Oncogene (2006) 25, 599–608 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Overexpression of regenerating islet-derived 1 alpha and 3 alpha genes in human primaryliver tumors with b-catenin mutations C Cavard1,5, B Terris1,2,5, G Grimber1, L Christa3, V Audard1, B Radenen-Bussiere2, M-T Simon3, C-A Renard4, M-A Buendia4 and C Perret1 1De´partement GDPM, INSERM U-567, CNRS UMR 8104, Institut Cochin, Universite´ Paris 5, Paris, France; 2Service d’Anatomie Pathologique, Hoˆpital Cochin, Universite´ Paris 5, Paris Cedex, France; 3INSERM U-370, Institut Necker-Pasteur, Universite´ Paris 5, Paris Cedex, France and 4Unite d’Oncogene`se et Virologie Mole´culaire, INSERM U-579, Institut Pasteur, Paris Cedex, France The Wnt/b-catenin signaling pathwayis activated in many although several genetic alterations have been impli- human hepatocellular carcinomas (HCC). We tried to cated in at least three carcinogenesis pathways, p53, RB identifythe genes involved in carcinogenesis and progres- and the Wnt/b-catenin signaling pathway (Buendia, sion of HCC with b-catenin mutations. We used PCR- 2000; Thorgeirsson and Grisham, 2002). Deregulation based subtractive hybridization to compare gene expres- of the Wnt pathway appears to be the most frequent in sion between malignant and benign components of a human HCC and is detected in about 30–40% of these human HCC occurring in pre-existing adenoma activated patients (de La Coste et al., 1998; Buendia, 2000). for b-catenin. Two of the genes identified belong to the Mutations affecting two partners of the Wnt pathway Regenerating gene (REG) family. They encode the have been found in liver cancers. Activating mutations Regenerating islet-derived 3 alpha (REG3A/HIP/PAP/ in the b-catenin gene occur mainly in HBV-negative REG-III) and 1 alpha (REG1A) proteins, both involved in HCC (Laurent-Puig et al., 2001). The others are liver and pancreatic regeneration and proliferation. Using mutations that inactivate the AXIN 1, and more rarely siRNA directed against b-catenin, we demonstrated that the AXIN 2 gene (Satoh et al., 2000; Laurent-Puig et al., REG3A is a target of b-catenin signaling in Huh7 2001; Taniguchi et al., 2002). hepatoma cells. The upregulation of REG3A and REG1A b-catenin is central to the Wnt pathway. It has a expression is significantlycorrelated to the b-catenin structural role in cell–cell adhesion, but is also a status in 42 HCC and 28 hepatoblastomas characterized transcription cofactor with T-cell factor/lymphoid en- for their b-catenin status. Thus, we report strong evidence hancer factor (TCF/LEF) in the Wnt pathway. In the that both genes are downstream targets of the Wnt absence of Wnt signaling, the level of b-catenin is low. pathwayduring liver tumorigenesis. b-catenin is phosphorylated at critical NH2-terminal Oncogene (2006) 25, 599–608. doi:10.1038/sj.onc.1208860; residues by the glycogen synthase kinase 3-b (GSK3-b) published online 28 November 2005 bound to a scaffolding complex of axin and adenoma- tous polyposis protein, and subsequently, the phos- Keywords: b-catenin; REG1A; REG3A; hepatocellular phorylated protein is degraded by the ubiquitin– carcinoma; hepatoblastoma; Wnt signaling proteasome system. Wnt stimulation leads to the inactivation of GSK3-b, resulting in the stabilization of b-catenin in the cytoplasm such that it is available to bind the TCF/LEF family of transcription factors and induce target gene expression (Giles et al., 2003). The Introduction genetic program triggered by activation of b-catenin signaling depends on the cellular context (Polakis, 2000; Hepatocellular carcinoma (HCC) is the most common Giles et al., 2003). The identification of b-catenin target type of primary liver cancer. It is the fifth most frequent genes would therefore help understand how aberrant cancer worldwide, and its incidence is still rising in b-catenin signaling contributes to liver carcinogenesis. Western countries (Parkin et al., 2001; El-Serag, 2002). Several studies have investigated the downstream target The prognosis is poor. The molecular changes under- genes of this pathway. Most of these studies involved lying HCC remain largely unknown (Bruix et al., 2004), differential expression analyses using animal models of hepatocarcinogenesis (Cadoret et al., 2002; Ovejero Correspondence: Dr C Cavard, De´ partement GDPM, INSERM U- et al., 2004), human hepatomas cell lines (Yamamoto 567, CNRS UMR 8104, Institut Cochin, Universite´ Paris 5, 24 rue du et al., 2003) and ex vivo infection of primary human Faubourg Saint-Jacques, Paris 75014, France. hepatocytes (Levy et al., 2002), which expressed an E-mail: [email protected] 5These authors contributed equally to this work. oncogenic form of b-catenin. Very few genes specifically