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[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 antirabbit 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

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). Results In Vitro Protein-binding Assay. The entire coding regions of SIAH1 and Identification of a Novel Gene Frequently Up-Regulated in SIAH2 were amplified using primers 5Ј-CGCGAATTCCGCCCACA- GAAATGAGCC-3Ј and 5Ј-CATCTCGAGACATGGAAATAGTTACAT- HCCs. Using a genome-wide cDNA microarray consisting of 23,040 TGATGC-3Ј or 5Ј-TGCGAATTCCATGGTTGGTTCGGAGC-3Ј and 5Ј- genes, we identified a gene that was commonly up-regulated among a GTGCTCGAGGACAACATGTAGAAATAGTAAC-3Ј, respectively, and total of 20 HCCs (6). Elevated expression of PEG10 in the tumors was cloned into appropriate cloning sites of pET21b vector (Novagen, Madison, confirmed by TaqMan PCR (data not shown). A multitissue Northern WI) or pCMV-Flag5 (Sigma). Recombinant His-tagged SIAH-1 protein blot using the cDNA as a probe showed a 6.4-kb transcript predom- was prepared using the Xpress system (Invitrogen, Carlsbad, CA) accord- inantly expressed in placenta, testis, and ovary (Fig. 1A). Using the 5Ј ing to the manufacturer’s recommendations. ProBond histidine affinity rapid amplification of cDNA ends method, we obtained cDNA se- resin (Invitrogen) incubated with or without 10 ␮g of His-tagged SIAH-1 quences that covered almost the entire transcript of this gene (Gen- protein at 4°C for 1 h, followed by extensive washing with binding buffer Bank accession no. AB049150). Simple Modular Architecture Re- [20 mM NaH PO , 500 mM NaCl (pH 7.8)], was then incubated with 50 ␮g 2 4 search Tool (version 3)4 suggested that the predicted protein of cell lysates from SNU423-PEG10 cells exogenously overexpressing contained a coiled-coil motif (codons 1–50) as well as a zinc-finger PEG10 in NP40 lysis buffer [150 mM NaCl, 50 mM Tris (pH 8.0), and 1% motif (codons 294–310). NP40]. After the resin was washed with wash buffer (20 mM NaH2PO4 and 500 mM NaCl) twice each at pH 7.8, pH 6.0, and pH 5.5, protein was eluted with elution buffer (300 mM imidazole in wash buffer). The eluted proteins 4 Internet address: http://smart.embl-heidelberg.de. 3044

antisense plasmid clones, the PEG10 sense plasmid vector promoted colony formation in both cell lines (Fig. 2A). To additionally investigate the growth-promoting effects of PEG10, we generated stable transfectants using SNU423 cells in which endogenous PEG10 expression was absent (Fig. 2B). The PEG10 stable transfectant cells revealed significant growth promotion compared with the parental or mock cells (Fig. 2C). Under conditions of serum starvation (0.1% FBS), the mock cells rapidly underwent growth arrest, but stable PEG10-expressing cells continued to proliferate (Fig. 2D). Suppression of Growth of Hepatoma Cells by Antisense Oligo- nucleotides of PEG10. To examine whether suppression of PEG10 would retard growth and/or induce death of HCC cells, we designed various antisense S-oligonucleotides. Among them, antisense S- oligonucleotides encompassing the first exon-intron boundary, but not other antisense or control S-oligonucleotides, significantly decreased endogenous expression of PEG10 in Alexander and Huh7 cells that constitutively express abundant PEG10 (Fig. 2E for Huh7 and data not shown). Transfection of the antisense S-oligonucleotides significantly reduced number of viable cells in these two cell lines (Fig. 2F for Huh7, and data not shown), but no growth-suppressive effect was observed when we introduced the antisense S-oligonucleotides into SNU423 cells, which do not express endogenous PEG10 (data not shown). Interaction of PEG10 with SIAH-1 and SIAH-2. To examine the oncogenic mechanism of PEG10, we searched for PEG10- interacting proteins using a yeast two-hybrid screening system

because the NH2-terminal region of PEG10 also contains a coiled- coil motif that generally allows for protein-protein interactions. Among the clones identified, those homologous to Drosophila seven in absentia (SIAH1 and SIAH2) interacted with PEG10 by simultaneous transformation with pAS2.1-PEG10 and pACT2- SIAH1 or SIAH2 (Fig. 3A). To confirm the interaction of PEG10 with SIAH1, we prepared recombinant His-tagged SIAH1 protein and detected this association when PEG10 protein was expressed in mammalian cells (Fig. 3B). In addition, we demonstrated asso-

