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Therapeutics, Targets, and Chemical Biology

Activated ERBB2/HER2 Licenses Sensitivity to Apoptosisupon Endoplasmic Reticulum Stress through aPERK-Dependent Pathway

Rosa Martín-P�erez1, Carmen Palacios1, Rosario Yerbes1, Ana Cano-Gonz�alez1, Daniel Iglesias-Serret2,Joan Gil2, Mauricio J. Reginato3, and Abelardo L�opez-Rivas1

AbstractHER2/Neu/ERBB2 is a receptor tyrosine kinase overexpressed in approximately 20% of human breast tumors.

Truncated ormutant isoforms that show increased oncogenicity compared with the wild-type receptor are foundinmany breast tumors. Here, we report that constitutively active ERBB2 sensitizes human breast epithelial cells toagents that induce endoplasmic reticulum stress, altering the unfolded protein response (UPR) of these cells.Deregulation of the ERK, AKT, and mTOR activities elicited by mutant ERBB2 was involved in mediating thisdifferential UPR response, elevating the response to endoplasmic reticulum stress, and apoptotic cell death.Mechanistic investigations revealed that the increased sensitivity of mutant ERBB2-expressing cells to endo-plasmic reticulum stress relied upon a UPR effector signaling involving the PERK–ATF4–CHOP pathway,upregulation of the proapoptotic cell surface receptor TRAIL-R2, and activation of proapoptotic caspase-8.Collectively, our results offer a rationale for the therapeutic exploration of treatments inducing endoplasmicreticulum stress against mutant ERBB2-expressing breast tumor cells. Cancer Res; 74(6); 1766–77. �2014 AACR.

IntroductionIn response to different environmental and physiologic

stress conditions that increase the load of unfolded proteinsin the endoplasmic reticulum, protein sensors located in theluminal face of the endoplasmic reticulummembrane activatethe unfolded protein response (UPR; ref. 1). Activation of thissignaling pathway leads to a reduction in the influx of proteinsinto the endoplasmic reticulum, activates protein degradationpathways, and increases the folding capacity of the endoplas-mic reticulum (2). In vertebrates, 3 different types of endo-plasmic reticulum stress transducers have been identified.Each type defines a distinct branch of the UPR that is mediatedby protein kinase RNA–like endoplasmic reticulum kinase(PERK; ref. 3), inositol-requiring protein-1 (Ire1; ref. 4), or

activating transcription factor-6 (ATF6; ref. 5). In each case,an integral membrane protein senses the protein-folding sta-tus in the endoplasmic reticulum lumen and transmits thisinformation across the endoplasmic reticulum membrane tothe cytosol and nucleus (2). These mechanisms allow adaptiveand repair mechanisms that re-establish homeostasis. How-ever, above a certain threshold, unresolved endoplasmic retic-ulum stress results in apoptosis (6).

As a major regulator of cell growth, metabolism, and sur-vival, the mTOR pathway is commonly activated during onco-genesis (7). Thus, inactivating mutations or deletions of PTENlead to Akt and mTOR activation and occur frequently inhuman cancers (8). Furthermore, loss of the upstream regu-lators of the mTOR pathway TSC1 and TSC2 leads to consti-tutive activation of mTORC1 and results in the development oftumors (9). Interestingly, tumor cells harboring an activatedmTOR pathway are more sensitive to endoplasmic reticulumstress–induced cell death (10–12), although the mechanismunderlying this cell death process remains to be elucidated.

ERBB2 is a member of the ERBB receptor family, which alsoincludes the epidermal growth factor receptor (EGFR, ERBB1),ERBB3, and ERBB4. Ligand binding to the extracellulardomains of EGFR, ERBB3, and ERBB4 leads to the formationof catalytically active homo- and heterodimers towhich ERBB2is recruited as a preferred partner (13). Activation of the ERBBreceptors leads to receptor autophosphorylation of C-terminaltyrosines and recruitment to these sites of cytoplasmic signaltransducers that activate several downstream signaling path-ways, such as the extracellular signal-regulated kinase and thephosphoinositide-3-kinase/AKT/mTOR pathways (14). Ampli-fication of a genomic region containing the ERBB2 gene onchromosome 17q12 has been observed in approximately 25%of

Authors' Affiliations: 1Centro Andaluz de Biología Molecular y MedicinaRegenerativa-CSIC, CABIMER, Avda Am�erico Vespucio s/n, Sevilla;2Departament de Ci�encies Fisiol�ogiques II, Institut d'Investigaci�oBiom�edica de Bellvitge (IDIBELL)-Universitat de Barcelona, L'Hospitaletde Llobregat, Barcelona, Spain; and 3Department of Biochemistry andMolecular Biology, Drexel University College of Medicine, Philadelphia,Pennsylvania

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

C. Palacios and R. Yerbes contributed equally to this work.

Corresponding Author: Abelardo L�opez-Rivas, Centro Andaluz de Bio-logía Molecular y Medicina Regenerativa (CABIMER), Consejo Superior deInvestigaciones Científicas, Avenida Americo Vespucio s/n, 41092 Sevilla,Spain. Phone: 34-954-467997; Fax: 34-954-461664; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-13-1747

�2014 American Association for Cancer Research.

