國立臺灣大學醫學院免疫學研究所 碩士論文
Graduate Institute of immunity College of Medicine National Taiwan University
Master Thesis
STAT3 抑制劑在第一型干擾素反應扮演角色之研究 The role of STAT3 inhibitors in Type I IFN-mediated
signaling and antiviral responses
廖千慧 Chien-Hui Liao
指導教授：李建國 博士
Advisor: Chien-Kuo Lee, Ph.D.
中華民國 101 年 7 月
July, 2012
~ 1L~~#Jt k~~~ ±~1iLt~x.
o~i\4~ tr1fJt:f
STAT3 {Cr~~ 11r'J4 ~ -~ T?lt-- &4
'f .f)}~!i ~ ~~htf Jt
•*t~ 5c.1~JJ-t ~~ (~~»t
R99449001) 11-
@]lL
i:~~~.k~v-
~ r1(.~& ~hft1EPlT
7t~~,?;Ji! ±~1iLt~5c. '
~ ~@101 -4=-
07 }j 25 87f<- T 7
1J
:Aftt(~ ~ ~ ~i!i&l&.
[1tt(&.{% ' 4i- v:Gti sA
致謝
兩年的碩士生涯結束了，心中想要感謝的人有好多好多，因為有他們的指引與幫 助，我才能如期完成這篇論文。首先感謝我的指導教授李建國 老師，在他耐心 督促與毫無保留的指導之下，充實了我在研究中必需的知識，指引我實驗探討的 方向，讓我從一開始的生澀迅速成長茁壯。也感謝口試委員黃麗華 老師的建議 及溫馨的鼓勵，還有陳念榮 老師深入的見解與指教，他們點出了我實驗上的盲 點，更不吝嗇的分享資源，讓我的研究得以順利進行。除了師長們的指點，也要 感謝實驗室成員各方面的照顧。非常謝謝王偉蓓學姊在我還是小碩一時，不但在 忙碌之餘教導我實驗上的技巧，更與我分享待人處事上的經驗，這些真的讓我受 益良多。也感謝陳婷婷學姊辛苦管理實驗室大小事，還有蔡明勳學長超級無私的 幫助，總是熱心的幫忙處理問題，讓我的研究生活不僅充實而且歡樂。謝謝陳怡 伶學姊在實驗上的幫助，還有我厲害的好同學子珮包容我的無厘頭，在這一段承 受壓力的辛苦日子裡有她的陪伴與照應，真的非常感謝。也要謝謝活潑可愛的學 妹們，于婷、宛蓉以及郁萱她們的幫忙和互相照應。此外，也要感謝免疫所的正 彥學長、芷君學姊、榮辰學長以及彰憲學長，他們不僅在實驗方面給我指導，也 提供我實驗資源，給予我大大的幫助，還要謝謝我的同學們，婉珍、雨蓉、水盈、
莉苓、穎超、佳儒、杜杜和哲銘，大家互相的鼓勵與討論，真的給了我很多動力，
讓我可以不斷的突破難關。最要感謝我的母親，二十多年來辛苦養育我，在求學 的一路上都給予我最大的支持，在我遇到挫折時扮演嚴師的角色，督促我克服困 難，希望我的努力可以成為妳的驕傲。還要謝謝我的好同學智傑不斷鼓勵、包容 壞脾氣的我，幫助我順利完成了課業。這一切的一切，在此獻上最深的敬意與感 激，非常謝謝大家。
中文摘要
先天性免疫系統(innate immunity)中，第一型干擾素在幫助宿主對抗病毒及 細菌感染扮演很重要的角色。第一型干擾素會活化訊息傳導與轉錄子(STAT)蛋
白，包括 STAT1、STAT2 以及 STAT3。相較於 STAT1 和 STAT2 主要是促
進第一型干擾素引起的抗病毒反應，STAT3 在其中則是扮演了一個負調控的角 色。之前的研究報告指出，STAT3 基因剔除的鼠胚胎纖維母細胞(MEF)以及骨 髓衍生的巨噬細胞(BMM)可表現較強的第一型干擾素反應，所以如果能抑制 STAT3 功能將可以提升干擾素引起的抗病毒作用。因此，我們篩選不同的 STAT3 抑制劑並研究其在調控第一型干擾素的訊息及功能上所扮演的角色。我
們發現在第一型干擾素的刺激之下，WP1066 能有效的提高正常的小鼠胚胎纖維
母細胞(MEFs)及小鼠骨髓衍生的巨噬細胞(BMMs)的抗病毒相關基因的表現。而
這個現象在 STAT3 基因剔除細胞上是觀察不到的，這證實了抑制劑所造成的現
象是因為作用在 STAT3 上。我們更進一步利用腦心肌炎病毒(EMCV)感染小鼠
胚胎纖維母細胞(MEFs)，試驗 WP1066 對於第一型干擾素所引起抗病毒反應的
影響。隨著 WP1066 藥物濃度的增加，被感染細胞內的 EMCV 基因會逐漸下
降，這暗示了細胞的抗病毒反應有效被提升。此外，我們也發現在 STAT3 基因
轉殖的細胞中，WP1066 同樣可以消弭 STAT3 在第一型干擾素調控訊息中的負
調控機制。另一方面，我們去探討了 WP1066 的作用機制，根據實驗結果發現，
