Activation of the Aryl Hydrocarbon Receptor Leads to Resistance … · Biology of Human Tumors Activation of the Aryl Hydrocarbon Receptor Leads to Resistance to EGFR TKIs in Non–Small

Biology of Human Tumors

Activation of the Aryl Hydrocarbon ReceptorLeads to Resistance to EGFR TKIs in Non–SmallCell Lung Cancer by Activating Src-mediatedBypass SignalingMingxiang Ye1,2, Yong Zhang1, Hongjun Gao3, Yan Xu4, Pengyu Jing5, Jianxiong Wu1,Xinxin Zhang1, Jie Xiong1, Chenfang Dong6, Libo Yao2, Jian Zhang2, and Jian Zhang1

Abstract

Purpose: The aryl hydrocarbon receptor (AhR) has been gen-erally recognized as a ligand-activated transcriptional factor thatresponds to xenobiotic chemicals. Recent studies have suggestedthat the expression of AhR varies widely across different cancertypes and cancer cell lines, but its significance in cancer treatmenthas yet to be clarified.

Experimental Design: AhR expression in non–small cell lungcancer (NSCLC) was determined by Western blotting and IHCstaining. In vitro and in vivo functional experiments were per-formed to determine the effect of AhR on sensitivity to targetedtherapeutics. A panel of biochemical assays was used to elucidatethe underlying mechanisms.

Results: A high AhR protein level indicated an unfavorableprognosis for lung adenocarcinoma. Inhibition of AhR signaling

sensitized EGFR tyrosine kinase inhibitors (TKIs) in NSCLC cellsthat express high level of endogenous AhR protein. Notably,activation of AhR by pharmacologic and molecular approachesrendered EGFR-mutant cells resistant to TKIs by restoring PI3K/Akt andMEK/Erk signaling through activation of Src. In addition,we found that AhR acts as a protein adaptor to mediate Jak2–Srcinteraction, which does not require the canonical transcriptionalactivity of AhR.

Conclusions: Our results reveal a transcription-independentfunction of AhR and indicate that AhR may act as a proteinadaptor that recruits kinases bypassing EGFR and drives resis-tance to TKIs. Accordingly, targeting Src would be a strategyto overcome resistance to EGFR TKIs in AhR-activated NSCLC.Clin Cancer Res; 24(5); 1227–39. �2017 AACR.

IntroductionFor the past few decades, the aryl hydrocarbon receptor (AhR)

has been extensively recognized as a ligand-activated basic helix-loop-helix transcriptional factor that mediates metabolicresponse to environmental pollutants. In a ligand-free condition,

AhR protein is primarily localized in the cytoplasm andcomplexed with two Hsp90 molecules, the Hsp90-interactingprotein p23 and the AhR-interacting protein AIP (1). Ligandbinding causes the receptor to dissociate from its partner proteinsand leads to the release of the nuclear translocation signal (NLS)region, which triggers the shuttling of AhR protein from thecytoplasm to the nucleus and the formation of the AhR-ARNT(aryl hydrocarbon nuclear translocator) heterodimer. TheAhR–ARNT complex binds to xenobiotic response elements(XRE) in dioxin-responsive gene promoters to induce transcrip-tional activation of phase I metabolic enzymes (2, 3). However,recent studies have also suggested a transcription-independentfunction of AhR signaling. For example, AhR protein has beenproposed to regulate the balance between Treg and Th17 cells,suppress dendritic cell immunogenicity and maintain immunecell homeostasis (4–7). AhR protein also acts as a key componentof the Cul-4B E3 ubiquitin ligase complex to mediate a nonge-nomic pathway regulating estrogen receptor destruction (8, 9).The identification of kynurenine (Kyn) as an endogenousAhR ligand was a major impetus for in-depth investigation ofAhR's previously unknown function in physiologic and patho-logic events (10). Although recent studies have suggested apotential role of AhR in regulating cell proliferation, cell-cycledistribution, and cell apoptosis, the underlying mechanism islargely unknown (11).

The great success of targeted therapy in non–small cell lungcancer (NSCLC) is no doubt a model paradigm of precisionmedicine in cancer research and treatment. The clinical

1Department of Pulmonary Medicine, Xijing Hospital, Xi'an, China. 2State KeyLaboratory of Cancer Biology, Department of Biochemistry and MolecularBiology, Fourth Military Medical University, Xi'an, China. 3Department of Pul-monary Oncology, Affiliated Hospital of Academy of Military Medical Sciences,Beijing, China. 4Department of Respiratory Medicine, Peking Union MedicalCollege Hospital, Peking Union Medical College, Chinese Academy of MedicalSciences, Beijing, China. 5Department of Thoracic Surgery, Tangdu Hospital,Fourth Military Medical University, Xi'an, China. 6Department of Pathology andPathophysiology, Zhejiang University School of Medicine, Hangzhou, China.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

M. Ye, Y. Zhang, H. Gao, and Y. Xu contributed equally to this article.

CorrespondingAuthors: Jian Zhang, Department of Pulmonary Medicine, XijingHospital, Fourth Military Medical University, #169 West Changle Road, Xi'an710032, China. Phone: 8629-8477-1135; E-mail: [email protected];and Jian Zhang, State Key Laboratory of Cancer Biology, Department ofBiochemistry and Molecular Biology, Fourth Military Medical University, #169West Changle Road, Xi'an 710032, China. Phone: 8629-8477-4513; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-17-0396

�2017 American Association for Cancer Research.

ClinicalCancerResearch

www.aacrjournals.org 1227

on April 26, 2020. © 2018 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst December 11, 2017; DOI: 10.1158/1078-0432.CCR-17-0396

http://crossmark.crossref.org/dialog/?doi=10.1158/1078-0432.CCR-17-0396&domain=pdf&date_stamp=2018-2-26

http://clincancerres.aacrjournals.org/

application of EGFR inhibitors, anaplastic lymphoma kinase(ALK) inhibitors, and ROS1 kinase inhibitors markedly extendedthe progression-free survival (PFS) of patients with geneticallydefinedNSCLC (12–14).Unfortunately, targeted therapy failed toprolong the overall survival (OS) of these patients. This paradoxmay be attributed to the loss of responsiveness to the therapeuticdrugs, known as secondary resistance or acquired resistance.Resistance mechanisms that have been identified include theemergence of resistance mutations (15) and bypass signaling(16, 17). In the interest of overcoming resistance driven by thesemechanisms, increasing efforts have been focused on the devel-opment of more potent tyrosine kinase inhibitors (TKIs) andcombinational strategies, which ultimately suppress the majorcell survival–related output of tyrosine kinases, such as PI3K/AktandMEK/Erk signaling (18). However, themolecular mechanismof resistance is not fully understood; thus, elucidating resistancemechanisms would greatly help in guiding new treatment strat-egies to extend the clinical benefits of TKIs.

AhR is ubiquitously overexpressed in multiple cancer celllines and tumor samples (19). In addition, the AhR repressor(AhRR) and miRNAs posttranscriptionally targeting AhR are pre-dominantly suppressed in cancer (20–22). Direct evidence show-ing that AhR is an oncogenic driver gene was demonstrated by thefinding that transgenic mice with constitutively activated AhR(AhR CA) developed gastric cancer and hepatocellular carcinoma(23, 24). Although there is no direct evidence showing that theactivation of AhR leads to NSCLC tumorigenesis, AhR has attract-ed increasing attention in this type of cancer. For example, over-expression of AhR has been detected inmore than 50% of patientswith NSCLC, and polymorphisms of AhR gene are implicated inthe development of NSCLC (25–28). Activation of AhR signalingupregulates several growth factors, and NSCLC cells require ele-vated AhR for sustained proliferation (29, 30). Moreover, the AhRgene encodes several resistance proteins, drug metabolic enzymesand drug transporters, and it also negatively regulates the rate ofapoptosis (31–34). It is plausible that AhR is implicated in deter-mining the sensitivity of NSCLC to apoptosis-inducing agents,such as targeted therapeutics. In this study, we demonstrated thatactivation of AhR signaling leads to resistance to EGFR TKIs. Onceactivated, the AhR protein acts as an adaptor to recruit Src, whichbypasses EGFR to preserve PI3K/Akt and MEK/Erk signaling. Wetherefore propose AhR and Src kinase as novel therapeutic targetsto overcome resistance to EGFR TKIs in NSCLC.

