Post on 05-Oct-2020
1
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
2
@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: abelardo.lopez@cabimer.es
The authors disclose no potential conflicts of interest.
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
3
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.
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
4
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
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
5
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.
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
6
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
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
7
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).
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
8
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
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
9
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).
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
10
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
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
11
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
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
12
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
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
13
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
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
14
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.
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
15
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.
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
16
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
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
17
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
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
18
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
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
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.
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
20
References
1. Kozutsumi Y, Segal M, Normington K, Gething MJ, Sambrook J. The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature. 1988;332:462-4. 2. Walter P, Ron D. The unfolded protein response: from stress pathway to homeostatic regulation. Science. 2011;334:1081-6. 3. Harding HP, Zhang Y, Ron D. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature. 1999;397:271-4. 4. Wang XZ, Harding HP, Zhang Y, Jolicoeur EM, Kuroda M, Ron D. Cloning of mammalian Ire1 reveals diversity in the ER stress responses. Embo J. 1998;17:5708-17. 5. Yoshida H, Haze K, Yanagi H, Yura T, Mori K. Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins. Involvement of basic leucine zipper transcription factors. J Biol Chem. 1998;273:33741-9. 6. Tabas I, Ron D. Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. Nat Cell Biol. 2011;13:184-90. 7. Guertin DA, Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell. 2007;12:9-22. 8. Sansal I, Sellers WR. The biology and clinical relevance of the PTEN tumor suppressor pathway. J Clin Oncol. 2004;22:2954-63. 9. Crino PB, Nathanson KL, Henske EP. The tuberous sclerosis complex. N Engl J Med. 2006;355:1345-56. 10. Fang M, Shen Z, Huang S, Zhao L, Chen S, Mak TW, et al. The ER UDPase ENTPD5 promotes protein N-glycosylation, the Warburg effect, and proliferation in the PTEN pathway. Cell. 2010;143:711-24. 11. Kang YJ, Lu MK, Guan KL. The TSC1 and TSC2 tumor suppressors are required for proper ER stress response and protect cells from ER stress-induced apoptosis. Cell Death Differ. 2011;18:133-44. 12. Ozcan U, Ozcan L, Yilmaz E, Duvel K, Sahin M, Manning BD, et al. Loss of the tuberous sclerosis complex tumor suppressors triggers the unfolded protein response to regulate insulin signaling and apoptosis. Mol Cell. 2008;29:541-51. 13. Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol. 2001;2:127-37. 14. Citri A, Yarden Y. EGF-ERBB signalling: towards the systems level. Nat Rev Mol Cell Biol. 2006;7:505-16. 15. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987;235:177-82. 16. Anido J, Scaltriti M, Bech Serra JJ, Santiago Josefat B, Todo FR, Baselga J, et al. Biosynthesis of tumorigenic HER2 C-terminal fragments by alternative initiation of translation. Embo J. 2006;25:3234-44. 17. Christianson TA, Doherty JK, Lin YJ, Ramsey EE, Holmes R, Keenan EJ, et al. NH2-terminally truncated HER-2/neu protein: relationship with shedding of the extracellular domain and with prognostic factors in breast cancer. Cancer research. 1998;58:5123-9. 18. Wang SE, Narasanna A, Perez-Torres M, Xiang B, Wu FY, Yang S, et al. HER2 kinase domain mutation results in constitutive phosphorylation and activation of HER2