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invasive breast tumors (15). ERBB2 overexpression is frequent-ly accompanied by the occurrence of truncated forms of thereceptor that are characterized by enhanced oncogenic poten-tial (16, 17). In addition, somatic mutations in the ERBB2 genehave been reported in a number of tumors, including breastcarcinomas, some of which results in a gain-of-function com-pared with wild-type ERBB2 (18, 19).Herein, we have addressed the issue of the sensitivity to

endoplasmic reticulum stress of human breast epithelial cellsexpressing an activated form of the ERBB2 oncogene. We showthatmutant ERBB2 expression in breast epithelial cells leads toan overactivation of the UPR and markedly sensitizes thesecells to endoplasmic reticulum stress–induced apoptosis.Hyperactivation of the PERK/ATF4/CHOP pathway in mutantERBB2 cells following endoplasmic reticulum stress upregu-lates TRAIL-R2 expression, resulting in the induction of acaspase-8–mediated and mitochondria-operated apoptoticpathway. Importantly, deregulation of the PERK/ATF4/CHOP/TRAIL-R2 pathway and sensitivity of mutant ERBB2to endoplasmic reticulum stress is abrogated by inhibition ofthe mitogen—activated protein kinase (MAPK)/ERK, Akt, ormTOR activities. These results point at endoplasmic reticulumstress as a biochemical target by which tumor cells expressinggain-of-function ERBB2 mutants may be approached for ther-apeutic intervention.

Materials and MethodsCell cultureMCF10A cells were maintained in Dulbecco's Modified Eagle

Medium (DMEM)/F12 supplemented with 5% donor horseserum (Gibco), 2 mmol/L L-glutamine, 20 ng of epidermalgrowth factor (EGF)/mL, 10 mg of insulin/mL, 100 ng of choleratoxin/mL, 0.5 mg of hydrocortisone/mL, 50 U of penicillin/mL,and 50 mg of streptomycin/mL at 37�C in a 5% CO2-humidified,95% air incubator. The human tumor cell lineMDA-MB231 wasmaintained inRPMI 1640medium supplementedwith 10%FBS,2 mmol/L L-glutamine, and 50 U of penicillin/mL, and 50 mg ofstreptomycin/mL. SKBr3 andBT-474 cell lines weremaintainedin DMEM supplemented with 10% FBS, 2 mmol/L L-glutamine,50 U of penicillin/mL, and 50 mg of streptomycin/mL.

Retroviral vectors and virus productionpbabe-NeuN vector (wild-type ERBB2) for stable gene

expression has been described previously (20). Constitutivelyactive ERBB2 mutant (pbabe-NeuT) was kindly provided by D.Carroll (Harvard Medical School, Boston, MA). pbabe-Bcl-xLwas a gift from Dr. C. Mu~noz (IDIBELL, Barcelona, Spain).Retroviruses for protein overexpression were produced bytransfection of HEK293-T cells by the calcium phosphatemethod with the corresponding retroviral vectors. Retrovi-rus-containing supernatants were collected 48 hours aftertransfection and concentrated by ultracentrifugation at22.000 rpm for 90 minutes at 4�C.

Generation of MCF10A cell linesCells were infected with the retroviruses mentioned above.

Stable populations were obtained by selection with 1.5 mg/mLpuromycin during 48 hours.

Determination of apoptosisCells (3� 105/well) were treated in 6-well plates as indicated

in the figure legends. After treatment, hypodiploid apoptoticcells were detected by flow cytometry according to publishedprocedures (21). Briefly, cells were washed with PBS, fixed incold 70% ethanol, and then stained with propidium iodidewhile treating with RNase. Quantitative analysis of sub-G1 cellswas carried out in a FACSCalibur cytometer using the CellQuest software (Becton Dickinson). In BT-474 cells, phospha-tidylserine exposure on the surface of apoptotic cells wasdetected by flow cytometry after staining with Annexin-V-FLUOS (Boehringer).

Immunoblot analysis of proteinsCells (3 � 105) were washed with PBS and lysed in radio-

immunoprecipitation assay buffer. Protein content was mea-suredwith the Bradford reagent (Bio-Rad Laboratories), beforeadding Laemmli sample buffer. Proteins were resolved on SDS-polyacrylamide minigels and detected as described previously(22). Tubulin and glyceraldehyde-3-phosphate dehydrogenasewere used as protein loading controls.

Reverse transcriptase and PCR assaysTotal RNA was isolated from MCF10A cells with the TRizol

reagent (Life Technologies) as recommended by the supplier.Total RNA was used as a template for cDNA synthesis using areverse transcriptase (RT)-PCR Kit (Perkin-Elmer). PCRs werecarried out using specific primers (Supplementary Materials).RT-PCR product of b-actin was used as a control for mRNAinput.

Real-time PCRmRNA expression was analyzed in triplicate by RT-qPCR

on the ABI Prism7500 sequence detection system usingpredesigned assay-on-demand primers and probes (AppliedBiosystems). Hypoxanthine-guanine phosphoribosyltrans-ferase (HPRT1 Hs01003267_m1) was used as an internalcontrol and mRNA expression levels of CHOP, TRAIL, andTRAIL-R2 were given as fraction of mRNA levels in controlcells. Primers and probes used were: ATF4 (Hs00909568_g1),CHOP (Hs01090850_m1), TRAIL (Hs00921974_m1), andTRAIL-R2 (Hs00366278_m1).

RNA interferencesiRNAs against TRAIL-R2, Bid, Ire1, ATF4, ATF6, caspase-8,

Raptor, Rictor, Noxa, Bim, and nontargeting scrambled control(Supplementary Materials) were synthesized by Sigma. Flex-itube siRNAs against PERK, CHOP, and TRAIL were purchasedfrom Qiagen. Cells were transfected with siRNAs using Dhar-maFECT-1 (Dharmacon) as described by the manufacturer.After 6 hours, transfection medium was replaced with regularmedium and cells were further incubated for 42 hours beforefurther analysis.