Materials and MethodsCell lines and reagents

The human NSCLC cell lines HCC827, PC-9, H358, H292,A549, SPC-A1,H1975,H3122, andHCC78have been extensivelydescribed. All the NSCLC cell lines were obtained between 2012and 2015, authenticated by STR profiling, tested for mycoplasmacontamination and used within 10 passages. Cells were culturedin RPMI1640 (Corning) with 10% FBS (Gibco) and 1% penicil-lin/streptomycin (Invitrogen). Beas2B and HEK293 cells werecultured in DMEM (Invitrogen) with 10% FBS and 1% penicil-lin/streptomycin.

Gefitinib, afatinib, AZD9291, crizotinib, dasatinib, PP2, andruxolitinib were purchased from Selleck and dissolved in DMSOas stock solutions. a-NF and b-NF (Sigma) were dissolved inethanol. G418, doxycycline, and Kyn (Sigma) were dissolved inPBS. TCDD solution was purchased from Sigma.

Constructs, mutants, and lentivirus productionpLKO.1-shRNAs against AhR, ARNT, cyp1a1, cyp1b1, Src, and

Jak2 were obtained from the National Institute of BiologicalSciences (NIBS, Beijing, China; Supplementary Table S1). shRNAlentiviruses were produced in HEK293 cells according to thestandard protocol. Cells were infected with the indicated lenti-viruses and selected with 2 mg/mL puromycin for 2 weeks. Togenerate doxycycline-inducible shRNAs, we cloned complemen-tary DNA oligos for AhR into the pSingle-tTS-shRNA vector(Clontech). Cells were transfected with the pSingle-tTS-AhRshRNA (Dox-on/AhR shRNA) plasmid and selected with100mg/mL G418. To conditionally knock down AhR expression,we added doxycycline at a final concentration of 1mg/mL to cellculture medium supplemented with 10% doxycycline system-approved FBS (Clontech).

The AhR ORF with an N-terminal Flag tag was inserted into thePstI/XhoI restriction sites of the pcDNA4 vector. The AhR WTDNLS, AhR CA, AhR CA DNLS, AhR WT DSH2, DAA2–200,DAA201–400, DAA401–600, and DAA601–800 mutants weregenerated with a site-directed mutagenesis kit (SupplementaryTable S2). To generate an AhR lentivirus, we subcloned the Flag-tagged AhR fragment into a pWPI vector (laboratory of Feng Shaoat NIBS, Beijing, China) at the PmeI restriction site. Lentivirusproduction, cell infection, and selection were performed asdescribed elsewhere. The Myc-tagged Jak2 plasmid was generatedby standard PCR amplification and was sequenced in full.HA-tagged Src WT and Src Y527F mutants were obtained fromSiyuan Zhang (University of Notre Dame, Notre Dame, IN).

RNA isolation and qPCRRNA extraction and cDNA synthesis were performed following

the standard protocol. qPCRwas performedwith SYBR Premix ExTaq (Takara). The relative amount of each gene transcript wasnormalized to the amount of b-actin transcript. The qPCR primersequences are listed in Supplementary Table S3.

Protein extraction, Western blot, kinase array, andimmunoprecipitation

Total cell protein was extracted using RIPA lysis buffer (Pierce)supplemented with a protease inhibitor cocktail (Roche). Forthe extraction of membrane protein, the cell pellet was treatedwith Mem-PER Membrane Protein Extraction Reagent (Pierce)following manufacturer's instructions. Protein concentration was

Translational Relevance

Recent studies have suggested that AhR functions in mul-tiple pathways outside of its canonical role in detoxification.Here, we described AhR as a novel therapeutic target for casesof NSCLC that express high levels of endogenous AhR. Acti-vation of AhR promotes resistance to EGFR TKIs by activatingSrc-mediated bypass signaling. In this model of resistance,AhR acts as a protein adaptor to recruit Src and Jak2 kinases,rescuing PI3K/Akt andMEK/Erk signaling. Taken together, ourresults provide proof-of-principle insights into a novel resis-tance mechanism driven by AhR and suggest that targeting Srcrepresents a new strategy to overcome resistance to targetedtherapy.

Ye et al.

Clin Cancer Res; 24(5) March 1, 2018 Clinical Cancer Research1228




measured with a bicinchoninic acid kit (Clontech), and theprotein was boiled in SDS-PAGE loading buffer. The kinase arraywas performed with a Phospho Kinase Array Kit (R&D Systems,#ARY003B) following the manufacturer's instructions. Briefly, atotal quantity of 300 mg of whole-cell lysate (WCL) was incubatedwith nitrocellulose membranes containing 43 phosphorylatedkinase antibodies printed in duplicate overnight at 4�C on arocking platform shaker. Themembranes werewashedwithWashBuffer, and then incubated at room temperature with DetectionAntibody Cocktail A/B for 2 hours and Streptavidin-HRP Reagentfor 30minutes at room temperature. The phosphorylated proteinsignal was developed with Chemi Reagent Mix.

An immunoprecipitation assay was performed as describedelsewhere. Briefly, the lysates were precleared and incubatedwith the indicated primary antibodies overnight with gentlerotation at 4�C. The immunocomplex was pelleted with ProteinA/G agarose beads (Santa Cruz Biotechnology), then resus-pended, boiled in SDS-PAGE loading buffer, and subjected toWestern blot analysis.

Equal amounts of all protein samples were loaded and runon SDS-PAGE gel, transferred onto a nitrocellulose membrane(Millipore), blocked with skim milk, and incubated with theindicated antibodies overnight. The membrane was washedwith TBST and incubated with HRP-conjugated IgG. Proteinbands were visualized by enhanced chemiluminescence(Millipore). The source and dilution of antibodies were listedin Supplementary Table S4.

Xenograft modelsApproximately 5 � 106 cells were suspended in 100 mL of

Matrigel (BD Biosciences) and subcutaneously injected into theflanks of 6-week-old athymic nude mice (Laboratory AnimalCenter of Fourth Military Medical University, Xi'an, China;FMMU).When the tumors reached appropriate size, the indicatedtreatments were initiated. Tumor growth was monitored andrecorded every three days. At the end of the experiment, the micewere humanely sacrificed, and the tumors were carefully isolatedand processed for histologic studies. All the animal experimentswere conducted in compliance with institutional guidelines andapproved by the Animal Care and Use Committee of FMMU(Xi'an, China).

Bioinformatic analysis and patient samplesTo assess the association between AhR mRNA expression

and NSCLC grade, metastasis, and prognosis, we performedbioinformatics analysis following the guidance of the TCGAdatabase.

Fifty-five patients who underwent surgical resection of lungadenocarcinoma from 2005 to 2013 were followed up andretrospectively screened for AhR protein expression. Four pairedsamples of primary lung adenocarcinoma (not overlapping withthe cases used for the tissue array) and metastatic lymph nodeswere obtained from patients undergoing lung cancer surgery atTangdu Hospital (Fourth Military Medical University, Xi'an, Chi-na). Another five patients with EGFR-mutant NSCLC whorelapsed on EGFR TKIs and underwent repeated biopsy were alsoenrolled. Expression of AhR protein was determined by IHC andWestern blotting. The experiment using human specimens wasapproved by the Ethics Committee of FMMU.Awritten documentillustrating the experimental design and purpose was sent to eachparticipant so that informed consent could be obtained.

Immunofluorescent staining and IHCAfter the indicated treatments, cells were fixed with 4% para-

formaldehyde and permeabilized with Triton X-100. The cellswere then incubated with primary antibodies overnight at 4�C,then with Cy3-conjugated IgG for 2 hours at room temperature.The cells were counterstained with DAPI, and fluorescent signalwas detected by a confocal laser microscope (Nikon).

