Loss of CDX1 expression in colorectal carcinoma: Promoter methylation, mutation, and loss of heterozygosity analyses of 37 cell lines

Abstract

Expression of the homeobox protein CDX1 is lost or reduced in a significant proportion of colorectal carcinomas (CRCs) but the underlying mechanism for this is unclear. We have demonstrated absence of CDX1 mRNA expression in 7 of 37 CRC cell lines and shown that all 7 cell lines have a methylated CDX1 promoter. Twenty-five cell lines showed both CDX1 mRNA expression and an unmethylated CDX1 promoter. The five remaining cell lines had a partially methylated CDX1 promoter and all expressed CDX1 mRNA; when treated with the demethylating agent, 5-aza-2-deoxycytidine, these five cell lines all showed increased CDX1 expression. No mutations were found in the promoter and coding regions of CDX1. One polymorphism was demonstrated in each of the promoter, 5 UTR, and coding region of exon 1 of CDX1, but there were no associations between CDX1 mRNA expression and different polymorphic genotypes. Similarly, there was no association between CDX1 mRNA expression and loss of heterozygosity at the CDX1 locus. In conclusion, absence or reduction of CDX1 expression in CRC seems to be primarily regulated by promoter methylation and is probably selected for because of its impact on the differentiation of colonocytes. C DX1 is a homeobox protein involved in regulating normal development of intestinal epithelium in utero (1). In adulthood, CDX1 expression is maintained in small and large intestinal epithelium (1, 2) although its exact physiological role at this time remains uncertain. That the protein has an oncosuppressor function is supported by in vitro and in vivo evidence of loss of CDX1 expression in colorectal carcinogenesis (3, 4). The mechanism underlying this loss is, however, unclear. Possible roles for genetic and͞or epigenetic controls must therefore be considered. There is an ever growing list of oncosuppressor genes whose expressions are silenced through specific methylation of gene promoter CpG islands (CGIs). These genes all show 5Ј regions that are rich in CGIs (5). In sharing this feature, CDX1 represents a prime candidate for promoter methylation control. Indeed, half way through our study, Suh et al. published preliminary data supporting this hypothesis (6).The most comprehensive way of analyzing a gene for evidence of promoter methylation control is by direct sequencing after bisulfite modification of target DNA (so called ''bisulfite sequencing'') (7). Ideally, this analysis should be accompanied by looking for reexpression of the silenced gene after treatment with the demethylating agent, 5-azacytidine or the less toxic 5-aza-2Ј-deoxycytidine (5aza2). Suh et al. (6) showed CDX1 promoter methylation status to explain CDX1 mRNA expression among eight colorectal carcinoma (CRC) cell lines and also successfully used 5aza2 treatment to induce or increase CDX1 mRNA expression in six of the cell lines. Nonetheless, two important questions regarding control of CDX1 expression in CRC remain unanswered. First, possible contributions of gene mutation and͞or loss of heterozygosity (LOH) have not yet been excluded. Secon...

Gastrointestinal differentiation marker Cytokeratin 20 is regulated by homeobox gene CDX1

“…As suggested by Wong et al (19), selection for reduced expression of CDX1 by methylation in colorectal cancers (17)(18)(19) is likely to be associated with a reduced capacity of the cancer stem cells to differentiate and so to contribute to an increased rate of growth. Studying the patterns of expression of CDX1 and its downstream-regulated differentiation markers, such as KRT20, should therefore enable better characterization of cancer stem cells both in CRC cell lines and in primary CRCs.…”

“…We have previously shown that the mRNA expression of CDX1 in partially methylated cell lines could be increased by 5-aza-2Ј deoxycytidine treatment and that ''postconfluent'' growth of these partially methylated cell lines, such as LOVO, led to an increase in CDX1 expression that was correlated with a decrease in methylation of a key region in the CDX1 promoter (19). It is now widely accepted that cancer stem cells are the driving cells of a cancer, which will also contain the differentiated cells derived from them (43).…”

“…The overall data are consistent with the idea that CDX1 expression is a key switch for intestinal epithelial differentiation, as suggested by Wong et al (29) with respect to the development of intestinal metaplasia in Barrett's oesophagus, and presumably also in gastric mucosa (31), where intestinal metaplasia is assumed often to be a precursor to the development of gastric carcinomas. The differential expression of CDX1 appears to be controlled by promoter methylation (19,29).…”

“…Our previous work has shown that about 20% of CRC cell lines have low or absent CDX1 mRNA expression because of hypermethylation of the CDX1 promoter region (19). In this study we examined the expression of CDX1 in an additional 32 CRC cell lines, as well as in 6 that had been studied previously, using semiquantitative RT-PCR.…”

“…CDX1 plays a role in intestinal epithelial cell differentiation (16) and is down-regulated in a number of CRC-derived cell lines as well as in patient samples (17)(18)(19). This suggests that absence of CDX1 expression is an advantage for the development of colorectal carcinomas.…”