Epithelial -to-Mesenchymal Transition Activates PERK–eIF2a ... · constitutively activate the PERK–eIF2α axis of the unfolded protein response (UPR). Protein kinase RNA-like

702 | CANCER DISCOVERY�JUNE 2014 www.aacrjournals.org

RESEARCH ARTICLE

Epithelial -to-Mesenchymal Transition Activates PERK–eIF2a and Sensitizes Cells to Endoplasmic Reticulum Stress Yu-xiong Feng 1 , Ethan S. Sokol 1 , 2 , Catherine A. Del Vecchio 1 , Sandhya Sanduja 1 , Jasper H.L. Claessen 1 , Theresa A. Proia 1 , Dexter X. Jin 1 , 2 , Ferenc Reinhardt 1 , Hidde L. Ploegh 1 , 2 , Qiu Wang 6 , and Piyush B. Gupta 1 , 2 , 3 , 4 , 5

Authors’ Affi liations: 1 Whitehead Institute for Biomedical Research; 2 Department of Biology, Massachusetts Institute of Technology; 3 Koch Institute for Integrative Cancer Research; 4 Harvard Stem Cell Institute; 5 Broad Institute, Cambridge, Massachusetts; and 6 Department of Chemis-try, Duke University, Durham, North Carolina

Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/).

Corresponding Author: Piyush B. Gupta, Whitehead Institute for Biomedi-cal Research, 9 Cambridge Center, Cambridge, MA 02142. Phone: 617-258-7778; Fax: 617-258-7226; E-mail: [email protected]

doi: 10.1158/2159-8290.CD-13-0945

©2014 American Association for Cancer Research.

ABSTRACT Epithelial-to-mesenchymal transition (EMT) promotes both tumor progression and

drug resistance, yet few vulnerabilities of this state have been identifi ed. 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–eIF2α axis of the unfolded protein response (UPR). Protein kinase

RNA-like ER kinase (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–eIF2α genes, but not

with other branches of the UPR. Taken together, our fi ndings 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 fi ndings identify a previously unrecognized vulnerability

of cancer cells that have undergone an EMT: sensitivity to ER stress. We also fi nd that PERK–eIF2α

signaling, which is required to maintain ER homeostasis, is also indispensable for EMT cells to invade

and metastasize. Cancer Discov; 4(6); 702–15. ©2014 AACR.

Research. on December 10, 2020. © 2014 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from

Published OnlineFirst April 4, 2014; DOI: 10.1158/2159-8290.CD-13-0945

http://cancerdiscovery.aacrjournals.org/

JUNE 2014�CANCER DISCOVERY | 703

INTRODUCTION

Carcinoma cells acquire key malignant traits by reprogram-

ming their differentiation state via an epithelial-to-mesen-

chymal transition (EMT; refs. 1, 2 ). This transdifferentiation

program, which was fi rst described in developmental contexts, is

phenotypically characterized by repression of epithelial markers,

upregulation of mesenchymal markers, and changes in mor-

phology associated with cell migration. EMT can be induced

experimentally by overexpression of transcription factors, such

as Snail or Twist, and, in some contexts, by treatment with

TGFβ. Cancer cells that undergo an EMT become invasive and

drug-resistant; such cells also effi ciently seed primary and meta-

static tumors, making them functionally indistinguishable from

tumor-initiating cells or cancer stem cells (TIC or CSC; refs. 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, fi bronectin ( FN1 ), plas-

minogen activator inhibitor 1 ( PAI1 ), and periostin ( POSTN ;

ref. 6 ), interact to form networks that provide tensional

forces and signals that are essential for migration. These qua-

ternary interactions are often initiated within the cell before

secretion. For example, collagens are partially assembled into

triple-helical fi bers within the endoplasmic reticulum (ER)

before their secretion into the extracellular space.

Cells have evolved several quality control pathways that

maintain ER homeostasis, collectively termed the unfolded

protein response (UPR; ref. 7 ). The UPR is activated by mis-

folded proteins within the ER, which accumulate upon nutri-

ent deprivation, hypoxia, oxidative stress, or viral infection

( 8–13 ). UPR signaling is initiated by three receptors local-

ized to the ER membrane—ER-to-nucleus signaling 1 (ERN1/

IRE1α), protein kinase RNA-like ER kinase (PERK), and ATF6

( 14, 15 ). These receptors converge on shared downstream fac-

tors that increase ER protein-folding capacity, including BiP/

GRP78 and GRP94; they also have unique signaling effects,

namely, activated IRE1α 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)];

and activated PERK phosphorylates eIF2α, inducing an inte-

grated 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 progres-

sion 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.





