Monensin Inhibits Epidermal Growth Factor Receptor Trafﬁcking … · Small Molecule Therapeutics...

Small Molecule Therapeutics

Monensin Inhibits Epidermal Growth Factor ReceptorTrafficking and Activation: Synergistic Cytotoxicity inCombination with EGFR Inhibitors

Khalil Dayekh1,2, Stephanie Johnson-Obaseki3, Martin Corsten3, Patrick J. Villeneuve1,4,Harmanjatinder S. Sekhon5, Johanne I. Weberpals1,6, and Jim Dimitroulakos1,2

AbstractTargeting theEGFR,with inhibitors such as erlotinib, represents a promising therapeutic option in advanced

head and neck squamous cell carcinomas (HNSCC). However, they lack significant efficacy as single agents.

Recently,we identified the ability of statins to induce synergistic cytotoxicity inHNSCCcells through targeting

the activation and trafficking of the EGFR. However, in a phase I trial of rosuvastatin and erlotinib, statin-

induced muscle pathology limited the usefulness of this approach. To overcome these toxicity limitations, we

sought to uncover other potential combinations using a 1,200 compound screen of FDA-approved drugs.

We identified monensin, a coccidial antibiotic, as synergistically enhancing the cytotoxicity of erlotinib in two

cell linemodels ofHNSCC, SCC9 and SCC25.Monensin treatmentmimicked the inhibitory effects of statins on

EGFR activation anddownstream signaling. RNA-seq analysis ofmonensin-treated SCC25 cells demonstrated

a wide array of cholesterol and lipid synthesis genes upregulated by this treatment similar to statin treatment.

However, this pattern was not recapitulated in SCC9 cells as monensin specifically induced the expression of

activation of transcription factor (ATF) 3, a key regulator of statin-induced apoptosis. This differential response

was also demonstrated in monensin-treated ex vivo surgical tissues in which HMG-CoA reductase expression

and ATF3 were either not induced, induced singly, or both induced together in a cohort of 10 patient samples,

including fourHNSCC.These results suggest thepotential clinical utility of combiningmonensinwith erlotinib

in patients with HNSCC. Mol Cancer Ther; 13(11); 2559–71. �2014 AACR.

IntroductionHead and neck squamous cell carcinoma (HNSCC) is

the sixth most common cancer and accounts for approx-imately 90% of the cancers occurring in the mucosa of thehead and neck (1). Presently, early-stage HNSCC tumorsare treated with radiation or surgery (2) and in advancedcases, a multimodal approach is used often incorporatingchemotherapy (3). Unfortunately, long-term survivalremains relatively unchanged (4) and new therapeutic

options are required to enhance overall survival (OS) ofpatients with HNSCC. Expression of the EGFR is associ-atedwithHNSCCpathogenesis and isupregulated in 90%of these tumors (5, 6). EGFR expression is associated withpoor prognosis (7–9). Preclinical studies that target theEGFR in HNSCC models have shown promising results(10, 11); however, ineffective receptor inhibition andderegulated downstream signaling pathways can lead toresistance to these agents (12). Thus, targeting the EGFR incombinationwith other strategies that can overcome theseresistant mechanisms is critical to enhance the efficacy ofthis approach.

Tyrosine kinase inhibitors (TKI) are a class of therapeu-tic agents that consist of low molecular weight ATPmimics that bind the intracellular kinase domain of recep-tors like the EGFR (13). TKIs compete with and reversiblybind to the ATP-binding site preventing the activation ofthe receptor and its downstream signal transduction cas-cades (13). Gefitinib (ZD 1839, Iressa) and erlotinib (OSI774, Tarceva) are themost clinically advancedEGFR–TKIs(13, 14). Interestingly, recent studies have revealed that asubset of patients with non–small cell lung cancer(NSCLC) respond more robustly to treatment with thesedrugs due to the presence of mutations in the kinasedomain of the EGFR (15, 16). These mutations stabilizeATP binding conferring enhanced activity to thismutated

1Centre for Cancer Therapeutics, the Ottawa Hospital Research Institute,The University of Ottawa, Ottawa, Ontario, Canada. 2Faculty of Medicineand the Department of Biochemistry, The University of Ottawa, Ottawa,Ontario, Canada. 3Department of Otolaryngology, The Ottawa Hospital,Ottawa, Ontario, Canada. 4Department of Thoracic Surgery, The OttawaHospital, Ottawa, Ontario, Canada. 5Department of Pathology and Labo-ratory Medicine, The Ottawa Hospital, Ottawa, Ontario, Canada. 6Depart-ment of Gynaecologic Oncology, The Ottawa Hospital, Ottawa, Ontario,Canada.

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

Corresponding Author: Jim Dimitroulakos, Centre for Cancer Therapeu-tics, Ottawa Hospital Research Institute, 501 Smyth Road, 3rd FloorGeneral Division, Box 926, Ottawa, ON, K1H 8L6. Phone: 613-737-7700, ext. 70335; Fax: 613-247-3524; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-13-1086

�2014 American Association for Cancer Research.

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on July 3, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst September 4, 2014; DOI: 10.1158/1535-7163.MCT-13-1086

http://mct.aacrjournals.org/

receptor; however, TKIs can also demonstrate enhancedbinding to the ATP-binding pocket rendering theseNSCLC cells more sensitive to these agents (15, 16).

Monensin is an antibiotic secreted by the bacteriaStreptomyces cinnamonensis and classified as a poly-ether antibiotic or ionophore possessing a cyclic con-formation with protruding alkyl groups making themolecule highly lipid-soluble (17). This allows monen-sin to freely pass across the lipid bilayer of the cyto-plasmic membrane or cellular organelles transportingions along by passive diffusion (17). During ion trans-port, monensin crosses the lipid membrane, loses theproton on the carboxylic group, and chelates a cationand then it crosses back to the opposite side of themembrane losing the ion in exchange for a proton andthe cycle repeats (18). During the past decade, monensinhas been the focus of many cancer studies in which itseffectiveness in targeting cell lines derived from manycancer types, including renal cell carcinoma, colon can-cer, myeloma, lymphoma, and prostate has been dem-onstrated (19–22). Furthermore, Ketola and colleagues(23) compared the cytotoxic effect of monensin betweenmalignant versus nonmalignant cell lines and conclud-ed that nonmalignant cell lines are more than 20-foldmore resistant than their malignant counterparts. Fur-thermore, it has been shown to have a positive safetyprofile in veterinary medicine as it has been used incattle and poultry feed for more than 40 years (17, 18).

