A Comprehensive Evaluation of Biomarkers Predictive of ...The PI3K/AKT/mTOR pathway is frequently activated in head and neck squamous cell carcinoma (HNSCC), but pathway inhibition

Companion Diagnostics and Cancer Biomarkers

A Comprehensive Evaluation of Biomarkers Predictive ofResponse to PI3K Inhibitors and of Resistance Mechanismsin Head and Neck Squamous Cell Carcinoma

Tuhina Mazumdar1, Lauren A. Byers1,2, Patrick Kwok Shing Ng3, Gordon B. Mills2,3, Shaohua Peng1,Lixia Diao4, You-Hong Fan1, Katherine Stemke-Hale5, John V. Heymach1,2, Jeffrey N. Myers2,6,Bonnie S. Glisson1, and Faye M. Johnson1,2

AbstractThe PI3K/AKT/mTOR pathway is frequently activated in head and neck squamous cell carcinoma

(HNSCC), but pathway inhibition has variable efficacy. Identification of predictive biomarkers and mechan-

isms of resistancewould allow selection of patientsmost likely to respond andnovel therapeutic combinations.

The purpose of this study was to extend recent discoveries regarding the PI3K/AKT/mTOR pathway in

HNSCCbymore broadly examining potential biomarkers of response, by examining pathway inhibitorswith a

diverse range of targets, and by defining mechanisms of resistance and potential combination therapies. We

used reverse-phase protein arrays (RPPA) to simultaneously evaluate expression of 195 proteins; SNP array to

estimate gene copy number; and mass array to identify mutations. We examined altered signaling at baseline

and after pathway inhibition. Likewise, we examined the activation of the PI3K/AKT/mTOR pathway in

HNSCC tumors by RPPA. Cell lines with PIK3CA mutations were sensitive to pathway inhibitors, whereas

amplification status did not predict sensitivity.Whilewe identified a set of individual candidate biomarkers of

response topathway inhibitors, proteomic pathway scoresdidnot correlatewith amplification ormutation and

did not predict response. Several receptor tyrosine kinases, includingEGFRandERK,were activated following

PI3K inhibition in resistant cells; dual pathway inhibition of PI3K and EGFR or MEK demonstrated synergy.

Combined MEK and PI3K inhibition was markedly synergistic in HRAS-mutant cell lines. Our findings

indicate that clinical trials of single-agent PI3K/AKT/mTORpathway inhibitors in selectedpopulations andof

PI3K/EGFR or PI3K/MEK inhibitor combinations are warranted; we plan to conduct such trials. Mol Cancer

Ther; 13(11); 2738–50. �2014 AACR.

IntroductionHead and neck squamous cell carcinoma (HNSCC) is a

common and deadly disease in the United States andworldwide; in patients with this disease, the rate of treat-ment-related morbidity is high and recurrence is com-

mon. Although the unbiased genomic characterization ofmultiple cancers has fundamentally changed ourapproach to cancer therapy and translational research,targeted therapy based on biomarkers does not yet existfor HNSCC. Genomic characterization of HNSCC hasdemonstrated 4 common driver signaling pathways inthe HNSCC oncogenome: mitogenic signaling, NOTCH/differentiation, cell cycle, and p53/apoptosis (1–4). Of themitogenic pathways affected, PI3K/AKT/mTOR is by farthe pathway most often altered in HNSCC, with about80% of HNSCC tumors containing molecular alterationsin one or more components of the pathway (2, 5). Thefrequent activation of the PI3K/AKT/mTOR pathway inHNSCC, the availability of pharmacologic inhibitors, andthe pathway’s importance in cancer cell signaling makethis pathway one of the most promising targets for des-perately needed improvement in systemic therapy. Fur-thermore, the pathway’s activation by identifiable molec-ular alterations suggests the possibility of using suitablebiomarkers for predicting response to pathway-inhibitingtherapy.

The PI3K/AKT/mTOR signaling pathway is critical forthe regulation of multiple cellular processes, including

1Department of Thoracic/Head andNeckMedicalOncology, TheUniversityof Texas MD Anderson Cancer Center, Houston, Texas. 2The University ofTexas Graduate School of Biomedical Sciences, Houston, Texas. 3Depart-ment of Systems Biology, The University of Texas MD Anderson CancerCenter, Houston, Texas. 4Department of Bioinformatics and Computation-al Biology, The University of Texas MD Anderson Cancer Center, Houston,Texas. 5Department of Translational Research, The University of TexasMDAnderson Cancer Center, Houston, Texas. 6Department of Head and NeckSurgery, The University of Texas MD Anderson Cancer Center, Houston,Texas.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Faye M. Johnson, The University of Texas MDAnderson Cancer Center, 1515 Holcombe, Box 432, Houston, TX 77030.Phone: 713-792-6363. Fax: 713-792-1220; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-13-1090

�2014 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 13(11) November 20142738

