A Mouse Model of Cholangiocarcinoma Uncovers a Role for Tensin-4 in Tumor Progression.

BACKGROUND AND AIMS: Earlier diagnosis and treatment of intrahepatic cholangiocarcinoma (iCCA) are necessary to improve therapy, yet limited information is available about initiation and evolution of iCCA precursor lesions. Therefore, there is a need to identify mechanisms driving formation of precancerous lesions and their progression toward invasive tumors using experimental models that faithfully recapitulate human tumorigenesis. APPROACH AND RESULTS: To this end, we generated a mouse model which combines cholangiocyte-specific expression of KrasG12D with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet-induced inflammation to mimic iCCA development in patients with cholangitis. Histological and transcriptomic analyses of the mouse precursor lesions and iCCA were performed and compared with human analyses. The function of genes overexpressed during tumorigenesis was investigated in human cell lines. We found that mice expressing KrasG12D in cholangiocytes and fed a DDC diet developed cholangitis, ductular proliferations, intraductal papillary neoplasms of bile ducts (IPNBs), and, eventually, iCCAs. The histology of mouse and human IPNBs was similar, and mouse iCCAs displayed histological characteristics of human mucin-producing, large-duct-type iCCA. Signaling pathways activated in human iCCA were also activated in mice. The identification of transition zones between IPNB and iCCA on tissue sections, combined with RNA-sequencing analyses of the lesions supported that iCCAs derive from IPNBs. We further provide evidence that tensin-4 (TNS4), which is stimulated by KRASG12D and SRY-related HMG box transcription factor 17, promotes tumor progression. CONCLUSIONS: We developed a mouse model that faithfully recapitulates human iCCA tumorigenesis and identified a gene cascade which involves TNS4 and promotes tumor progression.

High fetal hemoglobin level is associated with increased risk of cerebral vasculopathy in children with sickle cell disease in Mayotte.

BACKGROUND: Understanding the genetics underlying the heritable subphenotypes of sickle cell anemia, specific to each population, would be prognostically useful and could inform personalized therapeutics.The objective of this study was to describe the genetic modulators of sickle cell disease in a cohort of pediatric patients followed up in Mayotte. METHODS: This retrospective cohort study analyzed clinical and biological data, collected between January1st2007 and December 31st2017, in children younger than 18 years. RESULTS: We included 185 children with 72% SS, 16% Sß0-thalassemia and 12% Sß + thalassemia. The average age was 9.5 years; 10% of patients were lost to follow up. The Bantu haplotype was associated with an increase in hospitalizations and transfusions. The alpha-thalassemic mutation was associated with a decrease of hemolysis biological parameters (anemia, reticulocytes), and a decrease of cerebral vasculopathy. The Single Nucleotide Polymorphisms BCL11A rs4671393, BCL11A rs11886868, BCL11A rs1427407 and HMIP rs9399137 were associated with the group of children with HbF > 10%. Patients with HbF > 10% presented a significant risk of early onset of cerebral vasculopathy. CONCLUSIONS: The most remarkable result of our study was the association of SNPs with clinically relevant phenotypic groups. BCL11A rs4671393, BCL11A rs11886868, BCL11A rs1427407 and HMIP rs9399137 were correlated with HbF > 10%, a group that has a higher risk of cerebral vasculopathy and should be oriented towards the hemolytic sub-phenotype.

Role of ß-catenin in development of bile ducts.

Beta-catenin is known to play stage- and cell-specific functions during liver development. However, its role in development of bile ducts has not yet been addressed. Here we used stage-specific in vivo gain- and loss-of-function approaches, as well as lineage tracing experiments in the mouse, to first demonstrate that ß-catenin is dispensable for differentiation of liver precursor cells (hepatoblasts) to cholangiocyte precursors. Second, when ß-catenin was depleted in the latter, maturation of cholangiocytes, bile duct morphogenesis and differentiation of periportal hepatocytes from cholangiocyte precursors was normal. In contrast, stabilization of ß-catenin in cholangiocyte precursors perturbed duct development and cholangiocyte differentiation. We conclude that ß-catenin is dispensable for biliary development but that its activity must be kept within tight limits. Our work is expected to significantly impact on in vitro differentiation of stem cells to cholangiocytes for toxicology studies and disease modeling.

Cdx2 determines the fate of postnatal intestinal endoderm.

