Tumour suppressor p53 regulates aromatase and is inhibited by prostaglandin E₂ in adipose stromal cells in obesity and breast cancer

Oestrogen-dependent postmenopausal breast cancer is associated with the increased expression of aromatase in breast adipose stromal cells (ASCs). Androgens are converted into oestrogens by aromatase and tumour-derived factors such as prostaglandin E2 (PGE2) stimulate aromatase expression via the activation of the proximal promoter PII. As a tumour suppressor, p53 is often mutated in breast cancer. However, mutations in p53 in ASCs are infrequent. This study aimed to determine the effect of PGE2 on p53 and to examine the role of p53 in regulating aromatase expression in human breast ASCs in the context of obesity related postmenopausal breast cancer. Primary human breast ASCs were isolated from breast tissue following breast reduction surgery. ASCs were treated with 1μM PGE₂ or the PGE₂ mimetic forskolin (FSK; 25μM)/phorbol ester (PMA; 4nM), and/or RITA (1μM; to stabilize p53). The effect of PGE₂ on p53 expression, subcellular localization and activity was examined by real-time PCR, Western blotting and immunofluorescence (IF). Results demonstrate that p53 transcript and nuclear protein expression are significantly decreased in ASCs in response to PGE₂. Moreover, PGE₂ also reduces p53 phosphorylation at serine 15 (Ser15) and inhibits its nuclear localisation in ASCs. The effect of p53 on aromatase transcript and activity was examined in RITA-treated ASCs by real-time PCR, and tritiated-water release assays. RITA significantly decreased the PGE2-mediated mRNA and protein expression and activity of aromatase. To examine whether p53 binds to PII directly, ChIP and reporter assays were also performed to examine p53 binding and regulation of aromatase PII. ChIP results demonstrate that p53 binds to a region on PII 438-418bp upstream of the transcription start site and that this interaction is significantly decreased in the presence of PGE2. QPCR and reporter assays demonstrate that RITA also significantly decreases the PGE2-stimulated activity of aromatase PII expression and activity. IF performed on clinical samples of breast tissue from cancer-freewomen and women with breast cancer demonstrates that nuclear p53 expression is significantly decreased in the latter group. We also found that the ratio of perinuclear to nuclear p53 is increased in tumour-associated ASCs and a positive correlation between perinuclear (inactive) p53 and aromatase was also observed. In conclusion, our findings demonstrate that p53 inhibits aromatase expression via directly binding to PII, and that this inhibition is alleviated in the presence of PGE₂. This study therefore reveals an unconventional role for p53 as a tumour suppressor in breast cancer.