Ah receptor antagonism represses head and neck tumor cell aggressive phenotype

Abstract

The aryl hydrocarbon receptor (AHR) has been shown to play a role in an increasing number of cellular processes. Recent reports have linked the AHR to cell proliferation, cytoskeletal arrangement, and tumor invasiveness in various tumor cell types. The AHR plays a role in the de-repression of the IL6 promoter in certain tumor cell lines, allowing for increased transcriptional activation by cytokines. Here, we show that there is a significant level of constitutive activation of the AHR in cells isolated from head and neck squamous cell carcinoma (HNSCC) patients. Constitutive activation of the AHR in HNSCC cells was blocked by antagonist treatment, leading to a reduction in IL6 expression. Additionally, the AHR exhibits a high level of expression in HNSCC cells compared to normal keratinocytes. These findings led to the hypothesis that the basal AHR activity in HNSCC cells plays a role in the aggressive phenotype of these tumors, and that antagonist treatment could mitigate this phenotype. This study provides evidence that antagonism of the AHR in HNSCC tumor cells, in the absence of exogenous receptor ligands, has a significant effect on tumor cell phenotype. Treatment of these cell lines with the AHR antagonists 6, 2′, 4′-trimethoxyflavone, or the more potent GNF351, decreased migration, and invasion of HNSCC cells and prevented benzo[a]pyrene-mediated induction of the chemotherapy efflux protein ABCG2. Thus, an AHR antagonist treatment has been shown to have therapeutic potential in HNSCC through a reduction in aggressive cell phenotype.

INTRODUCTION

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that has been largely regarded as a mediator of xenobiotic metabolism for decades (1). Unliganded AHR is typically found in a cytoplasmic complex with the 90 kDa heat shock protein (hsp90) and the X-associated protein 2 (XAP2). Agonist binding leads to nuclear translocation of the receptor, where it releases the chaperone complex and heterodimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT). Prototypical AHR agonists include a variety of xenobiotics, including polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (B[a]P) and 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). PAHs are common environmental pollutants resulting from car exhaust, manufacturing, and cigarette smoke, in addition to other sources. The xenobiotic response initiated by the AHR centers on its ligand-mediated binding to dioxin response elements (DREs) in the promoters of CYP1A1, CYP1B1, and CYP1A2, which express enzymes that act in phase I xenobiotic metabolism, as well as other target genes.

Recent research has provided evidence that there are a myriad of endogenous roles for the AHR, both in the presence and absence of exogenous ligands. Examples of physiological activities in which the AHR plays a part include, attenuation of the acute phase response, cytokine signaling, Th17 immune cell differentiation, modulation of NFκB activity, and regulation of hormonal signaling (2–7). This multifaceted aspect of endogenous AHR activity arises not only through the AHR binding to its cognate response element, but also through protein-protein interactions. This latter mechanism can mediate transcription factor sequestering away from a gene promoter or tethering of the AHR to a transcription factor on a promoter. In support of the concept of endogenous ligands as a source of receptor activation, a growing list of endogenous ligands have been identified, such as the uremic toxin, 3-indoxyl sulfate, transient metabolites of the arachidonic acid pathway, and byproducts of the tryptophan oxidation pathway (8–10). This implies that there is potential for constitutive or transient in vivo activation of the receptor in certain tissue types, resulting in a wide range of effects.

We have previously shown in the MCF-7 breast cancer cell line that activation of the AHR by TCDD treatment induces binding of the receptor to DREs, ~3 kb upstream from the transcription start site of the IL6 promoter. This has the effect of priming the DNA for IL1B-mediated NFκB binding and a subsequent increase in transcription. In this context, the binding of the AHR coincides with derepression of the gene by dismissal of histone deacetylases (HDACs) from the proximal promoter (11, 12). In the absence of AHR expression, IL1B only poorly induces IL6 expression. Our research has focused on squamous cell carcinoma of the head and neck (HNSCC), which often shows constitutively high cytokine expression regardless of the tissue of origin (13–15). Analysis of the IL6 promoter in multiple HNSCC cell lines revealed a high level of AHR presence in the absence of exogenous ligand, apparently maintaining the promoter in the derepressed state. For this reason, basal IL6 production was higher than in MCF-7 cells, and IL1B readily induced IL6 transcription on its own. Treatment of HNSCC cells with the AHR antagonist 6, 2′, 4′-trimethoxyflavone (TMF) for 12 h or longer resulted in a significant reduction in the level of AHR found at the IL6 promoter and a corresponding increase in the amount of HDAC1 present (12). This reversal of constitutive de-repression through removal of the AHR from the IL6 promoter led to decreases in both basal and IL1B-induced IL6 transcription and subsequent IL6 secretion. Thus, AHR antagonist treatment has proven to be a viable method to decrease pro-growth IL6 in HNSCC cell culture models.

