Regular Article Oestrogen induced downregulation of TFPI expression is mediated by ERα Huda Omar Ali a,b,d , Benedicte Stavik a,b , Elisabeth Dørum a,b , Nina Iversen c , Per Morten Sandset a,b,d , Grethe Skretting a,b, ⁎ a Department of Haematology, Oslo University Hospital, Oslo, Norway b Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway c Department of Medical Genetics, Oslo University Hospital, Oslo, Norway d Institute of Clinical Medicine, University of Oslo, Oslo, Norway abstract article info Article history: Received 13 February 2014 Received in revised form 4 April 2014 Accepted 4 April 2014 Available online xxxx Keywords: TFPI oestrogen ERα fulvestrant raloxifene FXa Introduction: Oestrogens influence the pathophysiology and development of hormone-sensitive cancers, such as breast cancer. Tissue factor pathway inhibitor (TFPI) is a serine protease inhibitor of the extrinsic coagulation pathway and has recently been associated with breast cancer cell development. Moreover, reduced TFPI levels have been reported in plasma of healthy post-menopausal women receiving hormone replacement therapy, indicating a possible link between oestrogen and TFPI. In our study, we aimed to examine the effects of oestrogen and oestrogen analogues on TFPI expression in breast cancer cells and to identify underlying mechanism(s). Methods: Oestrogen receptor alpha (ERα) positive MCF7 and negative MDA-MB-231 cells were treated with 17- β-oestradiol, 17-β-ethinyloestradiol, raloxifene and fulvestrant. TFPI mRNA and protein was measured using qRT-PCR and ELISA, respectively. Transient ERα knockdown was achieved using siRNA. Results: In ERα expressing MCF7 cells, but not in MDA-MB-231 cells, the TFPI mRNA and protein levels were sig- nificantly downregulated by more than 50% after four or six hours of incubation with 17-β-ethinyloestradiol and 17-β-oestradiol, respectively. Moreover, a significant increase in FXa generation was detected in response to oestrogens. Breast tissue ER antagonists, raloxifene and fulvestrant, did not affect TFPI mRNA, however, fulvestrant blocked oestrogen mediated reduction of TFPI mRNA. Transient knockdown of ERα abolished the oestrogenic effect on TFPI and co-treatment of MCF7 cells with the protein synthesis inhibitor cycloheximide and 17-β-oestradiol also led to reduction of TFPI mRNA. Conclusion: Our data establish a direct and time dependent regulation of TFPI expression by oestrogens through the ERα at the transcriptional level. © 2014 Elsevier Ltd. All rights reserved. Introduction Thrombosis is the second leading cause of death in cancer patients and a link between cancer and the haemostatic system has been known for many years [1]. Oestrogens can influence the pathology and development of hormone-sensitive cancers, such as breast-, endometrial-, ovarian- and prostate cancers [2]. Oestrogenic effects on breast tumours are usually mediated through ligand-dependent nuclear receptors that possess the ability to regulate transcription of genes and exist in different structural isoforms, namely oestrogen receptor alpha (ERα) and beta (ERβ). They are part of the genomic or classical path- ways where the ligand bound receptors translocate to the nucleus and bind to oestrogen response elements (EREs) in DNA and mediate cellu- lar responses over the course of hours. In normal breast tissue, both ER isoforms are expressed at low levels, however, during cancer develop- ment the expression of ERα increases [3]. In fact, more than 70% of breast cancer cells express ERα [3,4], which mediates mitogenic signal- ling [5] and induces expression of genes that promote proliferation, differentiation, angiogenesis, invasion and metastasis [6]. Currently, there are many cancer preventive agents on the market to counteract the oestrogenic effects on breast tumour tissue. Fulvestrant is a widely approved chemopreventive breast cancer agent that exerts its effect by competitively inhibiting the ERα [7,8], while selective ER modulators, such as raloxifene, was previously used to prevent breast cancer. Hormone replacement therapy (HRT) contains oestrogen in combination with a progestogen and its use has been linked to an Thrombosis Research xxx (2014) xxx–xxx Abbreviations: ERα/β, Oestrogen rceptor alpha/beta; TFPI, Tissue factor pathway in- hibitor; TF, tissue factor; E2, 17-β-oestradiol; EE2, 17-β-ethinyloestradiol; FUL, fulvestrant; CHX, cycloheximide; ERE, oestrogen response element; HRT, hormone re- placement therapy; PR, progesterone receptor; HUVEC, human umbilical vein endothelial cells; HAEC, human aortic endothelial cells; HMEC-1, human dermal microvascular endo- thelial cells. ⁎ Corresponding author at: Department of Haematology, Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, PO Box 4950 Nydalen, N-0424 Oslo, Norway. Tel.: +47 23073348; fax: +47 23073362. E-mail address: grethe.skretting@medisin.uio.no (G. Skretting). TR-05482; No of Pages 6 http://dx.doi.org/10.1016/j.thromres.2014.04.004 0049-3848/© 2014 Elsevier Ltd. All rights reserved. Contents lists available at ScienceDirect Thrombosis Research journal homepage: www.elsevier.com/locate/thromres Please cite this article as: Ali HO, et al, Oestrogen induced downregulation of TFPI expression is mediated by ERα, Thromb Res (2014), http:// dx.doi.org/10.1016/j.thromres.2014.04.004