CCAAT/Enhancer Binding Protein δ Up-regulates Aromatase Promoters I.3/II in Breast Cancer Epithelial Cells

Abstract

Aromatase is the enzyme responsible for the last step of estrogen synthesis. The female hormone, estrogen, is known to stimulate breast cancer cell growth. Because the expression of aromatase in breast cancer tissues is driven by unique promoters I.3 and II, a more complete understanding of the regulatory mechanism of aromatase expression through promoters I.3/II in breast tumors should be valuable in developing targeted therapies, which selectively suppress estrogen production in breast tumor tissue. Results from in vivo footprinting analyses revealed several protein binding sites, numbered 1 to 5. When site 2 (-124/-112 bp, exon I.3 start site as +1) was mutated, promoters I.3/II activity was dramatically reduced, suggesting that site 2 is a positive regulatory element. Yeast one-hybrid screening revealed that a potential protein binding to site 2 was CCAAT/enhancer binding protein delta (C/EBP delta). C/EBP delta was shown to bind to site 2 of aromatase promoters I.3/II in vitro and in vivo. C/EBP delta up-regulated promoters I.3/II activity through this site and, as a result, it also up-regulated aromatase transcription and enzymatic activity. p65, a subunit of nuclear factor-kappaB (NF-kappaB) transcription factor, inhibited C/EBP delta-up-regulated aromatase promoters I.3/II and enzymatic activity. This inhibitory effect of p65 was mediated, in part, through prevention of the C/EBP delta binding to site 2. This C/EBP delta binding site in aromatase promoters I.3/II seems to act as a positive regulatory element in non-p65-overexpressing breast cancer epithelial cells, whereas it is possibly inactive in p65 overexpressing cancer epithelial cells, such as estrogen receptor-negative breast cancer cells.