這個 STAT3 抑制劑並不會影響到第一型干擾素引發的 STAT1 和 STAT2 的
磷酸化以及入核的能力。總而言之，綜合以上的結果，我們推論出 WP1066 可
以專一抑制住STAT3 的活性，進而提升細胞的抗病毒反應。這些結果也提供了
一個治療病毒感染的模式，利用STAT3 抑制劑去強化第一型干擾素的抗病毒反
應。
Abstract
Type I interferons (IFNs) plays a key role in innate immunity to protect host from
viral and bacterial infections. Signal transducer and activator of transcription (STAT)
proteins, such as STAT1, STAT2, and STAT3, are activated by IFN-α/β.Unlike the
complex of STAT1 and STAT2, which promotes type I IFN-mediated antiviral
response, STAT3 negatively regulates type I IFN-mediated pathway. In previous
study, STAT3 knockout mouse fibroblast (MEFs) and primary bone-marrow-derived
macrophages (BMMs) showed enhanced IFN functions. We hypothesize that
targeting STAT3 function would enhance IFN-mediated antiviral response. Therefore,
we screened different STAT3 inhibitors and investigated their roles in IFN-mediated
signaling and functions. Among them, WP1066 was shown to induce higher
expression of antivirus-associated genes in both WT MEFs and BMMs in response to
IFN-α4 stimulation. Interestingly, the phenomenon was abolished in the absence of
STAT3, suggesting that the effect of the inhibitor was STAT3-dependent. The effect
of WP1066 in IFN-α4-mediated antiviral response was further examined by infecting
MEFs with encephalomyocarditis virus (EMCV). Enhanced antiviral response was
revealed by reduced expression of EMCV-specific gene in infected cells in a
dose-dependent manner. In addition, the suppressive effect of FL- and N-terminal
domain- (NTD) STAT3 in STAT3KO MEFs in response to type I IFN was reversed
by WP1066 treatment. We further addressed the mechanism of WP1066 activity and
found that the inhibitor did not affect phosphorylation and nuclear translation of
STAT1 and STAT2. Taken together, these results suggest that WP1066 may enhance
antiviral function of cells by targeting STAT3 function. This study provides a
therapeutic approach for virus infection by targeting STAT3 to enhance type I
IFN-induced antiviral response.
