HER-Family Ligands Promote Acquired Resistance to ...Cancer Biology and Translational Studies HER-Family Ligands Promote Acquired Resistance to Trastuzumab in Gastric Cancer A€ da

Cancer Biology and Translational Studies

HER-Family Ligands Promote AcquiredResistance to Trastuzumab in Gastric CancerA€�da Sampera1, Francisco Javier S�anchez-Martín1, Oriol Arpí1, Laura Visa2,

Mar Iglesias3,4, Sílvia Men�endez1, �Elisabeth Gaye5, Alba Dalmases1, Sergi Clav�e1,3,Mariona Gelabert-Baldrich1, Thomas Tuxen Poulsen6, Michael Kragh6,Beatriz Bellosillo1,3, Joan Albanell1,2,7, Ana Rovira1,2, and Clara Montagut1,2,7

Abstract

Despite the clinical benefit of trastuzumab, eventually allHER2-amplified gastric cancer tumors developdrug resistance.We aimed to identify molecular mechanisms of acquiredresistance to trastuzumab in gastric cancer by using well-established cell line–based preclinical models, as well assamples from patients with HER2-positive gastric cancer trea-ted with trastuzumab. We studied trastuzumab resistance inNCI-N87 and OE19, two gastric cancer cell lines that over-express HER2 receptor and are trastuzumab sensitive. Differ-ences at protein, DNA, and RNA levels between the parentaland resistant cells were characterized and functional studieswere performed. Paired pre- and post-trastuzumab blood andtissue samples from patients with gastric cancer treated withtrastuzumab were analyzed. We found that resistant cellswere associated with increased activation of MAPK/ERK andPI3K/mTOR pathways driven by SRC activation. Upstream,

resistant cells showed increased coexpression of multipleHER-family ligands that allowed for compensatory activationof alternative HER receptors upon HER2 blockade. Simulta-neous inhibition of EGFR, HER2, and HER3 by the novelantibody mixture, Pan-HER, effectively reverted trastuzumabresistance in vitro and in vivo. Similarly, an increase inHER-family ligands was observed in serum and tumor frompatients with gastric cancer after trastuzumab therapy. Wepropose that trastuzumab resistance in gastric cancer is medi-ated by HER-family ligand upregulation that allows acompensatory activation of HER receptors and maintainsdownstream signaling activation despite trastuzumab therapy.Resistance is reverted by simultaneous inhibition of EGFR,HER2, and HER3, thereby revealing a potential therapeuticstrategy to overcome trastuzumab resistance in patients withgastric cancer.

IntroductionGastric cancer is the fifth most common cancer worldwide and

the third leading cause of cancer-related cell death (1). At diag-nosis, most patients present inoperable locally advanced ormetastatic disease, with a median overall survival below 1year (2).

Approximately 20%of gastric cancer tumors, depending on thesubtype of the tumor, overexpress the HER2 (2–4). HER2 is a

member of the HER-family of receptors and a recognized keytherapeutic target in breast and gastric cancer. It is associated withtumor cell proliferation, survival, angiogenesis, migration, and itcorrelates with a poor outcome and amore aggressive disease (5).In the ToGA trial, the addition of trastuzumab, a mAb against theHER2 receptor, to chemotherapy improved overall survival (OS)of patients with HER2-positive gastric cancer in 4.2 months (2).This led to the approval of trastuzumab in combination withcisplatin plus fluoropyrimidine as first-line treatment for HER2-positive advanced gastric cancer or gastro-esophageal junctionadenocarcinoma (6). Trastuzumab inhibits tumor growthmainlythrough prevention of HER2 homodimerization and blockade ofHER2 downstream signaling (7), inhibition ofHER2 extracellularamino-terminal domain (ECD) shedding (8), and induction ofantibody-dependent cellular cytotoxicity (ADCC; refs. 9, 10).

Despite the significant survival benefit of trastuzumab inpatients with gastric cancer, drug resistance invariably devel-ops (2). Identification of molecular mechanisms of acquiredresistance to trastuzumab in gastric cancer is crucial to delineatemore effective therapeutic strategies in the clinical setting.Mechanisms of resistance to trastuzumab in gastric cancer arepoorly characterized. Preclinical evidence suggests alternativeactivation of downstream signaling pathways, such as overexpres-sion of FGF 3 (11). Recently, next-generation sequencing analysisrevealed secondary alterations in ERBB2 as well as mutations inRAS/PI3K downstream signaling pathways in post-trastuzumabsamples from patients with gastro-esophageal junction adeno-carcinoma (12). Remarkably, these molecular alterations only

1Cancer Research Program, Institut Hospital del Mar d'Investigacions M�ediques(IMIM)-CIBERONC, Barcelona, Spain. 2Department of Medical Oncology, Hos-pital del Mar-CIBERONC, Barcelona, Spain. 3Department of Pathology, Hospitaldel Mar, Barcelona, Spain. 4Universitat Autonoma de Barcelona, Spain. 5Onco-logie M�edicale, Institut de C�ancerologie et H�ematologie, Morvan, Brest, France.6Symphogen A/S, Ballerup, Denmark. 7Universitat Pompeu Fabra, Barcelona,Spain.

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

Current address for M. Gelabert-Baldrich: Centre for Genomic Regulation, TheBarcelona Institute for Science and Technology, Barcelona, Spain.

