Cdx Transcription Factors Research Articles

Optimization of Cdx Transcription Factors Characteristics

This study presents a new application of TOPSIS for the optimization of transcription factors characteristics. This application is essential as it can help compare the characteristics of these proteins and determine the optimized output of their comparison with this decision-making method. The hypothesis in this article was that according to the previous study of the Cdx transcription factors, as the Cdx2 transcription factor showed more robust characteristics than Cdx1 and Cdx4, the TOPSIS method would show a better rank position of these first proteins in comparison with the two other ones. Moreover, the engrailed repressor domain EnRCdx1 used in the plasmid showed the reduction of the pax3 gene expression in comparison with the induced regulation of the gene expression with the production of the Cdx1, Cdx2, and Cdx4 transcription factors using the corresponding plasmids, the worst rank position with TOPSIS was expected for this repressor domain. The results obtained with this ranking method showed that the rank positions of the transcription factors and the repressor domain corresponded to their compared properties. Moreover, the change in the weight values of the candidates showed the modification of their distances from the best and worst alternatives and closeness coefficients. However, as expected, the candidates’ rank positions were unchanged, and the Cdx2 transcription factor was still the best candidate. The results of this article can be used in computer engineering to improve biological applications of these proteins.

Cdx2 Regulates Intestinal EphrinB1 through the Notch Pathway.

The majority of colorectal cancers harbor loss-of-function mutations in APC, a negative regulator of canonical Wnt signaling, leading to intestinal polyps that are predisposed to malignant progression. Comparable murine APC alleles also evoke intestinal polyps, which are typically confined to the small intestine and proximal colon, but do not progress to carcinoma in the absence of additional mutations. The Cdx transcription factors Cdx1 and Cdx2 are essential for homeostasis of the intestinal epithelium, and loss of Cdx2 has been associated with more aggressive subtypes of colorectal cancer in the human population. Consistent with this, concomitant loss of Cdx1 and Cdx2 in a murine APC mutant background leads to an increase in polyps throughout the intestinal tract. These polyps also exhibit a villous phenotype associated with the loss of EphrinB1. However, the basis for these outcomes is poorly understood. To further explore this, we modeled Cdx2 loss in SW480 colorectal cancer cells. We found that Cdx2 impacted Notch signaling in SW480 cells, and that EphrinB1 is a Notch target gene. As EphrinB1 loss also leads to a villus tumor phenotype, these findings evoke a mechanism by which Cdx2 impacts colorectal cancer via Notch-dependent EphrinB1 signaling.

The Cdx transcription factors and retinoic acid play parallel roles in antero-posterior position of the pectoral fin field during gastrulation

The molecular regulators that determine the precise position of the vertebrate limb along the anterio-posterior axis have not been identified. One model suggests that a combination of hox genes in the lateral plate mesoderm (LPM) promotes formation of the limb field, however redundancy among duplicated paralogs has made this model difficult to confirm. In this study, we identify an optimal window during mid-gastrulation stages when transient mis-regulation of retinoic acid signaling or the caudal related transcription factor, Cdx4, both known regulators of hox genes, can alter the position of the pectoral fin field. We show that increased levels of either RA or Cdx4 during mid-gastrulation are sufficient to rostrally shift the position of the pectoral fin field at the expense of surrounding gene expression in the anterior lateral plate mesoderm (aLPM). Alternatively, embryos deficient for both Cdx4 and Cdx1a (Cdx-deficient) form pectoral fins that are shifted towards the posterior and reveal an additional effect on size of the pectoral fin buds. Prior to formation of the pectoral fin buds, the fin field in Cdx-deficient embryos is visibly expanded into the posterior LPM (pLPM) region at the expense of surrounding gene expression. The effects on gene expression immediately post-gastrulation and during somitogenesis support a model where RA and Cdx4 act in parallel to regulate the position of the pectoral fin. Our transient method is a potentially useful model for studying the mechanisms of limb positioning along the AP axis.

