MyD88-Dependent Signaling Decreases the Antitumor …cancerres.aacrjournals.org/content/canres/75/8/1657.full.pdf · Antitumor Efﬁcacy of Epidermal Growth Factor ReceptorInhibitioninHeadandNeckCancerCells

Therapeutics, Targets, and Chemical Biology

MyD88-Dependent Signaling Decreases theAntitumor Efficacy of Epidermal Growth FactorReceptor Inhibition in Head andNeck Cancer CellsAdam T. Koch1,2, Laurie Love-Homan1,2, Madelyn Espinosa-Cotton2,3, Aditya Stanam2,4,and Andrean L. Simons1,2,3,4,5

Abstract

EGFR is upregulated in the majority of head and necksquamous cell carcinomas (HNSCC). However, many patientswith HNSCC respond poorly to the EGFR inhibitors (EGFRI)cetuximab and erlotinib, despite tumor expression of EGFR.Gene expression analysis of erlotinib-treated HNSCC cellsrevealed an upregulation of genes involved in MyD88-depen-dent signaling compared with their respective vehicle-treatedcell lines. We therefore investigated whether MyD88-dependentsignaling may reduce the antitumor efficacy of EGFRIs inHNSCC. Erlotinib significantly upregulated IL6 secretion inHNSCC cell lines, which our laboratory previously reportedto result in reduced drug efficacy. Suppression of MyD88expression blocked erlotinib-induced IL6 secretion in vitro andincreased the antitumor activity of erlotinib in vivo. There waslittle evidence of Toll-like receptor or IL18 receptor involve-

ment in erlotinib-induced IL6 secretion. However, suppressionof IL1R signaling significantly reduced erlotinib-induced IL6production. A time-dependent increase of IL1a but not IL1bwas observed in response to erlotinib treatment, and IL1ablockade significantly increased the antitumor activity of erlo-tinib and cetuximab in vivo. A pan-caspase inhibitor reducederlotinib-induced IL1a secretion, suggesting that IL1a wasreleased because of cell death. Human HNSCC tumors showedhigher IL1a mRNA levels compared with matched normaltissue, and IL1a was found to be negatively correlated withsurvival in patients with HNSCC. Overall, the IL1a/IL1R/MYD88/IL6 pathway may be responsible for the reduced anti-tumor efficacy of erlotinib and other EGFRIs, and blockade ofIL1 signaling may improve the efficacy of EGFRIs in the treat-ment of HNSCC. Cancer Res; 75(8); 1657–67. �2015 AACR.

IntroductionEGFR is a receptor tyrosine kinase that activates numerous pro-

survival pathways including Akt and STAT3 signaling pathways(1). Given that EGFR signaling is upregulated in many cancersespecially head and neck squamous cell carcinoma (HNSCC),several drugs that target EGFR have been developed and approvedfor cancer therapy such as monoclonal antibodies that blockthe extracellular ligand binding domain (e.g., cetuximab, pani-tumumab) and small molecule tyrosine kinase inhibitors (TKI)that prevent activation of the cytoplasmic tyrosine kinase domain(e.g., gefitinib, erlotinib; ref. 1). To date, only cetuximab is FDA-approved for use in HNSCC; however, it should be noted thatresponse rates to cetuximab as a single agent are quite low (13%)

and of limited duration (2–3 months). Similarly, low responserates (4%–11%)have beenobserved in clinical trialswith patientswith HNSCC treated with gefitinib and erlotinib (2–5). Manydifferent mechanisms (e.g., existing/acquired mutations andalternative signaling pathways) have been proposed that mayreduce patient response to EGFRIs, but this knowledge has notimproved survival rates for patients with HNSCC to date (6–9).

Previous studies in our laboratory observed a significant upre-gulation in IL6 expression in HNSCC cell lines treated withEGFRIs (10). IL6 is a pleotropic cytokine with a wide range ofbiologic activities and is well known for its role in inflammation,tumor progression, and chemoresistance in HNSCC (11–14). Weadditionally demonstrated the ability of IL6 signaling to protectHNSCC against erlotinib treatment in vitro and in vivo (10)supporting prior reports showing that IL6 may be involved inresistance to EGFRIs (15–18).

A well-established mechanism of IL6 production involves thecytosolic adaptor protein myeloid differentiation primaryresponse gene 88 (MyD88), which acts through intermediariesto induce NF-kB activation (19). MyD88 is required for theactivity of members of the Toll/IL1 receptor (TIR) superfamily,which include Toll-like receptors (TLR), the IL1R, and IL18R (19).Activation of these receptors leads to the recruitment of MyD88via its TIR domain, resulting inNF-kB activation and expression ofproinflammatory cytokines, including IL6 (19). Here we showthat EGFR inhibition using erlotinib activates the IL1a/IL1R/MyD88/IL6 signaling pathway and this pathway may serve as anovel mechanism responsible for the poor long-term antitumorefficacy of EGFRIs in HNSCC therapy.

1Department of Pathology, The University of Iowa, Iowa City, Iowa.2Roy J. and Lucille A. Carver College of Medicine, The University ofIowa, Iowa City, Iowa. 3Free Radical and Radiation Biology Program,Department of Radiation Oncology,The University of Iowa, Iowa City,Iowa. 4Interdisciplinary Human Toxicology Program,The University ofIowa, Iowa City, Iowa. 5Holden Comprehensive Cancer Center, TheUniversity of Iowa, Iowa City, Iowa.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Andrean L. Simons, Department of Pathology, 1161Medical Laboratories, University of Iowa, Iowa City, IA 52242. Phone:319-384-4450; Fax: 319-335-8453; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-2061

�2015 American Association for Cancer Research.