Statistical analysisAll data are presented as the mean � SE of at least three

independent experiments. The differences among differentgroups were determined by the Student t test. P < 0.05 was

Mutant ERBB2 Promotes Sensitivity to Endoplasmic Reticulum Stress

www.aacrjournals.org Cancer Res; 74(6) March 15, 2014 1767




considered significant. �, P < 0.05; ��, P < 0.01; ��, P < 0.001. n.s.,not statistically significant.

ResultsEnhanced sensitivity of mutant ERBB2-expressing cellsto endoplasmic reticulum stress

Recent findings have demonstrated that the presence ofERBB2 somatic mutations is an alternative mechanism toactivate ERBB2 in breast cancer (19). Different somatic muta-tions of ERBB2 enhance the tyrosine kinase activity and theoncogenic potential of this protein (18, 19, 23). One of theconsequences of ERBB2 activation is the deregulated activa-tion of the MAPK/ERK and PI3K/Akt/mTOR pathways, whichpromotes cell growth, proliferation, increasedmetabolism, andmotility (13). Given the reported crosstalk between the UPRand mTOR signaling pathways (10–12, 24), we investigated theimpact of ERBB2 activation on the sensitivity of breast epi-thelial cells to endoplasmic reticulum stress. Results depictedin Fig. 1A indicate that human breast epithelial cells MCF10Aexpressing a constitutively active ERBB2 (NeuT; ref. 20) showedconstitutive phosphorylation of ERBB2 at Tyr1248 and were

markedly more sensitive to the endoplasmic reticulum stressinducer thapsigargin than control cells (pbabe) or cells expres-sing wild-type ERBB2 (NeuN). Time course experiments fur-ther confirmed the increased sensitivity of NeuT cells toendoplasmic reticulum stress (Supplementary Fig. S1A). Tuni-camycin, another endoplasmic reticulum stress inducer, alsoactivated apoptosis differentially inNeuT cells (SupplementaryFig. S1B). Furthermore, results shown in Fig. 1B demonstratethat the ERBB2 tyrosine kinase inhibitor lapatinib markedlyreduced thapsigargin-induced apoptosis in NeuT cells, strong-ly suggesting that sensitivity to endoplasmic reticulum stress isa result of the constitutive activation of ERBB2. In agreementwith the data of the lower sensitivity of NeuN cells, breasttumor cell lines overexpressing either naturally (BT-474,SkBr3) or ectopically (MDA-MB231) the wild type form ofERBB2 showed a reduced sensitivity to endoplasmic reticulumstress (Fig. 1C). Evaluation of sensitivity to endoplasmic retic-ulum stress in breast tumor cells ectopically overexpressingthe NeuT oncogene was not possible because these cellsunderwent premature senescence (Supplementary Fig. S1C),as it has been previously reported (25).

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Figure 1. Apoptotic response of ERBB2-expressing cells to endoplasmic reticulum stress. A, cells were treated with the indicated doses of thapsigarginfor 30 hours and apoptosis was determined as described in Materials and Methods. Inset shows the expression of total ERBB2 and p-ERBB2(Tyr1248). B, pbabe andNeuTcellswere incubatedwith orwithout lapatinib (5mmol/L) for 48hours and then treatedwith thapsigargin (100 nmol/L) for 30 hoursin the presence or the absence of lapatinib. Apoptosis was determined as described in Materials and Methods. Inset shows the expressionof p-ERBB2 (Tyr1248) after treatment with or without lapatinib (5 mmol/L) for 48 hours. Error bars, SD from three independent experiments. �, P < 0.05;��, P < 0.01; ��, P < 0.001. C, indicated cell lines were treated with 100 nmol/L thapsigargin during 30 hours and apoptosis (left) was measured asdescribed in Materials and Methods. Right panel shows the expression of p-ERBB2 (Tyr1248) and total ERBB2 in the different cell lines tested. Error bars,SD from three independent experiments. �, P < 0.05 and ��, P < 0.01 comparing the various cell lines with NeuT cells.

Martín-P�erez et al.

Cancer Res; 74(6) March 15, 2014 Cancer Research1768




Role of the UPR branches in endoplasmic reticulumstress–induced apoptosis inmutant ERBB2-transformedcellsTo investigate the mechanism of the enhanced sensitivity of

NeuT cells to endoplasmic reticulum stress, we examined therole of the UPR branches, which are known to regulate cell fateupon endoplasmic reticulum stress (2). Thus, we determinedthe processing by Ire1a of the mRNA encoding the transcrip-tion factor X box–binding protein 1 (XBP1) to generate theactive transcription factor XBP1s. In control cells, initial Ire1asignaling was substantially reduced upon prolonged endoplas-mic reticulum stress (Fig. 2A), as previously reported in othercell systems (26). In contrast, in NeuT cells Ire1a activityremained elevated as indicated by the sustained expressionof XBP1s upon endoplasmic reticulum stress. However, Ire1a

knockdown did not result in the abrogation of endoplasmicreticulum stress–induced apoptosis (Supplementary Fig. S2A).Likewise, ATF6 silencing did not reduce significantly apoptosisinduced by thapsigargin (Supplementary Fig. S2B).

