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Preclinical Development

Activation of the Insulin-like Growth Factor-1 ReceptorInduces Resistance to Epidermal Growth Factor ReceptorAntagonism in Head and Neck Squamous Carcinoma Cells

Mark J. Jameson1, Andrew D. Beckler1, Linnea E. Taniguchi1, Amir Allak1, Lisa B. VanWagner1, Nora G. Lee1,William C. Thomsen1, Matthew A. Hubbard1, and Christopher Y. Thomas2

AbstractEpidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) have poor efficacy in head and

neck squamous carcinoma cells (HNSCC). Because the IGF-1 receptor (IGF1R) generates potent prosurvival

signals and has been implicated in therapeutic resistance, its ability to induce resistance to EGFR-TKIs was

studied in vitro. Five HNSCC cell lines showed reduced sensitivity to the EGFR-TKI gefitinib when the IGF1R

was activated. In SCC-25 and Cal27 cells, gefitinib inhibited basal and EGF-stimulated EGFR, extracellular

signal–regulated kinase (Erk), and Akt phosphorylation and reduced cell number. This correlated with

initiation of apoptosis based on a 4-fold increase in PARP cleavage and a 2.5-fold increase in Annexin V

positivity. The apoptotic response and reduction in cell number were blocked by IGF1R activation, which

resulted in phosphorylation of both Erk and Akt. In both the cell lines, IGF1R-induced Erk, but not Akt,

activationwas eliminated by gefitinib. IGF1R-induced gefitinib resistancewas unaffected byMAP/Erk kinase

inhibition with U0126 but was partially impaired by inhibition of phosphoinositide-3-kinase with LY294002.

The IGF1R-TKI PQ401 inhibited growth of SCC-25 and Cal27 cells alone and also acted synergistically

with gefitinib. Thus, the IGF1R can make HNSCC cells resistant to EGFR-TKI treatment via a prosurvival

mechanism. Of the 8 HNSCC tumor samples studied, all samples expressed the IGF1R and 5 showed

detectable IGF1R phosphorylation, suggesting that this receptor may be relevant in vivo, and thus, combined

EGFR/IGF1R inhibition may be necessary in some patients for effective targeted molecular therapy.

Mol Cancer Ther; 10(11); 2124–34. �2011 AACR.

Introduction

In head and neck squamous carcinoma cells (HNSCC),the epidermal growth factor receptor (EGFR) has emergedas a potential therapeutic target; more than 90% ofHNSCCs overexpress the EGFR and elevated EGFRexpression predicts decreased survival. Several targetedanti-EGFR agents have been developed, but their efficacyin HNSCC has been limited, showing both intrinsic (lowinitial response rate) and acquired (short duration ofbenefit) resistance. Phase II clinical trialswith single-agentEGFR-tyrosine kinase inhibitors (TKI) showed responserates of only 5% to 15% (1, 2). Treatment failures do notresult from lack of EGFR expression or inability to blockreceptor activation in vivo, and EGFR expression level

does not predict effectiveness. To date,molecularmarkersthat predict response or resistance in HNSCC have notbeen identified (3).

IGF-1 and IGF-2 are ubiquitously produced proteinhormones that interact with the IGF-1 receptor (IGF1R)to regulate growth, differentiation, and survival. TheIGF1R activates both Ras/Erk- and PI3K/Akt-relatedsignal transduction pathways, which act to promote pro-liferation and prevent apoptosis (4). The IGFs are regu-lated extracellularly by 6 IGF-binding proteins (IGFBP)that buffer receptor activation. Thus, dysregulation of IGFproduction, IGF1R expression, or IGFBP secretion canalter growth regulation.

There is substantial evidence that the IGF1R plays acentral role in cancer development and tumor cell growth.The IGF1R is expressed by nearly all tumor types studiedand is activated in an autocrine or paracrine fashionwhentumor or stromal cells secrete IGFs (5, 6). In certain set-tings, the IGF1R is required for transformation by otheragents (including the EGFR; ref. 7), and the IGF1Rencourages and supports properties of the transformedphenotype. In addition, IGF-1 is also involved in otheraspects of cancer progression such as angiogenesis andinflammation (8). Recent studies have shown that elevat-ed serum IGF-1 levels are associatedwith increased risk of

Authors' Affiliations: Departments of 1Otolaryngology-Head and NeckSurgery and 2Internal Medicine, Division of Hematology and Oncology,University of Virginia Health System, Charlottesville, Virginia