Our laboratory has previously identified high-dosestatins, a widely prescribed family of agents used to treathypercholesterolemia (24), as potent inducers of tumor-specific apoptosis particularly in various pediatric malig-nancies, acute myelogenous leukemias and HNSCC cells(25–27). This led to a phase I study in squamous cellcarcinoma evaluating lovastatin as a single agent thatdemonstrated potential utility likely as part of a combi-nation-based regimen (28). We then demonstrated thepotential of lovastatin to induce synergistic cytotoxicityin combination with EGFR–TKIs (29). The mechanism ofaction revolves around the ability of lovastatin to targetthe activity of EGFR and other RTKs enhancing the cyto-toxicity of EGFR–TKIs in HNSCC cells (30, 31). Thesestudies eventually led to a phase I clinical trial of rosu-vastatin and erlotinib at our Institute that showed diseasestabilization, although the high dose of statins used hadundesirablemuscle pathologies in a significant number ofpatients (ClinicalTrials.gov Identifier: NCT00966472),suggesting an alternative approach to improve cancertargeting while minimizing the side effects of the treat-ment regimen. In this study, we performed a high-throughput analysis of 1,200 FDA-approved compoundsin combination with lovastatin or erlotinib and identifiedmonensin as a potent enhancer of their cytotoxicity in twoHNSCC (SCC9 and SCC25)-derived cell lines. Important-ly, monensin has been shown to similarly inhibit thetrafficking of the EGFR as well as other receptors andmay represent an alternative to statins with a similarmechanism of action.

Materials and MethodsTissue culture

The HNSCC SCC9 and SCC25 and the normal lungfibroblasts GM-38 cell lines were purchased from theATCC. The SCC9 and SCC25 cells lines were authenticat-ed using short tandem repeat genetic profiling (Centre forApplied Genomics). PCR analysis confirmed that all celllines showed undetectable mycoplasma contaminationusing routine methodology as outlined (32). All experi-ments were performed within 10 passages of SCC9 andSCC25 cells and within three passages of the GM-38 cells.Cells were grown and maintained in DMEM (Cellgro)supplemented with 1% (v/v) penicillin/streptomycin(Sigma-Aldrich) and10% (v/v) fetal calf serum (Hyclone).Incubation settings were maintained at 37�C and 5%CO2.Erlotinib (Tarceva) hydrochloride was purchased fromBioVision and resuspended and diluted to 10 mmol/Lstock inDMSO.Lovastatinwasobtained fromApotex andsuspended in ethanol to 10 mmol/L. Sodium salt ofmonensin was purchased from Sigma-Aldrich and sus-pended in ethanol to 10 mmol/L solution.

High-throughput chemical library screenThe SCC9 and SCC25 cell lines were treated with a

chemical library of 1,200 FDA-approved compounds (Pre-stwick Chemical; ref. 33). All compounds were suppliedin a 10mmol/L stock diluted inDMSOandwere used at afinal concentration of 1mmol/L.Base-line cytotoxicitywasevaluated for the drug library alone for both the 48- and72-hour time points to be comparedwith the combinationtreatments. Two combination screenswere performed (1),SCC9 andSCC25pretreatedwith 10mmol/L lovastatin for24 hours then exposed to the drug library at 1mmol/L for afurther 48 hours, and (2) SCC9 and SCC25 pretreatedwiththe drug library for 24 hours at 1 mmol/L followed by 10mmol/L erlotinib treatment for 48 hours. The MTT assaywas used to determine cell viability as described below.

2.4 (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

SCC9, SCC25, and GM-38 cell lines were seeded on 96-well flat-bottomed plates (Corning Costar #3595) at adensity of 5,000 cells per well. The next day, cells werepretreated with lovastatin for 24 hours followed by mon-ensin (0–5 mmol/L) for a further 48 hours or pretreatedwith monensin (0–5 mmol/L) for 24 hours followed byerlotinib (0, 1, 5, or 10 mmol/L) for 48 hours. This sequenceof treatments recapitulates the chemical library screenusedin this study. After 48 hours, 10 mg/mL MTT reagent(Sigma-Aldrich) dissolved in PBS was added for 2 hoursfollowed by cell lysis and MTT solubilization with 0.005NHCl (in 10%SDS) for 24hours.MTT activitywasmeasuredusing a BioTek Synergy MX plate reader at 570 nm andanalyzed using Gen5 software (BioTek). The combinationeffects of lovastatin or erlotinib in combination with mon-ensin were determined by the Chou–Talalay method (34)using CalcuSyn computer software (Biosoft). The dose-effect curves of each drug alone, and in combination, were

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produced byMTT assay. These data were entered into theCalcuSyn software and combination index (CI) valueswere graphed on fraction-affected CI (Fa-CI) plots. A CI< 1 is a synergistic interaction, CI¼ 1 is additive, andCI > 1is antagonistic.

Propidium iodide flow cytometryOne million SCC25 cells were seeded in 10-cm plates

and incubated overnight to allow for attachment andrecovery. The following day, cells were pretreated with0, 1, or 5 mmol/L monensin for 24 hours then treatedwith 10 mmol/L erlotinib alone or in combination withmonensin for a further 24 hours. Adherent and cells insuspension were collected by centrifugation and fixedin 3 mL of cold 80% ethanol overnight at �20�C. Beforeanalysis, cell pellets were washed with PBS resus-pended in staining buffer containing 25 mg/mL propi-dium iodide (Sigma-Aldrich) and 40 mg/mL RNase A(BioShop) and incubated for a minimum of 1 hour in thedark at room temperature. Data (10,000 events) wereacquired on an EPICS XL flow cytometer (BeckmanCoulter) and analyzed with Modfit software (VeritySoftware House).

