Activated HER2 licenses sensitivity to apoptosis upon ... · 22/01/2014 · 1 Activated HER2...

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Activated HER2 licenses sensitivity to apoptosis upon endoplasmic reticulum stress

through a PERK-dependent pathway

Rosa Martín-Pérez1, Carmen Palacios1*, Rosario Yerbes1*, Ana Cano-González1,

Daniel Iglesias-Serret2, Joan Gil2, Mauricio J. Reginato3 and Abelardo López-Rivas1@.

(*equal contribution)

1Centro Andaluz de Biología Molecular y Medicina Regenerativa-CSIC, CABIMER,

Avda Américo Vespucio s/n, 41092 Sevilla, Spain. 2Departament de Ciències

Fisiològiques II, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL)-Universitat

de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain. 3Department of

Biochemistry and Molecular Biology, Drexel University College of Medicine,

Philadelphia, USA.

Short title: Mutant ERBB2 promotes sensitivity to ER stress

Key words: ER stress, ERBB2, ERK, Akt, mTOR, TRAIL-R2

Financial support: This work was supported by grants SAF2009-07163 and SAF2012-

32824 (ALR) and SAF2010-20519 (JG) from Ministerio de Ciencia e Innovación, Red

Temática de Investigación Cooperativa en Cáncer (RTICC: RD06/0020/0068 and

RD12/0036/0026 to ALR and RD12/0036/0029 to JG), the European Community

through the regional development funding program (FEDER) and Junta de Andalucía

(P09-CVI-4497) to ALR. RMP, CP and RY were supported by contracts from

Ministerio de Economía y Competitividad (MINECO) and Junta de Andalucía,

respectively. ACG was supported by a FPI fellowship from MINECO. We thank FJ

Fernandez-Farrán for excellent technical assistance. We also thank Tania Sánchez-Pérez

for scientific discussions.

Research. on February 16, 2021. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 22, 2014; DOI: 10.1158/0008-5472.CAN-13-1747

http://cancerres.aacrjournals.org/

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@Correspondence address: Abelardo López-Rivas, PhD, Centro Andaluz de Biología

Molecular y Medicina Regenerativa (CABIMER), Consejo Superior de Investigaciones

Científicas, Avenida Americo Vespucio s/n, 41092 Sevilla, Spain. Phone: 34-954-

467997 Fax: 34-954-461664, e-mail: [email protected]

The authors disclose no potential conflicts of interest.




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Abstract

HER2/Neu/ERBB2 is a receptor tyrosine kinase overexpressed in approximately

20% of human breast tumors. Truncated or mutant isoforms which show increased

oncogenicity compared to the wild-type receptor are found in many breast tumors. Here

we report that constitutively active ERBB2 sensitizes human breast epithelial cells to

agents that induce endoplasmic reticulum (ER) stress, altering the unfolded protein

response (UPR) of these cells. Deregulation of the ERK, AKT and mTOR activities

elicited by mutant ERBB were involved in mediating this differential UPR response,

elevating the response to ER stress and apoptotic cell death. Mechanistic investigations

revealed that the increased sensitivity of mutant ERBB2-expressing cells to ER 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 ER stress against mutant ERBB2-

expressing breast tumor cells.

Abbreviations: ER, endoplasmic reticulum; UPR, unfolded protein response; PERK,

protein kinase RNA (PKR)-like ER kinase; Ire1α, inositol-requiring protein-1; ATF6,

activating transcription factor-6; mTOR, mammalian target of rapamycin; CHOP,

CAAT/enhancer binding protein homologous protein; TRAIL-R2, tumor necrosis

factor-related apoptosis-inducing ligand receptor 2; EGF, epidermal growth factor;

EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase;

FLIP, FLICE-inhibitory protein; DISC, death-inducing signaling complex.




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Introduction

In response to different environmental and physiological stress conditions that

increase the load of unfolded proteins in the ER, protein sensors located in the luminal

face of the ER membrane activate the unfolded protein response (UPR) (1). Activation

of this signaling pathway leads to a reduction in the influx of proteins into the ER,

activates protein degradation pathways and increases the folding capacity of the ER (2).

In vertebrates, three different types of ER stress transducers have been identified. Each

type defines a distinct branch of the UPR that is mediated by protein kinase RNA

(PKR)-like ER kinase (PERK) (3), inositol-requiring protein-1 (Ire1) (4) or activating

transcription factor-6 (ATF6) (5). In each case, an integral membrane protein senses the

protein-folding status in the ER lumen and transmits this information across the ER

membrane to the cytosol and nucleus (2). These mechanisms allow adaptive and repair

mechanisms that re-establish homeostasis. However, above a certain threshold,

unresolved ER stress results in apoptosis (6).

As a major regulator of cell growth, metabolism and survival, the mammalian

target of rapamycin (mTOR) pathway is commonly activated during oncogenesis (7).

Thus, inactivating mutations or deletions of PTEN lead to Akt and mTOR activation

and occur frequently in human cancers (8). Furthermore, loss of the upstream regulators

of the mTOR pathway TSC1 and TSC2 leads to constitutive activation of mTORC1 and

results in the development of tumors (9). Interestingly, tumor cells harbouring an

activated mTOR pathway are more sensitive to ER stress-induced cell death (10-12),

although the mechanism underlying this cell death process remains to be elucidated.

