Béatrice Romagnolo

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.

Early development of polycystic kidney disease in transgenic mice expressing an activated mutant of the β-catenin gene

Autosomal dominant polycystic kidney disease (ADPKD) is common and is a major cause of renal failure. Although the genetics of ADPKD are well known and have led to the discovery of polycystins, a new protein family, the pathogenesis of the disease remains largely unknown. Recent studies have indicated that the β-catenin signaling pathway is one of the targets of the transduction pathway controlled by the polycystins. We have generated transgenic mice that overproduce an oncogenic form of β-catenin in the epithelial cells of the kidney. These mice developed severe polycystic lesions soon after birth that affected the glomeruli, proximal, distal tubules and collecting ducts. The phenotype of these mice mimicked the human ADPKD phenotype. Cyst formation was associated with an increase in cell proliferation and apoptosis. The cell proliferation and apoptotic indexes was increased 4–5-fold and 3–4-fold, respectively, in cystic tubules of the transgenic mice compared to that of littermate controls. Our findings provide experimental genetic evidence that activation of the Wnt/β-catenin signaling pathway causes polycystic kidney disease and support the view that dysregulation of the Wnt/β-catenin signaling is involved in its pathogenesis.

Distinct ATOH1 and Neurog3 requirements define tuft cells as a new secretory cell type in the intestinal epithelium

Tuft cells represent a fourth type of intestinal secretory cell that constitutes the primary source of endogenous intestinal opioids and are the only epithelial cell that constitutively express cyclooxygenases.

Functional intestinal stem cells after Paneth cell ablation induced by the loss of transcription factor Math1 (Atoh1)

Intestinal epithelium has the capacity to self-renew and generate differentiated cells through the existence of two types of epithelial stem cells: active crypt base columnar cells (CBCs) and quiescent +4 cells. The behaviors of these cells are regulated both by intrinsic programs and by extrinsic signals sent by neighboring cells, which define the niche. It is clear that the β-catenin pathway acts as an essential intrinsic signal for the maintenance and proliferation of CBC, and it was recently proposed that Paneth cells provide a crucial niche by secreting Wingless/Int (Wnt) ligands. Here, we examined the effect of disrupting the intestinal stem cell niche by inducible deletion of the transcription factor Math1 (Atoh1), an essential driver of secretory cell differentiation. We found that complete loss of Paneth cells attributable to Math1 deficiency did not perturb the crypt architecture and allowed the maintenance and proliferation of CBCs. Indeed, Math1-deficient crypt cells tolerated in vivo Paneth cell loss and maintained active β-catenin signaling but could not grow ex vivo without exogenous Wnt, implying that, in vivo, underlying mucosal cells act as potential niche. Upon irradiation, Math1-deficient crypt cells regenerated and CBCs continued cycling. Finally, CBC stem cells deficient in adenomatous polyposis coli (Apc) and Math1 were able to promote intestinal tumorigenesis. We conclude that in vivo, Math1-deficient crypts counteract the absence of Paneth cell-derived Wnts and prevent CBC stem cell exhaustion.

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.

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.

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.)

Intestinal inhibition of Atg7 prevents tumour initiation through a microbiome-influenced immune response and suppresses tumour growth

Here, we show that autophagy is activated in the intestinal epithelium in murine and human colorectal cancer and that the conditional inactivation of Atg7 in intestinal epithelial cells inhibits the formation of pre-cancerous lesions in Apc+/− mice by enhancing anti-tumour responses. The antibody-mediated depletion of CD8+ T cells showed that these cells are essential for the anti-tumoral responses mediated by the inhibition of autophagy. We show that Atg7 deficiency leads to intestinal dysbiosis and that the microbiota is required for anticancer responses. In addition, Atg7 deficiency resulted in a stress response accompanied by metabolic defects, AMPK activation and p53-mediated cell-cycle arrest in tumour cells but not in normal tissue. This study reveals that the inhibition of autophagy within the epithelium may prevent the development and progression of colorectal cancer in genetically predisposed patients.

Autophagy, microbiota and intestinal oncogenesis

Comment on: Lévy et al., Nat Cell Biol. 2015 Aug; 17(8):1062-73.