Knock out of intestinal Cdx2 produces different effects depending upon the developmental stage at which this occurs. Early in development it produces histologically ordered stomach mucosa in the midgut. Conditional inactivation of Cdx2 in adult intestinal epithelium, as well as specifically in the Lgr5-positive stem cells, of adult mice allows long-term survival of the animals but fails to produce this phenotype. Instead, the endodermal cells exhibit cell-autonomous expression of gastric genes in an intestinal setting that is not accompanied by mesodermal expression of Barx1, which is necessary for gastric morphogenesis. Cdx2-negative endodermal cells also fail to express Sox2, a marker of gastric morphogenesis. Maturation of the stem cell niche thus appears to be associated with loss of ability to express positional information cues that are required for normal stomach development. Cdx2-negative intestinal crypts produce subsurface cystic vesicles, whereas untargeted crypts hypertrophy to later replace the surface epithelium. These observations are supported by studies involving inactivation of Cdx2 in intestinal crypts cultured in vitro. This abolishes their ability to form long-term growing intestinal organoids that differentiate into intestinal phenotypes. We conclude that expression of Cdx2 is essential for differentiation of gut stem cells into any of the intestinal cell types, but they maintain a degree of cell-autonomous plasticity that allows them to switch on a variety of gastric genes.

Cdx2 homeoprotein inhibits non-homologous end joining in colon cancer but not in leukemia cells.

Cdx2, a gene of the paraHox cluster, encodes a homeodomain transcription factor that plays numerous roles in embryonic development and in homeostasis of the adult intestine. Whereas Cdx2 exerts a tumor suppressor function in the gut, its abnormal ectopic expression in acute leukemia is associated to a pro-oncogenic function. To try to understand this duality, we have hypothesized that Cdx2 may interact with different protein partners in the two tissues and set up experiments to identify them by tandem affinity purification. We show here that Cdx2 interacts with the Ku heterodimer specifically in intestinal cells, but not in leukemia cells, via its homeodomain. Ku proteins do not affect Cdx2 transcriptional activity. However, Cdx2 inhibits in vivo and in vitro the DNA repair activity mediated by Ku proteins in intestinal cells. Whereas Cdx2 does not affect the recruitment of Ku proteins and DNA-PKcs into the DNA repair complex, it inhibits DNA-PKcs activity. Thus, we report here a new function of Cdx2, acting as an inhibitor of the DNA repair machinery, that may contribute to its tumor suppressor function specifically in the gut.

Fine-tuning and autoregulation of the intestinal determinant and tumor suppressor homeobox gene CDX2 by alternative splicing.

On the basis of phylogenetic analyses, we uncovered a variant of the CDX2 homeobox gene, a major regulator of the development and homeostasis of the gut epithelium, also involved in cancer. This variant, miniCDX2, is generated by alternative splicing coupled to alternative translation initiation, and contains the DNA-binding homeodomain but is devoid of transactivation domain. It is predominantly expressed in crypt cells, whereas the CDX2 protein is present in crypt cells but also in differentiated villous cells. Functional studies revealed a dominant-negative effect exerted by miniCDX2 on the transcriptional activity of CDX2, and conversely similar effects regarding several transcription-independent functions of CDX2. In addition, a regulatory role played by the CDX2 and miniCDX2 homeoproteins on their pre-mRNA splicing is displayed, through interactions with splicing factors. Overexpression of miniCDX2 in the duodenal Brunner glands leads to the expansion of the territory of these glands and ultimately to brunneroma. As a whole, this study characterized a new and original variant of the CDX2 homeobox gene. The production of this variant represents not only a novel level of regulation of this gene, but also a novel way to fine-tune its biological activity through the versatile functions exerted by the truncated variant compared to the full-length homeoprotein. This study highlights the relevance of generating protein diversity through alternative splicing in the gut and its diseases.

"OpenLAB": A 2-hour PCR-based practical for high school students.

The Strasbourg University PhD school in Life and Health Sciences launched an initiative called "OpenLAB." This project was developed in an effort to help high school teenagers understand theoretical and abstract concepts in genetics. A second objective of this program is to help students in defining their future orientation and to attract them to biology. The general idea is a 2-hour PCR-based practical that is developed around a fictitious criminal investigation. The practical is taught by PhD graduate students who bring all the required reagents and modern equipment into the classroom. Running the PCR provides free time dedicated to discussions with students about their future plans after the high school diploma. A specific website and a powerpoint presentation were developed to provide appropriate scientific information. Starting on a modest scale in Strasbourg in December 2008, "OpenLAB" was rapidly and well received all around, visiting 53 classes spread over a 200 km area in Alsace until May 2009. It permitted interactions with almost one thousand students in their last year of high school, with the prospect to visit 20% more classes this school year. Our experience, along with feedback from students and their teachers, suggests that it is possible to reach out to many students and have a strong impact with a rather limited budget.