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proliferation, growth, differentiation, migration, and sur-vival, in normal and cancer cells. Pathway componentsare frequently altered in cancer cells, leading to pathwayactivation and poor prognosis (6, 7). In HNSCC specifi-cally, there are gain-of-function mutations in PIK3CA(6%–13%), LOH of PTEN (8%), reduced PTEN proteinexpression (30%), PIK3CA gene overexpression (52%),PIK3CA amplification (20%),PIK3CGmutations (4%), andinactivating mutations in PTEN (4%; refs. 2–6, 8–10).AKT1, PIK3R1, and mTOR mutations occur rarely (5). Atthe protein level, nearly all HNSCC tissues show phos-phorylation of AKT and the downstream target S6, indi-cating pathway activation (11, 12).Despite the frequent activation of the PI3K/AKT/

mTOR pathway in HNSCC, inhibition of this pathwaywith a variety of inhibitors has had variable efficacyin vitro and in vivo (12–18). Identification of molecularmarkers able to predict benefit through identification ofeither sensitivity or resistancemechanisms couldmarked-ly improve the utility of PI3K/AKT/mTOR pathwayinhibitors. Several candidate biomarkers have been iden-tified throughourknowledge of thepathwayandfindingsin HNSCC and other cancers. Of these candidates,PIK3CA mutations are the ones most consistently relatedto sensitivity to pathway inhibitors as demonstrated in 2recent studies with HNSCC patient–derived xenografts(PDX; refs. 5, 19): a recent series of phase I studies (20) andcell line and PDXmodels frommultiple cancer histologies(19, 21, 22). Additional markers of sensitivity includeHER2 amplification, 4EBP1 expression, PTEN loss (23–25). In contrast, in vitro inhibition of both signaling andproliferation by a dual PI3K/mTOR inhibitor wasobserved in breast cancer cells independently of PIK3CAmutation and basal pathway activation (26). PTEN loss,rather than PIK3CAmutation, was closely linked to breastcancer cell sensitivity to a PI3K inhibitor (25, 27). Non-responding HNSCC tissue had higher levels of multiplesignaling components, including pSTAT3, EGFR, and c-Kit, than responding tumors tested ex vivo (18). Thus,while there are a number of potential markers of benefit,there is no consensus as to their utility and their applica-bility to HNSCC based on its underlying gene expressionpattern and the patterns of co-mutations that occur.Here, we build upon recent discoveries regarding the

PI3K/AKT/mTOR pathway in HNSCC by significantlyexpanding the examination of potential biomarkers toinclude PIK3CA amplification, PTEN loss, and the expres-sion andactivation of 195proteins; by examiningpathwayinhibitors with a diverse range of targets; and by identi-fying mechanisms of resistance that were previouslyunknown in HNSCC, leading to combination therapieswith a strong potential for high clinical efficacy.We testeda panel of 18 HNSCC cell lines with and without detectedPI3K/AKT/mTOR pathway alterations for sensitivity toPI3K, PI3K/mTOR, AKT, and mTOR catalytic inhibitors.In addition to studying the expected markers of sensitiv-ity, we used reverse-phase protein and phosphoproteinarrays (RPPA) as unbiased approaches in a panel of 60

HNSCC cell lines. We inhibited activated pathways toidentify several candidate drug targets for PI3K/AKT/mTOR pathway inhibitor combinations.

Materials and MethodsMaterials

All PI3K pathway inhibitors, MEK162, erlotinib,OSI906, cabozantinib, and dovitinibwere purchased fromSelleck Chemicals and prepared as 10 mmol/L stocksolutions in dimethyl sulfoxide. Antibodies against totaland phosphorylated AKT, ERK, pS6 and 4EBP1, c-Myc,cyclin D1, phosphorylated SGK3, and the PathScan RTKsignaling antibody array kit were purchased from CellSignaling Technology; antibody against b-actin was pur-chased from Sigma-Aldrich.

Cell cultureThe HOSC1 cell line was derived from an HNSCC

tumor implanted into a mouse as previously described(28). Apanel of 64HNSCCcell lines used in this studywasvalidated and obtained as previously described (29).

Cytotoxicity assayThe cytotoxicity of PI3K pathway inhibitors in HNSCC

cell lines was assessed using an MTT assay as describedelsewhere (28). Under each experimental condition, 4 inde-pendent wells were treated. We calculated IC50 and com-bination index (CI) values with methods previouslydescribed by Chou and using CalcuSyn software (Biosoft;ref. 30).CIvalues less thanonewereconsidered synergistic.

Western blottingWestern blot analysis was performed as described

previously (28). In brief, cells were subjected to lysis onice and the lysates to centrifugation at 20,000 � g for 10minutes at 4�C. Equal amounts of protein samples wereresolved by SDS-PAGE and then transferred to nitrocel-lulose membranes, immunoblotted with different prima-ry antibodies, and detected with a horseradish peroxi-dase–conjugated secondary antibody (Bio-Rad Laborato-ries) and the ECL reagent (Amersham Biosciences).

PhosphoScan analysisPhosphoScan analysis was done according to the pro-

tocol provided by the manufacturer (Cell Signaling Tech-nology). In brief, HNSCC cellswere subjected to lysis, and100 mg of lysate was added to a slide coated with target-specific capture antibodies. The slide was then incubatedwith a biotinylated antibody cocktail. Streptavidin-conju-gated HRP and LumiGLO reagent were used to visualizethe bound detection antibody by chemiluminescence.

