The aryl hydrocarbon receptor regulates focal adhesion sites through a non-genomic FAK/Src pathway

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The aryl hydrocarbon receptor (AhR) is commonly described as a transcription factor, 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 integrin 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 integrins. Xenobiotic exposure, thus, may contribute to novel cell-migratory programs.


The aryl hydrocarbon receptor (AhR) is a xenobiotic-activated transcription factor involved in the detoxication pathways.1 It belongs to the basic helix loop helix/Per AhR nuclear translocator Sim family. Pollutant ligands of the AhR include dioxins, furans, polychlorinated biphenlys and polycyclic aromatic hydrocarbons.2, 3 Ubiquitous in mammals, the AhR forms a cytoplasmic complex with heat shock proteins. Upon ligand binding, the AhR translocates into the nucleus where it interacts with AhR nuclear translocator. The AhR/AhR nuclear translocator heterodimer recognizes xenobiotic-responsive elements in the promoters of target genes and controls their expression.4 The AhR signaling pathway is best known for the transcriptional regulation of xenobiotic-metabolizing enzymes, which are involved in the metabolism of drugs and pollutants. This elegant adaptive pathway allows the coordinate detection and elimination of pollutants and, thus, protects organisms against foreign chemicals.4

Recently, however, other genes and alternative pathways have been identified as AhR targets.1, 5 Studies with gene-knockout models (in both vertebrates and invertebrates) have suggested that the AhR has other functions including the regulation of cell migration during development.6, 7, 8, 9, 10, 11, 12 Several groups, including our own, have shown that, upon xenobiotic binding, the AhR stimulates the migration and the invasion of several types of cells.6, 10, 13, 14, 15, 16, 17

Cellular migration is a consequence, in part, of a redistribution of the focal adhesion sites (FASs), which are composed of multiple transmembrane and cytoplasmic proteins (integrins, kinases…).18, 19 One component of the FASs, which has a crucial role in cellular migration, is the focal adhesion kinase (FAK). FAK is activated by autophosphorylation (tyrosine 397) following the clustering of integrins. This activation leads to the recruitment of SH2-domain-containing regulators, such as Src and other members of the SFK family (Src family of tyrosine kinases). These kinases phosphorylate multiple other sites on FAK.20, 21, 22 Ultimately, the phosphorylation of FAK leads to the dissociation of the FAS and cell migration.

TCDD (2,3,7,8-p-Tetrachlorodibenzo-p-dioxin) is one of the most potent ligands of the AhR. Genome-wide transcriptome analyses that explore the effects of TCDD on gene expression have shown that several other biological pathways are regulated in addition to xenobiotic metabolism.23, 24, 25 We have shown that TCDD induces the expression of the Hef1/Cas-L/Nedd9, Agr2 and Sos1 genes,13, 15, 26, 27 genes that are involved in the ‘epithelial mesenchymal transition’ pathway and survival and metastasis. The increase in the cellular mobility and morphological changes observed during the epithelial mesenchymal transition elicited following 24–72 h of treatment with TCDD and were found to depend upon the induction of the Hef1 gene.13, 28 However, in the present study, we found that subtle morphological changes in the FASs were observed after only 1–4 h of treatment with TCDD, well before any significant increase in the HEF1 protein occurred. We, therefore, hypothesized that those rapid effects, which require neither transcription nor translation, could be at the origin of the initial changes in the cell morphology. Further, we suspected that these effects could be synergistic with the gene-regulatory effects of late onset to yield, together, the full spectrum of morphological and migratory changes triggered by TCDD. Circumstantial support for this hypothesis comes from the observations (including a recent elegant study using fluorescence resonance energy transfer (FRET) technology) that the AhR, in the cytoplasm, is bound to several chaperone proteins and to the Src kinase.29, 30, 31 The binding of xenobiotics to the AhR not only initiates the effects of the AhR on gene expression but also, perhaps as importantly, leads to the release of Src. This rapid liberation of Src, which is a non-transcriptional event, may lead to its activation and to the subsequent phosphorylation of Src partners, for example, those at the FASs. In the present study, we show that the binding of xenobiotic to the AhR activates the redistribution of FAS and leads to cell plasticity through non-transcriptional events and that these events act in synergy to the transcriptional regulation of the major genes involved in cell migration.