Corresponding Author: Clara Montagut, Del Mar University Hospital, PasseigMaritim 25–29, Barcelona 08003, Spain. Phone: 34-9-3248-3137; Fax: 34-9-3248-3366; E-mail: [email protected]

Mol Cancer Ther 2019;18:2135–45

doi: 10.1158/1535-7163.MCT-19-0455

�2019 American Association for Cancer Research.

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explain trastuzumab resistance in a subset of patients. In thisstudy, we aimed to explore additional mechanisms of acquiredresistance to trastuzumab in gastric cancer by using a well-established cell line–based preclinical model, as well as clinicalsamples from gastric cancer patients treated with trastuzumab.

Material and MethodsCellular models and reagents

The human gastric cancer cell line NCI-N87 was purchasedfrom ATCC and the OE19 and OTR6 (OE19 derived trastuzumabresistant) cells were kindly provided by Symphogen A/S (13, 14).All cell lines wereMycoplasma free and authenticity was tested bySTR DNA Profiling analysis at the ATCC (October 2014) afterresistant cells were generated. The number of passages betweenthawing and use in the described experiments was 10 or less. Cellswere grown as reported previously (15). Trastuzumab (Herceptin,Genentech, Roche) was obtained from the Hospital del Marpharmacy. Everolimus (RAD001), pimasertib (AS-703026;ref. 16), and saracatinib (AZD0530; ref. 17) were purchased fromSelleckchem, and Pan-HER from Symphogen A/S (13). Humanrecombinant EGF was from Calbiochem (Merck KGaA) andneuregulin (NRG1) from Sigma Aldrich Co (Merck KGaA).

Generation of trastuzumab-resistant cellsNCI-N87 cells were cultured in the presence of 15 mg/mL of

trastuzumab until cells were considered resistant (7 months;cell viability reduction �20%; refs. 18, 19). Single-cell–derivedresistant clones were isolated from the resistant pools using8 mm � 8 mm diameter cloning cylinders (Millipore, MerckKGaA) and further propagated for 3 more months in mediacontaining 15 mg/mL trastuzumab (18, 20, 21). Trastuzumabresistance was validated after a cycle of freezing and thawing andafter drug withdrawal up to 6 months (22). Parental cells werecultured without trastuzumab to exclude that resistance was dueto long time culturing.

Cellular proliferation assayTo analyze the growth inhibitory effect of trastuzumab, alone

or in combination with pimasertib, everolimus, or ligands, orperform dose curves, with trastuzumab, Pan-HER, cetuximab, orpertuzumab, 8–12 � 103 cells per well in a 24-well plate weretreated for 7 days. Cells were stained for 1 hour with crystal violetsolution at 0.1%. Quantification was evaluated with ImageJsoftware. To perform growth curve experiments with lapatinib,we plated 5–7 � 103 cells per well in 96-well plate. Cell viabilitywas obtained using CellTiter 96 Aqueous One Solution CellProliferation Assay (MTS) following the manufacturer's proce-dures. IC50 values were calculated using CalcuSyn software. Eachexperiment was performed at least three times and results wereplotted as percentage of the control.

Trastuzumab binding by flow cytometryWe measured trastuzumab binding to HER2 receptor as

reported previously (15), incubating cells with trastuzumab100 ng/mL as the primary antibody.

FISHTo assess ERBB2 copy number alterations, we applied the

PathVysion HER-2 DNA FISH Probe Kit (Abbott Molecular Inc).FISH was performed in fixedmaterial obtained from the cell lines

after application of a conventional cytogenetic protocol. About100 nonoverlapping cells with hybridization signals were exam-ined for each case and amplificationwas defined as ERBB2/CEP17ratio �2.0 (23).

ImmunocytochemistryTo study HER2 protein expression, 1 � 107 of cells were

pelleted, fixed with formalin, and embedded with paraffin(FFPE). HER2 expression was evaluated using HercepTest. Sec-tions of 3 mmwere placed on plus charged glass slides. The HER2antibody used was PATHWAY anti-HER-2/neu (4B5) rabbitmonoclonal primary antibody (Ventana Medical Systems, Inc.)and revealed with ultraView Universal 3,30-diaminobenzidine(DAB) Detection Kit (Ventana Medical Systems, Inc.) as thechromogen and counterstained with hematoxylin. HER2 stainingwas performed and evaluated in the Pathology Department ofHospital del Mar according to NCCN Guidelines (24).

Protein detectionWhole-cell lysates were subjected to Western blot analyses as

reported previously (20). The primary antibodies used were:EGFR, HER3, phosphoERK1/2 (T202/Y204), ERK1/2, phos-phoAKT (S473), AKT, phosphoS6 (S235/236), S6, phosphoSRC(Y416), and SRC purchased from Cell Signaling Technology;HER2 from BioGenex (clone CB11); a-Tubulin and b-Actin fromSigma-Aldrich Co (Merck KGaA).

Phospho-receptor tyrosine kinase arrayWe screened the activity of different receptor tyrosine kinase

(RTK) and signaling nodes using PathScan RTK Signaling Anti-body Array Kit (Chemiluminescent Readout) catalog no. 7982 ofCell Signaling Technology following the manufacturer's proce-dures. Image acquisition was done with the Typhoon ScannerControl v5.0 software and quantified with the ImageQuant TLv7.0. The digitalization part was done at Centre for GenomicRegulation in theParc deRecerca Biom�edica deBarcelona (PRBB).