Abbreviations
BMM: Bone marrow-derived macrophage
CCD: Coiled-coil domain
ChIP: Chromatin Immunoprecipitation
CSF-1: Colony-stimulating factor-1
DBD: DNA binding domain
dsRNA: Double-stranded RNA
DTT: Dithiothreithol
EDTA: Ethylene diamine tetracetic acid
EMCV: Encephalomyocarditis virus
FBS: Fetal bovine serum
GAS: Gamma-IFN-activated sequence
IFIT: Interferon-induced protein with tetratricopeptide repeat
IFN: Interferon
iNOS: Inducible nitric oxide synthetase
IRF: Interferon regulatory factor
ISG: Interferon-stimulated gene
ISGF3: Interferon stimulated growth factor 3
ISRE: Interferon stimulated response element
JAK: Janus kinase
JH: JAK homology
KO: Knock out
MDA5: Melanoma differentiation-associated gene 5
MEF: Mouse embryonic fibroblast
MOI: Multiplicity of infection
NTD: Amino-terminal domain
OAS: Oligoadenylate synthetase
PCR: Polymerase chain reaction
PKR: RNA-dependent protein kinase
PMSF: Phenylmethylsulfonyl fluoride
RIG-I: Retinoic-acid-inducible gene I
RNaseL: Ribonuclease L
SH2: Src-homology 2
STAT: Signal transducer and activator of transcription
Table of Contents
致謝...i
中文摘要...ii
Abstract... iii
Abbreviations ...v
Chapter 1 Introduction...1
1.1 Type I IFNs ...1
1.2 JAK and STAT family...1
1.3 Type I IFN-mediated signaling ...3
1.4 Antiviral effects of type I IFN ...3
1.5 Functions of STAT3...5
1.6 STAT3 inhibitors...6
1.6.1 WP1066 ...7
1.6.2 FLLL32 ...8
1.6.3 LLL12...8
1.6.4 Stattic ...9
1.6.5 Cpd188 ...9
Chapter 2 Rationales and objectives ...10
Chapter 3 Materials and Methods...11
3.1 Materials ...11
3.1.1 STAT3 inhibitors ...11
3.1.2 Antibody...11
3.1.3 Recombinant IFN-α4...12
3.1.4 Cell lines...12
3.1.5 Culture medium ...12
3.1.6 Virus strains ...13
3.2 Methods...13
3.2.1 Western blotting analysis ...13
3.2.2 Preparation of cytosolic and nuclear extracts ...14
3.2.3 Preparation of bone marrow-derived macrophage (BMM) ..14
3.2.4 Quantitative RT-PCR...15
3.2.5 Chromatin Immunoprecipitation (ChIP) ...16
Chapter 4 Results...19
4.1 WP1066 enhances IFN-α4-mediated expression of ISGs ...19
4.2 WP1066 enhances IFN-α4-mediated antiviral response to EMCV....21
4.3 WP1066 promotes ISG gene induction independent of alteration of phosphorylation of STAT1 and STAT2...22 4.4 WP1066 does not affect nuclear translocation of STAT1 and
STAT2………...22
4.5 WP1066 affects the binding ability of ISGF3 to ISRE ...23
4.6 WP1066 blocks STAT3 NTD-mediated suppression on IFN-α-induced gene expression and antiviral responses ...24
Chapter 5 Discussion ...25
5.1 The inhibitory effect of WP1066...25
5.2 How does WP1066 affect the activity of STAT3 ...26
5.3 The clinical implication of WP1066...27
References ...28
Index of Figures
Figures 1. The effect of FLLL32 on ISG genes induction by IFN-α4 stimulation………...……….………...………35 Figures 2. The effect of LLL12 on ISG genes induction by IFN-α4 stimulation………...……….………...………36 Figures 3. The effect of Cpd188 on ISG genes induction by IFN-α4 stimulation………...………...……….……37 Figures 4. The effect of Stattic on ISG genes induction by IFN-α4 stimulation………...………...……….……38 Figures 5. WP1066 enhances ISG gene induction by IFN-α4in a dose dependent mannerin WT MEFs………...……….……….………39 Figures 6. Enhanced ISG gene expression by WP1066 is abrogated in STAT3KO
MEFs………....………...41
Figures 7. WP1066 enhances ISG gene induction by IFN-α4in a dose dependent mannerin WT BMMs………...………...……..…………43 Figures 8. Enhanced IFN-α4-stimulated ISG gene expression by WP1066 is abrogated in STAT3KO BMMs…...………...……….…….…………45 Figures 9 WP1066 enhances IFN-α4-mediated antiviral response to EMCV in WT MEFs and BMMs………...…………..…………...47
Figures 10. Enhancement of IFN-α4-mediated antiviral response to EMCV by WP1066 is abrogated in STAT3KO MEFs………..49 Figures 11. WP1066 does not alter IFN-α4-stimulated activation of STAT1, STAT2 and STAT3………...…...………...………50 Figures 12. WP1066 does not affect nuclear translocation of IFN-α4 activated STAT1, STAT2 and STAT3 in WT MEFs...………..………..………52 Figures 13. WP1066 does not affect nuclear translocation of IFN-α4 activated STATs in WT BMMs………...………53 Figures 14. WP1066 enhances the recruitment of transcription complex ISGF3 to ISRE of ISGs ……..……...………...……54 Figures 15. WP1066 reverses the suppression effect of full length and N-terminal domain of STAT3 on ISG induction…………..………...……57 Figures 16. WP1066 reverse the repression effect of full length and N-terminal domain of STAT3 on type I IFN-inducing antiviral function...……….……….59
Chapter 1 Introduction
1.1 Type I IFNs
IFNs are best known for their antiviral properties (Levy and Garcia-Sastre,
2001). The IFN family includes three main classes of related cytokines: type I, type
II and type III IFNs. Type I IFNs include IFN-α and IFN-β.Type II IFN consists of
IFN-γ. And type III IFNs include three IFN-λ geneproducts, namely IFN-λ1
(IL29), IFN-λ2(IL28A) and IFN-λ3 (IL28B) (Iversen and Paludan, 2010; Pestka et
al., 2004). While most types of cells can produce IFN-α, IFN-βand IFN-λ,only
certain immune cells produce IFN-γupon stimulation.
