Introduction

The aryl hydrocarbon receptor (AhR), a ligand-operated transcription factor, is a xenosensor traditionally associated with xenobiotic metabolism1. AhR facilitates tumor progression, disease tolerance defense, intestinal immunity, and B-cell proliferation2,3,4,5. Interestingly, AhR influences the major stages of tumorigenesis, and studies of aggressive tumors and tumor cell lines have shown increased levels of AhR protein and constitutive nuclear localization in cancer tissue, whereas in normal tissues AhR is mainly inactive and resides in the cytoplasm6,7.

The activation of nuclear factor (NF)-κB leads to a protumorigenic inflammatory microenvironment, and the IκB-kinase (IKK) complex, which consists of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ, tightly regulates the NF-κB pathway8,9. Whereas, in most malignancies, the classical IKKβ/IKKγ-dependent NF-κB activation controls key functions for tumor initiation, promotion, and progression in tumors10. The role of IKKα is more complex in noncanonical NF-κB pathway11,12. Depending on the type of malignancy, IKKα can provide both tumor-promoting and tumor-suppressive mechanisms that are in most instances cell autonomous30.

AhR, a widely expressed nuclear receptor that senses environmental stimuli and modulates target gene expression, plays a critical role in breast CSCs etc31,32. AhR influences the major stages of tumorigenesis and chemoresistance33,34,35, and studies of aggressive tumors and tumor cell lines have shown increased levels of AhR protein and constitutive nuclear localization in cancer tissue, whereas in normal tissues AhR is mainly inactive and resides in the cytoplasm6,7. Here we found elevated AhR level in the nucleus linked with radioresistance in LC and NPC cells, indicating that activation of AhR contributes to cancer progression, stem-like properties, and radioresistance. Nuclear IKKα transcriptionally upregulates several miRNAs, contributing to chemoresistance36. Recently, mixed lineage kinase 4 binds and phosphorylates IKKα, leading to activation of NF-κB signaling in glioma stem cells37. Here we show that nuclear IKKα targets the promoters of stem-like genes as a partner of AhR, contributing to radioresistance, indicating a critical role of nuclear IKKα in cancer progression and radioresistance. Moreover, we found that the protein level of AhR, not mRNA level, increased in radioresistant sublines.

There is an opposed effect of IKKα in several types of cancer in the process of carcinogenesis. For example, inhibition of IKKα prolongs survival and suppresses the occurrence of metastatic diseases in models of mammary, prostate, and colorectal cancers38,39,40,41,42, whereas, IKKα controls metastasis in breast and prostate cancers and is required for mammary tumorigenesis through expansion of tumor-initiating cells39,40,41. In contrast, IKKα acts as a tumor suppressor in models of skin or lung (Squamous cell carcinoma) SCC, while loss of IKKα enhances susceptibility to carcinogen-induced SCC in the skin and leads to the development of spontaneous lung SCC43,44. Recently, we show that IKKα is diversely expressed in keratinizing and non-keratinizing carcinomas in the same type of cancer13. We also provide evidence that IKKα is absent in SCC of the skin while it is expressed in (Basal-cell carcinoma) BCC, indicating the opposed roles of IKKα in skin cancer between SCCs and BCCs, which originate from both keratinocyte tumors25. Moreover, SCC development is generally associated with cell dedifferentiation that IKKα is reversely involved in the process. Here we show the critical role of IKKα in the regulation of stemness-related genes in radioresistant cells through an interaction with AhR.

Epithelial–mesenchymal transition (EMT) is a key factor of cancer progression, metastasis, and self-renewal of CSCs45,46,47. TGF-β tumor suppression functions through an EMT-mediated disruption of a lineage-specific transcriptional network48. Interestingly, EMT is not required for metastasis but contributes to chemoresistance49,50. However, we did not find the classical features of EMT and (mesenchymal-epithelial transition) MET in the stem-like radioresistant cells, but a hybrid EMT/MET state (data not shown). This EMT/MET hybrid state is thought to be bidirectional displaying a gradient of partial states toward either extreme EMT or extreme MET, and further supports the importance for metastasis and metastasis-initiating cells as well as circulating tumor cells in the epithelial and mesenchymal composition51,52,53,54, and it also supports that an intermediate EMT state may make cells more prone to exhibiting stem-like properties54. Our findings indicate that stemness seems to be better manifested at intermediate epitheloid states, consistent with those observed in cultured mammospheres. In particular, tumor-associated fibroblasts may be responsible for inducing EMT by secreting TGF-β that can stimulate proliferation and lead to expansion of the preexisting CSC pool55,56, even though we did not find that TGF-β changed in radioresistant cells.

