Chronic IL-1β-induced inflammation regulates epithelial-to-mesen-

chymal transition memory phenotypes via epigenetic modification in

non-small cell lung cancer

Rui Li1,2, Stephanie L. Ong3, Linh M. Tran1, Zhe Jing1, Bin Liu1, Stacy J. Park1,

Zi Ling Huang1, Tonya C. Walser1, Eileen L. Heinrich4, Gina Lee1,5, Ramin

Salehi-Rad1,5, William P. Crosson2, Paul C. Pagano2, Manash K. Paul1, Shili

Xu2, Harvey Herschman2, Kostyantyn Krysan1, Steven Dubinett1,2,3,4,5,*

1Pulmonary and Critical Care Medicine, David Geffen School of Medicine at UCLA, Los Angeles,

90025, California, USA

2Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at

UCLA, Los Angeles, 90025, California, USA

3Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, 90025, Cal-

ifornia, USA

4Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA,

Los Angeles, 90025, California, USA

5VA Greater Los Angeles Health Care System, Los Angeles, California, 90025, USA

*Correspondence: sdubinett@mednet.ucla.edu

mailto:sdubinett@mednet.ucla.edu

C D

A B

– + – +

A427 H460

IL-1β(48h)

γ-catenin

GAPDH

Vimentin

CK18

E-cadherin

– L H – L H – L H – L H

9d -6d -9d -12d

IL-1β

Vimentin

GAPDH

CK18

E-cadherin

A549

50μm

IL-1β ctrl

CK18 lower band

– + – p – p – p – + – p – p – p

21d -6d -12d -18d

H460

γ-Catenin

Vimentin upper band

IL-1β

GAPDH

21d -6d -12d -18d

A427

E

– + – + – + – + – + – +

A 42

7

IL-1β(48h)

γ-catenin

GAPDH

Vimentin

CK18

E-cadherin

H 21

22

H 15

68

H 15

7

H 29

2

H 16

50

– + – + – +

R U 68

6

R h2

H 12

99

F

**** ****

**

A549

N -C

ad he

rin

S M

A

0.0

0.5

1.0

1.5

2.0

2.5

ctrl IL-1b (48h)

R e

la ti

v e E

x p

re s s io

n

Fi br

on ec

tin

Figure S1

Figure S1 related to Figure 1. Chronic cytokine exposure leads to EMT memory in NSCLC.

A, Phase-contrast microscopy showed decreased cell-to-cell contacts and increased cell protru-

sions (indicated by arrow head) following 48-hour IL-1β exposure in A549 cells. B, Gene expres-

sion of indicated mesenchymal markers by RT-PCR. C, High dose of IL-1β exposure did not lead

to EMT memory following 9-day IL-1β exposure in A549 cells. D, A screening of EMT in indi-

cated NSCLC cell lines following 48-hour IL-1β treatment. E, EMT markers in A427 and H460

cell lines following 48-hour IL-1β exposure. E-cadherin was not detected in these two cell lines.

F, Prolonged EMT phenotype was observed in A427 and H460 following chronic IL-1β exposure.

L, low dose (1 ng/ml); H, high dose (10 ng/ml). “p”, previously treated with IL-1β. All results

were reported as mean ± SEM. ** P

0.6

0.4

0.2

0.0

0.8

Gilmore_Core NFκB Pathway

-0.2

0.0

0.2

Matthews_AP1 Targets

0.5

0.4

0.3

0.2

GO_Lung Cell Differentiation

Day

P a

th w

a y

S c o

re ctrl IL-1β

c’-PARP

GAPDH

IL-1β (48h) – + – + – + – +

Etop Doxr SAHACis

– + – + – + – +

Etop Doxr SAHACis

H460 A427

A

C

B PD-L1

0

5

10

15

20

25 ctrl

IL-1b (48h)

A427H460

****

n.s.