Having shown that AHR antagonism effectively limits the secretion of IL6 in HNSCC cell lines, we then focused on the phenotypic effects of AHR antagonism on HNSCC. HNSCC is regarded as an aggressive form of carcinoma, with a five year overall survival rate below 50% and high levels of metastasis in patients (16). Current treatment for HNSCC centers on radical neck dissection with or without adjuvant radiation therapy and/or chemotherapy. While high IL6 levels in HNSCC correlate with disease aggressiveness and poorer patient prognosis (17), it has not been proven to be a cause and effect relationship. The possibility remains that the higher IL6 levels are due in part to higher AHR activity, and this activated AHR itself has numerous other effects on cellular phenotype. In this context, we assessed the ability of AHR antagonist treatment to abrogate multiple aspects of the aggressive phenotype of HNSCC cells. Results presented here reveal that blocking AHR activity can, in a relatively short time frame, lead to decreased HNSCC migration, invasion, and proliferation.

Material and Methods

Cell culture

HN13, HN30, HN2095 head and neck squamous cell carcinoma (HNSCC) cell lines were maintained at 37°C, 5% CO₂ in a high glucose 1:1 DMEM:F12 (Sigma), supplemented with 10% fetal bovine serum (FBS; Hyclone Labs.), 1,000 units/ml penicillin, and 0.1 mg/ml streptomycin (Sigma). Human epidermal keratinocytes (HEK) were purchased from Cell Applications, Inc. (San Diego, CA). These cells were maintained in Adult Keratinocyte Growth Medium and passaged using the Subculture Reagent Kit (Cell Applications, Inc.).

Chemicals

6, 2′, 4′-trimethoxyflavone (TMF) was purchased from Indofine Chemical Company 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was kindly provided by Dr. Steve Safe, Texas A&M University. The synthesis of GNF351 (N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine) is described in the supplemental methods.

Gene expression

Treatment of cells was performed by diluting compounds to the desired working concentration in serum-free media supplemented with 5 mg/ml bovine serum albumin (BSA) or low serum (2%) media. Total RNA was extracted from the cells using TRI reagent (Sigma) as specified by the manufacturer. The ABI high-capacity cDNA archive kit (Applied Biosystems) was used to prepare cDNA from isolated RNA. mRNA expression was measured by quantitative real-time PCR using the Quanta SYBR Green kit on an iCycler DNA engine equipped with the MyiQ single color real-time PCR detection system (Bio-Rad). Expressed quantities of mRNA were normalized to GAPDH mRNA levels and plotted using GraphPad Prism 4.0 (GraphPad Software). Histograms are plotted as mean values of biological replicates; error bars represent the standard deviation of replicates. QRT-PCR primers used are presented in supplemental materials.

Immunoblotting

Whole cell extracts were prepared by lysing cells in radioimmunoprecipitation assay buffer [RIPA; 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 140 mM NaCl, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS] supplemented with 1% ipegal, 300 mM NaCl, and protease inhibitor cocktail (Sigma). Homogenates were centrifuged at 21,000×g for 30 min at 4°C, and the soluble fraction was collected as whole cell extract. Protein concentrations were determined using the DC protein assay kit (Bio-Rad). Protein samples were resolved by tricine SDS-PAGE and transferred to PVDF membrane. Primary antibodies used to detect specific proteins are shown in supplemental materials and were visualized using biotin-conjugated secondary antibodies (Jackson Immunoresearch) in conjunction with ¹²⁵I-streptavidin (Amersham). In the case of ABCG2 protein, protein was detected by enhanced chemiluminescence (Pierce, Rockford, IL).