IFN-αgenes can be divided into two groups: immediate-early response genes
(IFN-α4/β), which are induced rapidly without ongoing protein synthesis, and the
other IFN-αgenes display delayed induction and are synthesized more slowly and
required newly cellular protein synthesis, including IRF7, a member of IRF family
(Marie et al., 1998; Taniguchi and Takaoka, 2002).
1.2 JAK and STAT family
Janus kinases (JAKs) and signal transducer and activator of transcription
factors (STATs) are involved in IFN-mediated signaling pathway. There are four
mammalian JAKs have been identified, namely JAK1, JAK2, JAK3, and TYK2.
All of them belong to a family of non-receptor tyrosine kinase (Taniguchi, 1995).
JAK family contains seven highly conserved domains, ranging from JAK
homology domains 1 (JH1) to JH7. The JH1 possess the tyrosine kinase function
and the JH2 is tyrosine kinase-like domain (also known as pseudokinase domain),
which appears to be required for JH1 catalytic activity. Other portions of the JAKs
have been implicated in receptor association and non-catalytic activity, containing
an SH2-like domain (JH3-JH4) and a Band-4.1, ezrin, radixin, moesin (FERM)
homology domain (JH4-JH7). (Giordanetto and Kroemer, 2002; Kisseleva et al.,
2002; Yamaoka et al., 2004).
The mammalian STAT family has seven members: STAT1, STAT2, STAT3,
STAT4, STAT5a, STAT5b, and STAT6 (Kisseleva et al., 2002). These STATs are
highly homologous and share structurally and functionally conserved domains,
including an amino-terminal domain (NH2), a coiled-coil domain (CCD), a DNA
binding domain (DBD), a linker domain, a Src-homology 2 (SH2) domain and a
transactivation domain (TAD). Among them, SH2 domain plays an important role
in the activation and dimerization of STATs (Lim and Cao, 2006). A conserved Tyr
residue at the C-terminus of all STATs undergoes phosphorylation upon activation,
and interaction with the SH2 domain of their dimer partners.
1.3 Type I IFN-mediated signaling
In general, activation of IFN receptor (composed of IFNAR1 and IFNAR2) by
type I IFN binding induces receptor dimerization and receptor-associated JAKs
phosphorylation. These JAKs mediate phosphorylation at the specific receptor
tyrosine residues, which serve as docking sites for STATs and other signaling
molecules. After STAT recruitment, JAKs activated STATs by phosphorylating
STATs on their tyrosine residue (Tyr701 for STAT1, Tyr689 for STAT2 and
Tyr705 for STAT3). The phosphorylated STAT1 and STAT2 complex with IRF-9,
to form ISGF3, translocate to the nucleus and induce IFN-stimulated genes through
the binding to IFN-stimulated response elements (ISREs) in the promoters of ISGs,
leading to antiviral immunity. On the other hand, phosphorylated STAT1 and
STAT3 form STAT1:STAT3 heterodimer, STAT1:STAT1 or STAT3:STAT3
homodimers, which also translocate into the nucleus and bind to IFN-γ-activated
sequence (GAS), and promote gene expression (Katze et al., 2002; Uddin and
Platanias, 2004).