CorrespondingAuthor:Mark J. Jameson, Department ofOtolaryngology-Head and Neck Surgery, University of Virginia Health System, PO Box800713, Charlottesville VA 22908. Phone: 434-924-2040; Fax: 434-982-3965; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-0294

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 10(11) November 20112124

on July 19, 2020. © 2011 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst August 30, 2011; DOI: 10.1158/1535-7163.MCT-11-0294

http://mct.aacrjournals.org/

a variety of epithelial cancers (9–12) and that reducedIGF-1 levels may be protective (13). Thus, it has beenproposed that reduction of IGF signaling in some cancertypes may have therapeutic benefit (4, 14). Augmentingthis concept is the recent demonstration that the IGF1Rcan promote therapeutic resistance to multiple treatmentapproaches including radiation, cytotoxic chemotherapy,and molecular targeted therapy (15–18). The significanceof these findings in HNSCC is, as yet, unknown.In the present study, we show that activation of the

IGF1R in HNSCC cells can overcome growth inhibitioncaused by EGFR-TKIs via a primarily antiapoptoticmech-anism. This validates the concept that, in the context ofEGFRblockade, an alternate growth factor can continue tosustain tumor cell growth and suggests that IGF1R sig-naling may be a mechanism of resistance to targeted anti-EGFR therapy in vivo. Thus, coinhibition of the EGFR andthe IGF1R may lead to increased clinical response rates.

Materials and Methods

ReagentsRecombinant human IGF-1 was obtained from Santa

Cruz Biotechnology, des[1-3]IGF-1 from GroPep, EGFfrom Sigma, and FBS from Invitrogen. U0126, PD158780,and LY294002 were obtained from EMD Biosciences;gefitinib and erlotinib from LC Laboratories; and PQ401from Tocris Bioscience. Anti-IGF1Ra was acquired fromSanta Cruz Biotechnology; anti-p-Erk from Sigma; anti-p-Tyr and anti-PARP from BD Biosciences; and anti-Akt,anti-p—Akt (S473), anti-Erk, anti-p-IGF1R, and anti-p-EGFR from Cell Signaling Technology.

Tissue culture and human tissue specimensHNSCC cell lines included SCC-25, SCC-9, Cal27, and

FaDu cells obtained from American Type Culture Collec-tion, and SCC-61 andUNC—7 cells were kindly providedby Dr. Wendell Yarbrough (Vanderbilt University, Nash-ville, TN). These were selected to evaluate a variety ofanatomic sites in the upper aerodigestive tract andbecause they exhibit a wide range of IGF1R expression.None of these cell lines had detectable basal IGF1R acti-vation. They were grown in Dulbecco’s Modified Eagle’sMedia/F12 supplemented with 400 ng/mL hydrocorti-sone and 5% FBS at 37�C and 5% CO2. In vitro, cells werehistopathologically consistent with HNSCC on standardhematoxylin and eosin staining and were positive forcytokeratin. Rat1 cells overexpressing the IGF1R (RIG)cells were kindly provided by Dr. Michael Weber (Uni-versity of Virginia). Theywere grown in Dulbecco’sMod-ified Eagle’s Media and 5% FBS at 37�C and 5% CO2.Monolayers were starved in no or very low (0.5%) serumfor 24 hours before assays were conducted. All cell lineswere passaged for less than 6 months after resuscitation.Other than testing conducted by the providers and theimmunohistopathology described above, no additionalauthentication of these cell lines was conducted by theauthors. All cell lines were free of mycobacterial infection

and exhibited behaviors similar to those previouslypublished.

Human HNSCC tissue specimens were obtained withapproval of the UVA Institutional Review Board. Smallportions of surgically extirpated tumors were collectedimmediately after resection and snap frozen in liquidnitrogen.Thespecimenswere storedat�80�Cuntilproteinextraction was conducted on all specimens simultaneous-ly. Standard clinical histopathology on the surgical speci-mens confirmed all tumors as squamous cell carcinoma.

