Caudal-type Homeobox Transcription Factor Research Articles

Silibinin modulates caudal-type homeobox transcription factor (CDX2), an intestine specific tumor suppressor to abrogate colon cancer in experimental rats.

To authenticate the colon cancer preventive potential of silibinin, the efficacy of silibinin needs to be tested by evaluating an organ-specific biomarker. The aim of this study was to evaluate the impact of silibinin on the colonic expression of the caudal-type homeobox transcription factor (CDX2) an intestine specific tumor suppressor gene and its downstream targets in the colon of rats challenged with 1,2 dimethyl hydrazine (DMH). Rats of groups 1 and 2 were treated as control and silibinin control. Rats under groups 3 and 4 were given DMH (20 mg/kg body weight (b.w.) subcutaneously) once a week for 15 consecutive weeks from the 4th week of the experimental period. In addition, group 4 rats alone were treated with silibinin (50 mg/kg b.w. per os) everyday throughout the study period of 32 weeks. Histological investigation and messenger RNA and protein expression studies were performed in the colonic tissues of experimental rats. Findings of the study revealed that DMH administration significantly decreased the expression of CDX2 and Guanylyl cyclase C (GCC) in the colon of experimental rats. Further the decreased levels of CDX2 protein, colonic mucin content, and increased number of mast cells in the colon of DMH alone-administered rats reflects the onset of carcinogenesis. The pathological changes caused due to CDX2 suppression were attenuated by silibinin supplementation.

Distinct patterns of DNA methylation in conventional adenomas involving the right and left colon

Recent studies have shown two distinct non-CIMP methylation clusters in colorectal cancer, raising the possibility that DNA methylation, involving non-CIMP genes, may play a role in the conventional adenoma–carcinoma pathway. A total of 135 adenomas (65 left colon and 70 right colon) were profiled for epigenome-wide DNA methylation using the Illumina HumanMethylation450 BeadChip. A principal components analysis was performed to examine the association between variability in DNA methylation and adenoma location. Linear regression and linear mixed effects models were used to identify locus-specific differential DNA methylation in adenomas of right and left colon. A significant association was present between the first principal component and adenoma location (P=0.007), even after adjustment for subject age and gender (P=0.009). A total of 168 CpG sites were differentially methylated between right- and left-colon adenomas and these loci demonstrated enrichment of homeobox genes (P=3.0 × 10−12). None of the 168 probes were associated with CIMP genes. Among CpG loci with the largest difference in methylation between right- and left-colon adenomas, probes associated with PRAC (prostate cancer susceptibility candidate) gene showed hypermethylation in right-colon adenomas whereas those associated with CDX2 (caudal type homeobox transcription factor 2) showed hypermethylation in left-colon adenomas. A subgroup of left-colon adenomas enriched for current smokers (OR=6.1, P=0.004) exhibited a methylation profile similar to right-colon adenomas. In summary, our results indicate distinct patterns of DNA methylation, independent of CIMP genes, in adenomas of the right and left colon.

Transcriptional regulation of PCFT by KLF4, HNF4α, CDX2 and C/EBPα: Implication in its site-specific expression in the small intestine

Proton-coupled folate transporter (PCFT), which is responsible for the intestinal uptake of folates and analogs, is expressed only in the proximal region in the small intestine. The present study was to examine its transcriptional regulation, which may be involved in such a unique expression profile and potentially in its alteration, using dual-luciferase reporter assays in human embryonic kidney (HEK) 293 cells. The luciferase activity derived from the reporter construct containing the 5′-flanking sequence of −1695/+96 of the human PCFT gene was enhanced most extensively by the introduction of Krüppel-like factor 4 (KLF4). The KLF4-induced luciferase activity was further enhanced by hepatocyte nuclear factor 4α (HNF4α) synergistically. To the contrary, caudal-type homeobox transcription factor 2 (CDX2) and CCAAT/enhancer-binding protein α (C/EBPα) extensively suppressed the luciferase activity induced by KLF4 alone and also that induced by KLF4 and HNF4α. Western blot analysis using the rat small intestine indicated uniform expression of KLF4 along the intestinal tract, proximal-oriented expression of HNF4α, distal-oriented expression of CDX2 and C/EBPα. These results suggest that the activity of PCFT promoter is basically induced by KLF4 and the gradiented expression profile of PCFT may be at least in part accounted for by those of HNF4α, CDX2 and C/EBPα.