ERBB2 is a member of the ERBB receptor family, which also includes the

epidermal growth factor receptor (EGFR, ERBB1), ERBB3, and ERBB4. Ligand

binding to the extracellular domains of EGFR, ERBB3, and ERBB4 leads to the




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formation of catalytically active homo- and heterodimers to which ERBB2 is recruited

as a preferred partner (13). Activation of the ERBB receptors leads to receptor

autophosphorylation of C-terminal tyrosines and recruitment to these sites of

cytoplasmic signal transducers that activate several downstream signaling pathways,

such as the extracellular signal-regulated kinase and the phosphoinositide-3-

kinase/AKT/mTOR pathways (14). Amplification of a genomic region containing the

ERBB2 gene on chromosome 17q12 has been observed in approximately 25% of

invasive breast tumors (15). ERBB2 overexpression is frequently accompanied by the

occurrence of truncated forms of the receptor which are characterized by enhanced

oncogenic potential (16, 17). In addition, somatic mutations in the ERBB2 gene have

been reported in a number of tumors, including breast carcinomas, some of which

results in a gain-of-function compared with wild-type ERBB2 (18, 19).

Herein, we have addressed the issue of the sensitivity to ER stress of human

breast epithelial cells expressing an activated form of the ERBB2 oncogene. We show

that mutant ERBB2 expression in breast epithelial cells leads to an overactivation of the

UPR and markedly sensitizes these cells to ER stress-induced apoptosis.

Hyperactivation of the PERK/ATF4/CHOP pathway in mutant ERBB2 cells following

ER stress up-regulates TRAIL-R2 expression resulting in the induction of a caspase-8-

mediated and mitochondria-operated apoptotic pathway. Importantly, deregulation of

the PERK/ATF4/CHOP/TRAIL-R2 pathway and sensitivity of mutant ERBB2 to ER

stress is abrogated by inhibition of the MAPK/ERK, Akt or mTOR activities. These

results point at ER stress as a biochemical target by which tumor cells expressing gain-

of-function ERBB2 mutants may be approached for therapeutic intervention.




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Materials and Methods

Cell culture – MCF10A cells were maintained in DMEM/F12 supplemented with 5%

donor horse serum (Gibco), 2 mM L-glutamine, 20 ng of epidermal growth factor

(EGF)/ml, 10 µg of insulin/ml, 100 ng of cholera toxin/ml, 0.5 µg of hydrocortisone/ml,

50 U of penicillin/ml and 50 µg of streptomycin/ml at 37ºC in a 5% CO2-humidified,

95% air incubator. The human tumor cell line MDA-MB231 was maintained in RPMI

1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 50

U of penicillin/ml, and 50 µg of streptomycin/ml. SKBr3 and BT-474 cell lines were

maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%

fetal bovine serum, 2 mM L-glutamine, 50 U of penicillin/ml and 50 µg of

streptomycin/ml.

Retroviral vectors and virus production – pbabe-NeuN vector (wild type ERBB2) for

stable gene expression has been described previously (20). Constitutively active ERBB2

mutant (pbabe-NeuT) was kindly provided by Danielle Carroll (Harvard Medical

School, Boston, MA). pbabe-BCL-xL was a gift from Dr. Cristina Muñoz (IDIBELL,

Barcelona, Spain). Retroviruses for protein overexpression were produced by

transfection of HEK293-T cells by the calcium phosphate method with the

corresponding retroviral vectors. Retrovirus-containing supernatants were collected 48

hours after transfection and concentrated by ultracentrifugation at 22.000 rpm for 90

minutes at 4ºC.

Generation of MCF10A Cell Lines -Cells were infected with the retroviruses

mentioned above. Stable populations were obtained by selection with 1.5 µg/ml

puromycin during 48 hours.

Determination of apoptosis – Cells (3x105/well) were treated in 6-well plates as

indicated in the figure legends. After treatment, hypodiploid apoptotic cells were




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detected by flow cytometry according to published procedures (21). Briefly, cells were

washed with phosphate buffered saline (PBS), fixed in cold 70% ethanol and then

stained with propidium iodide while treating with RNAse. Quantitative analysis of

subG1 cells was carried out in a FACSCalibur cytometer using the Cell Quest software

(Becton Dickinson, Mountain View, CA). In BT-474 cells, phosphatidylserine exposure

on the surface of apoptotic cells was detected by flow cytometry after staining with

Anexin-V-FLUOS (Boehringer Mannheim, Germany).

Immunoblot analysis of proteins – Cells (3 x 105) were washed with phosphate-

buffered saline (PBS) and lysed in RIPA buffer. Protein content was measured with the

Bradford reagent (Bio-Rad Laboratories, USA), before adding Laemmli sample buffer.

Proteins were resolved on SDS-polyacrylamide minigels and detected as described

previously (22). Tubulin and GAPDH were used as protein loading controls.

Reverse transcriptase (RT) and PCR assays - Total 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 a RT-PCR kit (Perkin-Elmer).

PCRs were carried out using specific primers (Supplementary materials). RT-PCR

product of β-actin was used as a control for mRNA input.

Real-time -PCR - mRNA expression was analyzed in triplicate by RT-qPCR on the ABI

Prism7500 sequence detection system using predesigned Assay-on-demand primers and

probes (Applied Biosystems). Hypoxanthine-guanine phosphoribosyltransferase

(HPRT1 Hs01003267_m1) was used as an internal control and mRNA expression levels

of CHOP, TRAIL and TRAIL-R2 were given as fraction of mRNA levels in control

cells. Primers and probes used were: ATF4 (Hs00909568_g1), CHOP

(Hs01090850_m1), TRAIL (Hs00921974_m1) and TRAIL-R2 (Hs00366278_m1).




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RNA interference- siRNAs against TRAIL-R2, Bid, Ire1, ATF4, ATF6, Caspase-8,

Raptor, Rictor, Noxa, Bim and non-targeting scrambled control (Supplementary

materials) were synthesized by Sigma (St. Louis, MO). Flexitube siRNAs against

PERK, CHOP and TRAIL were purchased from Qiagen. Cells were transfected with

siRNAs using DharmaFECT-1 (Dharmacon) as described by the manufacturer. After 6

h, transfection medium was replaced with regular medium and cells were further

incubated for 42 h before further analysis.

