Inappropriate activation of the Wnt/b-catenin signaling has been implicated in the development of hepatocellular carcinoma (HCC), but exactly how b-catenin works remains to be elucidated. To identify, in vivo, the target genes of b-catenin in the liver, we have used the suppression subtractive hybridization technique and transgenic mice expressing an activated b-catenin in the liver that developed hepatomegaly. We identified three genes involved in glutamine metabolism, encoding glutamine synthetase (GS), ornithine aminotransferase (OAT) and the glutamate transporter GLT-1. By Northern blot and immunohistochemical analysis we demonstrated that these three genes were specifically induced by activation of the b-catenin pathway in the liver. In different mouse models bearing an activated bcatenin signaling in the liver known to be associated with hepatocellular proliferation we observed a marked upregulation of these three genes. The cellular distribution of GS and GLT-1 parallels b-catenin activity. By contrast no up-regulation of these three genes was observed in the liver in which hepatocyte proliferation was induced by a signal-independent of b-catenin. In addition, the GS promoter was activated in the liver of GS +/LacZ mice by adenovirus vector-mediated b-catenin overexpression. Strikingly, the overexpression of the GS gene in human HCC samples was strongly correlated with b-catenin activation. Together, our results indicate that GS is a target of the Wnt/b-catenin pathway in the liver. Because a linkage of the glutamine pathway to hepatocarcinogenesis has already been demonstrated, we propose that regulation of these three genes of glutamine metabolism by b-catenin is a contributing factor to liver carcinogenesis.

To clarify molecular mechanisms underlying liver carcinogenesis induced by aberrant activation of Wnt pathway, we isolated the target genes of ␤-catenin from mice exhibiting constitutive activated ␤-catenin in the liver. Adenovirus-mediated expression of oncogenic ␤-catenin was used to isolate early targets of ␤-catenin in the liver. Suppression subtractive hybridization was used to identify the leukocyte cell-derived chemotaxin 2 (LECT2) gene as a direct target of ␤-catenin. Northern blot and immunohistochemical analyses demonstrated that LECT2 expression is specifically induced in different mouse models that express activated ␤-catenin in the liver. LECT2 expression was not activated in livers in which hepatocyte proliferation was induced by a ␤-catenin-independent signal. We characterized by mutagenesis the LEF/TCF site, which is crucial for LECT2 activation by ␤-catenin. We further characterized the chemotactic property of LECT2 for human neutrophils. Finally, we have shown an up-regulation of LECT2 in human liver tumors that expressed aberrant activation of ␤-catenin signaling; these tumors constituted a subset of hepatocellular carcinomas (HCC) and most of the hepatoblastomas that were studied. In conclusion, our results show that LECT2, which encodes a protein with chemotactic properties for human neutro- H epatocellular carcinoma (HCC), the major primary liver cancer, is becoming increasingly common worldwide. 1 The prognosis for patients with HCC is rather poor. The molecular changes underlying HCC remain largely unknown despite the fact that major risk factors, such as chronic hepatitis B or C infection and exposure to hepatocarcinogens like aflatoxin B1, are well recognized. Several genetic changes have been implicated in at least 3 pathways of carcinogenesis, specifically, the p53, RB and Wnt/␤-catenin signaling pathways. 2 Deregulation of the Wnt pathway appears to be most frequent of these changes in human HCC; it occurs in about 30% to 40% of patients. 2,3 It also occurs in more than 90% of hepatoblastomas, which are rare embryonal liver tumors. 4 Mutations affecting 2 partners of the Wnt pathway have been found in liver cancers. One is a mutation that activates the ␤-catenin gene. Such mutations occur mainly in hepatitis B-negative HCC 5 and in more than 50% of hepatoblastomas. 6,7 The other is a mutation that inactivates the axin 1, and, less commonly, the axin 2 gene. 5,8,9 Mutations that activate the Wnt pathway result in ␤-catenin accumulation in the nucleus. This process, in association with LEF/TCF transcription factors, modulates the transcription of target genes. 10,11 It is now clear that the genetic program triggered by activation of ␤-catenin signaling depends on the cellular context. The ␤-catenin target genes c-myc and cyclin D1 are well