A new appreciation of the positive and negative effects of interferons in cancer

George Stark, Lerner Research Institute, Cleveland Clinic

• Physiological levels of IFNs inhibit tumor development.

Tumor incidence is increased in IFNGR-null mice and IFNAR-

null mice.

(Kaplan et al. PNAS. 1998, Dunn et al. Nat Immun. 2005)

• High doses of IFNs (IFNα and IFNγ) are used for

cancer therapyActivate anti-cancer immunity and increase pro-apoptotic, anti-proliferative, anti-angiogenic proteins in cancer cells.

Anti-cancer effects of IFNs

IRDS

IRDS = IFN‐related DNA damage resistance gene signature

• Identified in a wide variety of cancer types, including breast, head and neck, prostate, lung, glioma, myeloma.

• Cancer cells expressing this signature are resistant to radiation and a variety of chemotherapeutic agents (doxorubicin, etoposide, cisplatin, etc).

• A subset of interferon-stimulated genes highly expressed in radio- or chemo- resistant cancer. (Khodarev et al. PNAS. 2004, Weichselbaum et al. PNAS. 2008)

• Not including apoptotic or anti-proliferative proteins.

IRDS genes

n=140 n=242

Survival rate after surgery (Breast cancer)

n=286

No adjuvant Tx Adjuvant Chemo Adjuvant Radio

Metastasis

 free

 survival ra

te

IFN response: two phases

Cheon et al. (2013) EMBO J

IRDS

IRDS = U‐ISGF3‐induced genes

Cheon et al. (2013) EMBO

* = known anti-viral proteins

+ = up-regulated in DNA damage resistant cancer cells

IRDS

Chronic exposure to a low dose of IFNβ increases levels of U-ISGF3 and IRDS genes

BJ fibroblastsTreated with low dose of IFNβ (0.5 U/ml) every other day for 16 days

RNA expression data for P-ISGF3-mediated ISGs and U-ISGF3-mediatedIRDS genes. Data were extracted fromthe TCGA database for 110 normal-tumor paired breast cancer patients. Theratio of T/N was used to plot the heatmap on a log scale.

The levels of U‐ISGF3 proteins in cancer correlate with response to DNA damage   

Small Cell Lung Carcinoma

IRDS/U‐ISGF3 genes

• Which IRDS/U-ISGF3-induced gene(s) are responsible for the DNA damage-resistant phenotype?– Not much evidence for any IRDS/U-ISGF3

gene being involved in DNA damage response pathways.

– Our hypothesis:• 2’,5’-oligoadenylate synthetases (OAS1, OAS2,

OAS3, OASL) are implicated

2’,5’‐OAS

Courtesy of Bob Silverman

2’,5’‐OAS

OAS proteins (except OASL) can synthesize 2’,5’-linked phosphodiester bonds to polymerize ATP into oligomers.• Red domains =

catalytically inactive.

Sadler and Williams (2008) Nat Rev Immunol.

OAS mRNA levels in sensitive vs resistant SCLC

0

1

2

3

4

5

6

7

8

H146 H526 H1048 DMS114 SW1271 H196 H1688

OAS

1 ge

ne e

xpre

ssio

n re

lativ

e to

GAP

DH

(x10

00)

OAS1

0

5

10

15

20

25

30

H146 H526 H1048 DMS114 SW1271 H196 H1688

OAS2

gen

e ex

pres

sion

rela

tive

to G

APDH

(x

1000

)

OAS2

0

5

10

15

20

25

30

35

40

H146 H526 H1048 DMS114 SW1271 H196 H1688

OAS

3 ge

ne e

xpre

ssio

n re

lativ

e to

GAP

DH

(x

1000

)

OAS3

0

0.5

1

1.5

2

2.5

3

H146 H526 H1048 DMS114 SW1271 H196 H1688

OASL

gen

e ex

pres

sion

rela

tive

to G

APDH

(x

1000

)

OASL

Sensitive SensitiveResistant Resistant

Why OAS???

We need to take a short trip back in time to…

1984

•When monkey CV-1 cells are infected with SV40 (a DNA virus) followed by treatment with IFN, high concentrations of 2-5A accumulate, but these are not classical 2-5A.

•The unusual 2-5As do not activate RNase L and do not lead to the degradation of RNA.