Gene expression microarrayRNA isolation and gene expressionmicroarray were performed

using the Human Gene 2.0 ST Array GeneChip (Affymetrix) asdetailed in Supplementary Material and Methods.

qRT-PCRTotal RNA was extracted from cells in basal conditions and

treated with trastuzumab 15 mg/mL and Pan-HER 10 mg/mL for24hours. cDNAwas synthetized using randomprimers andHigh-Capacity cDNA Reverse Transcription Kit (Applied Biosystems).Samples from three independent RNA extractions were amplifiedusing specific primers and LightCycler 480 SYBR Green I Master(Roche) in the LightCycler 480 Real Time PCR-System device(Roche). The primers used were: EGF Forward: 50-CGTGTCGT-GAAGGTTTTATG-30 and EGF Reverse: 50-GTTCTTTAGAT-CAACTTCACC-30; AREG Forward: 50-GCATGATTGACAGTAGTT-TATC-30 and AREG Reverse: 50-TTCTAAGCTGGACTGTAATAAC-30; TGFA Forward: 50-TGTGTCTGCCATTCTGGGTA-30 and TGFAReverse 50-GACCTGGCAGCAGTGTATCA-30; HBEGF Forward:50-GGAGAATGCAAATATGTGAAG-30 and HBEGF Reverse: 50-TTCCACTGGGAGGCTCAG-30; NRG1 Forward: 50-CAGCCCAA-GAGCCTGTTAAG-30 andNRG1 Reverse: 50-ACTGCTCTGGGAG-CTTGTGT-30; and ATP5E Forward: 50-GATCTGGGAGTATCG-GATG-30 and ATP5E Reverse: 50-CCGGCGTCTTGGCGATTC-30.

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Gene expressionwas calculated as 2 to the power of�DDCt, whereDDCt ¼ ðCtGene � CtATP5E or ACTIN Þ Assay.

ELISA analysisCell culture medium from 1–5 � 106 cells plated in 100 mm

culture dishwith 10mLofmedium for 72hourswas concentratedusing Amicon Ultra-4 Centrifugal Filter Units (Millipore, MerckKGaA) following the manufacturer's procedures. Ligands' levelsin the medium were measured using the Human EGF DuoSet#DY236, Human Amphiregulin DuoSet #DY262, and HumanTGF-alpha DuoSet #DY239 ELISA (R&D Systems) according tothe manufacturer's procedures.

Subcutaneous tumorigenesisFive-week-oldmale FoxChase SCIDBeigemicewere purchased

from Charles River Laboratories and hosted in the pathogen-freeanimal facility at the PRBB. Animal treatments were doneaccording to institution-approved protocols. A total of 5 � 106cells were resuspended in sterile PBS with 50% of Matrigel (BDBiosciences) and subcutaneously injected into the flank of themice. Tumor volume was determined twice a week from calipermeasurements of tumor length (L) and width (W) according tothe formula (L �W2 � 3.1416)/6. When tumor volume reached200–300 mm3, mice were randomized to three groups with 10mice in each one. IgG isotype control, trastuzumab (20 mg/kg),and Pan-HER (60 mg/kg) were administered intraperitoneallythree times a week until significant effect on tumor growth wasobserved of the treatment versus control or until tumor volumewas too large, for ethical reasons (30 days for parental and 85daysfor resistant cells). Mice were euthanized and part of the tumorswas FFPE for IHC studies.

IHCParaffin sections were used for IHC analysis as described in

Immunocytochemistry section. Tumor sections were stained withhematoxylin and eosin (H & E) to assess histologic changes. Toevaluate proliferation and apoptosis, slices were incubated withthe antibodies Ki-67 (clone MIB-1, Dako, Agilent Technologies)mouse mAb at 1:100, anti-phosphorylated (Ser10) Histone 3(Cell Signaling Technology) rabbit polyclonal antibody at 1:100,and active caspase-3 [clone ASP175 (5A1), Cell Signaling Tech-nology] rabbit monoclonal antibody at 1:100. Antigen–antibodyreaction was detected by incubation with an anti-mouse/rabbitIg–dextran polymer coupled with peroxidase (Flexþ, Dako, Agi-lent Technologies). Sections were then visualized with DAB aschromogen and counterstainedwith hematoxylin. H& E and IHCstaining was performed and evaluated in the Pathology Depart-ment of Hospital del Mar. Data were presented as the percentageof positive tumor cells.

Patient samplesBlood samples and tumor specimens frompatients withHER2-

positive gastric cancer and gastro-esophageal junction adenocar-cinoma that received trastuzumab and chemotherapy treatmentwere collected at diagnosis (pre-trastuzumab) and after trastuzu-mab therapy (post-trastuzumab) at Hospital del Mar. Ligandlevels of serum samples were measured by ELISA as described inELISA analysis. In FFPE tumor tissue samples, RNA was extractedusing the MagMAX FFPE DNA/RNA Ultra Kit catalog no. A31881(Thermo Fisher Scientific) and EGF expression analysis was per-formed as described in qRT-PCR section.

Statistical analysisStatistical analyseswere carriedout usingone-wayANOVA tests

followed by post hoc Tukey adjustment or Student t test. Prism 5.0software (GraphPad) was used for the statistical analyses. Datashown is mean � SD of three independent replicates.