ImmunoblotCell monolayers were grown to 70% confluence and

serum starved for 24 hours, then washed with PBS andincubated with inhibitor or vehicle for 2 hours followedby treatment with growth factor for 5 to 10 minutes.Stimulated monolayers were washed with ice-cold PBScontaining 2 mmol/L sodium orthovanadate, collected,and resuspended in lysis buffer (50mmol/LHEPES, pH¼8.0, containing 10 mmol/L sodium pyrophosphate,100 mmol/L sodium fluoride, 4 mmol/L EDTA, 1%Triton X-100, 1 mmol/L phenylmethanesulfonylfluoride,2 mmol/L sodium orthovanadate, 100 mmol/L benzami-dine, 1 mg/mL aprotinin, 2 mmol/L pepstatin, and25 mmol/L leupeptin), vortexed, and incubated at 4�Cfor 30 minutes. Insoluble material was removed by cen-trifugation at 13,000� g for 15 minutes at 4�C and samplebuffer containing 0.1 mol/L dithiothreitol was added.Proteins were resolved on an 8% SDS-PAGE and thentransferred to a polyvinylidene fluoride membrane(Millipore). The polyvinylidene fluoride membrane wasquenched, incubated with primary antibody, washed,and incubated with secondary antibody according to themanufacturer’s instructions. Proteins were visualizedby reaction with Immobilon Western ChemiluminescentHorseradish Peroxidase Substrate (Millipore).

Flow cytometryMonolayers were grown to 70% confluence and then

incubated in 0.5% serum for 24 hours. Monolayers werewashedwith PBS and then incubated inmedium contain-ing inhibitor or vehicle for 2 hours followed by treatmentwith growth factor for 48 hours. The medium from eachwell was collected; cell monolayers were trypsinized,resuspended in the corresponding medium, centrifugedat 1,000 � g for 7 minutes at 4�C, washed once with coldPBS, and resuspended in Annexin V-fluorescein isothio-cyanate (FITC) and propidium iodide according to themanufacturer’s recommendations (Millipore). Flow cyto-metry was conducted in the UVA Flow Cytometry CoreFacility within 24 hours of completion of the stimulation.

CellTiter 96 aqueous cell proliferation assayCells were plated at 5,000 cells per well in a 96-well

plate, grown for 24 hours, and serum-starved for 24 hours.Cells were washed with PBS, incubated for 2 hours withinhibitor, and stimulated for 48 hours with growth factor.MTS and phenazine methosulfate were added to each

IGF1R Induces Resistance to EGFR Antagonism in HNSCC

www.aacrjournals.org Mol Cancer Ther; 10(11) November 2011 2125




well according to the manufacturer’s protocol (Promega).Cells were incubated for 4 hours at 37�C and the absor-bance at 490 nmwas recorded, correcting for backgroundabsorbance. Control studies were conducted to show thelinearity of this assay at the cell densities used.

alamarBlue assayCells were prepared and treated as for the MTS assay

above. alamarBlue (Invitrogen) was added to each wellaccording to the manufacturer’s protocol. Cells wereincubated for 3 to 4 hours at 37�C and the fluorescenceat 540 nm was recorded.

Drug combination analysisGrowth inhibition data resulting from coinhibition of

both the EGFR and the IGF1R were assessed by median-effect analysis as described by Chou and Talalay (19)using CalcuSyn version 2.0 (Biosoft). The combinationindex was calculated, and an isobologram was plottedfor multiple dose combinations. Combination indexvalues of <1, 1, and >1 indicated synergism, additivity,and antagonism, respectively.

Results

IGF1R-induced resistance to EGFR-TKIsPD158780, gefitinib, and erlotinib are well-character-

izedEGFR-TKIs; treatment ofHNSCCcell lineswith thesecompounds caused growth inhibition. Activation of theIGF1Rwas tested for its ability to alter HNSCC sensitivityto EGFR-TKIs (Fig. 1). IGFBP-2 and IGFBP-3 are secretedat high levels by some HNSCC cells (20) and can interferewith activation of the IGF1R by exogenously added freeIGF-1. Because of its dramatically reduced affinity for theIGFBPs, des[1-3]IGF-1 was used to activate the IGF1R.

Theoriginal observation of IGF1R-induced resistance toEGFR inhibition was made in SCC-25 cells treated withPD158780 (Fig. 1A). PD158780 reduced cell number by66%at 48 hours.Addition of exogenous EGF caused a 40%increase in cell number; this effect was abolished byPD158780. Activation of the IGF1R with des[1-3]IGF-1caused a similar increase in cell number that was onlyminimally affected by PD158780. Stimulation with FBSresulted in an 86% increase in cell number; this waspartially blocked by PD158780 (Fig. 1A). These resultsshow that activation of the IGF1R in SCC-25 cells confersresistance to the growth-inhibitory effects of EGFR inhi-bition. The protective effect of IGF1R activation wasmeasurable across a range of doses for multiple EGFR-TKIs includingPD158780, gefitinib, anderlotinib (Fig. 1B).Because it is most likely to have clinical applicability forHNSCC, gefitinib was tested in 4 additional HNSCC celllines (Fig. 1C); activation of the IGF1R reduced gefitinibsensitivity in all 4 cell lines.

