Association of estrogen receptors’ promoter methylation and clinicopathological characteristics in Iranian patients with breast cancer

Background:Estrogens play a substantial role in the proliferation, progression and treatment of breast cancer by binding with two estrogen receptors, alpha and beta (ERα and ERβ). Resistance to endocrine therapy is a major problem in the treatment of breast cancers and, in some cases, may be related to loss of ER gene expression. We have already showed that ERα methylation occurs in high frequency and may be one of the important mechanisms for ERα gene silencing in a subset of Iranian primary sporadic breast cancers. In the other hand, the CpG Island methylation status of ERβ and the relationship between clinicopathological features and the pattern of ERβ methylation in sporadic breast cancer are still unknown, especially in Iranian women. Methods: In this study, we examined the exact role of DNA methylation in the estrogen receptors, alpha and beta genes using Combined Bisulfite Restriction enzyme Analysis (COBRA) and Methylation specific polymerase chain reaction (MSP) methods in 34 tissue and 40 peripheral white blood cells in the breast cancers. Results and Conclusions: ERα promoter methylation was identified in 29(72.5%) tissue samples and 35(87.5%) peripheral blood. Among these ERα-methylated cases, the co-occurrentmethylation of ER promoter in peripheral blood and tissue samples was evident in 25 (71.4%) patient (P=0.56). Furthermore, ERβ promoter methylation was detected in 13(32.5%) tissue samples and 4(10.0%) peripheral blood specimens. Of these ERα-methylated cases, the co-ocurrent methylation of ERβ promoter in the peripheral blood and tissue samples was evident in 1(7.7%) patient (P= 0.11). Based on COBRA analysis the percentage of DNA methylation at methylation-sensitive BstUI restriction site of the ERα promoter A ranged from 1% to 91%. The percentages at promoters A region showed a borderline associations with lymph node involvement (P=0.079, r=0.55) and a significant correlation with the grade of tumors (p= 0.27, r=0.65). No significant relation was found between ERα promoter and ERβ promoter methylation (Odds ratio =2.82, 95%, CI =0.28–28.5, P=0.36). The methylation of promoter ON was observed in only a subset of tumors without ER by IHC. In addition, we did not find any significant correlationbetween the prognostic factors such as grade, tumor size, lymph node involvement, and methylation status of this promoter. Our results indicate that methylation of ERβ promoter ON is not responsible for the loss of gene expression in of all breast tumors.