Hypoxia inducible factor-1α increases promoter II-driven aromatase expression in postmenopausal breast cancer (#13)
Introduction: The majority of postmenopausal breast cancers are oestrogen-dependent. Tumour-derived factors such as prostaglandin E2 (PGE2) stimulate CREB binding to cAMP response elements (CREs) on aromatase promoter II (PII), leading to increased expression of aromatase and biosynthesis of oestrogens within breast adipose stromal cells (hASCs). Hypoxia inducible factor-1α (HIF-1α) is a key mediator of hypoxic responses in tumours. PGE2 has been shown to stabilise HIF-1α independent of oxygen availability in colon and prostate cancer cells. We have identified a consensus HIF-1α binding motif overlapping with the proximal CRE of PII. However, the regulation of aromatase expression by HIF-1α in breast cancer has not been characterised. We aimed to characterise the role of HIF-1α in regulating aromatase PII. Methods: HIF-1α expression and localisation were examined in primary hASCs using QPCR, western blotting, immunofluorescence and high content screening. Reporter assays and ChIP were performed to assess the effect of HIF-1α on PII activity and binding. Treatments included PGE2, forskolin/phorbol-ester (FSK/PMA; PGE2 mimetic) or DMOG (HIF-1α stabiliser). Double immunohistochemistry for HIF-1α and aromatase was performed on formalin-fixed, paraffin-embedded tissues from breast cancer and cancer-free patients. Results: PGE2 increases HIF-1α transcript and protein expression, nuclear localisation and binding to aromatase PII in hASCs. Reporter assays demonstrate that HIF-1α significantly increases PII activity in the presence of DMOG and/or FSK/PMA, and HIF-1α and CREB1 act co-operatively to increase PII activity. There is a significant increase in HIF-1α positive ASCs in breast cancer patients compared to cancer-free women. HIF-1α and aromatase double-positive, and single positive ASCs for HIF-1α or aromatase, are significantly increased in tumour patients compared to normal. Moreover, double negative ASCs are significantly decreased in tumour cases compared to normal. Conclusion: This study identifies HIF-1α as a modulator of PII-driven aromatase expression in postmenopausal breast cancer. Together with our on-going studies on the role of AMP Kinase in the regulation of breast aromatase, this work provides another link between disregulated metabolism and breast cancer.