Fig. 2. Effect of PEG10 on cell growth in human hepatoma cell lines. A, colony formation by SNU423 and SNU475 cells after PEG10 gene transfer. Relative number of colonies transfected with plasmids expressing PEG10 to mock vector was calculated (mean Ϯ SD). B, expression of PEG10 protein in stable transfectant (SNU423-PEG10) cells. The amount of protein applied in the SDS-PAGE was evaluated by Coomassie Brilliant Blue (CBB) staining. C, Growth curve of SNU423-PEG10 cells cultured in RPMI 1640 with 10% FBS. D, growth curve of SNU423-PEG10 cells under serum- starved conditions (cultured in RPMI 1640 with 0.1% FBS). E, reduced expression of PEG10 in Huh7 cells transfected with either sense or antisense S-oligonucleotides. F, relative number of viable Huh7 cells after transfection with the sense or antisense S-oligonucleotides. Relative number of viable cells was examined by MTT assay (mean Ϯ SD).

Expression of PEG10 in HCC Cell Lines and Primary HCCs. To investigate the role of PEG10 in HCCs, we generated rabbit polyclonal antibody to the gene product. Using this antibody, we evaluated expression of this protein in six hepatoma cell lines. HepG2,

Huh7, and Alexander cells constitutively expressed Mr 40,000 PEG10 protein (Fig. 1B), and immunohistochemical staining disclosed that PEG10 was located in both nucleus and cytoplasm in those cells (Fig. 1C). Among 16 primary HCCs that were different from the 20 HCCs used for the cDNA microarray analysis, we detected strong nuclear Fig. 3. Interaction between PEG10 and SIAH1/2 proteins. A, yeast two-hybrid experiment. pAS2-1 or pAS2-1 containing PEG10 were cotransfected into yeast strain AH109 and cytoplasmic staining of PEG10 in the tumor tissue from 15 cases with library vectors containing SIAH1 or SIAH2. B, in vitro protein-binding assay of but not in their corresponding normal tissues (Fig. 1D). PEG10 and SIAH1. Protein from cells expressing PEG10 was incubated with resin Promotion of Growth of Human Hepatoma Cells by PEG10. To conjugated with or without SIAH1. Eluted protein was analyzed by immunoblotting using anti-His and anti-PEG10 antibodies. In the last two lanes, his-tagged SIAH1 and cell test the effects of PEG10 gene transfer on growth of hepatoma cells, lysate containing PEG10 were loaded directly on the gel as controls. C, in vitro protein- we transfected an expression plasmid containing PEG10 to two cell binding assay of PEG10 and SIAH2. Protein from cells expressing flag-tagged SIAH2 was incubated with GST-PEG10 or GST and purified with Glutathione Sepharose beads. In the lines (SNU423 and SNU475), which had shown no endogenous right three lanes, GST-PEG10 fusion protein, GST, and cell lysate containing flag-tagged expression of PEG10 protein (Fig. 1B). Compared with mock or SIAH2 protein were loaded as controls. 3045

Fig. 4. Effects of SIAH1 gene transfer on viability of hepatoma cells. A, expression of SIAH1 in hepatoma cell lines examined by RT-PCR. mRNA extracted from normal liver tissue was used for comparison. B, expression of exogenous myc- tagged SIAH1 protein in SNU423 cells by adenovirus-mediated SIAH1 gene transfer (100 MOI). C, assessment of cell viability by MTT assay after infection with Ad-SIAH1 at MOIs of 20, 50, and 100. D, apoptosis induced by Ad-SIAH1 (100 MOI) in Huh7 cells; sub-G1 population of cells infected with Ad-SIAH1 or Ad-LacZ was examined 48 h after infection by fluorescence-activated cell sorting (left panels). Apoptotic cells were detected in green or yellow by terminal deoxynucle- otidyl transferase-mediated nick end labeling assay (right panels). E, Effect of PEG10 expression on cell death induced by SIAH1. Cell viability was examined by MTT assay 48 h after infection with Ad-SIAH1 in PEG10-expressing cells (SNU423- PEG10), the parental (SNU423), or control (SNU423-Mock) cells. Data (mean Ϯ SD) repre- sent MTT activity of cells with Ad-SIAH1 relative to those with Ad-LacZ.