Western blottingSCC9 and SCC25 cells were pretreated with 0, 0.1, or 1

mmol/Lmonensin (Sigma-Aldrich), for 22 hours followedby 2 hours of treatment with control, 10, 1, 0.1, 0.01, and0.001 mmol/L erlotinib under serum-free conditions. Cellswere then stimulated for 15 minutes with 50 ng/mL EGFbefore lysis with RIPA buffer (50 mmol/L Tris-Cl, 150mmol/L NaCl, 1 mmol/L EDTA, 1% (v/v) Triton X-100,0.25% (w/v) sodium deoxycolate, and 0.1% (w/v) SDSand pH 7.5) supplemented with protease inhibitor cock-tail (Sigma-Aldrich), 17.5 mmol/L beta-glyceropho-sphate, and 0.2 mmol/L Na3VO4 (Sigma-Aldrich). Thesamples’ protein contents were quantified using thePierce BCA protein assay protocol (Pierce). Proteinsamples were resolved by SDS-PAGE and transferredonto polyvinylidene fluoride membranes (Immobilon-P; Millipore). Blocking the membrane was performedwith 5% BSA (Sigma-Aldrich) in Tris-buffered Salinewith 0.1% Tween-20 (TBS-T) and Western blotted withthe following primary antibodies: EGFR, Akt, pAkt,ERK, pERK (Cell Signaling Technology) overnight at4�C and diluted 1/1,000, pY20 (BD Biosciences) over-night at 4�C and diluted 1/2,000, and actin antibody(Sigma-Aldrich) for 1 hour at room temperature anddiluted 1/10,000. Dilution of antibodies was made in 5%BSA 0.1% TBS-T. Blots were washed twice with 0.1%TBS-T and once with TBS for 5 minutes each wash, thenincubated for 1 hour at room temperature with theappropriate horseradish peroxidase–tagged secondaryantibody (anti-rabbit 1/2,500 and anti-mouse 1/300Jackson ImmunoResearch). Supersignal west picochemiluminescence substrate (Pierce) was applied tothe blot and developed using Syngene bioimaging sys-tem (Syngene Bio-imaging).

Densitometric analysis was performed using the Gene-Tools software from Syngene (Syngene Bio-imaging). Thedensity of the nonphosphorylated EGFR antibody wasnormalized with that of the actin antibody, then thedensity of the pY20 antibody of the 170 kDa band wasdivided by the normalized density of the nonphosphory-lated EGFR and taken as percentages. This was done intriplicate to obtain the SD of the mean.

Fluorescence microscopySCC9 and SCC25 cells were seeded in 6-well plates

containing a cover slip at a density of 2.5 � 105 cells perwell in complete media and incubated overnight andreplaced by serum-free media containing 0, 0.1, 1, 10mmol/L erlotinib, 10 mmol/L lovastatin, or 1 mmol/Lmonensin for 24 hours. The following day the cells weretreated with 50 ng/mL EGF that is conjugated to theAlexa-488 fluorophore (Molecular probes) for 30 minutesat 4�C to allow the ligand to bind to the receptor but notinternalize. After that, the cells were washed three timeswith 2 mL ice-cold PBS followed by 15 to 120 minutesincubation at 37�C to allow for internalization. Then, cellswere incubated on ice to stop internalization, rinsed threetimes with ice-cold PBS and then acid washed for 5minutes with 2 mL of acetic acid solution (0.2 mol/Lacetic acid and 0.5 mol/L NaCl, pH 2.8) to remove ligandbound receptor on the cell surface. Cells were then fixedwith 4% paraformaldehyde (Sigma-Aldrich; 4% w/vparaformaldehyde, 2 mmol/L MgCl2, 1.25 mmol/LEDTA in PBS, pH 7.3) for 15minutes at 37�Candmountedon a microscope slide with VectaShield mounting mediawith DAPI (Vector Laboratories). The slides were thenexaminedunder aZiess invertedmicroscope (Ziess) usingoil immersion microscopy. A similar procedure was fol-lowed for the low density lipoprotein (LDL) internaliza-tion using 1 mg/mL LDL-Alexa 488 (Molecular Probes).

Transcriptome analysis (RNAseq)Total RNA was isolated using the RNeasy Isolation

Kit (Qiagen). mRNA expression profiling was deter-mined using NuGen reagents (www.nugeninc.com).After amplification, libraries compatible with IlluminaNGS methods were prepared using the Ovation UltraLow Library Prep Kit (NuGen). The quality of eachlibrary was assessed using a Bioanalyzer 2100 (AgilentTechnologies). Kappa Library Quant Kits (KappaBio-systems; www.kapabiosystems.com) were used forlibrary quantitation. Cluster generation and 2 � 36 bppaired-end sequencing were performed over a singlelane using the Illumina GAIIx or HiSeq Genome Ana-lyzer workstation. We obtained approximately 42 mil-lion reads per sample with 89% to 90% of reads mappedback to the genome. The Illumina CASAVA pipelinewas used to generate fastq sequence files. TopHat/Cufflink pipeline was used for mapping reads to thegenome and quantification of gene expression. Compu-tational assessments were conducted by the OttawaBioinformatics Core Facility.

