Primary and Acquired Resistance of Colorectal Cancer Cells ... · Teresa Troiani, Stefania Napolitano, Donata Vitagliano, Floriana Morgillo, Anna Capasso, Vincenzo Sforza, Anna Nappi,

Cancer Therapy: Preclinical

Primary and Acquired Resistance of Colorectal Cancer Cellsto Anti-EGFR Antibodies Converge on MEK/ERK PathwayActivation and Can Be Overcome by Combined MEK/EGFRInhibition

Teresa Troiani, Stefania Napolitano, Donata Vitagliano, Floriana Morgillo, Anna Capasso,Vincenzo Sforza, Anna Nappi, Davide Ciardiello, Fortunato Ciardiello, and Erika Martinelli

AbstractPurpose: The EGFR-independent activation of the RAS/RAF/MEK/MAPKpathway is one of the resistance

mechanisms to cetuximab.

Experimental Design: We have evaluated, in vitro and in vivo, the effects of BAY 86-9766, a selective

MEK1/2 inhibitor, in a panel of human colorectal cancer cell lines with primary or acquired resistance to

cetuximab.

Results: Among the colorectal cancer cell lines, five with a KRAS mutation (LOVO, HCT116, HCT15,

SW620, and SW480) and one with a BRAFmutation (HT29) were resistant to the antiproliferative effects of

cetuximab, whereas two cells (GEO and SW48) were highly sensitive. Treatment with BAY 86-9766

determined dose-dependent growth inhibition in all cancer cells, including two human colorectal cancer

cells with acquired resistance to cetuximab (GEO-CR and SW48-CR), with the exception of HCT15 cells.

Combined treatmentwith cetuximab andBAY86-9766 induced a synergistic antiproliferative and apoptotic

effects with blockade in the MAPK and AKT pathway in cells with either primary or acquired resistance to

cetuximab. The synergistic antiproliferative effects were confirmed using other two selective MEK1/2

inhibitors, selumetinib and pimasertib, in combination with cetuximab. Moreover, inhibition of MEK

expression by siRNA restored cetuximab sensitivity in resistant cells. In nude mice bearing established

humanHCT15, HCT116, SW48-CR, and GEO-CR xenografts, the combined treatment with cetuximab and

BAY 86-9766 caused significant tumor growth inhibition and increased mice survival.

Conclusion: These results suggest that activation of MEK is involved in both primary and acquired

resistance to cetuximab and the inhibition of EGFR andMEK could be a strategy for overcoming anti-EGFR

resistance in patients with colorectal cancer. Clin Cancer Res; 20(14); 3775–86. �2014 AACR.

IntroductionColorectal cancer is a major cause of morbidity and

mortality throughout the world (1). The prognosis ofpatients diagnosed with metastatic colorectal cancer hasimproved markedly over the last 15 years, with an increasein median overall survival from 6 months with only best

supportive care to more than 2 years with the introductionof active chemotherapy drugs, such as fluropyrimidines,oxaliplatin, and irinotecan, and of molecular targeteddrugs, such as bevacizumab, cetuximab, panitumumab,aflibercept, and regorafenib (2, 3).

Cetuximab and panitumumab are 2 blocking anti-epi-dermal growth factor receptor (EGFR) monoclonal anti-bodies (mAb) that inhibit the activation of the EGFR and itsdownstream intracellular signals, the RAS–RAF–MEK–MAPK and the PTEN–PIK3CA–AKT pathways (4–6). Inparticular, cetuximab is an effective treatment as singleagent or in combination with standard chemotherapy regi-mens for a subset of patients with metastatic colorectalcancer (7). Resistance to anti-EGFR therapies is likelybecause of the constitutive activation in cancer cells ofsignaling pathways acting downstream and/or indepen-dently of EGFR. In fact, point mutations in codon 12 or13 within the exon 2 of the KRAS gene have been found asthemajor negative predictor of efficacy for cetuximab (8, 9).Therefore, cetuximab is currently used inmonotherapyor in

Authors' Affiliation: Oncologia Medica, Dipartimento Medico-Chirurgicodi Internistica Clinica e Sperimentale "F. Magrassi e A. Lanzara," SecondaUniversit�a degli Studi di Napoli, Via S. Pansini 5, Naples, Italy

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

T. Troiani and S. Napolitano contributed equally to this article.

Corresponding Author: Teresa Troiani, Dipartimento Medico-Chirurgicodi Internistica Clinica e Sperimentale "F. Magrassi e A. Lanzara," SecondaUniversit�adegli Studi diNapoli, ViaS. Pansini 5, 80131Naples, Italy. Phone:39-0815666725; Fax: 39-0815666732; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-2181

�2014 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 3775

on June 15, 2021. © 2014 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 8, 2014; DOI: 10.1158/1078-0432.CCR-13-2181

http://clincancerres.aacrjournals.org/

combination with chemotherapy only in patients withmetastatic colorectal cancer with KRAS wild-type tumors.