Erk and Akt activation by the IGF1RThe effect of gefitinib and IGF1R activation on extra-

cellular signal–regulated kinase (Erk) and Akt phos-

phorylation was assessed in SCC-25 and Cal27 cells byimmunoblot (Fig. 2). A small amount of Erk and Aktphosphorylation were present in unstimulated cells.Whereas EGF caused a large increase in p-Erk withlittle change in p-Akt, treatment with des[1-3]IGF-1stimulated a modest increase in p-Erk and a largeincrease in p-Akt. Addition of gefitinib eliminated base-line p-Erk and p-Akt, confirming that their constitutivephosphorylation depends primarily on EGFR activity.Treatment with gefitinib eliminated EGFR- and des[1-3]IGF-1–induced Erk activation, suggesting that phos-phorylation of Erk downstream of the IGF1R is EGFRdependent; this has been reported in other HNSCC celllines (20, 21). Notably, des[1-3]IGF-1–induced Akt phos-phorylation was unaffected by gefitinib in both celllines, indicating that the IGF1R is capable of providinga persistent prosurvival signal in the presence of anEGFR-TKI.

IGF1R inhibition of PD158780-induced apoptosisCleavage of PARP, a DNA repair enzyme, occurs in

response to caspase-3 activation, serving as an earlymarker of apoptosis. Figure 3A shows an immunoblotanalysis of SCC-25 cell lysates using an anti-PARPantibody that detects the initial PARP cleavage product(c-PARP). Treatment with gefitinib caused a dose-dependent increase in c-PARP that was completelyblocked by IGF1R activation. Addition of exogenousEGF did not counteract gefitinib-induced PARP cleav-age (Fig. 3B). The densitometric analysis in Fig. 3Bconfirms that IGF1R activation in SCC-25 cells inhibitsgefitinib-induced apoptosis. These data are in keepingwith the understanding that the IGF1R can act as apotent prosurvival agent. A similar response to gefitiniband IGF1R activation was also noted in 3 other HNSCCcell lines (Fig. 3C).

Florescence-activated cell sorting was used in conjunc-tionwith Annexin V-FITC and propidium iodide stainingto identify cells in early and late apoptosis (Fig. 3D). Asshown in the quantitative analysis in Fig. 3D, the basal rateof early apoptosis plus late apoptosis (necrosis) was 11%at 48 hours. Addition of gefitinib increased total apoptosisto 28%. Although activation of the IGF1R had little effecton the basal apoptotic rate (10%), it dramatically inhibitedthe gefitinib-induced apoptosis rate to 13%. The effect ofexogenous des[1-3]IGF-1 was blocked by addition of theIGF1R-TKI PQ401 (22, 23).

Prosurvival signaling from the IGF1RThe MAP/ERK kinase (MEK) inhibitor U0126 and the

phosphoinositide-3-kinase (PI3K) inhibitor LY294002were used to assess the roles of the Erk and Akt pathwaysin the antiapoptotic response to IGF1R activation. U0126and LY294002 treatment resulted in highly selective inhi-bition of Erk and Akt, respectively (Fig. 4A). U0126 aug-mented gefitinib-induced PARP cleavage, but des[1-3]IGF-1 still eliminated PARP cleavage in the presence ofboth inhibitors (gefitinib andU0126; Fig. 4B), showing that

Jameson et al.

Mol Cancer Ther; 10(11) November 2011 Molecular Cancer Therapeutics2126




IGF1R-induced resistance to gefitinib is not MEK/Erkdependent. Addition of LY294002 augmented gefitinib-induced PARP cleavage and reduced, but did notcompletely eliminate, the ability of des[1-3]IGF-1 to blockPARP cleavage (Fig. 4B). Although these data implyincomplete inhibition of PI3K by LY294002, activation ofAkt by an alternate (non-PI3K) pathway, or participationof additional signaling pathways in the prosurvival activ-ity of the IGF1R, they strongly suggest involvement of thePI3K/Akt pathway rather than theMEK/Erk pathway inthis process.

