Catherine Cavard

Mutations of β-Catenin in Adrenocortical Tumors: Activation of the Wnt Signaling Pathway Is a Frequent Event in both Benign and Malignant Adrenocortical Tumors

Adrenocortical cancer is a rare cancer with a very poor prognosis. The genetic alterations identified to date in adrenocortical tumors are limited. Activating mutations of the Wnt signaling pathway have been observed in more frequent cancers, particularly digestive tract tumors. We investigated whether Wnt pathway activation is involved in adrenocortical tumorigenesis. In a series of 39 adrenocortical tumors, immunohistochemistry revealed abnormal cytoplasmic and/or nuclear accumulation of beta-catenin in 10 of 26 adrenocortical adenomas and in 11 of 13 adrenocortical carcinomas. An activating somatic mutation of the beta-catenin gene was shown in 7 of 26 adrenocortical adenomas and in 4 of 13 adrenocortical carcinomas; these mutations were observed only in adrenocortical tumors with abnormal beta-catenin accumulation and most were point mutations altering the Ser45 of exon 3 (in the consensus GSK3-beta/CK1 phosphorylation site). Functional studies showed that the activating Ser45 beta-catenin mutation found in the adrenocortical cancer H295R cell line leads to constitutive activation of T-cell factor-dependent transcription. This is the first molecular defect to be reported with the same prevalence in both benign (27%) and malignant (31%) adrenocortical tumors. beta-Catenin mutations are also the most frequent genetic defect currently known in adrenocortical adenomas. In adrenocortical adenomas, beta-catenin alterations are more frequent in nonfunctioning tumors, suggesting that beta-catenin pathway activation might be mostly involved in the development of nonsecreting adrenocortical adenomas and adrenocortical carcinomas. The very frequent and substantial accumulation of beta-catenin in adrenocortical carcinomas suggests that other alterations might also be involved. This finding may contribute to new therapeutic approaches targeting the Wnt pathway in malignant adrenocortical tumors, for which limited medical therapy is available.

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

To clarify molecular mechanisms underlying liver carcinogenesis induced by aberrant activation of Wnt pathway, we isolated the target genes of β‐catenin from mice exhibiting constitutive activated β‐catenin in the liver. Adenovirus‐mediated expression of oncogenic β‐catenin was used to isolate early targets of β‐catenin in the liver. Suppression subtractive hybridization was used to identify the leukocyte cell‐derived chemotaxin 2 (LECT2) gene as a direct target of β‐catenin. Northern blot and immunohistochemical analyses demonstrated that LECT2 expression is specifically induced in different mouse models that express activated β‐catenin in the liver. LECT2 expression was not activated in livers in which hepatocyte proliferation was induced by a β‐catenin–independent signal. We characterized by mutagenesis the LEF/TCF site, which is crucial for LECT2 activation by β‐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 β‐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 neutrophils, is a direct target gene of Wnt/β‐catenin signaling in the liver. Since HCC develops mainly in patients with chronic hepatitis or cirrhosis induced by viral or inflammatory factors, understanding the role of LECT2 in liver carcinogenesis is of interest and may lead to new therapeutic perspectives. (HEPATOLOGY 2004;40:167–176.)

Combined hepatocellular-cholangiocarcinomas exhibit progenitor features and activation of Wnt and TGFβ signaling pathways

Intrahepatic malignant tumours include hepatocellular carcinomas (HCC), cholangiocarcinomas (CC) and combined hepatocholangiocarcinomas (cHCC-CC), a group of rare and poorly characterized tumours that exhibit both biliary and hepatocytic differentiation. The aim of the study was to characterize the molecular pathways specifically associated with cHCC-CC pathogenesis. We performed a genome-wide transcriptional analysis of 20 histologically defined cHCC-CC and compared them with a series of typical HCC and of CC. Data were analysed by gene set enrichment and integrative genomics and results were further validated in situ by tissue microarray using an independent series of 152 tumours. We report that cHCC-CC exhibit stem/progenitor features, a down-regulation of the hepatocyte differentiation program and a commitment to the biliary lineage. TGFβ and Wnt/β-catenin were identified as the two major signalling pathways activated in cHCC-CC. A β-catenin signature distinct from that observed in well-differentiated HCC with mutant β-catenin was found in cHCC-CC. This signature was associated with microenvironment remodelling and TGFβ activation. Furthermore, integrative genomics revealed that cHCC-CC share characteristics of poorly differentiated HCC with stem cell traits and poor prognosis. The common traits displayed by CC, cHCC-CC and some HCC suggest that these tumours could originate from stem/progenitor cell(s) and raised the hypothesis of a potential continuum between intrahepatic CC, cHCC-CC and poorly differentiated HCC.

Dispensable role of the NF-χB sites in the UV-induction of the HIV-1 LTR in transgenic mice

We have previously reported the epidermis-specific expression of the HIV-1 LTR in transgenic mice and its induction by UV-B rays. To dissect the underlying mechanism of the UV induction of the LTR in mice, we developed two approaches. We first demonstrated by gel mobility shift analysis, using mice epidermal extracts, that the NF-kappa B sites of the HIV-1 LTR were one of the targets of the UV induction. The Sp-1 sites and the potential AP-1 sites of the LTR were not involved in this phenomenon. The transient transfection assays of modified LTR in HeLa cells also demonstrated the involvement of the NF-kappa B sites in the UV induction and were consistent with previously published data. Secondly, to study the regulation acting on an integrated gene, we generated transgenic mice carrying the lacZ gene under the control of the partially deleted LTR. All the transgenic lines and unexpectedly those carrying the LTR deleted for the kappa B sites displayed a UV-inducible epidermal expression. This suggests that, in mice, the UV induction might be mediated through other sites than the kappa B sites and may also depend on changes of the chromatin state.