Oncogene (2013) 32, 1811–1820 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc

ORIGINAL ARTICLE The aryl hydrocarbon receptor regulates sites through a non-genomic FAK/Src pathway

C Tomkiewicz1,2, L Herry1,2,3, L-C Bui1,2,CMe´ tayer1,2, M Bourdeloux1,2, R Barouki1,2,3 and X Coumoul1,2

The aryl hydrocarbon receptor (AhR) is commonly described as a , which regulates xenobiotic-metabolizing enzymes. Recent studies have suggested that the binding of ligands to the AhR also activates the Src kinase. In this manuscript, we show that the AhR, through the activation of Src, activates focal adhesion kinase (FAK) and promotes clustering. These effects contribute to cell migration. Further, we show that the activation of the AhR increases the interaction of FAK with the metastatic marker, HEF1/NEDD9/CAS-L, and the expression of several . Xenobiotic exposure, thus, may contribute to novel cell-migratory programs.

Oncogene (2013) 32, 1811–1820; doi:10.1038/onc.2012.197; published online 4 June 2012 Keywords: AhR; cell plasticity; FAK; integrin; Src

INTRODUCTION Ultimately, the of FAK leads to the dissociation of The aryl hydrocarbon receptor (AhR) is a xenobiotic-activated the FAS and cell migration. transcription factor involved in the detoxication pathways.1 It TCDD (2,3,7,8-p-Tetrachlorodibenzo-p-dioxin) is one of the most belongs to the basic helix loop helix/Per AhR nuclear translocator potent ligands of the AhR. Genome-wide transcriptome analyses that Sim family. Pollutant ligands of the AhR include dioxins, explore the effects of TCDD on expression have shown that furans, polychlorinated biphenlys and polycyclic aromatic several other biological pathways are regulated in addition to 23–25 hydrocarbons.2,3 Ubiquitous in mammals, the AhR forms a xenobiotic metabolism. We have shown that TCDD induces the 13,15,26,27 cytoplasmic complex with heat shock . Upon ligand expression of the Hef1/Cas-L/Nedd9, Agr2 and Sos1 , binding, the AhR translocates into the nucleus where it interacts genes that are involved in the ‘epithelial mesenchymal transition’ with AhR nuclear translocator. The AhR/AhR nuclear translocator pathway and survival and metastasis. The increase in the cellular heterodimer recognizes xenobiotic-responsive elements in the mobility and morphological changes observed during the epithelial promoters of target genes and controls their expression.4 The mesenchymal transition elicited following 24–72 h of treatment with AhR signaling pathway is best known for the transcriptional TCDD and were found to depend upon the induction of the Hef1 13,28 regulation of xenobiotic-metabolizing enzymes, which are gene. However, in the present study, we found that subtle involved in the metabolism of drugs and pollutants. This elegant morphological changes in the FASs were observed after only 1–4 h of adaptive pathway allows the coordinate detection and elimination treatment with TCDD, well before any significant increase in the HEF1 of pollutants and, thus, protects organisms against foreign occurred. We, therefore, hypothesized that those rapid chemicals.4 effects, which require neither transcription nor translation, could be Recently, however, other genes and alternative pathways have at the origin of the initial changes in the cell morphology. Further, we been identified as AhR targets.1,5 Studies with gene-knockout suspected that these effects could be synergistic with the gene- models (in both vertebrates and invertebrates) have suggested regulatory effects of late onset to yield, together, the full spectrum of that the AhR has other functions including the regulation of cell morphological and migratory changes triggered by TCDD. migration during development.6–12 Several groups, including our Circumstantial support for this hypothesis comes from the own, have shown that, upon xenobiotic binding, the AhR observations (including a recent elegant study using fluorescence stimulates the migration and the invasion of several types of resonance energy transfer (FRET) technology) that the AhR, in the cells.6,10,13–17 cytoplasm, is bound to several chaperone proteins and to the Src 29–31 Cellular migration is a consequence, in part, of a redistribu- kinase. The binding of xenobiotics to the AhR not only initiates tion of the focal adhesion sites (FASs), which are composed of theeffectsoftheAhRongeneexpressionbutalso,perhapsas multiple transmembrane and cytoplasmic proteins (integrins, importantly, leads to the release of Src. This rapid liberation of Src, kinasesy).18,19 One component of the FASs, which has a crucial which is a non-transcriptional event, may lead to its activation and to role in cellular migration, is the focal adhesion kinase (FAK). FAK is the subsequent phosphorylation of Src partners, for example, those activated by autophosphorylation (tyrosine 397) following the at the FASs. In the present study, we show that the binding of clustering of integrins. This activation leads to the recruitment of xenobiotic to the AhR activates the redistribution of FAS and leads to SH2-domain-containing regulators, such as Src and other cell plasticity through non-transcriptional events and that these members of the SFK family (Src family of tyrosine kinases). events act in synergy to the transcriptional regulation of the major These kinases phosphorylate multiple other sites on FAK.20–22 genes involved in cell migration.

