CDX2 autoregulation in human intestinal metaplasia of the stomach: impact on the stability of the phenotype

Background and aims Intestinal metaplasia (IM) is a gastric preneoplastic lesion that appears following Helicobacter pylori infection and confers an increased risk for development of cancer. It is induced by gastric expression of the intestine-specific transcription factor CDX2. The regulatory mechanisms involved in triggering and maintaining gastric CDX2 expression have not been fully elucidated. The Cdx2(+/-) mouse develops intestinal polyps with gastric differentiation and total loss of Cdx2 expression in the absence of structural loss of the second allele, suggesting a regulatory defect. This putative haplo-insufficiency, together with the apparent stability of IM, led to the hypothesis that CDX2 regulates its own expression through an autoregulatory loop in both contexts.Methods Gastrointestinal cell lines were co-transfected with wild-type or mutated Cdx2 promoter constructs and CDX2 expression vector for luciferase assays. Transfection experiments were also used to assess endogenous CDX2 autoregulation, evaluated by RT-PCR, qPCR and western blotting. Chromatin immunoprecipitation was performed in a cell line, mouse ileum and human IM. Results CDX2 binds to and transactivates its own promoter and positively regulates its expression in gastrointestinal human carcinoma cell lines. Furthermore, CDX2 is bound to its promoter in the mouse ileum and in human gastric IM, providing a major contribution to understanding the relevance of this autoregulatory pathway in vivo.Conclusion The results of this study demonstrate another layer of complexity in CDX2 regulation by an effective autoregulatory loop which may have a major impact on the stability of human IM, possibly resulting in the inevitable progression of the gastric carcinogenesis pathway

Anchoring Secreted Proteins in Endoplasmic Reticulum by Plant Oleosin: The Example of Vitamin B12 Cellular Sequestration by Transcobalamin

BACKGROUND: Oleosin is a plant protein localized to lipid droplets and endoplasmic reticulum of plant cells. Our idea was to use it to target functional secretory proteins of interest to the cytosolic side of the endoplasmic reticulum of mammalian cells, through expressing oleosin-containing chimeras. We have designed this approach to create cellular models deficient in vitamin B12 (cobalamin) because of the known problematics associated to the obtainment of effective vitamin B12 deficient cell models. This was achieved by the overexpression of transcobalamin inside cells through anchoring to oleosin. METHODOLOGY: chimera gene constructs including transcobalamin-oleosin (TC-O), green fluorescent protein-transcobalamin-oleosin (GFP-TC-O) and oleosin-transcobalamin (O-TC) were inserted into pAcSG2 and pCDNA3 vectors for expression in sf9 insect cells, Caco2 (colon carcinoma), NIE-115 (mouse neuroblastoma), HEK (human embryonic kidney), COS-7 (Green Monkey SV40-transfected kidney fibroblasts) and CHO (Chinese hamster ovary cells). The subcellular localization, the changes in vitamin B12 binding activity and the metabolic consequences were investigated in both Caco2 and NIE-115 cells. PRINCIPAL FINDINGS: vitamin B12 binding was dramatically higher in TC-O than that in O-TC and wild type (WT). The expression of GFP-TC-O was observed in all cell lines and found to be co-localized with an ER-targeted red fluorescent protein and calreticulin of the endoplasmic reticulum in Caco2 and COS-7 cells. The overexpression of TC-O led to B12 deficiency, evidenced by impaired conversion of cyano-cobalamin to ado-cobalamin and methyl-cobalamin, decreased methionine synthase activity and reduced S-adenosyl methionine to S-adenosyl homocysteine ratio, as well as increases in homocysteine and methylmalonic acid concentration. CONCLUSIONS/SIGNIFICANCE: the heterologous expression of TC-O in mammalian cells can be used as an effective strategy for investigating the cellular consequences of vitamin B12 deficiency. More generally, expression of oleosin-anchored proteins could be an interesting tool in cell engineering for studying proteins of pharmacological interest

CDX2 expression in the hematopoietic lineage promotes leukemogenesis via TGFβ inhibition