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Materials and MethodsCells and culture conditions

Cal-27 and FaDu human HNSCC cells were obtained from theATCC. SQ20BHNSCC cells (20) were a gift fromDr. Anjali Gupta(Department of Radiation Oncology, The University of Iowa,Iowa City, IA). All HNSCC cell lines are EGFR-positive and aresensitive to EGFR inhibitors. All cell lines were authenticated bythe ATCC for viability (before freezing and after thawing), growth,morphology, and isoenzymology. Cells were stored according tothe supplier's instructions and used over a course of nomore than3 months after resuscitation of frozen aliquots. Cultures weremaintained in 5% CO2 and air humidified in a 37�C incubator.

In vitro drug treatmentErlotinib (Tarceva), anakinra (Kineret), and N-acetyl cysteine

(NAC; Acetadote) were obtained from the inpatient pharmacy attheUniversity of IowaHospitals andClinics. Drugs were added tocells at final concentrations of 5 mmol/L erlotinib, 10 or 50 ng/mLanakinra, and20mmol/LNAC.Human IgGandDMSOwereusedas controls and were obtained from Sigma-Aldrich. PEGylatedcatalase (CAT; Sigma-Aldrich) was used at a final concentration of100 U/mL. Human IL1a, IL1b, and IL18Ra neutralizing antibo-dies were obtained from R&D Systems and were used at aconcentration of 0.5 mg/mL. Recombinant human IL1a wasobtained from Life Technologies and administered at a concen-tration of 1 ng/mL. Ac-Y-VAD-cho (CalBioChem) was suspendedin DMSO and used at 5 mmol/L. Z-VAD-fmk (Promega) wasdiluted in DMSO and used at 20 mmol/L. TLR agonists wereused at the following concentrations: Pam3CSK4 (200 ng/mL),FSL-1 (100ng/mL), Poly I:C (20mg/mL), lipopolysaccharide (200ng/mL), flagellin (200 ng/mL), gardiquimod (1 mg/mL), CL075(1 mg/mL), and Escherichia coli DNA (1 mg/mL). All TLR agonistswere obtained from InvivoGen. The required volume of each drugwas added directly to complete cell culture media on cells toachieve the indicated final concentrations.

Microarray analysesGene expression analysis ofHNSCCcells treatedwithDMSOor

erlotinib (5 mmol/L, 48 hours) has been described previously(GeneBank accession no. GSE45891; ref. 10). Downstream path-way, network, process, and disease analyses of the resultant geneexpression data for all cell lines (n ¼ 3 experiments per cell line)was carried out usingMetacoreTM (GeneGo) using a threshold ofþ1.3 and a P value of 0.05. Enrichment analysis of the resultantgene expression profiles of SQ20B and Cal-27 HNSCC cellsexposed to erlotinib versus DMSO was performed by mappinggene IDs from the resultant dataset onto gene IDs in built-infunctional ontologies, which include cellular/molecular processnetworks, disease biomarker networks, canonical pathway maps,and metabolic networks.

Real-time quantitative PCRTotal RNA was extracted from cells after indicated time

points using RNeasy Plus mini kit (Qiagen). Conversion of RNAinto cDNAwas accomplished with the iScript cDNA Synthesis Kit(Bio-Rad) and a thermocycler with the following conditions:5 minutes at 25�C, 30 minutes at 42�C, and 5 minutes at 85�C.Subsequent RT-PCR analysis was performed in a 96-well opticalplatewith eachwell containing 6mLof cDNA, 7.5mLof SyBrGreenUniversal SuperMix (Bio-Rad), and 1.5 mL of oligonucleotide

primers (sense and antisense; 4 mmol/L) for a total reactionvolume of 15 mL. Oligonucleotide primers for human genes wereobtained from Integrated DNA Technologies (IDT) and are aslisted in Supplementary Table SI. RT-PCR was performed on ABIPRISM Sequence Detection System (model 7000, Applied Bio-systems) with the following protocol: 95�C for 15 seconds (dena-turing) and 60�C for 60 seconds (annealing), repeated for 40cycles. Threshold cycle (CT) values for analyzed genes (in dupli-cate) were normalized as compared to GAPDH (cell lines) or 18S(human samples)CT values. Relative abundancewas calculated as0.5^(DCT), with DCT being the CT value of the analyzed geneminus the CT value of the reference gene (GAPDH or 18S).

Western blot analysisCell lysates were standardized for protein content, resolved on

4% to -12% SDS-PAGE, and blotted onto nitrocellulose mem-branes. Membranes were probed with rabbit anti-MyD88 (1:500,Cell Signaling), anti-IL1R1 (1:500, Santa Cruz), and anti-b-actin(1:5,000, Thermo Scientific). Antibody binding was detected byusing an ECL Chemiluminescence kit (Amersham).

ELISALevels of IL6, IL1a, and IL1bof treated cells were determined by

ELISA. The culture media of the treated cells were harvested andeach cytokine was detected according to the manufacturer's pro-tocol using Human Quantikine ELISA Kits (R&D Systems).

