Ectopic CDX2 Expression Research Articles

Maternal exposure to hyperbaric oxygen at the preimplantation stages increases apoptosis and ectopic Cdx2 expression and decreases Oct4 expression in mouse blastocysts via Nrf2-Notch1 upregulation and Nf2 downregulation.

The environmental oxygen tension has been reported to impact the blastocyst quality and cell numbers in the inner cell mass (ICM) during human and murine embryogenesis. While the molecular mechanisms leading to increased ICM cell numbers and pluripotency gene expression under hypoxia have been deciphered, it remains unknown which regulatory pathways caused the underweight fetal body and overweight placenta after maternal exposure to hyperbaric oxygen (HBO). The blastocysts from the HBO-exposed pregnant mice revealed significantly increased signals of reactive oxygen species (ROS) and nuclear Nrf2 staining, decreased Nf2 and Oct4 expression, increased nuclear Tp53bp1 and active caspase-3 staining, and ectopic nuclear signals of Cdx2, Yap, and the Notch1 intracellular domain (N1ICD) in the ICM. In the ICM of the HBO-exposed blastocysts, both Nf2 cDNA microinjection and Nrf2 shRNA microinjection significantly decreased the ectopic nuclear expression of Cdx2, Tp53bp1, and Yap whereas increased Oct4 expression, while Nrf2 shRNA microinjection also significantly decreased Notch1 mRNA levels and nuclear expression of N1ICD and active caspase-3. We show for the first time that maternal exposure to HBO at the preimplantation stage induces apoptosis and impairs ICM cell specification via upregulating Nrf2-Notch1-Cdx2 expression and downregulating Nf2-Oct4 expression.

Transcription factor-mediated intestinal metaplasia and the role of a shadow enhancer.

Barrett's esophagus (BE) and gastric intestinal metaplasia are related premalignant conditions in which areas of human stomach epithelium express mixed gastric and intestinal features. Intestinal transcription factors (TFs) are expressed in both conditions, with unclear causal roles and cis-regulatory mechanisms. Ectopic CDX2 reprogrammed isogenic mouse stomach organoid lines to a hybrid stomach-intestinal state transcriptionally similar to clinical metaplasia; squamous esophageal organoids resisted this CDX2-mediated effect. Reprogramming was associated with induced activity at thousands of previously inaccessible intestine-restricted enhancers, where CDX2 occupied DNA directly. HNF4A, a TF recently implicated in BE pathogenesis, induced weaker intestinalization by binding a novel shadow Cdx2 enhancer and hence activating Cdx2 expression. CRISPR/Cas9-mediated germline deletion of that cis-element demonstrated its requirement in Cdx2 induction and in the resulting activation of intestinal genes in stomach cells. dCas9-conjugated KRAB repression mapped this activity to the shadow enhancer's HNF4A binding site. Altogether, we show extensive but selective recruitment of intestinal enhancers by CDX2 in gastric cells and that HNF4A-mediated ectopic CDX2 expression in the stomach occurs through a conserved shadow cis-element. These findings identify mechanisms for TF-driven intestinal metaplasia and a likely pathogenic TF hierarchy.

Clinicopathologic and molecular characteristics of gastric cancer showing gastric and intestinal mucin phenotype.

Gastric cancer (GC), one of the most common human cancers, can be classified into gastric or intestinal phenotype according to mucin expression. TP53 mutation, allelic deletion of the APC gene and nuclear staining of β-catenin are frequently detected in the intestinal phenotype of GC, whereas CDH1 gene mutation, microsatellite instability and DNA hypermethylation of MLH1 are common events in the gastric phenotype of GC. Our Serial Analysis of Gene Expression (SAGE) and Escherichia coli ampicillin secretion trap (CAST) analyses revealed that CDH17, REG4, OLFM4, HOXA10, DSC2, TSPAN8 and TM9SF3 are upregulated in GC and that CLDN18 is downregulated in GC. Expression of CDH17, REG4, HOXA10 and DSC2 and downregulation of CLDN18 are observed in the intestinal phenotype of GC. In contrast, OLFM4 is expressed in the gastric phenotype of GC. Expression of TSPAN8, TM9SF3 and HER2 are not associated with either gastric or intestinal phenotypes. Ectopic CDX2 expression plays a key function in the GC intestinal phenotype. MUC2, CDH17, REG4, DSC2 and ABCB1 are direct targets of CDX2. Importantly, these genes encode transmembrane/secretory proteins, indicating that the microenvironment as well as cancer cells are also different between gastric and intestinal phenotypes of GC.