In addition to KRAS gene mutations, a number ofretrospective studies have provided evidence that primaryresistance to EGFR inhibitors in colorectal cancer could becorrelated to mutations in other intracellular downstreameffectors of EGFR, such as BRAF, NRAS, and PIK3CA(exon 20) genes (10). However, even among the molec-ularly enriched subset of patients with colorectal cancerwith KRAS, BRAF,NRAS, and PIK3CA (exon 20) wild-typegenes, cetuximab is not always clinically effective, sug-gesting that there are other undefined mechanisms ofprimary resistance (11). Although the evaluation of thepresence of these gene mutations in the EGFR signalingpathways could identify the appropriate colorectal cancerpatient population to treat with cetuximab, all initiallyresponding patients will ultimately develop resistance tocetuximab (i.e., acquired resistance; ref. 12). The mechan-isms involved in the primary resistance to anti-EGFRdrugs are likely to play a role also in the acquired resis-tance. In this regard, it has been suggested that the onsetof acquired resistance to anti-EGFR treatments in patientswith metastatic colorectal cancer could be because of theemergence of KRAS-mutated cancer cell clones (13, 14).Moreover, HER2 gene amplification has been found tooccur in approximately 3% of unselected patients withmetastatic colorectal cancer and with a significantlyhigher frequency in patients with KRAS wild-type tumorsthat do not benefit of treatment with anti-EGFR drugs

(15). In alternative to HER2 amplification, in a subset ofpatients with cetuximab-resistant metastatic colorectalcancer, acquired resistance is probably because ofincreased levels of heregulin, a ligand that binds HER3and HER4 (16). Both HER2 gene amplification andincreased heregulin production in cancer cells couldcause acquired resistance to anti-EGFR MAb treatmentby leading to persistent activation of RAS–RAF–MEK–MAPK signaling (15, 16).

In this respect, the RAS–RAF–MEK–MAPK signaling path-way plays a central role in the intracellular transduction ofproliferative signals from activated cell membrane growthfactor receptors to the nucleus in both normal and cancercells. RAF is a serine/threonine kinase that activates down-stream signals in response to activated GTP-bound RAS byphosphorylating MEK1 and MEK2, which in turn phos-phorylate and activate MAPK (or ERK1 and ERK2). MAPKphosphorylates a number of cellular substrates with keyroles in cell proliferation and survival (17). Direct inhibi-tion of MEK is a promising strategy in the development ofcancer therapeutics to control tumor growth that is depen-dent on aberrant MEK pathway signaling. A potentialadvantage of targeting MEK is that the RAS/RAF/MEK/MAPK pathway is a convergence point where a number ofupstream signaling pathways can be blocked with the directinhibition of MEK. Most of the MEK inhibitors that arecurrently in early clinical development are selective forMEK1 and/or MEK2 and do not have off target effects onother kinases (18).

BAY 86-9766 is a highly selective, potent, orally avail-able, small-molecule non-ATP-competitive inhibitor ofMEK1/2 (19). This drug binds to an allosteric site adja-cent to the ATP-binding region and then interacts withATP, the activation loop, and other surrounding residuesto prevent binding of MEK to its substrate ERK, therebyblocking ERK phosphorylation (20). BAY 86-9766 inhib-ited cell proliferation in human cancer cell lines, includ-ing those harboring BRAF V600E mutations, and alsoexhibited potent antitumor activity in human xenograftmodels (19). The safety profile and tolerability of BAY 86-9766 has been evaluated in a multicenter phase I clinicalstudy. This trial showed that it was well tolerated, withgood oral absorption, dose proportional pharmacokinet-ics, target inhibition at the maximum tolerated dose(MTD), and some evidence of clinical benefit across arange of tumor types (20, 21).

Constitutive activation of RAS–RAF–MEK–MAPK signal-ing, one of the key pathway downstream of EGFR, couldcause primary and/or acquired resistance to anti-EGFRtreatment. Based on this hypothesis, we have evaluated theefficacy of BAY 86-9766 in overcoming the resistance tocetuximab by using human colorectal cancer models. Forthis purpose,wehave selected a panel of 8human colorectalcancer cell lines, which we have characterized for theirsensitivity to the antiproliferative effects of cetuximab andfor the gene mutation profile for KRAS, BRAF, PIK3CA, andNRAS and 2 cancer cell lines with acquired resistance tocetuximab that we have obtained following continuous

Translational RelevanceCetuximab, a blocking anti-EGFR monoclonal anti-

body, is effective in combination with chemotherapy oras single agent for the treatment of patients with KRASwild-type metastatic colorectal cancer. However, clinicaldata indicate that, in all initially responding patientswith colorectal cancer, cancer cell acquired resistancedevelops leading to treatment failure. This evidence hastriggered a series of studies on the molecular mechan-isms of primary and acquired resistance to cetuximab. Inthis study, we have demonstrated that, in human colo-rectal cancer cells with either primary or acquired resis-tance to cetuximab, EGFR-independent constitutive acti-vation of MEK signaling is observed with subsequentMAPK activation. This observation suggests that MEKactivation is a convergence point for a number of dif-ferent signaling pathways in EGFR inhibitor-resistantcolorectal cancer cells and that direct inhibition of MEKcould be of therapeutic relevance. In fact, here we showthat BAY 86-9766, a selective MEK1/2 inhibitor hasantiproliferative activity in cetuximab-resistant colorec-tal cancer cell lines and that the combined blockade ofboth EGFR and MEK could represent a therapeuticstrategy for preventing and/or overcoming cetuximabresistance in patients with colorectal cancer.

Troiani et al.