Adenoviral vectorsConstruction and characterization of adenoviral vectors encod-

ing wild-type and dominant-negative NADPH oxidase-4 (NOX4)have each been described previously (10, 21). An empty vectorlacking the NOX4 construct was used as a control. All vectors wereobtained from the University of Iowa Gene Vector Core. HNSCCcells in serum-free media were infected with 100 multiplicity ofinfection (MOI) of the above-described adenoviral vectors for 24hours. Biochemical analyses were performed 72 to 96 hours aftertransfection.

siRNA/shRNA transfectionMyD88, TLR2, TLR5, and control siRNA (Santa Cruz) were

transfected intoHNSCC cells at a concentration of 40 to 80nmol/Lwith equal volume Lipofectamine RNAiMAX (Invitrogen). Cellswere incubated in Opti-MEM for 4 hours before addition ofsiRNA and 16 hours after addition of siRNA. For shRNA transfec-tion, SQ20B cells were transfected with 1 mg/mL of psiRNA-h7SKGFPzeo, psiRNA-shMyD88, or psiRNA-shIL1R (Invivogen)in the presence of Opti-MEM and Lipofectamine RNAiMAX.Cells were allowed to recover 48 to 72 hours in antibiotic-freeDMEM with 10% FBS before 48-hour erlotinib treatment. Knock-down was confirmed by RT-PCR and/or Western blotting.

Clonogenic survival assayClonogenic survival was determined as previously described

(22). Individual assays were performed with multiple dilutionswith at least 4 cloning dishes per data point, repeated in at leastthree separate experiments.

Tumor cell implantationMale and female athymic nu/nu mice (4–5 weeks old) were

purchased from Harlan Laboratories. Mice were housed in a

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pathogen-free barrier room in the Animal Care Facility at theUniversity of Iowa and handled using aseptic procedures. Allprocedures were approved by the IACUC committee of theUniversity of Iowa and conformed to the guidelines establishedby the NIH. Mice were allowed at least 3 days to acclimate beforebeginning experimentation, and food andwater weremade freelyavailable. Tumor cells were inoculated into nude mice by subcu-taneous injection of 0.1-mL aliquots of saline containing 2� 106

SQ20B cells into the right flank using 26-gauge needles.

In vivo drug administrationMice started drug treatment 1week after tumor inoculation. For

the MyD88 knockdown experiments, female mice were random-ized into two treatment groups and orally administered eitherwater or 12.5 mg/kg erlotinib daily. For the IL1a neutralizationexperiments, male and female mice were randomized into fourtreatment groups as follows. Control group: Mice were adminis-tered water orally daily and 1 mg/kg IgG intraperitoneally onceper week. Neutralizing IL1a antibody (nIL1aab) group: A humanIL1a neutralizing antibody (XBiotech) was administered intra-peritoneally at 100 mg/mouse once per week. Erlotinib group:Erlotinib was administered orally 12.5 mg/kg daily. Erlotinib þnIL1aab group: Erlotinib was administered orally 12.5 mg/kgdaily in addition to nIL1aab administered intraperitoneally at100 mg/mouse once per week. For experiments involving cetux-imab, cetuximab was administered intraperitoneally 0.2 mgper mouse twice per week and control mice were given IgG twiceper week. All treatments were given for the duration of 3 weeks.Mice were evaluated daily and tumor measurements taken 3times per week using Vernier calipers. Tumor volumes were cal-culated using the formula: tumor volume ¼ (length � width2)/2where the length was the longest dimension and width was thedimension perpendicular to length. Mice were euthanized viaCO2 gas asphyxiation or lethal overdose of sodium pentobarbital(100 mg/kg) when tumor diameter exceeded 1.5 cm in anydimension.

BioinformaticsThe Cancer Genome Browser (University of California-Santa

Cruz; https://genome-cancer.ucsc.edu) was used to download thelevel 3 dataset HNSCC dataset (TCGA_HNSC_exp_HiSeqV2_PANCAN) from The Cancer Genome Atlas (TCGA). RNAseq datawas normalized across all TCGA cohorts and reported as log2values. Corresponding level 3 clinical datawere available formostof the 467 samples. Selected tumors (n ¼ 41) also had RNAseqdata for matched normal tissue. Matched tumor and normalsamples were analyzed. Linear fold change was calculated toemphasize difference between groups. Kaplan–Meier survivalcurves were generated by comparing survival of the highestquartile of expressing tumors (for indicated gene) against thelowest quartile. In some cases, Kaplan–Meier curves were gener-ated using an aggregate of several genes. The genes aggregatedare as follows: TLR (TLR1,TLR2, TLR4,TLR5,TLR6,TLR7,TLR8,TLR9,TLR10), IL18R (IL18Ra,IL18Rb), and IL1R (IL1R1,IL1RAP).Tumors were ranked according to expression of each gene, andranks were averaged to determine highest and lowest quartile oftumors expressing the given receptor family.

Statistical analysisStatistical analysis was done using GraphPad Prism version 5

for Windows (GraphPad Software). Differences between 3 or

more means were determined by one-way ANOVA with Tukeypost tests. Linear mixed-effects regression models were used toestimate and compare the group-specific change in tumor growthcurves. Differences in survival curves were determined byMantel–Cox test. All statistical analysis was performed at the P < 0.05 levelof significance.