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
21
and EGFR and resistance to EGFR tyrosine kinase inhibitors. Cancer Cell. 2006;10:25-38. 19. Bose R, Kavuri SM, Searleman AC, Shen W, Shen D, Koboldt DC, et al. Activating HER2 mutations in HER2 gene amplification negative breast cancer. Cancer Discov. 2013;3:224-37. 20. Haenssen KK, Caldwell SA, Shahriari KS, Jackson SR, Whelan KA, Klein-Szanto AJ, et al. ErbB2 requires integrin alpha5 for anoikis resistance via Src regulation of receptor activity in human mammary epithelial cells. J Cell Sci. 2010;123:1373-82. 21. Gong J, Traganos F, Darzynkiewicz Z. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Analytical biochemistry. 1994;218:314-9. 22. Yerbes R, Palacios C, Reginato MJ, Lopez-Rivas A. Cellular FLIP(L) plays a survival role and regulates morphogenesis in breast epithelial cells. Biochim Biophys Acta. 2011;1813:168-78. 23. Bargmann CI, Weinberg RA. Increased tyrosine kinase activity associated with the protein encoded by the activated neu oncogene. Proc Natl Acad Sci U S A. 1988;85:5394-8. 24. Appenzeller-Herzog C, Hall MN. Bidirectional crosstalk between endoplasmic reticulum stress and mTOR signaling. Trends Cell Biol. 2012;22:274-82. 25. Trost TM, Lausch EU, Fees SA, Schmitt S, Enklaar T, Reutzel D, et al. Premature senescence is a primary fail-safe mechanism of ERBB2-driven tumorigenesis in breast carcinoma cells. Cancer research. 2005;65:840-9. 26. Lin JH, Li H, Yasumura D, Cohen HR, Zhang C, Panning B, et al. IRE1 signaling affects cell fate during the unfolded protein response. Science. 2007;318:944-9. 27. Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Schapira M, et al. Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol Cell. 2000;6:1099-108. 28. Li J, Lee B, Lee AS. Endoplasmic reticulum stress-induced apoptosis: multiple pathways and activation of p53-up-regulated modulator of apoptosis (PUMA) and NOXA by p53. J Biol Chem. 2006;281:7260-70. 29. Puthalakath H, O'Reilly LA, Gunn P, Lee L, Kelly PN, Huntington ND, et al. ER stress triggers apoptosis by activating BH3-only protein Bim. Cell. 2007;129:1337-49. 30. Yamaguchi H, Wang HG. CHOP is involved in endoplasmic reticulum stress-induced apoptosis by enhancing DR5 expression in human carcinoma cells. J Biol Chem. 2004;279:45495-502. 31. Luo X, Budihardjo I, Zou H, Slaughter C, Wang X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell. 1998;94:481-90. 32. Walczak H, Degli-Esposti MA, Johnson RS, Smolak PJ, Waugh JY, Boiani N, et al. TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. EMBO J. 1997;16:5386-97. 33. Hu P, Han Z, Couvillon AD, Exton JH. Critical role of endogenous Akt/IAPs and MEK1/ERK pathways in counteracting endoplasmic reticulum stress-induced cell death. J Biol Chem. 2004;279:49420-9. 34. Inoki K, Li Y, Zhu T, Wu J, Guan KL. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol. 2002;4:648-57.
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
22
35. Ma L, Chen Z, Erdjument-Bromage H, Tempst P, Pandolfi PP. Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and cancer pathogenesis. Cell. 2005;121:179-93. 36. Zhou X, Tan M, Stone Hawthorne V, Klos KS, Lan KH, Yang Y, et al. Activation of the Akt/mammalian target of rapamycin/4E-BP1 pathway by ErbB2 overexpression predicts tumor progression in breast cancers. Clin Cancer Res. 2004;10:6779-88. 37. Thoreen CC, Kang SA, Chang JW, Liu Q, Zhang J, Gao Y, et al. An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1. J Biol Chem. 2009;284:8023-32. 38. Inoki K, Mori H, Wang J, Suzuki T, Hong S, Yoshida S, et al. mTORC1 activation in podocytes is a critical step in the development of diabetic nephropathy in mice. J Clin Invest. 2011;121:2181-96. 39. Kato H, Nakajima S, Saito Y, Takahashi S, Katoh R, Kitamura M. mTORC1 serves ER stress-triggered apoptosis via selective activation of the IRE1-JNK pathway. Cell Death Differ. 2012;19:310-20. 40. Han D, Lerner AG, Vande Walle L, Upton JP, Xu W, Hagen A, et al. IRE1alpha kinase activation modes control alternate endoribonuclease outputs to determine divergent cell fates. Cell. 2009;138:562-75. 41. Hetz C, Bernasconi P, Fisher J, Lee AH, Bassik MC, Antonsson B, et al. Proapoptotic BAX and BAK modulate the unfolded protein response by a direct interaction with IRE1alpha. Science. 2006;312:572-6. 42. Avivar-Valderas A, Salas E, Bobrovnikova-Marjon E, Diehl JA, Nagi C, Debnath J, et al. PERK integrates autophagy and oxidative stress responses to promote survival during extracellular matrix detachment. Mol Cell Biol. 2011;31:3616-29. 