Our findings demonstrate that expression of the tumor suppressor gene MED1 is decreased in sporadic colorectal cancer and that it can be attributed to promoter methylation. We suggest that MED1 is suppressed in a large proportion of sporadic colorectal cancers in the absence of known MED1 mutations. Additionally, there is a significantly lower incidence of MED1 promoter methylation in normal control patients when compared to patients with colorectal cancer. Our in vitro studies show that reversal of MED1 methylation leads to increased gene expression, suggesting that methylation does contribute to gene suppression. The importance of these findings stem from the fact that MED1 methylation and gene suppression seem to develop in normal colon mucosa prior to the development of the adenoma-carcinoma sequence. The epigenetic regulation of the tumor suppressor gene MED1 thus seems to be associated with tumor initiation rather than tumor progression. This is corroborated by the lack of a significant decrease in MED1 expression correlated to more advanced tumor stages that would imply a role in tumor progression. Gene suppression can be detected in the normal colon mucosa of tumor-bearing patients as well as in polyps and carcinomas from matched specimens.
promoter methylation observed in tumor tissue is also present in adjacent, matched normal mucosa. This phenomenon of methylated tumor suppressor genes in tumor and adjacent normal mucosa has previously been reported in sporadic CRC and may reflect a “field effect” involving the mucosal lining of CRC patients. Epigenetic changes have been implicated as an early event in CRC tumorigenesis.12,36
We propose that a similar mechanism involving MED1
plays a role in sporadic CRC tumor initiation. The resulting CRC patient decrease in MED1
gene expression can be readily detected in the associated normal mucosa when compared to control samples. Normal colonic mucosa MED1
gene methylation and suppression has also been reported for the MLH1
The 56% increase in the amount of MED1
promoter methylation observed in mucosa from cancer patients further supports the role of promoter methylation as a plausible cause of MED1
suppression in these patients.
Our in vitro
studies demonstrated the reversibility of MED1
promoter methylation restoring MED1 gene expression, also in concordance with the MLH1
The reversibility of MED1
expression in ovarian and CRC cell lines suggests that observed promoter methylation does affect gene expression in at least two different types of epithelial cancers. If tissue methylation causes an initial drop in MED1
expression within normal mucosa, it poses the question as to what additional changes are necessary to induce the downregulation of MED1
expression detected in carcinomas. As suggested by Jones and Laird, promoter methylation in MED1
may require a second “hit” to see the ultimate degree of gene suppression observed in tumors. We speculate that methylation may trigger the initial decrease in gene expression and this is followed by mutations or LOH causing greater gene silencing and the development of cancer. An initial decrease in MED1
expression is seen when DNA from normal mucosa is methylated. This process could enable subsequent genetic changes that would further decrease MED1
expression, as described in polyps and carcinomas.39
The importance of decreased MED1 in CRC tumorigenesis has been demonstrated in mouse models with MED1/MBD4
targeted gene inactivation.30,40
Mice with MED1
knockout in the setting of an Apc Min/+
background had a significantly reduced survival and increased CRC tumor burden compared to control mice.30,40
Work from our and other laboratories suggests an additional role for MED1
inactivation in tumorigenesis.20,21,41
These studies suggest that decreased MED1 protein levels have an inhibitory effect on apoptosis. These concepts support our findings. The early changes that we have observed in MED1
expression in colon mucosa from cancer patients suggest that either decreased genomic surveillance, decreased apoptosis, or both, could lead to initiation of tumorigenesis.
A high frequency of MED1
methylation in cancer patients and the ability to demethylate and restore gene expression in cell lines does implicate a role for methylation in the silencing of this putative tumor suppressor gene. Further, comparison of frequently methylated DNA from CRC patients with DNA from non-diseased patients and their consistently low frequency of methylation status suggest that hypermethylation of the MED1
promoter is an abnormal occurrence in CRC tumorigenesis. The significance of this finding is that it appears to be an early occurrence in the development of CRC similar to other genes involved in the development of sporadic colorectal cancer.12
In conclusion, two observations suggest that MED1 promoter methylation and gene suppression are important in CRC tumorigenesis. First, methylation occurs in cancer patients at higher frequencies than those found in patients without cancer. Second, and perhaps most importantly, MED1 suppression seems to occur first at the level of the normal mucosa. The suppression of MED1 follows the adenoma-carcinoma sequence and is observed in normal mucosa, adenomas, and adenocarcinoma of the colon suggesting an early and detectable change. Suppression of MED1 expression and early detection of promoter methylation may necessitate a more frequent and rigorous screening repertoire in this population of patients leading to earlier detection of malignant changes.