[CANCER RESEARCH 63, 3043–3048, June 15, 2003] Advances in Brief Involvement of PEG10 in Human Hepatocellular Carcinogenesis through Interaction with SIAH11 Hiroshi Okabe, Seiji Satoh, Yoichi Furukawa, Tatsushi Kato, Suguru Hasegawa, Yumi Nakajima, Yoshio Yamaoka, and Yusuke Nakamura2 Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639 [H. O., S. S., Y. F., T. K., S. H., Y. Nakaj., Y. Nakam.], and Department of Gastroenterological Surgery, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 [H. O., S. S., T. K., Y. Y.], Japan Abstract certain HCC cell lines that did not manifest endogenous expression of this gene. In addition, we demonstrated interaction of PEG10 protein Through a genome-wide cDNA microarray, we identified that the with SIAH proteins, which play important roles in apoptosis. Our data paternally expressed gene 10 (PEG10) was highly expressed in a great raise novel insights into mechanisms of hepatocarcinogenesis and majority of hepatocellular carcinomas, although its expression was absent in normal liver cells. Exogenous expression of PEG10 conferred oncogenic suggest that PEG10 might serve as a novel molecular target for activity and transfection of hepatoma cells with antisense S-oligonucleo- treatment of HCCs. tides suppressing PEG10 resulted in their growth inhibition. Additional Materials and Methods experiments revealed that PEG10 protein associated with SIAH1, a me- diator of apoptosis, and that overexpression of PEG10 decreased the cell Cell Lines and Tissue Specimens. HEK293 cells and human hepatoma death mediated by SIAH1. These findings suggested that development of cell lines HepG2, Huh7 and Alexander were obtained from the American Type drug(s) inhibiting PEG10 activity could be a novel approach for the Culture Collection (Manassas, VA). SNU423, SNU449, and SNU475 were treatment of hepatocellular carcinomas. obtained from the Korea cell line bank. All cell lines were grown in mono- Introduction layers in appropriate media supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Sigma, St. Louis, MO) and maintained at 37°C 3 HCC is one of the most common malignancies worldwide. Al- in air containing 5% CO2. All HCCs and corresponding noncancerous liver though several novel therapeutic modalities have been developed in tissues were obtained with informed consent from patients who underwent recent years, prognosis of advanced HCC remains poor. Molecular hepatectomy. RT-PCR. RT-PCR experiments were carried out in 20-␮l volumes of PCR investigations have disclosed involvement of alterations of TP53, buffer (TaKaRa, Tokyo, Japan), with 4 min at 94°C for denaturing followed by CTNNB1, and AXIN1 in hepatocarcinogenesis (1–3) but only in a 20 (for GAPDH) or 30 (for PEG10 and SIAH1) cycles of 94°C for 30 s, 56°C limited fraction of HCCs. Thus, discovery of new target molecules for 30 s, and 72°C for 30 s in the GeneAmp PCR system 9700 (Perkin-Elmer, that are critically involved in a majority of cases and expressed Foster City, CA). Primer sequences were as follows: for GAPDH, forward specifically in tumors will be essential for improving therapeutic 5Ј-ACAACAGCCTCAAGATCATCAG-3Ј and reverse 5Ј-GGTCCACCACT- intervention and prognosis of hepatic cancers. GACACGTTG-3Ј; for PEG10, forward 5Ј-AACAACAACAACAACTC- Microarray technologies have enabled researchers to obtain com- CAAGC-3Ј and reverse 5Ј-TCTGCACCTGGCTCTGCAG-3Ј; and for SIAH1, prehensive data about gene expression, not only in experimental forward 5Ј-TCCAACAATGACTTGGCGAGT-3Ј and reverse 5Ј-CTTTT- models but also in human cancers (4, 5). In a previous report (6), we TCTGTGTGTGGCAGAG-3Ј. compared expression profiles of 20 HCCs with their corresponding Northern Blot Analysis. Human multiple tissue blots (Clontech, Palo Alto, CA) were hybridized with a 32P-labeled PEG10 cDNA. Prehybridization, noncancerous liver tissues using a cDNA microarray consisting of hybridization, and washing were performed according to the supplier’s rec- 23,040 genes. Those experiments disclosed a number of genes that ommendations. The blots were autoradiographed with intensifying screens at appeared to be involved in hepatocarcinogenesis and revealed more- Ϫ80°C for 24 h. over that expression profiles were different between hepatitis B virus- Immunoblotting. The polyclonal antibody to PEG10 was purified from positive and hepatitis C virus-positive HCCs. sera of immunized rabbits with recombinant GST-PEG10 protein produced in To identify ideal therapeutic targets, we chose to investigate genes Escherichia coli. Cell extracts were prepared using lysis buffer [150 mM NaCl, that were commonly and exclusively up-regulated in HCCs, using 1% Triton X-100, 50 mM Tris-HCl (pH 7.4), and 1 mM DTT, with complete data obtained from the microarray. In the work reported here, we Protease Inhibitor Cocktail (Boehringer Mannheim, Mannheim, Germany). isolated the entire transcript of a gene that was selectively expressed Proteins were separated by 10% SDS-PAGE and