The IHC and TUNEL assays were performed as describedpreviously. The tumors were cut into 5-mm-thick sections, whichwere mounted on slides and incubated with the indicated anti-bodies or TUNEL reagents (Roche). The slides were then thor-oughly washed and visualized with DAB or FITC. The AhR IHCstaining density was scored as negative (score 0), weakly positive(score 1), moderately positive (score 2), and strongly positive(score 3). The percentage of cancer cells positive for AhR expres-sion was assigned a proportion score (<5%, score 0; 6%–25%,score 1; 26%–50%, score 2; 51%–75%, score 3;>75%, score 4). Toquantitative evaluate AhR protein expression, we multiplied theintensity score by the proportion score to yield the H-score. Themedian H-score was set as the cut-off value to separate NSCLCspecimens with low and high AhR expression.

Statistical analysisValues were expressed as the mean � SD. The software pro-

grams GraphPad Prism 5 and Origin 6.1 were used to analyze thedata. A paired t-test and one-way ANOVAwere employed to assessthe statistical significance of differences between different groups.A statistically significant difference was defined as �, P < 0.05 or ��,P < 0.01.

ResultsAhR protein predicts poor prognosis and constitutes atherapeutic target for NSCLC

To investigate whether AhR correlates with NSCLC prognosis,we initially examined AhR expression at the mRNA level in theTCGA database. AhR gene amplification was more frequent inNSCLC, especially in lung adenocarcinoma, than in SCLC andreached approximately 4% overall frequency (SupplementaryFig. S1A). However, AhR mRNA was not associated with lungadenocarcinoma stage, lymph node infiltration, metastasis, orrecurrence (Supplementary Fig. S1B). Consistently, AhR mRNAdid not correlate with lung adenocarcinoma patients' survival(Supplementary Fig. S1C). To investigate whether AhR at theprotein level carried prognostic value, we examined surgicallyresected lung adenocarcinoma samples from 57 patients forwhomup to 8 years of follow-updatawere available. IHC stainingof normal lung tissues was performed to assess the baselineendogenous expression of AhR protein. As a positive control,human placenta tissues from two individuals were used. Thepatients were well balanced in terms of their characteristics, whichare listed in Table 1. Twenty-six of the 57 cases (46%) showed ahigh level of AhR protein expression (Fig. 1A; SupplementaryTable S5), which is consistent with previous IHC studies reportingAhR overexpression in 40% to 50% of NSCLC cases (25–27, 30).Positive AhR staining was prominently observed in the nuclei ofthe NSCLC cells (Fig. 1A), indicating the activation of AhRsignaling in these patients. Correlation analysis revealed that highAhR expression was associated with an aggressive tumor pheno-type (Fig. 1B). Strikingly, seven of these 26 AhRhigh patients haddisease recurrence with distant metastasis, whereas none of

Aryl Hydrocarbon Receptor and Resistance to Targeted Therapy

www.aacrjournals.org Clin Cancer Res; 24(5) March 1, 2018 1229




the 31 AhRlow patients developed local recurrence or metastasis(Supplementary Table S5). Western blot analysis of anotherfour paired primary lung adenocarcinomas andmetastatic lymphnodes showed that AhR protein tended to increase moresharply in themetastatic region (Fig. 1C) than the primary region.Log-rank analysis further revealed that high expression of AhRprotein was associated with reduced overall survival in this smallcohort (Fig. 1D).

We next analyzed AhR expression in NSCLC cell lines(HCC827, PC-9, H358, H292, A549, SPC-A-1, and H1975). Thehuman normal bronchoalveolar epithelial cell line Beas-2B wasused. Western blot analysis showed a significant increase in AhRexpression in SPC-A-1 and H1975 cells, whereas its expression inHCC827, PC-9, H358, H292, and A549 cells was comparable tothat in Beas2B cells (Fig. 1E). These NSCLC cells were furtherdivided into AhRhigh (SPC-A-1 and H1975 cells) and AhRlow

(HCC827, PC-9, H358, H292, and A549 cells) subtypes, accord-ing to their endogenous levels of AhR. Notably, the AhRhigh cellsalso exhibited elevated expression of AhR downstream genes,including cyp1a1 and cyp1b1, while the AhRlow cells did not(Supplementary Fig. S1D). To determine the biological signifi-cance of AhR in NSCLC cell proliferation and survival, we treatedthe AhRhigh and AhRlow cells with the AhR antagonist a-naphtho-flavone (a-NF). Interestingly, the AhRhigh cells were sensitive toa-NF, with an IC50 less than 1 mmol/L. In contrast, the AhRlow

PC-9 cells were refractory to the same agent, with an IC50 over10mmol/L (Supplementary Fig. S1E).Moreover, activation ofAhRsignaling by its ligands tetrachlorodibenzo-p-dioxin (TCDD),b-naphthoflavone (b-NF), and Kyn promoted H1975 cell prolif-eration (Supplementary Fig. S1F) and rendered growth indepen-dent of anchorage (Supplementary Fig. S1G), indicating that AhRis a tumor-promoting signaling molecule in NSCLC.

Persistent PI3K/Akt and MEK/Erk signaling activation is prev-alent in tumors and contributes to cancer cell survival andoutgrowth. Indeed, inhibition of EGFR and ALK signaling bycorresponding TKIs efficiently abolished the phosphorylation ofAkt and Erk, thereby leading to cancer cell apoptosis. In contrast,selective inhibition of either pathway failed to elicit anapoptotic response (Supplementary Fig. S1H). To characterize

the underlying molecular alterations following AhR inhibition inNSCLC, we treated cells with increasing concentrations of a-NFand evaluated the phosphorylation levels of Akt and Erk byimmunoblotting. The AhRlow PC-9 cells were resistant to a-NF,and the treatment had no effect on the phosphorylation of Akt orErk (Fig. 1F). However, a-NF exhibited single-agent efficacy inPI3K/Akt andMEK/Erk signaling inhibition and apoptosis induc-tion in the AhRhigh SPC-A-1 and H1975 NSCLC cells. Consistentwith the observation that H1975 cells had higher levels of endog-enous AhR, H1975 cells tended to bemore sensitive to a-NF thanSPC-A-1 cells, as the treatment induced more cleaved form ofPARP and caspase-3 (Fig. 1F). This pharmacologic effect may notrely on interference with EGFR, as a-NF exerted minimal effectson EGFR phosphorylation in the three tested cell lines. Takentogether, these results suggested that AhR is a potential druggabletarget in NSCLC, in which inhibition of AhR signaling predom-inantly leads to AhRhigh cell apoptosis by reducing Akt and Erkphosphorylation.

Inhibition of AhR sensitizes H1975 cells to EGFR TKIsTo confirm the data obtained from pharmacologic experi-

ments, we next inhibited AhR expression in H1975 cells by useof shRNAs. In agreement with the potent oncogenic function ofAhR, two independent AhR shRNAs significantly reduced H1975cell proliferation (Supplementary Fig. S2A).Western blot analysisshowed that knockdown of AhR readily inhibited phosphoryla-tion of Akt and Erk but not EGFR (Fig. 2A).Moreover, conditionalknockdown of AhR in H1975 cells led to apoptosis (Supplemen-tary Fig. S2B), highlighting the pivotal role of AhR inmaintainingsurvival and proliferation in this AhRhigh NSCLC cell line.

Because theAhRhighH1975 cells also harbored anEGFRL858R/T790Mmutation, it is plausible that AhR cooperates with mutantEGFR to induce oncogenic dependence. Thus, simultaneous inhi-bition of both pathways is expected to lead to a completeabrogation of PI3K/Akt and MEK/Erk signaling and induce moreprofound apoptosis. As shown in Fig. 2B, afatinib and a-NFexerted single-agent efficacy in apoptosis induction; however,neither drug alone completely blocked PI3K/Akt and MEK/Erksignaling. Strikingly, their combination led to a complete block-age of Akt and Erk phosphorylation and a sharp increase inapoptosis induction. In further support of this notion, afatinibin doses as low as 0.1 mmol/L efficiently blocked PI3K/Akt andMEK/Erk signaling in AhR knockdown cells, whereas afatinib at1 mmol/L failed to do so in shRNA control cells (Fig. 2C),suggesting that inhibition of AhR signaling sensitized H1975cells to afatinib. This effect was recapitulated in vivo: AlthoughH1975 GFP shRNA xenograft tumors were responsive to afatinib,the AhR shRNA xenograft tumors exhibited a much more dra-matic response as early as day 6 and achieved nearly completeregression on day 21 (n ¼ 6 in each group, Fig. 2D). The AhRshRNA tumors also underwent a higher prevalence of apoptosis,as judged by TUNEL immunofluorescence (Fig. 2E). Together,these data point to inhibition of AhR signaling as a potentialstrategy to sensitize NSCLC cells to EGFR TKIs.