THE ROLE OF VENTX HOMEOBOX GENE DURING HUMAN HAEMATOPOIETIC DEVELOPMENT

The Ventx genes are non-clustered homeobox transcription factors that confer a ventral phenotype on mesodermal cells in the developing embryo. Ventx genes are conserved in vertebrates but have been lost in rodents. In the human haematopoietic system, VENTX promotes myeloid differentiation and is expressed in some acute myeloid leukaemias (AML). Using a DOX (Doxycycline) inducible VENTX expression system, we found that VENTX overexpression in hPSC impaired mesoderm formation, but VENTX enforced expression after mesoderm commitment resulted in the emergence of an increased percentage of immature blood cells that co-expressed CD90 and CD34. These cells displayed high clonogenic capacity in methylcellulose, but only after DOX was removed from the media. This suggested that VENTX expression held cells in a non-proliferative state. Transcriptional profiling revealed increased expression of HOXA genes in haematopoietic cells following VENTX induction, consistent with their immature phenotype. Conversely, genes involved in myeloid differentiation were downregulated during VENTX overexpression, as were genes involved in proliferation, such as MYC and MYB. ATAC-sequencing demonstrated that VENTX closes selected chromatin loci, in particular areas targeted by HOXB13 and CDX transcription factors. We hypothesise that VENTX might act as a transcriptional suppressor during haematopoietic differentiation and we are currently investigating the genomic targets of VENTX, combining ATAC-seq and ChIP-seq with the transcriptional profiling. In summary, VENTX overexpression generated immature blood cells in a quiescent state, preventing their proliferation and differentiation into myeloid lineages.

Cdx2 Regulates Gene Expression through Recruitment of Brg1-associated Switch-Sucrose Non-fermentable (SWI-SNF) Chromatin Remodeling Activity

The packaging of genomic DNA into nucleosomes creates a barrier to transcription that can be relieved through ATP-dependent chromatin remodeling via complexes such as the switch-sucrose non-fermentable (SWI-SNF) chromatin remodeling complex. The SWI-SNF complex remodels chromatin via conformational or positional changes of nucleosomes, thereby altering the access of transcriptional machinery to target genes. The SWI-SNF complex has limited ability to bind to sequence-specific elements, and, therefore, its recruitment to target loci is believed to require interaction with DNA-associated transcription factors. The Cdx family of homeodomain transcript ion factors (Cdx1, Cdx2, and Cdx4) are essential for a number of developmental programs in the mouse. Cdx1 and Cdx2 also regulate intestinal homeostasis throughout life. Although a number of Cdx target genes have been identified, the basis by which Cdx members impact their transcription is poorly understood. We have found that Cdx members interact with the SWI-SNF complex and make direct contact with Brg1, a catalytic member of SWI-SNF. Both Cdx2 and Brg1 co-occupy a number of Cdx target genes, and both factors are necessary for transcriptional regulation of such targets. Finally, Cdx2 and Brg1 occupancy occurs coincident with chromatin remodeling at some of these loci. Taken together, our findings suggest that Cdx transcription factors regulate target gene expression, in part, through recruitment of Brg1-associated SWI-SNF chromatin remodeling activity.

Essential roles for Cdx in murine primitive hematopoiesis

The Cdx transcription factors play essential roles in primitive hematopoiesis in the zebrafish where they exert their effects, in part, through regulation of hox genes. Defects in hematopoiesis have also been reported in Cdx mutant murine embryonic stem cell models, however, to date no mouse model reflecting the zebrafish Cdx mutant hematopoietic phenotype has been described. This is likely due, in part, to functional redundancy among Cdx members and the early lethality of Cdx2 null mutants. To circumvent these limitations, we used Cre-mediated conditional deletion to assess the impact of concomitant loss of Cdx1 and Cdx2 on murine primitive hematopoiesis. We found that Cdx1/Cdx2 double mutants exhibited defects in primitive hematopoiesis and yolk sac vasculature concomitant with reduced expression of several genes encoding hematopoietic transcription factors including Scl/Tal1. Chromatin immunoprecipitation analysis revealed that Scl was occupied by Cdx2 in vivo, and Cdx mutant hematopoietic yolk sac differentiation defects could be rescued by expression of exogenous Scl. These findings demonstrate critical roles for Cdx members in murine primitive hematopoiesis upstream of Scl.