immunoblotted with the in cancerous tissues. This gene was eventually found to be identical to rabbit anti-PEG10 antibody. Horseradish peroxidase-conjugated goat antirab- bit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) served as the secondary PEG10 (7). Exogenous expression of PEG10 promoted growth of antibody for the ECL Detection System (Amersham Pharmacia Biotech, Pis- cataway, NJ). Received 9/3/02; accepted 5/1/03. Immunohistochemical Staining. Cultured cells on chamber slides were The costs of publication of this article were defrayed in part by the payment of page fixed with PBS containing 4% paraformaldehyde for 15 min, then rendered charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. permeable with PBS containing 0.1% Triton X-100 for 2.5 min at room 1 This work was supported by “Research for the Future” Program Grant 00L01402 temperature. Frozen sections from primary HCCs and noncancerous liver from the Japan Society for the Promotion of Science. tissue were fixed with acetone for 15 min. The cells were incubated with 2% 2 To whom requests for reprints should be addressed, at Laboratory of Molecular BSA in PBS for 24 h at 4°C and hybridized with the anti-PEG10 antibody. Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5372; Fax: Antibodies were stained with fluorescent substrate-conjugated antirabbit sec- 81-3-5449-5433; E-mail: [email protected]. ondary antibody (ICN Pharmaceuticals, Costa Mesa, CA). Nuclei were coun- 3 The abbreviations used are: HCC, hepatocellular carcinoma; HEK293, human em- terstained with 4Ј,6-diamidino-2-phenylindole. Fluorescent images were ob- bryonic kidney 293; PEG10, the paternally expressed gene 10; GST, glutathione S- tained with an Eclipse E800 microscope (Nikon, Tokyo, Japan). transferase; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MOI, multiplicity of infection; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- Colony Formation Assay and Growth Suppression Assay. Cells trans- diphenyltetrazolium bromide. fected with plasmid vector expressing the entire coding region of PEG10 3043 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2003 American Association for Cancer Research. INVOLVEMENT OF PEG10 IN HEPATOCARCINOGENESIS Fig. 1. Expression of PEG10 in adult human tissues and primary HCCs. A, multiple tissue Northern blot analysis of PEG10 in adult human tissues. B, gene and protein expression of PEG10 in six hepatoma cell lines. RT-PCR was carried out using a PEG10-specific PCR primer set (top panel). GAPDH served as an internal control. Immunoblotting was performed using anti-PEG10 antibody. The amount of protein applied in the SDS-PAGE was evaluated by the Coomassie Brilliant Blue (CBB) staining. C, subcellular localization of PEG10 protein in HCC cell lines (magnification, ϫ600). D, immunohistochemical staining of PEG10 in a primary HCC and the corresponding noncancerous liver tissue. (magnification, ϫ600). using FuGENE6 reagent according to the supplier’s protocol (Boehringer were analyzed by immunoblotting using anti-His probe antibody (Santa Mannheim) were cultured with an appropriate concentration of geneticin Cruz Biotechnology) or anti-PEG10 antibody. Similarly, GST or GST- for 2 weeks, fixed with 100% methanol, and stained by Giemsa solution. PEG10 fusion protein, immobilized on Glutathione Sepharose 4B beads Colonies Ͼ 1 mm were counted 2 weeks after transfection of pcDNA (Amersham Pharmacia Biotech, Uppsala, Sweden), was incubated with 3.1(ϩ), pcDNA 3.1(Ϫ)/PEG10, or pcDNA 3.1(ϩ)/PEG10. Cells trans- lysates from HEK293-SIAH2 cells overexpressing Flag-tagged SIAH2. fected with sense (5Ј-CCTCGCGTGGTGAGTA-3Ј) or antisense (5Ј- Bound proteins were eluted with elution buffer [120 mM NaCl, 50 mM TACTCACCACGCGAGG-3Ј) S-oligonucleotides of PEG10 were stained Tris-HCl (pH 8.0), and 20 mM glutathione (Sigma)] and analyzed by in the same manner. immunoblotting using anti-Flag (Sigma) and anti-PEG10 antibody. Flow Cytometry. A total of 1 ϫ 105 cells was collected by trypsinization Construction of Adenovirus Expressing SIAH1. Generation and prepa- at the given time points and fixed in 70% cold ethanol. Cells treated with ration of adenovirus-expressing SIAH1 was achieved using the Adenovirus RNase and propidium iodide (50 ␮g/ml) in PBS were analyzed by a FACScan Expression Vector Kit (TaKaRa) according to the supplier’s protocol. First, the (Becton Dickinson, San Jose, CA). entire coding region of SIAH1 was amplified and cloned into an appropriate Yeast Two-Hybrid Experiment. A yeast two-hybrid assay was performed site of the pcDNA3.1/myc-C vector (Invitrogen). Subsequently, the fragment with the Matchmaker GAL4 Two-Hybrid System 3 according to the manu- of myc-tagged SIAH1 was cloned into the cosmid vector pAxCAwt supplied in facturer’s protocols (Clontech). We cloned the entire coding sequence of the kit. (TaKaRa). PEG10 into the EcoRI-SalI site of pAS2-1 vector as bait and screened a human testis cDNA library (Clontech).