Clin Cancer Res; 20(14) July 15, 2014 Clinical Cancer Research3776




treatment with cetuximab of 2 cetuximab-sensitive humancolorectal cancer cell lines (22, 23) to further elucidate themolecular mechanisms of primary and acquired resistanceto cetuximab.

Materials and MethodsDrugsCetuximab, an anti-EGFR human-mouse chimeric mAb

was kindly provided by Merck Serono Italy. BAY 86-9766,selumetinib and pimasertib, selective MEK1/2 tyrosinekinase inhibitors, were kindly provided by Bayer PharmaItaly, Astra Zeneca, and Merck-Serono, respectively. All 3MEK1/2 inhibitors were dissolved in sterile dimethylsulf-oxide (DMSO) and a 10 mmol/L working solution wasprepared and stored in aliquots at �20�C. Working con-centrations were diluted in culturemedium just before eachexperiment.

Cell linesThe human HT29, LOVO, HCT15, SW620 colorectal

cancer cell lines were obtained from the American TypeCulture Collection (ATTC) and have been authenticated byIRCCS "AziendaOspedaliera Universitaria SanMartino-ISTIstituto Nazionale per la Ricerca sul Cancro, Genova," Italy.The human SW48 (catalogue number: HTL99020),HCT116 (catalogue number: HTL95025), SW480 (cata-logue number: HTL99017) dimethylsulfoxide cell lineswere obtained from IRCCS "Azienda Ospedaliera Univer-sitaria San Martino-IST Istituto Nazionale per la Ricerca sulCancro, Genova," Italy. The human GEO colon cancer cellline was kindly provided by Dr. N. Normanno (NationalCancer Institute,Naples, Italy). GEO-CR and SW48-CR cellswere established as previously described (22, 23). GEO andGEO-CR cell lines were grown in DMEM (Lonza), supple-mented with 20% fetal bovine serum (FBS; Lonza) and 1%penicillin/streptomycin (Lonza). SW48, SW48-CR, LOVO,HCT15, HCT116, and SW480 cells were grown in RPMI-1640 (Lonza) supplemented with 10% FBS and 1% peni-cillin/streptomycin. SW 620 and HT29 cancer cells weregrown inMcCoymedium (Lonza) supplemented with 20%FBS (Lonza) and 1% penicillin/streptomycin (Lonza). Allcell lines were grown in a humidified incubator with 5% ofcarbon dioxide (CO2) and 95% air at 37

�C. All cell lineswere routinely screened for the presence of mycoplasma(Mycoplasma Detection Kit; Roche Diagnostics).

Proliferation assayCancer cell lines were seeded in 24-well plates and were

treated with different concentrations of cetuximab (range,0.01–20 mg/mL) alone or in combination with BAY 86-9766 (range, 0.01–5 mmol/L), or selumetinib (range, 0.01–5 mmol/L) or pimasertib (range, 0.01–5 mmol/L) for 96hours. Cell proliferation was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT). The IC50 was determined by interpolation from thedose–response curves. Results represent the median of 3separate experiments, each performed in quadruplicate.Results of the combination treatmentwere analyzed accord-

ing to the method of Chou and Talalay by using theCalcuSyn software programme (Biosoft).

Apoptosis assayHCT15, HCT116, SW48-CR, and GEO-CR cells were

seeded in 6-well plates, treated for 72 hours and stainedwithAnnexinV-fluorescein isothiocynate (FITC). Apoptoticcell deathwas assessed by counting the numbers of cells thatstained positive for Annexin V-FITC and negative for pro-pidium iodide (PI) using an Apoptosis Annexin V-FITC Kit(Invitrogen), coupled with fluorescence-activated cell sort-ing (FACS) analysis.

ImmunoblottingHCT15, HCT116, SW48-CR, and GEO-CR cells were

seeded into 100 mm3 dishes and treated with vehicle,cetuximab, BAY 86-9766, or their combination for 24hours. Protein lysates containing comparable amounts ofproteins, estimated by a modified Bradford assay (Bio-Rad), were subjected to Western blot analysis, as previ-ously described (24). Immuno-complexes were detectedwith the Enhanced Chemiluminescence Kit (Pierce Bio-technology Inc.). EGFR monoclonal antibody (#4267),phospho-EGFR monoclonal antibody (#3777), p44/42MAPK polyclonal antibody (#9102), phospho-p44/42MAPK monoclonal antibody (#9106), anti-AKT poly-clonal antibody (#9272), pAKT monoclonal antibody(#4060), and anti-PARP polyclonal antibody (#9542)were from Cell Signaling (Beverly). Caspase-3 monoclonalantibody (sc-65496) was from Santa Cruz Biotechnology.Monoclonal anti-a-tubulin antibody (T8203) was fromSigma Chemical Co. The following secondary antibodiesfrom Bio-Rad were used: goat anti-rabbit IgG and rabbitanti-mouse IgG. Immunoreactive proteins were visualizedby enhanced chemiluminescence (ECL plus; Thermo Fish-er Scientific). Each experiment was done in triplicate.