AhR activates early integrin clustering

The reorganization of FAS in HepG2 cells treated with TCDD was observed by immunostaining of paxillin (a component of FAS) and staining of actin. Morphological changes, which consisted of an increase in cell spreading, the formation of stress fibers, an extension of the FAS and the loss of cell contacts in the HepG2 cells, occurred rapidly (4-8 h) following treatment with TCDD (Figure 1a). All of these changes depend upon the activation of the AhR because they were inhibited by the AhR antagonist, α-naphthoflavone (aNF) (Figure 1b). Because the formation of new FAS during cell migration depends upon integrin clustering, we looked for changes in the distribution of integrins with specific staining. After only 2–4 h of treatment with TCDD, clusters of integrins associated with actin fibers were present at the cell membrane (Figure 1c). This clustering was also blocked by aNF (Figure 1d).

Figure 1

TCDD/AhR activates early morphological changes, redistribution of FASs and triggers integrin β1 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 μm. (b) HepG2 cells were treated with TCDD (25 nM) and/or aNF (5 μM) 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 μm. (c) HepG2 cells were treated with TCDD (25 nM) for 30, 120 or 240 min and immunostained to detect integrin β1 (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 μm. (d) HepG2 cells were treated with TCDD (25 nM) and/or aNF (5 μM) for 24 h and immunostained to detect integrin β1 (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 μm. A full colour version of this figure is available at the Oncogene journal online.

AhR regulates early FAK and Src activation

The FAK regulates FAS reorganization and integrin clustering.32 We, therefore, hypothesized that FAK is an early target of TCDD. Examination of the activation of FAK and Src, as measured by their profiles of phosphorylation, showed (Figure 2a) that the phosphorylation of FAK on tyrosine 397 (Y397) is significantly increased after only 1 h of exposure of the cells to TCDD. This is an autophosphorylation, which creates a binding site for Src. Src, also, is activated significantly after only 15 min of exposure of cells to TCDD (Figure 2b), in agreement with previous studies.30, 33, 34, 35, 36, 37, 38 The total amounts of FAK and Src remain constant during the course of those experiments.

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 μM) 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 μM) 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. **P<0.01 and *P<0.05 as compared with controls. A full colour version of this figure is available at the Oncogene journal online.

Increased phosphorylation of tyrosines 861 and 925 in FAK also was observed following treatment of the cells with TCDD. However, the increase in the phosphorylation of Y925, which was significant after only 2 h of exposure of cells to TCDD, was still slower than that of tyrosine 397 and 861 (Figure 2a), as well as that of Src (Figure 2b). The phosphorylation of Y925 has been described as a marker of FAS turnover.39, 40, 41 Supplementary Figure 1 shows complete kinetics of FAK phosphorylation. Most 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 also conserved (and even magnified in the human mammary cell line, MCF-7. Supplementary figure 2 shows that the clustering of the integrin β1, the spatial reorganization (as assessed by paxillin staining) of FASs and the phosphorylation events on Src and FAK are conserved in this cell line which also expresses the AhR.

We next analyzed the contribution of the AhR to these phosphorylation events by treating the cells concomitantly with aNF. We observed a significant reduction, in the presence of aNF, of the phosphorylation of Src, FAK pY397, pY861 and pY925 (Figures 2c and d). We observed similar events following transfection of a small interfering RNA targeting the AhR and treatment with TCDD (Supplementary figure 3).