The role of CDX2 in trophoblast differentiation from human pluripotent tsem

s / Placenta 34 (2013) A1–A99 A22 [1] Stanley et al 2012. Hypertension. 59(5): 1021-8. [2] Constancia et al 2002. Nature. 417, 945-8. http://dx.doi.org/10.1016/j.placenta.2013.06.065 P1.29. THE ROLE OF CDX2 IN TROPHOBLAST DIFFERENTIATION FROM HUMAN PLURIPOTENT TSEM Chi-Wei Lu, Yasunari Seita Rutgers University, New Brunswick, NJ, USA Objective The unique requirement for Cdx2 in the development of mouse placental trophoblasts has been well-studied, in genetic models and through manipulating its expression in embryonic stem cells. The role of CDX2 in human trophoblast differentiation remains elusive. MethodsWe studied the involvement of CDX2 in trophoblast differentiation fromhumanembryonic and induced pluripotent stem cells (ESCs and iPSCs). Results From BMP4-induced trophoblast differentiation, we observed that CDX2 expressed in nearly 100% of cells, and largely overlapping with OCT4 expression, suggesting the model of their mutual transcription repression seen in the mouse is not conserved in humans. We tested whether CDX2 expression alone is associated with a trophoblast progenitor fate, through generating iPS cell lines with ectopic CDX2 expression. Two CDX2 expressing cell lines were analyzed, both expressing telomerase activity and capable of differentiation into syncytiotrophoblast after switching into serum containing media. However, in the cell line expressing CDX2 at a very high level, differentiation into mesoderm and endoderm lineages was also observed, suggesting the effect of CDX2 for trophoblast differentiation is dose dependent. Repressing CDX2 expression by shRNA does not blockade BMP4-induced trophoblast differentiation. Conclusion Altogether, our results suggest that while the mechanism of transcription regulation is different from the mouse, CDX2 in the human plays a conservative role in trophoblast development as being not necessary for differentiation, but sufficient for inducing a trophoblastic-stem cell like phenotype from pluripotent stem cells. http://dx.doi.org/10.1016/j.placenta.2013.06.066 P1.30. ENDOTHELIAL DYSFUNCTION OF THE FETOPLACENTAL CIRCULATION CONTRIBUTES TO THE INCREASED VASCULAR RESISTANCE OBSERVED IN FETAL GROWTH RESTRICTION Sarah Jones, Helen Bischof, Edward Johnstone, Sue Greenwood, Mark Wareing, Colin Sibley, Paul Brownbill University of Manchester, Manchester, UK Fetal growth restriction (FGR) affects 3-8% of pregnancies and is associated with increased risk of neonatalmorbidityandmortality. A hallmark of severe FGR is increased vascular resistance identified clinically using umbilical arteryDopplermeasurements. However, the sites andunderlyingmechanisms of increased vascular resistance in the fetoplacental circulation remain unclear. In this studywe tested the hypothesis that endothelial dysfunction and altered nitric oxide (NO) production in placental vessels contribute to the pathology of FGR. Dual perfusion was performed on placental cotyledons from normal pregnancies and those complicated by idiopathic FGR ( 5th centile). Vascular resistance and shear stress-induced vasodilatation of the fetal vasculaturewasmeasured in response to increasingflow rates (2-12ml/ min), and the role of NOwas examined using the NO synthase inhibitor Nunitro-L-arginine (L-NNA). Incremental increases in fetoplacental flow (212ml/min) in normal placentas elevated vascular resistance in a stepwise manner. This was significantly amplified in perfused cotyledons from FGR pregnancy, compared to those fromnormal pregnancy, particularly at higher flow rates (76.5 9mmHg versus 45.4 6mmHg at 12ml/min respectively; mean sem; n1⁄410 per group). Inhibition of eNOS by L-NNA moderately increased the resistance at high flow rates in normal placentas. In contrast, steady state vascular resistance in FGRplacentaswas augmentedby L-NNAat all flow rates. We propose that this effect of L-NNA may relate to increased levels of NO being produced to compensate for the increased resistance; a theory supported by our work on isolated endothelial cells (see separate abstract). In normal placentas increases in fetal-side flow ratewere followed by significant shear-induced vasodilatationwhichwas inhibited by L-NNA. In FGR placentas responses to shear-mediated vasodilatation were absent. In conclusion, placental vessels from pregnancies complicated by fetal growth restriction exhibit endothelial dysfunction and fail to adapt to increases in flow, resulting in increased vascular resistance. http://dx.doi.org/10.1016/j.placenta.2013.06.067 P1.31. MATERNAL ADMINISTRATION OF SILDENAFIL CITRATE ALTERS FETAL VASCULAR FUNCTION IN A MOUSE MODEL OF FETAL GROWTH RESTRICTION Lewis Renshall, Elizabeth Cowley, Susan Greenwood, Mark Dilworth, Mark Wareing Maternal and Fetal Research Centre, The University of Manchester,

