Identification of the leukocyte cell-derived chemotaxin 2 as a direct target gene of β-catenin in the liver

Abstract

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

Differential effects of inactivated Axin1 and activated β-catenin mutations in human hepatocellular carcinomas

“…We demonstrated that among the various liver b-catenin target genes tested (Cadoret et al, 2002;Yamamoto et al, 2003;Colnot et al, 2004;Ovejero et al, 2004), three were good markers of active b-catenin signaling: GS, GPR49 and GLT-1. Immunohistochemical analyses showed that GS is a marker of b-catenin activation in human HCC.…”

“…Control non-mutated HCC cell lines did not have mutations in either b-catenin or Axin1 (Table 1). The seven b-catenin target genes included two canonical b-catenin target genes, c-myc and cyclinD1 (He et al, 1998;Tetsu and McCormick, 1999), and five recently identified b-catenin target genes overexpressed in human HCCs: glutamine synthetase (GS) (also known as GLUL1), the glutamate transporter (GLT)-1, ornithine aminotransferase (OAT) (Cadoret et al, 2002), an orphan G-protein-coupled receptor (GPR)49 (also known as LGR5) (Yamamoto et al, 2003) and a chemokine-like protein LECT2 (Ovejero et al, 2004). We assayed mRNA of these seven b-catenin target genes in 45 HCCs and in 21 of the matched non-tumor liver tissues (Figure 1).…”

“…Tumor RNA was extracted using the Rneasy kit (Qiagen). Real-time RT-PCR was carried out as described previously (Ovejero et al, 2004). (Satoh et al, 2000).…”

“…Thus, there is no doubt that Axin1 is a tumor suppressor gene. However, Immunostaining was performed on 5 mm sections of formalin-fixed, paraffinembedded liver samples, using monoclonal anti-b-catenin (1/100, BD Biosciences 610153, Grenoble, France) and monoclonal antiglutamine-synthetase (1/200, BD Biosciences, 610517) as described previously (Ovejero et al, 2004). Two observers (CP, PB-S) independently evaluated the immunostaining results.…”

Le microenvironnement immunitaire, complice de la voie Wnt/β-caténine dans le cancer du foie

“…La voie de signalisation Wnt/-caténine est constitutivement activée dans 30 à 40 % des CHC chez l'homme [3,8]. Récemment, nous avons identifié une cible moléculaire contrôlée par la -caténine appelée LECT2 pour leukocyte cell-derived chemotaxin 2 [10]. On connaît encore très peu les fonctions de LECT2, bien qu'un rôle immunomodulateur lui ait été attribué.…”