ResultsErlotinib induces processes involved in inflammation

Of the top 10 upregulated cellular process networks identi-fied by erlotinib treatment, 6 processes were related to immuneresponse or inflammation for both cell lines (Fig. 1A and B).The top 10 significant diseases that were identified from erlo-tinib treatment were predominantly systemic inflammatorydisorders in both cell lines such as rheumatic diseases/disorders(rheumatic arthritis, rheumatic fever, rheumatic heart disease;Fig. 1C and D). Similarly, the majority of the top 10 upregu-lated canonical pathways were immune response/inflamma-tion related in both cell lines, which included IL6 and IL1signaling in SQ20B cells (Fig. 2A) and TLR and IL1 signaling inCal-27 cells (Fig. 2B).

The top network identified for SQ20B and Cal-27 was theNF-kB, MyD88, I-kB, IRAK1/2, NF-kB2 (p100) network(Fig. 2C) and TRAF6, TAK1 (MAP3K7), NF-kB, I-kB, IKK-gnetwork (Fig. 2D), respectively. The genes and processes inthese networks were both related to MyD88-dependent TLRsignaling and NF-kB activity (Supplementary Tables S2 andS3). Altogether, the gene expression analyses suggested thaterlotinib activates inflammatory processes and pathways thatmay be mediated by MyD88.

Loss of MyD88 increases tumor sensitivity to erlotinibWe have previously shown that erlotinib induces the secretion

of IL6 and other proinflammatory cytokines via NF-kB activationin HNSCC cells (10), which supports the gene expression results(Figs. 1 and 2). Transient knockdown of MyD88 significantlysuppressed baseline and erlotinib-induced IL6 production inboth SQ20B (Fig. 3A) and Cal-27 cells (Fig. 3B). MyD88 stableknockout clones (shMyD88#2, shMyD88#9) also demonstratedsignificantly reduced IL6 in the absence and presence of erlotinibcompared with control (Fig. 3C) supporting the role of MyD88-dependent signaling in erlotinib-induced IL6 production. BothMyD88-knockout clones showed reduced tumor growth whentreated with erlotinib compared with erlotinib-treated controlxenografts (Fig. 3D–G).Notably, xenografts bearing the shMyD88#9 clone showed reduced tumor growth in both treated anduntreated groups (Fig. 3D and G). Altogether these results suggestthatMyD88-dependent signaling is involved in erlotinib-inducedIL6 secretion and suppresses the antitumor activity of erlotinib.

TLR5 signaling may be involved in erlotinib-induced IL6secretion

A general trend of increased TLR, IL1R, and IL18R RNA expres-sion was found in HNSCC human tumors [obtained fromthe Tissue Procurement Core (TPC) in the Department ofPathology] compared with matched normal tissue (Fig. 4A andB). Notably, both tumors showed large increases in expression ofTLR2 compared with normal matched tissue (Fig. 4A and B). IL6secretion was significantly increased after treatment with agoniststo TLR1/2, TLR2/6, and TLR3 in all 3 cell lines (Fig. 4C), although

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TLR5 appeared to be active in only SQ20B cells (Fig. 4C). Erlotinibincreased TLR8 expression in SQ20B cells and TLR10 in Cal-27cells, although the absolute levels of these TLRs were very low andmost likely not of biologic significance (Fig. 4D). As the TLR1/2and TLR2/6 dimers both depend on TLR2, the activity ofthese dimers were suppressed using siRNA targeted to TLR2 (Fig.4E and F). Knockdown of TLR2 expression did not decreaseerlotinib-induced IL6 (Fig. 4E). However, knockdown of TLR5expression partially but significantly suppressed erlotinib-induced IL6 secretion in SQ20B cells (Fig. 4G and H), which wasnot observed in Cal-27 cells (data not shown). TLR3, which isnot a MyD88-dependent receptor also, was not involved inerlotinib-induced IL6 in both cell lines (Supplementary Fig.S1). Altogether, these results suggest that of the TLRs, only TLR5signaling may contribute to IL6 secretion induced by erlotinib inselect HNSCC cell lines.

IL1 signaling is critical for erlotinib-induced IL6 expression inHNSCC cells

To investigate the contribution of other MyD88-dependentsignaling pathways, the IL18R and IL1R pathways were studied.Neutralization of IL18R in SQ20B (Fig. 4I) and Cal-27 (Fig. 4J)

failed to suppress erlotinib-induced IL6. However, anakinra, arecombinant IL1R antagonist (IL1RA/IL1RN) significantlyreduced baseline and erlotinib-induced IL6 in both SQ20B(Fig. 5A) and Cal-27 (Fig. 5B). In addition, transient (Supple-mentary Fig. S2) and stable knockdown of IL1R suppressederlotinib-induced IL6 (Fig. 5C), suggesting that IL1R signalingmay be involved in erlotinib-induced IL6. Sequenced HNSCCtumors and matched normal tissue (n ¼ 40) were analyzedfrom TCGA for mRNA levels of ligands of the IL1 pathway. IL1aand IL1b were found to be increased in tumors by 4.8- and 2.5-fold, respectively, compared with normal samples while IL1RA/IL1RN was decreased by 2.5-fold (Fig. 5D). IL1a was alsoupregulated in both HNSCC tumors analyzed in Fig. 4A andB while IL1b was only upregulated in one of these tumors(Supplementary Fig. S3). IL1a but not IL1b was detectable aftererlotinib treatment and increased across all time points mea-sured in both cell lines (Fig. 5E). Exogenous IL1a increased IL6secretion in the presence and absence of erlotinib (Fig. 5F) andblockade of IL1a abut not of IL1b activity significantly reducedIL6 secretion in the absence and presence of erlotinib (Fig. 5G),suggesting that IL1a release may be responsible for erlotinib-induced IL6 production.