EGFR and IGF1R coinhibitionTo determine the utility of IGF1R blockade, SCC-25

and Cal27 cells were treated with PQ401, an IGF1R-TKI(22, 23). PQ401 had an IC50 value of approximately 4 to5 mmol/L (Fig. 5A). Although p-IGF1R could not bedetected in unstimulated cells, PQ401 inhibiteddes[1-3]IGF-1–stimulated IGF1R phosphorylation anddownstream Akt phosphorylation in a dose-dependentmanner (Fig. 5B). In SCC-25 cells, PQ401 was shown toreduce basal Akt phosphorylation at lower concen-trations (Fig. 5C), suggesting that some basal Akt

Figure 1. Inhibition of HNSCC cellgrowth by EGFR-TKIs. A, chemicalstructures of inhibitors. B, after24 hours of serum starvation,SCC-25 cells were incubated for48 hours and MTS assay wasconducted. Graph shows averagefold change compared withcontrol (unstimulated, uninhibited)cells � SEM for 4 independentquadruplicate experiments.�, P < 0.01 versus unstimulated;þ, P < 0.01 versus uninhibited (theStudent t test). Under the sameconditions, cells were harvested5 minutes after additions andwhole-cell lysates wereimmunoblotted. C, SCC-25 cellswere incubated for 48 hours with anEGFR-TKIwith orwithout 10nmol/Ldes[1-3]IGF-1 and alamarBlueassay was conducted. Graphshows average percentage ofcontrol (unstimulated, uninhibited)cells � SEM for 3 independentquadruplicate experiments.D, 4 additional SCCHN cell lineswere treated with gefitinib �10 nmol/L des[1-3]IGF-1 andassessed (as in C).

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phosphorylation may be due to low-level constitutiveIGF1R activity. Figure 5D shows the effect of combinedEGFR and IGF1R inhibition with gefitinib and PQ401. Forthese studies, PQ401 and gefitinib were combined inseveral ratios (1:1, 2:1, 1:2, and 1:4). On the basis of thecombination indices and isobologram analyses, the 2drugs are synergistic at IC50 levels.

IGF1R expression in HNSCC tumorsFigure 6 shows immunoblot analysis of humanHNSCC

tumor specimens. The tumor specimens used representsmall biopsies of solid tumor tissue and the surroundingtissue was shown to be predominantly squamous carci-noma cells by standard clinical histopathology. Whenlysates of these specimens were immunoblotted, IGF1Rwas detectable in all tumors, and p-IGF1R was detectablein 5 of 8 specimens. IGF-1-stimulated RIG cells (24) wereused as a positive control. These data indicate that theIGF1R is present in HNSCC and often constitutivelyphosphorylated, providing preliminary data that theIGF1R may have an important clinical role.

Discussion

In HNSCCs, EGFR—TKIs can induce tumor regressionor stabilization andmayaugment the antitumor activity of

radiotherapy and cytotoxic chemotherapy, but the pro-portion of responsive tumors is small, and the response isgenerally not sustained (2). Thus, clinical targeted anti-EGFR therapy yields disappointing results due to bothintrinsic and acquired resistance. While EGFR mutationsimpact sensitivity to EGFR-TKIs in other tumor types,their role in HNSCC is not yet clear (25–27). BecauseEGFR-TKIsmay impact their function, expression of otherHER family members may also play an important role insensitivity to EGFR inhibition. Gefitinib is more than 100-fold less potent at inhibiting HER2 (IC50 > 3.7 mmol/L)than EGFR (IC50¼ 0.033 mmol/L) in in vitro kinase assays,but it is not known that how important the effect ofgefitinib on HER2 is in relation to growth inhibition. Theexpression and function of HER family members inHNSCC is even less well studied than in other epithelialtumors but could be of significant consequence for EGFR-TKI therapy.

Signaling from other receptor tyrosine kinases caneffectively substitute for EGFR activity; these may beconstitutively active redundant pathways (intrinsic resis-tance) or compensatory responses (acquired resistance).The first well-described example of this was c-Met (28),and other receptor tyrosine kinases, including the IGF1R,have since been implicated. In non-HNSCC cell lines,IGF1R activation has been shown to inhibit the apoptosis

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Figure 2. Effect of gefitinib on Erkand Akt phosphorylation. SCC-25andCal27 cells were incubatedwithgefitinib for 2 hours and thenstimulated with des[1-3]IGF-1 orEGF for 5 minutes. Immunoblotswere conducted on whole-celllysates.