Monensin Enhances Erlotinib Activity

www.aacrjournals.org Mol Cancer Ther; 13(11) November 2014 2561




RNA isolation and RT-PCR from cell linesSCC25 and SCC9 cell lines were treated with either 1

mmol/L monensin or control for 24 hours and mRNAextracted using the RNeasy Kit (Qiagen) following themanufacturer’s protocol. The concentration of RNA wasquantified using Take3 Micro-Volume plate and BioTekSynergy MX plate reader and analyzed using Gen5 soft-ware (BioTek). Of note, 1 mg of total RNA was used forreverse transcription to cDNA using a High CapacitycDNA reverse Transcription Kit (Applied Biosystems) inan ABI Thermal Cycler (Applied Biosystems). The syn-thesized cDNA was used to carry out real-time PCR (RT-PCR). The total reaction volume of 20 mL contained 2 mL ofcDNA, 1 mL TaqMan Gene-Expression Assay Primer/Probe (20�), 10 mL of TaqMan Universal PCRMaster Mix(2�; Applied Biosystems; 4304437), and 7 mL of RNase-free water. The endogenous control for the assay was thehousekeeping gene, human GAPDH (20�; Applied Bio-systems; HS4333764-F). The reaction was performed in a7500 Real-Time PCR system (Applied Biosystems). Taq-Man Primers/Probes (ATF3, HS00231069); DDIT4:HS01111686_G1; HMGCS1: HS00940429_M1; INSIG1:HS01650977_G1; HMGCR: HS00168352_M1; BNIP3:HS00969291_M1)

Ex vivo tumor analysisSurgical tissue from 4 HNSCC, a mucosal hyperplasia

and an oropharynx lymphoma all from the head and neckregion (HN), 2 lung cancer (LU), and 2 ovarian cancer(OV) patients undergoing routine surgical procedureswere obtained and analyzed in this study that wasapproved by the Ottawa Hospital Research Ethics Board(Protocol# 20120559-01H). Areas containing tumor wereidentified by gross pathologic examinations. Approxi-mately 2-mm cores were obtained using a sterile biopsypunch that was further sliced with a scalpel to obtainapproximately 2- � 1-mm tumor slices. The slices wererandomized and three slices were placed into eachwell of24-well plate and cultured in DMEM (HyClone) supple-mented with 10% heat-inactivated FBS (Medicorp) and100-U/mL antibiotic/antimycotic solution (Sigma). After48 hours drug treatments, the tumor sliceswere processedfor RNA extraction and RT-PCR analysis of HMG-CoAreductase and activation of transcription factor (ATF) 3mRNA levels as described above in triplicate. Initialtumor cell viability was assessed by addition of a 10%Alamar Blue (Invitrogen) solution and measured in Syn-ergy Mx Monochromator-Based Multi-Mode MicroplateReader using Gen5 software, both from Biotek Instru-ments at 560-nm excitation wavelength and 590-nm emis-sion wavelength.

ResultsMonensin synergistically enhances the cytotoxicityof lovastatin and erlotinib

Our goal is to identifymore specific agents that enhanceerlotinib activity without the toxicities associated withhigh-dose statin use in our clinical trials (28). Thus, a high-

throughput drug screen to identify potential enhancers ofboth lovastatin and erlotinib cytotoxicity was performed.Two HNSCC cell line models, SCC9 and SCC25, weretreated with a chemical library of 1,200 FDA-approvedcompounds all at a dose of 1 mmol/L, alone or in combi-nation with either 10 mmol/L lovastatin or 10 mmol/Lerlotinib and assayed for changes in cell viability using theMTTassay.Monensin, used as an antibiotic in animal feed(18), enhanced the cytotoxic action of both lovastatin anderlotinib treatments in both cell lines examined (Supple-mentary Table S1). Interestingly, the EGFR TKIs gefitiniband erlotinib were also enhancers of lovastatin treatment,whereas the mevalonate pathway inhibitors fluvastatinand alendronate enhanced erlotinib cytotoxicityhighlighting our previous identification of the synergisticcytotoxicity inducedby this combinationof agents (29).Asexpected, MTT assay results showed increased lovastatincytotoxicity (Fig. 1A) and erlotinib cytotoxicity (Fig. 1B)when combined with monensin in both SCC9 and SCC25cell lines. To understand the nature of the combinationeffect, the Chou–Talalay method was used to distinguishbetween additive and synergistic interactions (34). Frac-tion-affected (on cell viability) plots were generated toshow the combination effect (CI). CI values of 1 areadditive, less than 1 are synergistic, and more than 1 areantagonistic. In HNSCC cell lines, SCC9 and SCC25,monensin in combination with lovastatin or erlotinibresulted in a marked decrease in viability in a dose-dependent manner, which was also found to be a syner-gistic (Fig. 1C).

Monensin induces a potent apoptotic response incombination with erlotinib

To further confirm the ability of monensin to potentiatethe cytotoxicity of erlotinib, we performed propidiumiodide flow cytometric analysis to assess apoptosis induc-tion (26, 29). SCC25 and SCC9 cells were pretreatedwith 1or 5 mmol/L monensin for 24 hours, followed treatmentwith 10 mmol/L erlotinib alone or in combination withmonensin for 24 hours; each condition performed intriplicate. The histograms of the various treatment con-ditions with the percentage of cells in the subG1 apoptoticpeak are depicted in the upper left of each treatment forSCC25 cells (Fig. 2A). Untreated cells displayed 2.5%apoptosis; 48 hours treatment with 1 mmol/L monensinshowed 4.5% whereas 5 mmol/L monensin for 48 hoursinduced a greater apoptotic response (16.4%). The erloti-nib treatment response for 24 hours at a 10 mmol/L dosewas similar to control (3.4%); however, pretreatment witheither 1 or 5 mmol/Lmonensin for 24 hours followed by 10mmol/L erlotinib treatment for another 24 hours resultedin a marked increases in apoptotic events (14.6% and38.7%, respectively) when compared with either monen-sin or erlotinib treatments alone. In addition, combinationof 5 mmol/L monensin with 10 mmol/L erlotinib showedthe highest percentage of apoptosis (38.7%). These histo-grams also show an increase in S phase suggesting thepotential for monensin to induce an S phase cell-cycle

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arrest as well as inducing apoptosis in SCC25 cells. Theresults of these flow cytometric analyses are tabulatedin Fig. 2B showing a statistically significant induction ofapoptosis in the monensin and erlotinib combinationtreatments compared with either agent alone. In contrastin the normal fibroblast cell line GM-38, monensin aloneor in combination with erlotinib treatment did not show

significant effects on cell viability as assessed by the MTTassay (Fig. 2C, conditions identical to Fig. 1C).

