399 Xenoestrogens modulate vascular endothelial growth factor secretion in breast cancer cells through an estrogen receptor-dependent mechanism He´le`ne Buteau-Lozano1,2, Guillaume Velasco1,2, Monique Cristofari1,2, Patrick Balaguer3 and Martine Perrot-Applanat1,2 1INSERM, U553, He´mostase, Endothe´lium et Angioge´ne`se, Hoˆpital Saint Louis, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France 2IFR105 Universite´ Paris 7, 75000 Paris, France 3INSERM, U824, Signalisation hormonale, Environnement et Cancer, 34098 Montpellier, France (Correspondence should be addressed to M Perrot-Applanat; Email: [email protected]) Abstract Environmental chemicals may affect human health by disrupting 182 780. VEGF increase was not observed in ERa-negative endocrine function. Their possible role in the mammary gland MDA-MB-231 cells. Most substances increased VEGF tran- and breast tumors is still unknown. Previous studies have script levels in MELN cells. In contrast, g-hexachlorocyclohex- demonstrated that vascular endothelial growth factor (VEGF), a ane, vinclozolin, and the phthalates (mono-n-butyl ester key factor in angiogenesis and tumor progression, is an estrogen- phthalic acid, di-isononyle phthalate, and di-isodecyle phtha- regulated gene. We analyzed whether VEGF expression is late) were ineffective on both VEGF secretion and estrogenic regulated by differentxenoestrogens in several breast cancercells, luciferase induction in these cell lines. Specific kinase inhibitors MELN (derived from MCF-7) and MELP (derived from MDA- PD98059, SB203580, or LY294002 suppressed the xenoestro- MB-231) and stablyexpressing estrogen receptor a (ERa); these gen-induced VEGF response, suggesting activation of MEK, cell lines stably express estrogen response element (b-globin)- p38 kinase, and phosphatidylinositol-3-kinase pathways. Our luciferase. Genistein, bisphenol A (BPA), 4-(tert-octyl)phenol in vitro results show for the first time that genistein and (OP), dieldrin, and several phthalates, including benzyl butyl xenoestrogens (BPA, OP, dieldrin, BBP, and DEHP at high phthalate (BBP) and di-ethyl-2-hexyle phthalate (DEHP), were concentrations) up-regulate VEGF expression in MELN cells first shown to be estrogenic. These compounds induced a dose- by an ER-dependent mechanism. Since VEGF increases dependent increase of VEGF secretion in MELN and MCF-7 capillary permeability and breast tumor angiogenesis in vivo, cells; maximal effect was observed at 1–10 mMnon-cytotoxic the physiological relevance of these findings is discussed. concentrations and was inhibited by the antiestrogen ICI Journal of Endocrinology (2008) 196, 399–412 Introduction than endogenous estrogens when tested in biological assays, they are cause of concern due to their persistence in the environment, Over the past 20 years, a great deal of attention has focused on the resistance to chemical or enzymatic degradation, and sequestra- impact of endocrine disruptors released in the environment on tion and storage in the adipose tissue. Whether it can be implied animal and human health. A large number of these environ- that these substances in human female reproductive diseases mental pollutants, including alkylphenol compounds, poly- (precocious puberty and endometriosis, Te i l m a n n et al.2002)or chlorinated biphenyls (PCBs), polychlorinated dibenzodioxins, in steroid hormone-dependent cancers (breast, prostate) are a organochlorine pesticides, and bisphenol A (BPA), have been leading cause of mortality and morbidity in industrialized shown or suspected to disrupt endocrine functions in animals. countries, is still under debate (Sasco 2001, Birnbaum & Fenton Generally, endocrine disruptors have estrogenic activity 2003, Mitra et al. 2004, Sharpe & Irvine 2004, Safe 2005). (so-called xenoestrogens including pesticides, bisphenols and Development of the mammary gland and breast cancer in alkylphenols, some PCBs, and phthalates; Colburn 1995, Jobling women depends on diverse factors including exposure to et al. 1995, Soto et al.1995, Sonnenschein & Soto 1998, estrogens ( Jordan & Murphy 1990). Estrogens regulate cell Andersen et al. 1999), anti-androgenic activity (some pesticides; function through tissue-dependent specific intracellular Colburn 1995, Kelce et al. 1997), or disrupt thyroid function receptors (estrogen receptor, ER), acting as ligand-activated (PCBs, perchlorate, and some pesticides) (Koopman-Esseboom transcription factors. Activated ERs regulate the expression of et al. 1994). Although xenoestrogens are generally less potent many genes via direct interaction with an estrogen response Journal of Endocrinology (2008) 196, 399–412 DOI: 10.1677/JOE-07-0198 0022–0795/08/0196–399 q 2008 