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 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) 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 expression

correlates strongly with both ECM and PERK-eIF2 , 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

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

g/ml), hydrocortisone (0.5g/ml), EGF (10 ng/ml) and MEGM. The

HMLE/HMLER_shEcand cells were validated by loss of E-cadherin expression, and the

HMLE/HMLER_Twist cells were validated by overexpression of Twist. MCF7, T47D,

BT474, ZR-75-30, Hs578T, MDA-MB-157, and MDA-MB-231 cells were obtained from

American Type Culture Collection (ATCC), and were cultured in DMEM + 10% FBS.

BT549 and 4T1 cells (ATCC) were cultured in RPMI + 10% FBS. SUM159 cells were

obtained from Asterand, and were cultured in F12 + 5% FBS, insulin (10 g/ml), and

hydrocortisone (0.5g/ml). The cell lines from ATCC have not been independently

validated in our laboratory. PERK inhibitor was purchased from Toronto Research

Chemicals Inc (Cat G797800). Cmp302 (acyl hydrazone 1), Cmp308 (acyl hydrazone 2)

and Dev4 (ML239) have been previously reported (18) and were identified in a

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Molecular Libraries Probe Production Centers Network screen conducted at the Broad

Institute.

Mammosphere formation assays were performed as described (5), but with 0.6%

methylcellulose (Stem Cell Technologies) added to the medium. Five thousand cells were

plated per well in low-adherence, 24-well plates and cultured for 5–8 days before being

counted and photographed.

Gene Set Enrichment Analysis (GSEA)

For analysis of Cmp302-treated expression data, Tm, Tg, and Dox gene sets were defined

respectively as the top 100 genes induced by tunicamycin (GSE24500), thapsigargin

(GSE24500) or doxorubicin (GSE39042). The Hypoxia gene set consisted of the top 80

genes induced by both low Oxygen tension (1%) and DMOG treatment (GSE3188).

Microarray analysis

HMLE_shGFP and HMLE_Twist cells were treated with 5μM or 10 μM of Cmp302, or

DMSO solvent for 6 hours. Total RNA were extracted using Qiagen RNeasy kit, and the

integrity and quality of the RNA met the quality requirements for U133

Plus 2.0 arrays (Affymetrix, Inc., Santa Clara, CA) recommended by the company. All of

the subsequent experimental procedures, including labeling, hybridization and scanning,

were processed according to the standard Affymetrix protocols. Raw CEL files were

generated by Affymetrix GCOS 1.2 software, and the present/absent calls were defined

with global scaling to target value of 500. By R software, the CEL files were normalized

to a median-intensity array, and model-based expression values were calculated using

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PM/MM difference model. Alteration of gene expression by Cmp302 was calculated by

comparing the expression of each gene in DMSO and Cmp302-treated group, in both

HMLE_shGFP and HMLE_Twist cells. The gene-expression data have been deposited in

the public database GEO (GSE55604).

Electron Microcopy analysis

Cells were fixed in 2.5% gluteraldehyde, 3% paraformaldehyde with 5% sucrose in 0.1M

sodium cacodylate buffer (pH 7.4), pelletted, and post-fixed in 1% OsO4 in veronal-

acetate buffer. Cell pellet was dehydrated and embedded in Embed-812 resin. Sections

were cut on a Reichert Ultracut E microtome with a Diatome diamond knife at a

thickness setting of 50 nm, stained with uranyl acetate, and lead citrate. Sections were

examined using a FEI Tecnai spirit at 80KV and photographed with an AMT CCD

camera.

Detection of ER exit sites (ERES)

HMLE and HMLE_shEcad cells (2×105/well of a 6-well plate) were transfected with 1

g pmGFP-Sec16S (Addgene 15775) using 2.5 l of Fugene (Roche). 24 hour post-

transfection, cells were re-plated and allowed to adhere onto cover slides before fixation

with 4% paraformaldehyde, and confocal imaging. Images were captured in accordance

with manufacturer’s protocols (Perkin Elmer).

Animal Experiments

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NOD/SCID mice were purchased from Jackson Labs. All mouse procedures were

approved by the Animal Care and Use Committees of the Massachusetts Institute of

Technology. For lung metastasis analysis, 1×106 cancer cells were suspended in 100 l

PBS and injected into the tail vein of each mouse. Lung tissues from experimental

animals were harvested at the various time points indicated in the text. All animals were

randomized by weight.

