Synergistic effect between erlotinib and MEK inhibitors in KRAS wild-type human pancreatic cancer...

Cancer Therapy: Preclinical

Synergistic Effect between Erlotinib and MEK Inhibitors in KRASWild-Type Human Pancreatic Cancer Cells

Caroline H. Diep, Ruben M. Munoz, Ashish Choudhary, Daniel D. Von Hoff, and Haiyong Han

AbstractPurpose: The combination of erlotinib and gemcitabine has shown a small but statistically significant

survival advantage when compared with gemcitabine alone in patients with advanced pancreatic cancer.

However, the overall survival rate with the erlotinib and gemcitabine combination is still low. In this study,

we sought to identify gene targets that, when inhibited, would enhance the activity of epidermal growth

factor receptor (EGFR)-targeted therapies in pancreatic cancer cells.

Experimental Design: A high-throughput RNA interference (RNAi) screen was carried out to identify

candidate genes. Selected gene hits were further confirmed and mechanisms of action were further

investigated using various assays.

Results: Six gene hits from siRNA screening were confirmed to significantly sensitize BxPC-3 pancreatic

cancer cells to erlotinib. One of the hits, mitogen-activated protein kinase (MAPK) 1, was selected for

further mechanistic studies. Combination treatments of erlotinib and two MAP kinase kinase (MEK)

inhibitors, RDEA119 and AZD6244, showed significant synergistic effect for both combinations

(RDEA119–erlotinib and AZD6244–erlotinib) compared with the corresponding single drug treatments

in pancreatic cancer cell lines with wild-type KRAS (BxPC-3 and Hs 700T) but not in cell lines with mutant

KRAS (MIA PaCa-2 and PANC-1). The enhanced antitumor activity of the combination treatment was

further verified in the BxPC-3 andMIA PaCa-2mouse xenograft model. Examination of theMAPK signaling

pathway by Western blotting indicated effective inhibition of the EGFR signaling by the drug combination

in KRAS wild-type cells but not in KRAS mutant cells.

Conclusions:Overall, our results suggest that combination therapy of an EGFR andMEK inhibitors may

have enhanced efficacy in patients with pancreatic cancer. Clin Cancer Res; 17(9); 2744–56. �2011 AACR.

Introduction

With a 5-year survival rate of less than 5%, pancreaticcancer remains themost deadly of all major cancer types (1,2). Gemcitabine has been the standard first line therapy forpatients with advanced pancreatic cancer since 1996. Sincethen, multiple clinical trials combining gemcitabine withother chemotherapeutics have failed to show improvementin overall survival compared with gemcitabine alone (3–5).Until recently in 2005, the FDA (Food and Drug Admin-istration) approved the regimen of gemcitabine and erlo-tinib (Tarceva), an epidermal growth factor receptor(EGFR) inhibitor, based on increased survival.

The EGFR tyrosine kinase signaling pathway has beenimplicated in several cellular processes pertinent to cancerprogression including cell survival, proliferation, invasion,and metastasis. Dysregulation of EGFR signaling occurs inas much as one-half of all pancreatic cancers (6). Erlotinibis a small molecular weight inhibitor of the EGFR tyrosinekinase that has shown clinical activity in patients withadvanced non–small cell lung or pancreatic cancer. Patientswith advanced pancreatic cancer treated with the combina-tion of erlotinib and gemcitabine have shown statisticalsignificant survival advantage over patients treated withgemcitabine alone. However, the overall response andsurvival rate with the erlotinib and gemcitabine combina-tion is still low with the survival rate at one year increasedfrom 17% to 23% with the combination (7).

The RAS–RAF–MAPK (mitogen-activated protein kinase)pathway is one of the downstream effector pathways of theEGFR tyrosine kinase signaling. It is constitutively activatedin multiple human tumors due to gain-of-function muta-tions in RAS or RAF. Of the RAS family, KRAS is more oftenmutated with approximately 20% of all human tumorspossessing an activating mutation in codon 12, 13, andmore rarely 61 (8). KRAS mutations are the most prevalentin pancreatic ductal adenocarcinomas (70%–90%; ref. 9–11), followed by colorectal adenocarcinomas (35%) and

Authors' Affiliation: Clinical Translational Research Division, Transla-tional Genomics Research Institute, Scottsdale, Arizona

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

Corresponding Author: Haiyong Han, Clinical Translational ResearchDivision, Translational Genomics Research Institute, 13208 E. Shea BLVD,Scottsdale, AZ 85259. Phone: 602-343-8739; Fax: 602-358-8360; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-10-2214

�2011 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 17(9) May 1, 20112744

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lung carcinomas (17%; ref. 12). Of the RAF family, muta-tions only in BRAF have been reported but are mutuallyexclusive from a KRASmutation. It is a rare exception that atumor possesses both KRAS and BRAF mutations (13).Downstream of RAS and RAF is MEK1/2, which is critical

to transmitting signals to extracellular signal–regulatedkinase (ERK) and has become an attractive pharmaceuticaltarget in tumors harboring aberrant signaling in the MAPKpathway. AZD6244 (ARRY-142886) and RDEA119 (BAY869766) are 2 potent MEK1/2 inhibitors that are currentlyin clinical development. AZD6244 is a selective ATP-uncompetitive inhibitor of MEK1/2, which has beenreported to inhibit cellular growth and induce apoptosisin vitro and reduce tumor growth in the BxPC-3 and HT-29xenograft models (14). RDEA119 is an allosteric, selectiveinhibitor of MEK1/2, which has been reported to inhibitcell proliferation in vitro and reduce tumor growth invarious in vivo models (15). Clinically, RDEA119 is cur-rently being evaluated in at least 3 studies: a phase I dose-escalation study, a phase I monotherapy in Japanesepatients, and a phase I/II study in combination withsorafenib in advanced cancer patients (http://www.clinicaltrials.gov).In this study, we employed high-throughput RNA inter-

ference (RNAi) screening approach to identify targets thatwould enhance the activity of erlotinib in pancreatic cancercells. We determined that the combination of a MAP kinasekinase (MEK) inhibitor and erlotinib has significant anti-tumor activity in a subset of pancreatic cancer cells thatharbor wild-type KRAS in in vitro and in vivo models.