PCR sequencingTo confirmmutations foundbySequenom (seeResults),

the regions of interest were amplified using custom PCRprimers. Sanger sequencing was performed on a 3730xlDNA Analyzer (Applied Biosystems) using BigDye Ter-minator v3 chemistry (Applied Biosystems). Mutation

PI3K Pathway Inhibition and Resistance Mechanisms in HNSCC

www.aacrjournals.org Mol Cancer Ther; 13(11) November 2014 2739




analysis was performed using SeqScape Software v2.5(Applied Biosystems).

Hotspot mutational analysisCustomSequenomassays for somatic variants across 45

different genes were designed by using Sequenom’sAssay Design program, with in silico analysis to avoidknown SNP site interference (see Supplementary Table S1for a list of the mutations). Point mutations were assayedusing the Sequenom MassArray as described previously(27), with additional analysis comparing relative allelicfrequencies of duplicate samples.

RPPA analysisRPPA analysis and antibody validation were per-

formed as previously described (31). Briefly, proteinlysate was collected from subconfluent HNSCC cell cul-tures after 24 hours in full serum medium (10% FBS).Protein lysates were adjusted to a 1 mg/mL concentration,and a serial dilution of 5 concentrations was printed, with10% of the samples replicated for quality control (2470Arrayer; Aushon Biosystems) on nitrocellulose-coatedslides (Grace Bio-Labs). Immunostaining was performedusing a DakoCytomation-catalyzed system and diamino-benzidine colorimetric reaction. Spot intensities wereanalyzed and quantified using MicroVigene software(VigeneTech Inc.) to generate spot signal intensities.

Statistical and RPPA analysisThe software SuperCurveGUI (http://bioinformatics.

mdanderson.org/Software/supercurve/) was used toestimate the IC50 values of the proteins in each dilutionseries (on a log2 scale)using the nonparametric, monotoneincreasing B-spline model; a correction for spatial biasbeforemodel fitting; a quality controlmetric for each slide;and loading correction using median centering acrossantibodies as previously described (31). As a measure ofpathway activation, we calculated PI3K/Akt and TSC/mTOR scores; the loading-corrected data were first log2-transformed, then median-centered and normalized bySD for each protein across samples. The score wasobtainedby calculating the sumofprotein level of positivecomponentsminus the sumof thenegative components ofthe pathway. The components of the PI3K/Akt and TSC/mTOR scores are listed in Supplementary Table S2.

SNP arrayWhole-genome SNP array profiling was performed in

cell lines using the Illumina Human1M-Duo DNA Anal-ysis BeadChip (Illumina, Inc.). Before analysis, SNP datawere normalized to the regional baseline copy number toaccount for aneuploidy, with the regional baseline copynumber being assigned a value of 2.

ResultsPI3K pathway alterations are common in head andneck cancer cells and tumors

We designed an HNSCC Sequenom panel that incor-porates 191 mutations in 45 genes found in squamous cell

cancers, including many of those identified in recentlypublished whole-exome sequencing studies of HNSCCtumors and 24 PIK3CA mutations (refs. 3, 4; Supplemen-tary Table S1). We isolated gDNA from 64 validatedHNSCC cell lines (29) as well as control cell lines withknown mutations in several of the tested genes. Weidentified several mutations, including HRAS (2 of 61,3%), PIK3CA (5 of 61, 8%), NOTCH1 (1 of 61, 2%), EPHA2(2 of 61, 3%), EPHA3 (2 of 61, 3%), andMET (1 of 61, 2%).PIK3CA mutations were validated by PCR and includedthe knownmissense activatingmutationsH1047R, E542K,and E545K (Table 1). Two of these 5 HNSCC lines did nothave PIK3CA amplification (TR146, UMSCC19) and theother 3 did not have SNP array data. An analysis of 279HNSCC tumors from The Cancer Genome Atlas (TCGA)yielded results consistent with both our cell line data andresults of other recently published studies (2, 5), demon-strating that PI3K/AKT/mTOR pathway alterations arecommon in these cancers: 36% of the tumors had PIK3CAgene alterations (44 tumors with mutations, 43 withamplifications, and 14 with both), 2% had PTEN altera-tions (5 mutations and 1 deletion), 4% had AKT1 altera-tions (n ¼ 10), and 1% had mTOR alterations (n ¼ 4;Supplementary Fig. S1).

We identified 2 cell lines with PTEN loss and 7 withPIK3CA amplification (�5 copies) using the SNP array.Both cell lines with PTEN loss demonstrated a completelack of PTEN protein expression byWestern blotting, butthe cell lines with PIK3CA amplification did not consis-tently demonstrate higher PI3K p100a protein expressionthan HNSCC cells without amplification (Fig. 1). PIK3CAprotein levels are regulated bymultiple processes, includ-ing expression of PIK3R1, which is required for p110stability. To confirm the results of the SNP arrays, FISHwas performed on 8 HNSCC cell lines (SupplementaryMethods). The copy numbers determined by FISH wereconsistent with those determined by the SNP array (Sup-plementary Table S3 and Supplementary Fig. S2).