Dose-Response Assays

Cells were plated in 100 l of medium per well in 96-well plates, at a density of 3000

cells/well. 24 hrs after seeding, compounds were added at 8 different doses with three

replicates per dose per cell line. The same volume of DMSO was added in three

replicates per line as a control. Cell viability was measured after 72 hr with the CellTiter-

Glo AQueous Non-radioactive Assay (Promega). Paclitaxel, doxorubicin, tunicamycin,

thapsigargin, A23187, and DTT were purchased from Sigma Aldrich. Cmp302 and

Cmp308 were purchased from Biointerscreen.

In vitro wound-healing assay

7.5×105 cells were seeded on 3.5 cm plates 18 hours before wounding. Cells were washed

two times with PBS, re-fed with culture medium, and allowed to migrate for 7 hours

before visualization.

Western blot

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Western blotting was performed as previously described (5). Antibodies used for

immunoblotting were as follows: E-cadherin (BD Transduction, 610182), Fibronectin

(Abcam, Ab6328), Beta-Actin (Cell Signaling Technology, 12620), Beta-Tubulin (Cell

Signaling Technology, 5346), CK8/18 (Cell Signaling Technology, 4546), PERK (Cell

Signaling Technology, 9956), BiP (Cell Signaling Technology, 9956), eIF2 (Cell

Signaling Technology, 9722), p-eIF2 (Cell Signaling Technology, 3597), Caspase-3

(Cell Signaling Technology, 9665), CHOP (Cell Signaling Technology, 5554), p-PERK

(Santa Cruz Biotechnology, Sc-32577).

Flow cytometry analysis

Flow cytometry analysis was performed according to the manufacturer’s protocol (BD

Biosciences), with at least 10000 live events captured per analysis. Reagents used were as

follows: APC-conjugated anti-CD44 antibody (clone G44-26), PE-conjugated anti-CD24

antibody (clone ML5), and propidium iodide (5 g/ml) (BD Bioscience).

ATF6 reporter assay

p5xATF6-GL3 and hRluc constructs were obtained from Addgene (Plasmid 11976 and

24348). 24 hours after co-transfection of 0.3g p5xATF6-GL3 and 0.05g hRluc

plasmids, cells were treated with indicated compounds for an additional 6 hours, after

which ATF6 activity was measured by a dual luciferase assay (Promega).

35S-methionine/cysteine protein labeling

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Equal numbers of cells were cultured in the presence of 35S-methionine/cysteine in

medium with reduced methionine/cysteine content and 0.5% serum. At the indicated time

points, aliquots of medium were extracted for analysis. Medium was centrifuged at 800×g

for 2 min to pellet any whole cell contaminants. An equal volume of medium was

reduced in loading buffer, separated by SDS-PAGE, and analyzed by autoradiography.

Identification of major secreted proteins

10 million HMLE_shGFP and HMLE_shEcad cells were seeded in serum-free medium,

and culture medium was collected at 48 hours. Protein from the culture medium was

precipitated using 10% TCA on ice. After centrifuging at 15000g for 15 min, the pellet

was washed in acetone and dissolved in reducing loading buffer. After separation by

SDS-PAGE, the gel was silver stained, and bands were cut out for analysis by LC-

MS/MS (44).

EMT, ECM and UPR gene-expression correlation in human tumors

Gene-expression sets for correlation analyses were defined as follows: the EMT core

gene set consisted of the top 100 genes upregulated upon EMT induction; the ECM gene

set was obtained from MolSigDB; the XBP1 (GSE40515; (45)) and ATF4 (GSE35681;

(46)) gene sets consisted of the most down-regulated genes in XBP1 and ATF4 knockout

cell lines, relative to the corresponding controls. For every gene set, a composite

expression score was calculated for each sample by summing the log-normalized

expression of the genes in the set. Genes in the EMT gene set that were also present in

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the ECM, XBP1, or ATF4 gene sets were excluded to eliminate any overlaps prior to

calculating correlations. Tumors from five human cancer datasets were analyzed

(GSE41998, GSE37892, GSE26942, GSE4573, GSE11360). Spearman’s rho was used as

the measure of correlation, and for each comparison a p-value was empirically

determined by Monte Carlo sampling to generate a null distribution of 1000 correlations

from random gene sets of the same size as those being compared.

