Down-regulation Of CDX2 Research Articles

TEAD4 regulates trophectoderm differentiation upstream of CDX2 in a GATA3-independent manner in the human preimplantation embryo.

TEAD4 regulates trophectoderm differentiation upstream of CDX2 in a GATA3-independent manner in the human preimplantation embryo.

Wnt3a Activates the WNT-YAP/TAZ Pathway to Sustain CDX2 Expression in Bovine Trophoblast Stem Cells.

Trophoblast stem cells (TSCs), the precursors of placental cells, are effective for studying placental formation in vitro. Using a dual inhibition (2i) medium and mixed L-Wnt3a/mouse embryonic fibroblast feeder cells, we previously established the bovine trophoblast cell line BTS-1. In this study, we used bovine fetal fibroblasts and added Wnt3a to the 2i medium to establish another bovine TSC line (BTSW). BTSW cells expressed pluripotency markers, including NANOG, SOX2, OCT4, TRA-1-60, TRA-1-81, SSEA4, CDH1, and KRT18, and TSC markers CDX2, TEAD4, and ESRRB. Methylation sequencing of the promoter regions of NANOG, OCT4, and CDX2 revealed no significant differences between BTS-1 and BTSW cells. Removal of Wnt3a from the culture medium resulted in downregulation (p < 0.05) of NANOG, OCT4, CDX2, and TSC marker genes, and upregulation of TSC differentiation markers, including MASH2, GCM1, and PAG. Western blotting indicated activation of the WNT-YAP/TAZ signaling pathway in BTS-1 and BTSW cells, consequently activating TEAD4 transcription. However, this pathway was not activated in BCFF cells, an established bovine embryonic stem-like cell line that expresses OCT4, SOX2, and NANOG, but not CDX2. Thus, Wnt3a may play a critical role in bovine TSC maintenance by activating and regulating CDX2 expression through the WNT-YAP/TAZ signaling pathway.

Omeprazole prevents CDX2 and SOX9 expression by inhibiting hedgehog signaling in Barrett's esophagus cells.

Activation of hedgehog (Hh) signaling contributes to the progression of Barrett's esophagus (BE), which increases the risk of esophageal adenocarcinoma. Recent clinical studies revealed that proton-pump inhibitors (PPIs) but not H2 receptor antagonists (H2RAs) were associated with a decreased risk of esophageal adenocarcinoma. We would like to know whether PPIs interfere with BE progression during BE treatment. Here, we explored the role of omeprazole on Hh signaling and expression of two crucial biomarkers of BE, SOX9 and CDX2. We demonstrated that bile acids elevated expression of Hh pathway target genes, such as GLI1 and PTCH1, and induced SOX9 and CDX2 up-regulation in both CP-A and CP-B cells. Omeprazole, but not famotidine, down-regulated these genes induced by bile acids. In addition, omeprazole-induced down-regulation of SOX9 and CDX2 was mediated by Hh signaling. To explore the mechanisms by which omeprazole inhibits Hh signaling, we performed luciferase assay but did not find any effects of omeprazole on the activity of GLI1 promoter, the critical transcription factor of Hh signaling. Therefore, we used miRNA sequencing and a bioinformatics tool in our study to identify the differently expressed miRNAs in BE organoids treated with or without omeprazole, and we identified miR-2116-3p was involved in omeprazole-mediated inhibition of Hh signaling and subsequent down-regulation of SOX9 and CDX2. Collectively, our data indicate omeprazole inhibits Hh signaling and subsequent SOX9 and CDX2 expression via up-regulating miR-2116-3p. We have demonstrated a novel acid-independent mechanism of omeprazole that might yield valuable insight into clinical management of BE progression, irrespective of acid reflux symptoms.

Effect of chronic exposure to inorganic arsenic on intestinal cells.