The AhR pathway can cross talk with other major signaling pathways that might be modulated by oncometabolites that are critical in cancer progression21,22,23. Diverse metabolites serve as cofactors or substrates for enzymes that are involved in the deposition or exchange of epigenetic marks, driving a metabolite-driven pathway of gene regulation as well as distinct cancer tissue types24,57,58,59. Also, organ-specific differences were observed in the metabolite levels of the TCA cycle and other intermediates58,60. 2-HG is a competitive inhibitor of α-KG-dependent dioxygenases in gliomas and hematological malignancies that carry mutations of isocitrate dehydrogenase genes (IDH1 and IDH2)61,62. However, we did not find mutations of IDH1 and IDH2 genes and accumulation of 2-HG in NPC and LC (data not shown). We found that the level of kynurenine, which is involved in tryptophan catabolism, remained the same in radioresistant sublines compared to P sublines, indicating that kynurenine did not further activate AhR signaling pathway, but increased the generation of α-KG oncometabolite in radioresistant sublines, and in turn induced stem-like signature gene expression by AhR in a dependent manner. Here, we report that α-KG can activate the AhR signaling pathway leading to elevated stemness gene expression to influence the pluripotency state, and we provide evidence of a direct link between cellular metabolism, AhR signaling, and radioresistance associated with stem-like property.

Taken together, we elucidated the crucial role of AhR in the maintenance of radioresistance and stem-like property involving in direct activation of self-renewal via stemness genes, leading to metabolic reprogramming. Our data showed that stemness reprogramming controlled by metabolism can modulate radioresistance and tumor progression.

Material and methods

Cell culture, antibodies, plasmids, siRNAs, and chemicals

Lung adenocarcinoma cell lines A549-P/H358-P and its irradiation-resistant cell lines A549-IR/H358-IR were provided by **nming Deng (Emory University School of Medicine and Winship Cancer Institute of Emory University, Atlanta, USA). A549-P/IR cell lines were maintained in DMEM/F12 and RPMI 1640 medium, respectively, which were supplemented with 10% fetal bovine serum (FBS). NPC cell line HK1 was used to establish ionizing radiation resistant cell line (HK1-IR), as described63. Briefly, HK1 cells were serially irradiated with 4 Gy of X-rays to a final dose of 80 Gy using XRAD320 (Precision X-ray, Inc., North Branford, CT). HK1-P/IR cell lines were maintained in RPMI 1640 medium. All cell lines tested negative for mycoplasma contamination and were maintained at 37 °C with 5% CO2. All cell lines were authenticated by short tandem repeat profiling and were passaged less than ten times after initial revival from frozen stocks.

Primary antibodies as indicated for Western analysis were purchased from Cell Signaling (Danvers, MA). Primary antibodies for AhR and β-actin were purchased from Sigma-Aldrich (St. Louis, MO). GV248-shAhR lentivirus plasmids were purchased from Genechem (www.genechem.com.cn). RNAi sequences used in these studies are provided (shAhR-1: CACAACAATATAATGTCTT; shAhR-2: TTCTTTGATGTTGCATTAA; shAhR-3: AATGATTAAGACTGGAGAA; shAhR-4: ATAATAACTCCTCAGACAT). The chemicals, citrate, α-KG, 2-HG, fumarate, malate, succinate, oxalacetic acid, 2-isopropylmalic acid, methanol, N,O-bis(trimethylsilyl) trifluoroacetamide, and trimethylcholorosilane and its dimethyl products were purchased from Sigma-Aldrich (St. Louis, MO). The chemicals AhR agonist V, VAF347 (Cat# 182690), and AhR antagonist (Cat# 182705) were purchased from Calbiochem.

Western blot analysis and co-immunoprecipitation (Co-IP) assay

Details of the western blot analysis and Co-IP assay were described previously24,29. A detailed procedure is presented in the Supplementary Material and Methods section. The following list of antibodies was used for western blot detection: AhR and IKKα antibodies.