Statistical analysis

All data unless otherwise specified are presented as Mean+S.E.M. from at least 3

independent experiments. A Student’s t-test was performed for comparisons between two

groups of data. Two-way ANOVA tests were performed when comparing the responses

of different groups of cells to various drug treatment doses.

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Acknowledgements

We thank Dr. George Bell and Dr. Inmaculada Barrasa for assistance with dose-response

data analysis, Eric Spooner for mass-spectrometry analysis, Nicki Watson for electron

microscope analysis, Dr. Jan Reiling for helpful discussions and Tom DiCesare for

assistance with graphical design. The p-mGFP-Sec16S, plasmids were kindly provided

by Dr. Benjamin Glick. This research was supported by grants from the Richard and

Susan Smith Family Foundation and the Breast Cancer Alliance.

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Figure & Table Legends

Figure 1. Small molecules with EMT-selective toxicity induce ER stress.

(A) Schematic illustration of large-scale chemical screen.

(B) Microarray analysis was performed on HMLE_shGFP and HMLE_Twist cells treated

with or without Cmp302. GSEA was performed with 4 gene sets (see Methods for

details) on the microarray dataset where Cmp302-induced genes in HMLE_Twist cells

were ordered from largest to smallest.

(C) Expression of UPR pathway components in HMLER_shGFP and HMLER_Twist

cells that were treated with increasing concentrations of Dev2, Cmp302, Dev4 or DMSO

solvent for 6 h. Western blot analysis for phospho-eIF2, total eIF2, CHOP, and -

tubulin. RT-PCR analysis of XBP1 and XBP1 splice variant and GAPDH transcripts.

(D) ATF6 activation of HMLER_shGFP and HMLER_Twist cells in response to

increasing concentrations of Dev2, Cmp302, Dev4 or DMSO solvent for 6 hours. ATF6

activation was measured by an ATF6 reporter assay. Reporter activity for each cell line

was normalized relative to DMSO treatment.

(E) qPCR analysis for BiP expression in HMLE_shGFP, HMLE_Twist, and

HMLE_shEcad cells treated with 4M of Dev2, Cmp302, Dev4 or DMSO solvent for 30

hours. BiP expression was normalized relative to GAPDH for each sample.

* p<0.05; ** p<0.01

Figure 2. EMT sensitizes cells to chemicals that perturb ER function.

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(A) Western blot analysis of HMLER_shGFP and HMLER_Twist cells treated with

vehicle (DMSO) or increasing concentrations of tunicamycin (Tm), thapsigargin (Tg),

DTT or A23187 for 4 h and probed for phospho-eIF2, CHOP, and -tubulin (loading

control). RT-PCR analyses of XBP1 and XBP1 splice variant and GAPDH transcripts.

(B) qPCR analysis of BiP expression in HMLER_shGFP and HMLER_Twist cells

treated with increasing concentration of thapsigargin or DTT. BiP expression was

normalized relative to GAPDH for each sample.

(C) qPCR analysis of GADD34 expression in HMLER_shGFP and HMLER_Twist cells

treated with increasing concentration of thapsigargin or DTT. GADD34 expression was

normalized relative to GAPDH for each sample.

(D) Dose-response curves of HMLER_shGFP (blue circle), HMLER_shEcad (red

square), and HMLER_Twist (black diamond) cells treated with various concentrations of

tunicamycin, thapsigargin, DTT or A23187 for 3 days. Cell survival was determined

using an ATP-based luminescence assay.

(E) Representative fluorescence images and quantification of dsRed-labeled non-EMT

(HMLER_shGFP) and GFP-labeled EMT (HMLER_Twist) cells mixed in 1:1 ratio and

treated with 5 nM thapsigargin, 10 nM thapsigargin, or solvent control for 5 days. Scale

bar: 50 m.

(F) Dose-response curves of four luminal breast cancer cells, MCF7, MDA361, T47D

and ZR-75-3(blue curves), and six basal-B lines, BT549, 4T1, Hs578T, MDA231,

MDA436 and MDA157 (red curves) treated with various concentrations of tunicamycin,

thapsigargin, DTT or A23187 for 3 days. Cell survival was determined using an ATP-

based luminescence assay.

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* p<0.05; ** p<0.01

Figure 3. Cells that undergo an EMT are highly secretory

(A) Expression of twelve genes encoding ECM proteins in EMT cells (HMLE-Gsc,

HMLE-shEcad, HMLE-Snail, HMLE-TGFb, HMLE-Twist) relative to control HMLE

epithelial cells.