Chronic exposure to inorganic arsenic (As)-As(III)+As(V)-is associated with type 2 diabetes, vascular diseases and various types of cancer. Although the oral route is the main way of exposure to inorganic As, the adverse gastrointestinal effects produced by chronic exposure are not well documented. The aim of the present study is to evaluate the effect of chronic exposure to As(III) on the intestinal epithelium. For this purpose, NCM460 cells, non-transformed epithelial cells from the human colon, were exposed to As(III) (0.01-0.2mg/L) for 6months and monitored for acquisition of a tumor-like phenotype. Secretion of matrix metalloproteinases, histone modifications (H3 acetylation), hyperproliferation capacity, formation of floating spheres, anchorage-independent growth, release of cytokine interleukin-8 and expression of relevant genes in colon tumorigenesis were assessed. The results show a maintained proinflammatory response from the beginning, with an increase in interleukin-8 secretion (≤570%). Downregulation of CDX1 and CDX2 was also observed. After 14weeks of exposure, cells presented marked increases in matrix metalloproteinase-2 secretion and histone modifications. As(III)-treated cells were hyperproliferative, grew in low-serum media and were able to form free-floating spheres. Overall, these data suggest that exposure of human colon epithelial cells to As(III) facilitates acquisition of transformed cell characteristics.

Reciprocal regulation of TEAD4 and CCN2 for the trophectoderm development of the bovine blastocyst.

The first segregation at the blastocyst stage is the symmetry-breaking event to characterize two cell components; namely, inner cell mass (ICM) and trophectoderm (TE). TEA domain transcription factor 4 (TEAD4) is a well-known regulator to determine TE properties of blastomeres in rodent models. However, the roles of bovine TEAD4 in blastocyst development have been unclear. We here aimed to clarify the mechanisms underlining TE characterization by TEAD4 in bovine blastocysts. We first found that the TEAD4 mRNA expression level was greater in TE than in ICM, which was further supported by TEAD4 immunofluorescent staining. Subsequently, we examined the expression patterns of TE-expressed genes; CDX2, GATA2 and CCN2, in the TEAD4-knockdown (KD) blastocysts. These expression levels significantly decreased in the TEAD4 KD blastocysts compared with controls. Of these downregulated genes, the CCN2 expression level decreased the most. We further analyzed the expression levels of TE-expressed genes; CDX2, GATA2 and TEAD4 in the CCN2 KD blastocysts. Strikingly, the CCN2 KD blastocysts showed the downregulation of CDX2, GATA2 and TEAD4 Furthermore, the ratio of TE-to-ICM cell numbers in the CCN2 KD blastocysts significantly decreased compared to controls. To our knowledge, this is the first study showing the regulation of CCN2 expression thorough TEAD4 in mammalian embryos. Not only that, this study also provides evidence that reciprocal regulation of TEAD4 and CCN2 is required for TE development with appropriate gene expression in bovine blastocysts.

Reelin protects from colon pathology by maintaining the intestinal barrier integrity and repressing tumorigenic genes

We previously reported that reelin, an extracellular matrix protein first known for its key role in neuronal migration, reduces the susceptibility to dextran sulphate sodium (DSS)-colitis. The aim of the current study was to determine whether reelin protects from colorectal cancer and how reelin defends from colon pathology. In the colon of wild-type and of mice lacking reelin (reeler mice) we have analysed the: i) epithelium cell renewal processes, ii) morphology, iii) Sox9, Cdx2, Smad5, Cyclin D1, IL-6 and IFNγ mRNA abundance in DSS-treated and untreated mice, and iv) development of azoxymethane/DSS-induced colorectal cancer, using histological and real time-PCR methodologies. The reeler mutation increases colitis-associated tumorigenesis, with increased tumours number and size. It also impairs the intestinal barrier because it reduces cell proliferation, migration, differentiation and apoptosis; decreases the number and maturation of goblet cells, and expands the intercellular space of the desmosomes. The intestinal barrier impairment might explain the increased susceptibility to colon pathology exhibited by the reeler mice and is at least mediated by the down-regulation of Sox9 and Cdx2. In response to DSS-colitis, the reeler colon increases the mRNA abundance of IL-6, Smad5 and Cyclin D1 and decreases that of IFNγ, conditions that might result in the increased colitis-associated tumorigenesis found in the reeler mice. In conclusion, the results highlight a role for reelin in maintaining intestinal epithelial cell homeostasis and providing resistance against colon pathology.

