Author Manuscript Published OnlineFirst on April 4, 2014; DOI: 10.1158/2159-8290.CD-13-0945 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Epithelial-to-mesenchymal transition activates PERK-eIF2a and sensitizes cells to
endoplasmic reticulum stress
Yuxiong Feng1, Ethan S. Sokol1,2, Catherine A. Del Vecchio1, Sandhya Sanduja1, Jasper
H.L. Claessen1, Theresa Proia1, Dexter X. Jin1,2, Ferenc Reinhardt1, Hidde L. Ploegh1,2,
Qiu Wang3, Piyush B. Gupta1,2, 4, 5, 6,*
1Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA
02142, USA
2 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139,
USA
3 Department of Chemistry, Duke University, Durham, NC 27708, USA
4 Koch Institute for Integrative Cancer Research, Cambridge, MA 02142, USA
5 Harvard Stem Cell Institute, Cambridge, MA 02142, USA
6 Broad Institute, Cambridge, MA 02142, USA
* Corresponding Author:
Piyush B. Gupta, Whitehead Institute for Biomedical Research, 9 Cambridge Center,
Cambridge, MA 02142. Phone: 617-258-7778; E-mail: [email protected]
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Running title
EMT activates PERK and sensitizes cells to ER stress
Key words
EMT, UPR, ER stress, cell migration and invasion
Abbreviations
EMT: epithelial-to-mesenchymal transition
UPR: unfolded protein response
ER stress: endoplasmic reticulum stress
ECM: extracellular matrix
SPCG: secretory pathway components
Funding support
This research was supported by grants from the Richard and Susan Smith Family
Foundation and the Breast Cancer Alliance.
Conflicts of interest disclosure:
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No potential conflicts of interest were disclosed by the authors.
This manuscript contains 7 figures, 8 supplementary figures and 2 supplementary tables.
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Abstract
Epithelial-to-mesenchymal transition (EMT) promotes both tumor progression and drug
resistance, yet few vulnerabilities of this state have been identified. Using selective small
molecules as cellular probes, we show that induction of EMT greatly sensitizes cells to
agents that perturb endoplasmic reticulum (ER) function. This sensitivity to ER
perturbations is caused by the synthesis and secretion of large quantities of extracellular
matrix (ECM) proteins by EMT cells. Consistent with their increased secretory output,
EMT cells display a branched ER morphology and constitutively activate the PERK-
eIF2 axis of the unfolded protein response (UPR). PERK activation is also required for
EMT cells to invade and metastasize. In human tumor tissues, EMT gene expression
correlates strongly with both ECM and PERK-eIF2 genes, but not with other branches
of the UPR. Taken together, our findings identify a novel vulnerability of EMT cells, and
demonstrate that the PERK branch of the UPR is required for their malignancy.
Significance
EMT drives tumor metastasis and drug resistance, highlighting the need for therapies that
target this malignant subpopulation. Our findings identify a previously unrecognized
vulnerability of cancer cells that have undergone an EMT: sensitivity to ER stress. We
also find that PERK-eIF2a signaling, which is required to maintain ER homeostasis, is
also indispensable for EMT cells to invade and metastasize.
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Introduction
Carcinoma cells acquire key malignant traits by reprogramming their differentiation
state via an epithelial-to-mesenchymal transition (EMT) (1, 2). This transdifferentiation
program, which was first described in developmental contexts, is phenotypically
characterized by repression of epithelial markers, upregulation of mesenchymal markers,
and changes in morphology associated with cell migration. EMT can be induced
experimentally by over-expression of transcription factors such as Snail or Twist, and, in
some contexts, by treatment with TGF . Cancer cells that undergo an EMT become
invasive and drug-resistant; such cells also efficiently seed primary and metastatic
tumors, making them functionally indistinguishable from tumor-initiating or cancer stem
cells (TICs or CSCs)(3-5).