CDX2is an amplified lineage-survival oncogene in colorectal cancer

The mutational activation of oncogenes drives cancer development and progression. Classic oncogenes, such as MYC and RAS, are active across many different cancer types. In contrast, "lineage-survival" oncogenes represent a distinct and emerging class typically comprising transcriptional regulators of a specific cell lineage that, when deregulated, support the proliferation and survival of cancers derived from that lineage. Here, in a large collection of colorectal cancer cell lines and tumors, we identify recurrent amplification of chromosome 13, an alteration highly restricted to colorectal-derived cancers. A minimal region of amplification on 13q12.2 pinpoints caudal type homeobox transcription factor 2 (CDX2), a regulator of normal intestinal lineage development and differentiation, as a target of the amplification. In contrast to its described role as a colorectal tumor suppressor, CDX2 when amplified is required for the proliferation and survival of colorectal cancer cells. Further, transcriptional profiling, binding-site analysis, and functional studies link CDX2 to Wnt/β-catenin signaling, itself a key oncogenic pathway in colorectal cancer. These data characterize CDX2 as a lineage-survival oncogene deregulated in colorectal cancer. Our findings challenge a prevailing view that CDX2 is a tumor suppressor in colorectal cancer and uncover an additional piece in the multistep model of colorectal tumorigenesis.

Thyroid transcription factor-1 expression in colorectal adenocarcinomas

Although thyroid transcription factor-1 (TTF-1) immunoreactivity is considered as a specific marker of lung and thyroid neoplasms, it may be positive in a proportion of extrapulmonary adenocarcinomas. This study examined the expression of TTF-1 in 555 colorectal adenocarcinomas using three commercial monoclonal antibodies: clone SPT24 (Novocastra) and 8G7G3/1 (Dako and Cell Marque), and compared the TTF-1 staining with other immunohistochemical markers, cytokeratin (CK) 7, CK 20, caudal-type homeobox transcription factor 2 (CDX2), and MUC2. The clinicopathological prognostic factors were compared with the TTF-1 expression status. Nuclear TTF-1 staining was detected in 24 cases (4.3%) with the SPT24 antibody and 18 cases (3.2%) with the 8G7G3/1 antibody. All cases positive for 8G7G3/1 were also positive for SPT24. CDX2 was expressed constantly in all 24 TTF-1-positive colorectal cancers, whereas CK7, CK20, and MUC2 were detected in 2 (8.3%), 23 (95.8%), and 8 (33.3%) cases, respectively. There was no correlation between TTF-1 expression and clinicopathological significance. To avoid potential pitfalls, TTF-1 should be interpreted in conjunction with the clinical setting, histological features, and the results of other markers, such as CK7, CK20, and CDX2. MUC2 can be used as supplementary information to confirm difficult cases.

KRAS Mutation Analysis Helps to Differentiate Between Pulmonary Metastasis from Colon Adenocarcinoma In Situ and Primary Lung Adenocarcinoma