WNT/β-catenin signaling affects cell lineage and pluripotency-specific gene expression in bovine blastocysts: prospects for bovine embryonic stem cell derivation.

Despite many attempts, true bovine embryonic stem cells (bESC) still remain elusive. The WNT pathway has been associated with stem cell control in vertebrates and its role in pluripotency maintenance has been proven for several mammalian species, including rodents and primates. Thus, we have aimed to investigate the effect of WNT activation on pluripotency marker gene expression in the inner cell mass (ICM) and the trophectoderm (TE) and to study the derivation potential of primary bESC lines from blastocysts obtained in the presence of the glycogen synthase kinase 3 inhibitor (GSK3i). WNT activity clearly exerted a positive effect on pluripotency gene expression in developing bovine embryos, manifested by upregulation of OCT4, NANOG, REX1, SOX2, c-MYC, and KLF4 in the ICM and downregulation of CDX2 in the TE. However, the prolonged exposition of preimplantation embryos to the GSK3i resulted in reduced potential to form primary ESC-like colonies. The results of bESC derivation experiments allowed us to speculate that the derived cell lines may share features of both naïve and primed ESCs. Similar to mouse epiblast stem cells and human ESCs, the derived lines grew as flat monolayer colonies intolerant to passaging as single cells. JAK/STAT signaling was indispensable for proper colony formation and proliferation, yet LIF alone was inefficient to support self-renewal. Concomitant with the naïve state of mouse ESCs, WNT activity supported by LIF had beneficial effects on cell culture propagation, survival after passage, morphology, and pluripotency-related marker gene expression. Moreover, colonies derived in the presence of LIF and GSK3i maintained KLF4 transcription over several passages, whereas EpiSCs virtually do not express KLF4.

The basal position of nuclei is one pre-requisite for asymmetric cell divisions in the early mouse embryo

The early mouse embryo undertakes two types of cell division: symmetric that gives rise to the trophectoderm and then placenta or asymmetric that gives rise to inner cells that generate the embryo proper. Although cell division orientation is important, the mechanism regulating it has remained unclear. Here, we identify the relationship between the plane of cell division and the position of the nucleus and go towards identifying the mechanism behind it. We first find that as the 8-cell embryo progresses through the cell cycle, the nuclei of most – but not all – cells move from apical to more basal positions, in a microtubule- and kinesin-dependent manner. We then find that all asymmetric divisions happen when nuclei are located basally and, in contrast, all cells, in which nuclei remain apical, divide symmetrically. To understand the potential mechanism behind this, we determine the effects of modulating expression of Cdx2, a transcription factor key for trophectoderm formation and cell polarity. We find that increased expression of Cdx2 leads to an increase in a number of apical nuclei, whereas down-regulation of Cdx2 leads to more nuclei moving basally, which explains a previously identified relationship between Cdx2 and cell division orientation. Finally, we show that down-regulation of aPKC, involved in cell polarity, decreases the number of apical nuclei and doubles the number of asymmetric divisions. These results suggest a model in which the mutual interdependence of Cdx2 and cell polarity affects the cytoskeleton-dependent positioning of nuclei and, in consequence, the plane of cell division in the early mouse embryo.

Temporal and spatial expression of caudal-type homeobox gene-2 during hindgut development in rat embryos with ethylenethiourea-induced anorectal malformations.

The main aim of this study was to determine Cdx2 expression patterns during anorectal development in normal and anorectal malformation (ARM) embryos with a view to establishing the possible role of Cdx2 in ARM pathogenesis. ARM was induced with ethylenethiourea on the 10th gestational day (GD10) in rat embryos, and Cesarean deliveries were performed to harvest the embryos. The temporal and spatial expression of Cdx2 was evaluated in normal rat embryos (n = 303) and ARM embryos (n = 321) from GD13 to GD16. Immunohistochemical staining revealed that, in normal embryos, Cdx2 was mainly expressed on the epithelium of the urorectal septum (URS) and the hindgut on GD13. On GD14, Cdx2-immunopositive cells were extensively detected on the URS, hindgut, and cloacal membrane. On GD15, increased immunopositive tissue staining on the anal membrane was evident. In ARM embryos, the epithelium of the cloaca, URS, and anorectum were negative or faintly immunostaining for Cdx2. Analyses by Western blot and real-time reverse transcription plus the polymerase chain reaction revealed that, in the normal group, Cdx2 protein and mRNA expression showed time-dependent changes in the developing hindgut from GD13 to GD16. Upon the URS division of the cloaca into the primitive rectum and urogenital sinus (UGS) on GD15, Cdx2 expression began to decrease. Moreover, the Cdx2 expression level in the ARM group from GD13 to GD14 was significantly lower than that in the normal group (P < 0.05). In ARM embryos, an imbalance in the spatiotemporal expression of Cdx2 was noted during anorectal morphogenesis from GD13 to GD16. Downregulation of Cdx2 at the time of cloacal separation into the primitive rectum and UGS might thus be related to the development of ARM.