Materials and Methods

Cell line cultureThe pancreatic cancer cell lines BxPC-3, Hs 700T, MIA

PaCa-2, and PANC-1 were obtained from the AmericanType Culture Collection (ATCC). All cell lines were main-tained in RPMI 1640 supplemented with 10% FBS, peni-cillin (100 U/mL), and streptomycin (100 mg/mL). Cellswere cultivated in a humidified incubator at 37�C and 5%

CO2. Cells were harvested with 0.05% trypsin at 70% to80% cell density.

Cell line identities were verified by short tandem repeat(STR) profiling (16) using the AmpFISTR Identifiler PCRamplification Kit (Applied Biosystems). This methodsimultaneously amplifies 15 STR loci and amelogenin ina single tube, using 5 dyes, 6-FAM, JOE, NED, PET, and LIZwhich are then separated on a 3100 Genetic Analyzer(Applied Biosystems). GeneMapper ID v3.2 software wasused for analysis (Applied Biosystems). AmpFISTR controlDNA and the AmpFISTR allelic ladder were run concur-rently. Results were compared with published STRsequences from the ATCC. The STR profiling is repeatedonce a cell line has been passagedmore than 6months afterprevious STR profiling.

siRNA library screening and hit selectionAn RNAi screen using a library of siRNA duplex oligo-

nucleotides targeting 588 known human kinase genes (2siRNAs per gene; QIAGEN) was carried out to identifysensitizing targets for erlotinib using a reverse transfectionprotocol as described previously (17). Two nonsilencingsiRNAs were used as negative controls, whereas the AllStarsHs Cell Death Control (QIAGEN) was used as a positivecontrol. The siRNAs were first arrayed into 384-well platesfor a final assay concentration of 20 nmol/L in duplicates.The arrayed siRNAs were then incubated with 20 mL serum-free RPMI 1640 cell culture media (Invitrogen) containing0.04 mL siLentfect lipid reagent (Bio-Rad) at room tem-perature for 30 minutes. Next, BxPC-3 cells were plated tothe siRNA-transfection reagent mix at 1,200 cells per welland serum supplemented at a final concentration of 5%.The plates were incubated in a humidified incubator at37�C for 24 hours. Afterward, a serial dilution of erlotinib(6 concentrations between 0 and 100 mmol/L) was addedto the wells and incubated for 96 hours. Cell viability wasdetermined by CellTiter-Glo Luminescent Assay (Promega)and the luminescence was recorded with the Synergy HTMicroplate Reader (BioTek).

The percent cell survival of the siRNA and erlotinibcombination was normalized to the percent cell survivalof corresponding siRNA alone control. The IC50 values foreach siRNA and erlotinib treatment were calculated byfitting the data to a sigmoid dose–response model usingnonlinear regression with the Matlab Software (2007a; TheMathWorks Inc). Each siRNA was evaluated and ranked bythe following qualifiers as potential hits: (i) the R2 for thesigmoid fitting curve was greater than 0.9, (ii) the effect oncell viability of the siRNA itself was less than 50%, and (iii)2-fold or higher decrease in the IC50 value compared withthe Neg siRNA control. To further validate the positive genehits, an additional 2 siRNA sequences (4 total) wereobtained for each gene and subjected to a confirmationscreen with 8 erlotinib concentrations using the sameprocedures described above. Genes that have at least 2of 4 siRNA sequences showing 2-fold or higher reductionin erlotinib IC50 value compared with the Neg siRNAcontrol were selected as confirmed hits.

Translational Relevance

With a 5-year survival rate of less than 5%, pancreaticcancer is among themost lethal types of human cancers.Current therapies are mostly ineffective and new thera-pies are desperately needed. This article describes thesynergistic effect between erlotinib and MAP kinasekinase (MEK) inhibitors in pancreatic cancer cells andanimal models. We show that the combination oferlotinib, an epidermal growth factor receptor (EGFR)inhibitor, and several MEK inhibitors have enhancedefficacy in pancreatic cancer cells with wild-type KRAS.This work is highly translational, as further evaluationof the combination treatment may result in new andimproved treatment for patients with pancreatic cancer.

Synergistic Effect between Erlotinib and MEK Inhibitors

www.aacrjournals.org Clin Cancer Res; 17(9) May 1, 2011 2745




Verification of siRNA-mediated mRNA and proteinexpression knockdown using real-time PCR andWestern blotting

To evaluate the gene-specific knockdown of mRNAexpression by siRNA, cancer cells (250,000 cells per well)were reverse transfected with 20 nmol/L of the MAPK1siRNAs (QIAGEN) for 72 hours in 6-well plates. The cellswere then washed with Dulbecco’s PBS (DPBS) twice andharvested with trypsinization. For RNA extraction, theRNeasy Mini Kit (QIAGEN) was utilized following themanufacturer’s recommended protocol. One microgramof total RNA was used in a 20 mL cDNA synthesis reaction(Quanta Biosciences). One microliter of the cDNA reactionmix, 10 mL of FastStart SYBR Green Master mix (Roche),and 4 mL of MAPK1 primer mix (50-TCATCCTCGGAAAA-CAGACC-30 and 50-TCAATGCTGTGTGGACCTTC-30, finalconcentration 0.4 mmol/L each) were combined. A separatereaction for GAPDH (glyceraldehydes-3-phosphate dehy-drogenase) was included as a control and the primersused were 50-ATTGCCCTCAACGACCACTT-30 and 50-GGTCCACCACCCTGTTGC-30. The real-time quantitativePCR was carried out using the MyIQ Real-Time PCR Detec-tion System (Bio-Rad). The cycling program used was 95�Cfor 10 minutes, 95�C for 30 seconds, 58�C for 30 seconds,and 72�C for 30 seconds for 30 cycles. Each sample was runin duplicates, normalized to GAPDH, and analyzed by thecomparative CT method (18).