We next examined the activity of the PERK/ATF4/CHOPpathway in both control and NeuT cells upon thapsigargintreatment. We found no differences in the activation of PERKupon thapsigargin treatment, as determined by the phosphor-ylation of the eukaryotic initiation factor 2a (eIF2a; Fig. 2B).Likewise, inhibition of general protein synthesis followingendoplasmic reticulum stress occurred to the same extentand with similar kinetics in both control and NeuT cells(Supplementary Fig. 2C). Interestingly, although we foundno differences in ATF4 mRNA levels between control andNeuT cells following thapsigargin treatment (Supplementary

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Figure 2. Role of the PERK/ATF4/CHOP pathway in endoplasmic reticulum stress–induced apoptosis. pbabe and NeuT cells were treated with thapsigargin(100 nmol/L) for the indicated times. Following these treatments, XBP1 splicing was analyzed by RT-PCR (A); phosphorylation of eIF2a (B) and ATF4induction (D) was assessed by Western blotting; protein synthesis was measured by the incorporation of [3H]leucine (10 mCi/mL) into acid-precipitablematerial (C) and CHOP mRNA levels were determined by RT-qPCR (E). Quantification of RT-PCR and Western blot signals was performed bydensitometry with ImageQuant TL software after scanning the films on ImageScanner II (GEHealthcare). The relative expression of proteinswas normalized tothat of loading controls. Results are representative of three independent experiments. NeuT cells were transfected either with a scrambled oligonucleotide(Scr) or with siRNAs targeting PERK (F), ATF4 (G), or CHOP (H) for 48 hours. Cells were then treated with or without thapsigargin (100 nmol/L) for30 hours and apoptosiswas determined. Error bars, SD from three independent experiments. �,P <0.05 and ��,P <0.01. ATF4 knockdownwasdetermined byWestern blotting. Silencing of PERK was assessed by RT-PCR. CHOP levels were determined by RT-qPCR.






Fig. S2C), expression of ATF4 was significantly enhanced at theprotein level in NeuT cells as compared with control cells uponendoplasmic reticulum stress (Fig. 2D). In addition, expressionof the ATF4 target gene CHOP (27) was also markedly upre-gulated in NeuT cells as compared with control cells, uponendoplasmic reticulum stress (Fig. 2E). Regarding sensitivity toendoplasmic reticulum stress, a significant inhibition of endo-plasmic reticulum stress–induced CHOP expression and apo-ptosis was observed in NeuT cells following PERK knockdown(Fig. 2F). Further evidences for the role of the PERK pathway inendoplasmic reticulum stress–induced apoptosis in mutantERBB2-transformed cells were obtained in experiments silenc-ing the expression of downstream effectors of this pathway. Asshown in Fig. 2G, ATF4 knockdown caused amarked inhibitionof CHOP expression and apoptosis upon thapsigargin treat-ment. Similarly, silencing CHOP expression resulted in animportant inhibition of thapsigargin-induced apoptosis inNeuT cells (Fig. 2H).

TRAIL-R2–mediated apoptosis in NeuT cells uponendoplasmic reticulum stress

We next investigated the role of caspases and the mito-chondria in this cell death process. The pan-caspase inhibitorZ-VAD-fmk completely abrogated cell death induced by thap-sigargin (Fig. 3A). Furthermore, caspase-9 and caspase-3 pro-cessing was also clearly enhanced in NeuT cells as comparedwith control cells (Fig. 3B). Strikingly, in Bcl-xL–overexpressingNeuT cells thapsigargin-induced apoptosis was clearly inhib-ited (Fig. 3C), which demonstrated that the increased sensi-tivity of NeuT cells to endoplasmic reticulum stress wasbecause of the enhanced activation of a mitochondria-oper-ated apoptotic pathway.

Transcriptional induction of BH3-only proteins by CHOPplays a role in endoplasmic reticulum stress–induced apopto-

sis in different systems (28, 29). We determined by reversetranscriptase–multiplex ligation-dependent probe amplifica-tion the mRNA levels of twenty members of the Bcl-2 family,including 10 BH3-only members. Although small differenceswere found between control and NeuT cells in the mRNAexpression levels of Noxa and Bim upon endoplasmic reticu-lum stress (Supplementary Fig. S3A), knockdown of these BH3-only proteins did not inhibit thapsigargin-induced apoptosis inNeuT cells (Supplementary Fig. S3B). Collectively, these resultssuggested that activation of the mitochondrial pathway ofapoptosis in NeuT cells by endoplasmic reticulum stress wasindependent of the upregulation of BH3-only or downregula-tion of anti-apoptotic Bcl-2 proteins expression.

CHOP involvement in endoplasmic reticulum stress–induced apoptosis has been previously linked to upregulationof TRAIL-R2 expression and a potential CHOP-binding sitehas been identified in the 50-flanking region of the TRAIL-R2gene (30). Time course analysis of TRAIL-R2 levels indicatedthat CHOP-dependent TRAIL-R2 upregulation followingendoplasmic reticulum stress treatment was enhanced inNeuT cells compared with pbabe cells (Fig. 4A and Supple-mentary Figs. S4, S5A, and S5B). Moreover, TRAIL-R2 upre-gulation induced by endoplasmic reticulum stress wasaccompanied by a marked decrease in FLIPL expression (Fig.4B). Notably, caspase-8 activation was only observed in NeuTcells treated with thapsigargin (Fig. 4C). As FLIPL down-regulation was observed in both cell lines, these resultssuggested that the enhanced upregulation of TRAIL-R2 inNeuT cells may be an important event in endoplasmic retic-ulum stress–induced apoptosis in these cells. In this respect,TRAIL-R2 knockdown markedly reduced endoplasmic retic-ulum stress–induced apoptosis (Fig. 4D and SupplementaryFig. S5C). Furthermore, silencing caspase-8 expressionmarkedly inhibited endoplasmic reticulum stress–induced