HNSCC cells with PIK3CA mutations, but notamplification, are sensitive to pathway inhibitors

We tested 18 of the 64 HNSCC cell lines with PIK3CAmutations, PTEN loss, low PTEN protein expression,PIK3CA gene amplification, or no known pathway aber-rations for sensitivity to 1 PI3K inhibitor (GDC0941), 2dual PI3K/mTOR inhibitors (GDC0980 andGSK1059615),1 AKT inhibitor (GSK690693), and 1 mTOR inhibitor(AZD8055;Table 1). The cutoff for sensitivity was themaximum concentration in humans or animals whenavailable or the concentration at which pathway inhibi-tion was observed by Western blotting (SupplementaryTable S4; refs. 32–36). The 1 PI3K inhibitor and the 2 dualPI3K/mTOR inhibitors show similar trends in sensitivity(P < 0.05). Sensitivity to the AKT and mTOR inhibitorsshowed distinct trends that did not correlate with any ofthe other agents tested (Supplementary Table S5).

To confirm that the pathway inhibitors function at theconcentrations used, we tested their ability to inhibit the

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Mol Cancer Ther; 13(11) November 2014 Molecular Cancer Therapeutics2740




pathway over a wide concentration range in HNSCC celllines of various sensitivities. In Detroit562 cells (PIK3CA-mutant, sensitive), all inhibitors except AKT inhibitorGSK690693 decreased pAKT and pS6K levels at concen-trations of 100 nmol/L and 4EBP1 level at 500 nmol/L(Fig. 2A). As previously demonstrated, GSK690693 didnot result in a decrease in pAKT level despite AKTinhibition because of pathway feedback (32).We observeda decrease in the AKT substrate pGSK3b at 1 mmol/L,which is consistent with AKT inhibition. Similar resultswere demonstrated with intermediate and resistantHNSCC cell lines (Fig. 2A and B).The 5HNSCCcell lineswithPIK3CAmutationswere all

sensitive to most of the inhibitors, whereas those withPIK3CA gene amplification were predominantly resistant(Table 1). Of the only 2 HNSCC cells lines with PTEN lossin existence, one (UMSCC4) was sensitive to 3 of the 5inhibitors and theother (MDA886LN)was resistant to all 5inhibitors. Likewise, the HNSCC cells with low PTENprotein expression were predominantly resistant. Thecontrol cell lines, which did not have any known PI3K/AKT/mTOR pathway aberrations, hadmixed sensitivity.To test these correlations in a larger set of HNSCC cells,

we determined the IC50 of GDC0941 in all 64 HNSCC celllines and analyzed them in relation to the mutationalstatus and copy number of PI3K. As before, we observedthat cell lines with a PIK3CAmutationweremore likely torespond to PI3K inhibition than cell lines without PIK3CA

mutation (P ¼ 6.35 � 10�13; Fig. 3A) and those withamplification (copy number � 4) were less likely torespond (P¼ 0.365; Fig. 3B), although the latter differencefailed to reach statistical significance even with a copynumber cutoff of 3 or 5.

PI3K pathway and TSC/mTOR scores do notcorrelate with response to PI3K inhibition

We hypothesized that the basal state of the PI3K path-way would correlate with cell sensitivity to PI3K inhibi-tion. To test this hypothesis, we examined the basalactivation of the PI3K/AKT/mTOR pathway by RPPAusing 2 proteomic scores—the PI3K/AKT score (37) andTSC/mTOR score—in 60 HNSCC cell lines (Fig. 3C;Supplementary Fig. S3). Both scores measure pathwayactivation, but there are no overlapping proteins; thePI3K/AKT score measures proximal pathway compo-nents and the TSC/mTOR score measures componentsdistal to TSC1/2 (Supplementary Table S2). In addition,the TSC/mTOR score integrates information from theRAS/MAPK and LKB1/AMPK pathways. Nevertheless,neither score predicted sensitivity to GDC0941. Specifi-cally, we observed no correlation between sensitivity andthe PI3K/AKT score when a sensitivity cutoff of 650nmol/L (P ¼ 0.69) or 1,300 nmol/L (P ¼ 0.65) was usedor when cell lines with sensitivities in the top third andbottom third were compared (P ¼ 0.34). Similarly, therewas no correlation between sensitivity and the TSC/

Table 1. IC50 values of PI3K pathway inhibitors for HNSCC cell lines

IC50, mmol/L

Cell line PI3K pathway alteration

GDC0941(PI3Kinhibitor)

GSK1059615(PI3K/mTORinhibitor)

GDC0980(PI3K/mTORinhibitor)

AZD8055(mTORinhibitor)

GSK690693(AKTinhibitor)