For protein expression, the cells were washed with DPBStwice and subsequently lysed with RIPA (radioimmuno-precipitation assay) buffer (Cell Signaling Technology)supplemented with Complete Mini protease cocktail andPhoSTOP (Roche). The lysates were incubated on ice for 30minutes prior to centrifugation at 14,000 � g for 10minutes at 4�C. Protein concentration was determinedusing the bicinchoninic acid protein assay (Pierce Biotech-nology). A total of 30 mg of protein per lane was separatedby 10% SDS-PAGE electrophoresis (Invitrogen) and trans-ferred to nitrocellulose membrane blots. The membraneswere blocked for 1 hour with 5% fat-free milk at roomtemperature and then incubated with a rabbit polyclonalp42 MAP kinase (Erk2) antibody (1:1,000 dilution; CellSignaling Technology) overnight at 4�C. The blots werewashed with Tris-buffered saline containing 0.1% Tween-20 (TBST) buffer for 3 times and then exposed to anti-rabbit IgG HRP (horseradish peroxidase)-linked antibody(1:4,000 dilution; Cell Signaling Technology) at roomtemperature for 1 hour. The protein bands were visualizedusing the chemiluminescence detection method (Milli-pore).

Drug combination treatment and immunoblottinganalysis

Erlotinib, AZD6244, and RDEA119were purchased fromChemieTek and dissolved in DMSO (dimethyl sulfoxide)to make 50 mmol/L (erlotinib) or 20 mmol/L (AZD6244and RDEA119) stock solutions. Cells were seeded into 384-well plates at 1,200 cells per well, allowed to grow for 24hours, and then treated with drugs for another 96 hours.

Ten concentrations of AZD6244 and RDEA119 were madein 1:3 serial dilutions in serum-free medium. Three drugconcentrations of erlotinib were added in combination tothe MEK inhibitors at 1:1 ratio concurrently. At the end oftreatment, cell viability was determined by the CellTiter-Glo Assay. The percent cell survival of erlotinib alone at theindicated drug concentration was normalized to the per-cent cell survival of the respective drug combination. Thegrowth curves were plotted in the GraphPad Prism v5software and the IC50 was determined by the softwareusing the nonlinear regression (curve fit) analysis.

For Western blot analysis, 1 � 106 cells were seeded inT25 flasks for overnight growth. The cells were treated withdrugs in serum conditions similar to the 384-well assays.The drugs at concentrations indicated in the figure legendswere added directly to the medium for incubation at hoursindicated. Cell lysates were prepared as described pre-viously. The cell lysates (30 mg per lane, except 15 mg forAKT) were separated on NuPAGE protein precast gels(Invitrogen), transferred to nitrocellulose (Bio-Rad), andthen probed with specific antibodies using manufacturer’srecommended dilutions. The antibodies, phospho-MAPK(Thr202/Tyr204; pMAPK), MAPK, phospho-EGFR(Tyr1068; pEGFR), EGFR, phospho-AKT (Ser473; pAKT),AKT, BIM, PARP, cyclin D1, p27, and b-actin were pur-chased from Cell Signaling Technology.

Apoptosis and cell-cycle analysisApoptosis analysis was conducted using the Annexin V:

FITC Apoptosis Detection Kit II (BD Biosciences) followingthe manufacturer’s protocol. Briefly, cells were seeded andtreated with the drugs as described previously for the immu-noblotting studies. Afterward, the cells were washed twicewithDPBS and 1� 106 cells were resuspended in 1mLof 1�Annexin V–binding buffer. Cells undergoing apoptotic celldeathwas analyzedby counting the cells that stainedpositivefor Annexin V/FITC (fluorescein isothiocyanate) and nega-tive for propidium iodide (PI), and late stage of apoptosis,necrosis, or already dead as Annexin V/FITC and PI positiveusing FACSCalibur (BDBiosciences). Differences of the drugcombinationgroupcomparedwith the singledruggroupwasconfirmed using an independent 2-tailed t test and consid-ered statistically significant when P < 0.05.

For cell-cycle analysis, cells were treated with erlotinib (3and 12.5 mmol/L) and RDEA119 (100 nmol/L) alone or incombination as described above and harvested by trypsi-nization. The cells were resuspended and stained with PI(Sigma-Aldrich) in a modified Krishan buffer (19) for 1hour at 4�C. The PI-stained samples were then analyzedwith a FACScan flow cytometer (BD Biosciences Immuno-cytometry systems). Histograms were analyzed for cell-cycle compartments and the percentage of cells at eachphase of the cell cycle was calculated using FlowJo (TreeStar, Inc.) analysis software.

In vivo studiesAnimal studies were conducted at the Translational

Genomics Research Institute Drug Development Services

Diep et al.