(B) Confocal microscopy and quantification of Sec16-GFP localization to ER exit sites in

EMT (HMLE-shEcad) and control (HMLE-Ctrl) cells. Data was presented as Mean+S.D.

Nuclei counterstained with DAPI. Scale bar: 10 m.

(C) Autoradiograph showing 35S-methionine/cysteine-labeled secreted proteins in EMT

(HMLE_shEcad, HMLE_Twist) and control (HMLE_shGFP) cells. Secreted proteins

were harvested at the indicated time points. Quantification of signal in each lane is

provided in arbitrary units.

(D) Representative electron microscopy images and quantification of endoplasmic

reticulum branching in EMT (HMLE_Twist) and non-EMT (HMLE_shGFP) cells.

Arrows: examples of ER branch points in HMLE_Twist cells; scale bar: 500 nm.

(E) Expression of genes encoding secreted ECM proteins in a panel of luminal (N=13,

blue) and basal-B (N=9, red) human breast cancer lines. These data were derived from

GSE16795(24).

(F) Autoradiograph showing 35S-methionine/cysteine-labeled secreted proteins in

Luminal and Basal-B human breast cancer lines. Quantification of signal in each lane is

provided in arbitrary units.

* p<0.05

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Figure 4. ECM secretion upon EMT sensitizes cells to ER stress and promotes

migration

(A) Western blot analysis showing stable shRNA-mediated knockdown of FN1 and

PAI1, individually and in combination, in HMLE_shEcad and HMLE_Twist cells. Two

distinct hairpins were applied per gene. DK-1 refers to double knockdown of FN1 and

PAI1 by hairpins shFN1-1 and shPAI1-1, and DK-2 refers to double knockdown of FN1

and PAI1 by hairpins shFN1-2 and shPAI1-2.

(B) Migratory ability of HMLE_shEcad_shLuc and HMLE_shEcad_DK-1 cells,

HMLE_Twist_shLuc and HMLE_Twist_DK-1 cells was measured using an in vitro

wound-healing assay. Representative images and quantifications at 0 hours and 7 or 8

hours post-wounding are shown.

(C) Expression of UPR pathway components in HMLE_shEcad_shLuc and

HMLE_shEcad_DK-1 cells treated with increasing concentrations of Dev4, thapsigargin,

or DMSO solvent for 6 h. Western blot analysis is shown for phospho-eIF2, total eIF2,

and -tubulin. RT-PCR analysis is shown for unspliced XBP1, spliced XBP1, and

GAPDH transcripts.

(D) Similar analysis of (C) was applied to HMLE_Twist_shLuc and HMLE_Twist_DK-2

cells.

* p<0.05; ** p<0.01

Figure 5. EMT cells require higher BiP expression for their survival

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(A) Left panel, BiP mRNA expression levels in HMLE_ER_Twist cells transduced with

a control hairpin targeting LacZ or two different hairpins targeting BiP. Right panel,

representative growth curves of control (LacZ) or Bip-inhibited (shBip)

HMLE_ER_Twist cells treated with or without 125 nM 4-OHT to induce EMT.

(B) Representative brightfield images of the morphology of control (LacZ) or Bip-

inhibited (shBiP) HMLE_ER_Twist cells treated with or without 4-OHT to induce EMT.

Images were taken 4 days post 4-OHT treatment.

(C) Left panel, quantification of cell viability of non-EMT Luminal breast cancer cell

lines (MCF7, T47D) and EMT Basal-B cell lines (MDA231,BT549) transduced with

control or BiP targeted hairpins. Right panel, RT-PCR expression of CHOP mRNA

expression in cells of the left panel.

(D) Representative brightfield images of the morphology of cell lines in (C), 4 days post

hairpin transduction.

* p<0.05; ** p<0.01

Figure 6. PERK signaling is constitutively activated upon EMT and promotes

malignancy

(A) Western blot analysis of EMT (shEcad, Twist) or control (shGFP) HMLE cells,

luminal (MCF7, T47D, BT474, ZR-75-30) and basal-B human breast cancer cell lines

(SUM159, MDA-MB-231, MDA-MB-157, Hs578T, BT549) for UPR pathway

components; -tubulin is used as a loading control.

