Christine Perret

New targets of beta-catenin signaling in the liver are involved in the glutamine metabolism

Inappropriate activation of the Wnt/β-catenin signaling has been implicated in the development of hepatocellular carcinoma (HCC), but exactly how β-catenin works remains to be elucidated. To identify, in vivo, the target genes of β-catenin in the liver, we have used the suppression subtractive hybridization technique and transgenic mice expressing an activated β-catenin in the liver that developed hepatomegaly. We identified three genes involved in glutamine metabolism, encoding glutamine synthetase (GS), ornithine aminotransferase (OAT) and the glutamate transporter GLT-1. By Northern blot and immunohistochemical analysis we demonstrated that these three genes were specifically induced by activation of the β-catenin pathway in the liver. In different mouse models bearing an activated β-catenin signaling in the liver known to be associated with hepatocellular proliferation we observed a marked up-regulation of these three genes. The cellular distribution of GS and GLT-1 parallels β-catenin activity. By contrast no up-regulation of these three genes was observed in the liver in which hepatocyte proliferation was induced by a signal-independent of β-catenin. In addition, the GS promoter was activated in the liver of GS+/LacZ mice by adenovirus vector-mediated β-catenin overexpression. Strikingly, the overexpression of the GS gene in human HCC samples was strongly correlated with β-catenin activation. Together, our results indicate that GS is a target of the Wnt/β-catenin pathway in the liver. Because a linkage of the glutamine pathway to hepatocarcinogenesis has already been demonstrated, we propose that regulation of these three genes of glutamine metabolism by β-catenin is a contributing factor to liver carcinogenesis.

Beta-catenin mutations in hepatocellular carcinoma correlate with a low rate of loss of heterozygosity

To determine the frequency of Wnt/Wingless β catenin pathway alteration in human hepatocellular carcinoma, a β catenin and APC gene mutation screening was performed in a series of 119 tumors. An activating β catenin mutation in exon 3 was found in 18% of the cases. Among tumors lacking β catenin mutation, no APC mutation has been evidenced in a subset of 30 cases tested. The correlation between β catenin mutation status and chromosome segment deletions was studied on a set of 48 hyperploid tumors. Chromosome 1p, 4q and 16p deletions were significantly associated with the absence of β catenin mutation (P<0.05). Furthermore the Fractional Allelic Loss was significantly smaller in the β catenin mutated tumors than in the non-mutated tumors (0.12 versus 022). Taken together, these results suggest, the existence of two carcinogenesis mechanisms. The first mechanism implies a β catenin activating mutation associated with a low rate of loss of heterozygosity. The second mechanism, operating in a context of chromosomal instability, would involve tumor suppressor 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.

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.

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)

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.

Bmi1 is required for tumorigenesis in a mouse model of intestinal cancer

The epigenetic regulator BMI1 is upregulated progressively in a wide variety of human tumors including colorectal cancer. In this study, we assessed the requirement for Bmi1 in intestinal tumorigenesis using an autochthonous mouse model in which Apc was conditionally ablated in the intestinal epithelium. Germline mutation of Bmi1 significantly reduced both the number and size of small intestinal adenomas arising in this model, and it acted in a dose-dependent manner. Moreover, in contrast to wild-type controls, Bmi1−/− mice showed no increase in median tumor size, and a dramatic decrease in tumor number, between 3 and 4 months of age. Thus, Bmi1 is required for both progression and maintenance of small intestinal adenomas. Importantly, Bmi1 deficiency did not disrupt oncogenic events arising from Apc inactivation. Instead, the Arf tumor suppressor, a known target of Bmi1 epigenetic silencing, was upregulated in Bmi1 mutant tumors. This was accompanied by significant upregulation of p53, which was confirmed by sequencing to be wild-type, and also elevated apoptosis within the smallest Bmi1−/− adenomas. By crossing Arf into this cancer model, we showed that Arf is required for the induction of both p53 and apoptosis, and it is a key determinant of the ability of Bmi1 deficiency to suppress intestinal tumorigenesis. Finally, a conditional Bmi1 mutant strain was generated and used to determine the consequences of deleting Bmi1 specifically within the intestinal epithelium. Strikingly, intestinal-specific Bmi1 deletion suppressed small intestinal adenomas in a manner that was indistinguishable from germline Bmi1 deletion. Thus, we conclude that Bmi1 deficiency impairs the progression and maintenance of small intestinal tumors in a cell autonomous and highly Arf-dependent manner.