Jameson et al.





induced by cetuximab (29) and by the EGFR-TKI AG1478(30). After long-term gefitinib exposure, HN11 HNSCCcells showed elevated levels of activated IGF1R and Aktand increased sensitivity to IGFBP-3 (17). These findingssupport an important EGFR-IGF1R interaction, and,although the mechanism is not fully understood, theprosurvival activity of the IGF1R appears to be crucial.Chakravarti and colleagues (30) showed that resistance of

a glioblastoma cell line to EGFR-TKIs was mediated byincreased IGF1R expression with persistent PI3K signal-ing. In multiple breast cancer cell lines, IGF1R inhibitionsynergistically increased apoptosis when combined withgefitinib; in these cell lines, inhibition of Akt activityrequired combined EGFR/IGF1R inhibition (31). Similar-ly, in A431 cells with induced resistance to gefitinib, Guixand colleagues (17) showed that elimination of persistent

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Figure 3. Effect of gefitinib on apoptosis. After 2 hours of treatment with vehicle or gefitinib, cells were incubated for 6 hours (A–C) or 48 hours (D) with growthfactors and harvested. A–C, cells were lysed and immunoblotted for PARP. B, PARP cleavage was quantified by densitometry (c-PARP density/total PARPdensity) and is shown as average percentage ofmaximal (gefitinib alone)�SEM for 3 independent experiments. D, cells were treatedwith Annexin V-FITC andpropidium iodide (PI) and assessed by florescence-activated cell sorting. The average percentage of Annexin V–positive cells � SEM is shown for 3 similarexperiments. Early apoptosis, Annexin V–positive/PI-negative; late apoptosis/necrosis, Annexin V–positive/PI-positive; gefitinib, 5 mmol/L gefitinib; IGF,10 nmol/L des[1-3]IGF-1; PQ401, 5 mmol/L PQ401. �, P < 0.05 versus uninhibited (the Student t test).






IGF1R-induced Akt activity was required to reestablishgefitinib sensitivity. On the basis of these findings, per-sistent IGF1R activitymaypredict resistance to anti-EGFRtherapy. More broadly, IGF1R inhibition also increasesapoptosis in response to cytotoxic agents (32, 33), and thusIGF1R activity may also impact the effectiveness of non-targeted chemotherapeutics.

Constitutive activation of the IGF1R in human HNSCCtumor specimens (Fig. 6) suggests the existence of anautocrine or paracrine loop. This is consistent with otherstudies showing the production of IGF-1 and/or IGF-2 byhead and neck tumor cells (20). However, IGF productionby epithelial tumor cells may not be necessary to activatethe IGF1R; IGF-1 and/or IGF-2 produced by nearby stro-mal cells could serve as a paracrine growth stimulator.These data establish an important distinction betweenin vitro studies and the in vivo situation: While in vitroevaluation suggests no IGF1R phosphorylation in theabsence of exogenous IGF, there is a high likelihood thatHNSCC tumors in vivo are exposed to IGF-1 and/or IGF-2and that the IGF1R exhibits some degree of constitutiveactivation. Combinedwith ourfindings of IGF1R-inducedresistance to EGFR inhibition in multiple HNSCC celllines in vitro, this would predict potential-widespreadresistance to EGFR-TKIs in vivo. Because HNSCC arehighly heterogeneous tumors, a method for predictingIGF1R-based resistance to anti-EGFR therapy in the clin-ical setting will be crucial to evaluate the relevance of thismechanism in patients with HNSCC.

Accurate predictors of response to EGFR-TKIs inHNSCC have not been identified. A recent assessmentby Rogers and colleagues (34) of a panel of 18HNSCC celllines showed a correlation between sensitivity to gefitiniband degree of EGFR phosphorylation. Although Metexpression predicted response, Met knockdown had noeffect on gefitinib sensitivity. In their study, neither IGF1Rexpression level nor degree of phosphorylation predictedresponse to gefitinib (34). However, there was no assess-

ment of downstream targets in this study and thus noindication of the net impact of IGF1R level/activity.Unfortunately, it is unlikely that IGF1R-based resistancewill correlate simply with IGF1R expression; in additionto the level and affinity of the IGF1R, the degree of IGF1Ractivation is dependent on the abundance of IGF ligandsand IGFBPs, and the impact of IGF1R activity is highlycontext sensitive. For example, IGF1R activation can havea greater impact on cell growth when the EGFR is inhib-ited (Fig. 1C). In addition, themechanism(s) of interactionbetween the HER and IGF systems may be complex. Arecent study showed that, in breast cancer, trastuzumabregulates IGFBP-2 and IGFBP-3 expression, whichimpacts IGF1R downstream signaling (35). Cytotoxicagents can also regulate IGFBP secretion: Increased secre-tion of IGFBP-3 in prostate cancer cells treated with 5-fluorouracil plays an important role in its proapoptoticeffect (36). Thus, because of the complexities of the IGFsystem and its regulation, further investigation will berequired to identify a reliable marker of IGF1R-basedresistance to therapy.

