CDX2 VDR polymorphism and colorectal cancer.

Studies suggest that polymorphisms of the vitamin D receptor (VDR) gene may influence colorectal cancer risk. In this study, we examine the association of the CDX2 VDR polymorphism (rs11568820) located in the 5'-untranslated region of the gene, and VDR haplotypes, including this polymorphism, with colon and rectal cancer using data from two large case-control studies of colon (N = 1,574 cases and 1,970 controls) and rectal (n = 791 cases and 999 controls) cancer. The frequency of the A allele of the CDX2 polymorphism was 19% among non-Hispanic white, 21% among Hispanic, 76% among African American, and 47% among Asian controls. The CDX2 polymorphism was not independently associated with either colon or rectal cancer, nor did it modify associations of dietary calcium, vitamin D, or fat with colon or rectal cancer. However, the bLFA haplotype which occurred in 6.5% of non-Hispanic white participants and 41.2% of African American participants was associated with an increased risk of colon cancer, and an odds ratio of 2.4 [95% confidence intervals (CI), 2.45 (1.38-4.38)]. The BSfG haplotype was associated with a 1.61 (95% CI, 1.05-2.49) increased risk of rectal but not colon cancer, whereas the BSFA haplotype was associated with a significantly reduced risk of rectal (odds ratio, 0.71; 95% CI, 0.52-0.97) but not colon cancer. These data suggest that haplotype analysis that encompasses different domains of the VDR gene might further our understanding of associations between the VDR gene and colon and rectal cancer.     

Frequent mutation of beta-catenin and APC genes in primary colorectal tumors from patients with hereditary nonpolyposis colorectal cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is characterized by defective DNA mismatch repair, which results in genetic instability of tumors; however, only a few target genes have been recognized. Our previous study detected a low frequency of APC gene mutation (21%) in colorectal tumors from HNPCC patients, in contrast to a high frequency of APC gene alteration (>70%) in non-HNPCC tumors. Because both beta-catenin and ACP gene mutations have recently been shown to activate the same signaling pathway, we analyzed beta-catenin mutation in HNPCC tumors. A notable frequency of beta-catenin gene mutation (43%, 12 of 28) was found to occur in HNPCC colorectal tumors. Beta-catenin mutations were not detected in tumors with APC mutations. All beta-catenin mutations detected in HNPCC tumors existed within the regulatory domain of beta-catenin. Immunohistochemical staining of tumors with this mutation showed accumulation of beta-catenin protein in nuclei. These and previous data from our laboratory suggest that activation of the beta-catenin-Tcf signaling pathway, through either beta-catenin or APC mutation, contributes to HNPCC colorectal carcinogenesis in approximately 65% of cases.     

The APC/beta-catenin pathway in ulcerative colitis-related colorectal carcinomas: a mutational analysis

BACKGROUND: Although the APC/beta-catenin pathway is known to play a crucial role in sporadic colorectal carcinogenesis, its influence on ulcerative colitis (UC)-related neoplastic progression is unknown. To elucidate the role of the APC-/beta-catenin pathway in UC-related carcinogenesis, the authors identified APC and beta-catenin mutations in a set of UC-related and sporadic colorectal carcinomas. METHODS: The mutational cluster region of APC (codon 1267 to 1529) and exon 3 of the beta-catenin were directly sequenced. RESULTS: Only 1 of 30 UC-related tumors (3%) showed an APC mutation whereas 11 of the 42 sporadic carcinomas (26%) had mutations within the mutational cluster region. Within the sporadic carcinoma group, only 8% of the right-sided carcinomas showed APC mutations whereas 50% of the left-sided carcinomas had mutations within the mutational cluster region. None of the tumors in either group showed a beta-catenin mutation. CONCLUSIONS: Mutations of the APC and beta-catenin are rare in UC-related tumors. These genes may be altered because of mutations outside the regions studied, or by epigenetic silencing. Alternatively, other proteins involved in the APC/beta-catenin signaling cascade may be altered, or this pathway may be involved infrequently in UC-related carcinogenesis. The significant difference in frequency of APC mutations between right- and left-sided sporadic tumors suggests different molecular pathways in these two tumor sites.     