Activation of FAK/Src by the AhR regulates cell plasticity

We, next, examined the contribution of the activation of FAK and Src to the reorganization of the FAS and the integrins. To this end, we transfected the cells with expression vectors that encode a dominant negative fragment of FAK (FRNK42). We show that the expression of a dominant negative fragment of FAK sharply reduces the number of cells with a morphology that is characteristic of AhR activation after a long exposure course (>24 h) (Figure 3a).

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 μM) 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, 10 μM) and/or PP2 (d, 10 μM) 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 μm. (e) HepG2 cells were treated with TCDD (25 nM) and/or PP2 (10 μM) for 4 h and immunostained to detect integrin β1 (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 μm. (f) A Boyden chamber assay was used to measure cell migration following treatment with TCDD (25 nM) and/or PF-228 (10 μM). The quantification of three independent experiments is shown. **P<0.01 and *P<0.05 as compared with controls. A full colour version of this figure is available at the Oncogene journal online.

We also inhibited the activity of FAK with a competitive antagonist (PF-228; Pfizer, Paris, France), which blocks the active site of FAK. PF-228 decreased the phosphorylation of FAK at all three sites (Figure 3b) and decreased the extent of dissociation of the cell contacts observed following exposure of cells to TCDD (Figure 3c). Because the phosphorylation of FAK at several sites (and, therefore, the activity of FAK) depends upon the binding of a Src, which has a functional kinase activity, we blocked the activity of Src with a pharmacological agent, PP2.29 PP2 eliminated both the morphological changes (Figure 3d) and the clustering of integrins (Figure 3e) that are observed following exposure of cells to TCDD.

Finally, using Bowden chambers, we confirmed that TCDD significantly increased HepG2 cell migration. This increase in cell migration was blocked completely by PF-228 (Figure 3f), which demonstrates that the cell migration that is induced by TCDD depends upon FAK activity. Thus, both FAK and Src, through the action of TCDD on the AhR, are involved in the early stimulation of cell plasticity.

TCDD stimulates the recruitment of HEF1 on FAK

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 to interact with FAK and to regulate the integrity and the stability of the FAS. Additionally, FAK phosphorylates CAS proteins and creates a high affinity site for their binding to Src, which phosphorylates multiple tyrosine motifs on FAK and CAS proteins.

We also previously showed that the increased expression of HEF1, which is mediated by TCDD, stimulates cell spreading, migration and plasticity.13 Because of the early effects on HepG2 morphology and the activation of both FAK and Src following exposure of cells to TCDD, we hypothesized that TCDD also may have effects on HEF1 that are independent of both transcription and translation. We, therefore, investigated whether the treatment of cells with TCDD promotes the interaction between HEF1 and FAK. Following treatment of cells with TCDD, FAK with HEF1 co-immunoprecipitate together (Figure 4a). In addition, treatment with TCDD increases the expression of HEF1 isoforms, p105 and p115 (the larger molecular weight form results from phosphorylation of p105HEF1) (Figure 4b). Interestingly, inhibition of FAK by PF-228 significantly and specifically reduced p115 (upper band in Figure 4b). Moreover, PP2 eliminated the AhR-mediated increase in phosphorylation of FAK and HEF1 (Figure 4c). As expected, the inhibition of Src abolished the decrease in expression of E-cadherin following treatment of cells with TCDD.13 Thus, in addition to the transcription-based stimulation of expression of 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 critical for cell spreading.

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 μM) 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 μM) 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. **P<0.01 and *P<0.05 as compared with controls.

TCDD induces the expression of several integrins.