M F

I/ IS

O

Figure S2

C 1

(n =

1 3

7 )

C 2 (

n =

2 2

0 )

C 3 (

n =

2 1

8 )

C 4 (

n =

1 6

6 )

CXCL1 CXCL5 CXCL8 CCL2 TGFB1 CSF3

ITGB3 FOSL1 RUNX2 CD274 MAPK13

CDH1 CLDN2 SMAD6

SIK1 MAP4K1

6d 15d 21d -6d -15 -30d

KIF12

SHH

FLRT2

FOXA1

3210-1-2-3 log2 (ratio)

0.3

0.2

0.1

0.0

GO_Positive Regulation of Epithelial Cell Migration

7 14 21 -7 -14 -21 -28 7 14 21 -7 -14 -21 -28

7 14 21 -7 -14 -21 -287 14 21 -7 -14 -21 -28

D

Figure S2 related to Figure 2. EMT-associated phenotypes induced by chronic IL-1β expo-

sure are also memorized. A, Chemotherapy-induced apoptosis following the acute IL-1β expo-

sure in H460 and A427 cells. B, Surface PD-L1 expression following the acute IL-1β exposure in

H460 and A427 cells. C, A heatmap of differentially expressed genes at indicated time points

following IL-1β exposure, which were divided into four clusters. C1 and C4, genes sensitive to

IL-1β exposure; C2 and C3, genes memorizing their expression patterns following IL-1β with-

drawal. D, Pathway analysis in A549 cells showing that pathways, such as the NF-κB and AP-1

pathways, and phenotypes, such as cell differentiation and migration, are IL-1β sensitive. Cis, cis-

platin; Etop, etoposide; Doxr, doxorubicin. All results were reported as mean ± SEM. **** P

E

B

CA

– + – + – + – +

si ct

rl

si S LU

G #1

si S LU

G #2

si Z E B 2

Vimentin

GAPDH

IL-1β(48h)

E-cadherin

CK18

0.0

0.5

1.0

1.5

2.0

2.5 ctrl

IL-1b (48h)

R e

la ti

v e E

x p

re s s io

n * **

n.s

S N A IL

S LU

G

ZE B 2

ZE B 1

TW IS

T1

D

Vimentin

SLUG

SLUG dark

GAPDH

SLUG(oe)

21d -7d

E-cadherin

– –

21 -7 0.0

0.5

1.0

1.5 SLUG

E-cadherin/GAPDH

Day

*** **

F o

ld C

h a

n g

e

SLUG(oe)

T im

e

21d

-7d

200μm

Figure S3

21d

-7d

IL-1β HS IL-1β LSctrl

200μm

p p

GAPDH

c’-caspse 3

c’-PARP

Slug(oe) Etop Doxr SAHACis Etop Doxr SAHACis

21d -7d

– – – – – – – –

Surface PD-L1

SLUG

21 -7 0

2

4

6

8

10 *** ****

F o

ld C

h a

n g

e F

G

Figure S3

Day

Figure S3 related to Figure 3. Accumulation of SLUG is indispensable for the establishment

of the memory phenotypes. A, Relative expression of EMT-associated transcription repressors

by RT-qPCR following the acute IL-1β exposure. B, Knockdown of SLUG or ZEB2 is sufficient

to prevent E-cadherin downregulation following the acute IL-1β exposure. C, Morphological

changes of the untreated, LS, and HS cells by bright-field microscopy. D, Morphological changes

with various levels of SLUG at day 21 of SLUG overexpression (21d) and day 7 after the overex-

pression was terminated (-7d). E-G, Evaluation of EMT markers, apoptosis resistance, and surface

PD-L1 expression with various levels of SLUG overexpression at indicated time points. “p”, pre-

viously treated with IL-1β. “oe”, overexpression. Cis, cisplatin; Etop, etoposide; Doxr, doxorubi-

cin. All results were reported as mean ± SEM. * P

C

– + IL-1β

30m

c-JUN

p-c-JUN

p-Fra-1

Fra-1

GAPDH

D

A B

Vimentin

GAPDH

SLUG

IL-1β(48h)

U0126(µM) JNKi II(µM)

CK18

E-cadherin

0.0

0.5

1.0

1.5

2.0 ctrl IL-1b (48h)

n.d.

R e

la ti

v e E

x p

re s s io

n

Fr a-

1

Fr a-

2

FO S B

FO S

JU N B

JU N

**** **

G

IL-1β

p-JNK

p-p65

p-AKT

GAPDH

15m 30m 1h

p-p38

p-ERK1/2

– + – + – + – + – + – +

8h 24h 48h

p65

JNK

ERK1/2

Vimentin

GAPDH

Fra-1

E-cadherin dark

IL-1β(24d) – + – + – + – + – +

un tr ea

te d

U 01

26 JN

K i I

I

si F ra

-1

U +J

E-cadherin

CK18

CK18 dark

IL-1β 0d 21d 24d

IL-1β

Inhibitors

IL-1β

SLUG

p