UMSCC47 None known 3.0 0.9 1.2 0.01 1.0HN4 None known 2.0 1.3 2.3 0.85 11.2OSC19 None known 0.74 0.2 0.50 0.43 11.8UMSCC1 None known 0.8 0.8 0.50 0.51 1.94Detroit 562 PI3K mutation (H1047R) 0.40 0.34 0.23 0.35 1.8TR146 PI3K mutation (E545K) 0.46 0.01 0.01 0.27 4.0HOSC1 PI3K mutation (E542K) 0.34 0.11 0.23 0.37 2.8UMSCC19 PI3K mutation (H1047R) 0.55 0.18 0.63 1.2 2.5UMSCC85 PI3K mutation (H1047R) 0.5 0.11 0.38 0.71 2.5MDA886LN PTEN loss 12 3.7 1.8 5.3 8.8UMSCC4 PTEN loss 1.1 0.16 0.18 1.0 0.82MDA1986 LN Low PTEN expression 2.6 2.0 2.0 0.19 1.5MDA686 TU Low PTEN expression 2.2 0.28 0.22 1.9 0.31483 PI3K amplification 7.2 2 8.0 8.0 >20FADU PI3K amplification 1.0 0.33 0.19 2.7 6.4UMSCC10A PI3K amplification 0.89 0.72 0.44 0.09 2.9JHU022 PI3K amplification 15.2 3 12 0.89 3.55HN-5 PI3K amplification 2.0 0.7 1.7 4.0 3.25

NOTE: Values that fall below the cutoff for sensitivity (Supplementary Table S4) are highlighted in green, intermediate values are in greyand pink, and values above the cutoff for resistance are in red.






mTOR score when a sensitivity cutoff of 650 nmol/L (P¼0.89) or 1,300 nmol/L (P¼ 0.12) was used or when the topthird and bottom third were compared (P ¼ 0.37).

We also analyzed the sensitivities of the 18 HNSCC celllines to the 5 pathway inhibitors in relation to the expres-sion of individual pathway components measured by

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Figure 1 . Basal protein expression levels in HNSCC cell lines without PIK3CA mutation, mutated, or amplified PIK3CA or with PTEN loss were determined.HNSCC cells were subjected to lysis and lysates were resolved by SDS-PAGE followed by Western blot analysis using the indicated antibodies for PTEN,PIK3CA (P110 a), phosphorylated PDK1 (A) and multiple pathway components (B).

Mazumdar et al.





Western blotting (Table 1 and Fig. 1B). We measured theexpression levels of several pathway components, includ-ing pAKT, pS6, 4EPB1, and pERK, in these cells. Of these 4markers, pS6 was the best predictor of response, with aconsistent trend for all inhibitors, but the correlation

reached significance (P < 0.05) only with GSK690693 (Sup-plementary Table S6). Likewise, analysis of the 60HNSCCcell lines in the RPPA dataset did not show a correlationbetween basal expression of pS6 (S235/236) or pS6 (S240/242) andsensitivity toGDC0941 (SupplementaryTable S7).

GDC0941 GSK1059615 GSK690693GDC0980 AZD8055

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Figure 2. Five PI3K/AKT/mTOR pathway inhibitors inhibit pathway signaling in both sensitive and resistant cell lines. A, Detroit562 cells (sensitive) and 1483cells (resistant) were treatedwith all 5 inhibitors at the indicated concentrations for 3 hours, andWestern blot analysis was done using antibodies as indicated.B, Western blot analysis shows the effects of the indicated drugs on PI3K/AKT/mTOR pathway components in sensitive (green), intermediate (yellow), andresistant (red) cell lines treated for 3 hours.






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Figure 3. PI3K mutation—but not amplification, PI3K score, or mTOR score—correlates with sensitivity to GDC0941. Sixty HNSCC cell lines were testedfor sensitivity to GDC0941 using an MTT assay. Basal expression levels of 195 proteins and phosphoproteins were measured using RPPA, and theexpression levels of 16proteinswere used todefinePI3KandmTORscores (Supplementary TableS2).CopynumberwasdefinedusingSNParrays.A, dot plotcomparing GDC0941 IC50 values in HNSCC cells with and without a PIK3CA mutation. B, dot plot comparing GDC0941 IC50 values in HNSCC cellswith andwithoutPIK3CA amplification. C, heatmapof expression, IC50, copy number, PI3K/AKT score, and TSC/mTOR score sorted byGDC0941 sensitivity.D, dot plots comparing PI3K/AKT and TSC/mTOR scores to PI3K alteration (PTEN loss and/or PIK3CA mutant and/or PIK3CA amplified), amplification, ormutation in HNSCC tumors from the TCGA.

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An analysis of 160 proteins from the RPPA datarevealed several candidate biomarkers whose expres-sion correlated with sensitivity to GDC0941. These datawere analyzed using the IC50 values as continuousvariables, by comparing the most sensitive third to theleast sensitive third and by using cutoffs of 650 and1,300 nmol/L. The proteins whose expression correlat-ed with sensitivity with a P < 0.05 in at least 3 analyseswere VEGFR2, pCRAF, and PCNA (SupplementaryTable S7).

Genetic alterations in the PI3K pathway do not leadto pathway activationWe expected that HNSCC cell lines with PIK3CAmuta-

tions, PTEN loss, or PIK3CA amplification would havebasal activation of the PI3K/AKT pathway. However,Western blotting demonstrated that there was no consis-tent increase inpAKT,p70-S6, p4E-BP1, orpPDK1 in thosecell lines (Fig. 1). Neither PIK3CA amplification norPIK3CA mutation correlated with the PI3K/AKT score(P¼ 0.21 and 0.31). Likewise, PIK3CAmutation or ampli-fication did not correlate with the TSC/mTOR score (P ¼0.49,P¼ 0.58).Notably, the 2HNSCC cell lineswithPTENloss did have high levels of phosphorylated S6 and AKT(Fig. 1).Similarly, PIK3CA amplification did not correlate with

an increase in the scores in 200 HNSCC tumors from theTCGA (Fig. 3D). However, tumorswith PIK3CAmutationdid have a higher PI3K/AKT score (P ¼ 0.039), but notTSC/mTOR score, than those without PIK3CA mutation.There was no difference in the PI3K/AKT or TSC/mTORscores between 8 tumors on which we have RPPA dataand that have both PIK3CA amplification and mutationand HNSCC tumors without these genetic aberrations(P ¼ 0.226 and 0.556).

