Chronic IL-1β-induced inflammation regulates epithelial-to ......
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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: [email protected]
mailto:[email protected]
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