Lynch syndrome-associated breast cancers do not overexpress chromosome 11-encoded mucins

Mismatch repair-deficient breast cancers may be identified in Lynch syndrome mutation carriers, and have clinicopathological features in common with mismatch repair-deficient colorectal and endometrial cancers such as tumour-infiltrating lymphocytes and poor differentiation. Mismatch repair-deficient colorectal cancers frequently show mucinous differentiation associated with upregulation of chromosome 11 mucins. The aim of this study was to compare the protein expression of these mucins in mismatch repair-deficient and -proficient breast cancers. Cases of breast cancer (n=100) were identified from families where (1) both breast and colon cancer co-occurred and (2) families met either modified Amsterdam criteria or had at least one early-onset (<50 years) colorectal cancer. Tumour sections were stained for the epithelial mucins, MUC2, MUC5AC, MUC5B and MUC6, and the homeobox protein CDX2, a regulator of MUC2 expression. In all, 16 mismatch repair-deficient Lynch syndrome breast cancers and 84 non-Lynch breast cancers were assessed for altered mucin expression. No significant difference in the expression of MUC2, MUC5AC or MUC6 was observed between the mismatch repair-deficient and mismatch repair-proficient breast cancers; however, there was a trend for mismatch repair-deficient tumours to express high levels of MUC5B less frequently (P=0.07, OR=0.2 (0.0–1.0)). Co-expression of two or more gel-forming mucins was common. Ectopic expression of CDX2 was associated with expression of MUC2 (P=0.035, OR=8.7 (1.3–58.4)). Mismatch repair-deficient breast cancers do not show differential expression of the mucins genes on chromosome 11 when compared with mismatch repair-proficient breast cancers, in contrast with mismatch repair-deficient colorectal and endometrial cancers, which frequently have increased mucin protein expression when compared with their mismatch repair-proficient counterparts. In addition, ectopic CDX2 expression is positively associated with de novo MUC2 expression.

The Hippo Pathway Member Nf2 Is Required for Inner Cell Mass Specification

During mammalian development, the first two lineages to be specified are the trophectoderm (TE) and the inner cell mass (ICM). The Hippo pathway kinases Lats 1 and 2 (Lats1/2) and the transcriptional coactivator Yap play important roles in this specification process [1]. In outside cells of the embryo, Yap is nuclear localized and cooperates with Tead4 to induce the TE-specifying transcription factor Cdx2. In inside cells, Lats1/2 phosphorylate Yap and prevent its nuclear localization. The factors acting upstream of Lats1/2 and Yap in this context have not been identified. Here, we demonstrate that the upstream Hippo pathway member Nf2/Merlin is required for Lats1/2-dependent Yap phosphorylation in the preimplantation embryo. Injection of dominant-negative Nf2 mRNA causes Yap mislocalization and ectopic Cdx2 expression, effects that can be rescued by overexpression of Lats2 kinase. Zygotic Nf2 mutant blastocysts have mild defects in Yap localization and Cdx2 expression, but these become much more severe upon removal of both maternal and zygotic Nf2. The inside cells of maternal-zygotic mutants fail to establish a pluripotent ICM and form excess TE, resulting in peri-implantation lethality. Together, these data establish a clear role for Nf2 upstream of Yap in the preimplantation embryo and demonstrate that Hippo signaling is essential to segregate the ICM from the TE.