ResultsGeneration and characterization of trastuzumab-resistantgastric cancer cells

To identify potential mechanisms of acquired resistance totrastuzumab, we performed an extensive screening on gastriccancer cell lines that could be used to generate resistance totrastuzumab and mimic the clinical setting. The requirementswere: gastric cancer adenocarcinoma, HER2 receptor amplifica-tion and overexpression, and high sensitivity to trastuzumab.Only two cell lines (NCI-N87 and OE19) fulfilled these require-ments. NCI-N87 cells were selected to generate single-cell trastu-zumab-resistant derivatives and characterize the resistance(Fig. 1A and B). Then results were confirmed in the OTR6 cells(OE19-derived trastuzumab resistant). Differences in cell mor-phology were observed between parental and resistant cells,showing the latter a flatter morphologic appearance (Fig. 1B).To explore potential mechanisms of resistance, we performednext-generation sequencing to analyze hotspot regions of a panelof genes commonly altered in cancer and related to HER2 sig-naling, including ERBB2, EGFR, KRAS, NRAS, BRAF, and PIK3CA(listed in Supplementary Material and Methods). We did notidentify the emergence of mutations in these genes in resistantcompared with parental cells.

Trastuzumab-resistant cells grow independently of HER2To exclude the role of HER2 in the resistant phenotype, we

determined whether there were substantial differences in HER2receptor amplification, expression, and activation in resistantcompared with parental cells. Trastuzumab-resistant cells main-tained ERBB2 gene amplification similar to parental cells, asassessed by FISH analysis (Fig. 1B). Moreover, genomic analysisdid not detect pointmutations in ERBB2 gene. IHC showedHER23þ immunoscore in both parental and resistant cells, although asmall reduction in protein staining was observed in resistantcompared with parental cells that was confirmed byWestern blotanalysis (Figs. 1B, 2A, 3E, and 4B).

The HER2 inhibitor lapatinib did not affect the viability ofresistant cells compared with parental cells (Fig. 1C; Supplemen-tary Fig. S1), suggesting that resistant cells were no longer depen-dent on HER2 for their growth and survival. Although we hadexcluded the role of a secondary mutation in ERBB2 gene as amechanism of resistance, it remained possible that reduced drug-receptor binding affinity was involved in resistance (21, 25). Totest whether trastuzumab was actively recognizing the cell surfaceof HER2, we performed flow cytometry analysis which revealedthat trastuzumab was binding to 100% of the cells in both theparental and resistant cells (Fig. 1D).

Resistance to trastuzumab is associated with SRC-mediatedactivation of MAPK/ERK and PI3K/AKT pathways

To explore the role of HER2 cell signaling in resistance, wecharacterized HER2 downstream effectors in parental and resis-tant cells. Resistant cells had an increase in basal levels of SRC,ERK 1/2, and S6 phosphorylation, which was confirmed by a

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phospho-array analysis (Fig. 2A and B; Supplementary Fig. S2). Asexpected, trastuzumab treatment reduced phosphorylation ofHER2 downstream proteins SRC, AKT, ERK 1/2, and S6 inparental cells. However, HER2 downstream effectors persistedactivated in the resistant cells (Fig. 2A).

To establish whether the MAPK/ERK and PI3K/mTOR path-ways played a role in maintaining survival and cell growth in theresistant cells, functional pharmacologic studies were performedwith the MEK inhibitor pimasertib and the mTOR inhibitoreverolimus. Each drug effectively targeted ERK and S6, respec-tively, and partially inhibited cell viability of resistant cells. Ofnote, the addition of trastuzumab had no additional effect,

supporting the limited role of HER2 in driving resistance(Fig. 2C; Supplementary Figs. S3, S4A and S4B). Simultaneousinhibition of both ERK and PIK3 pathways by the combination ofpimasertib and everolimus resulted in higher cell viability inhi-bition compared with each drug alone, suggesting that bothMAPK/ERK and PIK3/mTOR pathways were critical in maintain-ing cell growth and survival in the resistant cells (Fig. 2C; Sup-plementary Fig. S3).

To assess whether persistent activation of MAPK/ERK andPI3K/mTOR pathways in resistant cells was driven by the com-mon upstream activator SRC, we treated resistant cells with thesmall molecule SRC kinase inhibitor saracatinib. Saracatinib

Figure 1.

Proliferation of trastuzumab-resistant cells is independent of HER2. A, Cell viability analyzed by staining the cells with crystal violet after treating the cells withtrastuzumab (15 mg/mL) for 7 days. �� , P < 0.001. B, Representative images of parental and resistant cells under light microscopy (top images). ERBB2amplification by FISH: ERBB2 gene in red and centromere of chromosome 17 (CEP17) in green (middle images). HER2 overexpression by IHC (bottom images).C,Dose curve of lapatinib by MTS assay after 72 hours. IC50 values of lapatinib: IC50 parental cells¼ 0.26 mmol/L (SD 0.09); IC50 values for resistant cells notreached. ��, P < 0.001. Molecular effects in Supplementary Fig. S1. D, Histogram representing mean fluorescence intensity of trastuzumab-PE (Tz-PE)-expressing cells corresponding to 10,000 events. Parental and resistant cells (in dark and light gray, respectively) were incubated with trastuzumab (100 ng/mL)and a goat anti-human IgG conjugated with phycoerythrin (PE) as secondary antibody.

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significantly decreased cell viability in the resistant cells (Fig. 2C;Supplementary Fig. S3), which was correlated with effectivesuppression of HER2 downstream effectors (SupplementaryFig. S4C). Of note, because SRC mutations have been shown todrive lapatinib resistance inagastric cancerpreclinicalmodel (26),we sequenced the whole SRC gene. SRC mutations were neitherdetected in parental nor resistant cells.