Monensin enhances the erlotinib inhibition of EGFRactivation

The synergistic effects of lovastatin in combinationwith EGFR–TKIs that we previously reported were due

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Figure 1. A, cells pretreated withlovastin for 24 hours combinedwith increasing concentrations ofmonensin for a further 48 hoursand assayed for cell viability usingMTT assay. Activity presented asthe percentage of control using anaverage of six replicates and errorbars represent the SD from themean. B, cells pretreated withmonensin for 24 hours combinedwith increasing concentrations oferlotinib for a further 48 hours andassayed by MTT as above. C,fraction-affected/CI plots, thesynergistic effect of monensin incombination with lovastatin (toprow) or erlotinib (bottom row) inSCC25 and SCC9 cells. CIs < 1 areconsidered synergistic, CIs > 1 areconsidered antagonistic, andCIs ¼ 1 are considered additive.






to its ability to inhibit EGFR activity (30) and to inducethe integrated stress response; particularly, the ATF3that regulates the apoptosis induction of this pathway(35, 36). We first evaluated the effect of monensintreatment alone and in combination with erlotinib onligand-induced EGFR activation and its downstreamsignaling pathways byWestern blot analysis. Phosphor-ylation status of EGFR and its downstream targets AKTand ERK proteins were assessed in serum starved SCC9cells treated with 10 mmol/L lovastatin for 24 hours (Fig.3A, left) or 1 mmol/L monensin for 24 hours (Fig. 3A,right) each compared with untreated control cells and

10 mmol/L erlotinib treatment for 2 hours. In untreatedcontrol cells, 50 ng/mL EGF addition for 15 minutesinduced significant activation of this pathway aspEGFR, pAKT, and pERK levels were increased. Treat-ment of SCC9 cells with erlotinib inhibited ligand-induced activation of all three proteins as pEGFR,pAKT, and pERK levels were undetectable by Westernblot analysis. Both 10 mmol/L lovastatin and 1 mmol/Lmonensin 24 hours treatments induced approximately a50% inhibition of EGF-treated SCC9 cells with respect topEGFR and its downstream targets pAKT and pERK(Fig. 3A).

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Figure 2. A, histograms ofpropidium iodide flow-cytometrydata in SCC25 cells showing, in thetop row from left to right, control,1 and 5 mmol/L monensin. Bottomrow, the addition of 10 mmol/Lerlotinib and its combination withthe above treatments.Combinations of monensin witherlotinib increased apoptosis inSCC25 cells. B, flow-cytometryquantification using a bar diagramthat summarizes the data from theflow-cytometry experiments byplotting the percentage ofapoptosis (sub-G1 peak) as afunction of treatment. Error bars,SD from the mean; n ¼ 3. �, P <0.001 when comparing monensintreatment alone with itscombination with erlotinib bystudent t test analysis. C, GM-38fibroblasts pretreated for 24 hourswith monensin and further treatedwith increasing concentrations oferlotinib for a further 48 hoursassayed for cell viability using MTTassay. Activity presented as thepercentage of control using anaverage of six replicates and errorbars represent the SD from themean.

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To determine the effect of monensin treatment onerlotinib-induced EGFR inhibition, we treated bothSCC9 and SCC25 cells with a wide range of erlotinibconcentrations (0.001–10 mmol/L, 24 hours) alone andin combination with either 0.1 or 1 mmol/L monensintreatments in combination for 24 hours (Fig. 3B). Mon-ensin treatments enhanced the inhibitory effects oferlotinib on ligand-induced EGFR activation as mea-sured by pEGFR levels assessed by Western blot anal-ysis. These results are supported by densitometricquantification shown in Fig. 3C in which pEGFRlevels normalized to EGFR levels are presented thatdemonstrate cooperativity of monensin and erlotinibtreatments with respect to EGFR inhibition. Bothcell lines express wild-type (WT) EGFR at similarlevels (29).

Our previous studies demonstrated that lovastatin-induced inhibition of EGFR activity results from its abilityto inhibit ligand-induced receptor trafficking and dimer-ization (30). Monensin is a recognized inhibitor of intra-cellular trafficking, including the ability to inhibit traf-ficking of the EGFR (37, 38), thus, may share a commonmechanism with lovastatin. To assess the effect of mon-ensin on the trafficking of the EGFR in SCC25 and SCC9cells, we used an Alexa-488–tagged EGF ligand and fol-lowed its trafficking using fluorescentmicroscopy. SCC25and SCC9 cells were either untreated, 0.1 to 10 mmol/Lerlotinib, 10 mmol/L lovastatin, or 1 mmol/Lmonensin for24 hours in serum-free media and then stimulated withthe 50 ng/mL of tagged EGF for 15 minutes. As shownin Fig. 4A, following 15 minutes of tagged EGF treatmentat 37�C, untreated cells display a uniform punctuate

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1010.10.010.001Erlotinib (µmol/L)

1010.10.010.001

Figure 3. A,Western blot analysis ofcontrol, 10 mmol/L erlotinib, and10 mmol/L lovastatin (left) andcontrol, 10 mmol/L erlotinib, and1 mmol/L monensin (right) in SCC9cells treated for 24 hourswith theseagents with or without EGFstimulation (50 ng/mL for15 minutes). Western blots ofphospho (p)-EGFR, pAkt, andpERKand their corresponding totallevels along with actin as loadingcontrols are depicted. B, Westernblot analysis of pEGFR and EGFRin SCC9 and SCC25 cells treatedwith 0, 10, 1, 0.1, 0.01, and 0.001mmol/L erlotinib without or with 0.1or 1 mmol/L monensin for 24 hours.In all cases, EGF stimulation(50 ng/mL for 15 minutes) wasperformed before proteinextraction. C, densitometryanalysis of pEGFR levelsnormalized to their correspondingEGFR levels. Monensin treatmentenhanced the ability of erlotinib toinhibit phosphorylation of theEGFR. Error bars, SD from themean of three replicates (n ¼ 3).