Abnormalities of the APC/beta-catenin pathway in endometrial cancer

The activation of the APC/beta-catenin signalling pathway due to beta-catenin mutations has been implicated in the development of a subset of endometrial carcinomas (ECs). However, up to 25% of ECs have beta-catenin nuclear accumulation without evidence of beta-catenin mutations, suggesting alterations of other molecules that can modulate the Wnt pathway, such as APC, gamma-catenin, AXIN1 and AXIN2. We investigated the expression pattern of beta- and gamma-catenin in a group of 128 endometrial carcinomas, including 95 endometrioid endometrial carcinomas (EECs) and 33 non-endometrioid endometrial carcinomas (NEECs). In addition, we evaluated the presence of loss of heterozygosity and promoter hypermethylation of the APC gene and mutations in the APC, beta- and gamma-catenin, AXIN1, AXIN2, and RAS genes, and phospho-Akt expression. No APC mutations were detected but LOH at the APC locus was found in 24.3% of informative cases. APC promoter 1A hypermethylation was observed in 46.6% of ECs, and was associated with the endometrioid phenotype (P=0.034) and microsatellite instability (P=0.008). Neither LOH nor promoter hypermethylation of APC was associated with nuclear catenin expression. Nuclear beta-catenin expression was found in 31.2% of EECs and 3% of NEECs (P=0.002), and was significantly associated with beta-catenin gene exon 3 mutations (P<0.0001). beta-catenin gene exon 3 mutations were associated with the endometrioid phenotype, and were detected in 14 (14.9%) EECs, but in none of the NEECs (P=0.02). gamma-catenin nuclear expression was found in 10 ECs; it was not associated with the histological type but was associated with more advanced stages (P=0.042). No mutations in gamma-catenin, AXIN1 and 2 genes were detected in this series. Neither RAS mutations nor phospho-Akt expression, which were found in 16 and 27.6% of the cases, respectively, were associated with beta-catenin nuclear expression. Our results demonstrated a high prevalence of alterations in molecules of the APC/beta-catenin pathway, but only mutations in beta-catenin gene are associated with aberrant nuclear localization of beta-catenin.     

WNT5A exhibits tumor-suppressive activity through antagonizing the Wnt/beta-catenin signaling, and is frequently methylated in colorectal cancer.

PURPOSE: Aberrant activation of the Wnt/beta-catenin signaling pathway is associated with multiple tumors including colorectal cancer (CRC). WNT5A is a member of the nontransforming Wnt protein family, whose role in tumorigenesis is still ambiguous. We investigated its epigenetic alteration in CRCs. EXPERIMENTAL DESIGN: We examined its expression and methylation in normal colon, CRC cell lines, and tumors. We also evaluated its tumor-suppressive function and its modulation to Wnt signaling in CRC cells. RESULTS: WNT5A is silenced in most CRC cell lines due to promoter methylation, but is expressed in most normal tissues including the colon, and is unmethylated in normal colon epithelial cells. WNT5A expression could be reactivated by pharmacologic or genetic demethylation, indicating that methylation directly mediates its silencing. WNT5A methylation was frequently detected in CRC tumors (14 of 29, 48%), but only occasionally in paired normal colon tissues (2 of 15, 13%; P = 0.025). Ectopic expression of WNT5A, but not its nonfunctional short-isoform with the WNT domain deleted, in silenced CRC cells resulted in substantial inhibition of tumor cell clonogenicity, which is associated with down-regulated intracellular beta-catenin protein level and concomitant decrease in beta-catenin activity. CONCLUSIONS: WNT5A is frequently inactivated in CRC by tumor-specific methylation, and thus, is a potential biomarker. WNT5A could act as a tumor suppressor for CRC by antagonizing the Wnt/beta-catenin signaling.     

Tissue microarray analysis of beta-catenin in colorectal cancer shows nuclear phospho-beta-catenin is associated with a better prognosis.

PURPOSE: Beta-catenin is involved in homotypic cell-cell adhesion and the wnt signaling pathway. Deregulation of beta-catenin levels, caused in part by mutations of the adenomatous polyposis coli gene, is thought to play a role in the development of colorectal and other cancers. To further elucidate their roles, the expression pattern of beta-catenin and phosphospecific beta-catenin was correlated with clinical outcome in a series of patients with colorectal cancer. EXPERIMENTAL DESIGN: Immunohistochemical analysis of a tissue microarray with 650 colorectal cancer specimens was performed to study the expression and subcellular localization of beta-catenin and phosphospecific beta-catenin. These results were correlated with other clinicopathological factors and with overall survival. RESULTS: The majority of cancers retained some degree of beta-catenin membranous staining, whereas cytoplasmic or nuclear expression was seen in 42.5% and 20.4% of specimens, respectively. Phospho-beta-catenin showed nuclear staining in 9.5% of specimens, and there was no apparent membranous or cytoplasmic staining. There was no significant association between beta-catenin or phospho-beta-catenin and grade or stage. However, there was a positive correlation between beta-catenin and phospho-beta-catenin (P = 0.039), with phospho-beta-catenin representing a subset of nuclear beta-catenin. Patients with nuclear expression of beta-catenin did not have an altered survival compared with those that did not (P = 0.5611). Nuclear expression of phospho-beta-catenin, however, was associated with an improved survival (P = 0.0006). In multivariate analysis, only stage and phospho-beta-catenin were independently predictive of overall survival (P < 0.001 and P = 0.0034, respectively). CONCLUSIONS: These findings support a role for beta-catenin overexpression in colorectal tumorigenesis and provide initial evidence that phospho-beta-catenin may be a marker for improved overall survival independent of stage and grade.     

