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Published on: December 23, 2022
Epigenetic silencing of CDX2 is a feature of squamous esophageal cancer
MingZhou Guo1, Michael G House, Hiromu Suzuki
1Department of Oncology, Cancer Biology Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21231-1000, USA.
Abstract:
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.
Insights
CDX2 gene inactivation, through DNA methylation, is linked to esophageal squamous cell carcinoma. Restoring CDX2 expression reactivates MUC2, suggesting a role in glandular differentiation and phenotype determination.
Area of Science:
- Molecular biology
- Cancer research
- Epigenetics
Background:
- CDX2 is crucial for intestinal phenotype development and gut epithelial expression.
- Genetic disruptions of CDX2 are not well-documented causes of gene function loss.
- Epigenetic alterations, specifically DNA methylation, are investigated as a mechanism for CDX2 dysregulation.
Purpose of the Study:
- To investigate the role of epigenetic mechanisms, particularly DNA methylation, in altering CDX2 expression in cancer.
- To determine the association between CDX2 expression, DNA methylation, and the phenotype of esophageal and colorectal cancers.
Main Methods:
- Analysis of CDX2 expression in esophageal and colorectal cancer cell lines.
- Treatment of cell lines with 5-aza-2'-deoxycytidine to assess reversibility of CDX2 silencing.
- Methylation-specific PCR and bisulfite sequencing to detect DNA methylation in the CDX2 5' region.
- Semi-quantitative RT-PCR to evaluate CDX2 expression levels in cell lines and primary tumors.
- Analysis of CDX2 target gene MUC2 expression following CDX2 restoration.
Main Results:
- Eleven of 17 squamous esophageal cancer cell lines showed absent CDX2 expression, which was restored by 5-aza-2'-deoxycytidine treatment.
- All colorectal cancer cell lines expressed CDX2.
- Loss of CDX2 expression correlated with DNA methylation in its 5' region.
- CDX2 methylation was frequent in esophageal squamous carcinoma (49%) but rare in colorectal (2%) and esophageal adenocarcinoma (5%) primary tumors.
- CDX2 expression was detected at low levels in normal esophagus, higher in esophageal adenocarcinoma, and absent in esophageal squamous carcinoma.
- Restoration of CDX2 in silenced cell lines led to MUC2 expression.
Conclusions:
- Inactivation of CDX2 via DNA methylation is a significant event in esophageal squamous cell carcinoma development.
- CDX2 methylation contributes to the loss of glandular differentiation and the development of a squamous phenotype.
- Epigenetic silencing of CDX2 may serve as a determinant for non-adenomatous phenotypes in esophageal cancer.
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