Relationship of CDX-2 with intestinal metaplasia and biliary tract tumor

Caudal-related homeobox transcription factor (CDX-2) is selectively localized in the fetal and adult mu-cosal epithelial nuclei of normal small and large intestines.CDX2 plays an important role in the regulation of cell proliferation and differentiation of the intestinal epithelium. Ec-topic expression of CDX-2 in the stomach, esophagus and other hollow organs might be a cause of intestinal metaplasia and adenocarcinoma. CDX-2 is expressed in both cultured and primary gallbladder carcinoma cells, and dysplasia.CDX2 is not detected in normal gallbladder and biliary tract mucosa. Ectopic CDX2 expression is an early event in gallbladder carcinogenesis. The frequency of CDX-2 expression is significantly higher in well-differentiated type than in the moderately and poorly differentiated tumors. The present study clearly showed that MUC2 expression in intestinal differentiation was dependent on the nuclear expression of CDX-2 in intraductal papillary neoplasia of the liver (IPNL)and mucinous intrahepatic cholangiocarcinoma (ICC) in the same manner as in the gastric mucosa and Barrett s esophagus. CDX-2 may also be a key factor in gallbladder carcinogenesis with intestinal differentiation and is a useful immuno-histochemical marker of intestinal differentiation in intestinal metaplasia of the gallbladder. CDX2-dependent regulation of cell proliferation may be an important factor in defining prognosis of patients with BTC. Key words: Gallbladder; Biliary tract; Intestinal metaplasia; Turner; CDX-2

Ectopic Cdx2 expression in murine esophagus models an intermediate stage in the emergence of Barrett's esophagus.

Barrett's esophagus (BE) is an intestinal metaplasia that occurs in the setting of chronic acid and bile reflux and is associated with a risk for adenocarcinoma. Expression of intestine-specific transcription factors in the esophagus likely contributes to metaplasia development. Our objective was to explore the effects of an intestine-specific transcription factor when expressed in the mouse esophageal epithelium. Transgenic mice were derived in which the transcription factor Cdx2 is expressed in squamous epithelium using the murine Keratin-14 gene promoter. Effects of the transgene upon cell proliferation and differentiation, gene expression, and barrier integrity were explored. K14-Cdx2 mice express the Cdx2 transgene in esophageal squamous tissues. Cdx2 expression was associated with reduced basal epithelial cell proliferation and altered cell morphology. Ultrastructurally two changes were noted. Cdx2 expression was associated with dilated space between the basal cells and diminished cell-cell adhesion caused by reduced Desmocollin-3 mRNA and protein expression. This compromised epithelial barrier function, as the measured trans-epithelial electrical resistance (TEER) of the K14-Cdx2 epithelium was significantly reduced compared to controls (1189 Ohm*cm2 ±343.5 to 508 Ohm*cm2±92.48, p = 0.0532). Secondly, basal cells with features of a transitional cell type, intermediate between keratinocytes and columnar Barrett's epithelial cells, were observed. These cells had reduced keratin bundles and increased endoplasmic reticulum levels, suggesting the adoption of secretory-cell features. Moreover, at the ultrastructural level they resembled “Distinctive” cells associated with multilayered epithelium. Treatment of the K14-Cdx2 mice with 5′-Azacytidine elicited expression of BE-associated genes including Cdx1, Krt18, and Slc26a3/Dra, suggesting the phenotype could be advanced under certain conditions. We conclude that ectopic Cdx2 expression in keratinocytes alters cell proliferation, barrier function, and differentiation. These altered cells represent a transitional cell type between normal squamous and columnar BE cells. The K14-Cdx2 mice represent a useful model to study progression from squamous epithelium to BE.

CDX2 Regulates Multidrug Resistance 1 Gene Expression in Malignant Intestinal Epithelium

The caudal-related homeobox transcription factor CDX2 has a key role in intestinal development and differentiation. CDX2 heterozygous mutant mice develop colonic polyps, and loss of CDX2 expression is seen in a subset of colon carcinomas in humans. Ectopic CDX2 expression in the stomach of transgenic mice promotes intestinal metaplasia, and CDX2 expression is frequently detected in intestinal metaplasia in the stomach and esophagus. We sought to define CDX2-regulated genes to enhance knowledge of CDX2 function. HT-29 colorectal cancer cells have minimal endogenous CDX2 expression, and HT-29 cells with ectopic CDX2 expression were generated. Microarray-based gene expression studies revealed that the Multidrug Resistance 1 (MDR1/P-glycoprotein/ABCB1) gene was activated by CDX2. Evidence that the MDR1 gene was a direct transcriptional target of CDX2 was obtained, including analyses with MDR1 reporter gene constructs and chromatin immunoprecipitation assays. RNA interference-mediated inhibition of CDX2 decreased endogenous MDR1 expression. In various colorectal cancer cell lines and human tissues, endogenous MDR1 expression was well correlated to CDX2 expression. Overexpression of CDX2 in HT-29 cells revealed increased resistance to the known substrate of MDR1, vincristine and paclitaxel, which was reversed by an MDR1 inhibitor, verapamil. These data indicate that CDX2 directly regulates MDR1 gene expression through binding to elements in the promoter region. Thus, CDX2 is probably important for basal expression of MDR1, regulating drug excretion and absorption in the lower gastrointestinal tract, as well as for multidrug resistance to chemotherapy reagent in CDX2-positive gastrointestinal cancers.