RNA interference and PCR analysisThe small inhibitor RNAs (siRNA) ON-target plus

SMARTpool MEK1/2 (human: #L-003571-00/L-003573-00)were fromDharmacon. The siCONTROLNon-targetingPool (#D-001206-13-05) was used as a negative (scram-bled) control. Cells were transfected with 100 nmol/LsiRNAs using Hiperfect reagent (Qiagen) following manu-facturer’s instructions. The day before transfection, the cellswere plated in 35 mm dishes at 40% of confluence inmedium supplemented with 5% FBS without antibiotics.Cells were harvested at different time points (24, 48, 72, and96 hours) after transfection. Western blot analysis forMEK1/2 expression was done. The siRNA effects on cellproliferation and on cell signaling were evaluated by MTTand Western blot analysis as previously described. Briefly,cells were seeded into 24-multiwell cluster dishes andtransfected with MEK1/2 siRNA. Forty-eight hours aftertransfection, cells treated with MEK1/2 siRNA received 5mg/mL of cetuximab and cell proliferation andWestern blotanalysis were determined 24 hours later (after 72 hours oftransfection).

MEK Inhibitor Overcomes Resistance to Cetuximab in Colorectal Cancer

www.aacrjournals.org Clin Cancer Res; 20(14) July 15, 2014 3777




Tumor xenografts in nude miceFour- to six-week-old female balb/c athymic (nuþ/

nuþ) mice were purchased from Charles River Laborato-ries. The research protocol was approved and mice weremaintained in accordance with the institutional guide-lines of the Second University of Naples Animal Care andUse Committee. Mice were acclimatized at the SecondUniversity of Naples Medical School Animal Facility for 1week before being injected with cancer cells and thencaged in groups of 5. Mice were injected subcutaneouslywith 2.5 � 106 HCT15, HCT116, GEO-CR, and SW48-CRcells that had been resuspended in 200 mL of matrigel (BDBiosciences). When the mean values of tumors werebetween 200 and 300 mm3, mice were randomly dividedin 4 groups (10 mice per group). BAY 86-9766 wasprepared in vehicle 1 (10% cremohor EL in saline) andwas administered through oral gavage daily (5 mg/kg) for3 weeks. Cetuximab at the dose of 1 mg was injectedintraperitoneally twice a week for 3 weeks. Monitoring oftumor growth was continued until tumors reachedapproximately 2,000 mm3 when mice were sacrificed.Tumor size was evaluated twice per week by calipermeasurements using the following formula: p/6 � largerdiameter � (smaller diameter)2. Student t test was used toevaluate the statistical significance of the results.

Statistical analysisThe statistical analyses of in vitro and in vivo data were

carried out using Prism version 4.02 (GraphPad Software,Inc.). The Student t test was used to evaluate the statisticalsignificance of the results. AllP values represent 2-sided testsof statistical significance with P value < 0.05.

ResultsEffectsof cetuximab treatmentoncell proliferationandon EGFR-dependent intracellular signaling in humancolorectal cancer cell lines

We first evaluated the sensitivity to the cell growth inhi-biting effects of the anti-EGFRMAb cetuximab in a panel of8 human colorectal cancer (GEO, SW48, HT29, LOVO,HCT116, HCT15, SW620, and SW480) cell lines, that wereselected for having different mutation profiles in KRAS,NRAS, BRAF, PIK3CA, and EGFR genes (SupplementaryTable S2). Cancer cells were treated with cetuximab atconcentrations ranging from0.01 to 20 mg/mL for 96 hours.The drug concentrations required to inhibit cell growth by50% (IC50) were determined by interpolation from thedose–response curves. As shown in Fig. 1A and Supplemen-tary Table S1A, there was a differential sensitivity to cetux-imab-induced cell growth inhibition. In fact, 6 colorectalcancer cell lines (HT29, LOVO, HCT116, HCT15, SW620,and SW480) were primarily resistant to cetuximab. LOVO,HCT116, HCT15, SW620, and SW480 cells have an acti-vating KRAS genemutation in either codon 12 or 13 withinexon 2, whereas HT29 cells have a BRAF gene mutation(V600E). Between the 2 cetuximab-sensitive colorectal can-cer cell lines, SW48 is "quadruple wild type" for KRAS,BRAF, NRAS, and PIK3CA genes, whereas GEO cells has a

KRAS gene codon12mutation. In particular, althoughGEOcells harbor a KRAS gene mutation, previous studies fromdifferent laboratories, including our own, have demonstrat-ed that this colorectal cancer cell line is one of the mostsensitive colorectal cancer cell lines to the in vitro and in vivoantitumor activity of cetuximab treatment (23, 25–27).Furthermore, Fig. 1A and Supplementary Table S1A showthat cetuximabwas also not effective inGEO-CR and SW48-CR cells, 2 cell lines models of acquired resistance, that wehave recently obtained following continuous cetuximabtreatment in vivo of nude mice bearing GEO tumor xeno-grafts or following continuous in vitro exposure to cetux-imab of SW48 cells (22, 23). These 2 cell lines have beenpreviously evaluated for the presence of mutations in boththe KRAS and the NRAS genes by next generation sequenc-ing using the Ion AmpliSeq cancer panel (22). No addi-tionalmutations were observed inKRAS andNRAS genes inGEO-CR and SW48-CR cells compared with the parentalcell lines (data not shown). As shown in Fig. 1B, cetuximabtreatment suppressed EGFR-induced intracellular signalingonly in the 2 colorectal cancer sensitive cell lines GEO andSW48, with a significant inhibition in the expression ofactive, phosphorylated MAPK and AKT proteins, whereas ithad little or no effect on the levels of active, phosphorylatedMAPK and AKT proteins in cetuximab-resistant colorectalcancer cell lines.

