Estrogen receptor α (ER), BRCA1 and FANCF promoter methylation occur in distinct subsets of sporadic breast cancers

PR-12

Women of African ancestry are more likely to develop ER-negative, PR-negative and HER2-negative basal-like breast cancers, with worse prognoses and lack of therapeutic targets (Polite and Olopade, 2005). The underlying biological mechanisms are still largely unknown. ER and its ligand estrogen play vital roles in the development, progression and treatment of breast cancer. A potential mechanism for hormone resistance is the acquired loss of ER gene expression at the transcriptional level during disease progression. Methylation of the CpG islands in the 5' regulatory region of the ER gene has been associated with loss of ER gene expression in ER-negative breast cancers (Lapidus RG et al., 1998). An increasing number of studies have also provided evidence linking disruption of the Fanconi anemia/BRCA cascade to breast cancer. BRCA1-mutated and promoter-methylated cancers are often of high grade and are ER-negative, suggesting that alterations of the BRCA1 or related pathways might contribute to some sporadic breast cancers (Wei M et al., 2005). Our objectives were to examine the methylation status and expression profiles of ER, correlate the findings with BRCA1 and FANCF methylation and map the critical CpGs for ER expression. In analyzing a subset of domestic samples, we found that the CpG islands in the 5' region of the ER gene are methylated in 59 of 120 (49.2%) primary breast cancers, including 45 of 59 ER-negative tumors (76.3%, P<0.00001). In addition, we observed a strong correlation between ER promoter and BRCA1 promoter methylation (odds ratio 3.12, 95% confidence interval 1.10-9.68, P=0.02). In contrast, FANCF methylation was rare in breast tumors: 1 of 120 (0.8%). ER methylation was associated with high tumor grade (60.4 % methylated vs. 39.6 % unmethylated in grade 3 tumors, P = 0.04) and tumor subtype (P = 0.03). Though small in number, all tumors of the medullary subtype were ER methylated. In contrast, the lobular subtype had the least methylation (23.1 % methylated vs. 76.9 % unmethylated). In addition, we analyzed promoter methylation of the BRCA1 and FANCF genes by Methylation Specific PCR in a subset of Nigerian breast cancers. We found a significant higher proportion of BRCA1 promoter methylation in Nigerian samples compared to domestic samples (37.2% vs 20%). This may partially explain the aggressive nature of breast cancer in Nigeria since basal-like subsets are over-represented in this population. FANCF methylation was also rare in the Nigerian breast cancer cases. After treatment of MDA-MB-231 cells with 5-aza-cytidine (5-aza-dC) and trichostatin (TSA), which resulted in re-expression of ER mRNA, we localized dramatic demethylation effects to CpG islands in positions +68, +165, +192, +195, +337, +341 and +405 relative to transcription start site of the ER promoter. Together, these data suggest that unlike FANCF, both ER and BRCA1 are specifically targeted for methylation in sporadic breast cancers, a phenomenon that should be explored for development of novel diagnostic and therapeutic approaches. Future work will investigate how gene expression is altered by environmental factors and how methylation of CpG islands affects transcription factor binding.