a new appreciation of the positive and negative effects of ... lecture - george...a new appreciation...
TRANSCRIPT
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A new appreciation of the positive and negative effects of interferons in cancer
George Stark, Lerner Research Institute, Cleveland Clinic
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• 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
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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).
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• 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
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IFN response: two phases
Cheon et al. (2013) EMBO J
IRDS
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IRDS = U‐ISGF3‐induced genes
Cheon et al. (2013) EMBO
* = known anti-viral proteins
+ = up-regulated in DNA damage resistant cancer cells
IRDS
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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
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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.
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The levels of U‐ISGF3 proteins in cancer correlate with response to DNA damage
Small Cell Lung Carcinoma
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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
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2’,5’‐OAS
Courtesy of Bob Silverman
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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.
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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
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Why OAS???
We need to take a short trip back in time to…
1984
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•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.
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Chemical Structure of NAD 2-5A
NAD
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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
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Non‐canonical 2‐5A is produced in response to DNA damage
•H196 cells (resistant SCLC, high OAS)
•+/- 50IU/mL IFN-β, 24hrs
•10Gy radiation
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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
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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
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Down regulation of OAS1 using a JAK inhibitor
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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.
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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.
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An important new pathway of NFκB activation, mediated by epidermal growth factor receptor (EGFR)
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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
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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)
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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.
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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)
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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
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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
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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
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EGFR knockdown decreases NFB activation in cancer cells
pIKK
pIκB
Total EGFR
SKOV3
-actin
A549
pIKK
pIκB
Total EGFR
-actin
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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
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Does signaling to NFB depend solely onEGFR, or is another receptor that is alreadyknown to activate NFB involved?
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Correlation between EGFR and TLR
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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
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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.
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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
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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
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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
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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
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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
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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
?
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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?
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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
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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
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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
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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
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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
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Acknowledgments
• HyeonJoo Cheon• Elise Holvey‐Bates• Sarmishtha De• Josephine Dermawan