Gastric cancer patients less than 50 years of age exhibit significant downregulation of E-cadherin and CDX2 compared to older reference populations

PurposeThere is an increasing need to identify molecular markers, which can be used to prognosticate patient populations in gastric cancer. Whereas a significant number have been identified, very few have been characterized in the context of their ability to discriminate between young and old age groups in which a survival difference clearly exists. Material/methodsIn this study, using immunohistochemistry, we evaluated three markers with proven involvement in gastric cancer. The p53 tumor suppressor, the cell adhesion glycoprotein epithelial cadherin (CDH1) and the caudal-related homeobox transcription factor (CDX2) all of these have important roles in the aetiopathogenesis and/or progression of gastric cancer. ResultsAfter adjustments for TNM stage, tumor grade, histopathological characteristics (Lauren classification), we found significant differences in the expression of these proteins, particularly E-cadherin and CDX2 between young and elderly patients. However, these differences did not amount to a significant difference in survival. ConclusionsThis study demonstrates that the protein expression of p53, CDH1 and CDX2 significantly discriminates young patients with gastric cancer who have a better prognostic outlook from older patients, but this difference in expression does not contribute to a survival benefit.

Sa1682 TNF-α-Mediated Down-Regulation of CDx2 Decreases the Expression of β-Catenin Degradation Complex Genes in Colorectal Cancer

Background: Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine that is highly up-regulated in inflammatory bowel disease and reduces the expression of the intestinal specific homeodomain transcription factor, CDX2. CDX2 is a master regulator of the intestinal homeostasis and is considered to be a tumor suppressor. WNT/ β-catenin signaling is critical for intestinal cell proliferation, but decreased CDX2 expression contributes to an abnormal increased activation of WNT signaling and progression of cancer. Furthermore, low CDX2 expression is associated with enhanced epithelial-to-mesenchymal transition (EMT) in colorectal cancer (CRC). Inflammation is often observed at the invasive front of the colon cancer, and it has been suggested that the inflammatory microenvironment around a tumor also induces EMT. Hence, it is likely that down-regulation of CDX2 expression in the invasive front in CRC contributes to enhanced tumorigenicity by regulating genes associated with the WNT/β-catenin pathway. Aim: To determine the role of TNFα in the regulation of CDX2 expression in CRC and its influence onWNT/β-catenin signaling. Methods: Carcinoma specimens were obtained from patients undergoing surgical resection of the rectum. Ten cases with tumor cell buddings were selected, and the expression patterns of TNFα and CDX2 at the invasive front were evaluated on immunostained slides. The mechanisms behind TNF-α-mediated down-regulation of CDX2 were investigated in vitro by selective inhibitors, and the impact of TNF-α on APC, AXIN2 and GSK3β expression were analyzed by quantitative real-time PCR (qPCR) in Caco-2 cells. Subsequently, in vivo CDX2-DNA interactions to APC, AXIN2 and GSK3β gene regulatory elements were investigated by chromatin immunoprecipitation in relation to the TNF-α status (presence vs. absence). Results: Immunohistochemical staining showed reduced CDX2 positive-cells in tumor buddings in areas with TNF-α expression in the surrounding inflammatory cells. TNF-α treatment showed a dosedependent decrease of CDX2 mRNA and protein expression dependent on p38 and NFκB, but not on JNK and ERK signaling pathways. Down-regulation of CDX2 leads to significantly decreased levels of APC (p,0.05), AXIN2 (p,0.01), and GSK3β (p,0.05) mRNA. In line with these results, TNF-α treatment impaired the ability of CDX2 to interact and activate the expression of APC (p,0.05), AXIN2 (p,0.01), and GSK3β (p,0.05). Conclusions: This study suggests that TNF-α has a tumor-promoting effect on CRC by influencing the activity of WNT signaling through down-regulation of CDX2 expression. These findings provide a novel insight into the molecular regulation of genes in the β-catenin degradation complex in response to the pro-inflammatory cytokine TNFα in cancer cells.