HN30 and human epidermal keratinocyte comparative analysis

AHR protein levels were assessed after lyses of cells in MENG (25 mM MOPS, 2 mM EDTA, 0.02% NaN₃, 10% glycerol, pH 7.5) plus 1% IGEPAL CA-630, 300 mM NaCl, 20 mM molybdate, and protease inhibitors. Cell lysates were centrifuged at 13,000g for 10 min at 4°C, and soluble extracts containing both nuclear and cytosolic proteins were collected. The extracts were resolved by 8% tricine-SDS-PAGE, and transferred to a PVDF membrane. Relative AHR and β-actin protein levels were determined. For the isolation of cytosolic and nuclear extracts, cells were collected and suspended in MENG plus 20 mM molybdate, pH 7.4 (molybdate buffer), with the addition of protease inhibitors. The cells were homogenized and centrifuged at 1000g for 20 min at 4°. The soluble phase was saved as the cytosolic fraction. The pellet was washed with molybdate buffer and centrifuged at 1000g for 5 min at 4°C for a total of 3 wash cycles. Molybdate buffer + 50 mM NaCl was added to the pellet and the extracts were centrifuged at 1000g for 20 min at 4°C. The pellet was incubated on ice for 1 h with high salt buffer (molybdate buffer, 500 mM NaCl, and protease inhibitors). The samples were then centifuged for 1 h at 100,000g at 4°C, and the nuclear fraction collected. Samples were resolved using tricine SDS-PAGE and protein transferred to a PVDF membrane. The blot was probed for AHR, Lamin A/C and β-actin using the appropriate primary antibodies, secondary biotinylated antibodies and ¹²⁵I-streptavidin.

Chromatin immunoprecipitation assays

HN13 cells were grown to approximately 90% confluency in 150 cm² dishes and serum-starved 18 h before treatment. Cells were treated in serum-free media supplemented with 5 mg/ml BSA by diluting compounds to the desired working concentrations. Chromatin immunoprecipitation assays were performed as previously described (11). The primers utilized for PCR analysis and antibodies used are given in the supplemental materials.

Gene silencing

Specific protein levels were decreased using the Dharmacon small interfering RNA (siRNA) [control oligo D-001810-0X, AHR oligo J-004990-07]. Electroporation/nucleofection was performed using the Amaxa nucleofection system essentially as described in manufacturer protocols. Briefly, cells were washed and suspended at a concentration of 2.0 × 10⁶ cells per 100 μL of nucleofection solution. Control or targeted siRNA was added to the cells for a final concentration of 1.5 μmol/L. Cells were electroporated using manufacturer’s MCF-7 high efficiency program.

Migration assay

All HNSCC cell lines were plated and treated in serum free media for 24 h, after which they were trypsinized, counted, and normalized for cell number between treatments. Cells were then subjected to migration assay through an 8 μm pore polycarbonate filter (Neuro Probe #PFB8) using the Neuro Probe Multiwell Chemotaxis Chamber according to manufacturer specifications. Briefly, lower well chambers were filled with either serum free or 10% FBS-containing media, along with vehicle or treatment compound. Polycarbonate membrane was then placed over the lower wells, with the upper well plate was secured on top. Upper wells were then filled with cell suspensions in serum free media, containing vehicle or treatment compound. Cells were incubated for 48 h, after which non-migratory cells were removed and membranes fixed and stained (Dade Behring Diff-Quik stain set). Membranes were analyzed under 3.2× and 10× objectives and imaged. Quantification is the result of cell counts on biological triplicates of imaged membranes.

Agarose spot migration assay

HNSCC cell lines were treated in low serum media for 24 h, after which they were trypsinized, counted, and normalized for cell number between treatments. Cells were then subjected to agarose spot assay as described in Wiggins and Rappoport (18). Briefly, 0.5% agarose solution was heated to dissolution, cooled, and mixed with PBS or EGF, and spotted onto glass cover slides at concentation of 300 ng EGF/spot. Cover slides were placed in 6 well culture plates, placed at 4°C to solidify, and cell suspensions were then transferred into wells. 48 h later, spots were imaged at 10× objective and migratory cells that had passed under the horizon of the agarose spot were counted.