A 69-year-old woman never-smoker was noted to have a solitary 4-cm lesion in the left upper lobe on routine preoperative chest x-ray before elective hip replacement. Chest computed tomography redemonstrated the spiculated 4 3.3-cm lesion. Positron emission tomography-computed tomography confirmed that the lesion was fluorodeoxyglucose-avid with no other sites of fluorodeoxyglucose avidity (Figure 1). The patient underwent a core needle biopsy of the lung lesion showing a cytokeratin (CK) 20 and caudal type homeobox transcription factor 2–positive adenocarcinoma, which was negative for CK7 and thyroid transcription factor-1. The expected immunohistochemical markers for a primary lung adenocarcinoma are CK7 and thyroid transcription factor-1, whereas the usual markers for a primary colorectal adenocarcinoma are CK20 and caudal type homeobox transcription factor 2. The patient underwent a video-assisted thoracoscopic lobectomy and mediastinal lymphadenectomy revealing a 3.8-cm adenocarcinoma with the same histologic (Figure 2A) and immunophenotype (Figure 3) as the previous biopsy. One inferior mediastinal lymph node (level 9L) was also involved with tumor, and levels 5, 10L, 11L, and 12L nodes were negative for disease. A colonoscopy, upper endoscopy, and capsule video enteroscopy did not show any evidence of a primary tumor. Seven years before the discovery of the lung mass, she had a 2-cm sigmoid adenoma with areas of high-grade dysplasia (Figure 2B) and carcinoma in situ resected endoscopically in a piecemeal fashion. Her resected lung tumor was sent for genetic testing and found to harbor a Kirsten ras sarcoma viral oncogene homolog (KRAS) gene codon 12 GGT to GTT (glycine to valine) missense mutation (KRAS G12V). The tumor was wild-type for the epidermal growth factor receptor gene. Given there was no evidence of primary colorectal tumor on endoscopy, the archived, paraffin-embedded sigmoid adenoma was also genotyped and found to carry the same KRAS codon 12 GTT transversion. This suggested that the lung tumor was a late recurrence from the original KRAS mutant sigmoid carcinoma in situ. She was subsequently found to have multiple new pulmonary lesions.

Abstract 2130: Genomic profiling identifies CDX2 as a lineage-dependent oncogene amplified in colorectal cancer

Abstract Somatic DNA alteration underlies tumor development and progression, and gives rise to tumors with diverse genetic contexts. Characterizing these contexts helps identify driving genetic lesions and potential therapeutic vulnerabilities. Here, we surveyed a collection of 29 colorectal cancer cell lines for DNA copy number alterations and identified in a subset of samples gain/amplification on chromosome 13, which was well represented among the genomic profiles of 344 primary colorectal tumors examined, but not in other tumor types. A minimal region of high-level amplification on 13q12.2, containing the caudal type homeobox transcription factor CDX2, was identified in one cell line and 7 of 123 (6%) primary colorectal tumors. A tissue microarray consisting of an independent set of 83 colorectal tumors confirmed the presence of a subset of tumors harboring copy number gain/amplification of 13q with increased CDX2 protein expression. In the context of genomic amplification, RNA interference experiments showed CDX2 is required for proliferation of colorectal cancer cells and promotes cell cycle progression without an effect on apoptosis. CDX2 is a known transcriptional regulator of normal gut development and intestinal epithelial cell differentiation and maintenance. Here we have shown that for a subset of lineage-derived colorectal tumors, cell proliferation is dependent on the abnormal amplification and overexpression of CDX2. Thus, we nominate CDX2 as a novel lineage-dependent oncogene deregulated in colorectal cancer. 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 2130.

Immunophenotype and molecular characterisation of adenocarcinoma of the small intestine

Background:Despite having a dramatically larger surface area than the large intestine, the small intestine is an infrequent site for the development of adenocarcinoma. To better understand the molecular abnormalities in small bowel adenocarcinoma (SBA), we characterised a number of candidate oncogenic pathways and the immunophenotype of this rare cancer.Methods:Tissue microarrays were constructed from tumour samples from 54 patients with all stages of the disease. Immunohistochemistry and microsatellite instability (MSI) testing were conducted.Results:The profile of cytokeratin 20 and 7 coexpression was variable, but expression of caudal type homeobox transcription factor 2 (CDX2) was present in 70% of cases. In this young population (median age 54 years), loss of mismatch repair (MMR) proteins occurred in 35% of patients, with confirmed MSI in 100% of tested cases. Expression of vascular endothelial growth factor-A (VEGF-A) and epidermal growth factor receptor (EGFR) was common, occurring in 96 and 71% of patients, respectively. Only one case showed HER2 expression and none showed loss of phosphatase and tensin homologue mutated on chromosome 10 (PTEN).Conclusions:These results suggest that alterations in DNA MMR pathways are common in SBAs, similar to what is observed in large bowel adenocarcinomas. Furthermore, the high percentage of tumours expressing both EGFR and VEGF suggests that patients with this rare cancer may benefit from therapeutic strategies targeting EGFR and VEGF receptor (VEGFR).