1INSERM UMR-S 747, Toxicologie Pharmacologie et Signalisation Cellulaire, Paris, France; 2Universite´ Paris Descartes, Sorbonne Paris Cite´, Centre Universitaire des Saints-Pe`res, Paris, France and 3AP-HP, Hoˆpital Necker Enfants Malades, Service de Biochimie Me´tabolique, Paris, France. Correspondence: Professor X Coumoul, Universite´ Paris Descartes, Sorbonne Paris Cite´, 45 rue des Saints-Pe`res, Paris 75006, France. E-mail: [email protected] Received 26 September 2011; revised 6 March 2012; accepted 30 March 2012; published online 4 June 2012 The AhR/Src pathway regulates integrin function C Tomkiewicz et al 1812 RESULTS AhR regulates early FAK and Src activation AhR activates early integrin clustering The FAK regulates FAS reorganization and integrin clustering.32 The reorganization of FAS in HepG2 cells treated with TCDD was We, therefore, hypothesized that FAK is an early target of TCDD. observed by immunostaining of paxillin (a component of FAS) and Examination of the activation of FAK and Src, as measured by their staining of actin. Morphological changes, which consisted of an profiles of phosphorylation, showed (Figure 2a) that the phos- increase in cell spreading, the formation of stress fibers, an phorylation of FAK on tyrosine 397 (Y397) is significantly increased extension of the FAS and the loss of cell contacts in the HepG2 after only 1 h of exposure of the cells to TCDD. This is an cells, occurred rapidly (4-8 h) following treatment with TCDD autophosphorylation, which creates a binding site for Src. Src, also, (Figure 1a). All of these changes depend upon the activation of is activated significantly after only 15 min of exposure of cells to the AhR because they were inhibited by the AhR antagonist, TCDD (Figure 2b), in agreement with previous studies.30,33–38 The a-naphthoflavone (aNF) (Figure 1b). Because the formation of new total amounts of FAK and Src remain constant during the course FAS during cell migration depends upon integrin clustering, we of those experiments. looked for changes in the distribution of integrins with specific Increased phosphorylation of tyrosines 861 and 925 in FAK also staining. After only 2–4 h of treatment with TCDD, clusters of was observed following treatment of the cells with TCDD. integrins associated with actin fibers were present at the cell However, the increase in the phosphorylation of Y925, which membrane (Figure 1c). This clustering was also blocked by aNF was significant after only 2 h of exposure of cells to TCDD, was still (Figure 1d). slower than that of tyrosine 397 and 861 (Figure 2a), as well as

Figure 1. TCDD/AhR activates early morphological changes, redistribution of FASs and triggers integrin b1 clustering in HepG2 cells. (a) HepG2 cells were treated for 4, 8 or 48 h with TCDD (25 nM) and stained with DAPI (blue, nucleus), phalloidin (green, actin) and immunostained to detect paxillin (red, FASs). Scale bar, 10 mm. (b) HepG2 cells were treated with TCDD (25 nM) and/or aNF (5 mM) for 24 h and immunostained to detect paxillin (white, FASs, upper part of the panel) stained with phalloidin (white, actin, middle part of the panel). The merged images are shown in the lower panel. Scale bar, 10 mm. (c) HepG2 cells were treated with TCDD (25 nM) for 30, 120 or 240 min and immunostained to detect integrin b1 (green, upper part of the panel) and stained with phalloidin (magenta, middle part of the panel). The merged images are shown in the lower panel. White arrows show integrin clusters linked by sub-membrane actin fibers. Scale bar, 10 mm. (d) HepG2 cells were treated with TCDD (25 nM) and/or aNF (5 mM) for 24 h and immunostained to detect integrin b1 (green, upper part of the panel) and stained with phalloidin (magenta, actin, middle part of the panel). The merged images are shown in the lower panel. White arrows show integrin clusters linked by sub-membrane actin fibers. Scale bar, 10 mm. A full colour version of this figure is available at the Oncogene journal online.