Integrin clustering promotes the reorganization of FAS and permits cell migration.32 Integrins are crucial for cell invasion and migration not only by physically tethering cells to the matrix but also by sending and receiving molecular signals for the regulation of these processes. Considering the rapid effect on integrin clustering following treatment with TCDD, we decided to investigate the localization and expression of several members of the integrin family after longer periods of exposure of cells to TCDD. In marked contrast to a transmembrane localization, integrins colocalized with GRP78, an endoplasmic reticulum chaperone protein, after cells were treated for 48 h with TCDD (Figure 5a). In order to determine whether this localization is due to the neosynthesis integrins, we measured, as a function of time, by quantitative real-time PCR, the mRNA levels of several isoforms of the integrin family (alpha, Figure 5b; beta, Figure 5c) following treatment of cells with TCDD. The expression of some isoforms did not vary at any time point (for example, integrin beta4). In contrast, the expression of several integrins, including alphaV, 6 and beta1, 3 and 5, all of which are associated with tumor progression, were markedly induced following exposure of cells to TCDD. Moreover, the expression of the protein beta1 integrin also increased (Figure 5d). Thus, exposure of cells to TCDD modifies the localization and synthesis of integrins in addition to activating cell-morphological changes.

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 β1 (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 β1. The lower panel shows the quantification of four independent experiments. **P<0.01. A full colour version of this figure is available at the Oncogene journal online.


The ligands for AhR are xenobiotics that are commonly found in our environment (air and food). Some of them are persistent organic pollutants. The increasing amounts of xenobiotics in our environment lead to our inevitable exposure to these ligands. Some of them (including TCDD) are stored in our adipose and liver tissues and probably, as well, in cell membranes.43

In this study, we demonstrate that the TCDD-mediated activation of AhR in HepG2 cells leads to rapid activation of FAK and Src, which has functional consequences for cell morphology, migration and integrin recycling. Our data strengthen the conclusions of previous studies, which suggest that the non-transcription-based pathways may be regulated by the AhR, notably one involving activation of Src.29 Those studies show that Src is released and activated following exposure of cells to even low doses of TCDD.30, 33, 34, 35, 36, 37, 38, 44 Further, this activation of Src has been linked to functional regulatory processes including inflammation, which is a hallmark of cancer and is closely associated with cellular migratory processes.45, 46, 47, 48, 49 Our observations, although preliminary, suggest that integrins form clusters at the plasma membrane following the activation of AhR and Src. We also found that the activation of AhR leads to the induction of expression of several isoforms of integrins. Several of these isoforms are known to be associated with tumor progression. The clustering of integrins is directly related to the activation of FAK.32 In mouse mammary fibroblasts, deficiency of AhR is associated with a reduction in the phosphorylation of FAK.10 Those results along with those of the present study suggest that the AhR might stimulate the activity of 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 inhibition but this occurs through a Src-independent pathway.50 A recent study also show that the AhR ligands can modulate cell responses at the cell membrane in an AhR-independent pathway.51 These observations need to be reconsidered in the context of previous studies on the alternative AhR signaling pathways such as those involved in the regulation of several kinases including p38.52, 53

The results of the current study shed also new light on the mechanisms that regulate cell plasticity following exposure of cells to ligands of the AhR. Interestingly, we previously show that HEF1/CAS-L/NEDD9 is a target gene of the AhR (regulated by the transcriptional pathway involving AhR nuclear translocator). It has been characterized as a metastatic marker that can regulate focal adhesion dynamics.28, 54 The elevation of HEF1/CAS-L/NEDD9 expression in cancer is believed to have a significant role in tumor progression.55, 56, 57 HEF1/CAS-L/NEDD9 regulates integrin signaling, cell migration, cilia formation, the activities of the aurora-A kinase and Src.58, 59, 60, 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 and Src favor the p115 (‘hyperphosphorylated’) isoform of HEF1/CAS-L/NEDD9. Little is known about the precise role of p115 but cell adhesion regulates the conversion of p105 to p115, which depends on the phosphorylation of the residue 369.62 Those observations are concordant with the fact that TCDD stimulates the formation of new FASs and the involvement of FAK and Src in this process. More interestingly, a recent work by Tikhmyanova et al.63 demonstrated that HEF1/CAS-L/NEDD9 decreases E-cadherin expression through a Src pathway, a result which is concordant with our experiments (see Figure 4b). Interestingly, active Src also triggers c-Jun N-terminal kinases (JNK) signaling at the FASs. We 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). Thus, this Src–JNK pathway further links our observations to the decreased levels of E-cadherin, which is a hallmark of epithelial mesenchymal transition along with increased cell migration.63 The interplay between HEF1/CAS-L/NEDD9, Src and the AhR might be interesting to exploit in a therapeutical manner. Indeed, the tumorigenic HEF1/CAS-L/NEDD9-deficient cells have low levels of activation of Src and their viability is significantly reduced in the presence of a Src kinase inhibitor.61 Considering that the AhR stimulates HEF1/CAS-L/NEDD9 expression and the interplay between Src, FAK and HEF1/CAS-L/NEDD9, blocking the AhR with pharmaceutical antagonists may be an appropriate way to 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 to induce death of ovarian and colorectal cancer cell lines (not of normal cells).64