Taken together, these results strongly suggested that sustainedactivation of both MAPK/ERK and PI3K/mTOR pathways medi-ated by SRC was critical to the maintenance of cell survival inresistant cells.

MultipleHER-family ligands areoverexpressed in trastuzumab-resistant cells

To explore upstreammechanismsof SRC-mediatedMAPK/ERKand PI3K/mTOR pathways activation in the trastuzumab-resistant cells, we analyzed expression of HER-family receptorsand related ligands. Gene expression array showed no significantdifferences in basal expression of total HER receptors in resistantcompared with parental cells. Following trastuzumab therapy, atrend toward an increase in total EGFR, ERBB3, and ERBB4 wasobserved compared with parental lines (Fig. 3A). Strikingly,HER-family ligands including epidermal growth factor (EGF),

amphiregulin (AREG), transforming growth factor alpha (TGFA),heparin-binding EGF-like growth factor (HBEGF), andneuregulin1 (NRG1) were significantly overexpressed in resistant comparedwith parental cells (Fig. 3A and B). In addition, increased levels ofAREG and TGFa were detected in the cell culture medium ofresistant cells compared with parental cells (Fig. 3C). NRG1 andEGF could not be assessed in medium because of a very low limitof detection. Hence, we hypothesized that simultaneous over-expression of several HER-family ligands allowed for a compen-satory effect to maintain the activation of downstream signalingpathways upon HER2 blockade in the resistant cells.

HER-family ligand overexpression protects from trastuzumabinhibition

To explore whether HER-family ligands were able to inducetrastuzumab resistance, we exposed the parental cells to highconcentrations of ligands. Incubationof parental cellswith EGForNRG1 stimulated cell growth (Fig. 3D). The addition of eitherligand reduced the inhibitory effect of trastuzumab (Fig. 3D;Supplementary Fig. S5). Accordingly, biochemical analysisrevealed an activation of HER downstream effectors followingligand stimulation, which was not completely abrogated by theaddition of trastuzumab (Fig. 3E).

Figure 2.

Resistance to trastuzumab is driven by activation of SRC and downstream MAPK and PIK3 signaling. A, Biochemical analysis of HER2 and downstream effectorsin basal conditions and following trastuzumab therapy. Cells were cultured in the presence of trastuzumab (15 mg/mL) for 24 hours. Immunoblots wereperformed using antibodies to the indicated proteins. B, Cell lysates of parental and resistant cells in basal conditions were incubated with a phospho-array.Antibodies against RTK and signaling nodes are spotted in duplicates. Gray squares indicate the most relevant changes in intensity. Quantification of thephospho-array is shown in Supplementary Fig. S2. C, Cell viability was analyzed by staining the cells with crystal violet after treating the cells with the indicateddrugs for 7 days. �� , P < 0.01; �� , P < 0.001. Representative images are shown in Supplementary Fig. S3 andmolecular effects in Supplementary Fig. S4.






To evaluate whether simultaneous complete blockade of allmembers of the HER-family of receptors could prevent ligand-induced downstream activation, we used Pan-HER: a novel anti-body mixture comprised of synergistic pairs of antibodies target-ing EGFR, HER2, and HER3 in nonoverlapping epitopes (13).Pan-HER significantly reduced cell viability of parental cells evenin the presence of ligands (Fig. 3D; Supplementary Fig. S5) andthis was correlated with effective inhibition of EGFR, HER2,HER3, and downstream signaling in biochemical analysis specif-ically pAKT/pS6/pERK for EGF and pAKT/pS6 for NRG1 (Fig. 3E).

Simultaneous inhibition of EGFR, HER2, and HER3 by Pan-HER overcomes acquired resistance to trastuzumab in vitro

Because Pan-HER was blocking HER-family downstream acti-vation even in the presence of ligands, we aimed to explorewhether Pan-HER could revert trastuzumab acquired resistancein our model. Notably, Pan-HER significantly reduced cell via-bility of trastuzumab-resistant cells and this was correlated with asuccessful depletion of EGFR, HER2, and HER3 as well as aninhibition of the downstream effectors (Fig. 4; SupplementaryFig. S7). To study whether triple blockade of EGFR, HER2, andHER3 was necessary to overcome trastuzumab resistance, wetreated resistant cells with specific EGFR (cetuximab) orHER2/HER3 (pertuzumab)-targeted therapies. Neither cetuxi-

mab nor pertuzumab were able to revert trastuzumab resistance(Fig. 4A; Supplementary Fig. S7). Such results, together with thesimultaneous upregulation of several HER-family ligands (Fig. 3BandC) confirmed that compensatory ligand-induced coactivationof alternative HER-family members is involved in driving resis-tance to trastuzumab. Therefore, complete and simultaneousblockade of all HER-family members is necessary to revert tras-tuzumab resistance in our model.

Mechanisms of resistance to trastuzumab are confirmed inanother preclinical model

Remarkably, results were confirmed in the OTR6-resistant cellsderived from the trastuzumab-sensitive HER2-amplified OE19cell line. Trastuzumab was less effective in reducing ERK 1/2 andS6 phosphorylation in OTR6-resistant cells compared with theparental cells (Supplementary Fig. S8A). In addition, NRG1,AREG, and EGF ligands were overexpressed in OTR6-resistantcells (Supplementary Fig. S8B), and a high increase in NRGexpression was observed upon trastuzumab treatment in OE19parental cells (Supplementary Fig. S8C). Similar toNCI-N87 cells,EGF and NRG1 stimulation reduced trastuzumab growth inhib-itory rate inOE19parental cells, and simultaneous blockade of allHER-family of receptors by Pan-HER was able to prevent ligand-induced downstream activation (Supplementary Fig. S9A and

Figure 3.