Feng et al.RESEARCH ARTICLE

RESULTS Chemical Probes Selectively Activate ER Stress in EMT Cells

To identify probes of EMT cell biology, we previously per-

formed a large-scale chemical screen for small molecules with

selective toxicity toward EMT cells ( 5 , 16–18 ). Of 315,000 com-

pounds tested, this screen identifi ed a few structurally related

small molecules (Cmp302, Cmp308, and Dev4) with EMT-

selective toxicity ( Fig. 1A ). These compounds exhibited between

20-fold and >100-fold selective toxicity toward nontumorigenic

(HMLE) and tumorigenic (HMLER) human mammary epithe-

lial 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

cocultures with Cmp302, Cmp308, or Dev4 selectively depleted

GFP-EMT cells from the cocultures, further confi rming the

selective toxicity of these compounds. In contrast, two common

chemotherapy drugs, paclitaxel and doxorubicin, caused enrich-

ment of GFP-EMT cells within cocultures ( Supplementary

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 four gene sets (see Methods for details) on the microarray dataset where Cmp302-induced genes in HMLE_Twist cells were ordered from largest to smallest. (continued on following page)

0.8

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Enrichm

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Gene set doxorubicin, P = 0.058


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A Compound library (~315,000)

EMT cellsNon-EMT cells

EMT-selective compounds

Cl

O

O

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NNH

Cl O

O

O

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NNH

Cl

Cl

O

O

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NH

N

Cl

Dev4Cmp308Cmp302

B

Gene set hypoxia, P = 0.19

Gene set tunicamycin, P < 0.001 Gene set thapsigargin, P < 0.001





EMT Activates PERK and Sensitizes Cells to ER Stress RESEARCH ARTICLE

Fig. S1C and S1D); this was consistent with previous reports

indicating that EMT cells resist chemotherapies ( 5 , 19 ). The sub-

stitution of a single atom in the pyrrolidine group of Cmp302

was suffi cient to completely abolish toxicity (compound Dev2;

Supplementary Fig. S1E and S1F; ref. 18 ).

We assessed whether these compounds were also selectively

toxic toward breast cancer cells induced into EMT without

any genetic modifi cations. Relative to cells in adherent cul-

ture, MDA-MB-157 cells cultured in suspension undergo an

EMT as gauged by epithelial marker repression, upregulation

of mesenchymal markers, acquisition of a mesenchymal mor-

phology, and expression of stem-like surface markers (Sup-

plementary Fig. S1G–S1I). In comparison with cells grown

in adherent culture, MDA-MB-157 cells induced through an

Dev2 (μmol/L)

Dev4 (μmol/L)

Cmp302 (μmol/L) 20

HM

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20

Figure 1. (Continued) C, expression of UPR pathway components in HMLER_shGFP and HMLER_Twist cells that were treated with increasing con-centrations of Dev2, Cmp302, Dev4, or DMSO solvent for 6 hours. Western blot analysis for phospho-eIF2α (p-eIF2α), total eIF2α, CHOP, and β-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 4 μmol/L 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.






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-selec-

tive compounds, we used microarrays to profi le 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; Supplementary Table

S1A). This suggested that Cmp302 was selectively inducing

ER stress in EMT cells. Gene set enrichment analysis (GSEA)

demonstrated that Cmp302 signifi cantly upregulated—selec-

tively 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 and Supplementary Table

S1B). In contrast, Cmp302 did not upregulate the expression

of genes induced by either hypoxia or doxorubicin treatment

( Fig. 1B , bottom and Supplementary Table S1B).

To more directly assess this hypothesis, we determined

whether compound treatment affected UPR signaling path-

ways 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, eIF2α 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 toward EMT cells. In contrast, the nontoxic struc-

tural analog, Dev2, did not activate UPR signaling in EMT

or non-EMT cells ( Fig. 1C–E and Supplementary Fig. S2C).

Collectively, these fi ndings strongly suggested that

Cmp302/Dev4 was 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 com-

pounds caused activation of the PERK and IRE1 branches

of the UPR at 8-fold to 100-fold lower doses in EMT versus

non-EMT cells, as gauged by phosphorylation of eIF2α and

splicing of XBP1 , respectively ( Fig. 2A ). All four ER stres-

sors also activated the downstream UPR factors CHOP, BiP,

and GADD34 at lower doses in EMT versus 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 nontumori-

genic (HMLE) lines (∼10-fold for tunicamycin, ∼25-fold for

thapsigargin, ∼4-fold for DTT, and ∼8-fold for A23187; Fig.

2D ; Supplementary Fig. S2B). Cells induced to undergo EMT

by TGFβ treatment also showed increased sensitivity to tuni-

camycin and thapsigargin (Supplementary Fig. S2C). Thap-

sigargin also selectively eliminated EMT breast cancer cells

from cocultures of GFP-labeled EMT (HMLER_Twist_GFP)

and DsRed-labeled non-EMT cells (HMLER_shGFP_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 fi ndings, we assessed

whether sensitivity to ER stressors correlated with the dif-

ferentiation 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 sub-

types. Compared with the four luminal breast cancer lines,

the six basal-B cell lines were signifi cantly more sensitive to

tunicamycin, thapsigargin, DTT, and A23187 ( Fig. 2F and

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 an EMT.

