ATX-signaling in MCF-7 cells

Toxicology Letters 184 (2009) 26–32

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TCDD induces cell migration via NFATc1/ATX-signaling in MCF-7 cells夽 Anja Seifert a,∗ , Steffi Rau b , Gerhard Küllertz b , Bernd Fischer a , Anne Navarrete Santos a a b

Department of Anatomy and Cell Biology, Martin Luther University Faculty of Medicine, Grosse Steinstrasse 52, D-06097 Halle (Saale), Germany Research Unit for Enzymology of Protein Folding, Max Planck Society, Weinbergweg 22, D-06120 Halle (Saale), Germany

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Article history: Received 2 July 2008 Received in revised form 10 October 2008 Accepted 16 October 2008 Available online 6 November 2008 Keywords: NFAT autotaxin MCF-7 AhR HIF-1alpha

a b s t r a c t Breast cancer is characterized, among others, by the concurrence of lipophilic xenobiotica such as 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) with hypoxic tissue conditions. This condition activates the transcription factors hypoxia inducible factor-1alpha (HIF-1alpha) and aryl hydrocarbon receptor (AhR) that are known to promote tumor progression. An interrelation between these transcription factors and nuclear factor of activated T-cells (NFAT) was implied by gene array analysis. In the present study, the interplay of the three transcription factors was studied and correlated with the migration of MCF-7 cells in response to TCDD and/or hypoxia. An AhR-activation by 10 nM TCDD and HIF-1alpha activation by 5% oxygen induced activation of NFATc1. The effects were inhibited by cyclosporine A (CsA), suggesting that the activation of NFAT by AhR or HIF-1alpha signaling is calcineurin-dependent. The expression/activity of the NFAT target gene autotaxin (ATX) was increased. ATX is known to stimulate migration of tumor cells. The hydrolysis product of ATX, lysophosphatidic acid (LPA), increased the migration of MCF-7 cells under normoxia but not under hypoxia. This effect correlated with increased migration observed after TCDD treatment. Hypoxia did not promote migration of MCF-7 cells, suggesting that ATX down-stream signaling was inhibited by hypoxia. In conclusion, the TCDD-mediated activation of NFATc1 is suggested to promote cell migration via ATX/LPA-signaling. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Epidemiological studies have revealed a positive correlation between dioxin exposure and defects in immune, neurological and reproductive functions and in cancer incidence (Mandal, 2005). While an early report after the Seveso incident failed to see any increase in breast cancer incidence (Bertazzi et al., 1997), later studies showed that the serum 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) levels were correlated with an increased risk of all cancers (including breast cancer) after the accident (Mandal, 2005). A lower breast cancer incidence, however, was mentioned among women exposed to dioxin from municipal solid waste incinerators (Viel et al., 2008). The lipophilic TCDD accumulates in adipocyte

Abbreviations: TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; AhR, aryl hydrocarbon receptor; HIF-1alpha, hypoxia-inducible factor-1alpha; ANF, alphanaphthoflavone; ARNT, aryl hydrocarbon receptor nuclear translocator; VEGF, vascular endothelial growth factor; Cyp1A1, cytochrome 1A1; CsA, cyclosporine A; NFAT, nuclear factor of activated T-cells; ATX, autotaxin; DMSO, dimethylsulfoxid; LPC, lysophosphatidyl choline; LPA, lysophosphatidic acid. 夽 This work was supported by DFG GK 416/3 and the Wilhelm Roux Programme of the Martin Luther University Faculty of Medicine. ∗ Corresponding author. Tel.: +49 345 5571701; fax: +49 345 5571700. E-mail address: [email protected] (A. Seifert). 0378-4274/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2008.10.026

tissue in the mammary fat pad (Abraham et al., 1996). Effects of TCDD are mediated by the transcription factor aryl hydrocarbon receptor (AhR). The activated AhR binds, dimerized with aryl hydrocarbon receptor nuclear translocator (ARNT), to promoter regions of AhR target genes such as Cyp1 family members and several drug-metabolizing enzymes (Kewley et al., 2004). The tissue conditions in breast cancer are characterized by hypoxia, mainly caused by rapid growth and insufficient vascularization (Zhou et al., 2006). Hypoxia leads to stabilization of the hypoxia inducible factor-1alpha (HIF-1alpha) and subsequent signaling after nuclear translocation and ARNT dimerization. AhR and HIF-1alpha are members of the helix-loop-helix (bHLH)/Per-ARNT-SIM (PAS) transcription factor family (Kewley et al., 2004). We have recently described the interference of HIF-1alpha and AhR signaling in MCF-7 cells after exposure to TCDD and hypoxia. Mild hypoxia (5% oxygen) stabilizes the transcription factor HIF1alpha and induces target gene expression. TCDD clearly reduced the hypoxia-induced stabilization and activation of HIF-1alpha in an AhR-dependent manner. We could not find an influence of hypoxia on TCDD-mediated activation of AhR signaling in MCF-7 cells (Seifert et al., 2008). The mechanism of AhR- and HIF-1alpha-mediated tumor progression is still poorly understood. Evidence for tumor promotion and progression by AhR activation has been shown previously. Well