The APC gene, responsible for familial adenomatous polyposis, is mutated in human gastric cancer.

Although gastric cancer is the most common cancer in the world, genetic changes during its carcinogenesis are not well understood. Since some gastric cancers are considered to originate from the intestinal metaplasia, it is likely that the adenomatous polyposis coli (APC) gene, the mutation of which causes adenomatous polyps in the colon, is associated with carcinogenesis of gastric cancer. Based on this idea, DNAs isolated from gastric cancers were examined by means of a RNase protection analysis coupled with polymerase chain reaction followed by sequencing of the polymerase chain reaction products. By screening nearly one-half of the coding region of the APC gene in 44 tumors, somatic mutations were detected in three tumors: a missense mutation, a nonsense mutation, and a 5-base pair deletion resulting in a frame shift which causes truncation of the gene product. These results suggest that the mutation of the APC gene also plays an important role during the carcinogenesis of at least some gastric cancers.     

The APC gene in colorectal cancer

Mutations in the adenomatous polyposis coli (APC) gene are not only responsible for familial adenomatous polyposis (FAP), but also play a rate-limiting role in the majority of sporadic colorectal cancers. Colorectal tumours are known to arise through a gradual series of histological changes, the so-called 'adenoma-carcinoma' sequence, each accompanied by a genetic alteration in a specific oncogene or tumour suppressor gene. Loss of APC function triggers this chain of molecular and histological changes. In general, an intestinal cell needs to comply with two essential requirements to develop into a cancer: it must acquire selective advantage to allow for the initial clonal expansion, and genetic instability to allow for multiple hits at other genes responsible for tumour progression and malignant transformation. Inactivation of APC seems to fulfill both requirements. In this short review, I will discuss the role played by APC in providing, when mutated, selective advantage, through constitutional activation of the Wnt signal transduction pathway, and chromosomal instability to the nascent intestinal tumor cell.     

EphB2 is a prognostic factor in colorectal cancer.

A receptor tyrosine kinase for ephrin ligands, EphB2 is expressed in colorectal cancer and has been proposed as a target for immunoconjugate therapy. The aim of this study was to perform a detailed histologic analysis of EphB2 expression in normal and neoplastic colorectal tissues. In addition, we sought to evaluate EphB2 expression as a prognostic factor in colorectal cancer. Expression of EphB2 was examined in normal colon (n = 28), colorectal cell lines (n = 20), colorectal adenomas (n = 148), primary cancers (n = 28), and metastases (n = 39) using immunohistochemistry. In addition, a series of primary cancers and matched normal (n = 342) with outcome data were profiled in tissue microarrays. The intensity of EphB2 expression was assessed in the entire series by immunohistochemistry, and in a subset by in situ hybridization. Overall survival and recurrence-free survival were correlated with EphB2 protein expression in retrospective subset analyses. Epithelial EphB2 expression was shown at all stages of colorectal tumorigenesis, including the base of all normal crypts, 77% of adenomas, 82% of primary cancers, and 64% of metastases. Although homogeneous expression was observed in adenomas, the pattern of staining was focal (mean 25%) in most malignant lesions. Patients whose tumor stained 2+ for EphB2 expression (versus 0/1+) exhibited significantly prolonged overall survival: mean duration of survival, 2,514 versus 1,044 days; hazard ratio, 0.45; 95% confidence interval, 0.18 to 0.95 (P = 0.035). In summary, EphB2 is expressed in normal crypts, colorectal adenomas, primary cancers, and metastases. High levels of EphB2 expression are associated with a longer mean duration of survival in colorectal cancer.     

APC truncation and increased beta-catenin levels in a human breast cancer cell line

Mutations in the Adenomatous Polyposis Coli (APC) tumor suppressor gene or the beta-catenin gene are present in most colon cancers and less frequently in other tumor types. In this study, we screened 24 human breast cancer cell lines and three immortalized human breast epithelial cell lines for alterations in beta- and gamma-catenin and APC by western blotting, protein truncation assay and DNA sequence analysis. In one cell line (DU 4475), an APC mutation was identified (E1577stop) that resulted in expression of truncated APC. This mutation was associated with elevated cytosolic beta-catenin levels, probably due to loss of APC function, as in colon cancers. No mutations were found in exon 3 of the beta- or gamma-catenin genes. We conclude that APC mutations and beta-catenin upregulation may occur with low frequency in human breast cancer cells.     

