A new appreciation of the positive and negative
effects of interferons in cancer
George Stark, Lerner Research Institute,
Cleveland Clinic
Anti-cancer effects of IFNs
• Physiological levels of IFNs inhibit tumor development.
Tumor incidence is increased in IFNGR-null mice and IFNARnull mice.
(Kaplan et al. PNAS. 1998, Dunn et al. Nat Immun. 2005)
• High doses of IFNs (IFNα and IFNγ) are used for
cancer therapy
Activate anti-cancer immunity and increase proapoptotic, anti-proliferative, anti-angiogenic proteins in
cancer cells.
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).
IRDS genes
A subset of interferonstimulated 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.
No adjuvant Tx
Adjuvant Chemo
Adjuvant Radio
n=286
n=140
n=242
Metastasis free survival rate
•
Survival rate after surgery (Breast cancer)
IFN response: two phases
IRDS
Cheon et al. (2013) EMBO J
IRDS = U‐ISGF3‐induced genes
* = known anti-viral
proteins
IRDS
+ = up-regulated in
DNA damage
resistant cancer cells
Cheon et al. (2013) EMBO
Chronic exposure to a low dose of IFNβ increases levels of
U-ISGF3 and IRDS genes
BJ fibroblasts
Treated with low dose of
IFNβ (0.5 U/ml) every
other day for 16 days
RNA expression data for P-ISGF3mediated ISGs and U-ISGF3-mediated
IRDS genes. Data were extracted from
the TCGA database for 110 normaltumor paired breast cancer patients. The
ratio of T/N was used to plot the heat
map 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 damageresistant 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
OAS1
OAS2 gene expression relative to GAPDH
(x1000)
OAS1 gene expression relative to GAPDH
(x1000)
OAS2
8
30
7
25
6
20
5
4
15
3
10
2
5
1
0
0
H526
H1048
DMS114
SW1271
H196
H146
H1688
H526
H1048
DMS114
SW1271
H196
H1688
SW1271
H196
H1688
OASL
OAS3
OASL gene expression relative to GAPDH
(x1000)
OAS3 gene expression relative to GAPDH
(x1000)
H146
40
35
3
2.5
30
25
20
2
1.5
15
10
1
0.5
5
0
H146
H526
H1048
Sensitive
DMS114
SW1271
H196
H1688
Resistant
0
H146
H526
H1048
DMS114
Sensitive
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
hTERT-HME cells overexpressing OAS1
120
Relative cell survival (%)
100
80
60
p3XFLAG
OAS1
40
20
0
0.03μM0.1μM 0.3μM 1μM 0.3μM 1μM
Control
Doxorubicin
3μM 10μM 2Gy
Etoposide
5Gy 10Gy 20Gy
Radiation
Down regulation of OAS1 using a JAK inhibitor
Summary
• OAS genes are induced in cancer cells by UISGF3.
• 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)
Background
 NFB is an important mediator of the normal
response to inflammatory signals.
 NFB is activated by many different stimuli.
 Deregulated constitutive activation of NFB is a
hallmark of most cancers; the mechanisms are
diverse and have not been well defined.
 Production of IFN is a major
consequence of activating NFkB
downstream
Activation of NFκB (p65/p50)
Activating Signal
IKK
S‐P
IB S‐P
IB
P50
P50
P65 Phosphorylation
of IB
cytoplasm
P50
Nucleus
P50
P50 P65
P65
P65
DNA
P65
Degradation
of IB
IB S‐P
Transcriptional
activation
Epidermal Growth Factor Receptor Signaling
EGFR
Ligand (EGF)
• 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.