Statistical analysis - All data are presented as the mean ± SE of at least three

independent experiments. The differences among different groups were determined by

the Student’s t test. P<0.05 was considered significant. *P<0.05; **P<0.01;

***P<0.001. n.s. not statistically significant.

Results

Enhanced sensitivity of mutant ERBB2-expressing cells to ER stress

Recent findings have demonstrated that the presence of ERBB2 somatic

mutations is an alternative mechanism to activate ERBB2 in breast cancer (19).

Different somatic mutations of ERBB2 enhance the tyrosine kinase activity and the

oncogenic potential of this protein (18, 19, 23). One of the consequences of ERBB2

activation is the deregulated activation of the MAPK/ERK and PI3K/Akt/mTOR

pathways, which promotes cell growth, proliferation, increased metabolism and motility

(13). Given the reported crosstalk between the UPR and mTOR signaling pathways (10-

12, 24) we investigated the impact of ERBB2 activation on the sensitivity of breast

epithelial cells to ER stress. Results depicted in figure 1A indicate that human breast

epithelial cells MCF10A expressing a constitutively active ERBB2 (NeuT) (20) showed

constitutive phosphorylation of ERBB2 at Tyr1248 and were markedly more sensitive




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to the ER stress inducer thapsigargin than control cells (pbabe) or cells expressing wild

type ERBB2 (NeuN). Time course experiments further confirmed the increased

sensitivity of NeuT cells to ER stress (Fig. S1A). Tunicamycin, another ER stress

inducer, also activated apoptosis differentially in NeuT cells (Fig. S1B). Furthermore,

results shown in figure 1B demonstrate that the ERBB2 tyrosine kinase inhibitor

lapatinib markedly reduced thapsigargin-induced apoptosis in NeuT cells strongly

suggesting that sensitivity to ER stress is a result of the constitutive activation of

ERBB2. In agreement with the data of the lower sensitivity of NeuN cells, breast tumor

cell lines overexpressing either naturally (BT-474, SkBr3) or ectopically (MDA-

MB231) the wild type form of ERBB2 showed a reduced sensitivity to ER stress (Fig.

1C). Evaluation of sensitivity to ER stress in breast tumor cells ectopically

overexpressing the NeuT oncogene was not possible because these cells underwent

premature senescence (Fig. S1C), as it has been previously reported (25).

Role of the UPR branches in ER stress-induced apoptosis in mutant ERBB2-

transformed cells

To investigate the mechanism of the enhanced sensitivity of NeuT cells to ER

stress we examined the role of the UPR branches which are known to regulate cell fate

upon ER stress (2). Thus, we determined the processing by Ire1α of the mRNA

encoding the transcription factor X box-binding protein 1 (XBP1) to generate the active

transcription factor XBP1s. In control cells, initial Ire1α signaling was substantially

reduced upon prolonged ER stress (Fig. 2A), as previously reported in other cell

systems (26). In contrast, in NeuT cells Ire1α activity remained elevated as indicated by

the sustained expression of XBP1s upon ER stress. However, Ire1α knockdown did not

result in the abrogation of ER stress-induced apoptosis (Fig. S2A). Likewise, ATF6

silencing did not reduce significantly apoptosis induced by thapsigargin (Fig. S2B).




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We next examined the activity of the PERK/ATF4/CHOP pathway in both

control and NeuT cells upon thapsigargin treatment. We found no differences in the

activation of PERK upon thapsigargin treatment, as determined by the phosphorylation

of the eukaryotic initiation factor 2 α (eIF2α) (Fig. 2B). Likewise, inhibition of general

protein synthesis following ER stress occurred to the same extent and with similar

kinetics in both control and NeuT cells (Fig. 2C). Interestingly, although we found no

differences in ATF4 mRNA levels between control and NeuT cells following

thapsigargin treatment (Fig. S2C), expression of ATF4 was significantly enhanced at

the protein level in NeuT cells as compared to control cells upon ER stress (Fig. 2D). In

addition, expression of the ATF4 target gene CHOP (27) was also markedly up-

regulated in NeuT cells as compared to control cells, upon ER stress (Fig. 2E).

Regarding sensitivity to ER stress, a significant inhibition of ER stress-induced CHOP

expression and apoptosis was observed in NeuT cells following PERK knockdown (Fig.

2F). Further evidences for the role of the PERK pathway in ER stress-induced apoptosis

in mutant ERBB2-transformed cells were obtained in experiments silencing the

expression of downstream effectors of this pathway. As shown in figure 2G, ATF4

knockdown caused a marked inhibition of CHOP expression and apoptosis upon

thapsigargin treatment. Similarly, silencing CHOP expression resulted in an important

inhibition of thapsigargin-induced apoptosis in NeuT cells (Fig. 2H).

TRAIL-R2-mediated apoptosis in NeuT cells upon ER stress

We next investigated the role of caspases and the mitochondria in this cell death

process. The pan-caspase inhibitor Z-VAD-fmk completely abrogated cell death

induced by thapsigargin (Fig. 3A). Furthermore, caspase-9 and caspase-3 processing

were also clearly enhanced in NeuT cells as compared to control cells (Fig. 3B).

Strikingly, in Bcl-xL-overexpressing NeuT cells thapsigargin-induced apoptosis was




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clearly inhibited (Fig. 3C), which demonstrated that the increased sensitivity of NeuT

cells to ER stress was due to the enhanced activation of a mitochondria-operated

apoptotic pathway.

Transcriptional induction of BH3-only proteins by CHOP plays a role in ER

stress-induced apoptosis in different systems (28, 29). We determined by reverse

transcriptase-multiplex ligation-dependent probe amplification (RT-MLPA) the mRNA

levels of twenty members of the Bcl-2 family, including ten BH3-only members.