Selective activation of tumor growth-promoting Ca2+ channel MS4A12 in colon cancer by caudal type homeobox transcription factor CDX2

Colon cancer-associated MS4A12 is a novel colon-specific component of store-operated Ca2+ (SOC) entry sensitizing cells for epidermal growth factor (EGF)-mediated effects on proliferation and chemotaxis. In the present study, we investigated regulation of the MS4A12 promoter to understand the mechanisms responsible for strict transcriptional restriction of this gene to the colonic epithelial cell lineage. DNA-binding assays and luciferase reporter assays showed that MS4A12 promoter activity is governed by a single CDX homeobox transcription factor binding element. RNA interference (RNAi)-mediated silencing of intestine-specific transcription factors CDX1 and CDX2 and chromatin immunoprecipitation (ChIP) in LoVo and SW48 colon cancer cells revealed that MS4A12 transcript and protein expression is essentially dependent on the presence of endogenous CDX2. In summary, our findings provide a rationale for colon-specific expression of MS4A12. Moreover, this is the first report establishing CDX2 as transactivator of tumor growth-promoting gene expression in colon cancer, adding to untangle the complex and conflicting biological functions of CDX2 in colon cancer and supporting MS4A12 as important factor for normal colonic development as well as for the biology and treatment of colon cancer.

Expression of Cdx2 and Ll-cadherin in intestinal metaplasia of gastric mucosa and gastric adenocarcinoma of the intestinal type

Objective To explore the expression of caudal type homeobox transcription factor 2 （Cdx2） and liver intestinal cadherin （LI-CD） in intestinal metaplasia （IM） of gastric mucosa and gastric adenocarcinoma of the intestinal type （GAIT）. Methods By using immunohistoehemical method, the expressions of Cdx2 and LI-CD were detected in biopsy sample including 30 cases of GAIT, 30 IM and 30 normal controls. Results The positive rate of Cdx2 expression was 0, 93.3% and 46.7% in normal stomach mucosa, IM and GAIT, respectively. The positive rates of Cdx2 expression in IM and GAIT were significantly higher than that in normal stomach mucosa （ P 〈 0. 005 ） , and the expression of Cdx2 in IM was significantly higher than that in GAIT （P 〈0. 005）. Similarly, LI-CD was also negative in normal stomach mucosa, and positive rates were 96. 7% and 73.3% in IM and GAIT group, which were significantly higher than that in normal stomach mucasa （ P 〈 0. 005 ） , and LI-CD expression was also significantly higher in IM than in GAIT （P 〈 0. 05 ）. In addition, the staining intensity of Cdx2 and LI-CD expressions in IM exhibited significant correlation （ r =0. 827, P 〈 0. 01 ）, but those in GAIT did not （ r = 0. 438, P = 0. 117 ）. Conclusion Eetopic expression of Cdx2 and LI-CD can be found in IM and GAIT tissues, which could be an early marker in the disease progression. There is correlation between Cdx2 and LI-CD exorcssion in IM tissue of human, and presumably, Cdx2 regulates LI-CD in IM development. Key words: Cdx-2 protein ; LI cadherin liver-intestine ） protein ; Human ; Stomach ; Metaplasia ; Adenocareinoma

Identification of microRNA-181 by genome-wide screening as a critical player in EpCAM-positive hepatic cancer stem cells