Dysregulation of AhR signaling leads to EGFR TKI resistanceHaving established that inhibition of AhR signaling impaired

the phosphorylation of Akt and Erk and sensitized H1975 cells toafatinib, we next investigated whether activation of AhR signalingin EGFR-mutant NSCLC would render resistance to EGFR TKIs.IHC staining of AhR protein was performed in five pairs of

Table 1. Baseline characteristics of patients with lung adenocarcinoma

GenderMale 32Female 25

AgeMin 29Max 76Average 56.4

Smoking statusSmokers 21Nonsmokers 36

GradeI 15II 20III 22

Lymph nodeN0 29N1 19N2 9

Follow-upAlive 26

Died 31Total 57

Ye et al.





paraffin-embedded lung adenocarcinoma samples, with each pairconsisting of a treatment-na€�ve biopsy sample and a repeatedbiopsy sample. All the enrolled patients were positive for EGFRmutation and received first-line EGFR TKI treatment. Repeatedbiopsy was performed when the patients developed diseaseprogression; in our cases, an average PFS of 10.6 months was

achieved. Patients No. 1, No. 3, and No. 4 were found to have aT790M resistance mutation. Interestingly, IHC staining showedincreased AhR H-scores in the repeated biopsy samples of patientNo. 3 and patient No. 4, whereas patient No. 1 had comparableAhR expression in the treatment-na€�ve and repeated biopsy sam-ples. More strikingly, patients No. 2 and No. 5 had no evidence of

Figure 1.

AhR protein as an unfavorable prognostic factor and therapeutic target for NSCLC. A, Representative images of AhR IHC staining in placenta, lung andlung adenocarcinoma tissue. B, Quantitative analysis of AhR protein expression in relation to lung adenocarcinoma stage and lymph node infiltration.C, Western blot analysis of AhR protein expression in primary lung adenocarcinoma and paired metastatic lymph node. An equal amount of b-actin was usedas the loading control. D, Log-rank survival analysis of AhR protein expression and lung adenocarcinoma patients' overall survival (OS). E, Western blotanalysis of AhR, pSrc, Src, pJak2, and Jak2 expression in NSCLC cell lines. An equal amount of b-actin was used as the loading control. G, The AhRlow (PC-9)cells and AhRhigh (SPC-A-1 and H1975) cells were treated with increasing concentrations of an AhR inhibitor, a-NF (0, 1, 5 and 10 mmol/L), for 24 hours andevaluated for apoptosis and EGFR signaling.






T790M mutation or c-Met amplification as potential causes ofresistance, while patient No. 5 also had increased AhR expressionin the repeated biopsy samples (Fig. 3A).

To further investigate the biological consequences of AhRoverexpression, we established cell lines stably overexpressingAhR. In comparison with a control cell line overexpressing GFP(termed PC-9 GFP cells), a cell proliferation assay showed accel-erated growth of PC-9 cells overexpressing AhR (termed PC-9 AhRWT cells, Supplementary Fig. S3A); thus, we proposed that AhRsignaling may rescue EGFR-mutant PC-9 cells from EGFR inhi-bition. Indeed, the PC-9 GFP cells remained responsive to gefi-tinib at nanomole potency, whereas the PC-9 AhR WT cells wereinsensitive to the same drug, as judged by a short-term cellviability assay (Fig. 3B) and a long-term colony formation assay(Fig. 3C). Furthermore, resistance driven by AhR was specific toTKIs because stable overexpression of AhR did not alter sensitivityto chemotherapeutics, such as cisplatin and paclitaxel (Supple-mentary Fig. S3B). To our initial surprise, cells overexpressing theconstitutively activated form of AhR (termed PC-9 AhR CA cells)failed to show resistance to gefitinib (Fig. 3B and C). This paradoxled us to evaluate EGFR and downstream PI3K/Akt and MEK/Erksignaling across the tested cell lines. It was noted that overexpres-sion of AhR WT efficiently increased the baseline levels of phos-phorylated Akt and Erk without affecting the phosphorylation

level of EGFR. However, overexpression of AhR CA marginallyenhanced PI3K/Akt andMEK/Erk signaling (Fig. 3D). Consistent-ly, gefitinib at 0.1 mmol/L readily abolished Akt and Erk phos-phorylation in PC-9 GFP and PC-9 AhR CA cells, whereas Akt andErk phosphorylation persisted after gefitinib treatment in PC-9AhR WT cells (Fig. 3E).

Apart from overexpression, the hyperactivation of AhR signal-ing also results from elevated content of AhR ligands. To deter-mine whether AhR ligand treatment also elicits a TKI-resistantphenotype, we treated H1975 cells with b-NF and Kyn. Althoughthemost potent EGFR TKI, AZD9291, is designed to overcome theT790M-resistant mutation, AhR ligand preconditioning renderedH1975 cells resistant to AZD9291, as determined by PARPand caspase-3 cleavage (Fig. 3F). A consensus finding was theincrease in Akt and Erk phosphorylation, which could not befully inhibited by AZD9291 (Fig. 3F). Collectively, these datahighlighted activation of AhR signaling as a resistancemechanismin NSCLC, presumably through the restoration of PI3K/Akt andMEK/Erk signaling.

AhR activates Src kinase bypass signaling independent of itstranscriptional activity

To eliminate any possible "off-target" effect of the AhR ligand,we tested whether AhR was required for the maintenance of

Figure 2.

Inhibition of AhR sensitizes NSCLC cellsto EGFR TKIs. A, AhR expression inH1975 cells was downregulated byshRNAs, and the resulting cells wereevaluated for EGFR signaling and Srcphosphorylation. B, H1975 cells weretreated with 1 mmol/L afatinib, 5 mmol/La-NF or both drugs for 24 hours andprobed with the indicated antibodies.C, Cells were treated with increasingconcentrations of afatinib (0, 0.1,0.5, and 1 mmol/L) for 6 hours, andphosphorylation of EGFR, Akt, andErk were determined by Western blotanalysis. D and E, Six-week-old athymicnude mice were subcutaneouslyinjected with H1975 GFP shRNA andH1975 AhR pLKO.1-shRNA.1 cells andtreated with vehicle or 100 mg/kgafatinib (n ¼ 6 in each group). Tumorsize was measured for approximately3 weeks. Representative images oftumor-bearing mice are shown. At theend of the experiment, xenografttumors were isolated and analyzedfor apoptosis by TUNEL.Scale bar ¼ 200 mm.

Ye et al.





Figure 3.

AhR activation confers resistance to EGFR TKIs in NSCLC. A, Representative images of AhR staining in five pairs of EGFR-mutant treatment-na€�ve andEGFR-TKI–relapsed NSCLC samples. Scale bar ¼ 200 mm. B and C, Evaluation of the effects of AhR WT and AhR CA on sensitivity to gefitinib by short-termcell viability assay and long-term colony formation assay, respectively. D, AhR WT and AhR CA mutant were stably overexpressed in PC-9 cells; theresulting cells were lysed and probed with indicated antibodies. PC-9 GFP cells were used as an overexpression control. E, PC-9 cells overexpressing GFP,AhRWT or AhR CA were treated with increasing concentrations of gefitinib (0, 0.1, 0.5, and 1 mmol/L) for 6 hours and analyzed for EGFR and downstream signaling.F, H1975 cells were treated with 0.1 mmol/L AZD9291 for 24 hours. Two hours prior to AZD9291 treatment, cells were treated with 10 mmol/L b-NF or 100 mmol/LKyn to activate AhR signaling. At the end of these treatments, cells were evaluated for apoptosis and EGFR signaling by Western blot.