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known are TCDD-mediated effects on cell cycle (Marlowe et al., 2004). Depletion of E2F by TCDD prevents Rb phosphorylation and inhibits cell cycle progression. Furthermore, reduced cell proliferation and hypoxia-induced G1 phase arrest in human cancer cells (Box and Demetrick, 2004). AhR could play a role in cell migration and angiogenesis. In a wound healing approach constitutive expression and activation of AhR increased cell motility (Diry et al., 2006). Mulero-Navarro and coauthors (Mulero-Navarro et al., 2005) showed a lower migration in collagen-I and decreased lamellipodia formation in immortalized mouse mammary fibroblasts lacking AhR. Also a down-regulation of the VEGF receptor was observed. Vascular endothelial growth factor (VEGF) regulates cell migration and blood vessel formation. Ablation of AHR in mice resulted in enhancement of ischemia-induced angiogenesis that was attributable in part to the associated enhancement of VEGF expression (Ichihara et al., 2007). In addition, TCDD increased the migration associated with an increased protein expression of matrix metalloproteinases in U937 macrophages (Vogel et al., 2004). The NFAT family comprises four classical members, the transcription factors NFAT1-4. They are regulated by the calcium/calcineurin signaling pathway. The calcium-regulated phosphatase calcineurin directly dephosphorylates NFAT proteins, leading to rapid translocation, binding to its target sequence and initiation of transcription. Nuclear localization is counteracted by NFAT kinase(s) which rephosphorylate NFAT following cytoplasmatic export (Rao et al., 1997). NFATs are expressed in the immune system (Graef et al., 2001), in heart (de la Pompa et al., 1998), brain (Plyte et al., 2001) and muscle tissue (Boss et al., 1998). In all examined cell types except immune cells, NFAT regulates the expression of genes related to cell cycle progression, angiogenesis, tumorigenesis, and cell differentiation (Baksh et al., 2002; Graef et al., 2003; Neal and Clipstone, 2003; Zaichuk et al., 2004). The significance of the NFAT signaling pathway in cancer progression has previously been shown. Yiu and Toker (2006) described that NFAT promoted breast cancer cell invasion by induction of cyclooxygenase-2. Also alpha6beta4-integrin signaling targeted NFAT, inducing the NFAT target gene autotaxin (ATX) and followed by breast carcinoma invasion (Jauliac et al., 2002; Chen and O’Connor, 2005). Trying to mimic a pathophysiological condition as close as possible, the breast carcinoma cell line MCF-7 was exposed to TCDD and hypoxia. A gene expression analysis previously performed on MCF-7 cells in our laboratory disclosed a TCDD- and hypoxiadependent regulation of NFAT. An activation of NFAT signaling by AHR and HIF-1alpha is so far unknown. Therefore we analyzed the activation of NFAT, the expression and activity of the NFAT target gene ATX in response to treatment with TCDD and/or hypoxia. Because of the known cell migration-promoting effect of ATX we analyzed cell motility after exposure to TCDD and/or hypoxia in the human breast cancer cell line MCF-7. TCDD-mediated activation of NFATc1 is suggested to promote cell migration of breast cancer cells via ATX/lysophosphatidic acid (LPA)-signaling. Hypoxia inhibited TCDD-induced NFAT signaling and cell migration of MCF-7 cells.