1.4 Antiviral effects of type I IFN
STAT1 and STAT2 heterodimers associate with IRF-9 leads to the induction
of more than 300 IFN-stimulated genes ISGs. Among the ISGs, melanoma
differentiation-associated gene 5 (MDA5) and retinoic-acid-inducible gene I
(RIG-I) are members of cytosolic pattern-recognition receptor, and IFN regulatory
factor (IRF) family are involved in the amplification and regulation of the IFN
response. Other ISGs like protein kinase R (PKR), 2’,5’-oligoadenylate synthetase
(OAS), ribonuclease L (RNaseL) and members of IFIT1 family are involved in
antiviral mechanisms that interfere with the life cycle of individual viruses (Bowie
and Unterholzner, 2008).
PKR is a double-stranded RNA-dependent protein kinase. It is activated
directly by viral RNAs, and then autophosphorylated and dimerize to form an
activated PKR, which in turn, phophostylates EIF-2α, leading to inhibition of
protein synthesis. OAS in combination with RNaseL constitutes an antiviral RNA
decay pathway. When OAS is activated by viral double-stranded RNA (dsRNA),
the enzyme forms atetramerthatsynthesizes2′,5′-oligoadenylates on itself , which
in turn, activates RNaseL. RNaseL then dimerize through their kinase-like domains
and cleave viral RNAs (Sadler and Williams, 2008) .
The Interferon-induced protein with tetratricopeptide repeat 1 (IFIT1) family
is induced strongly in response to virus infection, IFNs and dsRNA. In mouse, this
family comprises three members, IFIT1 (ISG56), IFIT2 (ISG54) and IFIT3
(ISG49), which encode the corresponding proteins p56, p54, and p49, respectively.
The respective protein product would interact with different subunits of translation
initiation factor 3 (eIF3), achieving an inhibition of translation and leading
inhibition of various cellular and viral processes (Fensterl and Sen, 2011; Fensterl
et al., 2008). The member of IRF family such as IRF1, which is first identified as a
regulator of the IFN-α/βgene promoter. Besides, IRF3 and IRF7 regulate IFN-α/β
production during virus infection (Lin et al., 2000; Sato et al., 1998).
1.5 Functions of STAT3
STAT3 is transiently activated by a large number of different ligands
including IL-6, leukemia-inhibitory factor (LIF), Epidermal Growth Factor (EGF),
platelet derived growth factor (PDGF) and IL-10 other than type I IFNs. Unlike
other STATs, ablation of STAT3 leads to embryonic lethality (Takeda et al., 1997).
As revealed by condition knockout mice, the in vivo functions of STAT3 vary in
different tissues and organs. For example, STAT3 maintains T lymphocytes
survival in thymus (Takeda et al., 1998), participates in acute-phase response in the
liver during inflammation (Alonzi et al., 2001) and positively regulates T cells
differentiation in the thymus (Levy and Lee, 2002).
Interestingly, different from STAT1 and STAT2, STAT3 has been found to be
a negative regulator of type I IFN-mediated signaling. Overexpression of STAT3
downregulates IFN-α-induced induction of ISGs (Ho and Ivashkiv, 2006).
Knockout of STAT3 results in enhanced ISG genes induction and antiviral activity
in response to type I IFN. The phenotype can be attenuated by restoring STAT3
back to STAT3KO MEFs. In addition, N-terminus (1-134 aa) of STAT3 was
sufficient to antagonize IFN response, suggesting that suppressive effect of STAT3
is independent of its DNA-binding and transactivation ability (Wang et al., 2011).
1.6 STAT3 inhibitors
It has been reported that STAT3 is continuously activated in different tumor
cell lines (Yu et al., 2007). STAT3 drives malignant progression through the
dysregulation of key proteins, including Bcl-xL, c-myc, and vascular endothelial
growth factor (VEGF), which promote cell survival, proliferation and angiogenesis,
respectively (Fletcher et al., 2008). STAT3 also inhibits the expression of
mediators, which are necessary for immune activation against tumour cells ( Yu et
al., 2009). Until now, many STAT3 inhibitors are developed as therapeutic agents
for cancer cells. There are several designs of STAT3-targeting drugs, including
directly targeting to the SH2 domain, the DNA-binding domain and antisense
approaches to inhibit STAT3 (Haftchenary et al., 2011; Turkson, 2004).