The intestine-specific caudal-related homeobox gene-2 (CDX2) homeobox gene, while being a tumor suppressor in the gut, is ectopically expressed in a large proportion of acute leukemia and is associated with poor prognosis. Here, we report that turning on human CDX2 expression in the hematopoietic lineage of mice induces acute monoblastic leukemia, characterized by the decrease in erythroid and lymphoid cells at the benefit of immature monocytic and granulocytic cells. One of the highly stimulated genes in leukemic bone marrow cells was BMP and activin membrane-bound inhibitor (Bambi), an inhibitor of transforming growth factor-β (TGF-β) signaling. The CDX2 protein was shown to bind to and activate the transcription of the human BAMBI promoter. Moreover, in a leukemic cell line established from CDX2-expressing mice, reducing the levels of CDX2 or Bambi stimulated the TGF-β-dependent expression of Cd11b, a marker of monocyte maturation. Taken together, this work demonstrates the strong oncogenic potential of the homeobox gene CDX2 in the hematopoietic lineage, in contrast with its physiological tumor suppressor activity exerted in the gut. It also reveals, through BAMBI and TGF-β signaling, the involvement of CDX2 in the perturbation of the interactions between leukemia cells and their microenvironment

Contribution of Soft Substrates to Malignancy and Tumor Suppression during Colon Cancer Cell Division

In colon cancer, a highly aggressive disease, progression through the malignant sequence is accompanied by increasingly numerous chromosomal rearrangements. To colonize target organs, invasive cells cross several tissues of various elastic moduli. Whether soft tissue increases malignancy or in contrast limits invasive colon cell spreading remains an open question. Using polyelectrolyte multilayer films mimicking microenvironments of various elastic moduli, we revealed that human SW480 colon cancer cells displayed increasing frequency in chromosomal segregation abnormalities when cultured on substrates with decreasing stiffness. Our results show that, although decreasing stiffness correlates with increased cell lethality, a significant proportion of SW480 cancer cells did escape from the very soft substrates, even when bearing abnormal chromosome segregation, achieve mitosis and undergo a new cycle of replication in contrast to human colonic HCoEpiC cells which died on soft substrates. This observation opens the possibility that the ability of cancer cells to overcome defects in chromosome segregation on very soft substrates could contribute to increasing chromosomal rearrangements and tumor cell aggressiveness

Control of differentiation-induced calbindin-D 9k gene expression in Caco-2 cells by cdx-2 and HNF-1α

International audienceCalbindin D 9k (CaBP) is critical for intestinal calcium absorption; its in vivo expression is restricted to differentiated enterocytes of the small intestine. Our goal was to identify factors controlling the transcriptional regulation of this gene in the human intestine. Both the natural gene and a 4600-bp promoter construct were strongly regulated by differentiation (>100-fold) but not by treatment with 1,25(OH) 2 vitamin D (<2-fold) in the Caco-2 clone TC7. Deletion-mutation studies revealed that conserved promoter sequences for cdx-2 (at −3158 bp) and hepatocyte nuclear factor (HNF)-1 (at −3131 and at −98 bp) combined to control CaBP expression during differentiation. Other putative response elements were not important for CaBP regulation in TC7 cells (CCAAT enhancer binding protein, pancreatic duodenal homebox-1 (pdx-1), a proximal cdx-2 element). Mutation of the distal HNF-1 site had the greatest impact on CaBP gene expression through disruption of HNF-1α binding; both basal and differentiation-mediated CaBP expression was reduced by 80%. In contrast, mutation of the distal cdx-2 element reduced only basal CaBP expression. Whereas a 60% reduction of CaBP mRNA in the duodenum of HNF-1α null mice confirmed the physiological importance of HNF-1α for CaBP gene regulation, additional studies showed that maximal CaBP expression requires the presence of both HNF-1α and cdx-2. Our data suggest that cdx-2 is a permissive factor that influences basal CaBP expression in enterocytes and that HNF-1α modulates CaBP gene expression during cellular differentiation

Pathophysiology of intestinal metaplasia of the stomach: emphasis on CDX2 regulation. Biochem Soc Trans 38