Invasion assays

HN30 cells were plated and treated in serum free media for 24 h, after which they were trypsinized, counted, and normalized for cell number between treatments. Cells were then subjected to one of two invasion assays. Cells were either plated in serum free media into the Cell Biolabs CBA-110 CytoSelect Basement Membrane boyden chamber, or plated in serum free media into Corning Costar transwell plates (#3422) containing 8 μm polycarbonate filter coated in 0.5× basement membrane extract (Trevigen #3455-096-02, 3455-096-03). In both cases, samples were plated with and without 10% FBS in the lower chamber as a chemoattractant, and all samples and treatments were plated in triplicate. Treatment compounds were contained in the media of both the upper and lower chambers. 48 h following upper well seeding, non-migratory cells and basement membrane coating were removed, inserts were stained, rinsed, imaged, and cells were dissociated from the membrane. Colorimetric absorbance of stained cell containing solution was read at 570 nm.

LDH assay

Cyotoxicity of AHR ligands was measured by treating HNSCC cell lines for 24 or 48 h and subjecting culture media and cell lysates to Sigma-Aldrich Tox-7 lactic dehydrogenase assay, as per manufacturer’s specifications.

Proliferation

HNSCC cells were counted and normalized, and plated at 5% confluency in 10% serum containing media in 6-well culture plates. Treatments were added 18 h after seeding, once cells were adherent. Cells were grown in a reduced serum medium (2% fetal bovine serum) from time of initial treatment up to 48 h. Cells were treated every 12 h with either 10 μM TMF or 100 nM GNF351 and at 24 h the media was changed. Cells were trypsinized and counted using a hemocytometer. HN30 cells were also grown in 16-well plates and growth monitored using an xCELLigence RTCA DP instrument (Roche) with the same media conditions.

Zymography

HN13 cells were plated in 100 mm culture plates for 24 h, changed to serum-free media and treated for 48 h, after which media was collected and cells were lysed. Amicon Centricon centrifugal filtration columns were used to concentrate media. Gelatin zymography was conducted using a modified version of the protocol described by Troeberg and Nagase (19). Briefly, samples were denatured using only SDS and run on an 8% polyacrylamide gel containing 1 mg/ml gelatin. The gel was then washed in 2.5% Triton X-100 to remove SDS, and enzymes were reactivated and allowed to digest gelatin overnight. Gel was then stained with coomassie blue and destained over 36 h.

Statistical analysis

All experiments treatments were performed in triplicate and the experiments repeated at least twice. Statistical analyses were performed using Prism 5 graphing and statistical analysis software (GraphPad Software Inc., San Diego, CA). Data were analyzed by either one-way analysis of variance, Tukey’s multiple comparison or student’s t-tests. P values <0.05 were considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001).

RESULTS

The AHR antagonist GNF351 effectively represses constitutive AHR activity in HNSCC cells

We have previously shown that multiple HNSCC cell lines exhibit a constitutively active AHR that is able to maintain the IL6 promoter in a state of de-repression by preventing HDAC occupancy (17). Treatment of these cells with 10 μM of the AHR antagonist 6,2′,4′-trimethoxyflavone (TMF) reduced basal IL6 and CYP1A1 expression. Chemical screens conducted by the Genomics Institute of the Novartis Research Foundation lead to the development of a number of high-affinity AHR antagonists, one of which is the compound GNF351 (20). Prior studies have shown that GNF351 is a more potent AHR antagonist when compared to previously available AHR antagonists. Treatment of HN30 cells with 100 nM GNF351 for 24 h led to a significant decrease in IL6 transcription (Fig 1A), similar to results seen after treatment with 10 μM TMF (11). Thus, the higher affinity AHR antagonist exhibited a similar effects on constitutive IL6 expression at a one hundred-fold lower dose. In addition, basal CYP1A1 activity is almost completely ablated following GNF351 treatment (Fig 1B). Treatment of HN2095 cells with 100 nM GNF351 for 12 h revealed identical effects on protein occupancy of the IL6 promoter as shown in our previous studies with TMF (Fig 1C) (12). The constitutive presence of the AHR upstream from the IL6 transcription start site acts to dismiss the presence of transcriptionally repressive HDAC1, and thus allows for robust NFκB-driven transcription, as shown by the enhanced presence of p65 on the IL6 promoter in the absence of GNF351 treatment. Antagonist treatment of HNSCC cells prevents the nuclear localization of AHR and subsequent heterodimerization with ARNT, leading to AHR dismissal from and return of HDAC1 to the IL6 promoter with a subsequent loss of transcriptional potential. HEK were also examined for constitutive level of IL6 expression and the effect of GNF. Results reveal that relative to HN30 cells NEK cells exhibit extremely low levels of IL6 expression with no significant effect of GNF351 on expression, in contrast to AHR antagonist repression of IL6 expression (supplemental figure S1).