Received 2 March 2005; revised 9 May 2005; accepted 19 May 2005; induced in the liver in response to b-catenin have been published online 28 November 2005 identified (Cadoret et al., 2002; Yamamoto et al., 2003; Overexpression of REG genes in human primary liver tumors C Cavard et al 600 Ovejero et al., 2004). Among them, the glutamine- synthetase gene (GS) appears the most reliable marker (Cadoret et al., 2002; Zucman-Rossi et al., submitted). To identify novel candidate b-catenin/TCF transcrip- tional targets likely to contribute to liver carcinogenesis, we used PCR-based subtractive hybridization to com- pare gene expression between the different components of a particular human HCC occurring in pre-existing adenoma activated for b-catenin. We report the identification of two genes that belong to the family of the Regenerating (REG) genes that encode growth- promoting lectins: the Regenerating islet-derived 3 alpha (REG3A) (also named HIP/PAP for Hepatocarcinoma– Intestine–Pancreas/Pancreatitis Associated-Protein) and the Regenerating islet-derived 1 alpha (REG1A) genes (Zhang et al., 2003). We tested whether the REG3A and REG1A genes are downstream targets of b-catenin and analysed their expression in a panel of primary liver tumors, both HCC and hepatoblastomas (HB), char- acterized for the presence of b-catenin mutations, and also in colon cancers associated with aberrant b-catenin signaling, from familial adenomatous polyposis (FAP) patients. Figure 1 Description of the tumors used for the SSH procedure. (a) Macroscopic view of the HCC (HCC) in pre-existing Results adenoma (Ad). These tumors arose in non-cirrhotic liver (NT). (b) Immunostaining for b-catenin (magnification  200). Intense Cloning of REG3A and REG1A genes from a rare case of nuclear staining was observed in HCC. (c) Immunostaining for GS (magnification  100). Intense cytoplasmic staining is present in HCC occurring in a b-catenin-mutated adenoma the adenoma component. The adjacent non-tumoral liver displayed We studied a particularly rare case of HCC to assess the usual staining around the central vein. (d) RNAs used for the the molecular consequences of a dysregulation of subtracted libraries was analysed for GS mRNA by Northern the Wnt pathway from benign to malignant stages of blotting. NT: non-tumoral; Ad: adenoma; HCC: hepatocellular hepatic tumor development. This male patient displayed carcinoma. The blot was standardized with a human GAPDH probe. an HCC in a pre-existing liver adenoma in a morpho- logically normal liver (Figure 1a). In both components, adenoma and HCC, Wnt/b-catenin signaling was activated as evidenced by the presence of b-catenin specific for the HCC stage as HCC cDNAs have been not only at the membrane but also in the cytoplasm subtracted by sequences expressed in the adenomatous and more infrequently in the nucleus of tumor tissue. cells (Figure 1b). The GS gene is a reliable marker of In all, 78 positive clones corresponding to nine Wnt/b-catenin pathway activation in HCC (Cadoret independent genes were isolated from the adenoma- et al., 2002; Zucman-Rossi et al., submitted), and we enriched library and of 115 positive clones corresponding confirmed activation of the b-catenin pathway by to 27 independent genes from the carcinoma-enriched immunohistochemistry and Northern blot analyses library. The reidentification of the GS gene (gene ID and observed GS overexpression in both stages (Figures 2752) in our suppression subtractive hybridization 1c and d). approach validated the subtraction procedure and served The activation of the Wnt pathway in the adenoma as a positive control. Some of the genes were analysed and HCC was owing to an in-frame 15 base deletion by real-time RT–PCR to confirm their differential involving exon 3 (Dcodons 32–36) eliminating the expression in the tissues from which the RNA had residues that are targets for the destruction of b-catenin been extracted for library construction (data not shown). (amino acids D32, S33, G34) (Table 1, case no. 5). These They included the genes encoding the chitinase 3-like 1 tumor specimens were suitable tools to study the altered (gene ID 1116), the serine protease inhibitor Kazal- gene expression owing to aberrant Wnt signaling from type 1 SPINK1/TATI (gene ID 6690), ubiquitin D the benign to the malignant stages of the liver tumor. (gene ID 10537) and the secreted phosphoprotein 1 or Using the suppression subtractive hybridization tech- osteopontin (gene ID 6696) (Table 2, Supplementary nique, two libraries were constructed and screened on Information). the basis of the overexpression in the respective tester Two genes were found to be common to both tissue. One was expected to be enriched in adenoma- libraries. They encoded REG3A