Figure 1.Process network and disease analyses of erlotinib-treated HNSCC cells. Shown are the top 20 upregulated cellular/molecular processes (A and B) and diseases (Cand D) from differentially regulated transcripts comparing microarray data from erlotinib-treated (5 mmol/L, 48 hours) SQ20B (A and C) and Cal-27(B and D) HNSCC versus DMSO-treated cells.

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Erlotinib-induced cell death triggers IL1a releaseIL1a unlike IL1b is not secreted but is typically released by

cell death. To confirm this, we showed that Z-VAD-fmk(ZVAD), a pan-caspase inhibitor, significantly reduced base-line and erlotinib-induced levels of IL1a (Fig. 6A) andblocked erlotinib-induced cell death (Supplementary Fig.S4), suggesting that IL1a is likely released because of erloti-nib-induced cell death. These results were not observed withthe caspase-1 inhibitor, Ac-Y-VAD-cho (YVAD, Fig. 6A). Ourlaboratory has previously shown that erlotinib induces celldeath via H2O2-mediated oxidative stress due to NOX4 activity(23). To confirm that oxidative stress is involved in IL1arelease, we showed that the antioxidants NAC and CAT signif-icantly suppressed erlotinib-induced IL1a in addition to IL6 inboth SQ20B (Fig. 6B) and Cal-27 cells (Fig. 6C). We havepreviously shown that these antioxidants significantly protectthese HNSCC cell lines from erlotinib-induced cytotoxicity(23). Moreover, overexpression of dominant-negative NOX4

(N4dn) decreased erlotinib-induced IL1a, IL6 production (Fig.6D and E) and cytotoxicity (Fig. 6F) in both SQ20B (Fig. 6Dand F) and Cal-27 (Fig. 6E and F). The opposite results wereobserved with wild-type NOX4 (N4wt; Fig. 6D–F). The abilityof N4wt (and not N4dn) to significantly induce oxidative stressin these cell lines has been demonstrated in our previouspublications (10, 21). Altogether, these results suggest thaterlotinib-induced oxidative stress (via NOX4) results in celldeath, leading to IL1a release resulting in activation of IL1Rsignaling in unaffected/surviving cells leading to IL6 expressionand secretion.

IL1a is negatively correlated with survival in HNSCCSequenced HNSCC tumors (TCGA, n ¼ 467) with high

expression of MyD88, TLRs, IL1R, IL18R, IL1a, IL1b, and IL1RAwere plotted for survival against low expressing tumors(Fig. 7A–H). MyD88, TLRs, IL18R, IL1b, and IL1RA were notsignificantly correlated with survival (Fig. 7A–C, G, and H).

Figure 2.Pathway and network analysis of erlotinib-treated HNSCC cells. Shown are the top 10 upregulated pathways (A and B) and top upregulatedinflammation-related networks (C and D) constructed from differentially regulated transcripts comparing microarray data from erlotinib-treated(5 mmol/L, 48 hours) SQ20B (A and C) and Cal-27 (B and D) HNSCC versus DMSO-treated cells. Upregulated genes are marked with red circles anddownregulated with blue circles. The "checkerboard" color indicates mixed expression for the gene between cell lines.






High IL1R expressing tumors showed a trend (P ¼ 0.06) towarda negative correlation with survival (Fig. 7D) while IL1amRNAexpression was negatively correlated (P ¼ 0.04) with survival(Fig. 7E). Selected tumors from patients who received targetedmolecular therapy (TMT, n ¼ 40) showed an increased negativecorrelation with survival (P ¼ 0.02, Fig. 7F), suggesting thatIL1a expression may be an important prognostic marker inHNSCC.

Finally, we showed that SQ20B cells treated with an IL1aneutralizing antibody (XBiotech; ref. 24) in combination witherlotinib displayed a significant reduction in survival comparedwith the other treatment groups in vitro (Fig. 7I) and in vivo(Fig. 7J). Similar results were observed with cetuximab in vivo(Fig. 7K), suggesting that blockade of the IL1 pathway mayincrease the sensitivity of erlotinib and other EGFRIs. Altogether,our results and previous findings suggest that erlotinib (and

Figure 3.KnockdownofMyD88 reduces IL6 and tumor growth inHNSCCcells. SQ20B (A) andCal-27 (B) cellswere transfectedwith scrambled siRNAcontrol (siCON)or siRNAtargeted against MyD88 (siMyD88). Cells were treated with DMSO (black bars) or erlotinib (ERL; 5 mmol/L; gray bars) for 48 hours and IL6 measured byELISA. SQ20B cells were transfected with an shRNA targeted against MyD88 (shMyD88) or a control plasmid (shCON) and selected with zeocin. Clones wereanalyzed for MyD88 levels by Western blotting (C, inset) and IL6 in the presence of DMSO and 5 mmol/L erlotinib (C). D–G, above clones were injected intothe right flank of athymic nu/numice. Tumor growthwas measured over a 3-week treatment period (12.5 mg/kg erlotinib or water daily; D and E). Tumor volume atday 17 is shown for clone #2 (F) and clone #9 (G). n ¼ 11–13. Error bars, SEM. � , P < 0.05 versus control; �� , P < 0.05 versus erlotinib.