Sa1686 Characteristics of and Associations Among the HER2 Signal-Related Factors Thought to Be Predictive Factors for the Effectiveness of Trastuzumab Therapy in Gastric Cancer

Background: Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine that is highly up-regulated in inflammatory bowel disease and reduces the expression of the intestinal specific homeodomain transcription factor, CDX2. CDX2 is a master regulator of the intestinal homeostasis and is considered to be a tumor suppressor. WNT/ β-catenin signaling is critical for intestinal cell proliferation, but decreased CDX2 expression contributes to an abnormal increased activation of WNT signaling and progression of cancer. Furthermore, low CDX2 expression is associated with enhanced epithelial-to-mesenchymal transition (EMT) in colorectal cancer (CRC). Inflammation is often observed at the invasive front of the colon cancer, and it has been suggested that the inflammatory microenvironment around a tumor also induces EMT. Hence, it is likely that down-regulation of CDX2 expression in the invasive front in CRC contributes to enhanced tumorigenicity by regulating genes associated with the WNT/β-catenin pathway. Aim: To determine the role of TNFα in the regulation of CDX2 expression in CRC and its influence onWNT/β-catenin signaling. Methods: Carcinoma specimens were obtained from patients undergoing surgical resection of the rectum. Ten cases with tumor cell buddings were selected, and the expression patterns of TNFα and CDX2 at the invasive front were evaluated on immunostained slides. The mechanisms behind TNF-α-mediated down-regulation of CDX2 were investigated in vitro by selective inhibitors, and the impact of TNF-α on APC, AXIN2 and GSK3β expression were analyzed by quantitative real-time PCR (qPCR) in Caco-2 cells. Subsequently, in vivo CDX2-DNA interactions to APC, AXIN2 and GSK3β gene regulatory elements were investigated by chromatin immunoprecipitation in relation to the TNF-α status (presence vs. absence). Results: Immunohistochemical staining showed reduced CDX2 positive-cells in tumor buddings in areas with TNF-α expression in the surrounding inflammatory cells. TNF-α treatment showed a dosedependent decrease of CDX2 mRNA and protein expression dependent on p38 and NFκB, but not on JNK and ERK signaling pathways. Down-regulation of CDX2 leads to significantly decreased levels of APC (p,0.05), AXIN2 (p,0.01), and GSK3β (p,0.05) mRNA. In line with these results, TNF-α treatment impaired the ability of CDX2 to interact and activate the expression of APC (p,0.05), AXIN2 (p,0.01), and GSK3β (p,0.05). Conclusions: This study suggests that TNF-α has a tumor-promoting effect on CRC by influencing the activity of WNT signaling through down-regulation of CDX2 expression. These findings provide a novel insight into the molecular regulation of genes in the β-catenin degradation complex in response to the pro-inflammatory cytokine TNFα in cancer cells.

Sa1683 Five Different Subclasses of Colorectal Neoplasias Identified by Integrated Genetic and Epigenetic Analysis: Association With Polypoid and Nonpolypoid Carcinogenesis