In general, the effectiveness of EGFR-TKIs in vivo hasbeen based on assessment of EGFR phosphorylation. InSCC-25 and Cal27 cells treated with des[1-3]IGF-1, wedetected persistent downstream signaling even whenEGFR phosphorylation was undetectable (Fig. 2). Resid-ual downstream target phosphorylation may be due toincomplete EGFR blockade or the activity of other recep-tors such as the IGF1R. Janmaat and colleagues (37) haveshown that failure of gefitinib to inhibit cell growth iscorrelatedwith persistent Erk andAkt activity. Therefore,complete assessment of the effectiveness of EGFR-TKIsboth in vitro and in vivo requires analysis of phosphory-lation of downstream targets.

SCC-25 and Cal27 cells exhibit high EGFR and IGF1Rexpression and constitutive EGFR, Erk, and Akt phos-phorylation. Gefitinib induced apoptosis in a dose-depen-dent fashion; this was eliminated when IGF1R was

c-PARPPARP

Gefitinib (5 μmol/L) + + - -- - + ++ + - -- - + + - - + +- - + +

+ - + - +- + - +- + - + - + - +- + - +des[1-3]IGF-1 (10 nmol/L)

- -- - - -- -U0126 (10 μmol/L) + + + ++ + + +- -- - - -- -

LY294002 (40 μmol/L) - - + +- - + +- - + +- - + + - -- - - -- -

76 kDa102 kDa

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Figure 4. Role of MEK and PI3K ingefitinib-induced apoptosis andrescue by IGF1R activation. After24 hours of serum starvation,SCC-25 cells were incubated for5 minutes (A) or 48 hours (B) withinhibitors with or without growthfactor. Cell lysates wereimmunoblotted. B, average PARPcleavage (c-PARP density/totalPARP density) � SEM is shownrelative to gefitinib alone (100%) for3 independent experiments.

Jameson et al.





activated. On the basis of inhibitor studies, this effect is atleast partially related to persistent Akt signaling, which isconsistent with the well-established role of Akt as aprosurvival effector of the IGF1R. Addition of the PI3K

inhibitor LY294002 reduced, but didnot eliminate, IGF1R-based resistance to gefitinib-induced apoptosis suggest-ing incomplete PI3K inhibition, PI3K-independent Aktactivation, or an alternate (Akt-independent) prosurvival

SCC-25

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des[1-3]IGF-1 (10 nmol/L) - + + + + +PQ401 (μmol/L) - - 15 30 60 0

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Figure 5. IGF1R inhibition by PQ401 and synergism with gefitinib. A, SCC-25 and Cal27 cells were incubated for 48 hours with PQ401 and alamarBlue assayconducted. Graph shows average percentage of control (unstimulated, uninhibited) cells� SEM for 3 independent quadruplicate experiments. B, SCC-25 andCal27 cells were incubated for 2 hours with PQ401 (0, vehicle only) and then for 5 minutes with des[1-3]IGF-1 or vehicle, lysed and immunoblotted. C,unstimulated SCC-25 cells were incubated for 2 hours with PQ401 and then lysed and immunoblotted. D, SCC-25 and Cal27 cells were incubated for 48 hourswith gefitinib (Gef) and/or PQ401 (PQ) and alamarBlue assay conducted. Growth inhibition was calculated and average results of 4 independent quadruplicateexperiments (SEM < 5%) were analyzed for drug synergism using median-effect approach with CalcuSyn software. The graphs indicate dose response,combination index versus fractional effect, and conservative ED50 isobologram curves for the individual drugs and fixed-ratio combinations as indicated.






pathway from the IGF1R (as suggested by other authors;ref. 24). Activation of Erk, as observed in other contexts(20), is EGFR dependent in SCC-25 and Cal27 cells, thuseliminating it as an antiapoptotic agent when gefitinib ispresent. This was confirmed using the MEK inhibitorU0126. The remaining possibilities are incomplete PI3Kblockade or an alternate non-Erk, non-Akt–dependentprosurvival pathway.