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perhaps other EGFRIs) induce cell death via H2O2-mediatedoxidative stress due to NOX4 activity leading to IL1a release andactivation of the IL1R/MyD88/NF-kB signaling axis on surviv-ing tumor cells resulting in IL6 secretion (Fig. 7L). Our resultsalso propose that another unidentified DAMP may be releasedthat activates the TLR5/MyD88/NF-kB signaling axis, resultingin IL6 secretion. This IL6 signaling is believed to reduce theantitumor activity of EGFRIs and promote tumor progression(Fig. 7L).

DiscussionOur laboratory has previously shown that EGFRIs increased IL6

secretion and that IL6 levels played a critical role in the antitumoreffect of erlotinib in vitro and in vivo (10), which has beensupported and studied in depth by other groups (15–18). Thestudies presented here now indicate that MyD88-dependent IL1Rsignaling is most likely responsible for the IL6 productioninduced by EGFRIs. Therefore, targeting IL1 signaling may be anovel strategy to increase the antitumor efficacy of erlotinib andother EGFRIs in HNSCC.

We have observed that the majority of cellular processes andpathways upregulated by erlotinib treatment was related toimmune response and inflammation (Figs. 1 and 2). These

observations support one other study showing that the EGFRIPD153035 upregulated genes related to inflammation andinnate immunity (25). Interestingly, the inflammatory profiledisplayed by erlotinib treatment was remarkably similar tothat of rheumatic diseases and other systemic inflammatorydisorders (Fig. 1C and D). In fact, inhibition of the IL1pathway is a well-documented strategy for the treatment ofrheumatoid arthritis (RA), as IL1R ligands (IL1a and IL1b) areparticularly abundant in the synovial lining of the joint (26).Anakinra is a humanized recombinant IL1R antagonist(IL1RA) that is FDA-approved for use in the treatment of RA.IL1RA is an IL1R ligand that inhibits the IL1 pathway throughcompetition with the other IL1R ligands (27). In support ofthis, we have shown that anakinra effectively blocked erloti-nib-induced IL6 in HNSCC cell lines (Fig. 5A and B) implyingthat IL1 pathway-targeting drugs used for the management ofRA (and other systemic inflammatory disorders) could beinvestigated as a potential adjuvant to EGFRIs in the treatmentof HNSCC.

Of the ligands in the IL1 family, IL1b is the most well-studiedand its production is dependent on inflammasome-mediatedcaspase-1 activity (28). In the present studies, we believethat IL1a and not IL1b is involved in the activation of theIL1R/MyD88/IL6 pathway by erlotinib, as we were unable to

Figure 4.Role of TLR signaling in erlotinib (ERL)-induced IL6 in HNSCC cells. A and B, RNA isolated from two HNSCC tumors [#9 (A) and #13 (B); gray bars] andmatched normal tissue (black bars) was analyzed for TLR1–10, IL1R, and IL18R gene expression by RT-PCR. C, SQ20B, Cal-27, and FaDu cells were treated with TLRagonists as described in Materials and Methods. Secreted IL6 was measured by ELISA. D, SQ20B and Cal-27 were treated with DMSO or 5 mmol/L erlotinib for 48hours. Cells were analyzed by RT-PCR for the expression of TLR genes. Values were normalized to 18S mRNA levels and reported as fold change over DMSO(set at 1, dotted line). E–H, SQ20B or Cal-27 cells were transfected with scrambled siRNA control (siCON), siRNA targeted against TLR2 (siTLR2; E and F), or siRNAtargeted against TLR5 (siTLR5; G and H), treated with DMSO or 5 mmol/L erlotinib, and then analyzed for IL6. Knockdown of respective TLRs was confirmedby RT-PCR (F and H). SQ20B (I) and Cal-27 (J) cells were treated with IgG or an IL18R neutralizing antibody (nIL18Rab, 0.5 mg/mL) for 2 hours before DMSO orerlotinib (5 mmol/L) before IL6 analysis. n¼ 3; errors bars, SEM. �, P < 0.05 versus control; �� , P < 0.05 versus erlotinib.






detect any secreted IL1b and suppression of IL1b using aneutralizing IL1b antibody or a caspase-1 inhibitor did notaffect erlotinib-induced IL6 (Figs. 4E and G and 6A). On theother hand, we were able to detect IL1a (Fig. 5E) and suppres-sion of IL1a significantly blocked erlotinib-induced IL6 (Fig.5G), suggesting that IL1a was the ligand responsible for acti-vating the IL1 pathway.

Unlike IL1b, IL1a is not secreted from the cell but is releasedduring cell death and acts as a DAMP (29). It is likely that thecell death induced by erlotinib treatment resulted in IL1arelease, as the use of ZVAD blocked erlotinib-induced cell death(Supplementary Fig. S4) and IL1a release (Fig. 6A). Further-more, our laboratory has previously shown that erlotinibinduces cell death via H2O2-mediated oxidative stress due toNOX4 activity (23). We have now extended these findings toshow that IL1a release in addition to downstream IL6 secretionis mediated by erlotinib-induced cell death due to NOX4-induced oxidative stress (Fig. 6B–F).