In summary, our studies show that both transcriptional- (HEF1/CAS-L/NEDD9 induction) and non-transcriptionally (Src activation and its interplay with FAK and HEF1/CAS-L/NEDD9) mediated pathways converge to regulate FAS and cell migration (Supplementary figure 1). Strikingly, other targets of the AhR (for example, AGR2 as a direct target gene and a metastatic marker, E-cadherin as a secondary target) have been involved in tumor progression. Additional work is necessary to understand the functional interplay of these pathways. In addition to the mediation of non-transcriptional effects of the AhR, Src is able to phosphorylate tyrosine residues of the AhR and to control its effects on gene expression.45, 49 We conclude that a combination of non-transcriptional and transcriptional AhR pathways might contribute to previously unidentified toxicities of its ligands. This combination probably facilitates the temporal sequence of events that is essential for the multiple steps leading to increased cell migration.

Materials and methods

Reagents and antibodies

Antibodies to integrin beta 1 (ab24693 and ab52971), paxillin (ab32084), HEF-1(ab18056), FAK (ab40794) and phospho Tyr397 FAK (ab4803) were from Abcam (Paris, France). Antibodies to E-cadherin (4065), phospho Tyr925 FAK (3284), phospho Tyr416 Src (2101) and Src (2109) were from Cell Signaling (Saint Quentin Yvelines Cedex, France) those against phospho Tyr861 FAK (44–626G) from Biosource (Life Sciences Technologies, Saint Aubin, France), those against actin (A2066) from Sigma-Aldrich (Saint-Quentin Fallavier, France), those against normal IgG (sc-2027) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and others against HEF-1 (IQ297) from ImmunoQuest (GENTAUR, rue Lagrange, Paris). Alkaline phosphatase-linked secondary antibody (T2191 or T2192) was from Applied Biosystems (Courtaboeuf, France) FITC-conjugated phalloidin was from Sigma-Aldrich, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), an inhibitor of Src phosphorylation, was from Calbiochem (San Diego, CA, USA), the FAK inhibitor PF-573,228 (3,4-dihydro-6-[[4-[[[3-(methylsulfonyl)phenyl]methyl]amino]-5-(trifluoromethyl)-2-pyrimidinyl]amino]-2(1H)-quinoli none) was supplied by Pfizer and aNF, an AhR antagonist, was from Sigma-Aldrich. TCDD was from LGC Promochem (Molsheim, France).

Cell culture

Human hepatocarcinoma HepG2 cells were cultured in Dulbecco's minimal essential medium (DMEM), 10% fetal bovine serum, supplemented with non-essential amino acids, 200 U/ml penicillin, 50 μg/ml streptomycin (Invitrogen, Cergy-Pontoise, France) and 0.5 mg/ml amphotericin B (Bristol-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 DMEM without phenol red supplemented with 3% charcoal-treated (desteroidized) calf serum and maintained in the medium during all the subsequent treatments.