Cdx is crucial for the timing mechanism driving colinear Hox activation and defines a trunk segment in the Hox cluster topology

Cdx and Hox transcription factors are important regulators of axial patterning and are required for tissue generation along the vertebrate body axis. Cdx genes have been demonstrated to act upstream of Hox genes in midgestation embryos. Here, we investigate the role of Cdx transcription factors in the gradual colinear activation of the Hox clusters. We found that Hox temporally colinear expression is severely affected in epiblast stem cells derived from Cdx null embryos. We demonstrate that after initiation of 3′ Hox gene transcription, Cdx activity is crucial for H3K27ac deposition and for accessibility of cis-regulatory elements around the central – or ‘trunk’ – Hox genes. We thereby identify a Cdx-responsive segment of HoxA, immediately 5′ to the recently defined regulatory domain orchestrating initial transcription of the first Hox gene. We propose that this partition of HoxA into a Wnt-driven 3′ part and the newly found Cdx-dependent middle segment of the cluster, forms a structural fundament of Hox colinearity of expression. Subsequently to initial Wnt-induced activation of 3′ Hox genes, Cdx transcription factors would act as crucial effectors for activating central Hox genes, until the last gene of the cluster arrests the process.

Induction and dorsal restriction of Paired-box 3 (Pax3) gene expression in the caudal neuroectoderm is mediated by integration of multiple pathways on a short neural crest enhancer

Pax3 encodes a paired-box transcription factor with key roles in neural crest and neural tube ontogenesis. Robust control of Pax3 neural expression is ensured by two redundant sets of cis-regulatory modules (CRMs) that integrate anterior–posterior (such as Wnt–βCatenin signaling) as well as dorsal–ventral (such as Shh–Gli signaling) instructive cues. In previous work, we sought to characterize the Wnt-mediated regulation of Pax3 expression and identified the Cdx transcription factors (Cdx1/2/4) as critical intermediates in this process. We identified the neural crest enhancer-2 (NCE2) from the 5′-flanking region of Pax3 as a Cdx-dependent CRM that recapitulates the restricted expression of Pax3 in the mouse caudal neuroectoderm. While this is consistent with a key role in relaying the inductive signal from posteriorizing Wnt ligands, the broad expression of Cdx proteins in the tailbud region is not consistent with the restricted activity of NCE2. This implies that other positive and/or negative inputs are required and, here, we report a novel role for the transcription factor Zic2 in this regulation. Our data strongly suggests that Zic2 is involved in the induction (as a direct Pax3NCE2 activator and Cdx neural cofactor) as well as the maintenance of Pax3 dorsal restriction (as a target of the ventral Shh repressive input). We also provide evidence that the inductive Cdx–Zic2 interaction is integrated on NCE2 with a positive input from the neural-specific transcription factor Sox2. Altogether, our data provide important mechanistic insights into the coordinated integration of different signaling pathways on a short Pax3 CRM.

Abstract 2732: The role of Cdx transcription factors in intestinal tumorigenesis.