Our gene expression analyses also implicated TLR/MyD88signaling (especially TLR2) as a possible mediator of erlotinib-induced IL6 (Fig. 2); however, we found no evidence of TLR2involvement despite TLR2 being present and active on HNSCCtumors and cell lines (Fig. 4A–C). Surprisingly, we found thatTLR2 knockdown increased IL6 secretion (Fig. 4E). An explana-tion for these results is unclear, although one prior report hasshown that activation of TLR2 resulted in decreasedNF-kB activityvia increased miR-329 leading to decreased IL6 expression inhuman trophoblast cells (30). Perhaps in our HNSCC cell model,inhibition of TLR2 expression decreased levels of miR-329 result-ing in increased NF-kB and IL6 secretion, which would be con-

sistent with the previous findings in trophoblast cells (30).Interestingly, TLR5 was active in only SQ20B cells (Fig. 4C) andTLR5 knockdown partially but significantly suppressed erlotinib-induced IL6 production in this cell line only, suggesting that TLR5activity may be important in select HNSCC cell lines (Fig. 4G andH). At this time, endogenous DAMPs capable of activation ofTLR5 are unknown; therefore, we are unclear as to how erlotinibinduces TLR5.

Given that IL1a appears to be the ligand that triggers theIL1R/MyD88/IL6 cascade that we believe is responsible forpoor response to EGFRIs, then in theory, neutralization of IL1ashould increase the antitumor efficacy of EGFRIs in the samemanner as blockade of IL6 as previously shown by our labo-ratory (10, 15–18). Indeed we observed that IL1a neutraliza-tion significantly increased the antitumor efficacy of erlotinib(Fig. 7J) in addition to cetuximab (Fig. 7K) in SQ20B cells.These exciting results suggest that IL1a plays an important rolein response to EGFRIs. Moreover, we want to highlight that theobserved effects of erlotinib in our studies are believed to bedirectly due to cell death mediated by EGFR inhibition and notdue to off-target effects of the drugs as (i) we are using clinicalachievable doses (31) and (ii) we have already confirmed theability of EGFR knockdown (using siRNA targeted to EGFR) toinduce oxidative stress, cell death, and cytokine secretion(10, 23).

To further stress the importance of IL1a in the managementof HNSCC, we found that HNSCC tumors expressed highlevels of IL1a compared with matched normal tissue (Fig.5D) and high IL1a-expressing tumors have worse prognosisthan low IL1a-expressing tumors (Fig. 7E). Furthermore, when

Figure 5.Role of IL1 signaling in erlotinib (ERL)-induced IL6 secretion. SQ20B (A) and Cal-27 (B) cells were treated with DMSO (CON) or 50 and 10 ng/mL, respectively,of anakinra (IL1RA) for 2 hours, followed by 48-hour treatment with DMSO or erlotinib (5 mmol/L), and then analyzed for IL6 secretion by ELISA. C, SQ20Bcells were transfected with shRNA targeted against IL1R1 (shIL1R1) or a control plasmid (shCON/shGFP) and selected with zeocin. Clones were analyzed for IL1R1levels byWestern blotting (C, inset) and IL6 levels. A dataset (n¼41) of HNSCC tumors (T) andmatched normal tissue (N) fromTCGAwas analyzed for expression ofIL1a, IL1b, and IL1RA mRNA. Linear fold change (tumor over normal) is reported (D). E, cell lines were treated with DMSO or 5 mmol/L erlotinib for the indicatedtime points and analyzed for IL1a by ELISA. F, cells were treated with PBS or 1 ng/mL human recombinant IL1a for 2 hours, treated with DMSO or erlotinib,and then analyzed for IL6 secretion. G, cells were treated with anti-IL1a or anti-IL1b neutralizing antibodies for 2 hours before treatment with DMSO or erlotinib andthen analyzed for IL6. n ¼ 3; errors bars, SEM. � , P < 0.05 versus control; �� , P < 0.05 versus erlotinib.

Koch et al.





we selected for tumors from patients receiving TMT, we foundan increased separation and significance between the survivalcurves (Fig. 7F), suggesting that IL1a expression may not onlypredict overall survival in HNSCC but also predict response toTMT. Unfortunately, the clinical information associated withthe tumors from patients that received TMT did not reveal whattreatment regimen was administered; therefore, we cannotmake firm conclusions from this analysis. However, as the onlyTMT currently used in HNSCC is EGFR-targeting drugs and theonly approved EGFRI for HNSCC to date is cetuximab, it ismore likely than not that the TMT involved cetuximab in ouranalysis.

Suppression of MyD88 effectively blocked erlotinib-inducedIL6 production and suppressed tumor growth in the presenceof erlotinib (Fig. 3), which is likely due to the ability ofMyD88 knockdown to block all potential proinflammatorysignaling from MyD88-dependent receptors. It is unclear whycontrol-treated shMyD88 #9 tumors displayed such a pro-nounced inhibition of tumor growth (Fig. 3E) compared withcontrol-treated shMyD88 #2 tumors (Fig. 3D). Previousreports have shown that MyD88 signaling may induce EGFRligands such as amphiregulin (AREG) and epiregulin (EREG),resulting in the activation of EGFR (32). Perhaps knockdownof MyD88 expression in the shMyD88 #9 clone led to theinhibition of EGFR via downregulation of AREG/EREG inaddition to suppression of IL6, which may explain our obser-vations. Nevertheless, these results suggest that MyD88 inhi-

bition may also be a promising strategy to increase the effect oferlotinib.

It should be noted that global inhibition of MyD88, IL1a, orany factor in the IL1R/MyD88/IL6 signaling axis in vivomay haveunexpected results. Ourmodel takes into account only the activityof MyD88 or IL1a within cancer cells. Inhibition of these inflam-matory components in innate immune cells may change theinflammatory microenvironment especially in an immunocom-petentmousemodel, conceivably altering recruitment of immunecells andunpredictably altering growth of the tumor. This remainsto be studied.