Fig. 1. GNF351 is a potent AHR antagonist that reduces IL6 expression via transcriptional repression.

Serum starved HN2095 cells were treated with vehicle or 100 nM GNF351 for 12 h. The relative levels of IL6 (A) and CYP1A1 (B) mRNA were determined by quantitative real-time PCR. (C) ChIP analysis of the IL6 promoter in HN2095 cells following 12 h treatment with 100 nM GNF351.

HNSCC30 cells exhibit elevated constitutive nuclear levels of AHR in comparison to HEK

The ability of HN30 cells to constitutively express IL6 is in part due to the presence of AHR at the IL6 promoter. We wanted to assess whole cell extracts, cytosolic, and nuclear levels of the AHR in HN30 cells in reference to HEK (Fig. 2). The results revealed that HN30 cells have 7-fold more AHR and exhibited significant levels of AHR in the nucleus. In contrast, nuclear AHR was essentially undetectable in nuclear extracts of HEK under the experimental conditions used. This would suggest that AHR antagonist treatment would have a greater effect on HNSCC cells than normal keratinocytes. This underscores the potential of AHR antagonists as a targeted treatment of tumor cells relative to the surrounding tissue.

Fig. 2. HN30 cells express high overall levels of AHR, with significant levels detected in nuclei, when compared to human epidermal keratinocytes.

A, Whole cell extracts were prepared from HNSCC30 cells and HEK in triplicate. Levels of AHR and β-actin were assessed by western blot. B, Quantification of AHR protein levels shown in panel A. C, Nuclear and cytosolic fractions were isolated from HNSCC30 cells and HEK in triplicate. Levels of AHR, lamin A/C and β-actin were assessed by Western blot.

AHR antagonism has modest effects on HNSCC cellular proliferation and viability

Evidence suggests that the AHR can play a role in cellular proliferation through numerous mechanisms, including affecting cell cycle progression, pro-growth signaling, and anti-apoptotic pathways, although species- and tissue-specific differences have been noted (21). The use of potent antagonists will allow for investigations into the manner in which constitutively active AHR augments the typically aggressive phenotype of human HNSCC cells. Our initial experiments utilized a real time cell analzer to measure proliferation of HN30 cells in a dose-response study with the AHR antagonist TMF (Fig. 3A). The data revealed that TMF had no significant repressive effect on the proliferative ability through the rapid growth phase. However, treatment with 10 μM TMF did have an effect on cells after they reached the stationary phase. Since we were addressing the question as to whether AHR antagonist had an effect on rapid proliferation additional studies were performed at 24 and 48 h. Further assessments of AHR antagonism on the phenotype of HNSCC cell lines utilized the HN13 and HN30 cell lines, Cells were treated with either 10 μM TMF or 100 nM GNF351, an inhibition of proliferation was only seen with TMF at 48 h in NH13 cells (Fig. 3B,C). In order to rule out the possibility that AHR antagonism exerts a negative effect on proliferation simply by being cytotoxic, the culture supernatants from the HN13 and HN30 cells were subjected to LDH assay, a marker of cell damage (22). Neither TMF nor GNF351 showed any change in LDH levels in the culture media of HN30 over 24 or 48 h at the doses used in the proliferation study (Fig. 4). In contrast, TMF induced a significant increase in LDH levels in the media after 24 h, while at 48 h there was no additional increase in activity. No change in LDH activity was observed in HN13 cells after GNF treatment. These results would suggest that AHR antagonism does not effect cellular proliferation to a significant extend.

Fig. 3. AHR antagonist treatment fail to inhibit HNSCC proliferation.

A, HN30 cells were plated a low density into E-plates 16 and cell growth monitored using an xCELLigence system over time in the presence of various concentrations of TMF. HN30 (B) and HN30 (C) cells were plated at low confluency for 18 h, then placed in low serum media and treated with either carrier solvent (control) or 10 μM TMF or 100 nM GNF351 for 24 or 48 h. After multiple doubling times, cells were trypsinized and counted.

Fig. 4. AHR antagonists are not cytotoxic.

HN30 and HN13 cells were treated with vehicle or 10 μM TMF or 100 nM GNF for 24 or 48 h, the media was collected from the experiment in figure 3 and LDH activity levels were determined.