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Figure 3. Endoplasmic reticulumstress activates caspase-dependent cell death through amitochondria-operated apoptoticpathway. A, apoptosis wasdetermined in pbabe and NeuTcells treated for 30 hours withthapsigargin (100 nmol/L) in thepresence or absence of z-VAD-fmk (10 mmol/L). B, pbabe andNeuT cells were treated with100 nmol/L thapsigargin for theindicated times. Activation ofcaspase-9 and caspase-3 wasassessed by Western blotting.Results are representative of twoindependent experiments. C,apoptosis was assessed inBcl-xL–overexpressing NeuT cellstreated with thapsigargin(100 nmol/L) for 30 hours. Errorbars, SD from three independentexperiments. �, P < 0.001.Bcl-xL overexpression wasmeasured by Western blotting.






apoptosis in NeuT cells (Fig. 4E and Supplementary Fig. S5C),revealing a pivotal role of caspase-8 activation in endoplasmicreticulum stress–induced apoptosis in these cells. Activationof caspase-8 leads to the processing of the BH3-only substrateBid generating a 15-kDa fragment that translocates to mito-chondria to promote the release of apoptogenic factors (31).In NeuT cells, silencing Bid expression significantly reducedapoptosis induced by endoplasmic reticulum stress (Fig. 4F).Collectively, these results and data shown in Fig. 3C suggest adifferential activation of a TRAIL-R2–mediated, mitochon-dria-operated apoptotic pathway in NeuT cells upon endo-plasmic reticulum stress.

To determine the role of potentially secreted TRAIL inthe apoptosis induced by endoplasmic reticulum stress, weused a recombinant TRAIL-R2/Fc chimeric protein that hasbeen shown to potently neutralize the apoptotic activity ofexogenous TRAIL (32). However, at doses 5 times higherthan those required to inhibit apoptosis by exogenouslyadded TRAIL (Supplementary Fig. S6A, right), TRAIL-R2/Fcdid not inhibit apoptosis induced by thapsigargin (Supple-mentary Fig. S6A, left). Furthermore, silencing TRAILexpression before endoplasmic reticulum stress treatmentdid not inhibit apoptosis by thapsigargin (SupplementaryFig. S6B), which suggested the lack of involvement

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Figure 4. Involvement of TRAIL-R2and caspase-8 in endoplasmicreticulum stress–inducedapoptosis in NeuT cells. Cells weretreated with 100 nmol/Lthapsigargin for the indicatedtimes. Following these treatments,TRAIL-R2 induction (A) wasdetermined by RT-qPCR andWestern blotting. FLIPL levels (B)and caspase-8 activation (C) wereexamined by Western blotting.Results are representative of twoindependent experiments. D–F,NeuT cells were transfected eitherwith a scrambled oligonucleotideor siRNA oligonucleotidestargeting TRAIL-R2 (D), caspase-8(E), or Bid (F) for 48 hours asdescribed in Materials andMethods. Cells were thenincubated in the presence orabsence of thapsigargin(100 nmol/L) for 30 hours. Proteinknockdown and apoptosis weredetermined as previouslydescribed. Error bars, SDfrom three independentexperiments. �, P < 0.05;��, P < 0.01; ��, P < 0.001.






of endogenous TRAIL in endoplasmic reticulum stress–induced apoptosis.

Signaling pathways regulation upon endoplasmicreticulum stress in control and NeuT cells

To further investigate the mechanism of endoplasmic retic-ulum stress–induced apoptosis in NeuT cells, we first exam-ined the activation of Akt and ERK in both pbabe and NeuTcells treated with thapsigargin. Basal levels of phosphorylatedAkt were markedly different in pbabe and NeuT cells (Fig. 5A),as expected from the constitutive activation of the PI3K/Aktpathway by the mutant ERBB2 protein. As previously reportedin other cell types (33), there was an acute activation of Akt inboth pbabe andNeuT cells upon thapsigargin treatment, whichwas followed by a decrease in phosphorylated Akt to levelsbelow the basal level in pbabe cells. In contrast, levels ofphosphorylated Akt protein remained elevated after the initialpeak of activation in NeuT cells treated with thapsigargin (Fig.5A). Similarly to what was observed for Akt, basal levels ofactivated ERK were highly elevated in NeuT cells as comparedwith pbabe cells (Fig. 5B). Furthermore, in pbabe cells, endo-plasmic reticulum stress caused a marked inhibition of ERKphosphorylation starting at 7 hours after the addition ofthapsigargin (Fig. 5B). Remarkably, in NeuT cells we observeda sustained activation of ERK upon endoplasmic reticulumstress that remained phosphorylated for up to 20 hours ofthapsigargin treatment (Fig. 5B). The observed correlationbetween the sustained activation of the Akt and ERK pathwaysand the increased apoptosis of NeuT cells upon endoplasmicreticulum stress prompted us to assess the effect of specificinhibitors of these pathways on the apoptosis induced bythapsigargin. Incubation of NeuT cells either with an Akt(GSK690693) or a MEK1 (U0126) inhibitor partially inhibitedapoptosis (Fig. 5C). Strikingly, in the presence of both inhibi-tors apoptosis was markedly inhibited, suggesting that thesustained activation of the Akt and ERK pathways observed

upon endoplasmic reticulum stress played a key role in theinduction of apoptosis.

