung Memorial Hospital-Kaohsiung Medical Center. All of the HCC slides were reviewed by a pathologist (C-C.H.) and representative paraffin-embedded blocks consisting of tumor and adjacent nontumor parts were retrieved for immunohisto-