Cells That Undergo an EMT Are Highly Secretory To identify molecular factors underlying this increased ER

stress sensitivity, we compared global transcriptional profi les

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 ). ECM and secreted col-

lagen gene sets were the most signifi cantly 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 involved

in secretory pathway components (SPCG; ref. 30 ) were upreg-

ulated in at least four of the fi ve EMT lines relative to non-

EMT controls ( P < 1 × 10 −10 with sign test; Supplementary Fig.

S3B). Moreover, in highly secretory cells, increased ER capac-

ity gene expression occurs together with increased vesicular

transport from the ER to cis- Golgi. To assess vesicular fl ux,

we transiently expressed GFP fused with Sec16, a core com-

ponent of ER exit sites (ERES; ref. 31 ), and visualized ERES

by confocal microscopy ( 32, 33 ). Quantifi cation of Sec16-GFP

foci revealed a signifi cant 3-fold increase in ERES in EMT cells

relative to controls, indicative of increased ER-to-cis-Golgi

vesicular fl ux ( Fig. 3B and Supplementary Fig. S3C).

To directly quantify secreted proteins, we used 35 S-methio-

nine/cysteine to label secreted proteins, which were then

harvested from the culture medium and visualized by gel elec-

trophoresis and autoradiography. EMT cells (HMLE_shEcad,

HMLE_Twist) exhibited an approximately 10-fold to 14-fold

increase in total secreted proteins relative to isogenic con-

trol cells ( Fig. 3C ). As a control to confi rm 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 pro-

teins in the culture medium (Supplementary Fig. S3D).

Along with the altered protein secretion capacity between

EMT and control cells, signifi cant differences in ER morphol-

ogy 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






Figure 2. EMT sensitizes cells to chemicals that perturb ER function. 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 hours and probed for phospho-eIF2α (p-eIF2α), CHOP, and β-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 concentrations 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 concentrations 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 fl uorescence images and quantifi cation of dsRed-labeled non-EMT (HMLER_shGFP) and GFP-labeled EMT (HMLER_Twist) cells mixed in 1:1 ratio and treated with 5 nmol/L thapsigargin, 10 nmol/L thapsigargin, or solvent control for 5 days. Scale bar, 50 μm. 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. *, P < 0.05; **, P < 0.01.

A23187 [mol/L]DTT [mol/L]

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Figure 3. Cells that undergo an EMT are highly secretory. A, expression of 12 genes encoding ECM proteins in EMT cells (HMLE_Gsc, HMLE_shEcad, HMLE_Snail, HMLE_TGFβ, and HMLE_Twist) relative to control HMLE epithelial cells. B, confocal microscopy and quantifi cation of Sec16-GFP localization to ERES in EMT (HMLE_shEcad) and control (HMLE_Ctrl) cells. Data were presented as mean +/− SD. Nuclei counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 10 μm. C, autoradiograph showing 35 S-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. Quantifi cation of signal in each lane is provided in arbitrary units. D, representa-tive electron microscopy images and quantifi cation of ER 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 35 S-methionine/cysteine–labeled secreted proteins in luminal and basal-B human breast cancer lines. Quantifi cation of signal in each lane is provided in arbitrary units. *, P < 0.05.

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non-EMT cells had ER membranes with one or more branch

points ( Fig. 3D ). Because professional secretory cells (e.g., pan-

creatic β 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 whether increased ECM secretion is a general

feature of EMT, we examined the expression of ECM genes iden-

tifi ed to be upregulated upon EMT ( Fig. 3A ) in basal-B and lumi-

nal 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 signifi cantly

higher levels than luminal breast cancer lines ( n = 13; Fig. 3E ; ref.

24 ). In a subset of basal-B lines, EMT ECM genes were expressed

at 10-fold to 100-fold higher levels than in luminal cancer lines

(Supplementary Table S2; ref. 24 ). In contrast, ECM genes

not upregulated upon EMT did not exhibit increased expres-

sion ( COL4A3 , COL10A1 , COL13A1 , and COL15A1 ; Fig. 3E ). In

support of these observations, 35 S-methionine/cysteine labe-

ling showed that basal-B lines (Hs578T, BT549, MDA-MB-157,

SUM159, MDA-MB-231, and 4T1) also exhibited, on average, a

more than 40-fold increase in protein secretion relative to lumi-

nal lines (MCF7, T47D, BT474, and 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 activa-

tion in response to ER stressors. To examine this, we analyzed

the proteins secreted by two nontumorigenic EMT lines

(HMLE_shEcad and HMLE_Twist) that, by 35 S-methionine/

cysteine labeling, strongly upregulated secretion of a limited

number of proteins ( Fig. 3C ). Mass spectrometry of condi-

tioned medium from these two EMT-associated lines revealed

that, relative to the corresponding controls, they secreted two

major proteins, PAI1 and FN1.