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 10/01/2021 05:46:13AM via free access 400 H BUTEAU-LOZANO and others . VEGF expression by xenoestrogens in breast cells element (ERE) sequence located in the promoter or via induced by E2 in the uterus, vagina, and pituitary (Cullinan-Bove interaction with other transcription factors, such as members & Koos 1993, Shifren et al.1996, Bausero et al. 1998, Kazi et al. of activator protein-1 (AP-1), signal transducer and activator of 2005) and in rodent and primate models of mammary cancer transcription (STATs), nuclear factor-kappa B (NF-kB), and (Nakamura et al.1996, Dabrosin et al.2003). Interestingly, the specificity protein (SP-1) families (Stein & Yang 1995, Paech xenoestrogen BPA also increases VEGF expression in the uterus et al.1997). Selective effects of estrogens may be modulated by (Long et al.2001). However, whether the expression of VEGF, the receptor subtype (ERa or ERb), the relative expression of which is a key factor in tumor angiogenesis, is modulated by each receptor in a target tissue, and the promoter context of xenoestrogens in breast cancer is not known. Therefore, we estrogen-regulated genes (Carson-Jurica et al. 1990, Kuiper et al. hypothesized that xenoestrogens could modulate VEGF 1997, Paech et al.1997). The transcriptional modulation arises expression in estrogen-dependent breast cancer cells. from an interplay of interactions between co-activators and Inthepresentstudy,we tested several known and potential ER co-repressors that determine the specificityof hormone action in agonists (xenoestrogens) for their ability to induce VEGF the different tissues, a feature particularly highlighted by the expression in different breast epithelial cells. We used several puzzling pharmacology of selective ER modulators (SERMs), cellular models: i) MCF-7 cells; ii) MELN derived from MCF-7 acting as receptor agonist or antagonist depending on the target cells (Balaguer et al. 2001), which express endogenous ERa;and organ. Whether xenoestrogens, which can accumulate in iii) MELP derived from MDA-MB-231 cells, which stably adipose tissue, may play a role in the incidence or progression express ERa. Both MELN and MELP express ERE-coupled of breast tumors, is largely unknown. A few epidemiological luciferase gene, allowing us to combine the analysis of studies have reported a correlation between exposure to some endogenous VEGF expression and the induction of the ERE xenobiotics and increased incidence of tumors, including breast reporter gene by different compounds in the same cells and cancers (Hoyer et al. 1998, Mills 1998, Sasco 2001, Hope- between cells. We analyzed the effects of the phytoestrogen nhayn-Rich et al. 2002, Birnbaum & Fenton 2003, Mitra et al. genistein; BPA; 4-(tert-octyl)phenol (OP); several pesticides 2004). Prenatal exposure to endocrine disruptors, including including dieldrin, g-hexachlorocyclohexane (gHCH), vinclo- BPA, atrazine, and 2,3,7,8-tetrachlorodibenzo-p-dioxin zolin, and the herbicide atrazine; and several phthalates including (TCDD), has been shown to alter the development of the benzyl butyl phthalate (BBP), mono-n-butyl ester phthalic acid rodent mammary gland, and the susceptibility to further (MBuP), di-ethyl-2-hexyle phthalate (DEHP), di-isononyle carcinogenic exposure in adulthood (Brown et al. 1998, Markey phthalate (DINP), and di-isodecyle phthalate (DIDP). We et al.2001, Birnbaum & Fenton 2003, Fukamachi et al.2004). analyzed some of the mechanisms of VEGF regulation, such as The complexity of xenoestrogen action in vitro, and in several ER-dependent activation pathway and/or activation of kinases. physiopathological conditions, has not been well understood, especially due to the limited use and knowledge of estrogen- responsive genes. In vitro investigations of the molecular Materials and Methods mechanism of xenoestrogen effects have been focused on the proliferation test of ERa-expressing MCF-7 cells, binding Hormones and chemicals assays with ERs (a and b), reporter gene assays using ERE (Soto et al.1995, Kuiper et al.1997, Andersen et al.1999, Legler et al. E2, genistein (Gen), BPA (purity 99%), OP (purity 97%), 1999, Balaguer et al.2001, Diel et al.2002, Olsen et al.2003), or a lindane (1,2,3,4,5,6 gHCH, purity 97%), dieldrin (purity few estrogen target gene promoters (Hall & Korach 2002). 94%), vinclozolin (VIN, purity 99.6%), and atrazine (ATZ, Recently,a DNA microarray was also developed to evaluate the purity 99.2%) were purchased from Sigma or Sigma–Aldrich estrogenic activity of industrial chemicals (Terasaka et al.2004). (GmbH, D-30918). MBuP (purity 98%) was purchased from The identification of estrogen target genes is clearly needed to Interchim (Tokyo Chemical