Hypermethylation of the TPEF/HPP1 gene in primary and metastatic colorectal cancers

Abstract

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.

Figure 1

(A) TPEF gene methylation in the normal colon mucosa and matched colon cancers. (B) TPEF gene methylation in primary colon cancer and colon cancer metastasis.

Figure 2

Number of methylated genes per patient with primary colorectal cancer (A) and in metastasis (B).

Figure 3

Analysis of methylation with regard to clinicopathologic features of colorectal cancers. (A) In cancers of the colon including the sigmoid, methylation was significantly more frequent than in cancers of the rectum only. y-axis: number of patients; x-axis: number of methylated genes. (B) TPEF methylation was significantly more frequent in cancers of the colon including the sigmoid. Number of patients analyzed: colon cancer (without sigmoid colon): n = 23, sigmoid cancer: n = 13, rectal cancer: n = 10. One patient with recurrent cancer was not included in this analysis. y-axis: number of patients; x-axis: number of methylated genes.

Figure 4

(A) TPEF mRNA levels in two colon cancer cell lines (LoVo and DLD-1) after incubation with DMSO or the methylation inhibitor, 5-aza-2′-deoxycytidine (AZA). Treatment with 5-aza-2′-deoxycytidine restored TPEF mRNA levels in both cell lines. (-) Negative control; L, DNA ladder. β₂ Microglobulin levels were determined to allow comparison of TPEF mRNA levels (B2M). (B.) Methylight analysis revealed high levels of TPEF gene methylation in both cancer cell lines. (C) RT-PCR analysis of TPEF expression in nine human colorectal cancers revealed varying levels of TPEF mRNA. L: DNA ladder. β₂ Microglobulin levels were determined to allow comparison of TPEF mRNA levels (B2M). (D) Methylight analysis revealed a high degree of TPEF gene methylation in the majority of colorectal cancers. PMR, percentage of methylated reference; numbers correspond to the cases in panel C.