ciation of GST-PEG10 fusion protein with flag-tagged SIAH2 SNU475), each of which revealed transfection efficiencies of 67.3– protein expressed in HEK293 cells (Fig. 3C). 100% at an MOI of 100, with Ad-LacZ; immunoblot analysis using Effect of PEG10 on the Cell Death Induced by Gene Transfer of anti-myc antibody confirmed exogenous expression of myc-tagged SIAH1 in HCC Cells. Others had reported that expression of human SIAH1 protein after 24 h of infection (Fig. 4B). The increase in cell SIAH1 was increased during p53-dependent arrest of the cell cycle death was marked in cultures infected with Ad-SIAH1 in all five and that it induced apoptosis in several cell lines (8, 9). Hence, we cell-lines. Consistently, an MTT assay after 72 h of infection demon- hypothesized that PEG10 may affect the apoptosis mediated by strated a dose-dependent decrease of viability when cells were in- SIAH1. To examine this hypothesis, we generated recombinant ad- fected with Ad-SIAH1 (Fig. 4C). Flow cytometry demonstrated that enoviruses expressing myc-tagged SIAH1 protein (Ad-SIAH1) and induction of SIAH1 expression significantly increased the numbers of

LacZ (Ad-LacZ). Semiquantitative RT-PCR demonstrated that ex- cells in G2-M and sub-G1 populations, and a terminal deoxynucleoti- pression of SIAH1 was decreased in all six hepatoma cell lines dyl transferase-mediated nick end labeling assay corroborated that the examined, compared with normal liver tissue (Fig. 4A). We infected number of apoptotic cells infected with Ad-SIAH1 was significantly five hepatoma cell lines (HepG2, Huh7, Alexander, SNU423, and greater than cells with Ad-LacZ (Fig. 4D). To determine whether 3046

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2003 American Association for Cancer Research. INVOLVEMENT OF PEG10 IN HEPATOCARCINOGENESIS overexpression of PEG10 protein could protect hepatocytes from cell In our experiments, cells that stably expressed PEG10 revealed a death induced by SIAH1, we constructed SNU423-PEG10 cells, significant decrease in cell death in response to Ad-SIAH1, suggesting which stably express exogenous PEG10, and infected them with that imbalance between expression of PEG10 and SIAH1 may be Ad-SIAH1. Compared with the parental SNU423 or control SNU423- involved in hepatocarcinogenesis through inhibition of apoptosis. mock cells, SNU423-PEG10 cells showed significantly greater via- PEG10 was isolated as a paternally expressed gene from a newly bility 48 h after being infected with Ad-SIAH1 (Fig. 4E), indicating defined imprinted region at 7q21 (7). Loss of imprinting might be the protective role of PEG10 from SIAH1-mediated cell death. involved in the elevated expression of this gene in HCCs, although we found no evidence to support it. Hence, the actual mechanisms by Discussion which the expression of PEG10 is deregulated in HCCs remain to be investigated. Chronic hepatitis because of hepatitis B or hepatitis C virus is Finally, we have demonstrated that reduction of PEG10 expression considered a major risk factor for HCC, but each virus may contribute by treatment with antisense S-oligonucleotides decreases growth of to hepatocarcinogenesis via a different pathway (10, 11). For example, HCC cells significantly. Interestingly, the antisense sequences sup- our studies of gene expression profiles of HCCs using a genome-wide pressed growth only of HCC cells that endogenously expressed cDNA microarray have revealed different expression patterns between hepatitis B virus-positive and hepatitis C virus-positive tumors, PEG10, not in cell lines that did not. Because expression of this gene implying different character, although many genes are commonly was enhanced in the majority of HCC tissues and very low or absent up-regulated in both types of tumor. Because PEG10, a gene that was among all normal adult human tissues, except gonadal glands, sup- up-regulated in a great majority of HCCs (35 of 36 HCCs analyzed by pression of PEG10 might be an ideal therapeutic strategy for treating the cDNA microarray, RT-PCR, and/or immunostaining), was ex- primary HCCs. Although additional functional analysis of PEG10 is pressed at an undetectable level in matched noncancerous liver tis- required, the data provided here should contribute to a more profound sues, PEG10 could potentially serve as a diagnostic marker and might understanding of hepatocarcinogenesis and to development of novel be an ideal molecular target for development of drugs to treat patients therapeutic approaches. with primary HCC. Transfer of PEG10 into hepatoma cells that expressed no detectable References endogenous PEG10 protein elicited significant growth promotion 1. Tanaka, S., Toh, Y., Adachi, E., Matsumata, T., Mori, R., and Sugimachi, K. Tumor activity. Serum starvation did not suppress the growth of cells that progression in hepatocellular carcinoma may be mediated by p53 mutation. Cancer expressed PEG10 at high levels. Because transfer of PEG10 into Res., 53: 2884–2887, 1993. 2. 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Hiroshi Okabe, Seiji Satoh, Yoichi Furukawa, et al.

Cancer Res 2003;63:3043-3048.

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