MicroRNAs (miRNAs) are endogenous small noncoding RNAs that regulate gene expression with functional links to tumorigenesis. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, and it is heterogeneous in clinical outcomes and biological activities. Recently, we have identified a subset of highly invasive epithelial cell adhesion molecule (EpCAM)(+) HCC cells from alpha-fetoprotein (AFP)(+) tumors with cancer stem/progenitor cell features, that is, the abilities to self-renew, differentiate, and initiate aggressive tumors in vivo. Here, using a global microarray-based miRNA profiling approach followed by validation with quantitative reverse transcription polymerase chain reaction, we have demonstrated that conserved miR-181 family members were up-regulated in EpCAM(+)AFP(+) HCCs and in EpCAM(+) HCC cells isolated from AFP(+) tumors. Moreover, miR-181 family members were highly expressed in embryonic livers and in isolated hepatic stem cells. Importantly, inhibition of miR-181 led to a reduction in EpCAM(+) HCC cell quantity and tumor initiating ability, whereas exogenous miR-181 expression in HCC cells resulted in an enrichment of EpCAM(+) HCC cells. We have found that miR-181 could directly target hepatic transcriptional regulators of differentiation (for example, caudal type homeobox transcription factor 2 [CDX2] and GATA binding protein 6 [GATA6]) and an inhibitor of Wnt/beta-catenin signaling (nemo-like kinase [NLK]). Taken together, our results define a novel regulatory link between miR-181s and human EpCAM(+) liver cancer stem/progenitor cells and imply that molecular targeting of miR-181 may eradicate HCC.

Expression of guanylyl cyclase-c and caudal type homeobox transcription factor 2 in human gastric cancer and precursor lesions

To investigate the expression of guanylyl cyclase-C (GC-C) and caudal type homeobox transcription factor 2 (CDX2) in human gastric mucosa at different stages and the significance thereof. An Immunofluorescence method was used to detect the expression of GC-C and CDX-2 in 23 specimens of gastric carcinoma and matching noncancerous tissues. Western blotting was used to detect the protein expression of GC-C and CDX2 in the gastric carcinoma tissues and matching noncancerous tissues too. The GC-C and CDX2 expression rates were 39.1% and 39.1% respectively in the intestinal metaplasia specimens, 55.6% and 55.6% respectively in the dysplasia specimens, and 56.7 % and 60.0% in the gastric carcinoma specimens, all significantly higher than those in the normal mucosa specimens (all P = 0.000) without significant differences in the expression of GC-C and CDX-2 among the 3 pathological groups. The GC-C and CDX-2 expression was positively correlated with Lauren classification, The expression levels of GC-C and CDX-2 were significantly higher in the intestinal-type than in the diffuse-type gastric carcinoma (P < 0.05). The GC-C expression was positively correlated with the expression of CDX-2 in intestinal metaplasia and gastric carcinoma. Ectopic expression of GC-C and CDX2 in human gastric mucosa may play an important role in the carcinogenesis of intestinal-type gastric carcinoma. Detection of GC-C and CDX2 helps diagnose gastric carcinoma and precursor lesions.

Colonic‐type adenocarcinoma arising in a primary retroperitoneal mature cystic teratoma

A 47-year-old woman who presented with abdominal pain was found to have a 20 cm cystic retroperitoneal mass. Pathology indicated a colonic-type adenocarcinoma arising in a primary retroperitoneal mature cystic teratoma. The adenocarcinoma was predominantly intracystic with focal superficial invasion into the cyst wall but not beyond the teratoma capsule. Immunohistochemistry showed that the adenocarcinoma cells were diffusely positive for cytokeratin 20 (CK20) and caudal-type homeobox transcription factor-2 (CDX2) but negative for CK7, confirming the colonic phenotype. In addition, the adenocarcinoma was seen adjacent to teratomatous colonic-type mucosa with adenomatous change (i.e. adenoma), suggesting that it was probably arising from a colonic-type adenoma within the teratoma. The carcinoma had a higher Ki-67 proliferation index and had a higher percentage of cells stained for p53 than the adjacent adenomatous lesion. To the authors' knowledge this is the first documented case in which a colonic-type adenocarcinoma was seen arising from a precursor lesion (i.e. a colonic-type adenoma in a primary retroperitoneal mature cystic teratoma) and is the second case of intestinal-type adenocarcinoma arising in a primary retroperitoneal mature cystic teratoma.

