title: HIF-1α stimulates aromatase expression driven by prostaglandin E2 in breast adipose stroma.
abstract: BACKGROUND
  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-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.
  
  
  METHODS
  HIF-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.
  
  
  RESULTS
  Results 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.
  
  
  CONCLUSIONS
  This 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.
authors.forename: Nirukshi U
authors.initials: NU
authors.lastname: Samarajeewa
authors.forename: Fangyuan
authors.initials: F
authors.lastname: Yang
authors.forename: Maria M
authors.initials: MM
authors.lastname: Docanto
authors.forename: Minako
authors.initials: M
authors.lastname: Sakurai
authors.forename: Keely M
authors.initials: KM
authors.lastname: McNamara
authors.forename: Hironobu
authors.initials: H
authors.lastname: Sasano
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Fox
authors.forename: Evan R
authors.initials: ER
authors.lastname: Simpson
authors.forename: Kristy A
authors.initials: KA
authors.lastname: Brown
mesh_terms: D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D001141:Aromatase / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D018270:Carcinoma, Ductal, Breast / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D015232:Dinoprostone / Q000494:pharmacology*; D005260:Female; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D010120:Oxytocics / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D014407:Tumor Cells, Cultured
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D010120:Oxytocics; D012333:RNA, Messenger; D001141:Aromatase; D015232:Dinoprostone