SH2 domains are highly conserved in proteins, which can be found in various
families such as STATs, Src and JAK. All the SH2 domains examined contain a
basic“αβββα”structure(Gao et al., 2004). A pTyr-recognition site with two
positive charges is formed in its protein–protein interaction face, and one of the
positive charges comes from arginine located on thecentralβ strand (Arg609 in
STAT3-SH2). Most of STAT3 inhibitors are designed to target pTyr-recognition
site of the SH2 domain (Kasembeli et al., 2009; Park and Li, 2011). Other
inhibitors are targeting receptor associated cytoplasmic kinases, such as JAK,
which phosphorylates tyrosine(Y)residueson thetargetreceptor’scytoplasmic
tail. The inhibition of JAK activation disrupts the JAK-STAT pathway (Page et al.,
2011).
The following is a brief introduction of the small molecular STAT3 inhibitors
used in this thesis.
1.6.1 WP1066
WP1066 is an analog of AG490, which was originally selected from a group
of tyrphostins screened for their ability to block JAK2 activity. While AG490 has
limited activity in animal studies and must be used at high concentrations (~50 to
100 μM)to achieve inhibition, and low potency of AG490 is insufficient to warrant
clinical investigation of this compound for the treatment of cancer. WP1066
therefore synthesized based on the caffeic acid benzyl ester scaffold. WP1066
inhibits STAT3 activation potently and shows selective cytotoxicity toward cancer
cells at much lower doses than AG490 (Horiguchi et al., 2010; Iwamaru et al.,
2007).
1.6.2 FLLL32
FLLL32 is a curcumin-derived small molecule inhibitor of the JAK2/STAT3
pathway. Curcumin is the primary bioactive compound isolated from turmeric
which has been shown to inhibit several targets associated with cancer cell
proliferation, especially JAK2/STAT3 pathway. FLLL32 has the potential to
become a drug because of poor bioavailability of curcumin (Lin et al., 2010a).
Moreover, FLLL32 shows selective inhibition of STAT3 phosphorylation and its
DNA binding activities (Fossey et al., 2011).
1.6.3 LLL12
LLL12, which binds to the phosphoryl tyrosine 705 (pTyr705) binding site of
the STAT3 monomer, is developed by Dr. Jiayuh Lin at Ohio State University.
LLL12 inhibits STAT3 phosphorylation and STAT3 activities, as well as,
downregulates STAT3 downstream target genes (Lin et al., 2010b). In addition,
LLL12 specifically reduces phosphorylation of STAT3, but not STAT1 or STAT2
in multiple myeloma cells in response to IFN-αtreatment (Lin et al., 2011).
1.6.4 Stattic
Stattic (STAT3 inhibitory compound) is the first nonpeptidic small molecule
that inhibits the function of the STAT3 SH2 domain by blocking STAT3
phosphorylation in vitro (Schust et al., 2006). Stattic inhibited the binding of a
phosphotyrosine-containing peptide to the STAT3 SH2 domain in a strongly
temperature-dependent manner, suggesting that Static inhibits activation,
dimerization, and nuclear translocation of STAT3 (McMurray, 2006).
1.6.5 Cpd188
Cpd188 is targeting on phosphoryl tyrosine binding pocket of the STAT3 SH2
domain. Cpd188 inhibits the activation and nuclear translocation of phosphorylated
STAT3. It has better potency on STAT3 than STAT1 (Xu et al., 2009).
Chapter 2 Rationales and objectives
Clinically, IFN-αis used to treat patients with HCV infections. A common
property of IFNs is inducing antiviral-associated genes through JAK-STAT
signaling pathway. Unlike the complex of STAT1 containing ISGF3 complex,
which positively regulate ISGs expression, STAT3 plays a negative role in type I
IFN-mediated signaling and response. Deficiency of STAT3 enhances expressions
of antiviral genes and reduces the infection of EMCV and VSV (Wang et al., 2011).
Therefore targeting STAT3 function would promote the antiviral ability of IFN-α.
Here, we want to use small molecule inhibitors of STAT3 to block STAT3
function and enhance type I IFN-mediated signaling. The STAT3 inhibitors we
used including WP1066, FLLL32, LLL12, Cpd188 and Stattic. The aims of this
study are: (1) To screen candidate STAT3 inhibitors for enhancing IFN-α-mediated
antiviral response. (2) To understand the mechanism of STAT3 inhibitors in
enhancing antiviral response.
