Methylation of estrogen receptor 1 in colorectal adenomas is not age-dependent, but is correlated with K-ras mutation

Abstract

The promoter region of estrogen receptor 1 (ESR1) has been shown to be methylated in normal colorectal mucosa in an age-dependent manner. However, the methylation of this region in colorectal tumors has not sufficiently been investigated. The methylation status of ESR1 in 105 colorectal adenoma tissues was examined by MethyLight and presented as the percentage of methylated references (PMR). Factors that affect the PMR of ESR1 in adenomas were determined using parameters including patient age, sex, past history of malignancy, family history of colorectal cancer, smoking and drinking habits, clinical characteristics of adenomas (location, size, macroscopic appearance, and histology), and K-ras mutation. Multiple linear regression revealed that the PMR was not correlated with patient age. K-ras mutation was significantly correlated with the higher methylation status of ESR1 in adenoma (t-value = 3.21, P = 0.0018), whereas alcohol exposure was significantly correlated with lower methylation status (t-value = -2.37, P = 0.02). Because methylation of O6-methylguanine DNA methyltransferase (MGMT) has been reported to be correlated with K-ras G-to-A transition, methylation of ESR1 was compared with that of MGMT with regard to K-ras mutation. Contrary to expectations, methylation of MGMT was not significantly correlated with K-ras G-to-A transition, but that of ESR1 was strongly correlated with K-ras G-to-A transition. Thus, the methylation status of ESR1 in adenomas was not correlated with patient age, but was associated with K-ras mutation, suggesting that methylation of ESR1 in tumors functions differently from that in normal colon mucosa.

Figure 1

Methylation status of estrogen receptor 1 (ESR1) in adenomas and normal mucosa. (a) The percentage of methylated references (PMR) of ESR1 in 105 adenomas are shown and stratified by patient age. No significant correlation was observed between PMR and patient age. (b) The PMR of ESR1 in normal colon mucosa of 47 patients who provided normal samples are shown. PMR of normal mucosa were significantly correlated with patient age (P = 0.0004). (c) The PMR of ESR1 in adenoma and those in normal mucosa were compared in individual patients. No correlation was observed.

Figure 2

Bisulfite sequencing of the estrogen receptor 1 (ESR1) locus in representative patients. In normal mucosal samples, the percentage of methylated references (PMR) were relatively low (6.2 and 22.6) and methylation status shown with bisulfite sequencing was sparse, whereas in adenoma samples, the PMR were high (153.1 and 159.2) and the sequencing revealed that the locus was densely methylated.

Figure 3

Methylation of estrogen receptor 1 (ESR1) in adenomas stratified by K‐ras status and alcohol exposure. Adenomas were stratified into four groups according to K‐ras status (wild‐type or mutant) and alcohol exposure (+ or –). The Kruskal–Wallis rank test revealed that the differences in the percentage of methylated references (PMR) of ESR1 among the four groups were statistically significant (P < 0.0001). Adenomas derived from drinkers were likely to exhibit lower methylation of ESR1; in contrast, adenomas with K‐ras mutations were likely to exhibit higher methylation of ESR1. In particular, no adenomas with PMR below 47 carried a K‐ras mutation. Horizontal lines represent median methylation levels for each group.

Figure 4

Prevalence of the percentage of methylated reference (PMR) of estrogen receptor 1 (ESR1) and O⁶‐methylguanine DNA methyltransferase (MGMT) in adenomas according to K‐ras status. Adenoma samples were classified into three groups according to K‐ras status: adenomas with the G‐to‐A transition, those with other mutations, and wild‐type adenomas. The Kruskal–Wallis rank test revealed that the PMR of ESR1 was significantly correlated with K‐ras status (P = 0.0002). On the other hand, the PMR of MGMT was not significantly correlated with K‐ras status (P = 0.35). In addition, the PMR of ESR1 was significantly higher in adenomas with the K‐ras G‐to‐A transition than in wild‐type adenomas (P = 0.0003). Meanwhile, the PMR of MGMT was not significantly different between adenomas with the K‐ras G‐to‐A transition and wild‐type adenomas (P = 0.43). Horizontal lines represent median methylation levels for each group.