We next inhibited PAI1 and FN1, both singly and in com-

bination, 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 whether the reduction in PAI1 and FN1 levels was

biologically signifi cant, we assessed the migratory properties of

double-knockdown cells. Dual inhibition of PAI1 and FN1 also






signifi cantly 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 D , HMLE_shEcad and HMLE_Twist cells, respectively;

Supplementary Fig. S4C). These fi ndings 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 (4-OHT; HMLE_ER_Twist;

ref. 3 ). Using two different shRNAs, a 65% to 75% reduction

in BiP had negligible effects on the viability of this line in the

uninduced (non-EMT) condition ( Fig. 5A and B ). However,

induction of EMT in these shBiP lines caused signifi cant

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, although suffi cient for the needs of epithelial cells,

were not suffi cient 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 signifi cant death in

two basal lines (MDA-231 and BT549) together with CHOP

upregulation ( Fig. 5C and D ), suggesting that ER stress was

more readily induced in BiP-defi cient EMT cells. This was

confi rmed 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–eIF2a–ATF4 Signaling Is Activated upon EMT and Promotes Malignancy

Before their differentiation, progenitors of secretory cells

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 whether, in the absence

of ER stressors, they also activate one or more UPR pathways.

Compared with non-EMT cells, EMT cells had reduced PERK

protein mobility suggestive of its phosphorylation, increased

eIF2α 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 stressors (Supplementary Fig. S6A).

To confi rm that PERK was in fact phosphorylated in EMT cells,

we also performed phosphatase treatments and immunofl uo-

rescence with a phospho-specifi c PERK antibody. Treatment

of lysates with lambda phosphatase before Western blotting

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 quantifi cations at 0 hours and 7 or 8 hours postwounding 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 hours. Western blot analysis is shown for phospho-eIF2α (p-eIF2α), total eIF2α, and β-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 < 0.01.

FN1

HMLE_shEcad

HM

LE_s

hEca

d

HM

LE_T

wist

Ctrl CtrlshLuc

shLuc

shLuc

shFN1-1

shFN1-2

shPAI1

-1

shPAI1

-2

DK-1DK-1

DK-1

shLuc

DK-2

DK-2DK-2

HMLE_Twist

PAI1

Tubulin

0 h

7 h

0 h7 h

HMLE_shEcad_shLuc HMLE_shEcad_DK-1

0 h

8 h

0 h8 h

HMLE_Twist_shLuc HMLE_Twist_DK-1

p-eIF2α

eIF2α

Tubulin

Tg (nmol/L)

shLuc DK-1 shLuc DK-1

Dev4 (μmol/L)

2.5 5 10 20

5

1 2.5 5 10 1 2.5 5 10

10 20 40 5 10 20 40

2.5 5 10 20

XBP1

GAPDH

0

0.4

0.8

1.2

Rela

tive c

ell

mig

ration

shLuc DK-1

****

p-eIF2α

eIF2α

Tg (nmol/L)

shLuc DK-2 shLuc DK-2

Dev4 (μmol/L)

5 10 20 40 5 10 20 40

XBP1

GAPDH

A

B

C

D

Tubulin






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 thapsi-

gargin in both EMT and non-EMT cells ( Fig. 6C ). Immunofl uo-

rescence with a phosphorylation-specifi c antibody also showed

that PERK was constitutively activated upon EMT, but not in

non-EMT cells ( Fig. 6D ). Consistent with these fi ndings, cells

induced through an EMT by TGFβ treatment also activated

PERK but not IRE1 signaling (Supplementary Fig. S6B).

Because there are several kinases upstream of eIF2α, we

next examined whether its phosphorylation in EMT cells

was dependent on PERK. Suppression of PERK activity with

a specifi c chemical inhibitor strongly decreased both PERK

and eIF2α phosphorylation in EMT cells (Supplementary Fig.

S6C). Similarly, PERK inhibition by shRNA also decreased

eIF2α phosphorylation in two basal-B breast cancer lines

( Fig. 6E ). Collectively, these observations established that

the PERK–eIF2α–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 pro-

mote 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 thapsi-

gargin ( Fig. 6F ), indicating that activation of the PERK path-

way is adaptive and benefi cial for the survival of cancer cells

that have undergone an EMT. We next examined whether

PERK signaling also contributed to the malignant properties

of EMT cells. PERK inhibition strongly reduced the ability

of EMT cells to form tumorspheres ( Fig. 6G ) and migrate in

Transwell assays ( Fig. 6H ); at the same dose, the PERK inhibi-

tor minimally affected cell proliferation (Supplementary Fig.

S7A and S7B). Pretreatment of metastatic 4T1 cells with either

the PERK inhibitor or thapsigargin resulted in signifi cantly

Figure 5. EMT cells require higher BiP expression for their survival. A, left, BiP mRNA expression levels in HMLE_ER_Twist cells transduced with a control hairpin targeting LacZ or two different hairpins targeting BiP. Right, representative growth curves of control (LacZ) or BiP-inhibited (shBiP) HMLE_ER_Twist cells treated with or without 125 nmol/L 4-OHT to induce EMT. B, representative bright-fi eld 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 after 4-OHT treatment. C, left, quantifi cation of cell viability of non-EMT luminal breast cancer cell lines (MCF7 and T47D) and EMT Basal-B cell lines (MDA231,BT549) transduced with control or BiP-targeted hairpins. Right, RT-PCR expression of CHOP mRNA expression in cells of the left. D, representative bright-fi eld images of the morphology of cell lines in C, 4 days after hairpin transduction. *, P < 0.05; **, P < 0.01.

