INTRODUCTION: The majority of postmenopausal breast cancers are estrogen-dependent. Tumor-derived factors, such as prostaglandin E2 (PGE2), 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-1alpha (HIF-1alpha), 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-1alpha binding motif overlapping with the proximal CRE of aromatase PII. However, the regulation of aromatase expression by HIF-1alpha in breast cancer has not been characterized. This study aimed to characterize the role of HIF-1alpha in the activation of aromatase PII. METHODS: HIF-1alpha 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-1alpha on aromatase expression and activity. Reporter assays and chromatin immunoprecipitation (ChIP) were performed to assess the effect of HIF-1alpha on PII activity and binding. Treatments included PGE2 or DMOG ((dimethyloxalglycine), HIF-1alpha stabilizer). Double immunohistochemistry for HIF-1alpha and aromatase was performed on tissues obtained from breast cancer and cancer-free patients. RESULTS: Results indicate that PGE2 increases HIF-1alpha transcript and protein expression, nuclear localization and binding to aromatase PII in human breast ASCs. Results also demonstrate that HIF-1alpha significantly increases PII activity, and aromatase transcript expression and activity, in the presence of DMOG and/or PGE2, and that HIF-1alpha and CREB1 act co-operatively on PII. There is a significant increase in HIF-1alpha positive ASCs in breast cancer patients compared to cancer-free women, and a positive association between HIF-1alpha and aromatase expression. CONCLUSIONS: This study is the first to identify HIF-1alpha 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, post-menopausal 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.
Samarajeewa, N. U. G., Yang, F., Docanto, M., Sakurai, M., McNamara, K., Sasano, H., Fox, S. B., Simpson, E. R., & Brown, K. A. (2013). HIF-1alpha stimulates aromatase expression driven by prostaglandin E2 in breast adipose stroma. Breast Cancer Research, 15(2 (Art # R30)), 1 - 12. https://doi.org/10.1186/bcr3410