Oncogene (2013) 1811 – 1820 & 2013 Macmillan Publishers Limited The AhR/Src pathway regulates integrin function C Tomkiewicz et al 1813

Figure 2. TCDD triggers the early phosphorylation and activation of FAK/Src through an AhR-dependant pathway. (a and b) HepG2 cells were treated for increasing lengths of time (0,1 and 2 h) with TCDD (25 nM). Western blots were performed with cells extracts using (a) antibodies to: FAK total, FAK pY397, FAK pY861 or FAK pY925; (b) antibodies to: pan-Src or Src-pY416. The right panel shows the quantification of three independent experiments. (c) HepG2 cells were treated with TCDD (25 nM) and/or aNF (5 mM) for 24 h. Western blots were performed with cell extracts using antibodies to: FAK total, FAK pY397, FAK pY861 or FAK pY925. The right panel shows the quantification of three independent experiments. (d) HepG2 cells were treated with TCDD (25 nM) and/or aNF (5 mM) for 24 h. Western blots were performed with cell extracts using antibodies against pan-Src or Src-pY416. The right panel shows the quantification of three independent experiments. **Po0.01 and *Po0.05 as compared with controls. A full colour version of this figure is available at the Oncogene journal online.

that of Src (Figure 2b). The phosphorylation of Y925 has been (Figures 2c and d). We observed similar events following described as a marker of FAS turnover.39–41 Supplementary transfection of a small interfering RNA targeting the AhR and Figure 1 shows complete kinetics of FAK phosphorylation. Most treatment with TCDD (Supplementary figure 3). of those phosphorylation events appear early and remain unchanged following the 4-h treatment time point. Finally, we also showed that observations made in the HepG2 cells were Activation of FAK/Src by the AhR regulates cell plasticity also conserved (and even magnified in the human mammary We, next, examined the contribution of the activation of FAK and cell line, MCF-7. Supplementary figure 2 shows that the clustering Src to the reorganization of the FAS and the integrins. To this end, of the integrin b1, the spatial reorganization (as assessed by we transfected the cells with expression vectors that encode a paxillin staining) of FASs and the phosphorylation events on Src dominant negative fragment of FAK (FRNK42). We show that the and FAK are conserved in this cell line which also expresses expression of a dominant negative fragment of FAK sharply the AhR. reduces the number of cells with a morphology that is We next analyzed the contribution of the AhR to these characteristic of AhR activation after a long exposure course phosphorylation events by treating the cells concomitantly with (424 h) (Figure 3a). aNF. We observed a significant reduction, in the presence of aNF, We also inhibited the activity of FAK with a competitive of the phosphorylation of Src, FAK pY397, pY861 and pY925 antagonist (PF-228; Pfizer, Paris, France), which blocks the active

& 2013 Macmillan Publishers Limited Oncogene (2013) 1811 – 1820 The AhR/Src pathway regulates integrin function C Tomkiewicz et al 1814

Figure 3. Early activation of FAK/Src by TCDD is essential for cell plasticity and it triggers cell migration. (a) Quantification of fluorescent cells into normal or AhR activation-type morphologies. Cells were co-transfected with expression vectors encoding a dominant negative form of FAK(FRNK) or corresponding empty vector (pRKVSV) and expression vector enhanced green fluorescent protein (eGFP) for 48 h and treated 42 h with TCDD (25 nM). Cells were then fixed and quantified using a fluorescence microscope (see the image in the left part of the figure). A eGFP/(FRNK or pRKVSV) ratio of 1:10 was used to ensure that all the fluorescent cells expressed FRNK. (b) HepG2 cells were treated with TCDD (25 nM) and/or PF-228 (10 mM) for 24 h. Western blots were performed with cell extracts using antibodies to: FAK, FAK pY397, FAK pY861 or FAK pY925. The right panel shows the quantification of three independent experiments. (c and d) HepG2 cells were treated with TCDD (25 nM) and/or PF-228 (c,10mM) and/or PP2 (d,10mM) for 24 h and immunostained to detect paxillin (white, FASs, upper part of the panel) and stained with phalloidin (white, actin, middle part of the panel). The merged images are shown in the lower panel. Scale bar, 10 mm. (e) HepG2 cells were treated with TCDD (25 nM) and/or PP2 (10 mM) for 4 h and immunostained to detect integrin b1 (green, upper part of the panel) and stained with phalloidin (magenta, middle part of the panel). The merged images are shown in the lower panel. White arrows show integrin clusters linked by sub-membrane actin fibers. Scale bar, 10 mm. (f) A Boyden chamber assay was used to measure cell migration following treatment with TCDD (25 nM) and/or PF-228 (10 mM). The quantification of three independent experiments is shown. **Po0.01 and *Po0.05 as compared with controls. A full colour version of this figure is available at the Oncogene journal online.