•These studies were possible due to the generation of 2-5A-specific antibodies.

Chemical Structure of NAD 2-5A

NAD

2‐5A and DNA damage

• Is non-canonical 2-5A produced in response to DNA damage?– Collaboration with Silverman lab

• Babal Jha• Beihua Dong

• Two techniques to measure 2-5A levels– Fluorescence Resonance Energy Transfer

(FRET) Assay for 2-5A – detects canonical 2-5A– Competitive ELISA – measures all 2-5A

compounds

Non‐canonical 2‐5A is produced in response to DNA damage

•H196 cells (resistant SCLC, high OAS)

•+/- 50IU/mL IFN-β, 24hrs

•10Gy radiation

Non‐canonical 2‐5A is produced in response to DNA damage

•H196 cells (resistant SCLC, high OAS)

•+/- 50IU/mL IFN-β, 24hrs

•10uM etoposide or 1uM doxorubicin

Overexpression of OAS1 increases resistance to DNA damage 

0

20

40

60

80

100

120

0.03μM0.1μM 0.3μM 1μM 0.3μM 1μM 3μM 10μM 2Gy 5Gy 10Gy 20Gy

Control Doxorubicin Etoposide Radiation

Rel

ati

ve c

ell

surv

iva

l (%

)

hTERT-HME cells overexpressing OAS1

p3XFLAG

OAS1

Down regulation of OAS1 using a JAK inhibitor

Summary

• OAS genes are induced in cancer cells by U-ISGF3.

• OAS1-expressing cancer cells are more resistant to DNA damage.

• Non-canonical 2-5A molecules are made only by OAS1 in response to DNA damage.

• OAS1 (and not OAS2, 3, or L) is responsible for increasing resistance to DNA damage.

Future Directions

• What signal induced by DNA damage activates OAS1?

• Why does it make non-canonical rather than canonical 2-5A?

• What is non-canonical 2-5A?

• Prove that non-canonical 2-5A can induce resistance to DNA damage.

An important new pathway of NFκB activation, mediated by epidermal growth factor receptor (EGFR)

NFB is activated by many different stimuli.

NFB is an important mediator of the normalresponse to inflammatory signals.

Deregulated constitutive activation of NFB is ahallmark of most cancers; the mechanisms arediverse and have not been well defined.

Production of IFN is a major downstreamconsequence of activating NFkB

Background

IB

Phosphorylation of IB

IB

Degradationof IB

DNA

IKK S‐P

Transcriptional activation

cytoplasm

Nucleus

P50 P65

P50 P65

P50

S‐P

IB S‐P

P50 P65

P50 P65

P50 P65

Activating Signal

Activation of NFκB (p65/p50)

EGFR

K K

P P

AKT RAS

RAF

ERKmTOR

Cell survivalProliferation

Ligand (EGF)

Epidermal Growth Factor Receptor Signaling

• The epidermal growth factor receptor (EGFR,HER-1/ErbB1) is a receptor tyrosine kinase of the ErbB family.

• Upon ligand binding EGFR is activated, activated EGFR signals downstream to the PI3K/AKT and Ras/Raf/Erk pathways. 

• These intracellular signaling pathways regulate key cellular processes such as proliferation and survival.

EGFR‐driven NFκB activation in human mammary epithelial (HME) cells

EGF (min)0 5 15 30 60

pEGFR (Y1068)

Total EGFR

120

pIKK (S177/S181)

pIκBα (S32/S36)

Total IKKβ

β-actin

Total IκBα

Total AKT

β-actin

Total ERK

pERK (T202/Y204)

pAKT (S473)

EGFR

K K

P P

AKT RAS

RAF

ERKmTOR

Cell survivalProliferation

Ligand (EGF)

TKI Erlotinib

The kinase activity of EGFR is required for signaling to NFB 

pEGFR (Y1068)

pIKK (S177/S181)

pAKT (S473)

pERK

pIκB (S32/S36)

-actin

EGF

Erlotinib+

+

+

+

-

-

-

-

HME cells

0

10

20

30

40

50

60

70

% cell survival 

(com

pared to untreated

 con

trol)

NT shRNA shp65

PC9

PC9

p65

‐actin

A549

p65

‐actin

0

20

40

60

80A549

% cell survival 

(com

pared to untreated

 con

trol)