K K
P
AKT
P
RAS
RAF
mTOR
ERK
Cell survival
Proliferation
EGFR‐driven NFκB activation in human mammary
epithelial (HME) cells
0
5
15
30
60
120
EGF (min)
pEGFR (Y1068)
Total EGFR
pAKT (S473)
Total AKT
pERK (T202/Y204)
Total ERK
β-actin
pIKK (S177/S181)
Total IKKβ
pIκBα (S32/S36)
Total IκBα
β-actin
The kinase activity of EGFR is required for signaling to NFB
-
+
-
+
EGF
-
-
+
+
Erlotinib
EGFR
Ligand (EGF)
pEGFR (Y1068)
pIKK (S177/S181)
pIκB (S32/S36)
pAKT (S473)
Erlotinib
KTKIK
P
AKT
pERK
P
RAS
RAF
-actin
HME cells
mTOR
ERK
Cell survival
Proliferation
NFkB is essential for cancer cell survival
A549
PC9
p65
p65
‐actin
‐actin
60
80
PC9
50
40
30
20
10
0
NT shRNA shp65
% cell survival
(compared to untreated control)
% cell survival
(compared to untreated control)
70
A549
60
40
20
0
NT shRNA shp65
Erlotinib treatment decreases NFκB activation in cancer cells
A549 (NSCLC)
1
2
3
0
Erlotinib (20 μM)
OVCAR3 (Ovarian Ca)
pEGFR
pEGFR
pEGFR
pIκB
pIκB
pIκB
-actin
-actin
-actin
(h)
H1048 (SCLC)
1
2
3
0
Erlotinib (20 μM)
HCC827 (NSCLC)
1
2
3
0
Erlotinib (20 μM)
(h)
SW1271 (SCLC)
pEGFR
pEGFR
pIκB
pIκB
-actin
-actin
(h)
1
2
3
0
Erlotinib (20 μM)
(h)
0
15 30 60 (min)
Erlotinib (1 μM)
EGFR knockdown decreases NFB activation in cancer cells
A549
SKOV3
Total EGFR
Total EGFR
pIKK
pIKK
pIκB
pIκB
-actin
-actin
Summary
 EGF/EGFR drives NFB activation in non‐
malignant human epithelial cells.
EGFR
 In several cancer cell lines, treatment with
erlotinib or down regulation of EGFR
expression inhibits constitutive NFB
activation.
Erlotinib
Y‐P Y‐P
The efficacy of erlotinib in cancer treatment is
due not only to the suppression of the RAS/ERK
and PI3K/AKT pathways, but also due to
inhibition of NFB activation.
NFB activation
cancer cell suvival
Does signaling to NFB depend solely on
EGFR, or is another receptor that is already
known to activate NFB involved?
Correlation between EGFR and TLR
Toll‐like Receptors (TLRs)
Ligands
Toll-like
Receptors
TLRs are a family of pattern recognition receptor
expressed on various immune and non‐immune cells,
including epithelial cells.
They enable the innate immune system to recognize
pathogen‐associated molecular patterns (PAMPs).
NFB activation
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
TLR‐dependent pathways activate NFB.
TLR4 Signaling Pathway
LPS
Ectodomain
TLR4
TM
TLR4 is crucial for effective host cell
responses to Lipopolysaccharide (LPS) from
Gram‐negative bacteria.
TIR domain
MYD88
TRIF
p50 p65
Early phase NFB
p50 p65
IRF3
Upon LPS binding, TLR4 recruits adaptors to
its intracellular TIR signaling domains,
triggering downstream signaling
Late phase
NFB
Inflammatory
cytokines
IFN-B, IFN inducible
gene products
MYD88‐dependent
response
MYD88‐independent
response
Nature Reviews Immunology 4, 499–511, 2004.
Stimulation of TLR4 facilitates the activation
of two pathways: the MyD88‐dependent and
MyD88‐independent pathways.
EGFR stimulates TLR4 phosphorylation
EGF
HME
- 1
2
5
10 EGF(min)
pTLR4
EGFR
TLR4
TLR4
-actin
Y‐P
Y‐P
Y‐P Y‐P
A549
-
5
10 15 EGF(min)
pTLR4
TLR4
MYD88
erlotinib
TAK1
-actin
S‐P
IKK
IB S‐P
HME
- 5 10 - 5 10 EGF(min)
pTLR4
TLR4
-actin
control
Erlotinib
S‐P p65 p50
B B
S‐P
p65 p50
B B
active
NFB
TLR4 silencing abolishes EGF‐induced NFB activation
HME
A549
TLR4
-
TLR4
GAPDH
GAPDH
-
-
EGF (min)
pEGFR
EGF(min)
pEGFR
-
-
pIKK
pIKK
pIB
pIB
 actin
NT siRNA
IB
si TLR4
 actin
NT shRNA
sh TLR4
required for
for NFB
NFkB activation in response to LPS?
EGF
IsTLR4
EGFRisessential
EGF
LPS
EGFR
EGFR
TLR4
TLR4
Y‐P
Y‐P
Y‐P
Y‐P Y‐P
Y‐P
Y‐P Y‐P
?