distribution pattern corresponding to the ligand–receptorcomplex trafficking throughout the cell. Addition ofincreasing concentrations of erlotinib up to 10 mmol/L inboth cell lines for 2 hours resulted in absence of fluores-cence indicating lack of ligand internalization and traf-ficking. Labeled ligand at the cell surface was removed byacidwashingof these cells in all cases to limit visualizationto internalized ligand complexes only. In SCC25 cells,treatment with 10 mmol/L lovastatin or 1 mmol/L mon-ensin leads to the accumulation of the Alexa-488–taggedEGF ligandnear the cytoplasmicmembrane, indicating aninhibitory effect on its intracellular trafficking (Fig. 4A) . InSCC9 cells by contrast, similar lovastatin and monensintreatments showed inhibition of ligand internalizationas intracellular fluorescence was significantly reduced(Fig. 4A). To determine whether the inhibitory effectof monensin on EGF trafficking persists in SCC25cells, we evaluated later internalization times of up to120 minutes. In control-untreated SCC25 cells, EGFfluorescence was predominately located at the cell sur-face at 15 minutes but showed perinuclear localizationat the later time points clearly evident at 60 minutesEGF treatment (Fig. 4B). This likely demonstrates earlyendosome to lysosomal trafficking of this ligand as

previously reported (39). In 1 mmol/L 24 hours mon-ensin-treated SCC25 cells, the inhibitory effects on EGFtrafficking are present up to 30 minutes following ligandinternalization but progress to the typical perinuclearlocalization and later time points (Fig. 4B).

Monensin activates pathways implicated in lipidsynthesis and apoptosis

To gain further insight on the potential mechanism ofaction of monensin on EGFR inhibition and enhancementof erlotinib cytotoxicity in SCC25 cells, we performedRNA-seq full transcriptome analysis (40) of untreatedcontrols compared with 24 hours 1 mmol/L monensin-treated SCC25 cells. Significant differences in expressionof greater than 2-fold were 618 genes; to narrow the list ofhits to analyze, we selected the magnitude of differentialexpression to greater than 4-fold, limiting the number ofdifferentially expressed genes to 115 targets . Using theDAVID Bioinformatics Resources 6.7 (http://david.abcc.ncifcrf.gov/home.jsp) to perform functional annotationclustering of this gene set, a significant number of differ-entially regulated genes following monensin treatmentare involved in cholesterol and lipid synthesis pathways(Supplementary Table S2) as well as various apoptosis

Untreated 0.1 mmol/L Erlotinib 0.1 mmol/L Erlotinib 10 mmol/L Erlotinib 10 mmol/L Lovastatin 1 mmol/L Monensin

SCC25- 24 h treatments, 15 min EGF-Alexa 488, DAPI

SCC25 Untreated

SCC25 1 mmol/L Monensin 24 h

SCC9- 24 h treatments, 15 min EGF-Alexa 488, DAPI

50 ng/mLEGF-Alexa 488

15 min 30 min 60 min 90 min 120 min

A

B

Figure 4. A, the effect of 24 hours treatment of erlotinib (0.1, 1, and 10 mmol/L), 10 mmol/L lovastatin, and 1 mmol/Lmonensin on the ability of SCC25 and SCC9cells to uptake Alexa-488–conjugated EGF compared with untreated controls. Cell surface EGF was removed by acid wash before visualization. Bothtreatments with 10 mmol/L lovastatin or 1 mmol/L monensin for 24 hours lead to accumulation of the ligand adjacent to the cytoplasmic membrane in SCC25cells and an impairment of EGF internalization in SCC9 cells. Nuclei counterstainedwith DAPI. B, untreated SCC25 cells or SCC25 cells treatedwith 1mmol/Lmonensin for 24 hours treated with 50 ng/mL of Alexa-488–labeled EGF for 15 to 120 minutes to visualize ligand localization following internalization.

Dayekh et al.





regulators. The low-density lipoprotein (LDL) receptorbinds and transports LDLs that contain cholesterol andlipids into cells and is a mechanism to maintain choles-terol and cellular lipid pools (41). Previous studies haveshown that monensin treatment can also inhibit LDLreceptor trafficking (41), and we assessed the effect ofmonensin on LDL uptake in SCC25 cells that were treatedwith 0 and 1 mmol/L monensin for 48 hours. To visualizeLDL receptor trafficking, we added Alexa-488–taggedLDL for 15 minutes at 37�C. We found that monensintreatment inhibited the uptake of LDL, indicating that inSCC25 cells monensin treatment inhibits the cellular traf-ficking of LDL (Fig. 5A).We next used quantitative RT-PCR (qRT-PCR) to

validate the differential expression identified byRNA-seq in monensin-treated SCC25 cells (1 mmol/L,24 hours). Because of the fact that lovastatin also targetscholesterol synthesis and induces apoptosis in SCC25cells, we focused on the cholesterol synthesis enzymesHMG-CoA synthase, HMG-CoA reductase (target ofstatins) and INSIG1 and the apoptosis regulators BNIP3(42) and DDIT4 (43). All of these genes were signifi-cantly upregulated in SCC25 with 1 mmol/L 24 hoursmonensin treatment cells as predicted in our RNA-seqanalysis (Fig. 5B). However, when the expression ofthese genes was evaluated in SCC9 cells, similar mon-ensin treatments failed to demonstrate the induction ofthese genes, including the apoptosis regulators (Fig. 5B).Because both lovastatin and monensin induce apoptosisin SCC9 cells, we evaluated the expression of ATF3 inboth SCC25 and SCC9 cells following monensin treat-ment. Monensin induced a potent induction of ATF3 inSCC9 but not in SCC25 under these conditions (Fig. 5B),suggesting that monensin can induce apoptosis throughdifferent and independent mechanisms in these two celllines.