Optimal HNSCC cellular migration requires AHR expression

There has been a link suggested between the AHR and cellular motility or migration (23). In light of this information, AHR antagonists were analyzed for their ability to inhibit the highly migratory HN30 cell line in transwell assays. Using fetal bovine serum (FBS) as a chemoattractant, vehicle treated HN30 cells were found to undergo a significant increase in migration towards the lower chamber. Treatment with the potent agonist TCDD, had no effect on basal or chemoattractant-induced migration. In contrast, both TMF and GNF351 were able to significantly inhibit migration in the absence and the presence of FBS as a chemoattractant (Fig. 5A). In order to assess the role of the AHR in cellular migration, HN30 cells were transfected with AHR siRNA and subsequently plated for transwell migration assays. Nearly complete AHR protein ablation (Fig. 5B) resulted in a significant loss of migratory ability for HN30 cells, and one that is comparable to GNF351 treatment of control siRNA transfected cells (Fig. 5C). As expected, AHR siRNA transfection also resulted in a loss of GNF351-mediated repressive effects in FBS exposed samples.

Fig. 5. Antagonism of AHR inhibits cellular migration.

A, HN30 cells were serum starved and treated with control, TCDD, TMF, or GNF351 for 24 h, after which they were normalized and plated into a transwell migration assay using FBS as a chemoattractant. Cells were then allowed to migrate over a 48 h time period after which polycarbonate membranes were fixed, stained, and migratory cells counted. B, HN30 cells were subjected to electroporation with AHR siRNA oligonucleotides, after which cells were plated in full serum media for 24 h, then changed to serum free media for 48 h. Cells were then lysed and whole cell extract was subjected to western blotting for AHR and β actin protein levels. C, HN30 cells were electroporated with non-targeting or AHR-targeting siRNA and plated into a transwell migration assay with control or GNF351 treatment using FBS as a chemoattractant. Cells were then allowed to migrate over a 48 h time period after which polycarbonate membranes were fixed, stained, and migratory cells counted.

A cell culture based migratory assay that is more representative of a tumor microenvironment has been developed (18). Liquid agarose containing the chemoattractant EGF is spotted in cell culture plates and allowed to solidify, after which cells are plated around the spots. Given the reliance on EGF signaling for many characteristics of HNSCC cell line phenotype (24), the cells migrate towards and then under the agarose spots in an effort to reach the point of highest EGF density. HN30 cells show a significant reduction in their ability to migrate under EGF-containing agarose spots when treated with the AHR antagonists TMF or GNF351 (Fig. 6A). Note that the cells also appear more rounded after GNF351 treatment. HN13 and HN2095 also exhibited reduced migration in the agarose spot assay (Supplementary Fig. S2). Interestingly, TCDD also repressed migration in HN13 cells, but not in HN2095 cells.

Fig. 6. AHR antagonists inhibit HNSCC migration in agarose spot assay and in a transwell invasion assay.

A, HN30 cells were treated in low serum media with control, TCDD, TMF, or GNF351 for 24 h, after which they were trypsinized and plated in treatment-containing, serum free media into culture plates containing dried agarose spots with or without 300 ng EGF. Cells were then allowed to migrate towards and under agarose spots over 48 h, at the end of which the spots were imaged and migratory cells were counted. Data represents the mean of cell counts in four fields of view, with two biological replicates. B and C, HN30 cells in serum free media were treated with either TMF or GNF351 for 24 h, respectively. The cells were subsequently trypsinized and plated into transwell invasion assays. After 48 h, the number of cells on each side of the membrane was determined. D, HN13 cells were treated for 48 h in serum free media with vehicle or 10 μM TMF. After 48 h the media was collected, concentrated and protein content was determined. Samples were subjected to zymology analysis.

Treatment of HNSCC cells with AHR antagonists inhibits invasive potential

With the finding that AHR antagonist treatment of HNSCC cells prevented cellular migration, the focus then moved to determining the effect of GNF351 on invasive potential. Due to their lack of ability to migrate through a polycarbonate membrane containing 8 μm pores, HN13 cells were insufficient for use in transwell invasion assays. Therefore, HN30 cells were subjected to transwell assays in which a membrane was coated with basement membrane extract (BME). These cells showed a significant impairment in their ability to move through the BME layer and migrate through a polycarbonate filter in the presence of TMF (Fig. 6B) or GNF351 (Fig. 6C). This finding suggests that an AHR antagonist may be preventing expression of proteinases required to degrade BME components in addition to affecting migratory ability.