To invade, EMT cells must remodel the extracellular matrix (ECM) by secreting
matrix proteases and large scaffolding proteins that facilitate their migration. These
scaffolding proteins, which include collagens, fibronectin (FN1), plasminogen activator
inhibitor 1 (PAI-1) and periostin (POSTN) (6), interact to form networks that provide
tensional forces and signals that are essential for migration. These quaternary interactions
are often initiated within the cell prior to secretion. For example, collagens are partially
assembled into triple-helical fibers within the endoplasmic reticulum (ER) prior to their
secretion into the extracellular space.
Cells have evolved several quality control pathways that maintain ER homeostasis,
collectively termed the unfolded protein response (UPR) (7). The UPR is activated by
misfolded proteins within the ER, which accumulate upon nutrient deprivation, hypoxia,
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oxidative stress or viral infection (8-13). UPR signaling is initiated by three receptors
localized to the ER membrane – endoplasmic reticulum-to-nucleus signaling 1
(ERN1/IRE1 ), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and
ATF6 (14, 15). These receptors converge on shared downstream factors that increase ER
protein-folding capacity, including BiP/GRP78 and GRP94; they also have unique
signaling effects: activated IRE1 induces splicing of XBP1 mRNA, resulting in the
translation of a frame-shifted stable form of the protein that functions as a transcription
factor (XBP1(S)); activated PERK phosphorylates eIF2 , inducing an integrated stress
response associated with global translational repression and selective translation of repair
proteins (e.g., ATF4).
Because they play a major role in both tumor progression and drug resistance, there is
significant interest in finding vulnerabilities of cancer cells that have undergone an EMT.
In this study, we addressed this question by using selectively toxic small molecules to
probe EMT biology. This led to the discovery of a key vulnerability, and the finding that
EMT cells require UPR signaling for their malignancy.
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Results
Chemical probes selectively activate ER stress in EMT cells
To identify probes of EMT cell biology, we previously performed a large-scale
chemical screen for small molecules with selective toxicity towards EMT cells (5, 16-18).
Of 315,000 compounds tested, this screen identified a few structurally related small
molecules (Cmp302, Cmp308, Dev4) with EMT-selective toxicity (Fig. 1A). These
compounds exhibited between 20- to >100-fold selective toxicity towards non-
tumourigenic (HMLE) and tumourigenic (HMLER) human mammary epithelial cells
induced through an EMT by inhibition of E-cadherin (shEcad) or overexpression of Twist
(Supplementary Fig. S1A and S1B). Treatment of GFP-EMT and dsRed-non-EMT cell
co-cultures with Cmp302, Cmp308, or Dev4 selectively depleted GFP-EMT cells from
the co-cultures, further confirming the selective toxicity of these compounds. In contrast,
two common chemotherapy drugs, paclitaxel and doxorubicin, caused enrichment of
GFP-EMT cells within co-cultures (Supplementary Fig. S1C and D); this was consistent
with previous reports indicating that EMT cells resist chemotherapies (5, 19). The
substitution of a single atom in the pyrrolidine group of Cmp302 was sufficient to
completely abolish toxicity (compound Dev2, Supplementary Fig. S1E and S1F; (18)).
We assessed if these compounds were also selectively toxic towards breast cancer
cells induced into EMT without any genetic modifications. Relative to cells in adherent
culture, MDA-MB-157 cells cultured in suspension undergo an EMT as gauged by
epithelial marker repression, upregulation of mesenchymal markers, acquisition of a
mesenchymal morphology, and expression of stem-like surface markers (Supplementary
Fig S1G-I). In comparison to cells grown in adherent culture, MDA-MB-157 cells
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induced through an EMT by suspension culture exhibited increased sensitivity to
Cmp302 and Cmp308, and reduced sensitivity to paclitaxel (Supplementary Fig S1J and
S1K).