Effects of treatment with BAY 86-9766, a selectiveMEK1/2 inhibitor, on cell proliferation of humancolorectal cancer cell lines

Wenext evaluated the cell growth inhibiting effects of theselective MEK1/2 inhibitor BAY 86-9766 in the same panelof human colorectal cancer cell lines. Cancer cells weretreated with BAY 86-9766 at concentrations ranging from0.01 to 5 mmol/L for 96 hours. As illustrated in Fig. 2A andSupplementary Table S1B, BAY 86-9766 treatment caused adose-dependent cell growth inhibition in all colorectalcancer cell lines, with IC50 values ranging between 0.05mmol/L (HCT116) and 2 mmol/L (SW48-CR), except forHCT15 cells. The same effects on cell growth inhibitionwere obtained by using other 2 different MEK1/2 inhibitorssuch as selumetinib and pimasertib (Fig. 2B and C andSupplementary Table S1B).

Effects of cetuximab in combination with the selectiveMEK1/2 inhibitorBAY86-9766 in colorectal cancer cellsgrowth in vitro

All colorectal cancer cell lines were treated with differentconcentrations of cetuximab, of BAY 86-9766, or with thecombination of the 2 drugs (Fig. 3 and Supplementary Fig.S1A and S1B and Table S3). Combination index (CI) valueswere calculated according to the Chou and Talalay math-ematical model for drug interactions using the Calcusynsoftware, as previously described (25, 28). Both in thecolorectal cancer cell lines with primary resistance to cetux-imab (HT29, LOVO, HCT116, HCT15, SW620, andSW480) and in the colorectal cancer cell lines with acquiredresistance to cetuximab (GEO-CRandSW48-CR), treatment

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with the selective MEK1/2 inhibitor BAY 86-9766 deter-mined synergistic growth inhibitory effects in combina-tion with cetuximab. In fact, the CI values for the com-bined treatments were significantly 1.0), suggesting thatin EGFR inhibitor–sensitive colorectal cancer cell lines thecombined treatment with cetuximab and the MEK1/2inhibitor could negatively interfere on cell growth inhi-bition (Supplementary Fig. S1A and S1B and Table S3).To confirm the synergistic growth inhibitory effects ofcetuximab in combination with BAY 86-9766, we haveused other 2 selective MEK1/2 inhibitors, such as selu-metinib and pimasertib (24, 28). As depicted in Supple-mentary Table S1B, the combined treatment of cetuximab

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Figure 1. Effects of cetuximab oncell proliferation and on EGFR-dependent intracellular signaling ina panel of human colorectal cancercell lines. A, cells were treatedwith different concentrations ofcetuximab (range, 0.01–20 mg/mL)for 96 hours and evaluated forproliferation by MTT staining, asdescribed in Materials andMethods. The IC50was determinedby interpolation from the dose–response curves. Resultsrepresent the median of 3 separateexperiments, each performed inquadruplicate. B, analysis ofintracellular signaling pathways byWestern blotting in cancer cellstreated with cetuximab at theindicated dose for 24 hours. Totalcell protein extracts (50 mg) weresubjected to immunoblotting withthe indicated antibodies, asdescribed in the Materials andMethods.






with selumetinib or pimasertib induced a clearly syner-gistic antitumor activity only in the colorectal cancer celllines with either primary or acquired resistance to theanti-EGFR inhibitor. In contrast, the combined treatmentwas antagonistic on cetuximab sensitive cell lines, GEOand SW48 in accord to the results with BAY 86-9766 andcetuximab combinations (Supplementary Table S3).

Effects of cetuximab in combination with the selectiveMEK1/2 inhibitor BAY 86-9766 on induction ofapoptosis and on EGFR-dependent intracellularsignaling pathways in HCT15, HCT116, GEO-CR, andSW48-CR colorectal cancer cells

We next selected for further experiments 2 colorectalcancer cell lines with primary resistance to cetuximab(HCT15 and HCT116) and the 2 colorectal cancer cell lineswith acquired resistance to cetuximab (GEO-CR and SW48-CR) to determine whether the synergistic growth inhibitionthat was obtained by the combined treatment with theselective MEK1/2 inhibitor BAY 86-9766 and cetuximabwas because of a more effective inhibition of key intracel-lular signals for cell survival and proliferation. We, there-fore, measured the ability of these drugs (as single agents orin combination) to induce apoptosis by using Annexin V-FITC (Fig. 4A). Although cetuximab or BAY 86-9766 single-agent treatments resulted in little or no induction of apo-ptosis in all cancer cell lines, the combined treatmentdetermined a significant induction of apoptosis in bothcolorectal cancer cells with primary resistance to cetuximaband colorectal cancer cells with acquired resistance to cetux-imab (Fig. 4A). The combined treatment resulted also in theactivation of specific intracellular mediators of apoptosis,such as induction of cleaved PARP protein in all cancer celllines (Fig. 4B). To determine whether the combined inhi-bition of EGFR and MEK1/2 could be effective in blockingEGFR-activated intracellular pathways, Western blots wereperformed on protein extracts fromHCT15,HCT116, GEO-CR, and SW48-CR cells, which were treated for 24 hourswith cetuximab or BAY 86-9766 as single agents or incombination. The combined treatment resulted in effectiveinhibition of active, phosphorylated MAPK and AKT pro-

teins in all cancer cell lines (Fig. 4C and D and Supplemen-tary Fig. S2).