Oncogene (2013) 1811 – 1820 & 2013 Macmillan Publishers Limited The AhR/Src pathway regulates integrin function C Tomkiewicz et al 1815 site of FAK. PF-228 decreased the phosphorylation of FAK at all morphology and the activation of both FAK and Src following three sites (Figure 3b) and decreased the extent of dissociation of exposure of cells to TCDD, we hypothesized that TCDD also may the cell contacts observed following exposure of cells to TCDD have effects on HEF1 that are independent of both transcription (Figure 3c). Because the phosphorylation of FAK at several sites and translation. We, therefore, investigated whether the treatment (and, therefore, the activity of FAK) depends upon the binding of a of cells with TCDD promotes the interaction between HEF1 and Src, which has a functional kinase activity, we blocked the activity FAK. Following treatment of cells with TCDD, FAK with HEF1 co- of Src with a pharmacological agent, PP2.29 PP2 eliminated both immunoprecipitate together (Figure 4a). In addition, treatment the morphological changes (Figure 3d) and the clustering of with TCDD increases the expression of HEF1 isoforms, p105 and integrins (Figure 3e) that are observed following exposure of cells p115 (the larger molecular weight form results from phosphoryla- to TCDD. tion of p105HEF1) (Figure 4b). Interestingly, inhibition of FAK by Finally, using Bowden chambers, we confirmed that TCDD PF-228 significantly and specifically reduced p115 (upper band in significantly increased HepG2 cell migration. This increase in cell Figure 4b). Moreover, PP2 eliminated the AhR-mediated increase migration was blocked completely by PF-228 (Figure 3f), which in phosphorylation of FAK and HEF1 (Figure 4c). As expected, demonstrates that the cell migration that is induced by TCDD the inhibition of Src abolished the decrease in expression of depends upon FAK activity. Thus, both FAK and Src, through the E-cadherin following treatment of cells with TCDD.13 Thus, in action of TCDD on the AhR, are involved in the early stimulation of addition to the transcription-based stimulation of expression of cell plasticity. HEF1, the treatment of cells with TCDD stimulates, via its effects on the AhR, the recruitment and phosphorylation of HEF1 on FAK through non-transcription-based events. This cascade of events is TCDD stimulates the recruitment of HEF1 on FAK critical for cell spreading. We previously demonstrated that HEF1 is a transcriptional target of the AhR.13,15 HEF1 is a member of the Crk-associated substrate (CAS) family and several members of this family have been shown TCDD induces the expression of several integrins. to interact with FAK and to regulate the integrity and the stability Integrin clustering promotes the reorganization of FAS and of the FAS. Additionally, FAK phosphorylates CAS proteins and permits cell migration.32 Integrins are crucial for cell invasion creates a high affinity site for their binding to Src, which and migration not only by physically tethering cells to the matrix phosphorylates multiple tyrosine motifs on FAK and CAS proteins. but also by sending and receiving molecular signals for the We also previously showed that the increased expression of regulation of these processes. Considering the rapid effect on HEF1, which is mediated by TCDD, stimulates cell spreading, integrin clustering following treatment with TCDD, we decided to migration and plasticity.13 Because of the early effects on HepG2 investigate the localization and expression of several members of