To identify the intracellular effects of these EMT-selective compounds, we used
microarrays to profile global gene-expression in EMT and non-EMT cells after treatment
with Cmp302. This revealed that Cmp302 strongly induced expression of UPR genes in
EMT cells, but not in non-EMT cells (CHOP, ATF3, and GADD34; Table S1a). This
suggested that Cmp302 was selectively inducing ER stress in EMT cells. Gene set
enrichment analysis (GSEA) demonstrated that Cmp302 significantly upregulated --
selectively in cells that had undergone an EMT but not in those that had not -- genes
known from other work (20) to be induced by two well-established ER stressors,
thapsigargin and tunicamycin (Fig. 1B, top panels and Supplementary Table S1B). In
contrast, Cmp302 did not upregulate the expression of genes induced either by hypoxia or
doxorubicin treatment (Fig. 1B, bottom panels and supplementary Table S1B).
To more directly assess this hypothesis, we determined if compound treatment
affected UPR signaling pathways known to be activated by ER stress. In fact, Cmp302
and its more potent analog, Dev4, activated all three branches of UPR signaling in a
dosage-dependent manner -- causing increased XBP1 splicing and eIF2 phosphorylation
(Fig. 1C), and ATF6 activity (Fig. 1D); expression of downstream UPR factors, CHOP
and BiP, was also induced (Fig. 1C and E). Cmp302 and Dev4 activated the UPR at
lower doses in EMT cells relative to non-EMT cells; this paralleled their selective
toxicity towards EMT cells. In contrast, the non-toxic structural analog, Dev2, did not
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activate UPR signaling in EMT or non-EMT cells (Fig. 1C-E and Supplementary Fig.
S2C).
Collectively, these findings strongly suggested that Cmp302/Dev4 were selectively
causing cell death by selectively inducing ER stress in EMT cells.
EMT sensitizes cells to ER stress
The ability to selectively induce ER stress in EMT cells could be a unique feature of
Cmp302/Dev4, or might result from a generalized sensitivity of EMT cells to ER
stressors. To distinguish between these possibilities, we assessed whether EMT cells
were also selectively sensitive to four established chemical inducers of ER stress:
thapsigargin, tunicamycin, dithiothreitol (DTT), and A23187. Notably, all four
compounds caused activation of the PERK and IRE1 branches of the UPR at 8- to 100-
fold lower doses in EMT vs. non-EMT cells, as gauged by phosphorylation of eIF2 and
splicing of XBP1, respectively (Fig. 2A). All four ER stressors also activated the
downstream UPR factors, CHOP, BiP, and GADD34, at lower doses in EMT vs. non-
EMT cells (Fig. 2A-C and Supplementary Fig. S2A).
Moreover, EMT cells were markedly more sensitive to cell death caused by all four
ER stressors, and this was observed for both tumorigenic (HMLER) and non-tumorigenic
(HMLE) lines (~10-fold for Tunicamycin, ~25-fold for Thapsigargin, ~4-fold for DTT
and ~8-fold for A23187; Fig. 2D and supplementary Fig. S2B). Cells induced to undergo
EMT by TGF treatment also showed increased sensitivity to Tunicamycin and
Thapsigargin (Supplementary Fig. S2C). Thapsigargin also selectively eliminated EMT
breast cancer cells from co-cultures of GFP-labeled EMT (HMLER_Twist_GFP) and
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Dsred-labeled non-EMT cells (HMLER_shCntrl_DsRed), doing so in a dosage-
dependent manner (Fig. 2E). This was accompanied by increased cleavage of caspase-3,
indicating that EMT cells activated apoptosis in response to ER stress (Supplementary
Fig. S2D).
To evaluate the generality of these findings, we assessed whether sensitivity to ER
stressors correlated with the differentiation state of breast cancer lines. Breast cancers of
the basal-B subtype are more stem-like and display increased activation of the EMT
program relative to luminal subtype breast cancers (21-26). We therefore evaluated the
sensitivity of a panel of 10 breast cancer lines comprising these two subtypes. Compared
to the four luminal breast cancer lines, the six basal-B cell lines were significantly more
sensitive to tunicamycin, thapsigargin, DTT, and A23187 (Fig. 2F, Supplementary Fig.
