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Hepatitis B Virus-Related Insertional Mutagenesis Implicates SERCA1 Gene in the Control of Apoptosis

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Hepatitis B Virus-Related Insertional Mutagenesis Implicates SERCA1 Gene in the Control of Apoptosis Oncogene (2000) 19, 2877 ± 2886 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Hepatitis B virus-related insertional mutagenesis implicates SERCA1 gene in the control of apoptosis Mounia Chami1,7, Devrim Gozuacik1,7, Kenichi Saigo1,2,7, Thierry Capiod3, Pierre Falson4, Herve Lecoeur5, Tetsuro Urashima2, Jack Beckmann6, Marie-Lyse Gougeon5, Michel Claret3, Marc le Maire4, Christian Bre chot1 and Patrizia Paterlini-Bre chot*,1 1U-370 INSERM, Necker Institute, 75015 Paris, France; 2Second Department of Surgery, Chiba University, Chiba 263-8852, Japan; 3U-442 INSERM, Universite Paris Sud, 91405 Orsay, France; 4URA CNRS 2096, CEA Saclay, 91191 Gif sur Yvette, France; 5Pasteur Institute, 75015 Paris, France; 6Centre National GeÂnotypage, 91057 Evry, France We have used the Hepatitis B Virus DNA genome as a and Yun, 1998) and NF-kB signaling (Chirillo et al., probe to identify genes clonally mutated in vivo,in 1996). It has also been suggested that it directly human liver cancers. In a tumor, HBV-DNA was found interacts with cellular proteins controlling cell growth to be integrated into the gene encoding Sarco/Endoplas- (p53 (Truant et al., 1995; Wang et al., 1994a)), DNA mic Reticulum Calcium ATPase (SERCA), which pumps repair (ERCC (Wang et al., 1994b) and UVDDB (Lee calcium, an important intracellular messenger for cell et al., 1995; Sitterlin et al., 1997)), senescence (Sun et viability and growth, from the cytosol to the endoplasmic al., 1998), NFkB activity (Weil et al., 1999), basal reticulum. The HBV X gene promoter cis-activates transcription (RBP5 (Cheong et al., 1995) and RMP chimeric HBV X/SERCA1 transcripts, with splicing of (Dorjsuren et al., 1998)), signal transduction (ATF/ SERCA1 exon 11, encoding C-terminally truncated CREB (Williams and Andrisani, 1995)) and with SERCA1 proteins. Two chimeric HBV X/SERCA1 proteasome (Sirma et al., 1998). Depending on the proteins accumulate in the tumor and form dimers. In experimental conditions, HBV X has also been vitro analyses have demonstrated that these proteins reported to accelerate (Koike et al., 1994) or inhibit localize to the ER, determine its calcium depletion and cell cycle progression (Benn and Schneider, 1995), and induce cell death. We have also shown that these to induce (Chirillo et al., 1997; Sirma et al., 1999; biological eects are related to expression of the Terradillos et al., 1998) or inhibit apoptosis (Wang et SERCA, rather than of the viral moiety. This report al., 1995). Furthermore, HBV X induces HCC in involves for the ®rst time the expression of mutated certain transgenic mice (Kim et al., 1991). However, in SERCA proteins in vivo in a tumor cell proliferation and a dierent genetic context, HBV X transgenic mice in vitro in the control of cell viability. Oncogene (2000) exhibit only increased susceptibility to chemical 19, 2877 ± 2886. carcinogenes (Slagle et al., 1996), or an accelerated development of c-myc induced HCC (Terradillos et al., Keywords: SERCA1; Hepatitis B Virus; calcium; 1997). apoptosis; cancer In addition to these mechanisms, the clonal integra- tion of HBV-DNA into the host cell genome has been found in more than 90% of HBV-related HCCs Introduction (Paterlini and Bre chot, 1994). HBV-DNA integration may directly promote genetic instability or lead to cis- Chronic HBV infection is a major etiologic factor of activation of growth-related genes (Paterlini and hepatocellular carcinoma (HCC) (Chang et al., 1997; Bre chot, 1994). In certain isolated cases, the viral Hildt et al., 1996). Although no transforming viral genome was found to be integrated into the Retinoic oncogene has so far been identi®ed, HBV-induced acid receptor b gene (Dejean et al., 1986), and Cyclin chronic liver in¯ammation is thought to play an A2 gene (Wang et al., 1990), leading to the expression important role in carcinogenesis (Buendia, 1992; of chimeric transcripts and proteins, the transforming Paterlini and Bre chot, 1994). In addition, the expres- activity of which was subsequently proved (Berasain et sion of HBV X, PreS2/S (Kekule , 1994), and PreS1/ al., 1998; Garcia et al., 1993). Reports have also been PreS2/S (Chisari et al., 1989) viral proteins has been made of HBV-DNA integration in the gene encoding associated with the development of HCC. In particular, mevalonate kinase in the PLC/PRF/5 cell line (Graef et the 17 kDa HBV X encoded protein has been shown to al., 1994), and in the Carboxypeptidase N locus in a activate in trans a wide variety of cellular and viral human HCC (Pineau et al., 1996). In both instances, genes (reviewed in Weil et al., 1999). HBV X activates chimeric transcripts were identi®ed in the tumor cells, signal transduction pathways such as Ras/Raf/MAP but the eect of their