Treatment of ex vivo tumor tissue samples withmonensinTo assess the potential of monensin to induce either

HMG-CoA (Fig. 6A) reductase (Fig. 6A) and/or ATF3(Fig. 6B) mRNA expression in a more clinically relevantmodel, we assessed ex vivo HNSCC tumor tissue inculture from 4 different patients with HNSCC under-going surgery at our Institute (HN01-04), a noncancer-ous hyperplastic epithelial lesion (HN05) and an oro-pharynx lymphoma (HN06). We also similarly evaluat-ed 2 lung cancer and 2 ovarian cancer patient tumortissues. Tissue samples were processed following sur-gical excision into 2- � 1-mm slices, randomized withthree tissue slices evaluated per treatment (Fig. 6C) andanalyzed by qRT-PCR performed in triplicate. In ourprevious studies using lovastatin treatments, HMG-CoA reductase induction was generally to a lowerextent than ATF3 (29, 35).In the ex vivo tissue samples evaluated, seven of 10

showed induction of greater than 2-fold of HMG-CoAreductase mRNA levels following 1 and 10 mmol/L mon-

ensin treatments for 48 hours. This included three of thefour HNSCC, the hyperplastic lesion, and the lymphomahead and neck samples as well as one of two lung ade-nocarcinomas and one of two ovarian mucinous tumors(Fig. 6A). For ATF3 expression, five of 10 showed induc-tion of greater than 4-fold ofATF3mRNA levels followingmonensin treatments as above. This included two of thefour HNSCC and the hyperplastic lesion as well as one oftwo lung adenocarcinomas and one of two ovarian

SCC25

SCC25

SCC9

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1 mg/mL LDL (15 min)

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INSIG

1

BNIP3

DDIT4

ATF3

HMG-CoAS

HMG-CoAR

INSIG

1

BNIP3

DDIT4

A

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Figure 5. A, the effect of 1 mmol/L monensin treatment for 48 hours onthe uptake of LDL in vitro using the SCC25 cell line. Untreated cellsinternalized LDLas evidencedby intracellular punctate staining pattern ofthe labeled LDL, whereas treatment with monensin inhibited its uptake.Green color, Alexa-488–tagged LDL; blue, DAPI stain. B, confirmatoryqRT-PCR analysis of 1 mmol/L monensin treatment (black bars) for24 hours in SCC25 and SCC9 cells. Orange bars, controls. mRNA levelswere determined normalized to GAPDH levels. ATF3, HMG-CoAreductase, HMG-CoA synthase, INSIG1, BNIP3, and DDIT4 geneexpression was evaluated. The RT-PCR results show that the two celllines respond differently to monensin treatment as in SCC25 in whichcholesterol synthesis and BNIP3 and DDIT4 were upregulated. Incontrast, only the stress response gene ATF3 was upregulated in SCC9cells.






mucinous tumors (Fig. 6B). As in the SCC9 and SCC25 celllines, monensin treatments also displayed disparateresults with the induction patterns of the mRNA expres-sion of these two genes. For example, HMG-CoA reduc-tase (HN02, HN04, and HN06) or ATF3 (HN01) wereinduced alone, in concert (HN03,HN05, LU02, andOV01)

or showed no change in expression (LU01 and OV02). Inthis limited cohort of two HNSCC cell lines and a varietyof ex vivo tissue evaluated that included four HNSCCtumors, no associations with clinical parameters andresponse to monensin was evident (Supplementary TableS3; patient characteristics).

6

4

2

0

HMG-CoA reductase expressionUntreated1 mmol/L monensin

10 mmol/L monensin

Untreated1 mmol/L monensin

10 mmol/L monensin

HNSCC

HNSCC

HN01 HN02 HN03 HN04 HN05 HN06 LU01 LU02 OV01 OV02

HN01 HN02 HN03 HN04 HN05 HN06 LU01 LU02 OV01 OV02

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erpl

asia

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asia

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phom

aATF3 expression30

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~1-mm slices

Tumor slices randomlydistributed

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treatments

RQ

(G

AP

DH

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ized

)R

Q (

GA

PD

H n

orm

aliz

ed)

A

B

C

Figure 6. Levels of HMG-CoAreductase (A) and ATF3 (B) mRNAwere analyzed by real-time qRT-PCR following solvent control, 1and 10 mmol/L monensin in six HNtissues, including four HNSCC, amucosal hyperplasia and anoropharynx lymphoma, two lungadenocarcinomas (LU), and twoovarian mucinous carcinomas exvivo tissues. Fold changes werecalculated following normalizationto gapdH levels (DDCt) andexpressed as means (�SD; n ¼ 3).Error bars, SD from the mean ofthree replicates (n ¼ 3). C,schematic representation of exvivo tumor tissue processing andevaluation.

Dayekh et al.





DiscussionThe use of erlotinib in treatment regimens of cancers

with EGFR-activating mutation such as in 10% of patientswith NSCLC has demonstrated a promising althoughmodest improvement in patient outcomes (15, 16). In thesepatients, erlotinib shows enhanced binding affinity to theEGFR kinase domain demonstrating a more robust inhi-bition of this receptor (15, 16). In tumor types like HNSCCthat overexpressWTEGFR to enhance pathway activation,erlotinib treatments have not demonstrated significantclinical activity (6, 10). This is likely the result that erlotinibas a single agent may not inhibit WT EGFR sufficiently toinvoke a clinical response in these patients. Combiningerlotinib with agents that can co-operate to target EGFRmay enhance activity and uncover a novel therapeuticapproach. To this end, our previous work has identifiedthe mevalonate pathway as an important regulator ofEGFR activity and that inhibiting the rate-limiting enzymeof this pathway with statins inhibits EGFR activation andcan induce synergistic cytotoxicity with erlotinib in WTEGFRpossessingHNSCCandNSCLCcells (29, 30). In fact,a retrospectiveanalysisof theBR.21phase III clinical trial ofNSCLC treated with erlotinib as a single agent showed atrend to better OS in patients on statins for hypercholes-terolemia treatment (30). This has led to a phase I study atour Institute combining rosuvastatin and erlotinib in com-bination. Although durable stable disease was induced in20% of patients, these high rosuvastatin doses also pro-duced significant muscle toxicities, thereby limiting theefficacy of this approach (ClinicalTrials.gov Identifier:NCT00966472; G. Goss and colleagues, unpublished data).Thus, identifying more refined therapeutic approachesthat maintain the efficacywithout statin-induced toxicitiesis warranted. In this study, we identified monensin as anenhancerof the erlotinib-inducedHNSCCcell cytotoxicity.We also demonstrated a similar mechanism of action withrespect to EGFR inhibition as statin treatments. As such,monensin treatment impairs EGFR cellular trafficking,induced expression of similar apoptotic markers, inducedsynergistic cytotoxicity in combination with erlotinib, andimportantly activates pathways implicated in lipid andcholesterol synthesis.Monensin has been previously shown to induce cyto-