It is important to note that the chemical inhibitors usedin the present study are selective, and the results could beconfounded by "off-target" effects. As mentioned, gefiti-nib inhibits HER2 with 100-fold less potency than EGFR;while this is unlikely to play an important role in ourstudies, cell lines with high HER2 expression might showaltered responses when gefitinib is used at high concen-trations. Similarly, PQ401 is a selective IGF1R inhibitorthat undoubtedly also inhibits the insulin receptor (InsR),given the high homology between these 2 receptors.However, when stimulated with insulin, SCC-25 cellsshowed no response (i.e., no gefitinib resistance) at lowerdoses but showed similar behavior to IGF stimulation athigher doses (100-fold higher than IGF), indicating thatthe effect observed is because of IGF1R activation. Gableand colleagues (22) showed similar findings using PQ401:It inhibited both basal and IGF-stimulated MCF-7 breastcancer cell growth, with inhibition of IGF1R and Aktphosphorylation at similar concentrations to those shownin Fig. 5. As an inhibitor of IGF1R and Akt phosphoryla-tion, PQ401 has also been shown tomimic IGFBP-3 and anIGF1R-blocking antibody that does not bind the InsR (23).The ability of low-dose exogenous IGF to attenuate theeffects of gefitinib further substantiates the notion thatPQ401 is acting on-target when it augments the effects ofgefitinib. It is also noteworthy that inhibition of the InsRalong with the IGF1R (i.e., nonselectivity) may be clini-cally desirable due to the potential for the InsR to substi-tute for the IGF1R when the IGF1R is selectively inhibited(38). In addition to these predictable off-target effects,

there may be unexpected and as-yet unknown effectsresulting from interactions with unrelated proteins.Future studies should more extensively evaluate theimpact of these inhibitors on a broad range of moleculartargets in HNSCC.

Our data showing augmented growth inhibition ofSCC-25 and Cal27 cells with PQ401 provide preliminaryevidence that combined EGFR/IGF1R inhibition may bean effective treatment approach to HNSCCs. Otherauthors have used combined EGFR/IGF1R inhibition inlimited in vivomodels of HNSCC (39) but were unable toshow a synergistic effect of the 2 inhibitors. Wilsbacherand colleagues (40) showed that inhibition of EGFR andIGF1R acts synergistically in A431 cells; only the combi-nation eliminated Akt phosphorylation and introductionof constitutively active Akt resulted in resistance to thedrug combination. Erlotinib and the IGF1R-TKI PQIPwere noted by Buck and colleagues (41) to be synergisticin a range of epithelial tumors. Their data suggested thatcoinhibition was necessary due, at least in part, toincreased activation of the reciprocal receptorwhen eitherdrug was used alone.

It seems clear that, in most cancers, effective targetedtherapy will involve blockade of multiple targets. Thereis now mounting evidence that the IGF1R and/or itsdownstream signaling pathways will be of key impor-tance in conjunction with the EGFR-TKIs. The mostimportant element of attempting new combinations oftargeted therapies will be tumor/patient selection.Thus, correlative evidence is needed that shows biomo-lecular markers predictive of IGF1R-based EGFR-TKIresistance. It is currently unclear whether unique IGFsystem molecules (IGF1R, IGF-1 or IGF-2, IGFBP-1through IGFBP-6) or downstream signaling molecules,such as Akt or others, will be the most significantmarkers for exploitation and continued evaluation iswarranted.

Conclusion

The present study clearly shows that the IGF1R can actas a mechanism of resistance to EGFR-TKIs in an in vitromodel of HNSCC and highlights the potential utility ofcoinhibition of the EGFR and IGF1R.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

The study was supported by NIH grant K08-DE019477 and AmericanHead and Neck Society pilot grant to M.J. Jameson.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 24, 2011; revised July 14, 2011; acceptedAugust 22, 2011;published OnlineFirst August 30, 2011.

RIG

+ IG

F-1

Human tumor lysates

IGF1Rββ

β-Actin

p-IGF1Rβ

Figure 6. IGF1R in HNSCC tumor lysates. Tumor specimens were lysedand immunoblotted. RIG, Rat1 fibroblasts overexpressing humanIGF1R.

Jameson et al.





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2011;10:2124-2134. Published OnlineFirst August 30, 2011.Mol Cancer Ther Mark J. Jameson, Andrew D. Beckler, Linnea E. Taniguchi, et al. Head and Neck Squamous Carcinoma CellsResistance to Epidermal Growth Factor Receptor Antagonism in Activation of the Insulin-like Growth Factor-1 Receptor Induces

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