Abstract Mutations in the Adenomatous polyposis coli (APC) gene are an early causative event in human colorectal cancer (CRC). The Min mouse, which harbors mutations of APC, is a widely used model for CRC, although it presents with benign polyps restricted largely to the small intestine. The transcription factors Cdx1 and Cdx2 are expressed throughout the intestinal epithelium from early gut development, and persist throughout the life of the animal. Cdx1 null mice exhibit no intestinal phenotype while Cdx2 null mice die around embryonic day 3.5. Cdx2 heterozygotes, however, have an intestinal phenotype characterized by lesions in the pericaecal region and proximal colon suggestive of a role for Cdx2 in posteriorizing the intestinal tract. Consistent with this, recent studies using conditional knockout approaches show loss of Cdx2 from the developing or adult intestinal epithelium results in an anteriorized intestinal phenotype. Cdx2 loss has been detected in 30% of human CRC, and Cdx2 heterozygous-APC mutants exhibit an increase in polyp formation in the colon; similar results are seen with Cdx2 heterozygotes treated with the carcinogen azoxymethane. These studies suggest that Cdx2 has tumor suppressor properties, however the mechanism by which Cdx2 elicits this effect is unknown. We are using a Cre-lox system to conditionally delete Cdx2 from the adult intestine alone or in a heterozygous Min background. The tamoxifen-regulated Cre, under the spatial control of the villin promoter, is fused to a modified estrogen receptor ligand binding domain that responds only to tamoxifen. Sub-optimal tamoxifen treatment is used to effect partial loss of Cdx2 to circumvent the lethality inherent to Cdx2 loss in the adult intestine. Animals will be sacrificed commencing at 14 weeks post-treatment for analysis of intestinal lesions and compound mutants will be compared to mice deficient for Cdx2 or Min alone. Partial tamoxifen treatment was successful at yielding mice with chimeric loss of Cdx2. In contrast to germ-line Cdx2 heterozygotes, chimeric Cdx2 deletion did not produce hamartomas. In contrast, Cdx-Min compound mutants exhibited a faster trajectory of wasting compared to Min heterozygotes, necessitating endpointing by 14 weeks post-treatment. These compound mutant mice exhibited a three-fold increase in tumor incidence compared to Min controls, with an increased tumors size. Histologically, the small intestinal tumors that developed in the Cdx-Min background have an altered histological appearance compared to the adenomatous Min tumors. Proliferative cells and nuclear beta-catenin expression, seen within the Min tumors, were expanded in the Cdx-Min tumors, consistent with activation of the Wnt pathway in both tumor types. This work illustrates that the conditional Cdx2 allele is an effective tool for studying the role of this transcription factor in the adult intestine, and reaffirms an important role for Cdx2 in the genesis of intestinal cancers. Citation Format: Alexa Hryniuk, David Lohnes, Stephanie Grainger. The role of Cdx transcription factors in intestinal tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2732. doi:10.1158/1538-7445.AM2013-2732

Caudal-related Homeobox (Cdx) Protein-dependent Integration of Canonical Wnt Signaling on Paired-box 3 (Pax3) Neural Crest Enhancer

One of the earliest events in neural crest development takes place at the neural plate border and consists in the induction of Pax3 expression by posteriorizing Wnt·β-catenin signaling. The molecular mechanism of this regulation is not well understood, but several observations suggest a role for posteriorizing Cdx transcription factors (Cdx1/2/4) in this process. Cdx genes are known as integrators of posteriorizing signals from Wnt, retinoic acid, and FGF pathways. In this work, we report that Wnt-mediated regulation of murine Pax3 expression is indirect and involves Cdx proteins as intermediates. We show that Pax3 transcripts co-localize with Cdx proteins in the posterior neurectoderm and that neural Pax3 expression is reduced in Cdx1-null embryos. Using Wnt3a-treated P19 cells and neural crest-derived Neuro2a cells, we demonstrate that Pax3 expression is induced by the Wnt-Cdx pathway. Co-transfection analyses, electrophoretic mobility shift assays, chromatin immunoprecipitation, and transgenic studies further indicate that Cdx proteins operate via direct binding to an evolutionarily conserved neural crest enhancer of the Pax3 proximal promoter. Taken together, these results suggest a novel neural function for Cdx proteins within the gene regulatory network controlling neural crest development.

Regulation of the gene encoding the intestinal bile acid transporter ASBT by the caudal-type homeobox proteins CDX1 and CDX2

The apical sodium-dependent bile acid transporter (ASBT) is expressed abundantly in the ileum and mediates bile acid absorption across the apical membranes. Caudal-type homeobox proteins CDX1 and CDX2 are transcription factors that regulate genes involved in intestinal epithelial differentiation and proliferation. Aberrant expression of both ASBT and CDXs in Barrett's esophagus (BE) prompted us to study, whether the expression of the ASBT gene is regulated by CDXs. Short interfering RNA-mediated knockdown of CDXs resulted in reduced ASBT mRNA expression in intestinal cells. CDXs strongly induced the activity of the ASBT promoter in reporter assays in esophageal and intestinal cells. Nine CDX binding sites were predicted in silico within the ASBT promoter, and binding of CDXs to six of them was verified in vitro and within living cells by electrophoretic mobility shift assays and chromatin immunoprecipitation assays, respectively. RNAs were extracted from esophageal biopsies from 20 BE patients and analyzed by real-time PCR. Correlation with ASBT expression was found for CDX1, CDX2, and HNF-1α in BE biopsies. In conclusion, the human ASBT promoter is activated transcriptionally by CDX1 and CDX2. Our finding provides a possible explanation for the reported observation that ASBT is aberrantly expressed in esophageal metaplasia that also expresses CDX transcription factors.

