Epigenetic Control of Estrogen Synthesis in Human Preadipocytes and Breast Cancer Cells.

Abstract Estrogen plays a significant role in the development and progression of breast cancer (BC). Cytochrome aromatase p450, encoded by the gene CYP19, is the key enzyme catalyzing the synthesis of estrogens from androgens. In post-menopausal women, adipose tissue becomes the major site for estrogen production and an increase in CYP19 expression in mammary adipose is strongly linked to the hormone-dependent progression of BC. Therefore, understanding CYP19 regulation in BC is critical for the development of therapeutic measures. Previous studies of CYP19 regulation in BC have mainly focussed on hormone-induced regulation of transcription via upstream tissue- and promoter-specific regulatory regions. While this is clearly important, it is increasingly evident that epigenetic regulation of expression, such as DNA methylation, is also a common factor in the progression of cancers. The aim of this study was to investigate whether CYP19 expression is under epigenetic regulation and to determine if such mechanisms contribute to the tissue- and promoter-specific expression of CYP19 observed in BC.CYP19 transcripts in cancer-free primary human breast adipose fibroblasts (BAFs) stimulated with cytokines and glucocorticoids (e.g. TNFα and dexamethasone) are derived from the distal promoter 1.4, whereas breast tumour-derived factors (e.g. prostaglandin E2) induce a regulatory switch to proximal promoters 1.3 and II. DNA from BAFs maintained under these two conditions was treated with sodium bisulfite allowing for methylation analysis of CpG dinucleotides. Methylation mapping revealed a stochastic heterogeneous level of methylation among 9 CpG sites (promoter 1.4) and 11 CpG sites (promoters 1.3 and II) in untreated BAFs.No correlation was observed between promoter methylation and promoter activation after response to stimuli. However, treatment of BAFs with 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, increased total CYP19 mRNA expression up to 40-fold, primarily via promoter 1.4 thorugh up-regulation of trans-acting factors, suggesting CYP19 is under tonic inhibition by methylation in BAFs. Similar results were also obtained in four BC cell lines. Ongoing work has elucidated trans-acting factors of CYP19 epigenetically silenced in BAFs. These studies uncover a new layer of complexity in the regulation of aromatase expression in BC and may identify new targets for epigenetic therapy, including DNA methylation inhibitors. These findings will translate to BC by determining the methylation state of CYP19 in clinical BC specimens, and by correlation with clinicopathological parameters such as steroid receptor status, grade and stage. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1147.