The role of promoter methylation in the process of cancer cell metastasis has, 
however, not yet been studied. Recently, methylation of the TPEF (transmembrane 
protein containing epidermal growth factor and follistatin domain) gene was 
reported in human colon, gastric, and bladder cancer cells. Using the Methylight 
assay, TPEF/HPP1 gene methylation was assessed in primary colorectal cancers (n 
= 47), matched normal colon mucosa, as well as in the liver metastasis of 24 
patients with colorectal cancer, and compared to the methylation status of the 
TIMP-3, APC, DAPK, caveolin-2, and p16 genes. TPEF was frequently methylated in 
primary colorectal cancers (36 of 47) compared to the normal colon mucosa (1 of 
21) (P < .0001), and TPEF mRNA expression in colon cancer cell lines was 
restored after treatment with 5-aza-2'-deoxycytidine. The p16 and APC genes were 
also frequently methylated in primary colorectal cancers (P < .02) compared to 
the normal colon mucosa. Interestingly, promoter methylation was significantly 
more frequent in proximal, nonrectal cancers (P < .05). Furthermore, a high 
degree of methylation of the TPEF gene was also observed in liver metastasis (19 
of 24). In summary, we observed frequent TPEF methylation in primary colorectal 
cancers and liver metastases, indicating that epigenetic alterations are not 
only present in the early phases of carcinogenesis, but are also common in 
metastatic lesions. The high frequency of TPEF methylation in this series of 
colorectal cancers underscores the importance of epigenetic changes as targets 
for the development of molecular tests for cancer diagnosis.