Concerted involvement of Cdx/Hox genes and Wnt signaling in morphogenesis of the caudal neural tube and cloacal derivatives from the posterior growth zone

Decrease in Cdx dosage in an allelic series of mouse Cdx mutants leads to progressively more severe posterior vertebral defects. These defects are corrected by posterior gain of function of the Wnt effector Lef1. Precocious expression of Hox paralogous 13 genes also induces vertebral axis truncation by antagonizing Cdx function. We report here that the phenotypic similarity also applies to patterning of the caudal neural tube and uro-rectal tracts in Cdx and Wnt3a mutants, and in embryos precociously expressing Hox13 genes. Cdx2 inactivation after placentation leads to posterior defects, including incomplete uro-rectal septation. Compound mutants carrying one active Cdx2 allele in the Cdx4-null background (Cdx2/4), transgenic embryos precociously expressing Hox13 genes and a novel Wnt3a hypomorph mutant all manifest a comparable phenotype with similar uro-rectal defects. Phenotype and transcriptome analysis in early Cdx mutants, genetic rescue experiments and gene expression studies lead us to propose that Cdx transcription factors act via Wnt signaling during the laying down of uro-rectal mesoderm, and that they are operative in an early phase of these events, at the site of tissue progenitors in the posterior growth zone of the embryo. Cdx and Wnt mutations and premature Hox13 expression also cause similar neural dysmorphology, including ectopic neural structures that sometimes lead to neural tube splitting at caudal axial levels. These findings involve the Cdx genes, canonical Wnt signaling and the temporal control of posterior Hox gene expression in posterior morphogenesis in the different embryonic germ layers. They shed a new light on the etiology of the caudal dysplasia or caudal regression range of human congenital defects.

Interaction between Cdx transcription factors and the Retinoic Acid pathway in patterning the posterior neural plate

Interaction between Cdx transcription factors and the Retinoic Acid pathway in patterning the posterior neural plate

Modulation of Tcf3 repressor complex composition regulatescdx4expression in zebrafish

The caudal homeobox (cdx) gene family is critical for specification of caudal body formation and erythropoiesis. In zebrafish, cdx4 expression is controlled by the Wnt pathway, but the molecular mechanism of this regulation is not fully understood. Here, we provide evidence that Tcf3 suppresses cdx4 expression through direct binding to multiple sites in the cdx4 gene regulatory region. Tcf3 requires corepressor molecules such as Groucho (Gro)/TLE and HDAC1 for activity. Using zebrafish embryos and cultured mammalian cells, we show that the transcription factor E4f1 derepresses cdx4 by dissociating corepressor proteins from Tcf3 without inhibiting its binding to cis-regulatory sites in the DNA. Further, the E3 ubiquitin ligase Lnx2b, acting as a scaffold protein irrespective of its enzymatic activity, counteracts the effects of E4f1. We propose that the modulation of Tcf3 repressor function by E4f1 assures precise and robust regulation of cdx4 expression in the caudal domain of the embryo.

Regulation of the Gene Encoding the Intestinal Bile Acid Transporter ASBT by CDX Transcription Factors

Chemotactic migration classically exhibits a bell-shaped response over a narrow concentration range. Previous studies have shown the chemokine, CXCL12, exists in a monomer-dimer equilibrium with heightened concentrations resulting in preferential accumulation of homodimeric chemokine. Little is understood how CXCL12 oligomerization induces distinct cellular effects through the same cognent receptor, CXCR4. The objective of these studies is to determine the effects of monomeric and dimeric CXCL12 on colorectal cancer cell migration. HCT116 and HT29 human colonic carcinoma cells were utilized to examine both chemotactic and restitutive migration respectively. Previously engineered preferential monomeric CXCL12(H25R), locked dimer CXCL12(2), and wild-type CXCL12(WT) were utilized in our analyses. Transwell inserts and aspiration wounded monolayers demonstrated that CXCL12(WT) and CXCL12(H25R) induced a strong chemotactic and restitutive migration response. In contrast, CXCL12(2) dimer was unable to elicit cellular migration. In agreement with those data, filamentous actin formation was induced by CXCL12(WT) and CXCL12(H25R) but not dimer. Intracellular calcium flux, a classical hallmark of G-protein coupled receptor signaling, was elicited equally by each oligomeric variant. This response was blocked with addition of CXCR4-specific inhibitors indicating each of the variants engaged the same cognate receptor. Similarly, CXCL12(2) dose-dependently inhibited CXCR4, but not CXCR1, mediated chemotaxis. The lack of migration was subsequently shown using nuclear magnetic resonance analyses to reflect distinct interactions of the monomeric and dimeric ligand for CXCR4 homotypic receptor dimers. These latter data support the notion that double occupancy of dimeric receptor limits migration while single occupancy of the same receptors evokes strong migration responses. Together these data underlie the importance of CXCL12 oligomerization in dictating epithelial migration and suggest the CXCL12 dimer may serve as a potential inhibitor of colorectal cancer metastasis.