On the basis of these findings and our prior studies(10, 21, 23), we propose a model in which EGFR inhibitioncauses cell death and release of IL1a, which we believe binds itsreceptor IL1R on surviving cells, activates MyD88, and inducesIL6 secretion via NF-kB (Fig. 7L). IL6 signaling pathwaystypically lead to phosphorylation of STAT3, which is wellknown to compensating for the loss of EGFR signaling due tocross-talk (33). As such, we believe that the poor response andpossibly acquired resistance to erlotinib in the clinical settingmay be due to IL1R/MyD88/IL6 signaling triggered by releaseof IL1a from dying cells, which is different from other proposedmechanisms of poor response/acquired resistance (acquiredmutations, alternative signaling pathways; refs. 6–9). To ourknowledge, the studies presented here are the first to connectIL1a and MyD88-dependent signaling with response to EGFR-targeted therapy and this novel mechanism may offer insight

Figure 6.Erlotinib (ERL) increases IL1a secretion via oxidative stress–mediated cell death. A, SQ20B and Cal-27 cells were pretreated with Z-VAD-fmk (ZVAD) orY-VAD-fmk (YVAD) for 1 hour before 48-hour DMSO or 5 mmol/L erlotinib and then analyzed for IL1a by ELISA. SQ20B (B) and Cal-27 (C) cells were pretreated with20 mmol/L NAC or 100 U/mL PEGylated CAT for 1 hour before treatment with erlotinib and then analyzed for IL1a and IL6 secretion by ELISA. D–F, SQ20B(D) and Cal-27 (E) were transfected with empty (EMP), N4wt, or dominant-negative NOX4 (N4dn) adenoviral vectors before treatment with DMSO or erlotinib andthen analyzed for IL1a and IL6 secretion by ELISA (D and E) and clonogenic survival (F). n ¼ 3; errors bars, SEM. �, P < 0.05 versus control; �� , P < 0.05versus erlotinib.






into why other methods of overcoming EGFRI resistance havefailed and proposes new clinical targets that may enhance theefficacy of EGFRIs in HNSCC.

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

Authors' ContributionsConception and design: A.T. Koch, A.L. SimonsDevelopment of methodology: A.T. Koch, L. Love-HomanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.T. Koch, L. Love-Homan, M. Espinosa-Cotton,A. StanamAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.T. Koch, L. Love-Homan, A. Stanam, A.L. SimonsWriting, review, and/or revision of the manuscript: A.T. Koch, A. Stanam,A.L. SimonsAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. Love-HomanStudy supervision: A.L. Simons

AcknowledgmentsThe authors thank Dr. Thomas Bair in the Bioinformatics Division at The

University of Iowa for his assistance in analyzing the microarray studies andDr. C. Michael Knudson, Rita Sigmund, and Joe Galbraith from the TPC in theDepartment of Pathology for providing theHNSCC tumor samples. The authorsalso thank Dr. Sushma Shivaswamy and John Simard for kindly providingthe human neutralizing IL1a antibody for use in our in vivo studies. They alsothank Nicholas Borcherding and Drs. Weizhou Zhang, Fayyaz Sutterwala, andHasem Habelhah for their helpful suggestions and discussions on this article.

Grant SupportThis work was supported by grantsNIHR01DE024550,NIHK01CA134941,

and IRG-77-004-34 from the American Cancer Society, administered throughthe Holden Comprehensive Cancer Center at the University of Iowa.

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 July 11, 2014; revised January 12, 2015; accepted February 8, 2015;published OnlineFirst February 20, 2015.

Figure 7.IL1a expression affects response to EGFR inhibitors in HNSCC. A dataset (n ¼ 88) of HNSCC tumors from TCGA was analyzed for MyD88 (A), TLRs (B), IL18R(C), IL1R (D), IL1a (E), IL1b (G), and IL1RN (H) expression. A dataset (n¼ 48) of HNSCC tumors from patients who received TMTwas also analyzed for IL1a expression(F). The highest quartile of expressing tumors was plotted against the lowest quartile in Kaplan–Meier survival curves. SQ20B cells were treated with IL1a(anti-IL1a) or IL1b (anti-IL1b) neutralizing antibodies for 2 hours before treatment with DMSO (black bars) or erlotinib (ERL; 5 mmol/L, gray bars) for 48 hoursand then analyzed for clonogenic survival, n ¼ 3 (I). J and K, athymic (nu/nu) mice bearing SQ20B xenograft tumors were treated as described in Materials andMethods. Data points represent the average tumor volume values for 10 to 11 mice (J and K). L, schematic representing the proposed role of IL1 signaling inthe reduced effect of erlotinib in HNSCC. Error bars, SEM. � , P < 0.05 versus control; ��, P < 0.05 versus erlotinib.

Koch et al.





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2015;75:1657-1667. Published OnlineFirst February 20, 2015.Cancer Res Adam T. Koch, Laurie Love-Homan, Madelyn Espinosa-Cotton, et al. Cancer CellsEpidermal Growth Factor Receptor Inhibition in Head and Neck MyD88-Dependent Signaling Decreases the Antitumor Efficacy of

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MyD88-Dependent Signaling Decreases the Antitumor …cancerres.aacrjournals.org/content/canres/75/8/1657.full.pdf · Antitumor Efﬁcacy of Epidermal Growth Factor ReceptorInhibitioninHeadandNeckCancerCells