Figure 4. TCDD promotes the interaction of FAK with HEF1 and the phosphorylation of HEF1 through a Src/FAK pathway. (a) HepG2 cells were treated or not with TCDD (25 nM) for 24 h. Western blots of cell extracts (input) or cell extracts immunoprecipitated with FAK, HEF1 or IgG antibodies were performed with a HEF1 antibody. (b) HepG2 cells were treated with TCDD (25 nM) and/or PF-228 (10 mM) for 24 h. Western blots of cell extracts were performed with antibodies against actin or HEF1 (upper band: p115 and lower band: p105). (c) HepG2 cells were treated with TCDD (25 nM) and/or PP2 (10 mM) for 24 h. Western blots were performed with cell extracts using antibodies to: FAK pY397, FAK pY861, FAK pY925, HEF1 or E-cadherin. The right panel shows the quantification of three independent experiments. **Po0.01 and *Po0.05 as compared with controls.

& 2013 Macmillan Publishers Limited Oncogene (2013) 1811 – 1820 The AhR/Src pathway regulates integrin function C Tomkiewicz et al 1816 the integrin family after longer periods of exposure of cells to DISCUSSION TCDD. In marked contrast to a transmembrane localization, The ligands for AhR are xenobiotics that are commonly found in integrins colocalized with GRP78, an endoplasmic reticulum our environment (air and food). Some of them are persistent chaperone protein, after cells were treated for 48 h with TCDD organic pollutants. The increasing amounts of xenobiotics in our (Figure 5a). In order to determine whether this localization is due environment lead to our inevitable exposure to these ligands. to the neosynthesis integrins, we measured, as a function of time, Some of them (including TCDD) are stored in our adipose and liver by quantitative real-time PCR, the mRNA levels of several isoforms tissues and probably, as well, in cell membranes.43 of the integrin family (alpha, Figure 5b; beta, Figure 5c) following In this study, we demonstrate that the TCDD-mediated treatment of cells with TCDD. The expression of some isoforms did activation of AhR in HepG2 cells leads to rapid activation of FAK not vary at any time point (for example, integrin beta4). In and Src, which has functional consequences for cell morphology, contrast, the expression of several integrins, including alphaV, 6 migration and integrin recycling. Our data strengthen the and beta1, 3 and 5, all of which are associated with tumor conclusions of previous studies, which suggest that the non- progression, were markedly induced following exposure of cells to transcription-based pathways may be regulated by the AhR, TCDD. Moreover, the expression of the protein beta1 integrin also notably one involving activation of Src.29 Those studies show increased (Figure 5d). Thus, exposure of cells to TCDD modifies the that Src is released and activated following exposure of cells to localization and synthesis of integrins in addition to activating even low doses of TCDD.30,33–38,44 Further, this activation of Src cell-morphological changes. has been linked to functional regulatory processes including

Figure 5. Treatment of cells with TCDD is associated with late integrin neosynthesis. (a) HepG2 cells were treated for 48 h with TCDD (25 nM) and stained with TO-PRO-3 (blue, nucleus) and immunostained to detect integrin b1 (magenta) and GRP78 (green). The merged images are shown in the lower panel. (b and c) HepG2 cells were treated at different time points with TCDD (25 nM); total RNA were extracted and the expression of integrins (alpha: b and beta: c) was measured by quantitative real-time PCR. (d) HepG2 cells were treated with TCDD (25 nM)for 48 h. Western blots were performed with cell extracts using antibodies against actin or integrin b1. The lower panel shows the quantification of four independent experiments. **Po0.01. A full colour version of this figure is available at the Oncogene journal online.