CD8+ T cells against multiple tumor-associated antigens in peripheral blood of midgut carcinoid patients.

The aim of the study was to identify immunogenic HLA-A*0201-binding epitopes derived from a number of classical midgut carcinoid-associated proteins. CD8(+) T cells recognizing tumor-associated antigen (TAA) epitopes are of great interest for the establishment of immunotherapy as a novel treatment for this type of malignancy. Midgut carcinoid tumor specimens were microdissected and expression levels of potential TAAs were investigated by quantitative real time PCR. HLA-A*0201-binding motifs were selected using HLA peptide binding prediction algorithms and stabilization of HLA-A*0201 was verified using TAP-deficient T2 cells. Peripheral blood of midgut carcinoid patients was analyzed for peptide epitope recognition and the feasibility of generating peptide-reactive CD8(+) T cells in healthy blood donors was examined by an in vitro stimulation protocol using mature DCs. Activation of patient and healthy donor CD8(+) T cells was analyzed by intracellular flow cytometry staining of interferon gamma. Chromogranin A (CGA), tryptophan hydroxylase 1 (TPH-1), vesicular monoamine transporter 1 (VMAT-1), caudal type homeobox transcription factor 2 (CDX-2), and islet autoantigen 2 (IA-2) are properly expressed by midgut carcinoid tumor cells, with CGA mRNA expressed to greatest level. Midgut carcinoid patients have increased frequencies of peripheral blood CD8(+) T cells recognizing a pool of HLA-A*0201 peptides derived from these proteins compared to healthy age-matched individuals. Activated peptide-specific CD8(+) T cells could also be generated in healthy blood donors by in vitro stimulation. We have identified a number of immunogenic midgut carcinoid-associated peptide epitopes recognized by CD8(+) T cells. We show that midgut carcinoid patients display immune recognition of their tumors. Memory CD8(+) T cells in patient blood are of great interest when pursuing an immunotherapeutic treatment strategy.

Directed differentiation of human embryonic stem cells towards a pancreatic cell fate

The relative lack of successful pancreatic differentiation of human embryonic stem cells (hESCs) may suggest that directed differentiation of hESCs into definitive endoderm and subsequent commitment towards a pancreatic fate are not readily achieved. The aim of this study was to investigate whether sequential exposure of hESCs to epigenetic signals that mimic in vivo pancreatic development can efficiently generate pancreatic endodermal cells, and whether these cells can be further matured and reverse hyperglycaemia upon transplantation. The hESCs were sequentially treated with serum, activin and retinoic acid (RA) during embryoid body formation. The patterns of gene expression and protein production associated with embryonic germ layers and pancreatic endoderm were analysed by RT-PCR and immunostaining. The developmental competence and function of hESC-derived PDX1-positive cells were evaluated after in vivo transplantation. Sequential treatment with serum, activin and RA highly upregulated the expression of the genes encoding forkhead box protein A2 (FOXA2), SRY-box containing gene 17 (SOX17), pancreatic and duodenal homeobox 1 (PDX1) and homeobox HB9 (HLXB9). The population of pancreatic endodermal cells that produced PDX1 was significantly increased at the expense of ectodermal differentiation, and a subset of the PDX1-positive cells also produced FOXA2, caudal-type homeobox transcription factor 2 (CDX2), and nestin (NES). After transplantation, the PDX1-positive cells further differentiated into mature cell types producing insulin and glucagon, resulting in amelioration of hyperglycaemia and weight loss in streptozotocin-treated diabetic mice. Our strategy allows the progressive differentiation of hESCs into pancreatic endoderm capable of generating mature pancreatic cell types that function in vivo. These findings may establish the basis of further investigations for the purification of transplantable islet progenitors derived from hESCs.

