HIF-1α stimulates aromatase expression driven by prostaglandin E2 in breast adipose stroma

Introduction: The majority of postmenopausal breast cancers are estrogen-dependent. Tumor-derived factors, such as prostaglandin E-2 (PGE(2)), stimulate CREB1 binding to cAMP response elements (CREs) on aromatase promoter II (PII), leading to the increased expression of aromatase and biosynthesis of estrogens within human breast adipose stromal cells (ASCs). Hypoxia inducible factor-1 alpha (HIF-1 alpha), a key mediator of cellular adaptation to low oxygen levels, is emerging as a novel prognostic marker in breast cancer. We have identified the presence of a consensus HIF-1 alpha binding motif overlapping with the proximal CRE of aromatase PII. However, the regulation of aromatase expression by HIF-1 alpha in breast cancer has not been characterized. This study aimed to characterize the role of HIF-1 alpha in the activation of aromatase PII. Methods: HIF-1 alpha expression and localization were examined in human breast ASCs using quantitative PCR (QPCR), Western blotting, immunofluorescence and high content screening. QPCR and tritiated water-release assays were performed to assess the effect of HIF-1 alpha on aromatase expression and activity. Reporter assays and chromatin immunoprecipitation (ChIP) were performed to assess the effect of HIF-1 alpha on PII activity and binding. Treatments included PGE(2) or DMOG ((dimethyloxalglycine), HIF-1 alpha stabilizer). Double immunohistochemistry for HIF-1 alpha and aromatase was performed on tissues obtained from breast cancer and cancer-free patients. Results: Results indicate that PGE(2) increases HIF-1 alpha transcript and protein expression, nuclear localization and binding to aromatase PII in human breast ASCs. Results also demonstrate that HIF-1 alpha significantly increases PII activity, and aromatase transcript expression and activity, in the presence of DMOG and/or PGE(2), and that HIF-1 alpha and CREB1 act co-operatively on PII. There is a significant increase in HIF-1 alpha positive ASCs in breast cancer patients compared to cancer-free women, and a positive association between HIF-1 alpha and aromatase expression. Conclusions: This study is the first to identify HIF-1 alpha as a modulator of PII-driven aromatase expression in human breast tumor-associated stroma and provides a novel mechanism for estrogen regulation in obesity-related, postmenopausal breast cancer. Together with our on-going studies on the role of AMP-activated protein kinase (AMPK) in the regulation of breast aromatase, this work provides another link between disregulated metabolism and breast cancer.