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