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to attach for 24 h in a humidified atmosphere with 20% O2 , 5% CO2 at 37 ◦ C (normoxic condition). They were treated with 10 nM TCDD. This concentration had been selected in previous study (Seifert et al., 2008). For double exposure (TCDD and hypoxia) the TCDD-treated cells were cultured under hypoxic conditions. For hypoxic conditions, media were equilibrated for 6 h under hypoxic conditions. The cells were sealed in a hypoxic chamber equilibrated with 5% O2 , 5% CO2 and 37 ◦ C and fed with the equilibrated medium. The final concentration of 0.001% DMSO, employed in all TCDD and inhibitor studies, was used as vehicle control. For inhibition of the AhR or NFAT, the specific inhibitors ANF (10 ␮M) or CsA (1 ␮M) were used. MCF-7 cells were incubated for 4 h (ANF) or 1 h (CsA) prior to stimulation with TCDD and/or hypoxia. 2.3. RNA preparation and real time RT-PCR Isolation of total cellular RNA, synthesis of cDNA, Real-time PCR amplification and analysis was described previously (Seifert et al., 2008). The primers used were as follows: human ATX (NCBI, acc. NM 006209): forward 5 -GTTGCAAGGAAACCTTTGGA-3 and reverse 5 -AACTTCCTCTGGCATGGTTG-3 ; ribosomal 18S (NCBI, acc. X00686): forward 5 -GTTGGTGGAGCGATTTGTCTGG-3 and reverse 5 -AGGGCAGGGACTTAATCAACGC-3 . Dilution series of the corresponding standard were used to obtain standard curves for ATX amplification (Kehlen et al., 2004). 2.4. Immunoblotting The nuclear protein fractions of stimulated MCF-7 cells were isolated using the Nuclear Extract Kit (Active Motif, Rixensart, Belgium) according to the manufacturer’s instructions. Proteins were separated in 12% SDS polyacrylamide gels, transferred onto nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany). The membranes were blocked in 5% non-fat dry milk/TBS and probed against NFATc1 (1:200, Santa Cruz Biotechnology). The antigen–antibody complexes were detected using a chemiluminescence kit (Amersham Biosciences). Signals were analyzed with computer-assisted densitometry (SilverFast Mikrotek software). The protein amounts were evaluated by stripping the membranes. For this the blots were rinsed with 2% SDS, 60 mM Tris–HCl, pH 6–7, and 100 mM beta-mercaptoethanol at 50 ◦ C for 30 min and re-blotted with mouse monoclonal anti-actin antibody (1:5.000, Sigma–Aldrich, Taufkirchen, Germany). 2.5. Transient transfection methods MCF-7 (5 × 104 cells) were transfected with 30 ␮g of pEGFP/NFATc1 (generously provided by Dr. Erdmann, Research Unit for Enzymology of Protein Folding, Max Planck Society, Halle) using calcium phosphate method as described previously (Seifert et al., 2008). After overnight incubation cells were treated, as described above, for 2–3 h. Thereafter cells were washed twice with PBS and stained with 7-amino-actinomycin D for 20 min. For reporter gene experiments MCF-7 (5 × 104 cells) were cotransfected with 1.5 ␮g of pTal-luc/NFAT (Stratagene, Amsterdam) and pRLSV40 (Promega). Transfection and measurement of luciferase activity using the Dual luciferase kit (Promega) was described previously (Seifert et al., 2008). 2.6. Lyso-PLD enzyme assay Quantification of the Lyso-PLD activity has been described previously (Kehlen et al., 2004). Cell culture supernatants were incubated for 12 h with 1 mM lysophosphatidyl choline (LPC; Sigma) in 50 mM Tris–HCl at pH 9.0, 5 mM MgCl2 , 5 mM CaCl2 . Liberated choline was detected by an enzymatic colorimetric method using 0.8 U choline oxidase, 1 U peroxidase and 0.05 M aminoantipyrene and 0.21 M phenol (all from Sigma) in a reaction volume of 100 ␮l. Absorbance was read at 500 nm after 30 min at 37 ◦ C. 2.7. Transwell migration assay

The chemicals used were obtained from the following companies: 2,3,7,8TCDD (>99% pure, Amchro, Hattersheim, Germany); alpha-naphthoflavone (ANF), dimethylsulfoxid (DMSO) by Sigma (Taufkirchen, Germany). TCDD and ANF were dissolved in DMSO. Cyclosporin A (CsA), purchased from Sigma, was solved in 50% ethanol.