Interactions between Cdx genes and retinoic acid modulate early cardiogenesis

Cdx transcription factors regulate embryonic positional identities and have crucial roles in anteroposterior patterning (AP) processes of all three germ layers. Previously we have shown that the zebrafish homologues cdx1a and cdx4 redundantly regulate posterior mesodermal derivatives inducing embryonic blood cell fate specification and patterning of the embryonic kidney. Here we hypothesize that cdx factors restrict formation of anterior mesodermal derivatives such as cardiac cells by imposing posterior identity to developing mesodermal cells. We show that ectopic expression of Cdx1 or Cdx4 applied during the brief window of mesoderm patterning in differentiating murine embryonic stem cell (ESC) strongly suppresses cardiac development as assayed by expression of cardiac genes and formation of embryoid bodies (EB) containing “beating” cell clusters. Conversely, in loss-of-function studies performed in cdx-deficient zebrafish embryos, we observed a dose-dependent expansion of tbx5a+ anterior-lateral plate mesoderm giving rise to cardiac progenitors. However, further cardiac development of these mesodermal cells required additional suppression of the retinoic acid (RA) pathway, possibly due to differential activity of inhibitory RA signals in cdx mutants. Together, our data suggest that cdx proteins affect cardiogenesis by regulating the formation of cardiogenic mesoderm and together with the RA pathway control the early development of cardiac precursor cells.

Cdx mutant axial progenitor cells are rescued by grafting to a wild type environment

Cdx transcription factors are required for axial extension. Cdx genes are expressed in the posterior growth zone, a region that supplies new cells for axial elongation. Cdx2+/−Cdx4−/− (Cdx2/4) mutant embryos show abnormalities in axis elongation from E8.5, culminating in axial truncation at E10.5. These data raised the possibility that the long-term axial progenitors of Cdx mutants are intrinsically impaired in their ability to contribute to posterior growth. We investigated whether we could identify cell-autonomous defects of the axial progenitor cells by grafting mutant cells into a wild type growth zone environment. We compared the contribution of GFP labeled mutant and wild type progenitors grafted to unlabeled wild type recipients subsequently cultured over the period during which Cdx2/4 defects emerge. Descendants of grafted cells were scored for their contribution to differentiated tissues in the elongating axis and to the posterior growth zone. No difference between the contribution of descendants from wild type and mutant grafted progenitors was detected, indicating that rescue of the Cdx mutant progenitors by the wild type recipient growth zone is provided non-cell autonomously. Recently, we showed that premature axial termination of Cdx mutants can be partly rescued by stimulating canonical Wnt signaling in the posterior growth zone. Taken together with the data shown here, this suggests that Cdx genes function to maintain a signaling-dependent niche for the posterior axial progenitors.

S1056 The Prevalence of Dysplasia is Increasing in Patients With Newly Diagnosed Barrett's Esophagus (BE): Secular Trends From a Large, Multicenter, Cohort Study