S2E and S2F). Taken together, these data indicated that increased sensitivity to ER stress
is a general characteristic of cells that have undergone EMT.
Cells that undergo an EMT are highly secretory
To identify molecular factors underlying this increased ER stress sensitivity, we
compared global transcriptional profiles of EMT and non-EMT breast epithelial cells
(17). We analyzed 956 sets of functionally annotated genes for enrichment in cells that
have undergone an EMT (27). Extracellular matrix and secreted collagen gene sets were
the most significantly enriched in EMT cells (p<10-3), with many individual secreted
genes being highly upregulated (Supplementary Fig. S3A and Fig. 3A).
Secretory cells often upregulate ER protein-folding and transport capacity to sustain
their increased output (28, 29). Consistent with this, expression of 18 genes critically
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involved in secretory pathway components (SPCG) (30) were upregulated in at least 4 of
the 5 EMT lines relative to non-EMT controls (p< 1x10-10 with sign test, supplementary
Fig. S3B). Moreover, in highly secretory cells, increased ER capacity gene expression
occurs together with increased vesicular transport from the ER to cis-Golgi. To assess
vesicular flux, we transiently expressed GFP fused with Sec16, a core component of ER
exit sites(31), and visualized ER exit sites by confocal microscopy(32, 33).
Quantification of Sec16-GFP foci revealed a significant 3-fold increase in ER exit sites in
EMT cells relative to controls, indicative of increased ER-to-cis-Golgi vesicular flux
(Fig. 3B and Supplementary Fig. S3C).
To directly quantify secreted proteins, we used 35S-methionine/cysteine to label
secreted proteins, which were then harvested from the culture medium and visualized by
gel electrophoresis and autoradiography. EMT cells (HMLE_shEcad, HMLE_Twist)
exhibited a ~10- to ~14-fold increase in total secreted proteins relative to isogenic control
cells (Fig. 3C). As a control to confirm that the detected protein was secreted rather than
being released from dying cells, we treated cells with the secretion inhibitor Brefeldin-A,
which completely abrogated accumulation of labeled proteins in the culture medium
(Supplementary Fig. S3D).
Along with the altered protein secretion capacity between EMT and control cells,
significant differences in ER morphology were revealed using electron microscopy: in
EMT cells, 75% of ER membranes had one or more branch points, with 30% having over
10 branch points; in contrast, only 10% of non-EMT cells had ER membranes with one or
more branch points (Fig. 3D). Since professional secretory cells (e.g., pancreatic beta
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cells) often display a highly developed ER network (34), this further suggested that as
part of their function EMT cells also have an increased demand for protein secretion.
To determine if increased ECM secretion is a general feature of EMT, we examined
the expression of ECM genes identified to be upregulated upon EMT (Fig. 3A) in basal-B
and luminal breast cancer lines (35). Basal-B cancer lines (n=9) expressed many EMT
ECM genes – including FN1, COL1A1, COL1A2, COL4A1, COL5A1, POSTN, FBN1 and
COL6A1 – at significantly higher levels than luminal breast cancer lines (n=13) (Fig. 3E;
(24)). In a subset of basal-B lines, EMT ECM genes were expressed at 10- to 100-fold
higher levels than in luminal cancer lines (Supplementary Table S2; (24)). In contrast,
ECM genes not upregulated upon EMT did not exhibit increased expression (COL4A3,
COL10A1, COL13A1, COL15A1; Fig. 3E). In support of these observations, 35S-
methionine/cysteine labeling showed that basal B lines (Hs578T, BT549, MDA-MB-157,
SUM159, MDA-MB-231, 4T1) also exhibited, on average, more than 40-fold increase in
protein secretion relative to luminal lines (MCF7, T47D, BT474, ZR-75-3) (Fig. 3F).