Cell migration was analyzed using Transwell chamber membrane filter inserts in 12-well tissue culture plates (Corning Costar, Schiphol-Rijk, Netherland). The upper chamber (filter insert) was filled with 600 ␮l of cell suspension (2 × 105 cells) in medium with 10 nM TCDD or vehicle control, while the lower chamber contained 1200 ␮l of the same cell-free medium with 1.3 ng/ml collagen type I (BD Bioscience, Heidelberg, Germany). Plates were cultured under normoxic or hypoxic conditions. After incubating for 24 h, non-migrating cells were removed with a flattened swab from the upper surface of the membrane. Cells on the underside of the membrane were counted with the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Mannheim, Germany) according to the manufacturer’s instructions. The results were normalized by a seed control of 2 × 105 cells growing in a 12-well tissue culture plate.

2.2. Cell line, cell exposure

2.8. Statistical analysis

The ER-positive human MCF-7 cell line was obtained from German Resource Centre for Biological Material (Braunschweig, Germany). Culture of MCF-7 cells was performed as described before (Seifert et al., 2008). Cells were plated and allowed

The results were compared with ANOVA for all pairwise multiple comparisons and with the Student’s t-test. Statistically significant differences were set at p-value <0.05 (*) and <0.001 (**).

2. Materials and methods 2.1. Chemicals

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3. Results 3.1. Effects of TCDD or hypoxia exposure on NFATc1 translocation Upon activation by TCDD or hypoxia NFAT translocates into the nucleus. In normoxic controls the green fluorescence of the GFPtagged NFAT protein was localized in the cytoplasm of MCF-7 cells. Surprisingly, no translocation of NFAT was observed after simultaneous exposure to TCDD and hypoxia. ANF treatment inhibited the TCDD-mediated translocation and CsA abolished the translocation of NFATc1 in both TCDD or hypoxia exposed MCF-7 cells (Fig. 1). Western blot analysis of nuclear cell fractions showed several NFATc1 bands due to multiple dephosphorylation sites of NFAT by calcineurin. Exposure to TCDD or hypoxia but not simultaneous exposure increased NFATc1 protein in the nuclear fraction. Inhibition with CsA prevented TCDD- or hypoxia-induced increase in NFATc1 protein in the nuclear fraction of MCF-7 cells (Fig. 2A–C). 3.2. Regulation of NFAT mediated target gene expression To test whether the observed changes in NFATc1 translocation does influence the NFAT-mediated gene expression; the activation of NFAT-regulated luciferase reporter was measured. Reporter gene activity was up-regulated to approximately 140% by TCDD treatment. Additional ANF treatment inhibited this induction. Hypoxia alone increased NFAT-mediated luciferase activity to 150%, whereas simultaneous exposure of TCDD and hypoxia did not change

the reporter gene activity. Again, NFAT-mediated reporter gene activity was prevented by CsA under single exposure conditions (Fig. 2D). Exposure to TCDD or hypoxia induced ATX transcription whereas simultaneous exposure to TCDD and hypoxia did not significantly change the ATX mRNA amount (Fig. 3A). ATX activity was measured by absolute turnover of choline. Choline turnover, caused by ATX lysophospholipase D activity, was induced by TCDD treatment or hypoxia. Simultaneous exposure of TCDD and hypoxia reduced the induction of ATX activity. Treatment of ANF inhibited TCDD-mediated effects. ANF treatment prevented the reduction under simultaneous exposure to TCDD and hypoxia. CsA did not significantly change the ATX lysophospholipase D activity compared to normoxic or hypoxic control (Fig. 3B). 3.3. Effects of TCDD and hypoxia on cell migration of MCF-7 cells TCDD-treated cells showed a 70% increase in migration compared to non-treated and vehicle controls. Hypoxic cells did not exhibit any increase in motility, neither under hypoxia alone nor after simultaneous exposure to TCDD and hypoxia. CsA significantly changed the cell migration of MCF-7 cells. The CsA-treated vehicle controls cultured under normoxia or hypoxia were decreased in migration down to 70 or 50% (Fig. 4A). Finally, we measured the cell migration of MCF-7 cells after treatment with the ATX substrate LPC and the ATX enzyme product LPA to determine ATX caused effects. Cell migration was specifically induced by LPA but not by LPC under normoxic conditions (Fig. 4B). Under hypoxic condition,

Fig. 1. Localization of NFATc1 in MCF-7 cells. Immunofluorescence of NFATc1-GFP fusion protein. TCDD-treated MCF-7 cells were cultured in 5% and 20% oxygen, respectively, with or without addition of the inhibitors ANF or CsA. Nuclei were stained with 7-amino-actinomycin (7-AAD).