Clin Cancer Res; 17(9) May 1, 2011 Clinical Cancer Research2746




(TD2) under IACUC (Institutional Animal Care and UseCommittee)-approved protocols. Female ICR-SCID (severecombined immunodeficient) mice (IcrTac: ICR-Prkdcscid;Taconic) were inoculated subcutaneously in the right flankwith 0.1mL of a 50%RPMI/50%Matrigel (BD Biosciences)mixture containing a suspension of BxPC-3 (1 � 107 cellspermouse) or MIA PaCa-2 (5� 106 cells per mouse) tumorcells. Four days following inoculation, tumors were mea-sured using calipers and tumor weight was calculated usingthe Study Director V.1.6.80a Software (20). Tumor bearingmice were pair-matched into the 6 groups (8 mice pergroup) by random equilibration using Study Director(day 1). Body weights were recorded when the mice werepair-matched and were taken twice weekly thereafter inconjunction with tumor measurements. On day 1,RDEA119 (6 mg/kg), vehicle control (0.4% carboxymethylcellulose), and erlotinib (50 mg/kg) were administeredorally, whereas gemcitabine (BxPC-3, 40 mg/kg; MIA-PaCa-2, 20 mg/kg) was administered by intraperitonealinjection. RDEA119 and vehicle control were administeredtwice daily for 11 days. Erlotinib was dosed daily for 21days. Gemcitabine was dosed 3 times per day for 4 days.The study was terminated when tumor burden exceeded1,000 mg in the vehicle control group.Mean tumor growth inhibition (TGI) was calculated

utilizing the following formula, with X as the mean tumorweight:

TGI ¼ ½1�ðXTreatedðFinalÞ�XTreatedðDay 1Þ ÞðXControlðFinalÞ�XControlðDay 1Þ Þ

�100%

All statistical analyses in the xenograft study were con-ducted with GraphPad Prism v4 software. Differences infinal tumor weights were confirmed using an independent1-tailed t Test and considered statistically significant whenP < 0.05.

Results

Inhibition of MAPK1 expression by siRNA sensitizespancreatic cancer cells to erlotinibTo identify gene targets that, when inhibited, sensitize

pancreatic cancer cells to the treatment of erlotinib, wecarried out siRNA screening in the presence of a serialdilution of erlotinib in BxPC-3 cells using a kinase-focusedsiRNA library (see Materials and Methods). A total of 6genes were confirmed (Table 1). Of the 6 genes, the siRNAsilencing of GCK and MAPK1 (ERK2) had the highestsensitizing effects on erlotinib (6- to 16-fold and 6- to 8-fold reduction in IC50, respectively; SupplementaryFig. S1A and B), whereas BMPR2 (bone morphogeneticprotein receptor), BRAF, FLT3, and PRKAB2 had moderateeffects (ranging from 2- to 7-fold reduction in IC50; Sup-plementary Fig. S1C–F).Due to the high sensitizing effect of its siRNAs and its

well known involvement in cancer, MAPK1 was selected forfurther validation. The specific knockdown of MAPK1expression by the siRNAs was evaluated by quantitative

PCR and Western blotting analysis. Two MAPK1 siRNAsequences (MAPK1_1 and MAPK1_2) were able to achievemore than 80% efficiency in reducing mRNA transcriptexpression (data not shown) and protein expressionlevels at 72 hours in all 4 cell lines (Fig. 1A and Supple-mentary Fig. S3). The MAPK1 siRNA and erlotinib treat-ment in BxPC-3 cells were further repeated with 10 serialdilutions of erlotinib, nontargeting siRNA control (NegsiRNA), and a drug alone control. The MAPK1 siRNA anderlotinib combinations yielded an IC50 of a 6-fold dif-ference compared with both controls (Fig. 1B). The siRNAand erlotinib treatment was repeated in additional well-characterized pancreatic cancer cell lines, Hs 700T, MIAPaCa-2, and PANC-1, to determine if the sensitizingeffects of MAPK1 siRNA were cell line specific. Of the 3cell lines, Hs 700T had an IC50 fold difference of 2.3 and1.4 compared with the Neg siRNA control (Supplemen-tary Fig. S2A), whereas MIA PaCa-2 and PANC-1 exhib-ited no sensitizing effects in either MAPK1 siRNA(Supplementary Fig. S2B and C).

It is interesting to note that BxPC-3 and Hs 700T havepreviously been reported to have wild-type KRAS, whereasMIA PaCa-2 and PANC-1 harbor a mutation at codon 12(21). Sequencing of the KRAS open reading frame (ORF) ofthe 4 cell lines used in this study confirms the mutationalstatus (data not shown). We therefore hypothesized thatthe sensitizing effect of MAPK1 siRNA might be specific tocells harboring wild-type KRAS.

MEK inhibitors synergize with erlotinib to inhibit theproliferation of KRAS wild-type pancreatic cells

Because MEK proteins phosphorylate and activateMAPK1, we postulated that small molecular weightinhibitors of MEK1/2 might be able to sensitize pancreatic

Table 1. Top-ranked gene hits identified fromsiRNA screening

siRNAsequencesa

Erlotinib IC50,mmol/L

IC50 fold differenceto Neg siRNA

Neg siRNA 37.9 –

BMPR2_4 9.2 4.1BMPR2_6 5.3 7.2BRAF_3 6.7 5.7BRAF_4 6.2 6.1FLT_3 1.4 2.7FLT_6 8.9 4.3GCK_3 5.6 6.8GCK_4 2.3 16.5MAPK1_1 4.5 8.4MAPK1_2 6.2 6.1PRKAB2_5 8.4 4.5PRKAB2_6 7.1 5.4

aNumber after underscore indicates the siRNA sequencenumber designated by QIAGEN.






cancer cells to the treatment of erlotinib. We obtained 2MEK inhibitors, RDEA119 and AZD6244, and evaluatedtheir synergism with erlotinib inmultiple pancreatic cancercell lines. The synergistic effect of the combination ofRDEA119 and erlotinib was evaluated in the same 4 pan-creatic cancer cell lines used in siRNA studies. It is notedthat BxPC-3 and Hs 700T are moderately sensitive toerlotinib, whereas MIA PaCa-2 and PANC-1 are relativelyless sensitive, which is consistent with other studies (Sup-plementary Fig. S4; ref. 22). As to RDEA119, PANC-1 is theleast sensitive with an IC50 of 1.5 mmol/L, BxPC-3 and Hs700T are moderately sensitive at with IC50 ranging between300 and 700 nmol/L, and MIA PaCa-2 is exquisitely sensi-tive with an IC50 of 50 nmol/L (15). Similar sensitivityprofiles were observed for AZD6244 in these pancreaticcancer cell lines (data not shown).