Matrix metalloproteinases (MMPs) are a family of proteinases commonly studied in the context of their secretion by tumor cells for the purpose of degrading extracellular matrix proteins to allow for invasion into neighboring tissue (25, 26). MMPs have been shown to play a role in HNSCC disease progression, and MMP9 in particular has even been suggested as an indicator of relapse-free survival, where higher MMP9 expression correlates with a poorer disease prognosis (27). MMP9 is a gelatinase that has also been shown to be upregulated in certain tumor types by AHR activation (28–30). While real time quantitative PCR for MMP9 expression showed a relatively low, unchanging level of mRNA in HNSCC cell lines (data not shown), gelatin zymography was used to illustrate the role of the AHR in the secretion of active MMP9. HN13 cells treated with an AHR antagonist had lower levels of MMP9 secreted into the media over 48 h (Fig. 6D).

Treatment of HNSCC cells with an AHR antagonist inhibits ABCG2 induction

The sensitivity of tumor cells to chemotherapy treatment has been shown to decrease with increased expression of membrane pumps that work in drug efflux. One component of this protein network is ATP binding cassette sub family G member 2 (ABCG2/BCRP). The AHR has been shown to be a ligand-activated transcription factor that can mediate induction of human ABCG2 transcription (31, 32). Pre-treatment of HN30 cells for 6 h with GNF351 prevented benzo[a]pyrene (B[a]P)-mediated ABCG2 transcription after an 18 h exposure (Fig. 7A). As a component of a membrane-bound drug efflux pump, ABCG2 protein levels take longer to increase in response to stimuli. Treatment of HN30 cells for 48 h with GNF351 followed by 48 h with 5 μM B[a]P revealed that GNF351 pre-treatment was also able to mitigate protein increase after 96 h (Fig. 7B).

Fig. 7. Antagonism of AHR can inhibit the upregulation of ABCG2.

A, HN30 cells were plated and serum starved with 6 h control or 100 nM GNF351 pretreatment, after which control or 5 μM B[a]P was added for a further 18 h. Total RNA was isolated, cDNA prepared, and relative ABCG2 mRNA measured by quantitative real-time PCR. B, HN30 cells were plated in low serum media with pretreatment of control or 100 nM GNF351. After 48 h, cells were further treated for 48 h with control or 500 nM B[a]P, at the end of which cells were lysed and whole cell extract was subjected to western blotting for ABCG2 and β actin protein levels.

DISCUSSION

Recent reports have shown that enhanced IL6 expression in head and neck tumor cells stimulates cell growth and epithelial-mesenchymal transition (33, 34). Our previous studies have investigated the role of the AHR in the expression of the pro-survival cytokine IL6 in tumor cells (12). We have shown in MCF-7 breast cancer cells that activation of the AHR with an exogenous ligand such as TCDD, can mediate synergistic induction of IL6 upon stimulation with IL1B (3). Several HNSCC cell lines were subsequently shown to exhibit constitutive AHR occupancy at the IL6 promoter, leading to higher basal and readily inducible IL6 expression patterns. Treatment of these cells with an AHR antagonist successfully reduced the expression of IL6 within 12 h of the initial dose (12). The potential for constitutively active AHR in HNSCC cells, in combination with the understanding that known direct AHR target genes represent only a small segment of receptor-affected genes, points to the AHR as a possible mediator of numerous pathways that enhance the aggressive nature of HNSCC cells beyond anti-apoptotic IL6 expression.

The ability of AHR antagonists to block endogenous and exogenous ligand-mediated (e.g. polycyclic aromatic hydrocarbons) receptor activity makes their use a promising method for dissecting the role of the receptor in the HNSCC phenotype. Additionally, the lack of cellular toxicity at higher doses coupled with long exposure times would suggest that TMF or GNF351 can be used in long-term experiments in vivo. Another layer of complexity is the role of the AHR in cell cycle progression and in intracellular pro-growth signaling. It is quite possible that both an AHR agonist and antagonist can alter their own unique subset of genes, which then leads to the same phenotype. The AHR has been shown to modulate the activity of the retinoblastoma protein and cyclin dependent kinase 2, both positively and negatively affecting cell cycle progression, respectively (35–37). Another example is the ability of liganded AHR to block p300 recruitment to cell cycle genes (38). Introduction of a constitutively active AHR leads to spontaneous stomach and liver tumors in mice, suggesting that sustained AHR activation leads to outgrowth of tumor cells (39, 40). Activation of the receptor through TCDD treatment has shown results similar to those seen with EGF ligand treatment, pointing to the AHR as a mediator of downstream kinase activity that mimics EGFR signaling (41, 42). The fact that EGFR signaling is amplified in numerous HNSCC cell lines points to the possibility that EGFR and AHR ligands can act cooperatively, mediating accelerated cellular growth.