Background: Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine that is highly up-regulated in inflammatory bowel disease and reduces the expression of the intestinal specific homeodomain transcription factor, CDX2. CDX2 is a master regulator of the intestinal homeostasis and is considered to be a tumor suppressor. WNT/ β-catenin signaling is critical for intestinal cell proliferation, but decreased CDX2 expression contributes to an abnormal increased activation of WNT signaling and progression of cancer. Furthermore, low CDX2 expression is associated with enhanced epithelial-to-mesenchymal transition (EMT) in colorectal cancer (CRC). Inflammation is often observed at the invasive front of the colon cancer, and it has been suggested that the inflammatory microenvironment around a tumor also induces EMT. Hence, it is likely that down-regulation of CDX2 expression in the invasive front in CRC contributes to enhanced tumorigenicity by regulating genes associated with the WNT/β-catenin pathway. Aim: To determine the role of TNFα in the regulation of CDX2 expression in CRC and its influence onWNT/β-catenin signaling. Methods: Carcinoma specimens were obtained from patients undergoing surgical resection of the rectum. Ten cases with tumor cell buddings were selected, and the expression patterns of TNFα and CDX2 at the invasive front were evaluated on immunostained slides. The mechanisms behind TNF-α-mediated down-regulation of CDX2 were investigated in vitro by selective inhibitors, and the impact of TNF-α on APC, AXIN2 and GSK3β expression were analyzed by quantitative real-time PCR (qPCR) in Caco-2 cells. Subsequently, in vivo CDX2-DNA interactions to APC, AXIN2 and GSK3β gene regulatory elements were investigated by chromatin immunoprecipitation in relation to the TNF-α status (presence vs. absence). Results: Immunohistochemical staining showed reduced CDX2 positive-cells in tumor buddings in areas with TNF-α expression in the surrounding inflammatory cells. TNF-α treatment showed a dosedependent decrease of CDX2 mRNA and protein expression dependent on p38 and NFκB, but not on JNK and ERK signaling pathways. Down-regulation of CDX2 leads to significantly decreased levels of APC (p,0.05), AXIN2 (p,0.01), and GSK3β (p,0.05) mRNA. In line with these results, TNF-α treatment impaired the ability of CDX2 to interact and activate the expression of APC (p,0.05), AXIN2 (p,0.01), and GSK3β (p,0.05). Conclusions: This study suggests that TNF-α has a tumor-promoting effect on CRC by influencing the activity of WNT signaling through down-regulation of CDX2 expression. These findings provide a novel insight into the molecular regulation of genes in the β-catenin degradation complex in response to the pro-inflammatory cytokine TNFα in cancer cells.

Retrotransposon Activates Ectopic Ptf1 Expression: A Short Tail

Retrotransposon Activates Ectopic Ptf1 Expression: A Short Tail

Reversal of multidrug resistance in gastric cancer cells by CDX2 downregulation

To explore the role of CDX2 in the multi-drug resistance (MDR) process of gastric cancer in vitro and in vivo. A cisplatin-resistant gastric cancer cell line with stable downregulation of CDX2 was established. mRNA and protein expression levels of CDX2, survivin, cyclin D1, and c-Myc were detected by western blotting and semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). The influence of downregulation of CDX2 on MDR was assessed by measuring IC50 of SGC7901/DDP cells to cisplatin, doxorubicin, and 5-fluorouracil, rate of doxorubicin efflux, apoptosis, and cell cycle progression detected by flow cytometry. In addition, we determined the in vivo effects of CDX2 small interfering RNA (siRNA) on tumor size, and apoptotic cells in tumor tissues were detected by deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and hematoxylin and eosin staining. CDX2 siRNA led to downregulation of endogenous CDX2 mRNA (0.31 ± 0.05 vs 1.10 ± 0.51, 0.31 ± 0.05 vs 1.05 ± 0.21, P = 0.003) and protein (0.12 ± 0.08 vs 0.51 ± 0.07, 0.12 ± 0.08 vs 0.55 ± 0.16, P = 2.57 × 10(-4)) expression. It significantly promoted the sensitivity of SGC7901/DDP cells to cisplatin (0.12 ± 0.05 vs 0.33 ± 0.08, 0.12 ± 0.05 vs 0.39 ± 0.15, P = 0.001), doxorubicin (0.52 ± 0.13 vs 4.11 ± 1.25, 0.52 ± 0.13 vs 4.05 ± 1.44, P = 2.81 × 10(-4)), and 5-fluorouracil (0.82 ± 0.13 vs 2.81 ± 0.51, 0.82 ± 0.13 vs 3.28 ± 1.03, P = 1.71 × 10(-4)). Flow cytometry confirmed that the percentage of apoptotic cells increased after CDX2 downregulation (32.15% ± 2.15% vs 17.63% ± 3.16%, 32.15% ± 2.15% vs 19.3% ± 2.25%, P = 1.73 × 10(-6)). This notion was further supported by the observation that downregulation of CDX2 blocked entry into the S-phase of the cell cycle (31.53% ± 3.78% vs 65.05% ± 7.25%, 31.53% ± 3.78% vs 62.27% ± 5.02%, P = 7.55 × 10(-7)). Furthermore, downregulation of CDX2 significantly increased intracellular accumulation of doxorubicin (0.21 ± 0.06 vs 0.41 ± 0.11, 0.21 ± 0.06 vs 0.40 ± 0.08, P = 0.003). In molecular studies, semiquantitative RT-PCR and western blotting revealed that CDX2 downregulation could inhibit expression of c-Myc, survivin and cyclin D1. CDX2 may be involved in regulating multiple signaling pathways in reversing MDR, suggesting that CDX2 may represent a novel target for gastric cancer therapy.