When fixed concentrations of erlotinib (12.5, 3.0, or 0.8mmol/L) were added to a dose–response curve of RDEA119,a significant synergistic effect was observed in BxPC-3 andHs 700T cells (Fig. 2A and B). As the concentration oferlotinib decreased from 12.5 to 0.8 mmol/L, the level ofsynergy (left shift of the dose–response curves) decreased aswell, indicating that the level of synergy was dose depen-dent between erlotinib and RDEA119. The synergistic effectis muchmore dramatic in BxPC-3 cell line (672-fold at 12.5mmol/L of erlotinib) than in Hs 700T (14-fold at 12.5mmol/L of erlotinib; Table 2). No significant synergy wasobserved in MIA PaCa-2 and PANC-1 cells at the same drugconcentrations (Fig. 2C and D). Higher concentrations oferlotinib (up to 50 mmol/L) did not show any significantsynergy with RDEA119 (data not shown) in these 2 celllines. Table 2 lists the IC50 values of RDEA119 alone andRDEA119 and erlotinib as well as the fold reduction whencompared with RDEA119 alone in the 4 pancreatic cancercell lines. Other pancreatic cancer cell lines, such as AsPC-1,CFPAC-1, and L3.6pl, are similar in sensitivity to EGFRinhibitors as BxPC-3 and Hs 700T, yet contain mutant

KRAS (23, 24). No significant synergy was observed inthese KRAS mutant cell lines at the same drug combinationconcentrations (Supplementary Fig. S5A–C).

Similar to RDEA119, AZD6244 showed a significantsynergistic effect with erlotinib in BxPC-3 and Hs 700T(Supplementary Fig. S6A and B). The synergism betweenAZD6244 and erlotinib is also dose dependent, although itis more apparent in Hs 700T than in BxPC-3. Interestingly,the synergistic effect between AZD6244 and erlotinib ismore dramatic in Hs 700T cells than in BxCP-3 cells (64- vs.15-fold at 12.5 mmol/L of erlotinib; SupplementaryTable S1). There was no significant synergy between the2 drugs in MIA PaCa-2 and PANC-1 cells (SupplementaryFig. S6C and D).

On the basis of our current observations, we hypothe-sized that the efficacy of the EGFR and MEK inhibitorcombination was limited to cells with wild-type KRAS. TodeterminewhethermutantKRASwas thedetermining factorfor the lack of synergy, siRNA targeting the specific KRASmutation in MIA PaCa-2 and PANC-1 (25) was transfectedin combination with the treatment of erlotinib andRDEA119. In MIA PaCa-2, the inhibition of mutant KRASyielded a synergistic effect between RDEA119 and 50 mmol/L of erlotinib (2.4-fold compared with erlotinib andRDEA119; Supplementary Fig. S7A). As the dose of erlotinibwas reduced to 25 mmol/L (Supplementary Fig. S7B), thesynergistic effect was lost. In PANC-1, sensitivity with theKRAS mutant–specific siRNA and drug combination wasrestored to by 1.5-fold compared RDEA119 in combinationwith erlotinib at 50 mmol/L (Supplementary Fig. S7C), butno difference at 25 mmol/L (Supplementary Fig. S7D).

Together, these results indicate that the synergismbetween erlotinib and the MEK inhibitors is restricted topancreatic cancer cell lines with wild-type KRAS, furthersupporting the hypothesis that inhibition of MAPK1function specifically sensitizes KRAS wild-type pancreaticcancer cells to the treatment of erlotinib.

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Figure 1. Effect of MAPK1knockdown by siRNAoligonucleotides on theantiproliferation activity of erlotinibin BxPC-3 cells. A, two siRNAsequences (MAPK1_1 andMAPK1_2) effectively knockeddown the expression of MAPK1protein in BxPC-3 cell–detectedWestern blotting. A nontargetingsiRNA (Neg siRNA) was used as anegative control. B, treatment ofBxPC-3 cells with MAPK1 siRNAcaused a left shift of the erlotinibdose–response curve. Thepercent cell viability of the siRNA-treated cells was normalized tothat of the siRNA only treatment.Data are means � SD from 3independent experiments andeach experiment was carried outwith 4 replicates per drugconcentration.

Diep et al.





The erlotinib and MEK inhibitor combinationdisrupts a negative feedback loop of the EGFR–MAPKsignaling pathwaysOn the basis of the observed synergistic effect of the

erlotinib and RDEA119 combination in inhibiting cellproliferation, we assessed the phosphorylation activitieswithin the EGFR–MAPK signaling pathway. The MAPKsignaling pathway regulates cell proliferation and survi-val, and we hypothesized that the synergy observed in theKRAS wild-type cells were, at least in part, due to theincreased inhibition of the MAPK signaling. To examinethe effect of drug treatment on MAPK signaling, bothBxPC-3 and MIA PaCa-2 cells were treated with 12.5mmol/L of erlotinib, 100 nmol/L of RDEA119, or thecombination for a time course of 8, 24, and 48 hours.The erlotinib concentration used in our experiments(12.5 mmol/L) is comparable to the pharmacokineticvariables documented in patients, which showed the

trough, peak plasma, and average steady-state plasmaconcentrations of 9.6, 15.1, and 11.0 mmol/L, respectively(26–28). The 100 nmol/L concentration of the MEKinhibitor is below pharmacokinetic plasma concentra-tions obtained in recent clinical studies (29, 30).