Upregulated ECM secretion following EMT sensitizes cells to ER stress
We next considered the possibility that increased ECM secretion by EMT cells was
directly responsible for their increased sensitivity to ER stressors. If this were indeed the
case, then reducing ECM levels would attenuate UPR activation in response to ER
stressors. To examine this, we analyzed the proteins secreted by two non-tumorigenic
EMT lines (HMLE_shEcad, HMLE_Twist) which, by 35S-methionine/cysteine labeling,
strongly upregulated secretion of a limited number of proteins (Fig. 3C). Mass-
spectrometry of conditioned medium from these two EMT-associated lines revealed that,
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relative to the corresponding controls, they secreted two major proteins: plasminogen
activator inhibitor 1 (PAI1) and fibronectin (FN1).
We next inhibited PAI1 and FN1, both singly and in combination, with multiple
shRNAs (Fig. 4A). Consistent with their abundance by mass spectrometry, dual
inhibition of FN1 and PAI1 greatly reduced the total protein secreted by EMT cells into
conditioned medium (Supplementary Fig. S4A). To examine if the reduction in PAI1 and
FN1 levels was biologically significant, we assessed the migratory properties of double-
knockdown cells. Dual inhibition of PAI1 and FN1 also significantly reduced the
migration of both the HMLE_shEcad and HMLE_Twist EMT cells (Fig. 4B and
Supplementary Fig. S4B), consistent with prior reports (36). Dual inhibition of PAI1 and
FN1 also strongly abrogated UPR induction in response to either Dev4 or thapsigargin
treatment (Fig. 4C and 4D, HMLE_shEcad and HMLE_Twist cells respectively;
Supplementary Fig. S4C). These findings indicated that ECM secretion was required for
EMT cells to migrate, while also increasing their sensitivity to ER perturbations.
EMT increases dependence on the ER chaperone BIP
Nascent polypeptides en route to secretion are folded by critical chaperone proteins that
reside within the ER. Because EMT cells are more secretory and therefore have a higher
ER load, we hypothesized that they might also be more sensitive to reductions in
chaperone proteins. To test this, we used shRNAs to inhibit the key ER chaperone, BiP
(37), in a cell line model in which EMT could be induced within 3 days by addition of 4-
hydroxytamoxifen (HMLE_ER_Twist; (3)). Using two different shRNAs, a 65-75%
reduction in BiP had negligible effects on the viability of this line in the uninduced (non-
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EMT) condition (Fig. 5A and 5B). However, induction of EMT in these shBiP lines
caused significant reduction of cell growth (8-fold less in mesenchymal vs. epithelial
cells), and the surviving cells were clustered in epithelial islands (Fig. 5B). This indicated
that the reduced BiP levels, while sufficient for the needs of epithelial cells, were not
sufficient for cells to survive EMT. Inhibition of BiP also differentially affected the
viability of basal-B (EMT like) and luminal (non-EMT like) breast cancer lines.
Although BiP inhibition only modestly affected the viability of two luminal lines (MCF7,
T47D), it caused significant death in two basal lines (MDA-231, BT549) together with
CHOP upregulation (Fig. 5C and 5D), suggesting that ER stress was more readily
induced in BiP-deficient EMT cells. This was confirmed by examining UPR signaling,
which revealed that the UPR was activated upon BiP inhibition in the basal-B cancer
cells, but not the luminal cancer cells (Supplementary Fig. S5).
PERK-eIF2 -ATF4 signaling is activated upon EMT and promotes malignancy
Prior to their differentiation, progenitors of secretory cell types activate UPR
pathways in anticipation of an increased ER load(38, 39); this UPR activation is not a
response to ER stress but rather a means of preventing it. Because EMT cells are also
highly secretory, we examined if, in the absence of ER stressors, they also activate one or
more UPR pathways.