RNA extraction, reverse transcription and quantitative real-time PCR

Total RNAs were extracted using the RNeasy mini kit (Qiagen, Les Ulis, France) and reverse transcription was performed using the cDNA high-capacity archive kit (Applied Biosystems) as previously described. Gene-specific primers used for the real-time PCR were designed using the OLIGO Explorer software (Molecular Biology Insights, Inc., Cascade, CO, USA) and are available on request. Quantitative real-time PCR was carried out in a 10-μl reaction volume containing 40 ng of cDNA, 300 nM of each primer and ABsolute QPCR SYBR Green (Abgene, Villebon sur Yvette, France) using an ABI Prism 7900 Sequence Detector system (Applied Biosystems). PCR cycles consisted of the following steps: Taq activation (15 min, 95 °C), denaturation (15 s, 95 °C) and annealing and extension (1 min, 60 °C). The threshold cycle (Ct) was measured as the number of cycles for which the reporter fluorescent emission first exceeds the background. The relative amounts of mRNA were estimated using the ΔΔCt method with RPL13A for normalization.

Cell transfection

To examine the contribution of the activation of FAK to the reorganization of the FAS and the integrins, we transfected the cells with expression vectors that encode a dominant negative fragment of FAK (FRNK42). In order to monitor which cells have been transfected, enhanced green fluorescent protein was co-transfected and an empty vector was used as a control. On the day before transfection, HepG2 cells (4 × 105 cells per well) were seeded into six-well plates. On the day of transfection, the medium was replaced with DMEM without phenol red supplemented with 3% charcoal-treated (desteroidized) calf serum and then the cells were transfected with the FRNK expression vector pRKVSV-FRNK or pRKVSV empty vector (1 μg) and a green fluorescent protein expression vector, enhanced green fluorescent protein (200 ng), using Lipofectamine 2000 reagent following the protocol provided by the supplier (Life Technologies, Rockville, MD, USA). After 8 h of incubation at 37 °C, the medium was replaced and the cells were treated, or not, with 25 nM TCDD. Forty-eight hours later after the transfection, the cells were fixed in 4% paraformadehyde for 20 min. Following exposure of the cells to TCDD, or not, and fixation, fluorescent images were evaluated (on a Nikon Labophot 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 shape or dissociation of cell contacts as shown in Figure 1a).

Cell lysis, immunoprecipitation and immunoblotting

Cells were scraped into M-PER Mammalian Protein Extraction Reagent containing a protease and phosphatase inhibitor cocktail (Sigma-Aldrich) and clarified by centrifugation. For immunoprecipitation, 200 μg of cell lysates were incubated with specific antibodies (at dilutions recommended by the manufacturers) for 3 h at 4 °C with continuous shaking followed by the addition of Bio-Adembeads PAG magnetic beads (Ademtech) and incubation overnight. The beads were collected, washed 4 × with M-PER Mammalian Protein Extraction Reagent and then resuspended in Laemmli buffer. For Western blots, equal amounts of total protein were separated by SDS–PAGE and transferred onto nitrocellulose membranes. Blocking of the membrane was done in 0.2% I-Block (Life Technologies) solution containing 0.1% Tween-20 for 1 h at room temperature followed by incubation overnight at 4 °C with primary antibody. After three washes with 0.1% Tween-20 phosphate-buffered saline (PBS), the membrane was incubated with the corresponding alkaline phosphatase-conjugated secondary antibody or IRdye 800 and IRdye 680 (ScienceTec, Courtaboeuf Cedex, France). After the final washes, signals 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).