Chapter 3 Materials and Methods
3.1 Materials
3.1.1 STAT3 inhibitors
Inhibitors Stock conc. Working conc. Source WP1066 50 μM 1.25-5 μM (MEFs)
2.5-10 μM (BMMs)
Merck
LLL12 20 μM 0.4-1.2 μM Dr. Jiayuh Lin, OSU, USA
FLLL32 20 μM 5-20 μM Dr. Jiayuh Lin, OSU, USA
Cpd188 100 μM 50-60 μM Merck
Stattic 100 μM 2.5-10 μM Merck
All inhibitors are solved in DMSO (Merck).
3.1.2 Antibody
Name Source Catalogue NO.
Anti-STAT1 Home-made -
Anti-STAT2 Home-made -
Anti-STAT3 Home-made -
Anti- Tyr701 pSTAT1 Invitrogen 33-3400
Anti- Tyr689 pSTAT2 Millipore 07-224
Anti- Tyr705 pSTAT3 Epitomics 2236-1
Anti-α-tubulin Epitomics 2871-1
Anti-lamin B2 Santa Cruz sc-30267
3.1.3 Recombinant IFN-α4
Recombinant IFN-α4 was generated by stable transfection of HEK293 cells
with pcDNA3.1-mIFN-α4-Ig, a construct containing neomycin resistance gene and
mouse IFN-α4 fused to mouse IgG1. HEK293 cells were transfected by calcium
phosphate precipitation. The medium was refreshed with G418-containg
(700μg/ml) medium after 6 hours of transfection and cultured for 7 days to
eliminate nontransfected cells. mIFN-α4-Ig was purified from culture supernatant
by protein A beads. The titer of the home-made IFN-α4-Ig was determined by
ISRE-luc reporter activity assay using commercial mouse IFN-α(Merck) as a
standard.
3.1.4 Cell lines
Cell type Source
L929 Mouse fibroblast Dr. Betty Wu-Hsieh, NTU STAT3KO MEF Mouse fibroblast Dr. David E Levy, NYU, USA VERO Monkey kidney cells Dr. Lih-Hwa Hwang, NYMU WT MEF Mouse fibroblast Dr. David E Levy, NYU, USA
3.1.5 Culture medium
DMEM medium supplemented with 10% FBS (GIBCO) and 10 ng/ml
gentamicin (GIBCO). Medium was stored under sterile conditions at 4℃.
3.1.6 Virus strains
Encephalomyocarditis virus (EMCV) was a gift from Dr. Lih-Hwa Hwang, at
Graduate Institute of Microbiology and Immunity, National YangMing University.
EMCV was propagated in Vero cell line and viral supernatant was stored at –80℃.
Viral titer was determined by plaque formation assay in Vero cells.
3.2 Methods
3.2.1 Western blotting analysis
Treated cells were pelleted and re-suspended in lysis buffer (300 mM NaCl,
50 mM HEPES pH 7.6, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100, 10 mM
NaPyrPO4, 20 mM NaF, 1 mM EGTA, 0.1 Mm EDTA, 1 mM dithio-threithol, 1
mM phenylmethylsulfonyl fluoride and 1 mM Na4VO3) at 4ºC for 15 mins. Cell
extracts were collected by centrifugation at 12,000 x g for 20 mins and the protein
concentration was measured by Bradford method (Bio-rad). Equal amount of
whole cell extracts were analyzed by electrophoresis using 7% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the
nitrocellulose filter (Millipore). For immunoblotting, the membrane was blocked
with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 and then
blotted with antibody against the indicated proteins. After washing, the membrane
The membrane was then incubated with ECL (Millipore) and exposed to X-ray
film.
3.2.2 Preparation of cytosolic and nuclear extracts
After treatment, cells were washed with PBS and then scraped into RSB buffer
(10 mM Tris pH 7.4, 10 mM NaCl and 3 mM MgCl2). The cell pellet was obtained
by centrifugation at 300x g for 5 mins and resuspended in 0.1ml of RSB-G40 buffer
(10 mM Tris pH 7.4, 10 mM NaCl and 3 mM MgCl2, 10% glycerol, 0.25% NP40,
0.5 mM PMSF, 0.5 mM DTT) for 5 mins. The supernatant obtained after
centrifugation (10,000 x g, 5mins, 4ºC) was used as cytosolic extract. The nuclear
pellet was further resuspended in 50 μlof nuclear extraction buffer (20 mM HEPES
pH 7.9, 420 mM NaCl, 0.5 mM EDTA, 25% glycerol, 0.5 mM PMSF, 0.5 mM
DTT) and incubated on ice for 30 min. The supernatant obtained after
centrifugation (13,000 x g, 5 mins, 4ºC) was used as nuclear extracts. Protein
concentration was measured by the Bradford method.