Abstract 1520: CDX2 regulates Multidrug Resistance 1 gene expression in malignant intestinal epithelium

Abstract The intestine-specific caudal-related homeobox transcription factor CDX2 is important for intestinal development and differentiation. CDX2 heterozygous mice form colonic polyps, and loss of CDX2 expression is seen in some poorly differentiated colon carcinoma in humans. Conversely, ectopic CDX2 expression in the stomach of transgenic mice promotes intestinal metaplasia, and CDX2 expression is often seen in stomach and esophageal metaplasia. We sought to define CDX2-regulated genes to enhance the knowledge of CDX2 function. HT-29 colorectal cancer cells with minimal endogenous CDX2 expression were generated to express exogenous CDX2, and the gene expression changes were assessed by microarrays. Activation of CDX2 rapidly induced Multidrug Resistance 1 (MDR1/P-glycoprotein/ABCB1) gene expression in HT-29 cells, even in the presence of protein synthesis inhibitor, and RNA interference-mediated inhibition of CDX2 decreased MDR1 expression. In other colorectal cancer cell lines and human tissues, endogenous MDR1 expression was well correlated to CDX2 expression. Analysis with MDR1 reporter gene constructs and chromatin-immunoprecipitation assays suggested that CDX2 directly regulates MDR1 transcription. Overexpression of CDX2 in HT-29 cells revealed increased resistance to MDR1's known substrate, vincristine and paclitaxel, which was reversed by an MDR1 inhibitor, verapamil. These data indicate that CDX2 directly regulates MDR1 gene expression via binding to elements in the promoter region. Thus, CDX2 is probably important for basal expression of MDR1, regulating drug excretion and absorption in the lower gastrointestinal tract, as well as for multidrug resistance to chemotherapy reagent in CDX2-positive gastrointestinal cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1520.

Ectopic Expression of CDX2 Perturbs HOX Gene Expression in Murine Progenitors Depending on Its N-Terminal Transactivation Domain and Is Closely Correlated with Deregulated HOX Gene Expression in Human Acute Myeloid Leukemia.

Deregulated homeobox gene expression characterizes more than 60% of all acute myeloid leukemia (AML) patients, particular in patients with normal karyotype (NK). So far it is largely unknown how the aberrant expression of homeobox genes is initiated in the malignant clone. The ParaHox gene Cdx2 was shown to act as positive upstream regulator of Hox genes in embryogenesis. We have previously shown that ectopic Cdx2, which normally is not expressed in hematopoietic cells, can be the key event in the development of AML in mice (Rawat et al., PNAS 2004). In our present study we now demonstrate that ectopic expression of Cdx2 in murine hematopoietic progenitors induced significant up-regulation of Hox genes with leukemogenic potential such as HoxA9, Hoxa10, HoxA5, Hoxa7, Hoxb6, Hoxb8. Deletion of the N-terminal transactivation domain of Cdx2 abrogated its ability to perturb Hox gene expression and eliminated its leukemogenic potential in vivo (n=13), whereas inactivation of the putative Pbx binding site of the protein did not prevent Cdx2 induced leukemogenesis. As we showed that Cdx2 upregulated leukemogenic Hox genes and caused AML in the murine model we analyzed 115 AML patients for a correlation between the expression levels of CDX2 and deregulated HOX gene expression. A total of 71 patients with normal karyotype (AML NPMc+ = 45 cases; NPMc– = 26 cases) was analyzed for CDX2 expression. 89% of the AML NPMc+ cases showed ectopic expression of CDX2 as well as 88% of the cases without the NPM1 mutation. We extended this analysis to 44 patients with abnormal karyotype and detected ectopic CDX2 expression in 64% (28 out of 44) of the cases: 12 of 24 patients with t(8;21), 10 of 10 patients with t(15;17). Importantly, when the expression level of CDX2 was compared between AML cases with normal and abnormal karyotype, there was 14fold higher expression level in the patient group with NK (n=63) compared to the group with aberrant karyotype (n=28) (ØΔCT 7.72 vs. ØΔCT 11.62, respectively; p>0.001). By using oligonucleotide microarray analysis, we confirmed that high Cdx2 expressing AML-NK patients with (n=12) or without NPM1 mutation (n=12) were characterized by aberrant expression of multiple HOXA and B cluster genes such as HOXA10, HOXA9 and HOXB3, HOXB6 in contrast to AML cases expressing the PML-RARA or AML1-ETO fusion gene or normal healthy donors. Three NPMc- cases with normal karyotype showed the same low level of expression of CDX2 (ΔCT 10.55–11.55) as AML cases with aberrant karyotype. Of note, these three cases did not show any perturbation of HOX gene expression and thereby fell into the same cluster as AML cases with t(8;21) or t(15;17) in the microarray data set evaluating HOX gene expression in the different AML subtypes. All AML-NK patients tested were negative for CDX1 and CDX4 expression. These data link the leukemogenic potential of Cdx2 to its ability to dysregulate Hox genes. They furthermore correlate the level of CDX2 expression with HOX gene expression in human AML and support a potential role of CDX2 in the development of human AML with aberrant Hox gene expression.