MYD88
MYD88
erlotinib
erlotinib
TAK1
TAK1
IKK
S‐P
IKK
IB S‐P
IB S‐P
S‐P p65 p50
B B
S‐P
p65 p50
B B
active
NFB
S‐P
S‐P p65 p50
B B
S‐P
p65 p50
B B
active
NFB
EGFR is essential for LPS‐induced activation of NFB
HME
-
5
15
45
-
5
15
45
LPS(min)
pEGFR
EGFR
pIKK
pIB
IB
-actin
NT shRNA
A549
-
5
15 45 60
EGFR
sh EGFR
-
5 15 45 60 LPS(min)
pEGFR
-actin
pIKK
pIB
IB
-actin
NT shRNA
sh EGFR
Down regulation of LYN impairs EGF‐induced NFB activation
HME
A549
0 5 15 45 0 5 15 45 EGF(min)
LYN
pEGFR
β-actin
0
5
0
5
EGF (min)
pEGFR
LYN
pIKK
pIKK
β-actin
IB
pIB
β-actin
NTshRNA
β-actin
shLYN
NTsiRNA
si LYN
LPS stimulates the recruitment of LYN to EGFR and TLR4
LPS
HME
EGFR
LPS (min)
LPS (min)
-
5
15
45
-
5
15
TLR4
45
EGFR
Y‐P
LYN
TLR4
LYN
Y‐P Y‐P
?
MYD88
LYN
IP: LYN
erlotinib
Input
TAK1
A549
LPS (min)
-
5
LPS (min)
15
-
5
15
LPS (min)
-
5
15
LPS (min)
-
5
IKK
15
S‐P
IB S‐P
EGFR
S‐P p65 p50
B B
TLR4
LYN
Erlotinib -
-
-
+
IP: LYN
+
+
-
-
-
+
Input
+
+
S‐P
p65 p50
B B
Y‐P
active
NFB
Is EGFR required for LPS‐induced NFκB activation in vivo?
C57BL/6 mice (6-8 weeks old)
vehicle
media
(i.p.)
vehicle
100 mg/kg Erlotinib treatment (16 h)
10 mg/kg LPS (i.p.)
+ vehicle
10 mg/kg LPS (i.p.)
+ 100 mg/kg Erlotinib
Collect plasma and
splenocytes at 6 h
ELISA for cytokines
qPCR
Erlotinib blocks LPS‐induced cytokine production in vivo
IL6
P=0.0004
250
80
IL-6 (ng/ml)
IL-6 (ng/ml)
100
60
40
20
200
150
100
50
0 (▲)
Control
TNFα
LPS
0 (▲)
Control
LPS+Erl
P=0.0005
600
400
200
0 (▲)
Control
LPS
LPS+Erl
LPS+Erl
P=0.06
1500
TNF (pg/ml)
800
LPS
TNFα
1000
TNF (pg/ml)
P=0.04
IL6
1000
500
0 (▲)
Control
LPS
LPS+Erl
Erlotinib blocks LPS‐induced cytokine expression in vivo
3
2
1
0
5
5
P=0.017
4
3
2
1
0
P=0.009
P=0.01
Fold Change relative to actin
P=0.004
CXCL1
IL6
Fold Change relative to actin
Fold Change relative to actin
TNFα
4
4
3
2
1
0
Mouse splenocytes were assayed after 6 h
P=0.17
P=0.08
Erlotinib protects mice from LPS‐mediated lethality
100
Percent survival
80
LPS
60
Erlotinib+LPS
40
20
0
0
50
100
Post LPS (hrs)
150
Summary
 Activation of NFB by EGF requires TLR4, and activation by LPS requires EGFR.
 The SRC family member LYN is involved in the cross talk between EGFR and
TLR4, leading to downstream signaling.
 Treatment of mice with erlotinib, a well‐known drug used extensively in cancer
treatment, is also beneficial in suppressing the inflammatory signal triggered by
LPS.
DAMPS
TLRs, EGFR
Erlotinib
DNA damage
NFκB
IRF3
cytoplasmic
RNA, DNA
(Sting etc.)
IFNβ
tumor
inhibition
full ISG
response
partial IRDS
response
resistance to drugs, IR
Acknowledgments
•
•
•
•
HyeonJoo Cheon
Elise Holvey‐Bates
Sarmishtha De
Josephine Dermawan