A

D

B

C

0

4

8

12

MCF7 T47D MDA-231 BT549

CH

OP

exp

ressio

n

0

7

14

0 3 6 9

Nu

mb

er

of

ce

lls (

Lo

g2)

Days

shLacZ

shBiP-1

shBiP-2

shLacZ_4OHT

shBiP-1_4OHT

shBiP-2_4OHT

0

0.4

0.8

1.2

HMLE_ER_Twist

BiP

exp

ressio

nshLacZ

shBiP-1

shBiP-2

0

0.4

0.8

1.2

MCF7 T47D MDA-231

****

****

**

**

*

*

BT549

Re

lative

ce

ll su

rviv

al

shLuc shBiP-1 shBiP-2 shLuc shBiP-1 shBiP-2

shLacZ shBiP-1 shBiP-2

DM

SO

4-O

HT

shLuc shBiP-1 shBiP-2

MC

F7

MD

A-2

31

diminished metastatic capacity, as assessed by lung tumor bur-

den 15 days after tail-vein injection ( Fig. 6I ). Collectively, these

fi ndings indicated that disruption of the PERK pathway sig-

nifi cantly compromises the malignant phenotype of EMT can-

cer 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 fi nd-

ings by assessing primary human tumors. Primary cancer

cells (<3 passages) from breast tumors expressing EMT mark-

ers had elevated PERK and BiP expression, and increased

eIF2α phosphorylation, when compared with primary breast

cancer cells that did not express EMT markers ( Fig. 7A and B ).

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 signifi cantly reduced viability upon treatment

with ER stressors ( Fig. 7C and Supplementary Fig. S8).

To assess whether these fi ndings extended to other tumor

types, we analyzed gene-expression microarray data from

patient tumors to test for associations between the expres-

sion 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 fi ve datasets spanning 792 breast,

colon, 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), whereas a signifi cant

correlation was not observed between the expression of EMT

and XBP1 genes (mean corr. = −0.14; Fig. 7D ). These fi ndings

established that EMT is strongly associated with PERK but

not IRE1 signaling across a spectrum of tumor types.






Figure 6. PERK signaling is constitutively activated upon EMT and promotes malignancy. A, Western blot analysis of EMT (shEcad and Twist) or control (shGFP) HMLE cells, luminal (MCF7, T47D, BT474, and ZR-75-30) and basal-B human breast cancer cell lines (SUM159, MDA-MB-231, MDA-MB-157, Hs578T, and BT549) for UPR pathway components; β-tubulin is used as a loading control. B, expression of GADD34 in EMT (shEcad and Twist) or control (shGFP) HMLE cells (left), luminal (MCF7, T47D, BT474, and ZR-75-3) and basal-B (SUM159, MDA-MB-231, MDA-MB-157, Hs578T, and BT549) human breast cancer lines (right). C, cell lysates from control (HMLE_shGFP) and EMT (HMLE_shEcad) cells treated with or without thapsigargin were col-lected. 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 nmol/L thapsigargin for 2 hours, or 1 μmol/L PERK inhibitor (PERKi) for 24 hours. Phosphorylated PERK (p-PERK) protein was analyzed by immunofl uorescence staining. E, Western blotting for PERK and phospho-eIF2α expres-sion in cell lysates from the Hs578T and SUM159 lines infected with control (shCtrl) or PERK-specifi c (shPERK) hairpins. F, non-EMT (HMLE_shGFP) and EMT (HMLE_shEcad and HMLE_Twist) cells were cotreated with 1.5 nmol/L thapsigargin and 0, 0.5 or 1 μmol/L of PERK inhibitor for 6 days. Surviving cells were quantifi ed by cell counting. Data are represented as mean + SE from three replicates. G, HMLE_shGFP and HMLE_shEcad cells were pretreated with 1 μmol/L PERK inhibitor or vehicle control (DMSO) for 2 days before tumorsphere formation assay. The PERK inhibitor–pretreated cells were then cultured in tumorsphere- forming condition for another 4 days in the presence of 1 μmol/L PERK inhibitor, while the vehicle-pretreated cells were cultured in drug-free conditions for the same period of time. Representative bright-fi eld images and quantifi cation were shown. Scale bar, 50 μm. H, representative images of crystal violet staining of HMLE_shGFP and HMLE_shEcad cells pretreated with or without 1 μmol/L PERK inhibitor for 2 days following Transwell migra-tion assay. Cells that migrated within 8 hours following seeding into 8-μm pore inserts were visualized and quantifi ed. Scale bar, 50 μm. I, 4T1 cells were treated with DMSO control, 1 μmol/L PERK inhibitor for 3 days, or 2 nmol/L thapsigargin for 3 days followed by a 4-day recovery period in drug-free media. A total of 2 × 10 6 cells were injected into NOD/SCID mice via the tail vein, and lung tissues were harvested 15 days after injection. Representative images of mouse lung tissue stained with H&E are shown. Quantifi cation of metastasis is also shown (5 mice per condition). *, P < 0.05; **, P < 0.01.

