Abstract
IntroductionThe 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-1α (HIF-1α), 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-1α binding motif overlapping with the proximal CRE of aromatase PII. However, the regulation of aromatase expression by HIF-1α in breast cancer has not been characterized. This study aimed to characterize the role of HIF-1α in the activation of aromatase PII.MethodsHIF-1α 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-1α on aromatase expression and activity. Reporter assays and chromatin immunoprecipitation (ChIP) were performed to assess the effect of HIF-1α on PII activity and binding. Treatments included PGE2 or DMOG ((dimethyloxalglycine), HIF-1α stabilizer). Double immunohistochemistry for HIF-1α and aromatase was performed on tissues obtained from breast cancer and cancer-free patients.ResultsResults indicate that PGE2 increases HIF-1α transcript and protein expression, nuclear localization and binding to aromatase PII in human breast ASCs. Results also demonstrate that HIF-1α significantly increases PII activity, and aromatase transcript expression and activity, in the presence of DMOG and/or PGE2, and that HIF-1α and CREB1 act co-operatively on PII. 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.ConclusionsThis study is the first to identify HIF-1α 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.
Highlights
The majority of postmenopausal breast cancers are estrogen-dependent
Results indicate that prostaglandin E2 (PGE2) increases Hypoxia inducible factor-1a (HIF-1a) transcript and protein expression, nuclear localization and binding to aromatase promoter II (PII) in human breast adipose stromal cells (ASC)
Results demonstrate that HIF-1a significantly increases PII activity, and aromatase transcript expression and activity, in the presence of DMOG and/or PGE2, and that HIF-1a and cAMP response elements (CREs) binding protein 1 (CREB1) act co-operatively on PII
Summary
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). One of the transcription factors shown to be involved in this process is cAMP response element (CRE) binding protein-1 (CREB1) which binds to the proximal and distal CREs on PII, and stimulates the expression of aromatase [6]. HIF-1 is stabilized and binds to core hypoxia response elements (HREs) containing the 5’-RCGTG-3’ sequence [19] with other transcription factors, such as CBP/p300 via its CH1 domain [20], which results in the transcriptional activation of hypoxia-regulated genes including vascular endothelial growth factor (VEGF), known to promote angiogenesis (reviewed in [21]). In PC-3 ML human prostate cancer cells [22] and in HCT116 human colon carcinoma cells [23], it was demonstrated that PGE2 and hypoxia act both independently and synergistically to increase HIF-1a protein levels, and further demonstrated the time-dependent nuclear accumulation of HIF-1a in response to PGE2
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