Resistant cells overexpress multiple HER-family ligands, and HER-family ligand overexpression protects from trastuzumab inhibition. A, Heatmap representationof 15 selected gene expressions. Columns represent the mean of the intensities in the groups of triplicates parental control, parental trastuzumab, resistantcontrol, and resistant trastuzumab. Cells were cultured in the presence of trastuzumab (15 mg/mL) for 48 hours. Columns are clustered with ward. D2 method androws are clustered with averagemethod using in both cases correlation distances. Color intensity means degree of gene expressionmodulation. B, Analysis ofindicated gene expression by qRT-PCR. Gene expression levels were normalized to ATP5E as the housekeeping gene. Resistant cell data were normalized to therespective parental cell expression level (set at 1, dotted line). ��, P < 0.001. C, Levels of AREG and TGFa protein in cell culture medium of resistant cellsnormalized to the respective parental levels (set at 1, dotted line) measured by ELISA. � , P < 0.05. D, Cell viability was analyzed by staining the cells with crystalviolet after treating the cells with trastuzumab (15 mg/mL), Pan-HER (10 mg/mL), EGF (1 nmol/L), or NRG1 (5 nmol/L) during 7 days. �, P < 0.05 and �� , P < 0.001.Representative images in Supplementary Fig. S5. E, Biochemical analysis of HER-family receptors and downstream effectors following ligand stimulation. Cellswere cultured in the presence of trastuzumab (15 mg/mL) or Pan-HER (10 mg/mL) for 24 hours, followed by 15 minutes with EGF (1 nmol/L) or NRG1 (5 nmol/L).Immunoblots were performed using antibodies to the indicated proteins.

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S9B). At amolecular level, Pan-HERhad amore profound effect inreducing EGFR, HER2, HER3, and downstream effectors phos-phorylation compared with trastuzumab, even in the presence ofligands (Supplementary Fig. S9C). Moreover, Pan-HER signifi-cantly reduced cell viability of OTR6 trastuzumab–resistant cellsand this was correlated with a successful depletion of EGFR,HER2, and HER3 and inhibition of the downstream effectors(Supplementary Fig. S10).

Triple inhibition of EGFR, HER2, and HER3 by Pan-HERovercomes trastuzumab resistance in vivo

To expand our studies to in vivomodels, the Pan-HER antibodymixture was tested in parental and trastuzumab-resistant–derivedxenografts. As expected, in parental xenografts both trastuzumaband Pan-HER significantly reduced tumor growth compared withthe control group (Fig. 5A). In resistant xenografts, resistance totrastuzumab was observed after 1.5 months of treatment, whileno tumor growth was observed under treatment with Pan-HERduring 3months (Fig. 5B).H&E staining of parental and resistantxenografts showed that Pan-HER–treated tumors were smallerwith less percentage of ischemic necrosis and more hyalinizedfibrosis compared with control or trastuzumab-treated tumors(Fig. 5C; Supplementary Table S1A). IHC analysis showed lowerstaining percentage of the proliferative markers Ki-67 and phos-

phorylated (S10) Histone H3 (pHist H3) in Pan-HER–treatedtumors compared with control or trastuzumab group (Fig. 5C;Supplementary Table S1B). Staining of the apoptosis markercleaved caspase 3 (c-casp 3) did not totally reflect Pan-HERsuperiority, probably because Pan-HER–induced apoptosisoccurred previous to mice sacrifice (Fig. 5C; SupplementaryTable S1B). Altogether, histologic analysis confirmed a higherPan-HER antitumor efficacy compared with control and trastu-zumab effect.

HER-family ligands are increased in clinical samples frompatients with gastric cancer treated with trastuzumab

To explore whether HER-family ligands were involved in tras-tuzumab resistance in patients, we analyzed pre-trastuzumaband post-trastuzumab paired samples from 5 patients withHER2-positive gastric cancer. Clinical characteristics of thepatients are summarized in Supplementary Table S2. Of note,rebiopsy and collection of post-trastuzumab samples was limitedbecause of patient fragility and difficulty in tumor access. Anincrease in median EGF, AREG, and TGFa concentration wasobserved in post-trastuzumab compared with pre-trastuzumabserum samples (Fig. 6A; Supplementary Fig. S11A). In tissuesamples, EGF expression was 2.7 times higher in post-trastuzumab tumor biopsy compared with pre-treatment tumor

Figure 4.

Simultaneous inhibition of EGFR, HER2, and HER3 is necessary to inhibit cell growth in trastuzumab-resistant cell lines. A, Cell viability analyzed by stainingresistant cells with crystal violet. Cells were treated with trastuzumab, cetuximab, pertuzumab, and Pan-HER dose curves for 7 days. IC50 values of resistant cells:IC50 Pan-HER¼ 26.20 mg/mL (SD 0.96); IC50 values of trastuzumab, cetuximab, or pertuzumab not reached. �� , P < 0.01; �� , P < 0.001. Representative imagesare shown in Supplementary Fig. S7. B, Biochemical analysis of HER-family receptors and downstream effectors following trastuzumab and Pan-HER treatment.Cells were cultured in the presence of trastuzumab (15 mg/mL) or Pan-HER (10 mg/mL) for 24 hours. Immunoblots were performed using antibodies to theindicated proteins.






sample (Fig. 6B; Supplementary Fig. S11B). Other markers couldnot be characterized because of insufficient tumor sampleavailability.