Cells were seeded onto glass coverslips at a concentration of approximately 5 × 105 cells per well in six-well plates. Cells were treated with TCDD (25 nM) for 48 h in DMEM without phenol red and supplemented with 3% charcoal-treated calf serum. For immunofluorescence, all the steps were carried out at room temperature. The coverslips were washed twice in 1 × PBS and then fixed in 4% paraformadehyde for 20 min. The cells were permeabilized for 10 min in PBS-Triton 0.3% and then incubated in PBS-bovine serum albumin 1% for 30 min. Incubations with the primary antibody were done for 1 h at room temperature in PBS-bovine serum albumin 1%. For staining of actin, FITC-conjugated phalloidin was included during the incubation with the secondary antibody. The coverslips were sealed with Dako Faramount Aqueous Mounting Medium Ready-to-use (Invitrogen) or VECTASHIELD Hard SetMounting Medium with DAPI (Vector Laboratories, CliniSciences SAS, Nanterre, France) or TO-PRO-3 (Invitrogen). Mounted cells were observed and images recorded using a Zeiss LSM 510 confocal microscope (Carl Zeiss Meditec France SAS, Le Pecq, France) using a × 40 Plan-Neofluar 1.3 NA oil objective and LSM Image Browser, or on a Nikon Eclipse TE-2000 E microscopy. Deconvolution and 3D reconstitution was performed with Autoquant imaging Autodeblur version X 1.4.1 (AutoDebur and Autovisualize; Mediacybernetics, Bethesda, MD, USA). ImageJ 1.37v software was used for analysis of the images.

Cell-migration assay

Cell migration was measured using a Boyden chamber assay, with a BD Falcon Cell Culture insert with transparent polyethylene terephthalate membranes (8.0 μm pores, Falcon; Becton Dickinson, Franklin Lakes, NJ, USA). HepG2 cells (5 × 104), which were resuspended in DMEM without phenol red and supplemented with 3% charcoal-treated calf serum, were added to the upper compartment of the chamber in the presence, or the absence, of 25 nM TCDD. Medium containing 10% FBS in the presence, or the absence, of 25 nM TCDD was placed in the lower compartment of the migration chamber to act as a chemo-attractant. After 48 h of incubation at 37 °C in a 5% CO2 incubator, migratory cells were labelled with calcein acetoxymethyl ester, detached from the underside of the polyethylene terephthalate membrane with trypsin-EDTA solution and, finally, quantified in a standard fluorescence microplate reader. For each condition, three cell culture inserts were used and the studies were repeated three times.

Statistical analysis

The data are the result of at least three independent experiments. The results were expressed as the mean±s.e. The differences between the groups were analyzed by Mann–Whitney's U-test (nonparametric comparison of two independent series) using StatEL software (Paris, France). A P-value <0.05 was considered as statistically significant.


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This work was supported by the ANSES (Agence Nationale de SEcurité 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 le Cancer, 3927 and SFI20101201842; all authors), CNRS (Center Nationale de la recherche scientifique), Fondation pour la Recherche Médicale, ‘Ecole Doctorale du Médicament’, Hospitals Européen Georges Pompidou and Necker Enfants Malade, INSERM (Institut National de la Santé et de la Recherche Médicale; all authors), Ligue contre le Cancer (post-doctoral fellowship), Ministère de l'enseignement supérieur et de la recherché, Région Ile de France (doctoral fellowship) and Université Paris Descartes, Paris Sorbonne Cité. The FRNK-expressing vector is a generous gift of Dr Kenneth M Yamada (NIDCR, NIH, USA) and Dr Bernard Rothhut (CNRS UMR 6237, Reims). We warmly thank Dr Lawrence Aggerbeck for his critical reading of this manuscript.

Authors Contributions: Céline Tomkiewicz-Raulet performed most of the experiments and wrote a significant part of the manuscript. Linh-Chi Bui initiated several key experiments including the immunofluorescence staining. Laurence Herry set up the immunoprecipitation and the transfection experiments. Charles Métayer set up some of the Western blot experiments. Mathilde Bourdeloux set up some of the immunofluoresence and the immunoprecipitation experiments. Robert Barouki and Xavier Coumoul raised funds for the project, supervised all of the experiments and wrote most of the manuscript.

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Correspondence to X Coumoul.

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Tomkiewicz, C., Herry, L., Bui, L. et al. The aryl hydrocarbon receptor regulates focal adhesion sites through a non-genomic FAK/Src pathway. Oncogene 32, 1811–1820 (2013) doi:10.1038/onc.2012.197

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  • AhR
  • cell plasticity
  • FAK
  • integrin
  • Src

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