43. Rzymski T, Milani M, Pike L, Buffa F, Mellor HR, Winchester L, et al. Regulation of autophagy by ATF4 in response to severe hypoxia. Oncogene. 2010;29:4424-35. 44. Burikhanov R, Zhao Y, Goswami A, Qiu S, Schwarze SR, Rangnekar VM. The tumor suppressor Par-4 activates an extrinsic pathway for apoptosis. Cell. 2009;138:377-88. 45. He Q, Lee DI, Rong R, Yu M, Luo X, Klein M, et al. Endoplasmic reticulum calcium pool depletion-induced apoptosis is coupled with activation of the death receptor 5 pathway. Oncogene. 2002;21:2623-33. 46. Shiraishi T, Yoshida T, Nakata S, Horinaka M, Wakada M, Mizutani Y, et al. Tunicamycin enhances tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human prostate cancer cells. Cancer research. 2005;65:6364-70. 47. Chan FK, Chun HJ, Zheng L, Siegel RM, Bui KL, Lenardo MJ. A domain in TNF receptors that mediates ligand-independent receptor assembly and signaling. Science. 2000;288:2351-4. 48. Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Baldwin D, et al. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science. 1997;277:818-21. 49. Yusta B, Baggio LL, Estall JL, Koehler JA, Holland DP, Li H, et al. GLP-1 receptor activation improves beta cell function and survival following induction of endoplasmic reticulum stress. Cell Metab. 2006;4:391-406. 50. Gentilella A, Thomas G. Cancer biology: The director's cut. Nature. 2012;485:50-1.
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
23
51. Lassot I, Segeral E, Berlioz-Torrent C, Durand H, Groussin L, Hai T, et al. ATF4 degradation relies on a phosphorylation-dependent interaction with the SCF(betaTrCP) ubiquitin ligase. Mol Cell Biol. 2001;21:2192-202. 52. Wek RC, Cavener DR. Translational control and the unfolded protein response. Antioxid Redox Signal. 2007;9:2357-71. 53. MacFarlane M, Ahmad M, Srinivasula SM, Fernandes-Alnemri T, Cohen GM, Alnemri ES. Identification and molecular cloning of two novel receptors for the cytotoxic ligand TRAIL. J Biol Chem. 1997;272:25417-20.
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
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
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
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
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
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.
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
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.
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
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
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
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 (%
)
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
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
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
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
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
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
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
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
control Rapa Torin
100
80
60
40
20
Ap
op
tosi
s (%
)
0
n.s.
**
Control
TG
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
- + - + - + - + GSK/UO
7 15 20 Time withTG (h)
GAPDH
TRAIL-R2
- + - + - + - + Torin 1
GAPDH
TRAIL-R2
7 15 20 Time with TG (h)
CH
OP
mR
NA
(fold
in
du
ctio
n)
400
800
1200
1600
0
0 7 15 20 Time with TG (h)
NT
GSK/U0126
Torin 1
TR
AIL
-R2
mR
NA
(fo
ld i
nd
uct
ion
)
0 7 15 20 Time with TG (h)
4
8
12
16
0
20NT
GSK/U0126
Torin 1
- + - + - + - + GSK/U0
4 7 15 Time with TG (h)
ATF4
GAPDH
- + - + - + - + Torin 1
4 7 15 Time with TG (h)
ATF4
GAPDH
Figure 7
A
B
C
DScr Ra Scr Ra siRNA
+TG
TRAIL-R2
GAPDHResearch.
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
Published OnlineFirst January 22, 2014.Cancer Res Rosa Martin-Perez, Carmen Palacios, Rosario Yerbes, et al. pathwayendoplasmic reticulum stress through a PERK-dependent Activated HER2 licenses sensitivity to apoptosis upon
Updated version
10.1158/0008-5472.CAN-13-1747doi:
Access the most recent version of this article at:
Material
Supplementary
http://cancerres.aacrjournals.org/content/suppl/2014/01/23/0008-5472.CAN-13-1747.DC1
Access the most recent supplemental material at:
Manuscript
Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
.pubs@aacr.orgDepartment at
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://cancerres.aacrjournals.org/content/early/2014/01/22/0008-5472.CAN-13-1747To request permission to re-use all or part of this article, use this link
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