Quantitative real-time PCR

Cells were harvested with Trizol (Invitrogen). cDNAs were synthesized with SuperScript III (Invitrogen) according to the manufacturer’s protocol. Real-time PCR analysis was performed using the Applied Biosystems 7500 Real-Time PCR System, according to the manufacturer’s instructions. The reactions were performed in triplicates for three independent experiments: the results were normalized to β-actin. For qPCR, an SYBR Green-based method was used and the relative quantitation of gene expression was determined using the comparative CT (ΔΔCT) method and normalized to the β-actin gene.

The PCR primer sequences used are given in the Supplementary Table S1. The mean ± SD of three independent experiments is shown.

Cell proliferation assay, migration and invasion assay, and plate-colony formation assay

Details of the cell proliferation assay were described previously29. For plate-colony formation assay, cells (2 × 103/ml/well) were seeded into 6-well plates and cultured in RPMI-1640 medium supplemented with 10% FBS. Colonies were fixed with methanol and stained with viola crystalline, then scored using a microscope and Image J software (1.47V, NIH, USA).

The migration assay was described previously24. Cells (5 × 105) were seeded onto the upper chamber in 200 μl of serum-free medium; the lower compartment was filled with 0.6 ml of DMEM media supplemented with 10% of FBS. After 24 h incubation, migrated cells on the lower surface of the filter were fixed and stained using propidium iodide. Cells on the upper side were removed using a rubber scraper. Fluorescent images were obtained. The reported data represent the counts of migrated cells. Experiments were performed in triplicates.

For the plate-colony formation assay, cells (1 × 103/ml/well) were seeded into 6-well plates and cultured in RPMI-1640 medium supplemented with 10% FBS. Colonies were fixed with methanol, stained with viola crystallina and scored using a microscope and ImageJ software (1.47V, NIH, USA). Surviving colonies were counted and the surviving fraction (SF) was calculated using the formula SF = treatment colony numbers/control colony numbers.

Soft-agar colony forming assay

To assess anchorage-independent growth, soft-agar colony forming assay was performed using 8000 cells per well. A layer of agar containing 3.0 ml of 0.6% soft agar (BD Biosciences, USA) in (basement membrane extract) BME was poured into wells of a 6-well cell culture dish and allowed to set at room temperature for 30 min. A second layer containing 1 ml 0.35% soft agar in BME containing cells (800 cells/ml) was placed on the top of the first layer and allowed to set at room temperature for 30 min. Cells were incubated in an incubator at 37 °C for 14 days, and the number of colonies were counted, and the images were captured using an Olympus microscope.

Oncosphere formation assay

Cells were seeded on ultra-low attachment culture dishes (Corning, Corning, NY) in serum-free DMEM-F12 medium containing 50 μg/ml insulin (Sigma-Aldrich St. Louis, MO), 0.4% Albumin Bovine Fraction V (Sigma-Aldrich St. Louis, MO), N-2 Plus Media Supplement (Life Technologies, Grand Island, NY), B-27 Supplement (Life Technologies, Grand Island, NY), 20 μg/ml EGF (PeproTech Rocky Hill, NJ), and 10 μg/ml basic FGF (PeproTech, Rocky Hill, NJ) to support the growth of undifferentiated oncospheres. Cells were incubated in a CO2 incubator for 1–2 weeks, and the numbers of oncosphere cells were counted under a microscope.

Immunofluorescence assay and Operetta® High Content Screening and High Content Analysis

Details of the immunofluorescence assays were described previously24,29, cells were cultured and fixed in 4% paraformaldehyde for 30 min. To identify the potential presence of AhR and IKKα, cells were incubated with an anti-AhR antibody (Sigma) and an anti-IKKα antibody (Active motif) and then with fluorescein isothiocyanate (FITC)-conjugated anti-IgG (Santa Cruz) and Cy3-conjugated anti-IgG (Sigma). To visualize the nuclei, the cells were stained with Hoechst (1:1000). Fluorescent images were observed and analyzed with a laser scanning confocal microscope (Bio-Rad MRC-1024ES).