HNSCC patients tend to present with later stage disease and are at a high risk of recurrence. Due to the proximal location of lymph nodes and the ability of primary tumors to migrate and metastasize, formation of secondary tumors is a common occurrence. Adjuvant treatment that can mitigate cellular migration and invasion to neighboring tissue would therefore have an impact on preventing disease progression. In two different migration assays, AHR antagonists were able to almost completely ablate migration of several HNSCC cell lines. While TCDD treatment had little effect on the movement of most HNSCC cell lines, both TMF and GNF351 prevented the ability of cell passage through an 8 μm pore-containing polycarbonate membrane, as well as cellular migration towards and under EGF-containing agarose spots. Knockdown of AHR protein in HN30 cells led to a similar outcome in the transwell migration assay, highlighting the effect of receptor expression on HNSCC cell movement. The role that the AHR plays in cytoskeletal organization and migratory ability remains unclear, but evidence has shown that AHR null fibroblasts have lower migration due to deregulated cytoskeletons, and treatment of cells with AHR ligands results in a decrease in cell-cell contact and an increase in cell-extracellular matrix contact (43, 44). One potential pathway through which these changes could occur is the AHR-mediated regulation of VAV3, which is itself a mediator of Rho GTPases and downstream cytoskeletal organization (45, 46). Clearly, the targets of AHR antagonism in HNSCC cells will need to be explored.

The AHR has been shown to affect the invasiveness of numerous cancer cells, such as the increase in ER negative breast cancer cell migration following B[a]P treatment (47). One mechanism by which this may occur centers on the effect of the AHR on MMP expression. Antagonist treatment of HN30 cells has a significant effect on their invasiveness as measured by ECM coated transwell assays. While TCDD treatment has been shown to have an effect on the expression of numerous MMPs, the most widely studied interaction involves the gelatinase MMP9 (28–30). Gelatin zymography highlights the decrease in secreted MMP9 following AHR antagonist treatment in HN13 cells. Considering the lack of a difference in mRNA levels of MMP9 upon GNF351 treatment, the mechanism of repressed MMP9 secretion would appear to be post-transcriptional.

Little progress has been made over the past decades with regard to treatment for HNSCC. The five year overall survival rate remains below 50%, and the heterogeneity of tumors under the HNSCC classification precludes a single breakthrough treatment that would target one pathway and affect all primary malignancies. HNSCC is classified as only being moderately radiosensitive, and the efficacy of chemotherapy is questionable. Recent research has shown that higher EGFR expression in HNSCC correlates with poorer efficacy of radiation treatment and assessments are ongoing using concomitant EGFR inhibitors (48). This treatment would, by definition, only be effective through one molecular mechanism and only in certain patients. However, the ability of AHR antagonist pretreatment to sensitize HNSCC cells to a more aggressive adjuvant therapy could have a large impact on treatment outcomes. For example, treatment of HNSCC cell lines with GNF351 dramatically attenuates increases in the drug efflux pump ABCG2 in the presence of benzo(a)pyrene, which is known to actively remove the chemotherapy drugs doxorubicin and mitoxantrone from the cell (49). In this way, enhanced expression of ABCG2 in cancer cells, such as in cigarette smokers, could be reduced prior to treatment, allowing for longer half-life of chemotherapeutics within the cell and/or lower chemotherapy dosages to achieve similar efficacy endpoints. Radiosensitization is another method by which nontoxic therapies can influence the outcome of HNSCC survival in patients. For example, EGFR inhibitors have been shown to increase the number of cells in G2-M phase, with S phase growth arrest following radiation treatment (50). Thus, other therapies will likely need to be coupled with AHR antagonist treatments to test whether synergistic tumor cell toxicity can be achieved.