Cécile Godard

Crypt-restricted proliferation and commitment to the Paneth cell lineage following Apc loss in the mouse intestine

Loss of Apc appears to be one of the major events initiating colorectal cancer. However, the first events responsible for this initiation process are not well defined and the ways in which different epithelial cell types respond to Apc loss are unknown. We used a conditional gene-ablation approach in transgenic mice expressing tamoxifen-dependent Cre recombinase all along the crypt-villus axis to analyze the immediate effects of Apc loss in the small intestinal epithelium, both in the stem-cell compartment and in postmitotic epithelial cells. Within 4 days, Apc loss induced a dramatic enlargement of the crypt compartment associated with intense cell proliferation, apoptosis and impairment of cell migration. This result confirms the gatekeeper role of Apc in the intestinal epithelium in vivo. Although Apc deletion activatedβ -catenin signaling in the villi, we observed neither proliferation nor morphological change in this compartment. This highlights the dramatic difference in the responses of immature and differentiated epithelial cells to aberrant β-catenin signaling. These distinct biological responses were confirmed by molecular analyses, revealing that Myc and cyclin D1, two canonical β-catenin target genes, were induced in distinct compartments. We also showed that Apc is a crucial determinant of cell fate in the murine intestinal epithelium. Apc loss perturbs differentiation along the enterocyte, goblet and enteroendocrine lineages, and promotes commitment to the Paneth cell lineage through β-catenin/Tcf4-mediated transcriptional control of specific markers of Paneth cells, the cryptdin/defensin genes.

Colorectal cancers in a new mouse model of familial adenomatous polyposis: influence of genetic and environmental modifiers

Murine models of familial adenomatous polyposis harbor a germinal heterozygous mutation on Apc tumor suppressor gene. They are valuable tools for studying intestinal carcinogenesis, as most human sporadic cancers contain inactivating mutations of APC. However, Apc+/− mice, such as the well-characterized ApcMin/+ model, develop cancers principally in the small intestine, while humans develop mainly colorectal cancers. We used a Cre-loxP strategy to achieve a new model of germline Apc invalidation in which exon 14 is deleted. We compared the phenotype of these ApcΔ14/+ mice to that of the classical ApcMin/+. The main phenotypic difference is the shift of the tumors in the distal colon and rectum, often associated with a rectal prolapse. Thus, the severity of the colorectal phenotype is partly due to the particular mutation Δ14, but also to environmental parameters, as mice raised in conventional conditions developed more colon cancers than those raised in pathogen-free conditions. All lesions, including early lesions, revealed Apc LOH and loss of Apc gene expression. They accumulated β-catenin, overexpressed the β-catenin target genes cyclin D1 and c-Myc, and the distribution pattern of glutamine synthetase, a β-catenin target gene recently identified in the liver, was mosaic in intestinal adenomas. The ApcΔ14/+ model is thus a useful new tool for studies on the molecular mechanisms of colorectal tumorigenesis.

Identification of the IFITM Family as a New Molecular Marker in Human Colorectal Tumors

We analyzed the expression profiles of intestinal adenomas from a new murine familial adenomatous polyposis model (Apc(delta14/+)) using suppression subtractive hybridization to identify novel diagnostic markers of colorectal carcinogenesis. We identified 18 candidate genes having increased expression levels in the adenoma. Subsequent Northern blotting, real-time reverse transcription-PCR, and in situ hybridization analysis confirmed their induction in beta-catenin-activated epithelial cells of murine adenomas. We showed that most of the genes also have altered expression levels in human colonic adenomas and carcinomas. We focused on the IFITM genes that encode IFN-inducible transmembrane proteins. Serial analyses of gene expression levels revealed high levels of expression in early and late intestinal neoplasm in both mice and humans. Using a conditional mouse model of Apc inactivation and a human colon carcinoma cell line, we showed that IFITM gene expression is rapidly induced after activation of the beta-catenin signaling. Using a large-scale analysis of human tumors, we showed that IFITM gene expression is significantly up-regulated specifically in colorectal tumors and thus may be a useful diagnostic tool in these tumors.

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.)

Wnt/β-catenin pathway in hepatocellular carcinoma pathogenesis and liver physiology

The Wnt/beta-catenin pathway is a key developmental pathway for which alterations have been described in various human cancers. The aberrant activation of this pathway is a major event in human hepatocellular carcinoma. Several laboratories have shown that the Wnt/beta-catenin pathway plays an essential role in all phases of liver development and maturation, and is required for the metabolic function of this organ. In this review, we summarize current knowledge regarding the role of the Wnt/beta-catenin pathway in hepatocellular carcinoma pathogenesis and liver biology, and the possibilities for developing new therapeutic interventions based on this knowledge.