DiscussionThe approval of trastuzumab for patients with HER2-positive

gastric cancer represented a breakthrough in the treatment of thisdisease. Unfortunately, responses are transient and resistance totrastuzumab invariably emerges. To study themolecularmechan-isms underlying trastuzumab resistance, we studied two trastu-zumab-resistant cell lines and as proof-of-concept confirmed thefindings in patients' samples. Our preclinical results showed thatacquired resistance to trastuzumab was driven by mRNA increaseof multiple HER-family ligands that allowed compensatory acti-vation of MAPK/PIK3K downstream signaling in the presence of

trastuzumab. The novel antibody mixture Pan-HER effectivelyreverted trastuzumab resistance both in vitro and in vivo.Accordingly, analysis of clinical samples showed an increase inHER-family ligands levels after treatment with trastuzumab.

To our knowledge, there is limited evidence on the role ofHER-family ligands in innate and acquired resistance to trastuzumab inpatients with gastric cancer. Preclinical models have shown apotential involvement inEGF ligandupregulation in trastuzumabresistance in patients with gastric cancer (27, 28). Similarly, inbreast cancer cell lines, ligand-induced activation of HER recep-tors has been linked to trastuzumab resistance (29). Also apotentially broad role of widely expressed receptor-tyrosinekinase ligands has been shown in innate and acquired resistanceto small-molecule tyrosine-kinase inhibitors (30). For the firsttime in gastric cancer, our findings are supported by proof-of-concept data from a small group of patients treated with

Figure 5.

Pan-HER in vivo efficacy on parental and resistant xenograft models.A and B, Effects of trastuzumab and Pan-HER on gastric cancer tumor xenografts. Micewere injected subcutaneously in the dorsal flank with parental or resistant cells. After tumor reached an average tumor size of 200–300mm3, mice were treatedintraperitoneally three times aweek with IgG isotype control, trastuzumab (20mg/kg), or Pan-HER (60mg/kg). Each group consisted of 10 mice. Tumorvolumeswere normalized individually to the volume at the first day of treatment. Pan-HER significantly reduced tumor volume compared with trastuzumab inboth xenografts. �, P < 0.05; �� , P < 0.01; �� , P < 0.001. C, Representative images of H & E staining and immunostaining for Ki-67, pHist H3, or c-casp 3 of parentaland resistant xenograft tumor sections. Scale bar is 5 mm in H & E and 200 mm in IHC images. Red arrows point to representative positive cells in IHC images.

Sampera et al.





trastuzumab, who showed an increase in HER-family ligands inserum and tumor tissue after trastuzumab-based therapy.

Coexpression of multiple RTK and compensatory downstreamsignaling activation has been shown to limit the efficacy of single-target drugs in other cancer types (29, 31–33). Of note, in breastcancer models, long-term treatment with trastuzumab leads tooverexpression of EGFR and HER3, which circumvents HER2inhibition (31). This suggests that inhibition of multiple RTKsis potentially necessary to reach a complete abrogation of redun-dant downstream signaling activation. According to the results inJacobsen and colleagues, simultaneously targeting of EGFR,HER2, andHER3was necessary to prevent trastuzumab resistanceinduced by EGF and NRG1 ligand stimulation and by the com-pensatory upregulation of HER-family of receptors in several celllines (13). Upon receptor binding, Pan-HER induces internali-zation and degradation of EGFR, HER2, and HER3, preventingligand binding to the receptors. Furthermore, in this study weshow that Pan-HER more effectively inhibits cell viability com-pared with trastuzumab alone in parental cells, suggesting theincreased benefit of triple inhibition of RTKs even in HER2-addicted cells. This concept is reinforced in the NCI-N87 trastu-zumab-resistant cells, where single RTK mAbs, cetuximab, tras-tuzumab, or pertuzumab, or dual RTK inhibitor, lapatinib, had alimited effect compared with the more powerful inhibitory effectof Pan-HER. Similar to our data, in breast cancer xenografts withtrastuzumab resistance, Pan-HER, but not trastuzumab emtan-sine (T-DM1) or the combination of trastuzumab with pertuzu-mab or lapatinib, was able to arrest tumor growth (33). In ourmodel, tumors derived from trastuzumab-resistant cells did notcompletely recapitulate the in vitro resistant phenotype after beinginoculated subcutaneously inmice. However, after 1.5months of

treatment, these tumors were able to grow acquiring resistance totrastuzumab. Differences between tumor cells behavior in 2Dcultures in vitro and in vivo exist even in terms of proliferationrate (34). Now it is accepted that there are a number of situationswhere a particular in vitro phenotype can only be reproduced insolid tumors when cells have grown as 3D multicellular tumorspheroids (35, 36). Moreover, resistance can even vary whenresistant xenografts are reimplanted into untreated mice (37). Inour resistant xenograft, trastuzumab resistance is not the result ofa permanent genetic change in the tumor cells, but rather ismediated by HER-family ligands upregulation. For this reason,resistance could be affected by reversible changes that likely tooccur in the tumor and/or its microenvironment. Therefore, itmight explain thedelayed trastuzumab resistance observed.Nota-bly, no tumor growth was observed under treatment with Pan-HER during 3 months indicating the role of HER-family ligandsand receptors in trastuzumab resistance.