Although small differences were found between control and NeuT cells in the mRNA

expression levels of Noxa and Bim upon ER stress (Fig. S3A), knockdown of these

BH3-only proteins did not inhibit thapsigargin-induced apoptosis in NeuT cells (Fig.

S3B). Collectively, these results suggested that activation of the mitochondrial pathway

of apoptosis in NeuT cells by ER stress was independent of the up-regulation of BH3-

only or down-regulation of anti-apoptotic Bcl-2 proteins expression.

CHOP involvement in ER stress-induced apoptosis has been previously linked to

up-regulation of TRAIL-R2 expression and a potential CHOP-binding site has been

identified in the 5'-flanking region of the TRAIL-R2 gene (30). Time course analysis of

TRAIL-R2 levels indicated that CHOP-dependent TRAIL-R2 up-regulation following

ER stress treatment was enhanced in NeuT cells compared to pbabe cells (Fig. 4A, S4,

S5A and S5B). Moreover, TRAIL-R2 up-regulation induced by ER stress was

accompanied by a marked decrease in FLIPL expression (Fig. 4B). Notably, caspase-8

activation was only observed in NeuT cells treated with thapsigargin (Fig. 4C). As

FLIPL down-regulation was observed in both cell lines, these results suggested that the

enhanced up-regulation of TRAIL-R2 in NeuT cells may be an important event in ER

stress-induced apoptosis in these cells. In this respect, TRAIL-R2 knockdown markedly

reduced ER stress-induced apoptosis (Fig. 4D and S5C). Furthermore, silencing




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caspase-8 expression markedly inhibited ER stress-induced apoptosis in NeuT cells

(Fig. 4E and S5C), revealing a pivotal role of caspase-8 activation in ER stress-induced

apoptosis in these cells. Activation of caspase-8 leads to the processing of the BH3-only

substrate Bid generating a 15 kDa fragment which translocates to mitochondria to

promote the release of apoptogenic factors (31). In NeuT cells, silencing Bid expression

significantly reduced apoptosis induced by ER stress (Fig. 4F). Collectively, these

results and data shown in figure 3C suggest a differential activation of a TRAIL-R2-

mediated, mitochondria-operated apoptotic pathway in NeuT cells upon ER stress.

To determine the role of potentially secreted TRAIL in the apoptosis induced by

ER stress we used a recombinant TRAIL-R2/Fc chimeric protein that has been shown to

potently neutralize the apoptotic activity of exogenous TRAIL (32). However, at doses

five times higher than those required to inhibit apoptosis by exogenously added TRAIL

(Fig. S6A, right panel), TRAIL-R2/Fc did not inhibit apoptosis induced by thapsigargin

(Fig. S6A, left panel). Furthermore, silencing TRAIL expression prior to ER stress

treatment did not inhibit apoptosis by thapsigargin (Fig. S6B), which suggested the lack

of involvement of endogenous TRAIL in ER stress-induced apoptosis.

Signaling pathways regulation upon ER stress in control and NeuT cells

To further investigate the mechanism of ER stress-induced apoptosis in NeuT

cells, we first examined the activation of Akt and ERK in both pbabe and NeuT cells

treated with thapsigargin. Basal levels of phosphorylated Akt were markedly different in

pbabe and NeuT cells (Fig. 5A), as expected from the constitutive activation of the

PI3K/Akt pathway by the mutant ERBB2 protein. As previously reported in other cell

types (33), there was an acute activation of Akt in both pbabe and NeuT cells upon

thapsigargin treatment, which was followed by a decrease in phosphorylated Akt to

levels below the basal level in pbabe cells. In contrast, levels of phosphorylated Akt




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protein remained elevated after the initial peak of activation in NeuT cells treated with

thapsigargin (Fig. 5A). Similarly to what was observed for Akt, basal levels of activated

ERK were highly elevated in NeuT cells as compared to pbabe cells (Fig. 5B).

Furthermore, in pbabe cells ER stress caused a marked inhibition of ERK

phosphorylation starting at 7h after the addition of thapsigargin (Fig. 5B). Remarkably,

in NeuT cells we observed a sustained activation of ERK upon ER stress which

remained phosphorylated for up to 20h of thapsigargin treatment (Fig. 5B). The

observed correlation between the sustained activation of the Akt and ERK pathways and

the increased apoptosis of NeuT cells upon ER stress prompted us to assess the effect of

specific inhibitors of these pathways on the apoptosis induced by thapsigargin.

Incubation of NeuT cells either with an Akt (GSK690693) or a MEK1 (U0126)

inhibitor partially inhibited apoptosis (Fig. 5C). Strikingly, in the presence of both

inhibitors apoptosis was markedly inhibited, suggesting that the sustained activation of

the Akt and ERK pathways observed upon ER stress played a key role in the induction

of apoptosis.

TSC2 phosphorylation by either Akt or ERK/RSK leads to disruption of the

TSC1/TSC2 complex, a negative regulator of mTOR activity (34, 35). Moreover, a

number of evidences have revealed the existence of a bidirectional cross-talk between

ER stress and mTOR signaling (24). In addition, a link between ERBB2 overexpression

and activation of the Akt/mTOR/4E-BP1 pathway has been reported in breast cancer

progression (36). As expected, basal levels of phosphorylated p70(S6K), a major

mTORC1 substrate, was higher in NeuT than in control cells (Fig. 6A). Upon ER stress

there was an acute activation of the mTORC1 pathway in both cell lines, with maximal

activity at 3h post-thapsigargin addition. Thereafter, phosphorylated p70(S6K) returned

to levels below the basal level in control cells. On the contrary, in NeuT cells the initial




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peak of p70(S6K) phosphorylation was followed by a sustained mTORC1 activity

which remained elevated for up to 20h (Fig. 6A). We next examined the impact of

sustained mTORC1 activity in the sensitivity of NeuT cells to ER stress. Intriguingly, at

a dose that strongly inhibits p70(S6K) phosphorylation (Fig. 6B, right panel) the

mTORC1 inhibitor rapamycin did not significantly reduce ER stress-induced apoptosis