In BxPC-3, erlotinib significantly decreased the pMAPKlevel over the time course, whereas in MIA PaCa-2, therewas little effect (Fig. 3), which is expected on the basis oftheir KRAS mutational status. With the treatment ofRDEA119 alone, the pMAPK level was effectively sup-pressed at 8 hours in both BxPC-3 and MIA PaCa-2. Astime progressed, the phosphorylation of MAPK wasrecovered in both cell lines but at a higher level inBxPC-3 to near basal levels by 48 hours. In the combina-tion treatment, BxPC-3 showed sustained ablation ofpMAPK activity throughout the time course, whereasMIA PaCa-2 recovered activity similar to RDEA119 aloneby 48 hours.

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RDEA + E(0.8 μmol/L) (V = 92%)

Figure 2. Combination treatment of RDEA119 and erlotinib (E) in pancreatic cancer cells. Three erlotinib concentrations (12.5, 3, and 0.8 mmol/L), eachof which was concurrently added on to a serial dilution of RDEA119 in 4 pancreatic cancer cell lines, BxPC-3 (A), Hs 700T (B), MIA PaCa-2 (C), and PANC-1 (D).The drug dose–response curves for the combinations have been normalized to the percent cell viability of corresponding erlotinib alone treatment (denoted asV ¼ % in the legends). Data are means � SD from 3 independent experiments and each of which had 4 replicates per drug concentration.






In addition, MEK inhibition led to a marked increase inpEGFR in BxPC-3 cells indicating a feedback between MEKinhibition and EGFR activation (Fig. 3A). This was mostapparent at 48 hours of RDEA119 treatment in BxPC-3,when the level of pEGFR dramatically increased comparedwith basal levels, whereas the total EGFR levels remain thesame. This observation was previously also reported inbreast and lung cancer cell lines when treated with anotherMEK inhibitor, AZD6244 (31). The level of pEGFR was notchanged in MIA PaCa-2 in any of the treatments. With theincreased activation of EGFR by MEK inhibitors, the ratio-nale of the combination of erlotinib and RDEA119 isevident in BxPC-3 as it inhibits both the activity fromthe receptor tyrosine kinase and its signaling cascade toMAPK.

It has been noted in the literature that disruption of theEGFR–MAPK signaling pathway can shift signaling to AKTas amode tomaintain cell survival (32–34). In either of thedrug treatments in MIA PaCa-2, pAKT levels did increasewhen compared with basal levels of the cells only incontrol, yet the levels were not differentially affected ineach respective group as time progressed (Fig. 3B). ForBxPC-3, the combination increased pAKT levels at 8 hoursof treatment by 20% above the basal control, but as timeprogressed to 48 hours, the combination reduced pAKTlevels by 20% compared with basal control levels.RDEA119 alone increased pAKT in BxPC-3, whereas theerlotinib treatment slightly reduced it (Fig. 3A).

The exquisite sensitivity of MIA PaCa-2 to the MEKinhibitors in our study is comparable to previous reports(35, 36). Results from a recent study reported that sensi-tivity to MEK inhibition was inversely correlated with thebasal level of pAKT (37). In MIA PaCa-2, the basal level ofpAKT was comparably lower than BxPC-3 (Fig. 3). Despite

its sensitivity, the combination of MEK inhibition anderlotinib was not synergistic in MIA PaCa-2.

Overall, these results showed that the synergy of the drugcombination observed in BxPC-3 is likely due to thecomplete repression of the EGFR–MAPK signaling pathwayand reduction of pAKT activity, whereas the lack of synergyin MIA PaCa-2 may be attributed to the residual activity ofpMAPK and the increased levels of pAKT.

Dual EGFR and MEK inhibition cooperatively inducesapoptotic cell death

To further understand the mechanism of the synergismbetween EGFR and MEK inhibitions, we evaluated thedegree of apoptosis with Annexin V/FITC flow cytometricanalysis and the expression of BIM and PARP in cellstreated with erlotinib, RDEA119, or the combination. Asshown in Figure 4A, the percentage of cells that wereundergoing apoptosis (Annexin V positive/PI negative)in BxPC-3 were significantly higher in the combination(28%) than the respective single drug treatments (erlotinib,14% and RDEA119, 12%). The combination of erlotiniband RDEA119 cooperatively induces and enhances apop-totic cell death more than the single agents. The percentageof BxPC-3 cells in late apoptosis or already dead (Annexin Vpositive/PI positive) was consistent with 26% with erloti-nib, 21% with RDEA119, and 41% with the combinationtreatment (Fig. 4B). In contrast, in MIA PaCa-2, the erlo-tinib treatment elicited 12% of the cell population toundergo apoptosis, whereas RDEA119 was 24% and thecombination was 26%. Similarly, the percentage of lateapoptosis was 19% with erlotinib, 58% with RDEA119,and 58% with combination treatment. ComparingRDEA119 and the combination, the level of apoptosisobserved in the combination treatment in MIA PaCa-2 is

Table 2. IC50 values of the RDEA119 and erlotinib combination treatments in pancreatic cancer cell lines

Cell line Treatments, mmol/L RDEA119 IC50, nmol/La Fold reduction of IC50

BxPC-3 RDEA119 268.6 –

RDEA þ erlotinib (12.5) 0.4 671.5RDEA þ erlotinib (3.0) 2.5 107.4RDEA þ erlotinib (0.8) 24.5 11.0

Hs 700T RDEA119 684.1 –

RDEA þ erlotinib (12.5) 48.3 14.2RDEA þ erlotinib (3.0) 166.8 4.1RDEA þ erlotinib (0.8) 459.1 1.5

MIA PaCa-2 RDEA119 53.3 –

RDEA þ erlotinib (12.5) 107 0.5RDEA þ erlotinib (3.0) 60.3 0.9RDEA þ erlotinib (0.8) 58.5 0.9

PANC-1 RDEA119 1,411.1 –

RDEA þ erlotinib (12.5) 1,620.6 0.9RDEA þ erlotinib (3.0) 1,608.1 0.9RDEA þ erlotinib (0.8) 1,597.2 0.9

aIC50 was calculated after the RDEA119 dose–response curve was normalized to the effect of the erlotinib only control.