toxicity in a variety of cancer-derived cell lines, includingprostate, colon, and leukemias (19–22). Moreover, mon-ensin’s cytotoxicity appeared to be cancer-specific asmonensin differential sensitivity was observed betweensensitive malignant versus resistant nonmalignant pros-tate-derived cell lines (23). In this study, we have shownthat monensin mimics the inhibitory effects of lovastatinonEGFRactivity.Monensin can inhibit cellular traffickingof both the EGFR and LDL receptor (37, 41) and in thisstudy, we confirmed this inhibition in HNSCC cells.Monensin treatments showed aberrant EGF trafficking inboth HNSCC cell lines and is a known inhibitor of traf-ficking on many levels due to its influence on the endo-plasmic reticulum and golgi bodies, pH of endosomes,and protein glycosylation (44, 45).

Monensin is used extensively in poultry and cattle feedas it can increasemuscle mass in livestock (18). Our RNA-Seq analysis demonstrated its ability to induce expressionof cholesterol and lipid synthesis pathways as well asapoptosis regulating pathways that was confirmed byqRT-PCR. Although monensin and lovastatin exert pleio-tropic effects on a variety of cellular pathways, there isoverlapwith respect to their effects on EGFR function andcholesterol and lipid metabolism regulation. Monensinalso exhibited a significant induction of apoptosiswith theinduction of disparate pathways that was cell line depen-dent. In the SCC25 cell line, monensin-induced apoptosisis likely regulated by BNIP3 and DDIT4 that was associ-ated with cholesterol pathway induction, includingHMG-CoA reductase, whereas in the SCC9 cell line, theeffect of monensin is likely regulated by ATF3 induction.Remarkably, the ex vivo tissue samples from HNSCC, ahyperplastic lesion, a lymphoma, lung, and ovarian can-cers also demonstrate disparate responses to these path-ways. These tissue samples showed either no effect ofmonensin treatment on HMG-CoA reductase or ATF3expression, the induction of only ATF3 or HMG-CoAreductase alone or the induction of both genes in the samesample. Of interest, both the hyperplastic noncancerouscells and the nonepithelial lymphoma cells were respon-sive to monensin treatment that requires further study todetermine the scope of this response. Furthermore, theheterogeneity of the tumor cells within these tissues,including the presence of stromal cells, may account forthe upregulation of both HMG-CoA reductase and ATF3expression in four of the 10 tissues evaluated followingmonensin treatment also requires further study.

The mechanism by which monensin enhances erlotinibcytotoxicity inHNSCCcells is unknown. Itmay involve itsability to further inhibit EGFR in an unrelatedmechanismto erlotinib enhancing EGFR inhibition and cytotoxicity assuggested in this study. Monensin may also enhanceerlotinib activity through its ability to inhibit autophagy,a strategy that has demonstrated the potential to enhanceerlotinib cytotoxicity in NSCLC cells (46). Furthermore,lipoprotein lipolysis can lead to the induction of ATF3resulting in apoptosis (47) and the potential of monensinto target lipid homeostasis suggest this as an alternativemechanism. Thus, combining monensin with erlotinibrequires further study to delineate themechanism of theirsynergistic cytotoxic response in HNSCC and to deter-mine their utilization as a potential novel therapeuticstrategy.

Disclosure of Potential Conflicts of InterestP.J. Villeneuve was a consultant/participant in an Advanced NSCLC

Multidisciplinary Meeting for Pfizer and received travel/course reim-bursement for training onMatrix rib fixation system fromDePuy Synthes.No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: K. Dayekh, H.S. Sekhon, J. DimitroulakosDevelopment of methodology: J. DimitroulakosAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Dayekh, S. Johnson-Obaseki, M. Corsten,P.J. Villeneuve, H.S. Sekhon, J.I. Weberpals






Analysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): K. Dayekh, J. DimitroulakosWriting, review, and/or revision of the manuscript: K. Dayekh, S. John-son-Obaseki, P.J. Villeneuve, H.S. Sekhon, J.I. Weberpals, J. DimitroulakosAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J.I. WeberpalsStudy supervision: S. Johnson-Obaseki, J. Dimitroulakos

AcknowledgmentsThe authors thank Janet Barber and Stephanie Reid for expert technical

assistance and the team at StemCore (Ottawa Hospital Research Institute)including Theodore Perkins and Pearl Campbell.

Grant SupportThe grant support from the Canadian Institute of Health Research

MOP-311725 (to J. Dimitroulakos) and the Joan Sealy Trust (to J. Dimi-troulakos) is greatly appreciated.

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.

ReceivedDecember 19, 2013; revisedAugust 4, 2014; acceptedAugust 5,2014; published OnlineFirst September 4, 2014.

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2014;13:2559-2571. Published OnlineFirst September 4, 2014.Mol Cancer Ther Khalil Dayekh, Stephanie Johnson-Obaseki, Martin Corsten, et al. Inhibitorsand Activation: Synergistic Cytotoxicity in Combination with EGFR Monensin Inhibits Epidermal Growth Factor Receptor Trafficking

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Monensin Inhibits Epidermal Growth Factor Receptor Trafﬁcking … · Small Molecule Therapeutics...

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Transcript of Monensin Inhibits Epidermal Growth Factor Receptor Trafﬁcking … · Small Molecule Therapeutics...