0

2

4

6

8

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12

14

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1

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2

2.5

*

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DD

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xp

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n

A

C

B

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PH

ML

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ist

p-PERK/DAPI

λ-Phosphatase

Thapsigargin – – – –

––– –

+ + + +

++++

PERK

Treatment with 1.5 nmol/L Tg

0

0.5

1

1.5

Ce

ll su

rviv

al

DMSO PERKi 0.5 μmol/L PERKi 1 μmol/L

** ** ** **

HMLE

PERK

p-eIF2α

eIF2α

Tubulin

Luminal Basal-B

0

5

10

15

Sphere

s/f

ield

DMSO

PERKi

**

DMSO PERKi

HM

LE

_shG

FP

HM

LE

_shE

cad

0

20

40

60

80

100

120

140

Rela

tive c

ell

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ration

DMSO

PERKi

**

DMSO PERKi

HM

LE

_shG

FP

HM

LE

_shE

cad

F

G

DMSO PERKi Thapsigargin

0 0.4 0.8 1.2

DMSO

PERKi

Thapsigargin

Relative area of metastasis

**

**

H

I

Hs578T SUM159

PERK

p-eIF2α

Tubulin

shG

FP

shC

trl

shPER

Ksh

Ctrl

shPER

K

HM

LE_s

hGFP

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LE_s

hGFP

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LE_s

hGFP

HM

LE_s

hEca

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LE_T

wist

HM

LE_s

hEca

d

HM

LE_s

hGFP

HM

LE_s

hEca

d

HM

LE_T

wist

MCF7

T47D

BT474

ZR-7

5-3

SUM

159P

T

MDA-M

B-231

MDA-M

B-157

Hs5

78T

BT549

HM

LE_s

hEca

dHM

LE_s

hGFP

HM

LE_s

hEca

d

shEca

dTw

ist

MC

F7

T47

D

BT47

4ZR

-75-

3SU

M15

9M

DA-2

31M

DA-1

57H

s578

TBT54

9






DISCUSSION

Given the central role of EMT in tumor metastasis and ther-

apy resistance, there is a vital need to identify pathways and proc-

esses 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 profi ling. This revealed that EMT cells,

by virtue of their increased secretion of ECM, are highly sensitive

to ER stress. This fi nding 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 infl u-

ence its sensitivity to chemicals that cause ER stress.

Our fi ndings are consistent with prior studies linking EMT

induction with ECM secretion. However, although the impor-

tance of ECM for tumor progression is well established ( 36 ),

our study is the fi rst to suggest that ECM secretion, while

promoting malignancy, also creates a key cellular vulnerabil-

ity. 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 activation

by EMT cells of PERK–eIF2α–ATF4 signaling, but not the IRE1

branch of the UPR, raises the possibility that this branch may

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 lysates 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 nmol/L of thapsigargin for 2 days for expression of PERK, BiP, phospho-eIF2α (p-eIF2α), and β-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 +/− SD from three replicates. D, correlation analyses of expression of EMT genes and ECM genes, XBP1-targeted genes, 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 of various origins (GSE11360; n = 187).

–5 5 10 15 20–1

1

2

3

4

5

–5 0 5 10 15 20

2

4

6

8

10

–5 5 10 15 20

–2

2

4

6

–10 –5 5 10 15 20

–6

–4

–2

2

4

–10 –5 5 10 15 20

–5

5

10

15

–10 –5 5 10 15 20 10 20 30

–5 –5

5

10

15

5

10

15

5

10

15ρ = 0.90, P < 0.001

–10 10 20 30

–4

–2

2

4

6

–10 0 10 20 30

5

10

15

20

–10

ρ = 0.93, P < 0.001

–10 10 20 30

–5

5

10

–10 10 20 30

–5

5

10

15

–5 0 5 10 15 20 25

ρ = 0.87, P < 0.001 ρ = 0.92, P < 0.001 ρ = 0.84, P < 0.001

ρ = –0.01, P = 0.945ρ = 0.08, P = 0.690ρ = –0.18, P = 0.388ρ = –0.42, P = 0.02ρ = –0.36, P = 0.062

ρ = 0.80, P < 0.001 ρ = 0.87, P < 0.001 ρ = 0.88, P < 0.001 ρ = 0.68, P < 0.001 ρ = 0.75, P < 0.001

–5 5 10 15 20

–6

–4

–2

2

4

–5 5 10 15 20

–5

5

10

15

–5 5 10 15 20

–5

5

10

EMT vs. ECM

EMT vs. XBP1

EMT vs. ATF4

Breast cancer Colon cancer Gastric cancer Lung cancer Metastasis mix

Ncad

Vimentin

Slug

CK14

PAI1

FN1

CK8/18

Tubulin

PERK

BT51

04BT50

94

BT51

04BT50

94

BT51

04BT51

04BT50

94BT50

94

BiP

p-eIF2α

CHOP

β-Actin

Thapsigargin (nmol/L) 0 0 5 10 5 10

Thapsigargin [mol/L]

