cAMP response element (CRE)-dependent regulation of aromatase in obesity and postmenopausal breast cancer

The risk of breast cancer rises dramatically with advancing age, and recently obesity has also been recognised as a significant contributor. In postmenopausal women with breast cancer, the inappropriately elevated aromatase expression is governed by enhanced usage of the proximal promoters, PII and PI.3 which leads to an increased oestrogenic drive as a result of tumour-derived factors such as prostaglandin E2 (PGE2) in breast adipose stromal cells (ASCs). This increased expression of aromatase PII is largely regulated by the transcription factor cAMP response element (CRE) binding protein-1 (CREB1) which binds to proximal and distal CREs on PII. Our group has demonstrated that the CREB co-activator CRTC2 can bind and activate aromatase PII via CREs and that its subcellular localisation is regulated by PGE2. There are three known members of the CRTC family (CRTC1-3) and the regulation of aromatase expression by other CRTC members is poorly understood. HIF-1α is a master regulator of adaptive responses to hypoxia and found to be regulated by PGE2 independent of hypoxia. We have identified a putative hypoxia response element (HRE) which overlaps with the proximal CRE of aromatase PII. However, the regulation of aromatase expression by HIF-1α in breast cancer had not yet been characterised. Our group has previously demonstrated that the commonly used antidiabetic drug metformin inhibits aromatase expression in ASCs via the inhibition of PII activity. This study therefore aimed to characterise the role of CRTCs and HIF-1α in the activation of aromatase PII, and to examine the effect of metformin on promoter-specific transcript expression in breast ASCs. The results indicated that PGE2 causes nuclear translocation of all three CRTCs and HIF-1α. Reporter assays demonstrated that basal PII activity is significantly increased with all CRTCs. FSK/PMA, to mimic PGE2, results in a further significant increase in PII activity with all CRTCs. HIF-1α significantly increases PII activity in the presence of PGE2. Interestingly, CRTCs via both proximal and distal CREs, or HIF-1α via the proximal CRE, act co-operatively with CREB1 to maximise PII activity. There is a significant increase in HIF-1α positive ASCs in breast cancer patients compared to cancer-free women, and a positive association between HIF-1α and aromatase expression. Metformin results in a dose-dependent inhibition of FSK/PMA-induced aromatase PII and PI.3 transcript expression and does not affect PI.4. In conclusion, this study identifies CRTCs and HIF-1α as modulators of aromatase PII, and metformin as a breast-specific inhibitor of aromatase in breast ASCs, thereby furthering our understanding of the complex nature of aromatase regulation in obesity-related, postmenopausal breast cancer