Up-Regulation of Cadherin 17 and Down-Regulation of Homeodomain Protein CDX2 Correlate With Tumor Progression and Unfavorable Prognosis in Epithelial Ovarian Cancer

Cadherin 17 (CDH17), belonging to the 7D-cadherin superfamily, represents a novel oncogene, which is involved in tumor invasion and metastasis. Its expression has been demonstrated to be regulated by caudal-related homeobox transcription factor CDX2. The roles of 2 biomarkers have been conflictingly explained. Therefore, the aims of this study were to investigate the expression patterns of CDH17 and CDX2 in human epithelial ovarian cancer (EOC) and to evaluate the clinical significance of these 2 markers in the progression and prognosis of EOC. CDH17 and CDX2 expressions in 182 paraffin-embedded EOC specimens were detected by immunohistochemical staining. Associations of their expression with clinical pathological factors and overall survival were statistically evaluated. Compared with normal surface ovarian epithelium tissues, CDH17 expression was upregulated and CDX2 expression was downregulated in EOC tissues. There was a negative correlation between CDH17 and CDX2 expression in EOC tissues (r = -0.76, P = 0.001). Tumors with high CDH17 expression were more likely to have advanced stage (P = 0.01) and higher grade (P = 0.03). Patients with low CDX2 expression were more frequently to be at the advanced stage of disease (P = 0.01). In addition, univariate analysis indicated that the patients with high CDH17 expression correlated with poor prognosis in patients with EOC (P = 0.001), as opposed to CDX2 (P = 0.003). Especially, the survival rate of patients with EOC with CDH17-high/CDX2-low expression was the lowest (P < 0.001). Multivariate statistical analysis showed that the conjoined expression of CDH17/CDX2 was an independent prognostic indicator of EOC (P = 0.01). Our data suggest that both the up-regulation of CDH17 and the down-regulation of CDX2 may be associated with the advanced stage of EOC. A conjoined detection of CDH17/CDX2 expression may be associated with unfavorable prognosis in patients with this disease.

Tu1928 TNF-a-Induced Down-Regulation of CDx2 Suppresses Mep1a Expression in Colitis: A Potential Pathogenetic Key Factor in IBD

Tu1928 TNF-a-Induced Down-Regulation of CDx2 Suppresses Mep1a Expression in Colitis: A Potential Pathogenetic Key Factor in IBD

Hypoxia-inducible factor-1α modulates the down-regulation of the homeodomain protein CDX2 in colorectal cancer