Inhibition of MEK1/2 expression by siRNA restoressensitivity to cetuximab

To determine if MEK1/2 expression could be involved inthe acquisition of primary and acquired cetuximab resis-tance, we investigated whether inhibition of MEK1/2expression could restore cetuximab sensitivity in HCT15and HCT116. Transfection with a specific MEK1/2 siRNAfor 72 hours significantly reduced MEK1/2 protein expres-sion in these cells, as shown in Fig. 5A. As illustrated in Fig.5B,MEK1/2 siRNA treatment slightly reduced cell growth inHCT15 and HCT116. Although single-agent cetuximabtreatment did not affect cell proliferation, cetuximab treat-ment in combination with MET silencing determined cellgrowth inhibition in HCT15 and HCT116. MEK silencingalso restored the ability of cetuximab to inhibit MAPKactivation in both cell lines as shown by downregulationof phospho-MAPK levels (Fig. 5C).

Effects of cetuximab in combination with the selectiveMEK1/2 inhibitor BAY 86-9766 on HCT15, HCT116,GEO-CR, and SW48-CR tumor xenografts

We finally investigated the in vivo antitumor activity ofcetuximab alone or in combination with BAY 86-9766 innude mice bearing cetuximab-resistant HCT15, HCT116,GEO-CR, or SW48-CR cells grown subcutaneously as tumorxenografts. After 10 days, when established HCT15,HCT116, GEO-CR, or SW48-CR tumors were detectable,mice were treated for 3 weeks with cetuximab, with BAY 86-9766 or with the combination of the 2 drugs. As shownin Fig. 6, treatment with cetuximab had little or no effect ontumor growth in all tumor xenografts, whereas BAY 86-9766 treatment caused approximately 40% to 50% reduc-tion in tumor growth in tumor xenograft models of bothprimary and acquired resistance to cetuximab. In contrast,the combined treatmentwith the 2 drugs suppressed almostcompletely HCT15, HCT116, GEO-CR, or SW48-CR tumorgrowth at the end of the 3 weeks of therapy (Fig. 6).Furthermore, the combined treatment with cetuximab and

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Figure 2. Effects ofMEK1/2 inhibitors, on cell proliferation in a panel of human colorectal cancer cell lines. A–C, cells were treatedwith different concentrationsof BAY 86-9766, selumetinib and pimasertib (range, 0.01–5 mmol/L for all 3 drugs) for 96 hours and evaluated for proliferation byMTT staining, as described inthe Materials and Methods.

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BAY 86-9766 determined a significant increase in micesurvival as compared with control and to single-agenttreatments in all tumor xenografts. Single-agent and com-bination treatment protocols were well tolerated by mice,with no weight loss or other signs of acute or delayedtoxicity (Supplementary Fig. S3).

DiscussionElucidating the mechanisms of cancer cell resistance to

anticancer drugs is critical for the development of moreeffective therapies. An extensive effort has been made to

understand cancer cell resistance mechanism(s) to EGFRinhibitors (6, 22, 29). Retrospective studies have revealedthat mutations of KRAS in hotspot regions of exons 2, 3, 4,BRAF, NRAS, and PIK3CA exon 20 genes are predictors oflack of efficacy of either cetuximab or panitumumab inpatients with chemorefractory metastatic colorectal canceras a result of EGFR-independent intracellular downstreamsignaling activation (8, 30–34).

In agreement with these findings, in this study we havefound that among the panel of human colorectal cancer celllines, those with primary resistance to cetuximab havemutations in the RAS/RAF/MEK/MAPK pathway. In

Figure 3. Effects of cetuximab in combination with the selectiveMEK1/2 inhibitor BAY 86-9766 in HCT15, HCT116, GEO-CR, and SW48-CR colorectal cancercells growth in vitro. The indicated colorectal cancer cell lines were treated every day with different concentrations of cetuximab (range, 0.01–20 mg/mL)and/or BAY-86-9766 (range, 0.01–5 mmol/L) for 96 hours at a fixed drug ratio of 1:1 and cell proliferation was evaluated by MTT assay. CI values werecalculated according to the Chou and Talalay mathematical model for drug interactions using the Calcusyn software, as described in the MaterialsandMethods. For each cell line, it is shown in the left panel the percent of cell growth inhibition determined by treatment with cetuximab, with BAY 86-9766 orwith their combination. For each cell line, in the right panel it is shown the CI for the combination of cetuximab plus BAY 86 9766. Results representthe median of 3 separate experiments, each performed in quadruplicate.






particular, LOVO, HCT116, HCT15, SW620, and SW480cells have an activatingKRAS genemutation in either codon12 or 13 within exon 2, whereas HT29 cells have a BRAFgene mutation (V600E).