NT shRNA shp65

NFkB is essential for cancer cell survival

Erlotinib treatment decreases NFκB activation in cancer cells

A549 (NSCLC)

1 2 3 (h)

pEGFR

pIκB

Erlotinib (20 μM)

-actin0

OVCAR3 (Ovarian Ca)

1 2 3 (h)

pEGFR

pIκB

Erlotinib (20 μM)

-actin0

H1048 (SCLC)

1 2 3 (h)

pEGFR

pIκB

Erlotinib (20 μM)

-actin0

SW1271 (SCLC)

1 2 3 (h)

pEGFR

pIκB

Erlotinib (20 μM)

-actin0

HCC827 (NSCLC)

15 30 60 (min)

pEGFR

pIκB

Erlotinib (1 μM)

-actin0

EGFR knockdown decreases NFB activation in cancer cells

pIKK

pIκB

Total EGFR

SKOV3

-actin

A549

pIKK

pIκB

Total EGFR

-actin

The efficacy of erlotinib in cancer treatment isdue not only to the suppression of the RAS/ERKand PI3K/AKT pathways, but also due toinhibition of NFB activation.

Summary

EGF/EGFR drives NFB activation in non‐malignant human epithelial cells.

In several cancer cell lines, treatment witherlotinib or down regulation of EGFRexpression inhibits constitutive NFBactivation.

EGFR

Y‐P Y‐P

Erlotinib

NFB activation

cancer cell suvival

Does signaling to NFB depend solely onEGFR, or is another receptor that is alreadyknown to activate NFB involved?

Correlation between EGFR and TLR

TLR1 Bacterial lipoproteins

TLR2 Peptidoglycan/bacterial lipoproteins

TLR3 dsRNA (Poly I:C)

TLR4 Lipopolysaccharide (LPS)

TLR5 Flagellin

TLR6 Diacyl Lipoprotein

TLR7 ssRNA

TLR8 ssRNA

TLR9 CpG DNA

TLRs are a family of pattern recognition receptorexpressed on various immune and non‐immune cells,including epithelial cells.

They enable the innate immune system to recognizepathogen‐associated molecular patterns (PAMPs).

Toll‐like Receptors (TLRs)

TLR‐dependent pathways activate NFB.

Toll-like Receptors

Ligands

NFB activation

TLR4 Signaling Pathway

TLR4 is crucial for effective host cellresponses to Lipopolysaccharide (LPS) fromGram‐negative bacteria.

Upon LPS binding, TLR4 recruits adaptors toits intracellular TIR signaling domains,triggering downstream signaling

MYD88

TLR4

Early phase NFB p50 p65

Inflammatory cytokines

TRIF

IRF3IRF3

IFN-B, IFN inducible gene products

Late phase NFB

MYD88‐dependent response 

MYD88‐independent response 

LPS

p50 p65

Nature Reviews Immunology 4, 499–511, 2004. 

Ectodomain

TM

TIR domain

Stimulation of TLR4 facilitates the activationof two pathways: the MyD88‐dependent andMyD88‐independent pathways.

MYD88

TLR4

IB

p65 p50B B

IKK

S‐P

S‐P

Y‐P Y‐P

S‐P

p65 p50B B

S‐P active NFB

EGFR

TAK1

Y‐P

Y‐P

EGF

erlotinib

A549

pTLR4TLR4-actin

5 10 15 EGF(min)-

HME

pTLR4

TLR4

-actin

1 2 5 10 EGF(min)-

HME

pTLR4TLR4-actin

5 10 5 10- EGF(min)-

control Erlotinib

EGFR stimulates TLR4 phosphorylation

TLR4GAPDH

TLR4GAPDH-

pEGFR

pIKK

pIB

NT siRNA si TLR4

actin

EGF (min)- - EGF(min)pEGFR

IB

pIB

actin

pIKK

NT shRNA sh TLR4

--

HME A549

TLR4 silencing abolishes EGF‐induced NFB activation

MYD88

TLR4

IB

p65 p50B B

IKK

S‐P

S‐P

Y‐P Y‐P

S‐P

p65 p50B B

S‐P active NFB

EGFR

TAK1

Y‐P

Y‐P

LPS

erlotinib

?