Hypoxia-inducible factor-1alpha (HIF-1alpha) is the main active subunit of HIF-1, which promotes tumor cell survival and critical steps involved in tumor progression and aggressiveness. To clarify the possible involvement of the HIF-1alpha subunit and homeodomain protein CDX2 in the development and progression of colorectal cancer (CRC), we examined in vivo the immunohistochemical expression of HIF-1alpha and its topological correlation with CDX2 expression in colorectal adenocarcinoma. We then examined the in vitro effect of hypoxia mimicked by CoCl2 on mRNA and protein expression of HIF-1alpha and CDX2 using two human colon cancer cell lines, SW480 and LS174T. In addition, hypoxia-induced changes in the mRNA expression of Snail were also analyzed. Of the 62 cases of CRC examined, 43 (69.4%) and 39 (62.9%) were positive for CDX2 and HIF-1alpha, respectively, such that their expression was correlated with differentiation grade, tumor stage and lymph node metastasis (chi2 test, p<0.01). Furthermore, HIF-1alpha expression observed in 10 of the 16 (62.5%) poorly differentiated CRCs showed a topological correlation with loss of CDX2 expression. Real-time PCR and western blotting demonstrated that CDX2 expression was decreased by CoCl2 (100 microM) in both SW480 and LS174T cells. mRNA and protein expression of HIF-1alpha and mRNA expression of Snail was increased by hypoxia in both colon cancer cell lines. In conclusion, the present observations support that hypoxia-inducible factor-1alpha induces down-regulation of CDX2 in colon carcinoma cells and that Snail may be involved in this regulation process. These findings suggest that hypoxia plays an important role in the malignant progression of CRC.

Down-regulation of Cdx2 in colorectal carcinoma cells by the Raf–MEK–ERK 1/2 pathway

Cdx2 is a homeodomain transcription factor that regulates normal intestinal cell differentiation. Cdx2 is frequently lost during progression of colorectal cancer (CRC) and is widely viewed as a colorectal tumour suppressor. A previous study suggested that activation of protein kinase C (PKC) may be responsible for Cdx2 down-regulation in CRC cells. Here we show that activation of PKC does indeed promote down-regulation of Cdx2 at both the mRNA and protein levels. However, PKC-dependent loss of Cdx2 is dependent upon activation of the Raf–MEK–ERK1/2 pathway. Indeed, specific activation of the ERK1/2 pathway using the conditional kinase ΔRaf-1:ER is sufficient to inhibit Cdx2 transcription. The Raf–MEK–ERK1/2 pathway is hyper-activated in a large fraction of colorectal cancers due to mutations in K-Ras and we show that treatment of CRC cell lines with MEK inhibitors causes an increase in Cdx2 expression. Furthermore, activation of the ERK1/2 pathway promotes the phosphorylation and proteasome-dependent degradation of the Cdx2 protein. The inhibitory effect of ERK1/2 upon Cdx2 in CRC cells is in sharp contrast to its stimulatory effect upon Cdx2 expression in trophectoderm and trophoblast stem cells. These results provide important new insights into the regulation of the Cdx2 tumour suppressor by linking it to ERK1/2, a pathway which is frequently activated in CRC.

Juvenile polyps have gastric differentiation with MUC5AC expression and downregulation of CDX2 and SMAD4

CDX2 is a homeobox transcription factor that works as a master gene in intestinal differentiation, both in the colon and in aberrant locations such as intestinal metaplasia (IM) of the stomach. Transgenic mice with Cdx2 expression in the stomach develop IM and Cdx2(+/-) mice develop hamartomatous polyps in the colon presenting gastric differentiation. We previously observed regulation of CDX2 by the BMP/SMAD pathway in the gastric context. Here, we hypothesized that juvenile polyps, which are hamartomatous polyps caused by mutations in members of the BMP/SMAD pathway, might recapitulate the gastric differentiation observed in Cdx2(+/-) mice due to SMAD4 and CDX2 downregulation. We characterized SMAD4 and CDX2 expression in a series of 18 solitary juvenile polyps and 2 polyps from juvenile polyposis (JP) patients, one with a germline SMAD4 mutation and one with a germline BMPRIA mutation, as well as the expression of an intestinal differentiation marker, MUC2 (by immunohistochemistry and in situ hybridization), and gastric differentiation markers, MUC5AC and MUC6 (by immunohistochemistry). We observed that juvenile polyps have a heterogeneous expression of CDX2, MUC2 and SMAD4, with negative areas, and 15 of the 18 solitary polyps and the JP case with SMAD4 mutation exhibit de novo expression of MUC5AC but not MUC6. In conclusion, juvenile polyps have gastric transdifferentiation associated with downregulation of CDX2 and SMAD4, lending support to the role of the BMP/SMAD pathway in CDX2 regulation.