(Fig. 6B, left panel). We also tested the effect of torin1, a highly potent and selective

ATP-competitive mTOR inhibitor which, unlike rapamycin, fully inhibits mTORC1 and

mTORC2 complexes (37). Torin1 completely inhibited both rapamycin-sensitive and –

insensitive mTORC1 activities in NeuT cells (Fig. 6B, right panel). Furthermore, torin1

also efficiently inhibited mTORC2 activity as indicated by the complete inhibition of

AktSer473 phosphorylation (Fig. 6B, right panel). Strikingly, we observed an almost

complete inhibition of apoptosis by torin1 (Fig. 6B, left panel), suggesting an important

role of mTOR in this cell death process. The role of mTORC1 and mTORC2 activities

in the sensitivity of NeuT cells to ER stress was further studied in experiments silencing

Raptor or Rictor expression with siRNA. Interestingly, Raptor knockdown significantly

reduced thapsigargin-induced apoptosis in NeuT cells (Fig. 6C, left panel). In contrast,

silencing Rictor expression did not result in a significant inhibition of ER stress-induced

apoptosis (Fig. 6C). Furthermore, a double Raptor/Rictor knockdown did not further

reduce ER stress-induced apoptosis over the effect of Raptor siRNA (Fig. 6C). In view

of these results, although inhibition of apoptosis by Raptor knockdown was not as

strong as that observed with torin1, it is possible that the residual Raptor protein may be

sufficient to sensitize the cells to thapsigargin (Fig. 6C, right panel). They also suggest

that inhibition of rapamycin-insensitive mTORC1 activities may be responsible for the

observed abrogation of ER stress-induced apoptosis by Torin1.




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Crosstalk between ERBB2-regulated signaling and the PERK/ATF4/CHOP/TRAIL-R2

pathway upon ER stress

To elucidate the step(s) in the PERK/ATF4/CHOP/TRAIL-R2 pathway that was

affected by ERBB2-activated signaling in NeuT cells we first studied the up-regulation

of ATF4 upon thapsigargin treatment. Of note, ATF4 induction by thapsigargin was

reduced in the presence of GSK690693 and U0126 (Fig. 7A, left panel) or Torin1 (Fig.

7A, right panel). Likewise, results shown in figure 7B (left panel) demonstrated that the

combination of GSK690693 and U0126 significantly inhibited CHOP expression upon

ER stress. In addition, mTOR inhibition by Torin1 reduced CHOP mRNA induction by

thapsigargin (Fig. 7B). Finally, we determined the effect of the various inhibitors on

TRAIL-R2 expression in ER-stressed NeuT cells. Upregulation of TRAIL-R2 mRNA

(Fig. 7B, right panel) and protein levels (Fig. 7C) by thapsigargin were considerably

abrogated by the inhibitors. Interestingly, Raptor knockdown by siRNA markedly

abrogated TRAIL-R2 up-regulation upon thapsigargin treatment (Fig. 7D), further

confirming the role of mTORC1 activity in ER stress-induced apoptosis (Fig. 6C).

Overall, our results support the model that sustained activation by mutant ERBB2 of

signaling pathways that converge in mTORC1 favours the transition from an adaptive

response to an ATF4/CHOP/TRAIL-R2-mediated apoptotic process in human breast

epithelial cells undergoing ER stress.




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Discussion

ERBB2 activation leads to dysregulation of intracellular signaling pathways that

control cell metabolism, growth and proliferation (13, 14). A number of ERBB2

somatic mutations found in ERBB2 gene amplification negative breast cancer patients

are activating mutations that likely drive tumorigenesis and may confer resistance to

ERBB2 tyrosine kinase inhibitors (19). The present study demonstrates that breast

epithelial cells expressing a constitutively active form of ERBB2 are markedly sensitive

to ER stress. Our work also identifies ERK, Akt and mTOR activities as responsible for

the enhanced sensitivity of these cells to ER stress. Recent studies have reported that

chronic activation of mTORC1 results in an increase in PERK activity and sensitivity to

ER stress although the molecular mechanism leading to cell death was not elucidated

(11, 12, 38, 39). Moreover, there are marked differences between our results in mutant

ERBB2-expressing cells and those reported in TSC1/2 deficient cells. Thus, TSC1/2

deficient cells showed elevated eIF2α phosphorylation upon ER stress and a truncated

UPR in which induction of other ER stress markers was severely compromised (11). In

contrast, we did not observe an increased PERK activity upon ER stress in ERBB2 cells

compared with control cells. Interestingly, we have found an enhanced induction of

ATF4 and CHOP and sustained activation of the Ire1α branch in mutant ERBB2-

expressing cells. Although certain links between Ire1α and ER stress-induced apoptosis

have been suggested (40, 41), our results indicate that Ire1α silencing did not reduce

apoptosis in ER-stressed ERBB2 cells, excluding a role of the Ire1α pathway in death

induced by ER stress in these cells.