Compared to non-EMT cells, EMT cells had reduced PERK protein mobility
suggestive of its phosphorylation, increased eIF2 phosphorylation (Fig. 6A), and
increased expression of the downstream gene GADD34 (Fig. 6B). In contrast, IRE1
signaling was not increased in EMT or non-EMT cells in the absence of exogenous ER
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stressors (Supplementary Fig. S6A). To confirm that PERK was in fact phosphorylated in
EMT cells, we also performed phosphatase treatments and immunofluorescence with a
phospho-specific PERK antibody. Treatment of lysates with lambda phosphatase prior to
Western blotting abolished the reduced PERK mobility present in EMT cells under basal
conditions; as a control, phosphatase treatment also abolished the reduced PERK mobility
caused by thapsigargin in both EMT and non-EMT cells (Fig. 6C). Immunofluorescence
with a phosphorylation-specific antibody also showed that PERK was constitutively
activated upon EMT, but not in non-EMT cells (Fig. 6D). Consistent with these findings,
cells induced through an EMT by TGF treatment also activated PERK but not IRE1
signaling (Supplementary Fig. S6B).
Because there are several kinases upstream of eIF2a, we next examined if its
phosphorylation in EMT cells was dependent on PERK. Suppression of PERK activity
with a specific chemical inhibitor strongly decreased both PERK and eIF2
phosphorylation in EMT cells (Supplementary Fig. S6C). Similarly, PERK inhibition by
shRNA also decreased eIF2 phosphorylation in two basal-B breast cancer lines (Fig.
6E). Collectively, these observations established that the PERK-eIF2 -ATF4 branch of
the UPR is selectively and constitutively induced by cells that have undergone an EMT.
Depending on the context, UPR signaling can either promote survival or induce
apoptosis in cells challenged with ER stress (7). Inhibition of PERK in EMT cells with a
chemical inhibitor dramatically increased their sensitivity to thapsigargin (Fig. 6F),
indicating that activation of the PERK pathway is adaptive and beneficial for the survival
of cancer cells that have undergone an EMT. We next examined if PERK signaling also
contributed to the malignant properties of EMT cells. PERK inhibition strongly reduced
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the ability of EMT cells to form tumorspheres (Fig. 6G) and migrate in transwell assays
(Fig. 6H); at the same dose, the PERK inhibitor minimally affected cell proliferation
(Supplementary Fig. S7A and S7B). Pretreatment of metastatic 4T1 cells with either the
PERK inhibitor or thapsigargin resulted in significantly diminished metastatic capacity,
as assessed by lung tumor burden 15 days post tail-vein injection (Fig. 6I). Collectively,
these findings indicated that disruption of the PERK pathway significantly compromises
the malignant phenotype of EMT cancer cells and further increases their sensitivity to ER
stressors.
EMT correlates with PERK but not IRE1 signaling in primary human tumors
We next examined the clinical relevance of the above findings by assessing primary
human tumors. Primary cancer cells (< 3 passages) from breast tumors expressing EMT
markers had elevated PERK and BiP expression, and increased eIF2 phosphorylation,
when compared to primary breast cancer cells that did not express EMT markers (Fig. 7A
and 7B). Primary cancer cells expressing EMT markers were also more sensitive to the
ER stressor thapsigargin as indicated by UPR pathway activation (Fig. 7B). Consistent
with this, these cells also exhibited significantly reduced viability upon treatment with
ER stressors (Fig. 7C and Supplementary Fig. S8).
To assess if these findings extended to other tumor types, we analyzed gene-
expression microarray data from patient tumors to test for associations between the
expression of EMT, ECM, and UPR pathway genes (see Methods for details). This
analysis revealed that the expression of EMT and ECM genes is strongly correlated
across patient tumors, and could be observed in 5 datasets spanning 792 breast, colon,
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gastric and lung tumors, as well as metastatic tumors (Fig. 7D). EMT and ATF4 genes
were also strongly correlated in their expression (mean corr. = 0.80), while a significant
correlation was not observed between the expression of EMT and XBP1 genes (mean
corr. = -0.14; Fig. 7D). These findings established that EMT is strongly associated with
PERK but not IRE1 signaling across a spectrum of tumor types.