TSC2 phosphorylation by either Akt or ERK/RSK leads todisruption of the TSC1/TSC2 complex, a negative regulator ofmTOR activity (34, 35). Moreover, a number of evidences haverevealed the existence of a bidirectional crosstalk betweenendoplasmic reticulum stress and mTOR signaling (24). Inaddition, a link between ERBB2 overexpression and activationof the Akt/mTOR/4E-BP1 pathway has been reported inbreast cancer progression (36). As expected, basal levels ofphosphorylated p70(S6K), a major mTORC1 substrate, washigher in NeuT than in control cells (Fig. 6A). Upon endo-plasmic reticulum stress there was an acute activation of themTORC1 pathway in both cell lines, with maximal activity at3-hour post-thapsigargin addition. Thereafter, phosphorylat-ed p70(S6K) returned to levels below the basal level in controlcells. On the contrary, in NeuT cells the initial peak of p70(S6K) phosphorylation was followed by a sustained mTORC1activity, which remained elevated for up to 20 hours (Fig. 6A).We next examined the impact of sustained mTORC1 activityin the sensitivity of NeuT cells to endoplasmic reticulumstress. Intriguingly, at a dose that strongly inhibits p70(S6K)phosphorylation (Fig. 6B, right) the mTORC1 inhibitor rapa-mycin did not significantly reduce endoplasmic reticulumstress–induced apoptosis (Fig. 6B, left). We also tested theeffect of torin1, a highly potent and selective ATP-competitivemTOR inhibitor that, unlike rapamycin, fully inhibitsmTORC1 and mTORC2 complexes (37). Torin1 completelyinhibited both rapamycin-sensitive and -insensitive mTORC1activities in NeuT cells (Fig. 6B, right). Furthermore, torin1also efficiently inhibited mTORC2 activity as indicated by thecomplete inhibition of AktSer473 phosphorylation (Fig. 6B,right). Strikingly, we observed an almost complete inhibitionof apoptosis by torin1 (Fig. 6B, left), suggesting an importantrole of mTOR in this cell death process. The role of mTORC1and mTORC2 activities in the sensitivity of NeuT cells to

p-AKT

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Figure 5. Signaling pathwaysactivated in cells undergoingendoplasmic reticulum stress.A and B, cells were treated with100 nmol/L thapsigargin for theindicated times. A, Akt activation(p-AktSer473). B, Erk activation(p-ERK) was assessed by Westernblotting. Results are representativeof three independent experiments.C, cells were treated withthapsigargin (100 nmol/L) for 30hours in the presence or absenceof GSK690963 (10 mmol/L), U0126(10 mmol/L), or both inhibitors, andapoptosis was determined. Errorbars, SD from three independentexperiments. �, P < 0.05;��, P < 0.01; ��, P < 0.001. Theefficacy of the inhibitors wasassessed by Western blotting.






endoplasmic reticulum stress was further studied in experi-ments silencing Raptor or Rictor expression with siRNA.Interestingly, Raptor knockdown significantly reduced thap-sigargin-induced apoptosis in NeuT cells (Fig. 6C, left). Incontrast, silencing Rictor expression did not result in asignificant inhibition of endoplasmic reticulum stress–induced apoptosis (Fig. 6C). Furthermore, a double Raptor/Rictor knockdown did not further reduce endoplasmic retic-ulum stress–induced apoptosis over the effect of RaptorsiRNA (Fig. 6C). In view of these results, although inhibitionof apoptosis by Raptor knockdown was not as strong as thatobserved with torin1, it is possible that the residual Raptorprotein may be sufficient to sensitize the cells to thapsigargin(Fig. 6C, right). They also suggest that inhibition of rapamy-cin-insensitive mTORC1 activities may be responsible for theobserved abrogation of endoplasmic reticulum stress–induced apoptosis by Torin1.

Crosstalk between ERBB2-regulated signaling and thePERK/ATF4/CHOP/TRAIL-R2 pathway uponendoplasmic reticulum stressTo elucidate the step(s) in the PERK/ATF4/CHOP/TRAIL-

R2 pathway that was affected by ERBB2-activated signaling inNeuT cells, we first studied the upregulation of ATF4 uponthapsigargin treatment. Of note, ATF4 induction by thapsi-gargin was reduced in the presence of GSK690693 and U0126

(Fig. 7A, left) or Torin1 (Fig. 7A, right). Likewise, results shownin Fig. 7B (left) demonstrated that the combination ofGSK690693 andU0126 significantly inhibitedCHOP expressionupon endoplasmic reticulum stress. In addition, mTOR inhi-bition by Torin1 reduced CHOP mRNA induction by thapsi-gargin (Fig. 7B). Finally, we determined the effect of the variousinhibitors on TRAIL-R2 expression in ER-stressed NeuT cells.Upregulation of TRAIL-R2 mRNA (Fig. 7B, right) and proteinlevels (Fig. 7C) by thapsigargin were considerably abrogated bythe inhibitors. Interestingly, Raptor knockdown by siRNAmarkedly abrogatedTRAIL-R2 upregulation upon thapsigargintreatment (Fig. 7D), further confirming the role of mTORC1activity in endoplasmic reticulum stress–induced apoptosis(Fig. 6C). Overall, our results support the model that sustainedactivation by mutant ERBB2 of signaling pathways that con-verge in mTORC1 favors the transition from an adaptiveresponse to an ATF4/CHOP/TRAIL-R2–mediated apoptoticprocess in human breast epithelial cells undergoing endoplas-mic reticulum stress.