Fra

ctio

n s

urv

ivin

g

–0.5

0.0

0.5

1.0

1.5

–0.5

0.0

0.5

1.0

1.5

Tunicamycin [mg/mL]

Fra

ctio

n s

urv

ivin

g

BT5104BT5094

BT5104BT5094

BA

D

C

α-SMA






be specifi cally required for ECM production. In support of this

notion, mouse models have shown that PERK defi ciency specifi -

cally compromised ECM production by osteoblasts ( 38 ); in con-

trast, XBP1 loss prevented the maturation of antibody-secreting

plasma cells ( 40 ). Because PERK is activated in both cancerous

and noncancerous cells following EMT, it may contribute to

normal (non-neoplastic) functions of the EMT state. For exam-

ple, during wound healing, epithelial cells must undergo EMT

to secrete new ECM and migrate to close the wound, and inter-

fering with EMT induction signifi cantly impairs this process

( 41 ). If PERK signaling is required for ECM secretion during

wound healing, its activation in EMT cancer cells may be a con-

sequence 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 profi ling of CSCs

has revealed signifi cant 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 eIF2α phosphorylation (data not shown).

The fi nding 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 mitigate tumor malignancy. In addition, ER

stressors may effectively target disseminated cancer cells that

have undergone EMT; they may also prove useful for eradicat-

ing 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.

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 laboratory, and maintained

in a 1:1 mixture of DMEM + 10% FBS, insulin (10 μg/mL), hydrocor-

tisone (0.5 μg/mL), EGF (10 ng/mL), and Mammary Epithelial Cell

Growth Medium (MEGM). The HMLE/HMLER_shEcad 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-75-3, Hs578T, MDA-MB-157, and MDA-MB-231 cells

were obtained from 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 μg/mL), and hydrocortisone (0.5 μg/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 pre-

viously reported ( 18 ) and were identifi ed in a Molecular Libraries

Probe Production Centers Network screen conducted at the Broad

Institute (Cambridge, MA).

Mammosphere formation assays were performed as described

previously ( 5 ), but with 0.6% methylcellulose (R&D Systems) added

to the medium. Five thousand cells were plated per well in low-

adherence, 24-well plates and cultured for 5 to 8 days before being

counted and photographed.

GSEA For analysis of Cmp302-treated expression data, Tm, Tg, and Dox

gene sets were defi ned 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 dimethyloxalylglycine

(DMOG) treatment (GSE3188).

Microarray Analysis HMLE_shGFP and HMLE_Twist cells were treated with 5 or 10

μmol/L of Cmp302, or DMSO solvent for 6 hours. Total RNA were

extracted using the Qiagen RNeasy Kit, and the integrity and quality

of the RNA met the quality requirements for Human Genome U133

Plus 2.0 arrays (Affymetrix, Inc.) recommended by the company.

All of the subsequent experimental procedures, including labeling,

hybridization, and scanning, were processed according to the stand-

ard Affymetrix protocols. Raw CEL fi les were generated by Affymetrix

GCOS 1.2 software, and the present/absent calls were defi ned with

global scaling to target value of 500. By R software, the CEL fi les were

normalized to a median-intensity array, and model-based expression

values were calculated using PM/MM difference model. Alteration

of gene expression by Cmp302 was calculated by comparing the

expression of each gene in the DMSO- and Cmp302-treated groups,

in both HMLE_shGFP and HMLE_Twist cells. The gene-expression

data have been deposited in the public database Gene Expression

Omnibus (GEO; GSE55604).

Electron Microcopy Analysis Cells were fi xed in 2.5% gluteraldehyde, 3% paraformaldehyde with

5% sucrose in 0.1 mol/L sodium cacodylate buffer (pH 7.4), pelletted,

and postfi xed in 1% OsO 4 in veronal-acetate buffer. The 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 80 kV and

photographed with an AMT CCD camera.

Detection of ERES HMLE and HMLE_shEcad cells (2 × 10 5 /well of a 6-well plate) were

transfected with 1-μg pmGFP-Sec16S (Addgene 15775) using 2.5 μL

of FuGENE (Roche). Twenty-four hours after transfection, cells were

replated and allowed to adhere onto cover slides before fi xation with

4% paraformaldehyde, and confocal imaging. Images were captured

in accordance with the manufacturer’s protocols (PerkinElmer).

Animal Experiments NOD/SCID mice were purchased from The Jackson Laboratory. All

mouse procedures were approved by the Animal Care and Use Com-

mittees of the Massachusetts Institute of Technology (Cambridge,

MA). For lung metastasis analysis, 2 × 10 6 cancer cells were suspended

in 100 μL 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 μL of medium per well in 96-well plates,

at a density of 3,000 cells per well. Twenty-four hours after seeding,






compounds were added at eight different doses with three repli-

cates 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 hours 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 Interbioscreen.