Moreover, MAPK activation, a major downstream effec-tor of EGFR pathway, is one of the potential mechanismsof resistance to anti-EGFR therapies. In fact, cetuximabtreatment was not effective in primary and acquired (GEO-CR and SW48-CR) cetuximab-resistant colorectal cancercell lines in which activation of MAPK and AKT, was notblocked, despite EGFR inhibition. The lack of phosphor-

ylated MAPK protein inhibition as a molecular finding ofacquired resistance to anti-EGFR therapies is in agreementwith other recent reports. In particular, Yonesaka andcolleagues have developed cetuximab-resistant clones ofGEO cells, in which activation of ERBB2 signaling, eitherthrough ERBB2 amplification or through heregulin proteinupregulation, leads to persistent, constitutive ERK1/2 acti-vation (16). Moreover, Normanno and colleagues haveshown, in human breast cancer cell lines models ofacquired resistance to the small molecule EGFR tyrosinekinase inhibitor (TKI) gefitinib, a role for constitutive

Figure 4. Effects of cetuximab in combination with the selective MEK1/2 inhibitor BAY 86-9766 on induction of apoptosis and on intracellular signalingpathways in HCT15, HCT116, GEO-CR, and SW48-CR colorectal cancer cells. A, apoptosis was evaluated with Annexin V staining, as describedin theMaterials andMethods. Cells were treatedwith cetuximab,with BAY 86-9766 or with their combinationwith the indicated doses for 72 hours. The rate ofapoptosis was expressed as a percentage of the total cells counted. Columns are the means of 3 independent experiments. B, cells were treated withcetuximab, with BAY 86-9766 or with their combination at the indicated doses for 24 hours. Expression of caspase 3 and of the cleaved form of PARP wereevaluated by immunoblotting, as described in the Materials and Methods. Antitubulin antibody was used for normalization of protein extract content.C andD, analysis of intracellular signaling pathways byWestern blotting in the indicated colorectal cancer cell lines treatedwith cetuximab, with BAY 86-9766or with their combination at the indicated doses for 24 hours. Total cell protein extracts (50 mg) were subjected to immunoblotting with the indicatedantibodies, as described in the Materials and Methods. Antitubulin antibody was used for normalization of protein extract content.

Troiani et al.





activation of MEK/MAPK signaling (35). In this respect, wehave recently developed human cancer cell lines withacquired resistance to erlotinib and gefitinib, 2 selectiveEGFR TKIs, following chronic exposure to these drugs ofCALU-3 lung adenocarcinoma and HCT116 colorectalcancer cells (36). We have found that EGFR inhibitoracquired resistance in these cells was predominantly drivenby the constitutive activation of the RAS/RAF/MEK/MAPKpathway and it could be overcome by treatment withsorafenib, which blocked RAF signaling (36). Furthermore,

we have demonstrated that a common phenotype corre-lated to acquired resistance to different TKIs is representedby a MEK-dependent transition from an epithelial tomesenchimal phenotype and could be overcome by usingselective MEK inhibitors (37). Moreover, a clone of GEOcells with acquired resistance to cetuximab were sensitiveto the growth inhibitory effects of the MEK1/2 inhibitorselumetinib (16). Taken together, these findings suggestthat MEK inhibition might be a potentially effective ther-apeutic strategy for preventing and/or overcoming cancercell resistance to different molecular targeted agentsthat block growth factor receptor–driven intracellularsignaling.

Based on these findings, we have explored whether theuse of MEK1/2 inhibitors could be able to overcome intrin-sic and acquired resistance to the anti-EGFR inhibitor. Forthis reason, we investigated the combination of cetuximaband MEK1/2 inhibitors (either BAY 86-9766, selumetiniband pimasertib) in a panel of human colorectal cancer celllines. Here we show a synergistic growth inhibition ofcetuximab-resistant colorectal cancer cell lines followingcombined treatment with the anti-EGFR monoclonal anti-body and selective MEK1/2 inhibitor. The synergistic anti-proliferative effects were observed only in cetuximab-resis-tant cell lines, whereas in cetuximab-sensitive GEO andSW48 cells, the combined treatment induced a clearlyantagonistic activity. These data may suggest that in anti-EGFR sensitive colorectal cancer cell lines the combinedtreatment with cetuximab and a MEK1/2 inhibitor couldnegatively interfere on cell growth inhibition, although thishypothesis needs further studies.

The results of this study demonstrate that the synergisticantiproliferative effect is correlated with an inhibition ofactivated pMAPK and pAKT, that leads to increased apo-ptosis, as shown by increased levels of cleaved PARP andcaspase-3 activation.

Collectively, these results suggest that RAS/RAF/MEK/MAPK pathway activation may play a relevant role indetermining resistance to cetuximab. To further validatethis hypothesis and verify that the effect of resistance tocetuximab is correlated to MEK activation, we have usedselective MEK1/2 siRNA. Inhibition of MEK1/2 correlatedwith a partially restored sensitivity to cetuximab in colo-rectal cancer cells resistant to anti-EGFR inhibitors. In fact,MEK silencing restores cetuximab ability to inhibit MAPKand cell proliferation.

Finally, the in vivo experiments on nude mice bearingtumor xenograft models of both primary and acquiredresistance to cetuximab with the combined treatmentwith cetuximab and BAY 86-9766 resulted in tumorgrowth inhibition with consequent increase in micesurvival.