HOX deregulation in acute myeloid leukemia

The deregulation of homeobox (HOX) genes in acute myeloid leukemia (AML) and the potential for these master regulators to perturb normal hematopoiesis is well established. To date, overexpression of HOX genes in AML has been attributed to specific chromosomal aberrations and abnormalities involving mixed-lineage leukemia (MLL), an upstream regulator of HOX genes. The finding reported in this issue of the JCI by Scholl et al. that caudal-type homeobox transcription factor 2 (CDX2), which is capable of affecting HOX gene expression during embryogenesis, is overexpressed in 90% of patients with AML and induces a transplantable AML in murine models provides an alternative mechanism for HOX-induced leukemogenesis and yields important insights into the hierarchy of HOX gene regulation in AML (see the related article beginning on page 1037).

COX-2, CDX2, and CDC2 immunohistochemical assessment for dysplasia-carcinoma progression in Barrett's esophagus

Background Immunohistochemical changes associated with development of cancer in Barrett's esophagus offer potential areas of intervention to prevent and manage esophageal cancer. Aims To assess the role of cyclooxygenase 2, caudal-type homeobox transcription factor 2 and cell division cycle 2/cyclin-dependent kinase 1 in the Barrett's metaplasia–dysplasia–adenocarcinoma sequence. Patients and methods Specimens from 46 patients with Barrett's esophagus (39% without dysplasia, 33% with dysplasia and 28% with adenocarcinoma) were stained for cyclooxygenase 2, caudal-type homeobox transcription factor 2 and cell division cycle 2. Results Cyclooxygenase 2: No expression differences between groups were found, except for adenocarcinomas ( p = 0.04). Caudal-type homeobox transcription factor 2: Nuclear positivity decreased from Barrett's esophagus without dysplasia (71.6%), to Barrett's esophagus with low grade dysplasia (35.3%), to Barrett's esophagus with high grade dysplasia (17.14%); in adenocarcinoma these percentages were intermediate between high and low grade dysplasia (30.5%). Cell division cycle 2: Expression on deeper glandular structures was 40% in Barrett's esophagus without dysplasia, 55.47% in Barrett's esophagus with dysplasia, and 63.84% in adenocarcinoma, with no statistical differences between groups. Concerning cells of the superficial layer, Barrett's esophagus with low grade dysplasia expressed focal positivity ( p = 0.0001 vs. no dysplasia); Barrett's esophagus with high grade dysplasia displayed diffuse positivity ( p = 0.0001 vs. no dysplasia and low grade dysplasia). A diffuse positivity was found in Barrett's esophagus with adenocarcinoma ( p = 0.0001 vs. no dysplasia and low grade dysplasia). Conclusions Further evaluation of cyclooxygenase 2, cell division cycle 2 and caudal-type homeobox transcription factor 2, in association with morphology, might help to improve the accuracy of diagnosis and be useful for the clinical–pathological assessment of patients with Barrett's esophagus.

Aberrant Expression of the Homeobox Gene CDX2 in Acute Myeloid Leukemia.

The caudal-type homeobox transcription factor 2 (CDX2) plays an important role in embryonic development and regulates the proliferation and differentiation of intestinal epithelial cells in the adult. Ectopic expression of CDX2 in the hematopoietic compartment was previously identified as the key pathogenetic event in a single patient with acute myeloid leukemia (AML) and t(12;13)(p13;q12). Using real-time quantitative PCR, we detected aberrant CDX2 expression in 153 (90%) of 170 patients with AML, in patients with high-risk myelodysplastic syndrome or advanced-stage chronic myeloid leukemia, and in several AML cell lines, but not in bone marrow derived from normal individuals. Expression of CDX2 was monoallelic in the majority of cases with informative single-nucleotide polymorphisms in the CDX2 coding region, but was not related to mutations in the CDX2 coding region or in the predicted CDX2 promoter sequence, gene-specific hypomethylation of the CDX2 promoter, or increased CDX2 gene copy numbers. Stable knockdown of CDX2 expression by lentivirus-mediated RNA interference inhibited the proliferation of various human AML cell lines exhibiting CDX2 transcript levels that were in the range of those observed in most primary AML samples, and strongly reduced their clonogenic potential in vitro. Primary murine hematopoietic progenitor cells transduced with Cdx2 acquired serial replating activity in vitro, could be continuously propagated in liquid culture, generated a fully penetrant and transplantable AML in vivo, and displayed dysregulated expression of Hox family members. Together, these results (i) demonstrate that aberrant expression of CDX2 is a frequent event in myeloid leukemogenesis, (ii) suggest a role for CDX2 as part of a common effector pathway that increases the proliferative capacity and promotes the self-renewal potential of hematopoietic progenitors, and (iii) support the unifying hypothesis that CDX2 is responsible, at least in part, for abnormalities in HOX gene expression in AML.