In Vitro Wound-Healing Assay A total of 7.5 × 10 5 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 Analysis Western blotting was performed as previously described ( 5 ). Antibodies

used for immunoblotting were as follows: E-cadherin (BD Transduction;

610182), fi bronectin (Abcam; Ab6328), β-actin (Cell Signaling Technol-

ogy; 12620), β-tubulin (Cell Signaling Technology; 5346), CK8/18 (Cell

Signaling Technology; 4546), PERK (Cell Signaling Technology; 9956),

BiP (Cell Signaling Technology; 9956), eIF2α (Cell Signaling Technol-

ogy; 9722), p-eIF2α (Cell Signaling Technology; 3597), caspase-3 (Cell

Signaling Technology; 9665), CHOP (Cell Signaling Technology; 5554),

and p-PERK (Santa Cruz Biotechnology; Sc-32577).

Flow Cytometry Analysis Flow cytometry analysis was performed according to the manu-

facturer’s protocol (BD Biosciences) with at least 10,000 live events

captured per analysis. Reagents used were as follows: allophycocyanin

(APC)-conjugated anti-CD44 antibody (clone G44-26), phycoeryth-

rin (PE)-conjugated anti-CD24 antibody (clone ML5), and propid-

ium iodide (5 μg/mL; BD Biosciences).

ATF6 Reporter Assay p5xATF6-GL3 and hRluc constructs were obtained from Addgene

(Plasmid 11976 and 24348). Twenty-four hours after cotransfection

of 0.3-μg p5xATF6-GL3 and 0.05-μg 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).

35 S-Methionine/Cysteine Protein Labeling Equal numbers of cells were cultured in the presence of 35 S-methio-

nine/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

minutes to pellet any whole-cell contaminants. An equal volume of

medium was reduced in loading buffer, separated by SDS-PAGE, and

analyzed by autoradiography.

Identifi cation of Major Secreted Proteins Ten 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%

trichloroacetic acid (TCA) on ice. After centrifuging at 15,000 × g for

15 minutes, the pellet was washed in acetone and dissolved in reduc-

ing 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 defi ned as fol-

lows: the EMT core gene set consisted of the top 100 genes upregu-

lated upon EMT induction; the ECM gene set was obtained from

MolSigDB; the XBP1 (GSE40515; ref. 45 ) and ATF4 (GSE35681; ref.

46 ) gene sets consisted of the most downregulated 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 the ECM,

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

before calculating correlations. Tumors from fi ve human cancer data-

sets were analyzed (GSE41998, GSE37892, GSE26942, GSE4573, and

GSE11360). Spearman ρ 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 1,000 correlations

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

Statistical Analysis All data unless otherwise specifi ed are presented as mean +/− SEM

from at least three independent experiments. A Student t test was

performed for comparisons between two groups of data. Two-way

ANOVA tests were performed when comparing the responses of dif-

ferent groups of cells to various drug treatment doses.

Disclosure of Potential Confl icts of Interest No potential confl icts of interest were disclosed.

Authors’ Contributions Conception and design: Y.-x. Feng, E.S. Sokol, Q. Wang, P.B. Gupta

Development of methodology: Y.-x. Feng, E.S. Sokol, H.L. Ploegh,

P.B. Gupta

Acquisition of data (provided animals, acquired and managed

patients, provided facilities, etc.): Y.-x. Feng, E.S. Sokol, C.A. Del Vecchio,

S. Sanduja, J.H.L. Claessen, T.A. Proia, D.X. Jin, H.L. Ploegh, Q. Wang

Analysis and interpretation of data (e.g., statistical analysis,

biostatistics, computational analysis): Y.-x. Feng, E.S. Sokol,

C.A. Del Vecchio, S. Sanduja, D.X. Jin, H.L. Ploegh, P.B. Gupta

Writing, review, and/or revision of the manuscript: Y.-x. Feng,

E.S. Sokol, S. Sanduja, D.X. Jin, H.L. Ploegh, P.B. Gupta

Administrative, technical, or material support (i.e., reporting or

organizing data, constructing databases): Y.-x. Feng, F. Reinhardt,

Q. Wang, P.B. Gupta

Study supervision: P.B. Gupta

Acknowledgments The authors 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 (University of Chicago).

Grant Support This research was supported by grants from the Richard and Susan

Smith Family Foundation and the Breast Cancer Alliance (to P.B.

Gupta), and the National Science Foundation Graduate Research

Fellowship (Grant No. 1122374; to E.S. Sokol).

Received December 2, 2013; revised March 28, 2014; accepted

March 31, 2014; published OnlineFirst April 4, 2014.

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2014;4:702-715. Published OnlineFirst April 4, 2014.Cancer Discovery Yu-xiong Feng, Ethan S. Sokol, Catherine A. Del Vecchio, et al. Sensitizes Cells to Endoplasmic Reticulum Stress

andαeIF2−Epithelial-to-Mesenchymal Transition Activates PERK

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