represent potential therapeutic targets for the treatment of esophageal malignancies. Methods: Esophageal cell lines HET-1A(normal-squamous), QhTRT(metaplastic), GOhTRT(dysplastic) and SKGT4(adenocarcinoma) were utilized. Cell viability was assessed using the MTT proliferation assay. Preliminary experiments demonstrated that specific inhibition using siRNA to BCL-XL sensitized GOhTRTs to DCA(10hours@500μM) induced cell death, decreasing cell viability by ~45%(p-value <0.0001). siBCL-XL was incorporated as a positive control in the screening protocol. An automated siRNA transfection protocol was developed using the Matrix Hydra II and Matrix WellMate liquid handling robots. Results: A significant level of resistance to DCA-induced cell death was observed in the GOhTRTs when compared to Het-1As(normal-squamous). An siRNA screen targeting GPCRs identified a number of genes whose specific inhibition resulted in a sensitization of the GOhTRT cell line to DCA induced cell death (10hours @ 500μM). The top 25 targets that reduced cell viability by greater than 25% include FZD3, GPSM2, GPR27, CCL25 and IL8Rα. Validation and further investigations will assess how these molecules mediate resistance to DCA induced cell death. Discussion: IL8Rα is a high affinity receptor for IL8 and mediates its angiogenic effect in intestinal microvascular endothelial cells. This may be important in progression through the esophageal carcinogenic sequence from Barrett's esophagus(BE) to adenocarcinoma. CCL25 is a chemokine ligand with chemotactic activity for dendritic cells, thymocytes and activated macrophages. Interestingly, the promoter region of CCL25 contains the binding site for the caudal-related homeobox(Cdx) transcription factors. This is significant as CDX2 is upregulated in BE and may drive an inflammatory response through CCL25. These findings have potential implications for pathogenesis and therapeutics of esophageal cancer. Future investigations will involve the implementation of this screening strategy with more comprehensive siRNA libraries to target a larger number of genes to potentially identify new therapeutic targets.

Chapter 10 - Cdx Genes, Inflammation, and the Pathogenesis of Intestinal Metaplasia

Intestinal metaplasia (IM) is a biologically interesting and clinically relevant condition in which one differentiated type of epithelium is replaced by another that is morphologically similar to normal intestinal epithelium. Two classic examples of this are gastric IM and Barrett's esophagus (BE). In both, a chronic inflammatory microenvironment, provoked either by Helicobacter pylori infection of the stomach or acid and bile reflux into the esophagus, precedes the metaplasia. The Caudal-related homeodomain transcription factors Cdx1 and Cdx2 are critical regulators of the normal intestinal epithelial cell phenotype. Ectopic expression of Cdx1 and Cdx2 occurs in both gastric IM as well as in BE. This expression precedes the onset of the metaplasia and implies a causal role for these factors in this process. We review the observations regarding the role of chronic inflammation and the Cdx transcription factors in the pathogenesis of gastric IM and BE.

CDX Transcription Factors Positively Regulate Expression of Solute Carrier Family 5, Member 8 in the Colonic Epithelium

Caudal-related homeodomain transcription factors CDX1 and CDX2 regulate gut development and differentiation of intestinal epithelial cells; they are candidate tumor suppressors of colorectal carcinomas. Because the functions of CDX1 and CDX2 in the colonic epithelium are not fully understood, we sought to identify genes that they target. We conducted a chromatin immunoprecipitation (ChIP) screen to identify genes that bind the CDX transcription factors. Expression of target genes was analyzed in colon cells and tissues from Cdx1(-/-), Cdx2(+/-), Apc(+/Delta716), and wild-type (control) mice. Using the ChIP screen, we identified solute carrier family 5, member 8 (SLC5A8, also known as SMCT1) as a direct target of CDX1 and CDX2. CDX transcription factors bind to the promoter region of SLC5A8 and transactivate SLC5A8 reporter constructs. Overexpression of Cdx1 or Cdx2 in human colon cancer cell lines induced expression of endogenous SLC5A8, whereas CDX1 and CDX2 knockdowns reduced its level. Consistently, Slc5a8 expression was significantly reduced in colons of Cdx1(-/-) or Cdx2(+/-) mice compared with wild-type mice. Slc5a8 levels were also reduced in colonic adenomatous polyps and hamartomas from Apc(+/Delta716) and Cdx2(+/-) mutant mice, respectively, compared with adjacent normal colon tissues. CDX1 and CDX2 bind the promoter region of SLC5A8 and up-regulate its expression in cultured cells and in colonic epithelium. SLC5A8 transports monocarboxylates such as pyruvate, lactate, and butyrate; CDX1 and CDX2 might therefore regulate the uptake of these substances in the colon.