in vitro expression on cell kinase (Benn and Schneider, 1994), Jak1-STAT (Lee phenotype has not been investigated so far. On the other hand, a number of studies have failed to identify HBV-DNA integration into cellular DNA coding sequences, so that insertional mutagenesis has been *Correspondence: P Paterlini-Bre chot, Unite INSERM 370, 156 rue considered as a rare event in HBV-related carcinogen- de Vaugirard, 75015 Paris, France 7The ®rst three authors contributed equally to this work esis (Koike, 1998). Received 12 November 1999; revised 3 April 2000; accepted 4 April In contrast, in the woodchuck animal model, 2000 Woodchuck hepadnavirus (WHV) integration into the HBV-related insertional SERCA1 mutagenesis in a HCC M Chami et al 2878 N-myc2 and c-myc oncogenes is a frequent event, (446 in Figure 1A), fused in frame to SERCA1 cDNA occurring in more than 70% of tumors, and its nucleotide 153 in exon 3. Interestingly, SERCA1 transforming eect has been well established (Buendia, cDNA was characterized by the splicing of exon 11, 1992). Interestingly, in some tumors, WHV-DNA leading to a 22 codon frameshift and a premature stop integration occurs in a locus called win, which is codon in exon 12. Three of eight clones exhibited an situated at more than 150 Kb from the N-myc2 gene additional splicing of SERCA1 exon 4. These two (Fourel et al., 1994). chimeric cDNAs and encoded proteins will be referred In HBV-related, human HCC, however, the actual to as Xt/St+4 and Xt/St74: Xt for HBV X truncated impact of insertional mutagenesis has not yet been sequence fused to truncated SERCA1 (St), with or explored exhaustively. This is due to technical limita- without exon 4. tions of the genomic libraries previously used for identifying HBV-DNA integration sites, which pre- Chimeric HBV X/SERCA1 proteins predicted structure cluded large scale screening. prevents calcium pumping Using an original, Alu-PCR-based approach devel- oped in our laboratory (Minami et al., 1995), we have According to the cDNA sequence, the chimeric initiated a screening program of HBV-related HCC, proteins have their SERCA1 N-terminal 51 aminoacids searching for HBV/cellular DNA junctions. In the replaced by 148 aminoacids of the HBV X protein and present study, we report on a liver tumor cell an additional residue encoded by the viral/cellular in proliferation carrying the HBV-DNA integration into frame junction (Figure 2A). Xt/St+4 protein lacks six the SERCA1 gene. putative SERCA1 transmembrane segments (M5-M10), SERCA proteins play a pivotal role in regulating which include ®ve of the six Ca2+ binding residues cellular calcium (Pozzan et al., 1994), which, in turn, (Glu-771, Asn-796, Thr-799, Asp-800, and Glu-908) acts as an intracellular messenger involved in a broad (MacLennan et al., 1997) as well as the cytoplasmic range of specialized and basic cellular activities, loop between transmembrane segments 6 and 7 that ranging from muscle contraction, transmission of also controls Ca2+ binding (Falson et al., 1997). A neuronal signal, cell proliferation and death (Berridge mutated peptide of 22 aminoacids is encoded at the C- et al., 1998). Three distinct SERCA genes encode a terminal end. In addition to that, Xt/St-4 protein lacks number of dierentially expressed isoforms (Wu et al., a peptide (aa. 74 ± 108) encoding the second putative 1995). SERCA1a and 1b are mainly found in fast- transmembrane segment (M2) and the last ®ve C- twitch skeletal muscle, SERCA2a in slow-twitch terminal residues of M1. The expected size of the Xt/ skeletal and cardiac muscle, SERCA2b is ubiquitous St+4 and Xt/St74 chimeric proteins is 56 and and SERCA3 which also displays isoforms is expressed 53 kDa, respectively. in several tissues, including hemopoietic, endothelial Thus, although these proteins retain the putative and secretory epithelial cells. We show here that HBV calcium binding residue Glu 309 and the phosphory- integration into the SERCA1 gene in a human liver latable Asp-351, consistent with previously reported tumor cis-activates SERCA1 chimeric transcripts mutants (MacLennan et al., 1998), they cannot encoding chimeric proteins. We also provide evidence function as calcium pumps. that the in vitro expression of these transcripts and of their SERCA moiety induces ER calcium depletion and Chimeric proteins are expressed and form dimers in the apoptosis. Our study therefore highlights the implica- tumor tion of SERCA1 gene mutation in clonal cell expansion and the control of cell viability. We then looked for these chimeric and truncated proteins in the tumorous tissue (T86) by Western blot. A chimeric protein (53/56 kDa), reacting with both anti-HBV X (anti-X) and anti-SERCA1 antibodies Results (Figure 2B), was clearly detectable in the tumorous tissue. High resolution analysis demonstrated two HBV-DNA integration into the SERCA1 gene drives the proteins, of 53 and 56 kDa (Figure 2C), which size expression of chimeric transcripts was consistent with the cDNA sequences.
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