The PERK/ATF4/CHOP branch of the UPR has a dual role in cells undergoing

ER stress. As part of the adaptive response to ER stress the PERK/ATF4/CHOP

pathway has been related to the activation of cytoprotective autophagy upon ER stress




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in different cellular models (42, 43). Although at present we could not exclude a role of

autophagy in the different sensitivity of mutant ERBB2-expressing cells to ER stress,

our data demonstrate that knockdown of any of the PERK/ATF4/CHOP axis proteins

resulted in a significant inhibition of ER stress-induced apoptosis in cells expressing

constitutively active ERBB2. The proapoptotic role of the PERK/ATF4/CHOP pathway

in ER-stress-induced apoptosis has been previously demonstrated. Thus, down-

regulation of anti-apoptotic Bcl-2 family members and up-regulation of proapoptotic

BH3-only proteins by CHOP have been frequently reported in the activation of

apoptosis upon ER stress (6, 29). However, our data do not support a similar mechanism

in mutant ERBB2-expressing cells. On the other hand, a number of evidences support

the involvement of death receptors and in particular TRAIL-R2 in the death of cells

undergoing ER stress (30, 44, 45). In addition, TRAIL-R2 up-regulation upon ER stress

treatments has been suggested to play a prominent role in the sensitization of tumor

cells to exogenous TRAIL by ER stress treatments (46). Furthermore, CHOP-dependent

up-regulation of TRAIL-R2 and a potential CHOP binding site in the TRAIL-R2

promoter have been reported (30). However, the molecular mechanisms controlling

TRAIL-R2 up-regulation by the PERK/ATF4/CHOP pathway have not been fully

elucidated. We found that dysregulated activation of the ERK, Akt and mTORC1 in

cells expressing a constitutively active form of ERBB2 critically enhances ATF4,

CHOP and TRAIL-R2 levels upon ER stress which lead to the activation of a caspase-

8-dependent, TRAIL-independent apoptotic process. Ligand-independent assembly of

the DISC has been demonstrated in the TNF family of death receptors, most likely due

to the homotypic association of receptors mediated by the pre-ligand-binding assembly

domain (PLAD) (47). Furthermore, ectopic TRAIL-R2 expression has been previously

demonstrated to be sufficient to induce apoptosis in the absence of ligand (48). As the




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DISC components may co-localize in an intracellular membrane fraction in breast

epithelial cells in the absence of TRAIL (22), the increased expression of TRAIL-R2

and down-regulation of cFLIP induced by ER stress in ERBB2-expressing cells could

result in the formation of a DISC containing TRAIL-R2, FADD and procaspase-8 in

which caspase-8 is activated. Our findings underscore the complexity of the

mechanisms involved in the apoptosis elicited by ER stress in mutant ERBB2-

expressing cells and warrant further investigation to characterize the site of caspase-8

activation in these cells.

Although we do not know the mechanism underlying the increased expression of

ATF4 protein in mutant ERBB2-expressing cells upon ER stress, available evidences

suggest that a critical step in the regulation of ATF4 protein levels is the ATF4

translational control at the 5’-leader of the ATF4 mRNA, a region containing two

upstream open reading frames (uORFs) that are well conserved among vertebrates (49).

Furthermore, regulatory elements in the 5�-untranslated region (5�-UTR) located

upstream of the translation start site (TSS) of certain mRNAs are also key components

of the translational response to mTOR activation (50). Alternatively, ATF4 is a short-

lived protein with a half-life of less than 30 min, whose degradation by the proteasome

depends on the interaction with the SCF/βTrCP E3 ubiquitin ligase (51). In addition to

an increase in ATF4 levels, other important mechanisms for posttranslational regulation

of ATF4 activity are phosphorylation or interaction with other transcription factors thus

increasing its transcriptional activity (52). Whether or not these mechanisms are

responsible for the enhanced ATF4 expression and activity in mutant ERBB2 cells is an

issue that requires further investigation. In addition, further studies to elucidate the role

of mTORC1 activity in the control of ATF4 levels would provide new insights into the




19

molecular basis of the the transition from an adaptive response to an apoptotic process

in human breast epithelial cells undergoing ER stress.

In vivo, tumor microenvironment is characterized by severe hypoxia, glucose

deprivation and acidosis. These combined factors lead to the accumulation of misfolded

proteins in the ER which results in ER stress, triggering the UPR to facilitate tumor

survival and growth. Chronic ER stress in tumor cells increases the expression of the

ER chaperones which provides a survival advantage to tumor cells in an adverse

microenvironment. Therefore, pharmacological interference or knockdown strategies to

abrogate chaperone expression or function may represent a potentially relevant strategy

to sensitize these cells to different chemotherapeutic agents. Alternatively, our results

suggest that overactivating the proapoptotic branches of the UPR in tumor cells that are

prone to ER stress due to environmental conditions or constitutive activation of

signaling pathways may modulate the expression of proteins of the TRAIL pathway and

activate a ligand-independent apoptotic program in these cells. This would be

particularly relevant in tumor cells with activating mutations in the ERBB2 gene or

expressing truncated p95ERBB2, which may be resistant to trastuzumab or tyrosine

kinase inhibitors.




20

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24

Figure Legends

Figure 1. Apoptotic response of ERBB2-expressing cells to ER stress.

(A) Cells were treated with the indicated doses of thapsigargin for 30 hours and

apoptosis was determined as described under Materials and Methods. Insert shows the

expression of total ERBB2 and p-ERBB2 (Tyr1248). (B) pbabe and NeuT cells were

incubated with or without Lapatinib (5 µM) for 48 hours and then treated with

thapsigargin (100 nM) for 30 hours in the presence or the absence of Lapatinib.

Apoptosis was determined as described under Materials and Methods. Insert shows the

expression of p-ERBB2 (Tyr1248) after treatment with or without Lapatinib (5µM) for

48 hours. Error bars represent S.D. from three independent experiments. *P<0.05,

**P<0.01, ***P<0.001. (C) Indicated cell lines were treated with 100 nM thapsigargin

during 30 h and apoptosis (left panel) was measured as described under Materials and

Methods. Right panel shows the expression of p-ERBB2 (Tyr1248) and total ERBB2 in

the different cell lines tested. Error bars represent S.D. from three independent

experiments. *P<0.05, **P<0.01, comparing the various cell lines with NeuT cells.

Figure 2. Role of the PERK/ATF4/CHOP pathway in ER stress-induced apoptosis.

pbabe and NeuT cells were treated with thapsigargin (100 nM) for the indicated times.