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Fig. 2. TCDD and hypoxia influence nuclear localization of NFATc1 (A–C) and NFAT-mediated promoter activity (D) in MCF-7 cells. The NFATc1 protein amount was shown by western blot in cells exposed to 10 nM TCDD under hypoxia and/or normoxia for 6 h. NFATc1 protein in TCDD or hypoxia exposed cells (A), in TCDD and hypoxia exposed cells (B) and in DMSO-treated cells (C) are shown. For inhibition of NFAT 1 ␮M CsA was used. The NFATc1 protein was normalized to actin. Data are represent results from at least two separate experiments and are shown as mean ± S.E.M. of three experiments (*p < 0.05 compared to non-treated normoxic control) (D) Cells were transiently transfected with a NFAT reporter gene construct and incubated for 24 h. Firefly luciferase activity was normalized to renilla, providing an internal control. Results are expressed in percent of the untreated control. Data are representative for comparable results from at least three separate experiments and are shown as mean ± S.E.M. of three experiments (*p < 0.05 compared to non-treated normoxic control).

however, neither LPA nor LPC could increase cell migration, suggesting that other processes downstream of the LPA prevents ATX mediated induction under hypoxic conditions.

4. Discussion The aim of our study was to show the effects of TCDD on NFAT signaling under different oxygen conditions. Recently, we showed that mild hypoxia (5% oxygen) – typical for breast carcinoma (stage T1B to T4) (Vaupel et al., 2002) – stabilizes the transcription factor HIF-1alpha and induces expression of its target genes (Seifert et al., 2008). The regulation of transcription factor NFAT by TCDD and/or hypoxia was implied by gene array analysis (data not shown). Consequences of NFAT regulation after exposure to TCDD and/or

hypoxia were analyzed by measurement of ATX expression/activity (NFAT target gene). ATX was described to stimulate random and directed motility in different cell lines derived from breast cancer, renal tumor, thyroid carcinomas, melanomas, and neuroblastomas (Kehlen et al., 2004). Therefore, we analyzed cell motility after exposure to TCDD and/or hypoxia in the human breast cancer cell line MCF-7. NFATc1 was activated after exposure to TCDD or hypoxia in MCF-7 cells. NFAT signaling is closely linked with calcineurin. Calcineurin is a Ca2+ /calmodulin dependent serine/threonine protein phosphatase that is an inherent part of numerous cellular processes (Blumenthal et al., 1986; Dawson et al., 1993; Sugimoto et al., 1997; Rusnak and Mertz, 2000; Lynch et al., 2005). Inhibition of the phosphatase activity of calcineurin by cyclosporine A (Huai et al., 2002; Jin and Harrison, 2002) prevents nuclear translocation

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Fig. 3. TCDD and hypoxia influence ATX mRNA amount (A) and choline turnover by autotaxin (B) in MCF-7 cells. (A) Absolute autotaxin mRNA amount was measured using quantitative RT-PCR. (B) Lysophospholipase D activity of TCDD and/or hypoxia-treated MCF-7 cells. Data are representative for comparable results from at least three separate experiments and are shown as mean ± S.E.M. of three experiments (*p < 0.05 compared to non-treated normoxic control).

Fig. 4. TCDD and hypoxia influence motility of MCF-7 cells. Cell migration assay was performed using transwell migration assays. The percent of migrated cells is shown related to untreated control. In (A) cells were treated with 10 nM TCDD and/or hypoxia. In (B) cells were stimulated with the ATX substrate LPC (10 ␮M) or the ATX product LPA (10 ␮M) under normoxic and hypoxic conditions. The values shown represent the mean ± S.E.M. of three independent experiments (*p < 0.05 compared to non-treated normoxic control).