PI3K inhibitors induce cell-cycle arrest in HNSCCcell lines with PIK3CA mutationsTo further characterize the biologic effects of PI3K

inhibition, HNSCC cells were treated with GDC0941 for72 hours. In the sensitive Detroit562 and TR146 cells, butnot in the resistant 1483 cells, GDC0941 caused cell-cycle arrest in G1 with a reduction of the number ofcells in S-phase (Supplementary Fig. S4). No significantapoptosis, mitotic catastrophe, or senescence wasobserved in any of the cell lines (all <5%, SupplementaryMethods).

Activation of the MEK/ERK pathway inducesresistance to PI3K inhibitorsFollowing incubation with GDC0941, pERK levels

increased in the resistant cell lines JHU022 and 1483,whereas remaining unchanged in the sensitive cell lineDetroit562 and the intermediate cell line OSC19 (Fig. 4A).Likewise, in the 2HNSCC cell lineswithPTEN loss, pERKwas inhibited in the cell line with intermediate sensitivitybut not in the resistant cell line (Fig. 4B). Treatment of bothsensitive and resistant HNSCC lines with a combination

of MEK and PI3K inhibitors led to additive or synergisticeffects in all cell lines except Detroit562 (Fig. 4C). Thecombination also led to enhanced cell-cycle arrest (Sup-plementary Fig. S5) in TR146 (sensitive) and 1483 (resis-tant) compared with untreated or GDC0941 alone. InDetroit562 (sensitive), GDC0941 alone and combinationshowed almost same degree of cell-cycle arrest. However,we did not see any significant cell-cycle arrest inMDA886LN. Because the combination of MEK and PI3Kinhibitors was particularly effective in a murine model ofKRAS-mutant adenocarcinoma (38), we tested it in the 2cell lines with HRAS mutations and found that the drugcombination was synergistic, with CIs of 0.015� 0.27 and0.096� 0.12 for UMSCC17A and HN31 cells, respectively(Fig. 4D). Target inhibitionwas confirmedbyWestern blotanalysis for both drugs (Fig. 4E).

Activation of multiple receptor tyrosine kinasesfollowing PI3K inhibition

To investigate potential pathways of resistance inHNSCC following PI3K inhibition, we incubated 2 sen-sitive and 2 resistantHNSCC cell lineswithGDC0941 andmeasured the levels of 39 phosphoproteins. We discov-ered that the phosphorylation of EGFR, RET, FGFR3, andM-CSFR was increased in resistant lines but not in sen-sitive ones following incubation with GDC0941 (Fig. 5A).Phosphorylation of IGF1Rwas increased in both sensitiveand resistant cells although to a lesser extent in thesensitive lines.

Inhibition of activated receptor tyrosine kinasepathwayspartially reversesPI3K inhibitor resistance

To explore the functional significance of the EGFR,IGF1R, RET, and FGFR3 activation in resistant cell lines,we combinedGDC0941with the EGFR inhibitor erlotinib,the IGF1R inhibitor OSI906, the RET inhibitor cabozanti-nib, or the FGFR inhibitor dovitinib (Fig. 5B). Only thecombination of erlotinib and GDC0941 was synergistic inboth resistant cell lines, with CIs of <0.01 and 0.096� 0.11.

DiscussionIn an effort to studymutation-directed targeted therapy

for HNSCC, we identified several missense mutations inHNSCC cell lines that mirror those found in HNSCCpatient samples; the mutations included HRAS, PIK3CA,NOTCH1, EPHA2, EPHA3, and MET (3, 4). Like patienttumors, our cell lines had other alterations in the PI3K/AKT/mTOR pathway, including PIK3CA amplificationand PTEN loss. HNSCC cell lines with PIK3CAmutationswere universally sensitive to inhibitors of PI3K,mTOR, orboth. Surprisingly, PIK3CA amplification, PTEN loss, andbasal pathway activity did not predict response. Therewas anunexpected, statistically nonsignificant correlationbetween PIK3CA amplification and resistance. All thePI3K, mTOR, and dual inhibitors tested were equallyefficacious in the 18 HNSCC cell lines tested; sensitivityto the PI3K and dual inhibitors correlated with each other






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Figure 5. Treatment with GDC0941activates EGFR, IGF1R, FGFR,RET, and M-CSFR in resistant celllines. A, 2 sensitive and 2 resistantHNSCCcell lineswere treatedwith500 nmol/L GDC0941 or vehiclealone for 6 hours and thenprocessed for PhosphoScananalysis as described in Materialsand Methods. Densitometricanalysis was done to measurechange in protein expression usingImageJ software. B, resistant cellswere incubated with GDC0941alone or in combination (1:1) witherlotinib, OSI906, cabozantinib, ordovitinib for 72 hours. i, inhibitor.Cell viability was estimated by anMTT assay. CIs are indicated.