T‐cell factor 4 and β‐catenin chromatin occupancies pattern zonal liver metabolism in mice

β‐catenin signaling can be both a physiological and oncogenic pathway in the liver. It controls compartmentalized gene expression, allowing the liver to ensure its essential metabolic function. It is activated by mutations in 20%‐40% of hepatocellular carcinomas (HCCs) with specific metabolic features. We decipher the molecular determinants of β‐catenin‐dependent zonal transcription using mice with β‐catenin‐activated or ‐inactivated hepatocytes, characterizing in vivo their chromatin occupancy by T‐cell factor (Tcf)−4 and β‐catenin, transcriptome, and metabolome. We find that Tcf‐4 DNA bindings depend on β‐catenin. Tcf‐4/β‐catenin binds Wnt‐responsive elements preferentially around β‐catenin‐induced genes. In contrast, genes repressed by β‐catenin bind Tcf‐4 on hepatocyte nuclear factor 4 (Hnf‐4)‐responsive elements. β‐Catenin, Tcf‐4, and Hnf‐4α interact, dictating β‐catenin transcription, which is antagonistic to that elicited by Hnf‐4α. Finally, we find the drug/bile metabolism pathway to be the one most heavily targeted by β‐catenin, partly through xenobiotic nuclear receptors. Conclusions: β‐catenin patterns the zonal liver together with Tcf‐4, Hnf‐4α, and xenobiotic nuclear receptors. This network represses lipid metabolism and exacerbates glutamine, drug, and bile metabolism, mirroring HCCs with β‐catenin mutational activation. (Hepatology 2014;59:2344–2357)

Antitumour activity of an inhibitor of miR-34a in liver cancer with β-catenin-mutations.

Objective Hepatocellular carcinoma (HCC) is the most prevalent primary tumour of the liver. About a third of these tumours presents activating mutations of the β-catenin gene. The molecular pathogenesis of HCC has been elucidated, but mortality remains high, and new therapeutic approaches, including treatments based on microRNAs, are required. We aimed to identify candidate microRNAs, regulated by β-catenin, potentially involved in liver tumorigenesis. Design We used a mouse model, in which β-catenin signalling was overactivated exclusively in the liver by the tamoxifen-inducible and Cre-Lox-mediated inactivation of the Apc gene. This model develops tumours with properties similar to human HCC. Results We found that miR-34a was regulated by β-catenin, and significantly induced by the overactivation of β-catenin signalling in mouse tumours and in patients with HCC. An inhibitor of miR-34a (locked nucleic acid, LNA-34a) exerted antiproliferative activity in primary cultures of hepatocyte. This inhibition of proliferation was associated with a decrease in cyclin D1 levels, orchestrated principally by HNF-4α, a target of miR-34a considered to act as a tumour suppressor in the liver. In vivo, LNA-34a approximately halved progression rates for tumours displaying β-catenin activation together with an activation of caspases 2 and 3. Conclusions This work demonstrates the key oncogenic role of miR-34a in liver tumours with β-catenin gene mutations. We suggest that patients diagnosed with HCC with β-catenin mutations could be treated with an inhibitor of miR-34a. The potential value of this strategy lies in the modulation of the tumour suppressor HNF-4α, which targets cyclin D1, and the induction of a proapoptotic programme.

Early steps of a thymic tumor in SV40 transgenic mice: hyperplasia of medullary epithelial cells and increased mature thymocyte numbers disturb thymic export.

Bone marrow progenitors migrate to the thymus, where they proliferate and differentiate into immunologically competent T cells. In this report we show that mice transgenic for SV40 T and t antigens under the control of the L-pyruvate kinase promoter develop, in a first step, thymic hyperplasia of both thymocytes and epithelial cells. Morphological studies (histology, immunohistolabeling and electron microscopy) revealed modifications of the thymic microenvironment and gradual expansion of medullary epithelial cells in 1 month-old mice, taking over the cortical region. Then, a thymic carcinoma develops. Two-color labeling of frozen sections identified the transgene in medullary epithelial cells. Flow cytometry analysis demonstrated a marked increase in mature CD4+ and CD8+ thymocytes in adult mice (39±10×106 in transgenic mice and 12±5×106 in age-matched controls). Furthermore, thymocyte export was disturbed.