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of NFAT (Rao et al., 1997). Effects of AhR-activation on NFAT signaling are unknown yet. Hypoxia and TCDD mediate an increase in intracellular Ca2+ (Puga et al., 1997; Toescu, 2004), suggest that the induction of NFAT signaling is caused by calcium-dependent activation of calcineurin. In this study we show that the expression and enzyme activity of the NFAT target gene ATX was induced after exposure to TCDD or hypoxia. The TCDD- or hypoxia-mediated induction of ATX via NFAT was unknown so far. Overexpression of ATX has previously been reported in melanoma, neuroblastoma, hepatoma, lung carcinoma, renal-cell carcinoma and breast carcinoma (Kehlen et al., 2004). It has been described that ATX is regulated by v-Jun (Black et al., 2004), by TNFalpha (Boucher et al., 2005) and alpha6beta4integrin (Chen and O’Connor, 2005). In addition, ATX has lyso-PLD activity, hydrolyzing lysophosphatidyl choline (LPC) and several other glycerophospholipids to LPA (Clair et al., 1997; Aoki et al., 2002; Tokumura et al., 2002). The product LPA of the ATX-mediated hydrolysis are known to stimulate random and directed motility (Kehlen et al., 2004). ATX enhance the tumorigenic capacity and metastatic potential of ras-transformed cells and seems to be an angiogenic factor (Nam et al., 2000, 2001). TCDD but not hypoxia increased NFAT-dependent cell migration of MCF-7 cells whereas NFAT signaling and ATX expression/activity were increased under both conditions. An AhR-dependent increase in cell migration after treatment with TCDD had previously been observed (Diry et al., 2006). Furthermore a lower migration combined with reduced response to angiogenic factors have been shown in immortalized mouse mammary fibroblasts lacking AhR (Mulero-Navarro et al., 2005). Our results involving inhibition with CsA suppose that a NFAT/ATX-dependent regulation is involved in this process. Furthermore, under normoxic conditions LPA is able to bind to the endothelial differentiation gene (EDG) receptors (Lynch, 2002) activating GTPases of the Rho family (rac1, cdc42 and rho). Downstream of the GTPases the focal adhesion kinase (FAK) is phosphorylated and the cytoskeleton is reorganized (Swarthout and Walling, 2000; Jung et al., 2002). We show that LPA responsiveness of motile MCF-7 cells was abolished under hypoxia. Corley and coauthors (Corley et al., 2005) describe a decreased phosphorylation of FAK and migration under hypoxia in vascular smooth muscle cells. It was also shown that ablation of AHR in mice resulted in enhancement of ischemia-induced angiogenesis that was attributable in part to the associated enhancement of VEGF expression (Ichihara et al., 2007). In line with these results, the increased expression of ATX and production of LPA in MCF7 cells were not able to induce migration due to blockage of the downstream target FAK under hypoxia. The response of CsA on cell migration could be explained by complexes that were formed with intracellular immunophilins modulating the activity of crucial signal transduction molecules within the cell (Walsh et al., 1992). Activation of both the AhR and HIF-1alpha pathway prevents the induction of the NFAT signal pathway and motility in MCF-7 cells. Most likely, the dephosphorylation of NFAT by calcineurin, regulated by single activation of AhR or HIF-1alpha signaling, was prevented after simultaneous activation of the AhR and HIF-1alpha pathway. In contrast to the extensively studied role of AhR activation for cancer initiation and promotion, up to now its role in tumor progression is only poorly understood. Our results indicate a regulatory function of the AhR in tumor progression. It has been shown that tobacco smoke induces activation of AhR (Port et al., 2004; Kitamura and Kasai, 2007; Baglole et al., 2008). Terry and Goodman (2006) observed a significantly positive associations between long smoking and breast cancer risk. In contrast to dioxin, which accumulates in adipose tissues and is resistant to AhR