Is EGFR essential for NFB activation in response to LPS?

MYD88

TLR4

IB

p65 p50B B

IKK

S‐P

S‐P

Y‐P Y‐P

S‐P

p65 p50B B

S‐P active NFB

EGFR

TAK1

Y‐P

Y‐P

EGF

erlotinib

TLR4 is required for NFkB activation in response to EGF

HME

EGFR

-actin

pEGFR5 15 45 5 15 45

pIKK

IB

pIB

LPS(min)

NT shRNA sh EGFR

- -

pEGFR

pIKK

IB

pIB

-actin

LPS(min)

NT shRNA sh EGFR

5 15 45 60 5 15 45 60- -A549

EGFR-actin

EGFR is essential for LPS‐induced activation of NFB

HME

shLYNNTshRNA

0 5 15 45 0 5 15 45 EGF(min)

pIKK

IB

pEGFR

β-actin

β-actinLYN pEGFR

pIKK

pIB

β-actin

si LYNNTsiRNA

EGF (min)0 5 0 5

LYN

β-actin

A549

Down regulation of LYN impairs EGF‐induced NFB activation

LPS stimulates the recruitment of LYN to EGFR and TLR4

HME

Input

EGFR

5 15 45 5 15 45

LPS (min) LPS (min)

IP: LYN

LYN 

TLR4

- -

A549

5 15 5 15 5 15 5 15

LPS (min)

Erlotinib --- +++--- +++

InputIP: LYN

- - - -

EGFR

LYN 

TLR4

LPS (min)LPS (min)LPS (min)

MYD88

TLR4

IB

p65 p50B B

IKK

S‐P

S‐P

Y‐P Y‐P

S‐P

p65 p50B B

S‐P active NFB

EGFR

TAK1

Y‐P

Y‐PLYN LYN

LPS

erlotinib

?

C57BL/6 mice (6-8 weeks old)

100 mg/kg Erlotinib treatment (16 h)vehiclevehicle

10 mg/kg LPS (i.p.) + vehicle

media (i.p.)

10 mg/kg LPS (i.p.) + 100 mg/kg Erlotinib

Collect plasma and splenocytes at 6 h

ELISA for cytokines qPCR

Is EGFR required for LPS‐induced NFκB activation in vivo? 

IL6IL

-6 (n

g/m

l)

0

20

40

60

80

100

LPS LPS+Erl(▲)Control

P=0.0004

IL-6

(ng/

ml)

IL6

0

50

100

150

200

250

(▲)Control LPS LPS+Erl

P=0.04

LPS0

200

800

1000TNFα

TNF

(pg/

ml)

LPS+Erl

600

400

(▲)Control

P=0.0005

TNF

(pg/

ml)

0

500

1000

1500

LPS LPS+Erl

TNFα

(▲)Control

P=0.06

Erlotinib blocks LPS‐induced cytokine production in vivo  

0

1

2

3

4

5

Fold Change relative to actin

P=0.17 P=0.08

CXCL1

0

1

2

3

4

5

Fold Change relative to actin P=0.009 P=0.01

IL6 

Fold Change relative to actin

0

1

2

3

4

P=0.004 P=0.017

TNFα

Erlotinib blocks LPS‐induced cytokine expression in vivo

Mouse splenocytes were assayed after 6 h

LPS

Erlotinib+LPS

0 50 100 1500

20

40

60

80

100

Post LPS (hrs)

Percen

t survival

Erlotinib protects mice from LPS‐mediated lethality  

Activation of NFB by EGF requires TLR4, and activation by LPS requires EGFR.

The SRC family member LYN is involved in the cross talk between EGFR andTLR4, leading to downstream signaling.

Treatment of mice with erlotinib, a well‐known drug used extensively in cancertreatment, is also beneficial in suppressing the inflammatory signal triggered byLPS.

Summary

TLRs, EGFR

Erlotinib

NFκB IRF3 cytoplasmic RNA, DNA(Sting etc.)

DNA damage

IFNβ

partial IRDSresponse

resistance to drugs, IR

full ISG response

tumor inhibition

DAMPS

Acknowledgments

• HyeonJoo Cheon• Elise Holvey‐Bates• Sarmishtha De• Josephine Dermawan