In line with our results of downstream signaling activationunder HER-family ligand stimulation, Wilson and colleaguesobserved that EGF preferentially activated the MAPK/ERK path-way, whereas NRG1 mainly mediated PI3K/mTOR pathway acti-vation (30). This suggests that dual activation of ERK1/2 and S6 inour gastric cancer trastuzumab-resistant model may potentiallybe caused by a combination of EGF, NRG1, or other HER ligandsupregulation. Moreover, it supports the concept that extensiveredundancy activation of RTKs signaling is observed in cancercells. RTKs downstream activation including activation of thePI3K/mTOR signaling pathway or the common-node SRC hasbeen linked to both preclinical and clinical resistance toHER2-targeted therapy in different cancer types (12, 26, 38–44).Similarly, in our gastric cancer trastuzumab-resistant model,

Figure 6.

HER-family ligands increase after trastuzumab treatment in clinical samples from patients with HER2-positive gastric cancer. A, Scatter plot of EGF, AREG, andTGFa serum concentration from paired pre- and post-trastuzumab samples from patients with HER2-positive gastric cancer (n¼ 5) measured by ELISA. B, EGFmRNA levels from FFPE tumor tissue from paired pre-trastuzumab and post-trastuzumab samples (n¼ 3) by qRT-PCR (�� , P < 0.01).






MAPK/ERK and PI3K/mTOR activation was mediated byincreased SRC phosphorylation, as a common node downstreamof RTKs ligand-induced activation. Drug inhibition of SRC (orconcomitant inhibition of ERK and PI3K) would therefore be agood therapeutic strategy to revert trastuzumab resistance, asshown in our preclinical cell-culture model. However, small-molecule inhibitors do not trigger the immune system, whereasPan-HER induces ADCC and enhanced complement-dependentcytotoxicity activation similar to trastuzumab (13). Therefore,Pan-HER would be a more powerful clinical therapeutic strategyto overcome trastuzumab resistance taking into consideration theessential role of the immune system in cancer therapy. Pan-HER isa promising candidate not only in reverting resistance to trastu-zumab but also as a targeted therapy against EGFR, HER2, orHER3. Clinical trials with Pan-HER are ongoing.

Taken together, the data presented herein suggests that ligand-induced redundant activation of HER-family of receptors is apotential mechanism of resistance to trastuzumab in gastriccancer, supported by proof-of-concept evidence in a small cohortof patients. Simultaneous inhibition of all members of the HER-family of receptors is therefore necessary to revert or preventtrastuzumab resistance. Potential clinical implications of ourfindings are (i) the need to dynamically evaluate levels of HERligands before, during, and after trastuzumab therapy and (ii) theneed to evaluate Pan-HER as a potential therapeutic strategy toovercome trastuzumab resistance in clinical trials.

Disclosure of Potential Conflicts of InterestB. Bellosillo has received speakers bureau honoraria from Qiagen, Thermo

Fisher Scientific, and Hoffman-LaRoche. J. Albanell has received speakersbureau honoraria from Roche, Amgen, Pfizer, and Novartis, and has consul-tant/advisory board relationships with Roche, Pfizer, and Amgen. C. Montagutreports receiving a commercial research grant from Symphogen, has receivedspeakers bureau honoraria fromRoche and Symphogen, and has an consultant/

advisory board relationship with Symphogen. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: A. Sampera, F.J. S�anchez-Martín, C. MontagutDevelopment ofmethodology: A. Sampera, O. Arpí, T.T. Poulsen, B. Bellosillo,C. MontagutAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Sampera, O. Arpí, L. Visa, M. Iglesias, S. Clav�e,M. Gelabert-Baldrich, B. Bellosillo, C. MontagutAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Sampera, F.J. S�anchez-Martín, O. Arpí, L. Visa,A. Dalmases, T.T. Poulsen, B. Bellosillo, C. MontagutWriting, review, and/or revision of the manuscript: A. Sampera, F.J. S�anchez-

Martín, L. Visa, �E. Gaye, T.T. Poulsen, M. Kragh, B. Bellosillo, J. Albanell,A. Rovira, C. MontagutAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Men�endez, S. Clav�e, M. Gelabert-Baldrich,M. Kragh, J. Albanell, C. MontagutStudy supervision: F.J. S�anchez-Martín, A. Rovira, C. Montagut

AcknowledgmentsThis work was supported by ISCiii (CIBERONC CB16/12/00481, PIE15/

00008, PI18/00006, and PI18/00323), Generalitat de Catalunya (2017 SGR507), andAECC (11964). C.Montagutwas supported byDepartament de Salut,Generalitat deCatalunya (PERIS SLT006/17/00055).MARBiobanc is supportedby ISCiii/FEDER (PT17/0015/0011) and the "Xarxa de Bancs de tumors"sponsored by Pla Director d' Oncologia de Catalunya (XBTC). We thankFundaci�o Cellex (Barcelona) for a generous donation to Hospital del MarMedical Oncology Service.

The costs of publicationof this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 2, 2019; revised July 28, 2019; accepted August 27, 2019;published first September 4, 2019.

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2019;18:2135-2145. Published OnlineFirst September 4, 2019.Mol Cancer Ther Aïda Sampera, Francisco Javier Sánchez-Martín, Oriol Arpí, et al. in Gastric CancerHER-Family Ligands Promote Acquired Resistance to Trastuzumab

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