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due to mostly the effect of RDEA119 alone. Erlotinib doesnot synergistically nor additively enhance apoptosis in thecombination in MIA PaCa-2.It has been reported that MAPK can inhibit the activity of

several proapoptotic proteins by promoting its ubiquitina-tion for proteasome-dependent degradation, more notablythe BIM protein (38–40). Several studies have reported thatBIM is a major target of MAPK-dependent survival signal-ing, and the upregulation of BIM was required for com-mitted apoptosis (41, 42). As single agents, erlotinib andRDEA119 had different effects on the proapoptotic BIMprotein levels (Fig. 4C). In the cell lines treated with theinhibitors, BIMEL was the predominant isoform expressed.BIMEL expression moderately increased in BxPC-3 and MIAPaCa-2 cells treated with either erlotinib or RDEA119, yetthe drug combination induced the dephosporylated, sta-bilized, active form at 48 hours more so in BxPC-3 than inMIA PaCa-2. Erlotinib or RDEA119 induced comparablelevels of BIM expression and moderate apoptosis, but thecombination is necessary to potently inhibit the EGFR–MAPK signaling pathway in BxPC-3 and dramatically

increase apoptotic cell death. The magnitude and durationof pMAPK inhibition committed BxPC-3 to proapoptoticsignaling as seen by the upregulation of BIM expression.

Next, the analysis of cells undergoing apoptosis wasevaluated by PARP cleavage. In MIA PaCa-2, there existsa basal level of PARP cleavage in the cells only control, butthere was not a significant differential change in PARPcleavage with either drug treatments (Fig. 4C). The basallevel of PARP cleavage in MIA PaCa-2 was similar in aprevious study examining the in vitro activity of AZD6244and rapamycin (43). It is of note that the study used a highdose of AZD6244 at 1 mmol/L to induce PARP cleavage inboth BxPC-3 and MIA PaCa-2. In this study, we used 100nmol/L of RDEA119, which increased PARP cleavageslightly in MIA PaCa-2 compared with those in the cellsonly control, erlotinib, and the combination. In contrast,PARP cleavage was observed only in the drug combinationin BxPC-3, which is consistent with the results from theAnnexin V/FITC analysis.

Finally, the effects of erlotinib and RDEA119 on the cell-cycle distribution were examined. Consistent with theapoptotic immunoblot profiles, a substantial increase inthe sub-G1 population was only observed in BxPC-3 in thecombination treatment (Supplementary Table S2). Asthe concentration of erlotinib was reduced from 12.5 to3 mmol/L, the differential affect on cell cycle was notsignificant. In MIA PaCa-2, the sub-G1 population wasminimal in all of the drug treatments but did exhibit somecell-cycle arrest with an increase of G0/G1 population(Supplementary Table S3). Accordingly, cyclin D1 levelswere downregulated with an increase of p27 (Supplemen-tary Fig. S8).

The combination of erlotinib and RDEA119 showsantitumor efficacy in vivo

The antitumor efficacy of the erlotinib and RDEA119combination was evaluated in BxPC-3 and MIA PaCa-2mouse xenografts. Consistent with in vitro observations, asignificant effect of tumor growth suppression was seenin BxPC-3 in the combination treatment of erlotinib at50 mg/kg and RDEA119 at 6 mg/kg with a TGI of 67%when compared with vehicle (P < 0.001), and single drugdose controls of erlotinib had a TGI of 42% (P < 0.05) andfor RDEA119 of 52% (P < 0.01) by day 24 (Fig. 5A). Similarantitumor effects were observed for a second dosing regi-men (erlotinib at 50 mg/kg and RDEA119 at 12 mg/kg;data not shown). In MIA PaCa-2, no significant effect oftumor growth suppression was observed in the drug com-bination compared with vehicle (P ¼ 0.18) and RDEA119(P ¼ 0.2; Fig. 5B).

Because the combination of erlotinib and gemcitabinehas shown small yet statistically significant survival advan-tage over patients treated with gemcitabine alone, weevaluated if the addition of gemcitabine to the erlotiniband RDEA119 combination treatment would enhancetumor growth suppression. The addition of gemcitabinesignificantly reduced tumor growth in BxPC-3 whencompared with the combination treatment (P < 0.01;

A

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Figure 3. The effect of erlotinib and RDEA119 treatment on thephosphorylation of MAPK, EGFR, and AKT in BxPC-3 (A) and MIA PaCa-2(B) cell lines. Cells were treated with erlotinib (12.5 mmol/L), RDEA119 (100nmol/L), or a combination of erlotinib and RDEA119 and harvested atindicated hours.






Supplementary Fig. S9A). In MIA PaCa-2, gemcitabine didnot enhance tumor suppression in addition to the erlotiniband RDEA119 combination treatment (P ¼ 0.13; Supple-mentary Fig. S9B).

The effects of drug treatment did not notably affect thebody weight of the mice during the treatment course. Alltreatment regiments were well tolerated with minimalweight losses (<10%) that are similar to that of vehiclecontrol (Fig. 5C and D and Supplementary Fig. S9Cand D).