PI3K/Akt and MEK/Erk signaling. To maximally reduce back-ground phosphorylation signal, we cultured the H1975 GFPshRNA and H1975 AhR shRNA cells in FBS-deprived mediumovernight and treated them with 10 nmol/L TCDD for 15 and30 minutes, respectively. As shown in Fig. 4A, phosphorylationof Akt and Erk increased sharply with as little as 15 minutes ofTCDD treatment, while this effect was entirely abolished inAhR-deficient cells. As expected, cyp1a1 and cyp1b1 mRNAtranscription levels at this early time point were unchanged(Supplementary Fig. S4A). Confocal laser scanning of H1975

GFP shRNA cells showed a cytoplasmic distribution pattern ofAhR protein following 15 and 30 minutes of TCDD treatment(Supplementary Fig. S4B), suggesting that the transcriptionalmachinery may not be dispensable for increased Akt and Erkphosphorylation. Consistently, abolishing AhR's transcription-al activity by removal of the AhR NLS region or knockdown ofARNT still facilitated TCDD-induced Akt and Erk phosphory-lation (Fig. 4B). Furthermore, shRNA-mediated knockdown ofAhR target genes, such as cyp1a1 and cyp1b1, had minimaleffects on pAkt and pErk induction (Fig. 4C). These results

Figure 4.

Activation of AhR activates Src independently of its transcriptional activity. A, H1975 cells carrying indicated shRNAs were treated with 10 nmol/L TCDD for15 and 30 minutes. Levels of phosphorylated Akt, Erk, and Src were determined by Western blot analysis. B, HEK293 cells were transfected with Flag-taggedAhR WT DNLS mutant or shRNA against ARNT, treated with 10 nmol/L TCDD for 30 minutes, and subjected to Western blotting. C, H1975 cells stablycarrying cyp1a1 or cyp1b1 shRNA were treated with 10 nmol/L TCDD for 12 hours and analyzed for Akt and Erk phosphorylation. D, PC-9 cells carrying mCherryor Src Y527F were treated with DMSO or 1 mmol/L gefitinib for 6 hours and subjected to Western blot analysis. E, PC-9 AhR WT cells were treated withgefitinib and AhR ligands with or without the Src kinase inhibitors dasatinib and PP2 for 24 hours. Cleavage of PARP and caspase-3 and phosphorylation ofSrc, Akt and Erk were determined by immunoblotting. F, Src expression in PC-9 AhR WT cells was knocked down by three independent shRNAs. Theresulting cells were treated with DMSO or 1 mmol/L gefitinib for 6 hours and subjected to Western blot analysis.

Ye et al.





strongly indicated that AhR is crucial for TCDD-induced Aktand Erk phosphorylation, independently of its canonical tran-scriptional activity.

The nonreceptor tyrosine kinase Src has been reported as acommon downstream node of multiple resistance pathways, andconcurrently targeting Src overcomes resistance to targeted ther-apy in breast cancer and chronic myeloid cell leukemia (35, 36).Although there was no difference in total Src protein expressionacross the tested cell lines, an impressive finding was that theAhRhigh cells had a considerable amount of phosphorylated Src atresidue Tyr416 (Y416) while the AhRlow cells did not (Fig. 1E). Inagreement with this, activation of the AhR pathway by over-expression assay or ligand treatment readily increased Src Y416phosphorylation (Fig. 3D), whereas AhR silencing compromisedthis effect (Fig. 2A). These findings imply that Srcmay be involvedin AhR-driven resistance to EGFR TKIs in NSCLC. To addresswhether Src activation is sufficient to confer TKI resistance, westably overexpressed its constitutively activated Src Y527Fmutantin three representative genetically defined NSCLC cell lines (PC-9cells harboring an EGFR delE746_A750 mutation, H3122 cellsharboring an EML4-ALK rearrangement and HCC78 cells harbor-ing an SLC34A2-ROS1 rearrangement). Compared with controlcells overexpressingmCherry, the Src Y527F cells exhibited hyper-phosphorylation of Src Y416 (Fig. 4D; Supplementary Fig. S4C).The Src Y527F cells responded poorly to TKIs (gefitinib andcrizotinib in this study), and the treatment failed to abolishPI3K/Akt and MEK/Erk signaling (Fig. 4D; SupplementaryFig. S4D). Thus, enhanced Src phosphorylation renders NSCLCresistant to targeted therapy, and it is plausible that Src is theintermediate signaling hub linking AhR to TKI resistance. Indeed,pharmacologic inhibition of the Src pathway diminished Akt andErk phosphorylation and overcame gefitinib resistance in PC-9AhR WT cells (Fig. 4E). shRNA-mediated Src silencing also sen-sitized cells to gefitinib and facilitated apoptotic response(Fig. 4F). Collectively, these data strongly indicated that enhancedSrc phosphorylation is a critical event following activation of AhRsignaling, which bypasses EGFR to restore PI3K/Akt andMEK/Erksignaling and contributes to EGFR TKI resistance.

Jak2 phosphorylates Src upon AhR activationWe have established that AhR restores Akt and Erk phosphor-

ylation through activation of Src kinase; however, how Srcbecomes phosphorylated in cells with active AhR signalingremains to be elucidated. Src kinase can bind to protein adaptorsthat present the Y-X-X-N motif (Supplementary Fig. S5A). Forexample, hepatocyte growth factor (HGF) binding to the c-Metreceptor tyrosine kinase leads Src to bind to Gab1, Gab2, Grb2,and other adaptors, thus initiating full activation of the c-Metsignaling cascade (37, 38). Alignment analysis of the AhR proteinsequence indicated a highly conserved putative SH2 domainbinding motif within its C-terminal proportion that may providedocking site(s) for Src recruitment (Fig. 5A). Immunoprecipita-tion assays with both endogenous AhR (Fig. 5B) and ectopicallyexpressed AhR (Fig. 5C) showed that binding between AhR andSrc was enhanced by TCDD, whereas this interaction was dra-matically diminished upon removal of the SH2 domain bindingmotif (Fig. 5C). While AhR is not a protein kinase, Src Y416phosphorylation cannot be a direct biological consequence of itsbinding to AhR. To investigate how Src became phosphorylated,we used phospho-protein kinase array profiling in AhR ligand–treated cells and vehicle-treated control cells. This analysis

revealed increased phosphorylation of 17 proteins (�2-foldchange) in the ligand-treated cells (Fig. 5D; Supplementary Fig.S5B). Consistent with our previous observation, the kinase arrayrevealed hyperactivation of Src, Akt, and Erk after AhR ligandtreatment. Strikingly, we found a significant increase in thephosphorylation of all the Stat family proteins, including Stat2,Stat3, Stat5b, and Stat6, in the AhR ligand–treated cells (Supple-mentary Fig. S5B). On the basis of these findings, we proposedthat protein kinases upstreamof the Stat pathway are very likely tomediate Src phosphorylation.

Numerous studies have shown that Stat proteins are primarilyphosphorylated by Janus kinase 2 (Jak2, not represented in thekinase array) (39–41). Thus, we determined whether Jak2 medi-ated Src phosphorylation. We found that activation of AhRsignaling readily increased Jak2 Y1007/1008 phosphorylation(Supplementary Fig. S5C). Inhibition of Jak2 phosphorylationby ruxolitinib, a selective orally available small-molecule inhib-itor evaluated in phase I/II clinical trials, abolished Src Y416phosphorylation (Fig. 5E). Moreover, knockdown of Jak2impaired TCDD-induced Src-Akt/Erk signaling in H1975 andPC-9 AhR WT cells (Supplementary Fig. S5D). These findingsindicate a causal relationship between Jak2 and Src phosphory-lation; therefore, we speculated that targeting Jak2 would blockAhR-induced Src phosphorylation and overcome TKI resistance.In PC-9AhRWT cells, the combinationof gefitinib and ruxolitinibresulted in cell apoptosis, whereas neither drug exerted single-agent efficacy in apoptosis induction (Supplementary Fig. S5E).Although ruxolitinib was capable of inhibiting Src bypass signal-ing, the PI3K/Akt and MEK/Erk signaling pathways persistedowing to mutant EGFR. The combination of gefitinib and rux-olitinib efficiently blocked "EGFR pathway signaling" and "Jak2-Src bypass signaling," leading to the blockade of Akt and Erk(Supplementary Fig. S5E). Taken together, these results stronglyindicated that Jak2 is one of the upstream kinases responsible forSrc phosphorylation. Targeting Jak2 would overcome TKI resis-tance in NSCLC activated by Src signaling.