DiscussionERBB2 activation leads to dysregulation of intracellular

signaling pathways that control cell metabolism, growth, andproliferation (13, 14). A number of ERBB2 somatic mutationsfound in patients with ERBB2 gene amplification–negativebreast cancer are activating mutations that likely drive

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Figure 6. Role of mTOR inendoplasmic reticulum stress–induced apoptosis. A, cells weretreated with 100 nmol/Lthapsigargin for the indicated times.P-p70S6K and p70S6K levels wereassessed by Western blotting. B,NeuT cells were treated with 100nmol/L thapsigargin for 30 hours inthe presence or absence ofrapamycin (500 nmol/L) or torin1(250 nmol/L). Apoptosis (left) wasmeasured as previously described.Error bars, SD from threeindependent experiments.��, P < 0.01. P-p70S6K, p70S6K, p-4E-BP1, AktSer473 phosphorylation,and Akt levels were assessed byWestern blotting (right). C, NeuTcells were transfected either with ascrambled oligonucleotide orsiRNA oligonucleotides targetingRaptor, Rictor, or both for 72 hours,and then treated with 100 nmol/Lthapsigargin during 30 hours.Apoptosis was measured aspreviously described. Error bars,SD from three independentexperiments. ��, P < 0.01. n.s., notstatistically significant. Raptor andRictor knockdown were assessedby Western blotting.






tumorigenesis and may confer resistance to ERBB2 tyrosinekinase inhibitors (19). This study demonstrates that breastepithelial cells expressing a constitutively active formof ERBB2are markedly sensitive to endoplasmic reticulum stress. Ourwork also identifies ERK, Akt and mTOR activities as respon-sible for the enhanced sensitivity of these cells to endoplasmicreticulum stress. Recent studies have reported that chronicactivation of mTORC1 results in an increase in PERK activityand sensitivity to endoplasmic reticulum stress, although themolecular mechanism leading to cell death was not elucidated(11, 12, 38, 39).Moreover, there aremarked differences betweenour results in mutant ERBB2-expressing cells and thosereported in TSC1/2-deficient cells. Thus, TSC1/2-deficient cellsshowed elevated eIF2a phosphorylation upon endoplasmicreticulum stress and a truncated UPR in which induction ofother endoplasmic reticulum stress markers was severelycompromised (11). In contrast, we did not observe an increasedPERK activity upon endoplasmic reticulum stress in ERBB2cells compared with control cells. Interestingly, we have foundan enhanced induction of ATF4 and CHOP and sustainedactivation of the Ire1a branch in mutant ERBB2-expressingcells. Although certain links between Ire1a and endoplasmicreticulum stress–induced apoptosis have been suggested (40,41), our results indicate that Ire1a silencing did not reduceapoptosis in ER-stressed ERBB2 cells, excluding a role of theIre1a pathway in death induced by endoplasmic reticulumstress in these cells.

The PERK/ATF4/CHOP branch of the UPR has a dual role incells undergoing endoplasmic reticulum stress. As part of theadaptive response to endoplasmic reticulum stress, the PERK/ATF4/CHOP pathway has been related to the activation ofcytoprotective autophagy upon endoplasmic reticulum stressin different cellular models (42, 43). Although at present wecould not exclude a role of autophagy in the different sensitivityof mutant ERBB2-expressing cells to endoplasmic reticulumstress, our data demonstrate that knockdown of any of thePERK/ATF4/CHOP axis proteins resulted in a significant inhi-bition of endoplasmic reticulum stress–induced apoptosis incells expressing constitutively active ERBB2. The proapoptoticrole of the PERK/ATF4/CHOP pathway in ER-stress–inducedapoptosis has been previously demonstrated. Thus, downre-gulation of anti-apoptotic Bcl-2 familymembers and upregula-tion of proapoptotic BH3-only proteins by CHOP have beenfrequently reported in the activation of apoptosis upon endo-plasmic reticulum stress (6, 29). However, our data do notsupport a similar mechanism in mutant ERBB2-expressingcells. However, a number of evidences support the involvementof death receptors and in particular TRAIL-R2 in the death ofcells undergoing endoplasmic reticulum stress (30, 44, 45). Inaddition, TRAIL-R2 upregulation upon endoplasmic reticulumstress treatments has been suggested to play a prominent rolein the sensitization of tumor cells to exogenous TRAIL byendoplasmic reticulum stress treatments (46). Furthermore,CHOP-dependent upregulation of TRAIL-R2 and a potential

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Figure 7. Crosstalk betweenmutant ERBB2-regulatedsignaling and the ATF4/CHOP/TRAIL-R2 pathway uponendoplasmic reticulum stress.NeuT cells were treated with 100nmol/L thapsigargin for theindicated times in the presenceor absence of GSK690963 (10mmol/L), U0126 (10 mmol/L), orTorin1 (250 nmol/L). ATF4 (A) andTRAIL-R2 protein levels (C) wereassessed by Western blotting.Results are representative of threeindependent experiments. B,NeuTcells were treated as in A andCHOP and TRAIL-R2 mRNA levelswere assessed by RT-qPCR asdescribed in Materials andMethods. D, NeuT cells weretransfected with either a siRNAoligonucleotide targetingRaptor ora scrambled oligonucleotide for 72hours and then treated with 100nmol/L thapsigargin for 15 hours.TRAIL-R2 levels were assessed byWestern blotting. Results arerepresentative of threeindependent experiments.