Regulation of Cdx2 expression by promoter methylation, and effects of Cdx2 transfection on morphology and gene expression of human esophageal epithelial cells

Caudal-related homeobox 2 (Cdx2) has been suggested as an early marker of Barrett's esophagus (BE), which is the premalignant lesion of esophageal adenocarcinoma (EAC). However, the mechanism of ectopic Cdx2 expression in the esophageal epithelial cells and its role in the development of BE remained unclear. RT-PCR, pyrosequencing and methylation-specific PCR were used to determine expression and promoter methylation of Cdx2 in human esophageal epithelial cells (HET1A and SEG1) after treatment with 5-aza-2'-deoxycytidine (DAC), acid, bile acids and their combination. HET1A cells with stable transfection of Cdx2 were characterized for morphology and gene expression profiles with Affymetrix array. We found Cdx2 was expressed in most human EAC cell lines, but not in squamous epithelial cell lines. DAC-induced demethylation and expression of Cdx2 in HET1A and SEG1 cells, and treatment with a DNA methylating agent counteracted the effect of DAC. Treatment of HET1A and SEG1 cells with acid, bile acids or both also resulted in promoter demethylation and expression of Cdx2. HET1A cells with stable transfection of human Cdx2 formed crypt-like structures in vitro. Microarray analysis and quantitative real-time PCR showed that stable transfection of Cdx2 up-regulated differentiation markers of intestinal columnar epithelial cells and goblet cells in HET1A cells. This may be partially due to modulation of Notch signaling pathway, as western blotting confirmed down-regulation of Hes1 and up-regulation of Atoh1 and Muc2. Our data suggest that exposure to acid and/or bile acids may activate Cdx2 expression in human esophageal epithelial cells through promoter demethylation, and ectopic Cdx2 expression in esophageal squamous epithelial cells may contribute to intestinal metaplasia of the esophagus.

CDX2-regulated expression of iron transport protein hephaestin in intestinal and colonic epithelium

The homeobox transcription factor CDX2 has a key role in intestinal development and differentiation. Mice heterozygous for Cdx2 inactivation develop colonic polyps with epithelial cells showing gastric or squamous differentiation. Loss of CDX2 expression is seen in some poorly differentiated colon carcinomas in humans. Conversely, ectopic CDX2 expression in the stomach of transgenic mice promotes intestinal metaplasia, and CDX2 expression often is seen in intestinal metaplasia in stomach and esophagus. To enhance knowledge of CDX2 function, we sought to define CDX2-regulated genes. HT-29 and WiDr colorectal cancer (CRC) cells with low endogenous CDX2 expression were transduced with a CDX2 expression vector, and gene expression changes were assessed by microarrays. The gene for ceruloplasmin-related iron transport protein hephaestin (HEPH) was induced by CDX2 in HT-29 and WiDr. In other CRC lines and human and mice tissues, endogenous HEPH expression was linked to CDX2 expression. Activation of CDX2 rapidly induced HEPH expression, and RNA interference-mediated inhibition of CDX2 led to lower HEPH expression. Studies with HEPH reporter gene constructs and chromatin-immunoprecipitation approaches suggested that CDX2 directly regulates HEPH transcription. In CRC cells, CDX2 induction suppressed intracellular iron levels, consistent with the view that HEPH regulates iron export. CDX2 expression was modulated in response to changes in intracellular iron levels, implying a regulatory pathway in which increased iron levels lead to increased expression of CDX2 and HEPH and enhanced iron export. CDX2 has a key role in regulating HEPH expression and iron levels in intestinal cells.