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DNA methylation down-regulates CDX1 gene expression in colorectal cancer cell lines
Eun Ran Suh1, Chong S Ha, Erinn B Rankin
1Department of Internal Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA. suher@mail.med.upenn.edu
Abstract:
CDX1 is a homeobox protein that inhibits proliferation of intestinal epithelial cells and regulates intestine-specific genes involved in differentiation. CDX1 expression is developmentally and spatially regulated, and its expression is aberrantly down-regulated in colorectal cancers and colon cancer-derived cell lines. However, very little is known about the molecular mechanism underlying the regulation of CDX1 gene expression. In this study, we characterized the CDX1 gene structure and identified that its gene promoter contained a typical CpG island with a CpG observed/expected ratio of 0.80, suggesting that the CDX1 gene is a target of aberrant methylation. Alterations of DNA methylation in the CDX1 gene promoter were investigated in a series of colorectal cancer cell lines. Combined Bisulfite Restriction Analysis (COBRA) and bisulfite sequencing analysis revealed that the CDX1 promoter is methylated in CDX1 non-expressing colorectal cancer cell lines but not in human normal colon tissue and T84 cells, which express CDX1. Treatment with 5'-aza-2'-deoxycytidine (5-azaC), a DNA methyltransferase inhibitor, induced CDX1 expression in the colorectal cancer cell lines. Furthermore, de novo methylation was determined by establishing stably transfected clones of the CDX1 promoter in SW480 cells and demethylation by 5-azaC-activated reporter gene expression. These results indicate that aberrant methylation of the CpG island in the CDX1 promoter is one of the mechanisms that mediate CDX1 down-regulation in colorectal cancer cell lines.
Insights
Aberrant DNA methylation silences the CDX1 gene in colorectal cancer. Reactivating CDX1 expression in cancer cells was achieved by inhibiting DNA methyltransferase, suggesting a therapeutic target.
Area of Science:
- Molecular Biology
- Epigenetics
- Cancer Biology
Background:
- CDX1 is a crucial homeobox protein regulating intestinal epithelial cell proliferation and differentiation.
- CDX1 expression is often reduced in colorectal cancers, but the regulatory mechanisms remain unclear.
Purpose of the Study:
- To investigate the molecular mechanisms regulating CDX1 gene expression in colorectal cancer.
- To determine if DNA methylation plays a role in CDX1 down-regulation.
Main Methods:
- Gene structure and promoter CpG island analysis of CDX1.
- DNA methylation analysis using COBRA and bisulfite sequencing in colorectal cancer cell lines and normal colon tissue.
- CDX1 expression induction using 5'-aza-2'-deoxycytidine (5-azaC), a DNA methyltransferase inhibitor.
- Reporter gene assays to assess de novo methylation and demethylation.
Main Results:
- The CDX1 promoter contains a CpG island, a potential target for aberrant methylation.
- CDX1 promoter methylation was observed in colorectal cancer cell lines lacking CDX1 expression, but not in normal colon tissue or CDX1-expressing cells.
- Treatment with 5-azaC reactivated CDX1 expression in methylation-silenced cancer cell lines.
- Reporter gene assays confirmed methylation and demethylation dynamics at the CDX1 promoter.
Conclusions:
- Aberrant methylation of the CDX1 promoter CpG island is a key mechanism for CDX1 down-regulation in colorectal cancer.
- Epigenetic silencing of CDX1 contributes to colorectal tumorigenesis.
- Targeting DNA methylation could be a potential therapeutic strategy for colorectal cancer.