Following these treatments, XBP1 splicing was analyzed by RT-PCR (A).

phosphorylation of eIF2alpha (B) and ATF4 induction (D) were assessed by western

blotting, protein synthesis was measured by the incorporation of [3H]leucine (10

μCi/ml) into acid-precipitable material (C) and CHOP mRNA levels were determined

by RT-qPCR (E). Quantification of RT-PCR and western blot signals was performed by

densitometry with ImageQuant TL software after scanning the films on ImageScanner II

(GE Healthcare). The relative expression of proteins was normalized to that of loading




25

controls. Results are representative of 3 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 o without

thapsigargin (100 nM) for 30 hours and apoptosis was determined. Error bars represent

S.D. from three independent experiments. *P<0.05, **P<0.01. ATF4 knockdown was

determined by western blotting. Silencing of PERK was assessed by RT-PCR. CHOP

levels were determined by RT-qPCR.

Figure 3. ER stress activates caspase-dependent cell death through a

mitochondria-operated apoptotic pathway.

(A) Apoptosis was determined in pbabe and NeuT cells treated for 30 hours with

thapsigargin (100 nM) in the presence or absence of z-VAD-fmk (10 µM). (B) pbabe

and NeuT cells were treated with 100 nM thapsigargin for the indicated times.

Activation of caspase-9 and caspase-3 were assessed by western blotting. Results are

representative of 2 independent experiments. (C) Apoptosis was assessed in Bcl-xL –

overexpressing NeuT cells treated with thapsigargin (100 nM) for 30 hours. Error bars

represent S.D. from three independent experiments. *P<0.001. Bcl-xL overexpression

was measured by western-blotting.

Figure 4. Involvement of TRAIL-R2 and caspase-8 in ER stress-induced apoptosis

in NeuT cells.

Cells were treated with 100 nM thapsigargin for the indicated times. Following these

treatments, TRAIL-R2 induction (A) was determined by RT-qPCR and western

blotting. FLIPL levels (B) and caspase-8 activation (C) were examined by western

blotting. Results are representative of two independent experiments. In (D), (E) and (F)

NeuT cells were transfected either with a scrambled oligonucleotide or siRNA

oligonucleotides targeting TRAIL-R2 (D), caspase-8 (E) or Bid (F) for 48 hours as




26

described in Methods. Cells were then incubated in the presence or absence of

thapsigargin (100 nM) for 30 h. Protein knockdown and apoptosis were determined as

previously described. Error bars represent S.D. from three independent experiments.

*P<0.05, **P<0.01, ***P<0.001.

Figure 5. Signaling pathways activated in cells undergoing ER stress.

(A,B) Cells were treated with 100 nM thapsigargin for the indicated times. (A) Akt

activation (p-AktSer473) or (B) Erk activation (p-ERK) were assessed by western blotting.

Results are representative of 3 independent experiments. (C) Cells were treated with

thapsigargin (100 nM) for 30 hours in the presence or absence of GSK690963 (10 µM),

U0126 (10 µM) or both inhibitors and apoptosis was determined. Error bars represent

S.D. from three independent experiments. *P<0.05, **P<0.01, ***P<0.001. The

efficacy of the inhibitors was assessed by western-blotting.

Figure 6. Role of mTOR in ER stress-induced apoptosis.

(A) Cells were treated with 100 nM thapsigargin for the indicated times. P-p70S6K and

p70S6K levels were assessed by western blotting. (B) NeuT cells were treated with 100

nM thapsigargin for 30 h in the presence or absence of Rapamycin (500 nM) or torin1

(250 nM). Apoptosis (left panel) was measured as previously described. Error bars

represent S.D. from three independent experiments. **P<0.01. P-p70S6K, p70S6K, p-

4E-BP1, Akt Ser473 phosphorylation and Akt levels were assessed by western blotting

(right panel). (C) NeuT cells were transfected either with a scrambled oligonucleotide

or siRNA oligonucleotides targeting Raptor, Rictor or both for 72 h, and then treated

with 100 nM thapsigargin during 30 h. Apoptosis was measured as previously

described. Error bars represent S.D. from three independent experiments. **P<0.01. n.s.

not statistically significant. Raptor and Rictor knockdown were assessed by western

blotting.




27

Figure 7. Crosstalk between mutant ERBB2-regulated signaling and the

ATF4/CHOP/TRAIL-R2 pathway upon ER stress

NeuT cells were treated with 100 nM thapsigargin for the indicated times in the

presence or absence of GSK690963 (10 µM), U0126 (10 µM) or Torin1 (250 nM)).

ATF4 (A) and TRAIL-R2 protein levels (C) were assessed by western blotting. Results

are representative of 3 independent experiments. (B) NeuT cells were treated as in (A)

and CHOP and TRAIL-R2 mRNA levels were assessed by RT-qPCR as described in

Methods. (D) NeuT cells were transfected with either a siRNA oligonucleotide targeting

Raptor or a scrambled oligonucleotide for 72 h, and then treated with 100 nM

thapsigargin for 15 h. TRAIL-R2 levels were assessed by westerm blotting. Results are

representative of 3 independent experiments.