Human acyl-CoA:cholesterol acyltransferase 2 gene expression in intestinal Caco-2 cells and in hepatocellular carcinoma.

Humans express two ACAT (acyl-CoA:cholesterol acyltransferase) genes, ACAT1 and ACAT2. ACAT1 is ubiquitously expressed, whereas ACAT2 is primarily expressed in intestinal mucosa and plays an important role in intestinal cholesterol absorption. To investigate the molecular mechanism(s) responsible for the tissue-specific expression of ACAT2, we identified five cis-elements within the human ACAT2 promoter, four for the intestinal-specific transcription factor CDX2 (caudal type homeobox transcription factor 2), and one for the transcription factor HNF1alpha (hepatocyte nuclear factor 1alpha). Results of luciferase reporter and electrophoretic mobility shift assays show that CDX2 and HNF1alpha exert a synergistic effect, enhancing the ACAT2 promoter activity through binding to these cis-elements. In undifferentiated Caco-2 cells, the ACAT2 expression is increased when exogenous CDX2 and/or HNF1alpha are expressed by co-transfection. In differentiated Caco-2 cells, the ACAT2 expression significantly decreases when the endogenous CDX2 or HNF1alpha expression is suppressed by using RNAi (RNA interference) technology. The expression levels of CDX2, HNF1alpha, and ACAT2 are all greatly increased when the Caco-2 cells differentiate to become intestinal-like cells. These results provide a molecular mechanism for the tissue-specific expression of ACAT2 in intestine. In normal adult human liver, CDX2 expression is not detectable and the ACAT2 expression is very low. In the hepatoma cell line HepG2 the CDX2 expression is elevated, accounting for its elevated ACAT2 expression. A high percentage (seven of fourteen) of liver samples from patients affected with hepatocellular carcinoma exhibited elevated ACAT2 expression. Thus, the elevated ACAT2 expression may serve as a new biomarker for certain form(s) of hepatocellular carcinoma.

Promoter methylation downregulates CDX2 expression in colorectal carcinomas

CDX2 (caudal type homeobox transcription factor 2) is a homeobox protein, which is expressed in intestinal epithelium. CDX2 has been considered to play a role as a tumor suppressor gene in colorectal cancer because its expression is lacking in colorectal carcinomas, but preserved in adenomas. The point mutations have been found to account for loss of CDX2 expression, but the main mechanism responsible for CDX2 gene inactivation is less understood. We analyzed methylation and expression status of CDX2 in colorectal cancer cell lines and primary tumors. There are two CpG-rich sites in promoter region of CDX2 gene, -1570 to -1200 and -220 to +880. By COBRA (combined bisulfite restriction analysis) assay, the upper CpG-rich site was heavily methylated in all cell lines, but the lower CpG-rich site was methylated in limited cell lines. Bisulfite sequencing analysis and RT-PCR revealed that methylation of the lower CpG site was associated with down-regulation of CDX2. In addition, gene expression was restored in COLO201, a methylated cell line with 5-aza-2'-deoxycytidine, confirming that methylation caused gene down-regulation. We also examined CDX2 promoter methylation of primary tumors by MSP (methylated-allele specific PCR) assay and found that nearly 40% of cases have a methylated CDX2 gene. Our results demonstrate that CDX2 methylation is frequently present in colorectal cancers and may play a key role in inactivating CDX2 expression.