Sabine Colnot

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.

The H19 locus acts in vivo as a tumor suppressor

The H19 locus belongs to a cluster of imprinted genes that is linked to the human Beckwith-Wiedemann syndrome. The expression of H19 and its closely associated IGF2 gene is frequently deregulated in some human tumors, such as Wilms tumors. In these cases, biallelic IGF2 expression and lack of expression of H19 are associated with hypermethylation of the imprinting center of this locus. These observations and others have suggested a potential tumor suppressor effect of the H19 locus. Some studies have also suggested that H19 is an oncogene, based on tissue culture systems. We show, using in vivo murine models of tumorigenesis, that the H19 locus controls the size of experimental teratocarcinomas, the number of polyps in the Apc murine model of colorectal cancer and the timing of appearance of SV40-induced hepatocarcinomas. The H19 locus thus clearly displays a tumor suppressor effect in mice.

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.

Oncogenic β-catenin triggers an inflammatory response that determines the aggressiveness of hepatocellular carcinoma in mice.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Its pathogenesis is frequently linked to liver inflammation. Gain-of-function mutations in the gene encoding β-catenin are frequent genetic modifications found in human HCCs. Thus, we investigated whether inflammation was a component of β-catenin-induced tumorigenesis using genetically modified mouse models that recapitulated the stages of initiation and progression of this tumoral process. Oncogenic β-catenin signaling was found to induce an inflammatory program in hepatocytes that involved direct transcriptional control by β-catenin and activation of the NF-κB pathway. This led to a specific inflammatory response, the intensity of which determined the degree of tumor aggressiveness. The chemokine-like chemotactic factor leukocyte cell-derived chemotaxin 2 (LECT2) and invariant NKT (iNKT) cells were identified as key interconnected effectors of liver β-catenin-induced inflammation. In genetic deletion models lacking the gene encoding LECT2 or iNKT cells, hepatic β-catenin signaling triggered the formation of highly malignant HCCs with lung metastasis. Thus, our results identify inflammation as a key player in β-catenin-induced liver tumorigenesis. We provide strong evidence that, by activating pro- and antiinflammatory mediators, β-catenin signaling produces an inflammatory microenvironment that has an impact on tumoral development. Our data are consistent with the fact that most β-catenin-activated HCCs are of better prognosis.

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.

A genetic study of the role of the Wnt/β-catenin signalling in Paneth cell differentiation

Wnt/beta-catenin signalling plays a key role in the homeostasis of the intestinal epithelium. Whereas its role in the maintenance of the stem cell compartment has been clearly demonstrated, its role in the Paneth cell fate remains unclear. We performed genetic studies to elucidate the functions of the Wnt/beta-catenin pathway in Paneth cell differentiation. We analysed mice with inducible gain-of-function mutations in the Wnt/beta-catenin pathway and mice with a hypomorphic beta-catenin allele that have not been previously described. We demonstrated that acute activation of Wnt/beta-catenin signalling induces de novo specification of Paneth cells in both the small intestine and colon and that colon cancers resulting from Apc mutations expressed many genes involved in Paneth cell differentiation. This suggests a key role for the Wnt/beta-catenin pathway in Paneth cell differentiation. We also showed that a slight decrease in beta-catenin gene dosage induced a major defect in Paneth cell differentiation, but only a modest effect on crypt morphogenesis. Overall, our findings show that a high level of beta-catenin activation is required to determine Paneth cell fate and that fine tuning of beta-catenin signalling is critical for correct Paneth cell lineage.

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.

A Complex Interplay between Wnt/β-Catenin Signalling and the Cell Cycle in the Adult Liver.

Canonical Wnt signalling, governed by its effector β-catenin, is known for a long time as playing an important role in development, tissue homeostasis, and cancer. In the liver, it was unravelled as both an oncogenic pathway involved in a subset of liver cancers and a physiological signalling identified as the “zonation-keeper” of the quiescent liver lobule. This duality has encouraged to explore the role of canonical Wnt in liver regeneration and liver-cell proliferation mainly using murine genetic models of β-catenin overactivation or inactivation. These studies definitely integrate Wnt signalling within the hepatic network driving regeneration and proliferation. We will review here the current knowledge concerning the mitogenic effect of Wnt, to switch on its specific role in the liver, which is quiescent but with a great capacity to regenerate. The duality of β-catenin signalling, associated both with liver quiescence and liver-cell proliferation, will be brought forward.