Finally, to address the significance of AhR in mediating theSrc–Jak2 interaction, we expressed Flag-tagged AhR, HA-taggedSrc, and Myc-tagged Jak2 in HEK293 cells. AhR activation byTCDD readily increased Src binding to Jak2, whereas this inter-action was lost upon AhR depletion (Fig. 5F). To map the regionresponsible for Jak2 binding, we transfected HEK293 cells withexpression vectors for Myc-tagged Jak2 together with Flag-taggedfull-length, DAA2–200, DAA201–400, DAA401–600, or DAA601–800 truncated forms of AhR. Immunoprecipitation of cell lysateswith anti-Flag antibody and probing of the resultant precipitateswith anti-Myc antibody revealed that TCDD triggeredMyc-taggedJak2 in association with substantial amounts of Flag-tagged AhRconstructs, with the exceptions of DAA2–200 and DAA201–400(Fig. 5G), indicating that AhR was implicated as an intermediateprotein linking Src to Jak2 through its SH2domain–bindingmotifand N-terminal segment of AA2–400, respectively.

DiscussionThis study describes a novel mechanism of resistance to tar-

geted therapy. We showed that activation of AhR signaling drivesresistance to EGFR TKIs in NSCLC independently of its canonicaltranscriptional activity. In this model, AhR acts as a proteinadaptor to mediate the cross-talk between Src and Jak2 kinases,which bypass EGFR to restore PI3K/Akt and MEK/Erk signaling






(Fig. 6). These findings serve as a new paradigm of nongenomiceffects of AhR and indicate a new strategy to overcome resistanceto targeted therapy.

In addition to mediating the cytotoxicity of TCDD and otherenvironmental pollutants, AhR protein also facilitates tumori-genesis throughmultiplemechanisms involving disruption of thecell cycle, evasion of apoptosis, and suppression of immunesurveillance. Therefore, inhibition of AhR signaling by directlytargeting the AhR protein or targeting its ligands is expected toinduce tumor regression. Indeed, the AhR antagonist a-NF(a pseudoligand that binds to the Ah receptor to form a non-functional complex) exhibits single-agent efficacy in inhibitingcell-cycle progression (42) and inducing apoptosis in cancer cellsexpressing high level of endogenous AhR (as shown in this study).In addition, the indoleamine 2,3-dioxygenase (IDO) and trypto-phan-2,3-dioxygenase (TDO) inhibitors that inhibit Kyn, anendogenous AhR ligand, potentiate chemotherapy and immuno-therapy, and clinical trials evaluating their safety and efficacyin cancers are currently underway (43–45). Interestingly, theAhRhigh cells tend to have a much higher magnitude of Kynsynthase expression than the AhRlow cells (Supplementary

Fig. S1D), enabling constitutive activation and amplification ofthe IDO/TDO-Kyn-AhR signaling loop. Testing AhR expressionand Kyn synthase activity may help to predict the responsivenessof cancers to IDO/TDO/AhR inhibitors and chemotherapy.

Our study implies a compelling role of AhR in determining thesensitivity of NSCLC to EGFR TKIs. This is a surprising findingbecause AhR is not a protein kinase and is not associated withresistance mutations, while activation of AhR restores PI3K/Aktand MEK/Erk signaling despite inhibition of EGFR. This is not anentire recapitulation of the c-Met amplification resistance mech-anism because AhR lacks kinase activity and aberrant phosphor-ylation of Akt and Erk may not be a direct consequence of AhRactivation. Our data reveal a rational explanation of how thekinase activity–defective AhR protein preserves PI3K/Akt andMEK/Erk signaling, in which AhR and targeted therapy resistanceconverge at the nonreceptor tyrosine kinase Src. As highlighted byZhang and colleagues, activation of Src is a common biologicalevent downstream of multiple resistance pathways; targetedtherapy resistance in ErbB2-positive breast cancer andBCR-ABL–positive chronic myeloid cell leukemia can be over-come by concurrently targeting Src (35, 36). We have found that

Figure 5.

AhR activates Src through Jak2 kinase. A, Alignment analysis of the AhR protein SH2 domain binding motif. B, After H1975 cells were treated with 10 nmol/LTCDD for 30 minutes, endogenous AhR and Src protein were coprecipitated and their interaction was determined by immunoblotting. C, Flag-tagged AhRWT or DSH2 mutant and HA-tagged Src were coexpressed in HEK293 cells. Cells were treated with 10 nmol/L TCDD for 30 minutes. Immunoblotting analysisof anti-HA tag precipitate was performed accordingly. D, Representative image of phospho-protein kinase array profiling of AhR-ligand-treated andnon-AhR-ligand–treated H1975 cells. Phospho-proteins with more than 2-fold changes are numbered. E, H1975 cells were treated with increasingconcentrations of ruxolitinib (0, 0.1, 0.5, and 1 mmol/L), an orally available Jak2 inhibitor, for 6 hours, and subjected to Western blot analysis. F, HEK293 cellswere transfected with HA-tagged Src, Myc-tagged Jak2 and Flag-tagged AhR plasmids and treated with 10 nmol/L TCDD for 30 minutes. WCL wasprecipitated with anti-HA tag antibody and probed with the indicated antibodies. G, Expression plasmids for Myc-tagged Jak2 and Flag-tagged AhR or itsdeletion mutants were introduced into HEK293 cells. Cells were treated with 10 nmol/L TCDD for 30 minutes, and WCL was subjected to immunoprecipitationwith anti-Flag tag antibody.

Ye et al.





Src phosphorylation markedly increases following the activationof AhR signaling and that Src inhibition resensitizes AhR WT–overexpressing cells to gefitinib. Thus, Src also emerges as atherapeutic target to overcome resistance driven by AhR inNSCLC.Althoughothers have alsodemonstrated that AhR ligandstreatment enhance Src phosphorylation (46–48), our study pro-vides a proof-of-principle insight into the molecular machineryresponsible for Src activation. In our model, the binding of AhRprotein to a ligand recruits cytosolic Src protein in a transcrip-tionally independent manner. The AhR–Src complex transientlytranslocates to the cell membrane, where AhR provides dockingsites for Jak2 to phosphorylate Src (Fig. 6; SupplementaryFig. S6A). This result is consistent with findings from recentstudies reporting that Jak2 directly phosphorylates Src andimpacts the behavior of cancer cells (49, 50). Importantly, wefurther demonstrated that the AhR protein adaptor is essential forJak2–Src interaction in NSCLC. In contrast to AhR WT, which isprominently localized in the cytoplasm, the AhR CAmutant doesnot require ligand treatment for translocation to the nucleus(Supplementary Fig. S6B), leading to the loss of its ability torecruit cytosolic Src kinase. The removal of NLS resulted inredistribution of the AhRCAprotein into the cytoplasm, enablingits binding to Src and the restoration of PI3K/Akt and MEK/Erksignaling (Supplementary Fig. S6B and S6C). These distributionpatterns explain the differential effects of AhRWT and AhR CA ongefitinib sensitivity. However, AhR protein membrane transloca-tion is an immediate and transient response to ligand-induced

signaling activation, Src phosphorylation persists regardless ofsubsequent AhR protein nuclear translocation. One possibleexplanation for this discrepancy is the structure and propertiesof Src kinase: once Src Y416 phosphorylation is triggered byupstream kinases, it undergoes autophosphorylation of this tyro-sine residue and persists in Src signaling activity (51). Thus,AhR-mediated Src phosphorylation by Jak2 is an initiation signalfor constitutively active Src signaling that bypasses EGFR to restorePI3K/Akt and MEK/Erk signaling (Supplementary Fig. S6D).Again, this places Src at a common signaling node for targetedtherapy resistance in NSCLC and may have important clinicalimplications. The availability of Src and Jak2 inhibitors makes itpossible to overcome EGFR TKI resistance in patients whosetumors express high endogenous levels of AhR and depend onJak2/Src kinase activity for persistent PI3K/Akt and MEK/Erksignaling activation.