but not with sensitivity to the mTOR or AKT inhibitor.More cell lines were resistant to the single AKT inhibitortested than to the other inhibitors tested, but this mayreflect thedifficulty in choosing a cutoff for this drug in theabsence of clinical pharmacokinetic data and feedbackpathway reactivation of AKT. We found that the phos-phorylation levels ofERK,EGFR, IGF1R,RET, FGFR3, andM-CSFRwere increased by PI3K inhibition in the resistantlines. This reactivation had biologic significance asdual inhibition ofMEKor EGFRwith PI3K led to synergy.In particular, the 2 HNSCC cell lines with HRAS muta-tions had the most marked synergy of PI3K and MEKinhibition.

Ours is not the first study to demonstrate a correlationbetween PI3K mutation and response to pathway inhibi-tors. In HNSCC, this finding is particularly intriguing, ashuman papilloma virus–positive tumors have a higherrate of PIK3CAmutation (5). However, early clinical datademonstrate that even mutant tumors are neither pro-foundly nor universally sensitive to pathway inhibitors(20). Our in vitro data support this clinical finding in thatthe sensitive cell lines underwent cell-cycle arrest but notapoptosis, demonstrating that resistance pathways needto be further defined.

Several mechanisms of resistance to these pathwayinhibitors have been elucidated. The best-studied inter-action is between PI3K/AKT/mTOR and receptor tyro-sine kinases (RTK); this interaction can subsequentlyactivate the RAS/RAF/MEK/ERKpathway. As expectedwith this model, AKT inhibition leads to the increasedexpression of multiple RTKs, and the combined admin-istration of ERK and PI3K inhibitors leads to higher ratesof cancer cell death (39). Likewise, we found that theaddition of anMEK inhibitor to a PI3K inhibitor enhancedefficacy in both sensitive and resistant cell lines. That thiswas particularly the case in HRAS-mutant HNSCC celllines is consistent with the findings in genetically engi-neered murine models of lung cancer with inducibleexpression of KRAS (G12D) or PIK3CA (H1047R; ref. 38).One potential mechanism leading to ERK activation is theloss of inhibition of the transcription factor FOXO3 fol-lowingAKT inhibition, leading to increased expression ofmultiple RTKs (40). Enhanced signaling via HER2 follow-ing PI3K inhibition has been reported to lead to ERKactivation (39). Similarly, we also found that PI3K inhi-bition in resistant HNSCC cells led to the activation ofmultiple RTKs and that EGFR and PI3K inhibitors weresynergistic inHNSCC cells consistentwith priorwork in 2HNSCCcell lines (41). This cross talkmaybebidirectional,as MEK inhibition leads to PI3K/AKT activation via theloss of HER3 inhibition (42).

Additional mechanisms of resistance include mTORinhibition leading to the induction of IRS-1 and subse-quent AKT activation in cancer cells and patient tumors(43). IGF1R inhibition sensitizes cells to mTOR inhibition(43). In a transgenic breast cancer model, both PIK3CA-independent (Myc amplification) and -dependent (Metamplification) resistance pathways were identified (44).

In addition, accumulation of nuclear b-catenin can sub-vert the proapoptotic signal of nuclear FOXO3a, inducedby PI3K inhibition, into a prometastatic signal (45). Inbreast cancer cells, PI3K inhibition leads to DNA damageand BRCA1/2 downregulation; PI3K inhibition or knock-down sensitizes cells to PARP inhibition (46).

Although the drugs we used potently inhibited thePI3K/AKT/mTOR pathway in both sensitive and resis-tant cells, distal pathway inhibition (pS6 and p4EBP1)occurred at lower drug doses and was more complete insensitive cell lines. Distal pathway components may bereactivated by ERK, RTKs, or AKT-independent path-ways downstream of PIP3, such as JNK (47) and STAT3(48). Our future studies will include RPPA as well assiRNA screens to identify targetable pathways thatenhance the efficacy of PI3K/AKT/mTOR pathwayinhibitors.

Multiple biomarkers for predicting drug sensitivitybeyond PI3K mutations have been proposed, includingPTEN mutations, PTEN loss, and AKT phosphorylation(21, 27); HER2 amplification (23, 24); 4EBP1 expression(25); NOTCH1 mutation (19); and pSTAT3, EGFR, and c-Kit expression (18). In a screenof theNCI-60human tumorcell line collection, the expression of several genes, includ-ingOGT andDDN, correlatedwith response to GDC0941;knockdown of either gene enhanced sensitivity to thisagent (49). The presence of KRAS mutation, BRAF muta-tion, or EGFR amplification correlates with resistance toPI3K/AKT/mTOR pathway inhibition (20, 22). pAKTlevels have been clinically validated as a biomarker ofprogression-free survival in 17 patients with pancreaticneuroendocrine tumors treated with everolimus (21). Weidentified 25 proteins whose expression correlates withresponse byat least onemethodof analysis and3 thatwereconsistently correlated by at least three methods. Ourfuture studies will involve testing these biomarkers in anindependent set and in clinical samples frompatientswithHNSCC treated with pathway inhibitors. Unlike breastcancer cell lines, HNSCC cell lines did not show a corre-lation betweenPTENprotein expression and sensitivity toPI3K inhibitors (27).