Oncogene (2013) 1811 – 1820 & 2013 Macmillan Publishers Limited The AhR/Src pathway regulates integrin function C Tomkiewicz et al 1817 inflammation, which is a hallmark of cancer and is closely associated example, AGR2 as a direct target gene and a metastatic marker, with cellular migratory processes.45–49 Our observations, although E-cadherin as a secondary target) have been involved in tumor preliminary, suggest that integrins form clusters at the plasma progression. Additional work is necessary to understand the membrane following the activation of AhR and Src. We also found functional interplay of these pathways. In addition to the mediation that the activation of AhR leads to the induction of expression of of non-transcriptional effects of the AhR, Src is able to phosphorylate several isoforms of integrins. Several of these isoforms are known to tyrosine residues of the AhR and to control its effects on gene be associated with tumor progression. The clustering of integrins is expression.45,49 We conclude that a combination of non- directly related to the activation of FAK.32 In mouse mammary transcriptional and transcriptional AhR pathways might contribute fibroblasts, deficiency of AhR is associated with a reduction in the to previously unidentified toxicities of its ligands. This combination phosphorylation of FAK.10 Those results along with those of the probably facilitates the temporal sequence of events that is essential present study suggest that the AhR might stimulate the activity of for the multiple steps leading to increased cell migration. FAK and regulate the processes associated with the remodeling of the cell membrane. Along the same lines as our study, TCDD has been found to induce a rapid release of WB-F344 cells from contact MATERIALS AND METHODS inhibition but this occurs through a Src-independent pathway.50 Reagents and antibodies A recent study also show that the AhR ligands can modulate cell Antibodies to (ab24693 and ab52971), paxillin (ab32084), HEF- 51 responses at the cell membrane in an AhR-independent pathway. 1(ab18056), FAK (ab40794) and phospho Tyr397 FAK (ab4803) were from These observations need to be reconsidered in the context Abcam (Paris, France). Antibodies to E-cadherin (4065), phospho Tyr925 FAK of previous studies on the alternative AhR signaling pathways (3284), phospho Tyr416 Src (2101) and Src (2109) were from Cell Signaling such as those involved in the regulation of several kinases (Saint Quentin Yvelines Cedex, France) those against phospho Tyr861 FAK including p38.52,53 (44–626G) from Biosource (Life Sciences Technologies, Saint Aubin, France), The results of the current study shed also new light on the those against actin (A2066) from Sigma-Aldrich (Saint-Quentin Fallavier, France), those against normal IgG (sc-2027) from Santa Cruz Biotechnology, mechanisms that regulate cell plasticity following exposure of cells Inc. (Santa Cruz, CA, USA) and others against HEF-1 (IQ297) from to ligands of the AhR. Interestingly, we previously show that HEF1/ ImmunoQuest (GENTAUR, rue Lagrange, Paris). Alkaline phosphatase-linked CAS-L/NEDD9 is a target gene of the AhR (regulated by the secondary antibody (T2191 or T2192) was from Applied Biosystems transcriptional pathway involving AhR nuclear translocator). It has (Courtaboeuf, France) FITC-conjugated phalloidin was from Sigma-Aldrich, been characterized as a metastatic marker that can regulate focal PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), an adhesion dynamics.28,54 The elevation of HEF1/CAS-L/NEDD9 inhibitor of Src phosphorylation, was from Calbiochem (San Diego, CA, expression in cancer is believed to have a significant role in USA), the FAK inhibitor PF-573,228 (3,4-dihydro-6-[[4-[[[3-(methylsulfonyl) tumor progression.55–57 HEF1/CAS-L/NEDD9 regulates integrin phenyl]methyl]amino]-5-(trifluoromethyl)-2-pyrimidinyl]amino]-2(1H)-quinoli signaling, cell migration, cilia formation, the activities of the none) was supplied by Pfizer and aNF, an AhR antagonist, was from Sigma- Aldrich. TCDD was from LGC Promochem (Molsheim, France). aurora-A kinase and Src.58–61 In the current study, we have found that, in cells exposed to TCDD, AhR increases the interaction of HEF1/CAS-L/NEDD913 with FAK. Moreover, the activities of FAK Cell culture and Src favor the p115 (‘hyperphosphorylated’) isoform of HEF1/ Human hepatocarcinoma HepG2 cells were cultured in Dulbecco’s minimal CAS-L/NEDD9. Little is known about the precise role of p115 but essential medium (DMEM), 10% fetal bovine serum, supplemented with cell adhesion regulates the conversion of p105 to p115, which non-essential amino acids, 200 U/ml penicillin, 50 mg/ml streptomycin depends on the phosphorylation of the residue 369.62 Those (Invitrogen, Cergy-Pontoise, France) and 0.5 mg/ml amphotericin B (Bristol- 1 observations are concordant with the fact that TCDD stimulates Myers Squibb Co., Stamford, CT, USA) at 37 C in a humidified atmosphere in 5% CO2. One day before treatment with TCDD, cells were cultivated in the formation of new FASs and the involvement of FAK and Src in DMEM without phenol red supplemented with 3% charcoal-treated this process. More interestingly, a recent work by Tikhmyanova (desteroidized) calf serum and maintained in the medium during all the 63 et al. demonstrated that HEF1/CAS-L/NEDD9 decreases E-cadherin subsequent treatments. expression through a Src pathway, a result which is concordant with our experiments (see Figure 4b). Interestingly, active Src also RNA extraction, reverse transcription and quantitative real-time triggers c-Jun N-terminal kinases (JNK) signaling at the FASs. We PCR demonstrated in a former study, a link between the JNK activity in MCF-7 cells and the decrease of E-Cadherin levels (Diry et al.15). Total RNAs were extracted using the RNeasy mini kit (Qiagen, Les Ulis, France) and reverse transcription was performed using the cDNA high-capacity Thus, this Src–JNK pathway further links our observations to the archive kit (Applied Biosystems) as previously described. Gene-specific decreased levels of E-cadherin, which is a hallmark of epithelial primers used for the real-time PCR were designed using the OLIGO Explorer 63 mesenchymal transition along with increased cell migration. The software (Molecular Biology Insights, Inc., Cascade, CO, USA) and are available interplay between HEF1/CAS-L/NEDD9, Src and the AhR might be on request. Quantitative real-time PCR was carried out in a 10-ml reaction interesting to exploit in a therapeutical manner. Indeed, the volume containing 40 ng of cDNA, 300 nM of each primer and ABsolute QPCR tumorigenic HEF1/CAS-L/NEDD9-deficient cells have low levels of SYBR Green (Abgene, Villebon sur Yvette, France) using an ABI Prism 7900 activation of Src and their viability is significantly reduced in the Sequence Detector system (Applied Biosystems). PCR cycles consisted of the presence of a Src kinase inhibitor.61 Considering that the AhR following steps: Taq activation (15 min, 95 1C), denaturation (15 s, 95 1C) and 1 stimulates HEF1/CAS-L/NEDD9 expression and the interplay annealing and extension (1 min, 60 C). The threshold cycle (Ct) was measured as the number of cycles for which the reporter fluorescent between Src, FAK and HEF1/CAS-L/NEDD9, blocking the AhR emission first exceeds the background. The relative amounts of mRNA were with pharmaceutical antagonists may be an appropriate way to estimated using the DDCt method with RPL13A for normalization. sensitize tumor cells expressing high levels of HEF1/CAS-L/NEDD9 (including metastatic cells) to Src inhibitors. A similar approach (dual inhibition of SRC and aurora kinases) has been recently used Cell transfection to induce death of ovarian and cell lines (not of To examine the contribution of the activation of FAK to the reorganization normal cells).64 of the FAS and the integrins, we transfected the cells with expression vectors that encode a dominant negative fragment of FAK (FRNK42). In In summary, our studies show that both transcriptional- (HEF1/ order to monitor which cells have been transfected, enhanced green CAS-L/NEDD9 induction) and non-transcriptionally (Src activation fluorescent protein was co-transfected and an empty vector was used as a and its interplay with FAK and HEF1/CAS-L/NEDD9) mediated control. On the day before transfection, HepG2 cells (4 Â 105 cells per well) pathways converge to regulate FAS and cell migration were seeded into six-well plates. On the day of transfection, the medium (Supplementary figure 1). Strikingly, other targets of the AhR (for was replaced with DMEM without phenol red supplemented with 3%