Taken together, our findings highlighted a novel resistancemechanism and identified Src activation as the key resistanceoutput of AhR signaling. More importantly, more treatmentoptions may be discovered by identifying protein adaptors, suchas AhR, that catalyze persistent PI3K/Akt and MEK/Erk signalingrather than solely focusing on the alteration of kinases. It will beinteresting to determine whether AhR activation also contributesto resistance to ALK TKIs, ROS1 TKIs, and other targeted thera-peutics in NSCLC.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: L. Yao, J. Zhang, J. ZhangDevelopment of methodology: M. Ye, Y. Zhang, P. Jing, L. Yao, J. Zhang,J. ZhangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Ye, Y. Zhang, H. Gao, Y. Xu, P. Jing, X. Zhang,J. ZhangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Ye, Y. Xu, J. Wu, C. Dong, J. ZhangWriting, review, and/or revision of the manuscript: Y. Xu, L. Yao, J. Zhang,J. ZhangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X. Zhang, J. Xiong, J. Zhang, J. ZhangStudy supervision: L. Yao, J. Zhang, J. Zhang

AcknowledgmentsThis work was sponsored by grants from the National Natural Science

Foundation of China (#81472192 and #81272518, to J. Zhang). We wouldlike to thank Jeffrey Engelman (Massachusetts General Hospital Cancer Center,Boston, MA) for supplying NSCLC cell lines and Siyuan Zhang (University ofNotre Dame, Notre Dame, IN) for supplying HA Src WT and HA Src Y527Fconstructs. We also express gratitude to Chia-Hsin Chan (Stony Brook Univer-sity, Stony Brook,NY) and Siyuan Zhang for helpful discussion and suggestions.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 9, 2017; revised July 8, 2017; accepted December 5, 2017;published OnlineFirst December 11, 2017.

References1. Carlson DB, Perdew GH. A dynamic role for the Ah receptor in cell

signaling? Insights from adiverse group of Ah receptor interacting proteins.J Biochem Mol Toxicol 2002;16:317–25.

2. Ikuta T, Eguchi H, Tachibana T, Yoneda Y, Kawajiri K. Nuclear localizationand export signals of the human aryl hydrocarbon receptor. J Biol Chem1998;273:2895–904.

Figure 6.

A schematic diagram of AhR protein adaptor–mediated Jak2-Src interaction.In the AhR OFF condition, the AhR protein is localized in the cytoplasm andforms an inactive complex with its partner proteins, including Hsp90, AIP, andp23. Cell-survival–related PI3K/Akt and MEK/Erk signaling in EGFR-mutantNSCLC could be blocked by the EGFR TKI gefitinib. In contrast, activation ofAhR signaling (AhR ON) leads to conformation changes of the AhR protein,which results in enhanced binding with the nonreceptor Src kinase. The AhRprotein transiently translocates to the cell membrane and acts as an adaptorto mediate the cross-talk between Jak2 and Src. Jak2 phosphorylates Srcto bypass mutant EGFR to restore PI3K/Akt and MEK/Erk signaling andelicits a TKI-resistant phenotype.






3. Kanno Y, Miyama Y, Takane Y, Nakahama T, Inouye Y. Identification ofintracellular localization signals and of mechanisms underlining thenucleocytoplasmic shuttling of human aryl hydrocarbon receptor repres-sor. Biochem Biophys Res Commun 2007;364:1026–31.

4. Veldhoen M, Hirota K, Westendorf AM, Buer J, Dumoutier L, Renauld JC,et al. The aryl hydrocarbon receptor links TH17-cell-mediated autoimmu-nity to environmental toxins. Nature 2008;453:106–9.

5. Quintana FJ, Basso AS, Iglesias AH, Korn T, Farez MF, Bettelli E, et al.Control of T(reg) and T(H)17 cell differentiation by the aryl hydrocarbonreceptor. Nature 2008;453:65–71.

6. Nguyen NT, Kimura A, Nakahama T, Chinen I, Masuda K, Nohara K, et al.Aryl hydrocarbon receptor negatively regulates dendritic cell immunoge-nicity via a kynurenine-dependent mechanism. Proc Natl Acad Sci U S A2010;107:19961–6.

7. Li Y, Innocentin S, Withers DR, Roberts NA, Gallagher AR, Grigorieva EF,et al. Exogenous stimuli maintain intraepithelial lymphocytes via arylhydrocarbon receptor activation. Cell 2011;147:629–40.

8. Ohtake F, Baba A, Takada I, Okada M, Iwasaki K, Miki H, et al. Dioxinreceptor is a ligand-dependent E3ubiquitin ligase.Nature2007;446:562–6.

9. WormkeM, StonerM, Saville B,Walker K,AbdelrahimM,Burghardt R, et al.The aryl hydrocarbon receptor mediates degradation of estrogen receptoralpha through activation of proteasomes. Mol Cell Biol 2003;23:1843–55.

10. Opitz CA, Litzenburger UM, Sahm F, Ott M, Tritschler I, Trump S, et al. Anendogenous tumour-promoting ligand of the human aryl hydrocarbonreceptor. Nature 2011;478:197–203.

11. Puga A, Ma C, Marlowe JL. The aryl hydrocarbon receptor cross-talks withmultiple signal transduction pathways. Biochem Pharmacol 2009;77:713–22.

12. Mok TS, Wu YL, Thongprasert S, Yang CH, Chu DT, Saijo N, et al. Gefitinibor carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med2009;361:947–57.

13. Shaw AT, Kim DW, Nakagawa K, Seto T, Crin�o L, Ahn MJ, et al. Crizotinibversus chemotherapy in advanced ALK-positive lung cancer. N Engl J Med2013;368:2385–94.

14. Shaw AT, Ou S-HI, Bang Y-J, Camidge DR, Solomon BJ, Salgia R, et al.Crizotinib in ROS1-rearranged non-small-cell lung cancer. N Engl J Med2014;371:1963–71.

15. Kobayashi S, BoggonTJ,DayaramT, J€annePA,KocherO,MeyersonM, et al.EGFR mutation and resistance of non-small-cell lung cancer to gefitinib.N Engl J Med 2005;352:786–92.

16. Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO, et al.MET amplification leads to gefitinib resistance in lung cancer by activatingERBB3 signaling. Science 2007;316:1039–43.

17. Ye M, Zhang X, Li N, Zhang Y, Jing P, Chang N, et al. ALK and ROS1 astargeted therapy paradigms and clinical implications to overcome crizo-tinib resistance. Oncotarget 2016;7:12289–304.

18. Engelman JA. TargetingPI3K signalling in cancer: opportunities, challengesand limitations. Nat Rev Cancer 2009;9:550–62.

19. Tsuchiya Y, Nakajima M, Itoh S, Iwanari M, Yokoi T. Expression of arylhydrocarbon receptor repressor in normal human tissues and inducibilityby polycyclic aromatic hydrocarbons in human tumor-derived cell lines.Toxicol Sci 2003;72:253–9.

20. Zudaire E, Cuesta N, Murty V,Woodson K, Adams L, Gonzalez N, et al. Thearyl hydrocarbon receptor repressor is a putative tumor suppressor gene inmultiple human cancers. J Clin Invest 2008;118:640–50.

21. Huang TC, Chang HY, Chen CY, Wu PY, Lee H, Liao YF, et al. Silencing ofmiR-124 induces neuroblastoma SK-N-SH cell differentiation, cell cyclearrest and apoptosis throughpromotingAHR. FEBS Lett 2011;585:3582–6.