Wedid not find a correlation betweenPIK3CAmutationor amplification and protein markers of pathway activa-tion. Likewise, pAKT, pS6, and p4EBP1were not elevatedin luminal/endoplasmic reticulum (ER)þ breast cancerwith PIK3CA mutation (50). In an independent set of 77breast tumors, there was no association between PIK3CAmutation and the phosphorylation of GSK3, AKT, S6, orAKT (27). In contrast, mRNA and protein signatures ofPI3K pathway activations were enriched in basal-likebreast cancer where PIK3CA mutations are uncommon.The activation of the PI3K/Akt/mTOR pathway in thebasal subtype may be due to loss of PTEN and INPP4Band/or amplification of PIK3CA rather than mutation ofPIK3CA (50). For amore direct comparison,we also used aprotein signature score identical to that used in breasttumors in the TCGA (50), which includes components ofboth the PI3K andmTOR scores presented above, and did

Mazumdar et al.





not find a correlation with PIK3CA mutation or amplifi-cation in HNSCC tumors from the TCGA (data notshown).Use of hotspot mutational analysis allows for screening

of many samples at specific relevant bases across manypatient samples; however, it can miss also relevant butrarermutations. In a recent study that usedwhole-genomesequencing of 151 HNSCC tumors, additional PIK3CAmutations were detected. These mutations (R115L,G363A, C971R, R975S) were each found in less than 1%of patients with HNSCC and were not included in ourstudy (5). Another limitation of our study was that we didnot conduct animal experiments, but the efficacy of a dualmTOR/PI3Kinhibitor inHNSCCPDXmodelshas recentlybeen demonstrated by 2 independent groups (5, 19).Despite the frequent activation and importance of the

PI3K/AKT/mTOR pathway, inhibition of this pathwayhas had variable efficacy in vitro, in vivo, and in clinicaltrials (12–18). Identification of biomarkers that predictsensitivity to these inhibitors could result in the firstbiomarker-driven targeted therapy for HNSCC. Further-more, the development of rational drug combinationsbased on a comprehensive understanding of HNSCC cellresistance mechanisms could significantly improve out-comes for patients with HNSCC. In this study, we haveidentified potential biomarkers and combination thera-pies that will be tested in future clinical studies.

Disclosure of Potential Conflicts of InterestG.B. Mills received a commercial research grant from AstraZeneca,

HanAlBio, andGSK; has ownership interest (including patents) in CatenaPharmaceuticals, PTV Ventures, and Spindle Top Ventures; and is aconsultant/advisory board member for AstraZeneca, Bind, Critical Out-

come Technologies, Han AlBio Korea, Nuevolution, and Symphogen.K. Stemke-Hale has ownership interest (including patents) in Glaxo-SmithKline and Exelixis. J.V. Heymach is a consultant/advisory boardmember for AstraZeneca, GlaxoSmithKline, Genentech, Boehringer Ingel-heim, Exelixis, and Synta. No potential conflicts of interest were disclosedby the other authors.

Authors' ContributionsConception and design: T. Mazumdar, J.N. Myers, B.S. Glisson,F.M. JohnsonDevelopment of methodology: T. Mazumdar, L.A. Byers, G.B. Mills,F.M. JohnsonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Mazumdar, L.A. Byers, Y.-H. Fan, K. Stemke-Hale, F.M. JohnsonAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): T. Mazumdar, L.A. Byers, P.K.S. Ng,G.B. Mills, S. Peng, L. Diao, K. Stemke-Hale, B.S. Glisson, F.M. JohnsonWriting, review, and/or revision of the manuscript: T. Mazumdar,L.A. Byers, G.B. Mills, K. Stemke-Hale, J.V. Heymach, J.N. Myers,B.S. Glisson, F.M. JohnsonAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): T. Mazumdar, G.B. Mills, S. Peng,F.M. JohnsonStudy supervision: F.M. Johnson

AcknowledgmentsThe authors thank Arthur Gelmis, Kathryn Hale, and the Department

of Scientific Publications for editorial assistance with the manuscript.They also thank Brenda Robinson for administrative assistance.

Grant SupportThis work was supported by the National Cancer Institute MD Ander-

son Cancer Center Head &Neck SPORE P50CA097007 (to J.N. Myers, B.S.Glisson, and F.M. Johnson). RPPA, mutational analysis, FISH, and DNAsequencing were supported by the National Cancer Institute CancerCenter Support Grant P30CA016672 (to The University of Texas MDAnderson Cancer Center).

Received January 6, 2014; revised August 8, 2014; accepted August 22,2014; published OnlineFirst September 5, 2014.

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Mazumdar et al.




2014;13:2738-2750. Published OnlineFirst September 5, 2014.Mol Cancer Ther Tuhina Mazumdar, Lauren A. Byers, Patrick Kwok Shing Ng, et al. Squamous Cell Carcinoma

Neckto PI3K Inhibitors and of Resistance Mechanisms in Head and A Comprehensive Evaluation of Biomarkers Predictive of Response

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