For Operetta® High Content Screening and High Content Analysis, cells were grown on the 96-well plate. After incubation overnight, the cells were washed with phosphate-buffered saline (PBS) and fixed with methanol for 10 min at 37 °C. The cells were incubated in PBS supplemented with 1% BSA for 1 h at room temperature. The cells were incubated overnight with E-cadherin mouse monoclonal antibody (1:100 dilution, abcam 1416) or Vimentin rabbit monoclonal antibody (1:200 dilution, CST 5741s) at 4 °C. The cells were washed three times in PBS and then stained with Alexa Fluor 594 Goat Anti-Mouse IgG or Alexa Fluor 594 Goat Anti-Rabbit IgG (1:4000 dilution, Invitrogen) for 1 h at room temperature. After incubation with DAPI to stain the nuclei, the cells were imaged on the Operetta® High Content Imaging System and analyzed using Harmony® High Content Image Analysis Software (USA).

ALDEFLUOR assay, flow cytometry, and cell sorting

Cells were stained directly using different antibodies (including CD338-PerCP-Cy™5.5, CD326-BV510) according to the manufacturer’s instructions. Labeled cells were detected using a FACSCalibur (BD Immunocytometry Systems, CA, USA) and analyzed with Flowjo software. IgG isotype controls corresponding to each directly conjugated fluorophore were utilized to identify, quantify, and positively select desired cell populations. Debris and cell clusters were excluded during side-scatter and forward-scatter analyses. For cell sorting, cells were stained and followed by sorting with FACS Aria III (BD Immunocytometry Systems, CA, USA). The separated populations were used for RNA and animal experiments.

The ALDEFLUOR kit (StemCell Technologies Inc.) was used as described previously64 and with the guidance of Dr. Suling Liu (Fudan University, Shanghai, China).

Chromatin immunoprecipitation (ChIP) assays

ChIP assays were essentially performed as previously described24,29 with modifications: 5 × 106 cells were fixed with formaldehyde (1% final volume concentration, Sigma), 10 min at room temperature. Fixation was stopped by the addition of 1/10 volume 1.25 M glycine and incubated for 5 min at room temperature. The sonication step was performed in a Qsonica sonicator (5 min, 20 s on, 20 s off), and 200 μg of protein–chromatin complex was used for each immunoprecipitation. Antibody–protein complex was captured with preblocked dynabeads protein G (Invitrogen). ChIP DNA was analyzed by qPCR with SYBR Green (Biorad) in ABI-7500 (Applied Biosystems) using the primers specified in Supplemental Table S2. The antibodies used are as indicated in Supplemental Table S3.

Nude mice and study approval

A xenograft tumor formation was essentially performed as previously described26, whereas the Hunan SJA laboratory Animal Co. Ltd. (http://www.hnsja.com) provided the mice. SCID mice were injected with indicated cells (1 × 106 cells/mouse or as indicated) via mammary fat pad or tail vein (6 mice/group or as indicated). Mice with A549-IR cells or indicated cells were imaged from dorsal and ventral views once per week. All mice (6 of 6) injected with cells via the tail developed the expected lung metastatic lesions within 8 weeks. Visible lung metastatic nodules were examined macroscopically or detected in paraffin-embedded sections stained with H&E. Data were analyzed using Student’s t-test; a p value < 0.05 was considered significant.

All procedures for animal study were approved by the Institutional Animal Care and Use Committee of the Central South University of **angya School of Medicine and conform to the legal mandates and federal guidelines for the care and maintenance of laboratory animals.

NCI-Frederick is accredited by AAALAC International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the “Guide for Care and Use of Laboratory Animals (National Research Council; 1996; National Academy Press; Washington, DC).

Immunohistochemistry (IHC) analysis and in situ hybridization of tumor biopsies

NPC biopsies, validated by pathologist Dr. Desheng **ao (**angya Hospital), were obtained from the Department of Pathology of **angya Hospital. The NPC tissue array was purchased from Pantomics (Richmond, CA, USA). IHC analysis of paraffin sections from NPC patient or xenograft samples was performed as described previously26,65. In situ hybridization was performed using the EBV-encoded RNA (EBER) HRP conjugated probe and DAB as substrate from the ISH kit (Life technologies), according to the manufacturer’s instructions.

Statistics

The experiments were repeated at least three times except the nude mice experiments. Results are expressed as mean ± SD or SEM as indicated. All statistical analyses were performed using Prism 6.0 GraphPad Software. Significant differences between two groups were compared using the Student’s t-test, and comparisons among more than two groups were performed using analysis of variance (ANOVA). The correlation analysis was conducted using Pearson’s correlation coefficient. A p value less than 0.05 was considered statistically significant.

Additional information can be found in the Supplementary Materials and Methods section.