Discussion
Given the central role of EMT in tumor metastasis and therapy resistance, there is a
vital need to identify pathways and processes that modulate either the survival or
malignancy of cancer cells that have undergone EMT. In this study, we have assessed the
effects of EMT-selective small molecule probes in the context of global transcriptional
profiling. This revealed that EMT cells, by virtue of their increased secretion of ECM, are
highly sensitive to ER stress. This finding is noteworthy because EMT cells are resistant
to a wide range of chemotherapies, and because the secretory output of a cell has not
previously been shown to influence its sensitivity to chemicals that cause ER stress.
Our findings are consistent with prior studies linking EMT induction with ECM
secretion. However, although the importance of ECM for tumor progression is well
established (36), our study is the first to suggest that ECM secretion, while promoting
malignancy, also creates a key cellular vulnerability. Thus, the acquisition of invasive
and metastatic ability – by virtue of increased ECM production – might invariably lead to
increased vulnerability to ER stress.
We have shown that EMT cells constitutively activate the PERK branch of the UPR,
which is required for them to invade, metastasize and form tumorspheres. The selective
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activation by EMT cells of PERK-eIF2 -ATF4 signaling, but not the IRE1 branch of the
UPR, raises the possibility that this branch may be specifically required for ECM
production. In support of this notion, mouse models have shown that PERK deficiency
specifically compromised ECM production by osteoblasts (38); in contrast, XBP1 loss
prevented the maturation of antibody-secreting plasma cells (40). Because PERK is
activated in both cancerous and non-cancerous cells following EMT, it may contribute to
normal (non-neoplastic) functions of the EMT state. For example, during wound healing,
epithelial cells must undergo EMT to secrete new ECM and migrate to close the wound,
and interfering with EMT induction significantly impairs this process (41). If PERK
signaling is required for ECM secretion during wound healing, its activation in EMT
cancer cells may be a consequence of the normal functional properties of the EMT state.
Cancer cells that undergo an EMT are, in many cases, functionally indistinguishable
from CSCs (3). This raises the possibility that CSCs may also exhibit increased
sensitivity to ER stressors. In support of this, expression profiling of CSCs has revealed
significant upregulation (relative to non-CSCs) of secreted ECM components also
upregulated upon EMT, including Col1A1 and Col1A2 (42); we have also observed that
CSC-enriched subpopulations from breast cancer cells display increased eIF2
phosphorylation (data not shown).
The finding that EMT cancer cells are vulnerable to ER stress has implications for the
treatment of malignant tumors. Investigational agents that cause ER stress(43) may be
most effective against tumors containing a high proportion of EMT cancer cells, such as
breast tumors of the basal-B subtype. In such tumors, ER stressors could directly cause
the death of EMT cancer cells, or interfere with their ability to secrete ECM and thereby
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mitigate tumor malignancy. In addition, ER stressors may effectively target disseminated
cancer cells that have undergone EMT; they may also prove useful for eradicating EMT
cells when they only constitute a small fraction of a tumor, provided that another therapy
is used to eradicate the bulk population. Because PERK pathway inhibitors strongly
abrogated the malignant traits of EMT cells, they also warrant further exploration as
potential cancer therapies.
Materials and Methods
Cell culture and reagents
HMLE and HMLER cells expressing shRNAs targeting GFP (shGFP), E-cadherin
(shEcad), or the coding sequence of Twist (Twist) were generated from Dr. Robert A.
Weinberg’s lab, and maintained in a 1:1 mixture of DMEM + 10% FBS, insulin (10