binding site in the TRAIL-R2 promoter have been reported (30).However, the molecular mechanisms controlling TRAIL-R2upregulation by thePERK/ATF4/CHOPpathway havenot beenfully elucidated. We found that dysregulated activation of theERK, Akt, and mTORC1 in cells expressing a constitutivelyactive form of ERBB2 critically enhances ATF4, CHOP, andTRAIL-R2 levels upon endoplasmic reticulum stress that leadto the activation of a caspase-8–dependent, TRAIL-indepen-dent apoptotic process. Ligand-independent assembly of theDISC has been demonstrated in the TNF family of deathreceptors, most likely because of the homotypic associationof receptors mediated by the pre-ligand–binding assemblydomain (47). Furthermore, ectopic TRAIL-R2 expression hasbeen previously demonstrated to be sufficient to induce apo-ptosis in the absence of ligand (48). As the DISC componentsmay colocalize in an intracellular membrane fraction in breastepithelial cells in the absence of TRAIL (22), the increasedexpression of TRAIL-R2 and downregulation of cFLIP inducedby endoplasmic reticulum stress in ERBB2-expressing cellscould result in the formation of a DISC containing TRAIL-R2,FADD, and procaspase-8 in which caspase-8 is activated. Ourfindings underscore the complexity of the mechanismsinvolved in the apoptosis elicited by endoplasmic reticulumstress in mutant ERBB2-expressing cells and warrant furtherinvestigation to characterize the site of caspase-8 activation inthese cells.Although we do not know the mechanism underlying the

increased expression of ATF4 protein in mutant ERBB2-expressing cells upon endoplasmic reticulum stress, availableevidences suggest that a critical step in the regulation of ATF4protein levels is the ATF4 translational control at the 50-leaderof the ATF4 mRNA, a region containing 2 upstream open-reading frames that are well conserved among vertebrates (49).Furthermore, regulatory elements in the 50-untranslatedregion located upstream of the translation start site of certainmRNAs are also key components of the translational responseto mTOR activation (50). Alternatively, ATF4 is a short-livedprotein with a half-life of less than 30 minutes, whose degra-dation by the proteasome depends on the interaction with theSCF/bTrCP E3 ubiquitin ligase (51). In addition to an increasein ATF4 levels, other important mechanisms for posttransla-tional regulation of ATF4 activity are phosphorylation orinteraction with other transcription factors thus increasingits transcriptional activity (52). Whether or not these mechan-isms are responsible for the enhanced ATF4 expression andactivity in mutant ERBB2 cells is an issue that requires furtherinvestigation. In addition, further studies to elucidate the roleofmTORC1 activity in the control of ATF4 levels would providenew insights into the molecular basis of the transition from anadaptive response to an apoptotic process in human breastepithelial cells undergoing endoplasmic reticulum stress.

In vivo tumor microenvironment is characterized by severehypoxia, glucose deprivation, and acidosis. These combinedfactors lead to the accumulation of misfolded proteins in theendoplasmic reticulum that results in endoplasmic reticulumstress, triggering theUPR to facilitate tumor survival andgrowth.Chronic endoplasmic reticulum stress in tumor cells increasesthe expression of the endoplasmic reticulum chaperones thatprovides a survival advantage to tumor cells in an adversemicroenvironment. Therefore, pharmacologic interference orknockdown strategies to abrogate chaperone expression orfunction may represent a potentially relevant strategy to sen-sitize these cells to different chemotherapeutic agents. Alterna-tively, our results suggest that overactivating the proapoptoticbranches of the UPR in tumor cells that are prone to endoplas-mic reticulum stress because of environmental conditions orconstitutive activation of signaling pathways may modulate theexpression of proteins of the TRAIL pathway (53) and activate aligand-independent apoptotic program in these cells. Thiswouldbe particularly relevant in tumor cells with activatingmutationsin the ERBB2 gene or expressing truncated p95ERBB2, whichmay be resistant to trastuzumab or tyrosine kinase inhibitors.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: R. Martín-P�erez, C. Palacios, A. L�opez-Rivas.Developmentofmethodology:R.Martín-P�erez, C. Palacios, R. Yerbes, A. Cano-Gonz�alez, D. Iglesias-Serret, J. GilAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Martín-P�erez, C. Palacios, R. Yerbes, A. Cano-Gonz�alez, D. Iglesias-Serret, J. GilAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Martín-P�erez, C. Palacios, R. Yerbes, A. Cano-Gonz�alez, D. Iglesias-Serret, J. Gil, A. L�opez-RivasWriting, review, and/or revision of the manuscript: R. Martín-P�erez,C. Palacios, M.J. Reginato, A. L�opez-RivasStudy supervision: A. L�opez-Rivas

AcknowledgmentsThe authors thank F.J. Fernandez-Farr�an for excellent technical assistance

and T. S�anchez-P�erez for scientific discussions.

Grant SupportThis work was supported by grants SAF2009-07163 and SAF2012-32824

(A. L�opez-Rivas) and SAF2010-20519 (J. Gil) from Ministerio de Ciencia eInnovaci�on, Red Tem�atica de Investigaci�on Cooperativa en C�ancer (RTICC:RD06/0020/0068 and RD12/0036/0026 to A. L�opez-Rivas and RD12/0036/0029 toJ. Gil), the European Community through the regional development fundingprogram (FEDER) and Junta de Andalucía (P09-CVI-4497) to A. L�opez-Rivas andNIH grant CA155413 to M.J. Reginato. R. Martín-P�erez, C. Palacios, and R. Yerbeswere supported by contracts from Ministerio de Economía y Competitividad(MINECO) and Junta de Andalucía, respectively. A. Cano-Gonz�alez was sup-ported by a FPI fellowship from MINECO.

The costs of publication of this article were defrayed in part 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 June 18, 2013; revised November 21, 2013; accepted December 11,2013; published OnlineFirst January 22, 2014.

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2014;74:1766-1777. Published OnlineFirst January 22, 2014.Cancer Res Rosa Martín-Pérez, Carmen Palacios, Rosario Yerbes, et al. PathwayEndoplasmic Reticulum Stress through a PERK-Dependent Activated ERBB2/HER2 Licenses Sensitivity to Apoptosis upon

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