& 2013 Macmillan Publishers Limited Oncogene (2013) 1811 – 1820 The AhR/Src pathway regulates integrin function C Tomkiewicz et al 1818 charcoal-treated (desteroidized) calf serum and then the cells were in a standard fluorescence microplate reader. For each condition, three cell transfected with the FRNK expression vector pRKVSV-FRNK or pRKVSV culture inserts were used and the studies were repeated three times. empty vector (1 mg) and a green fluorescent protein expression vector, enhanced green fluorescent protein (200 ng), using Lipofectamine 2000 Statistical analysis reagent following the protocol provided by the supplier (Life Technologies, Rockville, MD, USA). After 8 h of incubation at 37 1C, the medium was The data are the result of at least three independent experiments. The ± replaced and the cells were treated, or not, with 25 nM TCDD. Forty-eight results were expressed as the mean s.e. The differences between the hours later after the transfection, the cells were fixed in 4% groups were analyzed by Mann–Whitney’s U-test (nonparametric compar- paraformadehyde for 20 min. Following exposure of the cells to TCDD, or ison of two independent series) using StatEL software (Paris, France). not, and fixation, fluorescent images were evaluated (on a Nikon Labophot A P-value o0.05 was considered as statistically significant. 2 microscope, Nikon France S.A.S., Champigny sur Marne, France) as being normal or characteristics of AhR activation (Supplementary Figure 4) by observers who were naive as to the treatment of the cell (an extended ACKNOWLEDGEMENTS shape or dissociation of cell contacts as shown in Figure 1a). This work was supported by the ANSES (Agence Nationale de SEcurite´ Sanitaire de l’alimentation, de l’environnement et du travail; all authors), ANR (Agence Nationale de la Recherche, 06SEST26, Oncopop; all authors), ARC (Association pour la Recherche sur Cell lysis, immunoprecipitation and immunoblotting le Cancer, 3927 and SFI20101201842; all authors), CNRS (Center Nationale de la Cells were scraped into M-PER Mammalian Protein Extraction Reagent recherche scientifique), Fondation pour la Recherche Me´dicale, ‘Ecole Doctorale du containing a protease and phosphatase inhibitor cocktail (Sigma-Aldrich) and Me´dicament’, Hospitals Europe´en Georges Pompidou and Necker Enfants Malade, clarified by centrifugation. For immunoprecipitation, 200 mgofcelllysates INSERM (Institut National de la Sante´ et de la Recherche Me´dicale; all authors), Ligue were incubated with specific antibodies (at dilutions recommended by the contre le Cancer (post-doctoral fellowship), Ministe`re de l’enseignement supe´rieur et de manufacturers) for 3 h at 4 1C with continuous shaking followed by the la recherche´,Re´gionIledeFrance(doctoralfellowship)andUniversite´ Paris Descartes, addition of Bio-Adembeads PAG magnetic beads (Ademtech) and incubation Paris Sorbonne Cite´. The FRNK-expressing vector is a generous gift of Dr Kenneth M overnight. The beads were collected, washed 4  with M-PER Mammalian Yamada (NIDCR, NIH, USA) and Dr Bernard Rothhut (CNRS UMR 6237, Reims). We Protein Extraction Reagent and then resuspended in Laemmli buffer. For warmly thank Dr Lawrence Aggerbeck for his critical reading of this manuscript. Western blots, equal amounts of total protein were separated by SDS–PAGE Authors Contributions:Ce´line Tomkiewicz-Raulet performed most of the experi- and transferred onto nitrocellulose membranes. Blocking of the membrane ments and wrote a significant part of the manuscript. Linh-Chi Bui initiated several was done in 0.2% I-Block (Life Technologies) solution containing 0.1% Tween- key experiments including the immunofluorescence staining. Laurence Herry set up 20 for 1 h at room temperature followed by incubation overnight at 4 1Cwith the immunoprecipitation and the transfection experiments. Charles Me´tayer set up primary antibody. After three washes with 0.1% Tween-20 phosphate- some of the Western blot experiments. Mathilde Bourdeloux set up some of the buffered saline (PBS), the membrane was incubated with the corresponding immunofluoresence and the immunoprecipitation experiments. Robert Barouki and alkaline phosphatase-conjugated secondary antibody or IRdye 800 and IRdye Xavier Coumoul raised funds for the project, supervised all of the experiments and 680 (ScienceTec, Courtaboeuf Cedex, France). After the final washes, signals wrote most of the manuscript. were assessed using enhanced chemiluminescence (Tropix CDP-Star substrate; Life Technologies) and C-Extra films (Amersham, Courtaboeuf Cedex, France) or the Odyssey Infrared Imager (LI-COR, ScienceTec). REFERENCES 1 Barouki R, Coumoul X, Fernandez-Salguero PM. The aryl hydrocarbon receptor, more than a xenobiotic-interacting protein. FEBS Lett 2007; 581: Immunofluorescence 3608–3615. Cells were seeded onto glass coverslips at a concentration of approxi- 2 Ashida H, Nishiumi S, Fukuda I.. An update on the dietary ligands of the AhR. 5 mately 5  10 cells per well in six-well plates. Cells were treated with Expert Opin Drug Metab Toxicol 2008; 4: 1429–1447. 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Oncogene (2013) 1811 – 1820 & 2013 Macmillan Publishers Limited