Abstract IM (intestinal metaplasia) of the stomach is a pre-neoplastic lesion that usually follows Helicobacter pylori infection and that confers increased risk for gastric cancer development. After setting the role played by CDX2 (Caudal-type homeobox 2) in the establishment of gastric IM, it became of foremost importance to unravel the regulatory mechanisms behind its de novo expression in the stomach. In the present paper, we review the basic pathology of gastric IM as well as the current knowledge on molecular pathways involved in CDX2 regulation in the gastric context. Metaplasia: definition and examples Metaplasia is classically defined as a reversible change in which one epithelial or mesenchymal adult cell type is replaced by another adult cell type In epithelial tissues, metaplasia follows a glandularsquamous, a squamous-glandular or a glandular-glandular pathway. A frequent example of the first type can be found in the respiratory epithelium of cigarette smokers, whereas a squamous-glandular metaplasia is most frequently observed in Barrett&apos;s oesophagus, subsequent to gastro-oesophageal reflux disease. IM (intestinal metaplasia) of the stomach is the prototype of a glandular-glandular metaplasia. Nevertheless, the metaplastic lesions are not exclusive to epithelial tissues, and mesenchymal tissues, such as bone, cartilage and adipose tissue, can be encountered ectopically in soft tissues or other organs (e.g. heart). The present review examines several aspects of gastric IM, with an emphasis on the molecular pathways that lead to the CDX (Caudal-type homeobox) 2-dependent intestinalization of the gastric mucosa. Intestinal metaplasia of human stomach Gastric IM is a multifocal lesion characterized, morphologically, by the presence in the gastric mucosa, alone or in combination, of intestinal cell types such as goblet, Paneth and absorptive cells. The metaplastic glands show reorganization of the whole metaplastic crypt, with displacement of the proliferative zone from the neck region to the base of the crypt, and modification of the stroma that surrounds the metaplastic glands Characterization of gastric IM was initially based on histochemical methods that allowed the identification of three distinct types: Type I, containing absorptive cells, Paneth cells and goblet cells secreting sialomucins; Type II with goblet and columnar cells secreting sialomucins; and Type III, with goblet and columnar cells secreting sulfomucins We and others have contributed to describe the molecular alterations of IM beyond the classical morphological and histochemical characterization. We know more now about the differentiation markers that are lost and gained in the differentiation switch. On the basis of newly identified markers of gastric and intestinal differentiation, namely mucin markers, a new classification of IM emerged. The new approach defines two main IM types: complete, or intestinal IM, and incomplete, or gastrointestinal mixed IM Common to both types of IM is the de novo expression of CDX2, a transcription factor that, in normal conditions, is expressed exclusively in the intestine, but is present in every focus of aberrant intestinal differentiation occurring in the human body. It is thus considered the master gene for intestinal differentiation and it is reviewed in detail below

Wnt/β-Catenin/Tcf Signaling Induces the Transcription of Axin2, a Negative Regulator of the Signaling Pathway

Axin2/Conductin/Axil and its ortholog Axin are negative regulators of the Wnt signaling pathway, which promote the phosphorylation and degradation of β-catenin. While Axin is expressed ubiquitously, Axin2 mRNA was seen in a restricted pattern during mouse embryogenesis and organogenesis. Because many sites of Axin2 expression overlapped with those of several Wnt genes, we tested whether Axin2 was induced by Wnt signaling. Endogenous Axin2 mRNA and protein expression could be rapidly induced by activation of the Wnt pathway, and Axin2 reporter constructs, containing a 5.6-kb DNA fragment including the promoter and first intron, were also induced. This genomic region contains eight Tcf/LEF consensus binding sites, five of which are located within longer, highly conserved noncoding sequences. The mutation or deletion of these Tcf/LEF sites greatly diminished induction by β-catenin, and mutation of the Tcf/LEF site T2 abolished protein binding in an electrophoretic mobility shift assay. These results strongly suggest that Axin2 is a direct target of the Wnt pathway, mediated through Tcf/LEF factors. The 5.6-kb genomic sequence was sufficient to direct the tissue-specific expression of d2EGFP in transgenic embryos, consistent with a role for the Tcf/LEF sites and surrounding conserved sequences in the in vivo expression pattern of Axin2. Our results suggest that Axin2 participates in a negative feedback loop, which could serve to limit the duration or intensity of a Wnt-initiated signal