Epigenetic silencing of CDX2 is a feature of squamous esophageal cancer

CDX2, a mammalian homologue of the homeobox gene 'caudal,' is expressed in gut epithelia and plays an important role in establishing the intestinal phenotype during development. Mice heterozygously disrupted for CDX2 develop disorganized polypoid hamartomas with glandular epithelium and stratified squamous metaplasia resembling foregut mucosa. Since no genetic disruptions of CDX2 have been reported to explain loss of gene function, we examined whether epigenetic mechanisms altered CDX2 expression. Eleven of 17 squamous esophageal cancer cell lines lacked expression of CDX2 that was restored following treatment with 5-aza-2'-deoxycytidine, while all colorectal cancer cell lines expressed CDX2. Loss of expression was associated with DNA methylation in the 5' region of CDX2 determined by methylation specific PCR and bisulfite sequencing. Methylation of CDX2 was rare in primary colorectal (1 of 44 tumors, 2%) and esophageal adenocarcinoma neoplasms (2 of 43 tumors, 5%), but was common in esophageal squamous carcinoma (24 of 45 tumors, 49%). No CDX2 methylation was found in normal tissues. Using semi-quantitative RT-PCR, expression of CDX2 was found in low level in normal esophagus, at higher levels in primary adenocarcinoma of the esophagus, but not in primary squamous cancers of the esophagus. Restoration of CDX2 in silenced cell lines resulted in expression of the CDX2 target gene MUC2, a gene important in glandular differentiation. Our results suggest that the inactivation of CDX2 in esophageal cancer associated with DNA methylation may be an important determinant of the squamous or non-adenomatous phenotype.     

Clinical significance of CDX2-positive circulating tumour cells in colorectal cancer patients

Background:

Our recent work has shown the feasibility of using a refined immunomagnetic enrichment (IE) assay to detect cytokeratin 20-positive circulating tumour cells (CK20 pCTCs) in colorectal cancer (CRC) patients. We attempted to improve the sensitivity for CRC by detecting another intestinal-type differentiation marker, CDX2 pCTCs, using the same methodology.

Methods:

CDX2 pCTCs were detected in patients with CRC, colorectal adenoma (CAD), benign colorectal diseases (BCD), other common cancers (OCC) and normal subjects (NS). Statistical analysis was used to correlate CDX2 pCTCs to the clinicohistopathological factors, recurrence, metastasis and survival after follow-up for 42 months in CRC patients.

Results:

CDX2 pCTCs were detected in 81% CRC patients (73 out of 90, median number=21.5 CTCs), 7.5% CAD patients (3 out of 40), 0% patients with BCD (0 out of 90), 2.5% patients with OCC (2 out of 80) and 0% NS (0 out of 40). Furthermore, statistical analysis showed that CDX2 pCTC numbers were associated with tumour- node-metastasis stage and lymph node status. Using the median CDX2 pCTC numbers as the cutoff points, stratified groups of CRC patients had significant differences in their recurrence and survival.

Conclusions:

This study showed that the refined IE assay can detect CDX2 pCTCs with high sensitivity and that CDX2 pCTCs can generate clinically important information for CRC patients.     

Prognostic significance of beta-catenin in colorectal cancer with liver metastasis

AimsA decreased expression of β-catenin has been known to be associated with tumour metastasis. The prognostic value of β-catenin expression in colorectal cancer (CRC) patients with liver metastasis was evaluated.Materials and methodsSeventy patients who underwent curative resection for CRC with liver metastasis were included. Tissue samples from normal colon mucosa, primary CRC and metastatic liver lesions were prepared in tissue microarrays, and were stained by immunohistochemistry with β-catenin antibody. The β-catenin expression of primary CRC tissues and metastatic liver tissues was analysed.ResultsA high expression of β-catenin (score > 6) was observed in 42.0% and 21.9% of primary colorectal tissues and metastatic liver tissues, respectively. The β-catenin expression in metastatic liver tissues was significantly lower than in primary CRC tissues (P = 0.022). The patients were classified into two groups according to the difference in the β-catenin expression score between the primary CRC and the liver metastasis. Group A was defined as patients showing a remarkably decreased expression of β-catenin in their metastatic liver tissue and group B was defined as patients showing a maintained or increased β-catenin expression in their metastatic liver tissue in comparison with their primary CRC. The overall survival and disease-free survival rates were better in group B than in group A, and this was statistically significant (P = 0.02, P = 0.002).ConclusionThe decreased expression of β-catenin in a metastatic liver lesion may be a poor prognostic marker in CRC with liver metastasis and further investigation is necessary.