Discussion

There have been multiple investigations of chemother-apeutics that target EGFR and thereby inhibiting the EGFR–MAPK signaling pathway in solid tumors. However,response rates for single agent remain relatively modestunless the EGFR-targeted therapy is combined with otherchemotherapeutics or radiation (44). The use of high-throughput siRNA (HT-siRNA) screening is a powerfulmethod in the development of therapeutic compounds,

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Figure 4. Induction of apoptosisby erlotinib and RDEA119 inpancreatic cancer cells. A and B,apoptosis levels on erlotinib andRDEA119 treatment detected byAnnexin V assay in BxPC-3 andMIA PaCa-2 cells. E þ R, erlotinib(12.5 mmol/L) þ RDEA119 (100nmol/L). C, effect of erlotinib andRDEA119 treatment on BIMexpression and PARP cleavage inBxPC-3 and MIA PaCa-2 cells.Data are means � SD from 3independent experiments.

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from target discovery to validation and elucidatingmechanisms of action (45). In this study, we presentedevidence of how efficacious the HT-siRNA screen was indiscovering a rational drug combination to erlotinib inpancreatic cancer cells. Results from the screen identifiedseveral gene targets, which when inhibited, would enhancethe cytotoxic activity of erlotinib. The targets identified inthe screen may also present potential use molecular targetsand provide interesting insight into molecular interactionsof EGFR signaling inhibition in pancreatic cancer. BMP-2and its receptors BMPR1 and BMPR2 are overexpressed inpancreatic cancer cells. The aberrant activation of BMP-2and its receptors have been linked to significantly activateMAPK1 in pancreatic cancer cell lines, and treatment of aMAPK inhibitor blocks the BMP-2 mitogenic effects (46).Recently, in BRAF wild-type cells, RAF inhibitors canincrease CRAF activity resulting in an increase of MEK–ERK phosphorylation and enhanced growth (47). Theidentification of the inhibition of BMPR2 and BRAF as

enhancers to erlotinib further highlights how the activationof alternative protein kinases in the EGFR–MAPK pathwayenables cancer cells to maintain the oncogenic potentialwhen EGFR is inhibited.

Sunitinib, a multitargeted receptor tyrosine kinase inhi-bitor, FLT3 among them (48), has been reported to sensi-tize pancreatic cancer cells to ionizing radiation byattenuating pAKT and pERK levels (49). GCK phosphor-ylates glucose to provide glucose-6-phosphate for thesynthesis of glycogen and preferentially expresses in hepa-tocytes and pancreatic beta cells. PRKAB2 is a regulatorysubunit of AMP-activated protein kinase, which regulatesthe intercellular metabolism of fatty acids and glycogen.Although both GCK and PRKAB2 have not been studied inthe context of pancreatic cancer, both genes have beenassociated to diabetes (50–52).

The use ofMEK inhibitors to perturb the activity ofMAPKhas been reported to have efficacy in pancreaticmodels (36,53). Other groups have reported on the synergistic effect of

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Figure 5. Inhibition of tumor growth by erlotinib and RDEA119 as single agents or in combination in the BxPC-3 and MIA PaCa-2 xenograft model.Tumor growth curves for the different treatment groups in BxPC-3 (A) andMIA PaCa-2 (B). Mouse bodyweight curves for different treatment groups in BxPC-3(C) and MIA PaCa-2 (D). Arrows indicate the treatment start and end dates.






EGFR and MEK inhibition. Jimeno and colleagues reportedon the efficacy of the combined EGFR andMAPK inhibitorsin biliary and pancreatic cancer. Their discovery was basedon global analysis of gene expression profiles on biliarycancer cell lines that were resistant to EGFR-targeted ther-apeutics, gefitinib and erlotinib, and then confirmed theactivity in vivo in human pancreatic cancer xenografts withminimal mechanistic studies to explain the synergy in theirpancreatic cancer model (54). In EGFR-dependent lungcancer cell lines, Balko and colleagues observed synergisticcytotoxicity with the combination of EGFR and MEK inhi-bitors only in cells with wild-type KRAS (55). Yoon andcolleagues recently reported the synergistic effect betweengefitinib and AZD6244 in gastric and lung cancer cells (56,57). The authors found that the combination treatmentrepressed AKT and ERK activation and consequentlyenhanced apoptosis.

The occurrence of somatic KRAS mutations is a highlypredictive marker of resistance to anti-EGFR chemothera-pies. In colorectal cancer, KRAS mutations were associatedwith resistance to anti-EGFR therapies (cetuximab andpanitumumab) in patients withmetastatic disease, whereasKRAS wild-type predicted efficacy in terms of tumorresponse and patient survival (58–60). In non–small celllung cancer, KRAS mutations are attributed to the poorresponse to erlotinib and gefitinib (61). In pancreaticcancer, the combination of gemcitabine and erlotinibexhibited modest (6.24 vs. 5.91 months) but statisticallysignificant improvement in overall survival (7). As KRAS

mutations are reported so prevalently in pancreatic cancer,our study does raise the issue of the status of KRAS forpatients that may benefit from the treatment of erlotiniband a MEK inhibitor.

In summary, we have shown a synergistic effect betweenerlotinib and 2 clinically relevant MEK inhibitors,RDEA119 and AZD6244, in a subset of KRAS wild-typepancreatic cancer cells in vitro and in vivo. Our resultsprovide a clear biological rationale for the investigationof erlotinib in combination with a MEK inhibitor in KRASwild-type pancreatic cancer.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Jill Muehling and Kristen Bisanz for assistance with theSTRprofilingof the cell lines,Carole Viso and TammyBrehm-Gibson for cell-cycle analysis, and Paul Gonzales and Mario Sepulveda for animal studies.

Grant Support

This work was supported by funding from the National Foundation forCancer Research (NFCR) and a National Cancer Institute grant CA109552.

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 August 17, 2010; revised January 25, 2011; accepted February27, 2011; published OnlineFirst March 8, 2011.

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2011;17:2744-2756. Published OnlineFirst March 8, 2011.Clin Cancer Res Caroline H. Diep, Ruben M. Munoz, Ashish Choudhary, et al. KRAS Wild-Type Human Pancreatic Cancer CellsSynergistic Effect between Erlotinib and MEK Inhibitors in

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