Figure 1

A

p-ERBB2

(Tyr1248)

ERBB2

Tubulin

NeuNpbabe NeuT

p-ERBB2 (Tyr1248)

ERBB2

Tubulin

NeuN NeuT SkBr3 BT474 NeuNpbabe

MCF10A MDA-MB231

C

B

0

20

40

60

80

Control Lapatinib TG Lapatinib

+TG

Ap

op

tosi

s (%

)

pbabe

NeuT

p-ERBB2

GAPDH

pbabe NeuT

- + - + Lapatinib

***

0

20

40

60

80

100

0 10 25 50 100 150

Thapsigargin (nM)

Ap

op

tosi

s (%

)

*

*

***

**

*** ***

*****NeuT

pbabe

NeuN

MCF10A MDA-MB231

***

**

** **

0

20

40

60

80

100

NeuN NeuT SKBR3 BT474 pbabe NeuN

Ap

op

tosi

s (%

)

Control

TG




0 2 4 6 8 15 0 2 4 6 8 15

1 87 93 92 90 46 1 88 90 91 90 91 % XBP1s

pbabe NeuT

Time with TG (h) CH

OP

mR

NA

(fold

in

du

ctio

n)

1.5 3 6 8 15

Time with TG (h)

200

300

400

500

100

NeuT pbabe

A

E

XBP1s

XBP1un

0 3 7 15

pbabe NeuT

GAPDH

ATF4

1 55 39 93 2 91 105 170 ATF4 (fold induction)

D

0 3 7 15

p-eIF2a

Tubulin

pbabe NeuT

eIF2a

1 6.3 4.8 8 0.8 5.5 5.7 8.3 p-eIF2a (Fold induction)

B

C

Figure 2

pbabe NeuT

D

C

0

20

40

60

80

100

120

0 3 7 15 Time with TG (h)

Pro

tein

syn

thes

is (

%)

pbabe

NeuT

pbabe NeuT

C

D

Time with TG (h)

Time with TG (h)

0 3 7 15 0 3 7 15

F

PERK

Actin

None Scr PERK siRNA

*

0

100

200

300

Scr ATF4 siRNA

0

20

40

60

80

100

Ap

op

tosi

s (%

)

0

20

40

60

Ap

op

tosi

s (%

)

Scr CHOP siRNA

**

Control

TG

Control TG

CH

OP

mR

NA

(fold

in

du

ctio

n)

G

H

Scr

PERK

0

20

40

60

80 *

Scr PERK siRNA

Scr ATF4

- -+ +

ATF4

GAPDH

TG

siRNA

0

100

200

300

Control

CH

OP

mR

NA

(fold

in

du

ctio

n)

0

100

200

300

Control TG

CH

OP

mR

NA

(fold

in

du

ctio

n)

Scr

ATF4

Scr

CHOP

TG

Control

TG

Control

TG

Ap

op

tosi

s (%

)




Figure 3

80

60

40

20

Control TG z-VAD TG+

z-VAD

Ap

op

tosi

s (%

)

pbabe

NeuTCaspase -9

Caspase -3

GAPDH

0 7 15 20 0 7 15 20 Time with TG (h)

pbabe NeuT

A

NeuT NeuT/Bcl-xL

100

80

60

40

20Ap

op

tosi

s (%

)

Control

TGNeuT

NeuT/

BCL-xL

ERBB2

BCL-xL

Tubulin

***

B

C




B

pB pB pBNeuT NeuT NeuT

7 15 20

C-8

pBNeuT

GAPDH

C-8 p18

C

Figure 4

A

FLIPL

0 3 7 15 20 - 3 7 15 20

pbabe NeuT

GAPDH

Time with TG (h)

0 7 15 200

6

2

4

8

10

12

14

TR

AIL

-R2 m

RN

A

(fo

ld i

nd

ucti

on

)

time with TG (h)

TRAIL-R2

GAPDH

pB pB pBNeuT NeuT NeuT

7 15 20

pBNeuT

Time with TG (h)

Time with TG (h)

pbabe

NeuT

Scr TRAIL-R2 siRNA

TRAIL-R2

GAPDH

0

20

40

60

80 **

siRNA Scr TR2

Ap

op

tosi

s (%

)

D

Control

TG

Scr C8 siRNA

GAPDH

procaspase-8

Ap

op

tosi

s (%

)

0

***

siRNA Scr Caspase-8

20

40

60

80E

Control

TG

Bid

Tubulin

Scr Bid siRNA

0

20

40

60

80

siRNA Scr Bid

Ap

op

tosi

s (%

)

*F

ControlTG

0

0




Figure 5

p-AKT

AKT

Tubulin

- 3 7 15 20 - 3 7 15 20 time with TG (h)

pbabe NeuTA

- 3 7 15 20 - 3 7 15 20 time with TG (h)

pbabe NeuT

p-ERK

ERK

Tubulin

B

C

- GSK UO126 GSKUO126

***

Ap

op

tosi

s (

%)

20

40

60

80 *

**

Control

TG

0

p-ERK

p-sust. AKT

GAPDH

TG

con

trol

GS

K

UO

126

GS

K+

UO

con

trol

GS

K

UO

126

GS

K+

UO

Control

ERK




Tubulin

p-p70/S6K

p70/S6K

- 3 7 15 20 - 3 7 15 20 time with TG (h)

pbabe NeuTA

Figure 6

B

p-p70/S6K

p70/S6K

Tubulin

p-4E-BP1

TG

p-AKT

AKT

GAPDH

Control

TG

0

20

40

60

80

Scr Raptor

Rictor

Raptor

+Rictor

Ap

op

tosi

s (%

)

Raptor

Rictor

GAPDH

C Ra Ri RaRi C Ra Ri RaRi siRNA

+TGn.s.

n.s.

**

C

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100

80

60

40

20

Ap

op

tosi

s (%

)

0

n.s.

**

Control

TG




- + - + - + - + GSK/UO

7 15 20 Time withTG (h)

GAPDH

TRAIL-R2

- + - + - + - + Torin 1

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7 15 20 Time with TG (h)

CH

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mR

NA

(fold

in

du

ctio

n)

400

800

1200

1600

0


NT

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TR

AIL

-R2

mR

NA

(fo

ld i

nd

uct

ion

)


4

8

12

16

0

20NT

GSK/U0126

Torin 1

- + - + - + - + GSK/U0


ATF4

GAPDH

- + - + - + - + Torin 1


ATF4

GAPDH

Figure 7

A

B

C

DScr Ra Scr Ra siRNA

+TG

TRAIL-R2

GAPDHResearch.

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