34

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(B) Expression of GADD34 in EMT (shEcad, Twist) or control (shGFP) HMLE cells (left

panel), luminal (MCF7, T47D, BT474, ZR-75-3) and basal-B (SUM159, MDA-MB-231,

MDA-MB-157, Hs578T, BT549) human breast cancer lines (right panel).

(C) Cell lysates from control (HMLE_shGFP) and EMT (HMLE_shEcad) cells treated

with or without thapsigargin were collected. The lysates were then treated with or without

lambda-phosphatase and PERK protein expression was analyzed by Western blotting.

(D) Non-EMT (HMLE_shGFP) and EMT (HMLE_Twist) cells were treated with DMSO

control, 40 nM thapsigargin for 2 hours, or 1 μM PERK inhibitor (PERKi) for 24 hours.

Phosphorylated PERK protein was analyzed by immunofluorescence staining.

(E) Non-EMT (HMLE_shGFP) and EMT (HMLE_shEcad, HMLE_Twist) cells were co-

treated with 1.5 nM thapsigargin and 0, 0.5 μM or 1 μM of PERK inhibitor (PERKi) for

6 days. Surviving cells were quantified by cell counting. Data are represented as

Mean+S.E. from three replicates.

(F) HMLE_shGFP and HMLE_shEcad cells were pretreated with 1 M PERK inhibitor

or vehicle control (DMSO) for 2 days prior to tumoursphere formation assay. The PERK

inhibitor –pretreated cells were then cultured in tumoursphere-forming condition for

another 4 days in the presence of 1 M PERK inhibitor, while the vehicle-pretreated cells

were cultured in drug-free condition for the same period of time. Representative bright

field images and quantification were shown. Scale bar: 50 m

(G) Representative images of crystal violet staining of HMLE_shGFP and

HMLE_shEcad cells pretreated with or without 1 M PERK inhibitor for 2 days

following transwell migration assay. Cells that migrated within 8 hour following seeding

into 8 m pore inserts were visualized and quantified. Scale bar: 50 m

35

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(H) 4T1 cells were treated with DMSO control, 1 M PERK inhibitor for 3 days, or 2 nM

thapsigargin for 3 days followed by a 4 day-recovery period in drug-free media. 2×106

cells were injected into NOD/SCID mice via the tail vein, and lung tissues were

harvested 15 days post injection. Representative images of mouse lung tissue stained with

H&E were shown. Quantification of metastasis is also shown (5 mice per condition).

* p<0.05; ** p<0.01

Figure 7. EMT correlates with PERK but not IRE1 signaling in primary human

tumors

(A) Two primary breast cancer cells (BT5104 and BT5094) were freshly collected from

patient ascites, and cell lysate were collected and analyzed by western blotting for

differentiation markers.

(B) Western blot analysis of non-EMT-like BT5104 cells and EMT-like BT5094 cells

treated with 0, 5, and 10 nM of thapsigargin for 2 days for expression of PERK, BiP,

phos-eIF2, eIF2 and -actin.

(C) Dose-response curves of non-EMT-like BT5104 and EMT-like BT5094 cells treated

with increasing concentrations of tunicamycin or thapsigargin for 3 days. Cell survival

was determined using an ATP-based luminescence assay. Data are represented as

Mean+S.D. from three replicates.

(D) Correlation analyses of expression of EMT genes and ECM genes, XBP1, or ATF4-

targeted genes in breast cancers (GSE41998, n=255), colon cancers (GSE37892, n=130),

gastric cancers (GSE26942, n=90), lung cancers (GSE4573, n=130) and metastatic

cancers with various origins (GSE11360, n=187).

36

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34 4 10 BiP expre ADD 5 2 G

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Fig. 6

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Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2014 American Association for Cancer Research. Fig. 7

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15 15 rho=0.90, p<0.001 15 rho=0.93, p<0.001 rho=0.87, p<0.001 6 rho=0.92, p<0.001 10 rho=0.84, p<0.001

10 10 10 4 EMT vs ECM 5 5 5 2 5

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5 2 -10 -5 5 10 15 20 -10 10 20 30 -5 5 101520 -10 0 10 20 30 -5 0 5 10 15 20 -5 -5 -5 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 Feng, Ethan S Sokol, Catherine A Del Vecchio, et al.

Cancer Discovery Published OnlineFirst April 4, 2014.

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