22. Oshida K, Vasani N, Thomas RS, Applegate D, Gonzalez FJ, Aleksunes LM,et al. Screening a mouse liver gene expression compendium identifiesmodulators of the aryl hydrocarbon receptor (AhR). Toxicology 2015;336:99–112.

23. Andersson P, McGuire J, Rubio C, Gradin K, Whitelaw ML, Pettersson S,et al. A constitutively active dioxin/aryl hydrocarbon receptor inducesstomach tumors. Proc Natl Acad Sci U S A 2002;99:9990–5.

24. Moennikes O, Loeppen S, Buchmann A, Andersson P, Ittrich C, PoellingerL, et al. A constitutively active dioxin/aryl hydrocarbon receptor promoteshepatocarcinogenesis in mice. Cancer Res 2004;64:4707–10.

25. Lin P, ChangH, TsaiWT,WuMH, Liao YS, Chen JT, et al. Overexpression ofaryl hydrocarbon receptor in human lung carcinomas. Toxicol Pathol2003;31:22–30.

26. Lin P, Chang H, Ho WL, Wu MH, Su JM. Association of aryl hydrocarbonreceptor and cytochrome P4501B1 expressions in human non-small celllung cancers. Lung Cancer 2003;42:255–61.

27. Tsay JJ, Tchou-Wong KM, Greenberg AK, Pass H, Rom WN. Arylhydrocarbon receptor and lung cancer. Anticancer Res 2013;33:1247–56.

28. Kim JH, Kim H, Lee KY, Kang JW, Lee KH, Park SY, et al. Aryl hydrocarbonreceptor gene polymorphisms affect lung cancer risk. Lung Cancer2007;56:9–15.

29. Wang CK, Chang H, Chen PH, Chang JT, Kuo YC, Ko JL, et al. Arylhydrocarbon receptor activation and overexpression upregulated fibro-blast growth factor-9 in human lung adenocarcinomas. Int J Cancer 2009;125:807–15.

30. Chang JT, Chang H, Chen PH, Lin SL, Lin P. Requirement of arylhydrocarbon receptor overexpression for CYP1B1 up-regulation andcell growth in human lung adenocarcinomas. Clin Cancer Res 2007;13:38–45.

31. Tan KP, Wang B, Yang M, Boutros PC, Macaulay J, Xu H, et al. Arylhydrocarbon receptor is a transcriptional activator of the human breastcancer resistance protein (BCRP/ABCG2). Mol Pharmacol 2010;78:175–85.

32. Quadri SA, Qadri AN, Hahn ME, Mann KK, Sherr DH. The bioflavonoidgalangin blocks aryl hydrocarbon receptor activation and polycyclic aro-matic hydrocarbon-induced pre-B cell apoptosis. Mol Pharmacol 2000;58:515–25.

33. Marlowe JL, Fan Y, Chang X, Peng L, Knudsen ES, Xia Y, et al. The arylhydrocarbon receptor binds to E2F1 and inhibits E2F1-induced apoptosis.Mol Biol Cell 2008;19:3263–71.

34. ChopraM, SchrenkD.Dioxin toxicity, aryl hydrocarbon receptor signaling,and apoptosis-persistent pollutants affect programmed cell death. Crit RevToxicol 2011;41:292–320.

35. Zhang S, Huang W-C, Li P, Guo H, Poh S-B, Brady SW, et al. Combatingtrastuzumab resistance by targeting SRC, a common node downstream ofmultiple resistance pathways. Nat Med 2011;17:461–9.

36. PuttiniM, Coluccia AM, Boschelli F, Cleris L,Marchesi E, Donella-Deana A,et al. In vitro and in vivo activity of SKI-606, a novel Src-Abl inhibitor,against imatinib-resistant Bcr-Ablþ neoplastic cells. Cancer Res 2006;66:11314–22.

37. Bolanos-Garcia VM. MET meet adaptors: functional and structural impli-cations in downstream signalling mediated by the Met receptor. Mol CellBiochem 2005;276:149–57.

38. Chan PC, Chen YL, Cheng CH, Yu KC, Cary LA, Shu KH, et al. Srcphosphorylates Grb2-associated binder 1 upon hepatocyte growth factorstimulation. J Biol Chem 2003;278:44075–82.

39. Quintas-Cardama A, Verstovsek S. Molecular pathways: Jak/STATpathway: mutations, inhibitors, and resistance. Clin Cancer Res 2013;19:1933–40.

40. Vainchenker W, Constantinescu SN. JAK/STAT signaling in hematologicalmalignancies. Oncogene 2013;32:2601–13.

41. Yu H, Pardoll D, Jove R. STATs in cancer inflammation and immunity: aleading role for STAT3. Nat Rev Cancer 2009;9:798–809.

42. Reiners JJ, Clift R, Mathieu P. Suppression of cell cycle progression byflavonoids: dependence on the aryl hydrocarbon receptor. Carcinogenesis1999;20:1561–6.

43. Muller AJ, DuHadaway JB, Donover PS, Sutanto-Ward E, Prendergast GC.Inhibition of indoleamine 2,3-dioxygenase, an immunoregulatory targetof the cancer suppression gene Bin1, potentiates cancer chemotherapy. NatMed 2005;11:312–9.

44. Dolusic E, Larrieu P, Moineaux L, Stroobant V, Pilotte L, Colau D, et al.Tryptophan 2,3-dioxygenase (TDO) inhibitors. 3-(2-(pyridyl)ethenyl)indoles as potential anticancer immunomodulators. J Med Chem2011;54:5320–34.

45. UyttenhoveC, Pilotte L, Th�eate I, Stroobant V, ColauD, Parmentier N, et al.Evidence for a tumoral immune resistance mechanism based on trypto-phan degradation by indoleamine 2,3-dioxygenase. Nat Med 2003;9:1269–74.

46. DongB,ChengW, LiW, Zheng J,WuD,Matsumura F, et al. FRET analysis ofprotein tyrosine kinase c-Src activation mediated via aryl hydrocarbonreceptor. Biochim Biophys Acta 2011;1810:427–31.

47. Randi AS, SanchezMS, Alvarez L, Cardozo J, Pontillo C, Kleiman de PisarevDL. Hexachlorobenzene triggers AhR translocation to the nucleus, c-Src


Ye et al.




activation and EGFR transactivation in rat liver. Toxicol Lett 2008;177:116–22.

48. Backlund M., Ingelman-Sundberg M. Regulation of aryl hydrocarbonreceptor signal transduction by protein tyrosine kinases. Cell Signal2005;17:39–48.

49. Liang K, Esteva FJ, Albarracin C, Stemke-Hale K, Lu Y, Bianchini G, et al.Recombinant human erythropoietin antagonizes trastuzumab treat-

ment of breast cancer cells via Jak2-mediated Src activation and PTENinactivation. Cancer Cell 2010;18:423–35.

50. Ingley E, Klinken SP. Cross-regulation of JAK and Src kinases. GrowthFactors 2006;24:89–95.

51. Roskoski R Jr. Src kinase regulation by phosphorylation anddephosphorylation. Biochem Biophys Res Commun 2005;331:1–14.






2018;24:1227-1239. Published OnlineFirst December 11, 2017.Clin Cancer Res Mingxiang Ye, Yong Zhang, Hongjun Gao, et al. Src-mediated Bypass Signaling

Small Cell Lung Cancer by Activating−to EGFR TKIs in Non Activation of the Aryl Hydrocarbon Receptor Leads to Resistance

Updated version

10.1158/1078-0432.CCR-17-0396doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2017/12/09/1078-0432.CCR-17-0396.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/24/5/1227.full#ref-list-1

This article cites 51 articles, 14 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/24/5/1227To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-17-0396

http://clincancerres.aacrjournals.org/content/suppl/2017/12/09/1078-0432.CCR-17-0396.DC1

http://clincancerres.aacrjournals.org/content/24/5/1227.full#ref-list-1

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/24/5/1227


Documents

Activation of the Aryl Hydrocarbon Receptor Leads to Resistance … · Biology of Human Tumors Activation of the Aryl Hydrocarbon Receptor Leads to Resistance to EGFR TKIs in Non–Small