More than 10 selective MEK inhibitors are currently inclinical development (17, 20, 38–42). So far, clinicalefficacy of MEK inhibitors as single agents has beendemonstrated in patients with metastatic melanoma withBRAF-mutated cancers (43), whereas it has been rarelyobserved in patients with unselected chemorefractory

Figure5. InhibitionofMEK1/2 expression restores cetuximabsensitivity inHCT15, HCT116, and colorectal cancer cells. A, HCT15 and HCT116cells were transfected with either specific siRNA targeting MEK1/2or with a control (scrambled) RNA sequence and harvested at 24, 48, 72,and 96 hours after transfection. Western blot analysis for MEK1/2expression was done as described in the Materials and Methods. B,HCT15 and HCT116 cells were transfected with a specific siRNAtargetingMEK1/2. Forty-eight hours after transfection, cells were treatedwith cetuximab, 5 mg/mL. Viable cells were counted after 24 hours oftreatment (after 72 hours of transfection) and plotted relative to untreatedcontrol. The results are average�SDof 3 independent experiments eachdone in duplicate. C, Western blot analysis of cell signaling proteins inHCT15, HCT116, GEO-CR, and SW48-CR cells transfected with aspecific siRNA targeting MEK1/2 or with a scrambled, control siRNA for48 hours and subsequently treated with the indicated dose of cetuximabfor 24 hours (after 72 hours of transfection). Total cell protein extractswere subjected to immunoblotting with the indicated antibodies, asdescribed in the Materials and Methods.






metastatic with other cancer types (44, 45). A recent phaseI study of selumetinib in combination with cetuximab inchemorefractory solid tumors, including KRAS-mutantmetastatic colorectal cancer, has been reported. The com-bination has been well tolerated, with preliminar evi-dence of antitumor activity in patients with metastaticcolorectal cancer with 2 partial responses and 2 diseasestabilization (46). Finally, a phase Ib/II clinical trial iscurrently ongoing in patients with BRAF-mutant meta-static colorectal cancer and it evaluates the combinationof LGX818, a MEK inhibitor, with cetuximab or withBYL719, a PIK3CA inhibitor (47).

Finally, to our knowledge, this study provides the firstexperimental evidence of the synergistic antitumor activityof the combination of cetuximab and a selective MEKinhibitor in cetuximab-resistant colorectal cancer cells witheither primary or acquired resistance in which cetuximabsingle-agent treatment fails to inhibit RAS-RAF-MEK-MAPKintracellular signaling. On the contrary, selective MEK inhi-bition by treatment with BAY 86-9766 significantly blocksMAPK activation, inhibits cell proliferation, and inducesapoptosis in almost all cetuximab-resistant colorectal can-cer cell lines. Finally, treatment of these cells with BAY 86-9766 restores cetuximab sensitivity, suggesting that thecombined inhibition of both EGFR and MEK could repre-sent a rational therapeutic strategy for preventing and/orovercoming cetuximab resistance in patients with metastat-ic colorectal cancer. In this respect, it could be clinicallyrelevant to target the MEK pathway in patients whosetumors develop resistance to anti-EGFR therapies or, even-

tually, to combine MEK1/2 inhibitors and anti-EGFR drugssuch as cetuximab to delay the emergence of resistance.

Disclosure of Potential Conflicts of InterestF. Ciardiello is a consultant/advisory board member for Bayer and Merck

Serono.No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: T. Troiani, S. Napolitano, A. Nappi, F. Ciardiello,E. MartinelliDevelopment of methodology: T. Troiani, D. Vitagliano, A. Capasso, A.Nappi, F. Ciardiello, E. MartinelliAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): T. Troiani, F. Morgillo, A. Nappi, D. Ciardiello, E.MartinelliAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): T. Troiani, F. Morgillo, A. Capasso, V.Sforza, A. Nappi, D. Ciardiello, F. Ciardiello, E. MartinelliWriting, review, and/or revision of the manuscript: T. Troiani, S. Napo-litano, V. Sforza, A. Nappi, F. Ciardiello, E. MartinelliAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): T. Troiani, D. Vitagliano, A. Nappi,F. Ciardiello, E. MartinelliStudy supervision: T. Troiani, F. Ciardiello, E. Martinelli

Grant SupportThis work was supported by a grant from Associazione Italiana per la

Ricerca sul Cancro (AIRC) and a grant from Ministero dell’ Istruzione,Universit�a e Ricerca (MIUR)-PRIN 2010–2011.

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 13, 2013; revised March 29, 2014; accepted April 17,2014; published OnlineFirst May 8, 2014.

Control Cetuximab BAY 86-9766 Combination

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Figure6. Effects of cetuximab incombinationwith theselectiveMEK1/2 inhibitor BAY86-9766onHCT15,HCT116,GEO-CR, andSW48-CR tumor xenografts.The indicated cancer cell lines were grown as subcutaneous tumor xenografts in nude mice. After tumor establishment (200–300 mm3), mice were treatedwith cetuximab (1 mg/dose twice a week intraperitoneally) and/or BAY 86-9766 (5 mg/kg/daily oral gavage) for 3 weeks. Animals were sacrificed whentumors achieved 2,000 mm3 in size. Each group consisted of 10 mice. For each colorectal cancer cell lines, the left graph shows tumor xenograft growth andthe right graph shows mice survival. ��, P < 0.0001.

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2014;20:3775-3786. Published OnlineFirst May 8, 2014.Clin Cancer Res Teresa Troiani, Stefania Napolitano, Donata Vitagliano, et al. and Can Be Overcome by Combined MEK/EGFR InhibitionAnti-EGFR Antibodies Converge on MEK/ERK Pathway Activation Primary and Acquired Resistance of Colorectal Cancer Cells to

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