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induced metabolism, AhR ligands in tobacco smoke are metabolized. Thus, exposure to dioxin or tobacco smoke represents two events of long term AhR activation. The significance of simultaneous activation of AhR and HIF-1alpha has not been investigated to date. Therefore, the interplay of angiogenic factors like ATX and VEGF activated by AhR or HIF-1alpha needs further investigation in view of tumor progression and survival. Taken together TCDD or hypoxia activates NFATc1 calcineurindependent followed by induction of ATX. Under normoxic conditions the TCDD-mediated induction of cell migration could be explained by activation of NFAT/ATX and the following production of LPA. Furthermore, the calcineurin-dependent action of TCDD apparently implied the connection of TCDD- and CsA-mediated effects on immunological response. Conflict of interest statement There are no conflicts of interest. References Abraham, K., Knoll, A., Ende, M., Papke, O., Helge, H., 1996. Intake, fecal excretion, and body burden of polychlorinated dibenzo-p-dioxins and dibenzofurans in breast-fed and formula-fed infants. Pediatr Res. 40, 671–679. Aoki, J., Taira, A., Takanezawa, Y., Kishi, Y., Hama, K., Kishimoto, T., Mizuno, K., Saku, K., Taguchi, R., Arai, H., 2002. Serum lysophosphatidic acid is produced through diverse phospholipase pathways. J. Biol. Chem. 277, 48737–48744. Baglole, C.J., Maggirwar, S.B., Gasiewicz, T.A., Thatcher, T.H., Phipps, R.P., Sime, P.J., 2008. The aryl hydrocarbon receptor attenuates tobacco smoke induced cyclooxygenase-2 and prostaglandin production in lung fibroblasts through regulation of the NF-kappa B family member RELB. J. Biol. Chem.. Baksh, S., Widlund, H.R., Frazer-Abel, A.A., Du, J., Fosmire, S., Fisher, D.E., DeCaprio, J.A., Modiano, J.F., Burakoff, S.J., 2002. NFATc2-mediated repression of cyclindependent kinase 4 expression. Mol. Cell 10, 1071–1081. Bertazzi, P.A., Zocchetti, C., Guercilena, S., Consonni, D., Tironi, A., Landi, M.T., Pesatori, A.C., 1997. Dioxin exposure and cancer risk: a 15-year mortality study after the “Seveso accident”. Epidemiology 8, 646–652. Black, E.J., Clair, T., Delrow, J., Neiman, P., Gillespie, D.A., 2004. Microarray analysis identifies autotaxin, a tumour cell motility and angiogenic factor with lysophospholipase D activity, as a specific target of cell transformation by v-Jun. Oncogene 23, 2357–2366. Blumenthal, D.K., Takio, K., Hansen, R.S., Krebs, E.G., 1986. Dephosphorylation of cAMP-dependent protein kinase regulatory subunit (type II) by calmodulindependent protein phosphatase. Determinants of substrate specificity. J. Biol. Chem. 261, 8140–8145. Boss, V., Abbott, K.L., Wang, X.F., Pavlath, G.K., Murphy, T.J., 1998. The cyclosporin A-sensitive nuclear factor of activated T cells (NFAT) proteins are expressed in vascular smooth muscle cells. Differential localization of NFAT isoforms and induction of NFAT-mediated transcription by phospholipase C-coupled cell surface receptors. J. Biol. Chem. 273, 19664–19671. Boucher, J., Quilliot, D., Praderes, J.P., Simon, M.F., Gres, S., Guigne, C., Prevot, D., Ferry, G., Boutin, J.A., Carpene, C., Valet, P., Saulnier-Blache, J.S., 2005. Potential involvement of adipocyte insulin resistance in obesity-associated up-regulation of adipocyte lysophospholipase D/autotaxin expression. Diabetologia 48, 569–577. Box, A.H., Demetrick, D.J., 2004. Cell cycle kinase inhibitor expression and hypoxiainduced cell cycle arrest in human cancer cell lines. Carcinogenesis 25, 2325–2335. Chen, M., O’Connor, K.L., 2005. Integrin alpha6beta4 promotes expression of autotaxin/ENPP2 autocrine motility factor in breast carcinoma cells. Oncogene 24, 5125–5130. Clair, T., Lee, H.Y., Liotta, L.A., Stracke, M.L., 1997. Autotaxin is an exoenzyme possessing 5 -nucleotide phosphodiesterase/ATP pyrophosphatase and ATPase activities. J. Biol. Chem. 272, 996–1001. Corley, K.M., Taylor, C.J., Lilly, B., 2005. Hypoxia-inducible factor 1alpha modulates adhesion, migration, and FAK phosphorylation in vascular smooth muscle cells. J. Cell Biochem. 96, 971–985. Dawson, T.M., Steiner, J.P., Dawson, V.L., Dinerman, J.L., Uhl, G.R., Snyder, S.H., 1993. Immunosuppressant FK506 enhances phosphorylation of nitric oxide synthase and protects against glutamate neurotoxicity. Proc. Natl. Acad. Sci. U.S.A. 90, 9808–9812. de la Pompa, J.L., Timmerman, L.A., Takimoto, H., Yoshida, H., Elia, A.J., Samper, E., Potter, J., Wakeham, A., Marengere, L., Langille, B.L., Crabtree, G.R., Mak, T.W., 1998. Role of the NF-ATc transcription factor in morphogenesis of cardiac valves and septum. Nature 392, 182–186. Diry, M., Tomkiewicz, C., Koehle, C., Coumoul, X., Bock, K.W., Barouki, R., Transy, C., 2006. Activation of the dioxin/aryl hydrocarbon receptor (AhR) modulates cell plasticity through a JNK-dependent mechanism. Oncogene.

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