3.2.3 Preparation of bone marrow-derived macrophage (BMM)
Bone marrow cells were taken from the femurs and tibiae of mice. After
removed RBC cells by ACK lysis buffer, cells were cultured with DMEM
containing 10% FBS overnight to remove stroma cells. Nonadherent bone marrow
cells were cultured in 10% L929 conditioned medium and fresh DMEM
supplemented with 10% FBS for another 5 days. L929 supernatant is the source of
colony-stimulating factor-1 (CSF-1), which is required for differentiation of
macrophages.
3.2.4 Quantitative RT-PCR
Total RNA was isolated with TRIzol reagent (Invitrogen). 3μg of RNA was
taken for reverse transcription. The cDNA prepared form the reaction was subjected
to quantitative PCR by iCycler IQ (Bio-rad) using the following primer sets.
β-actin: Forward,5’-GTGGGGGCGCCCCAGGCACCA-3’
Reverse,3’-CTCCTTATTGTCACGCACGATTTC-5’
EMCV 2A2B: Forward,5’-AATGCCCACTACGCTGGT-3’
Reverse,3’-GTCGTTCGGCAGTAGGGT-5’
IP-10: Forward,5’-TGAGCAGAGATGTCTGAATCCG-3’
Reverse,3’-TGTCCATCCATCGCAGCA-5’
IRF7: Forward,5’-GAGCAAGACCGTGTTTACGA-3’
Reverse,3’-CCATCTTCGACTTCAGCACT-5’
IFIT1: Forward,5’-AGAGCAGAGAGTCAAGGCAGGT-3’
Reverse,3’-TGGTCACCATCAGCATTCTCTCCCA-5’
IFIT2: Forward,5’-ATTGCGAACTACCGTCTG-3’
Reverse,3’-CTTCAGTGCCAAGAGGAC-5’
IFIT3: Forward,5’-GCTCAGCCCACACCCAGCTTT-3’
Reverse,3’-AGATTCCCGGTTGACCTCACTCAT-5’
MDA5: Forward,5’-GAGCCAGAGCTGATGARAGC-3’
Reverse,3’- TCTTATGWGCATACTCCTCTGG-5’
PKR: Forward,5’-GGGCAGACAATGTATGGTAC-3’
Reverse,3’-CAGCAGCTCGTCTATGACAA-5’
RIG-I: Forward,5’-GCATATTGACTGGACGTGGCA-3’
Reverse,3’-CAGTCATGGCTGCAGTTCTGTC-5’
RNaseL: Forward,5’-AGAGACTTGGAGGATCTTGG-3’
Reverse,3’-AGGTCCTTAGTCTCCTCATC-5’
3.2.5 Chromatin Immunoprecipitation (ChIP)
The ChIP protocol was adapted from the fast protocol (Nelson et al., 2006)
with some modifications. Briefly, cells were fixed in 1.42% formaldehyde at RT,
followed by quenching with 125 mM glycine. Cells were washed with cold PBS,
collected, and pelleted by centrifugation at 2000 x g for 5 min. Cells were then
resuspended in ChIP buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 mM EDTA,
0.5% NP-40, 1% Triton X-100, 0.5 mM PMSF, 10 mM NaF and 0.1 mM Na3VO4)
and ChIP lysis buffer (50 mM Tris-HCl pH 8.1, 10 mM EDTA, 1% SDS 1mM
DTT, 0.5mM PMSF, 0.1 mM Na3VO4) then incubated on ice for 10 min to lyse the
nuclei. Nuclear extracts were then sonicated with Vibra-Cell VCX 130 sonicator
(Sonics) to obtain ~200-500 bp fragments of chromatin. The sonication condition is
10s pulse on, 10s pulse off on ice, processed for 5 min. The efficiency of shearing
was verified by agarose gel electrophoresis. For immunoprecipitation, protein A
beads (Roche) preincubated with corresponding antibody overnight at 4°C were
added to nuclear extracts and a small amount of nuclear extracts was kept as input
control in quantitative PCR reactions. After beads-Ab-chromatin complexes
formation, immune complexes were then washed with ChIP buffer, high salt wash
buffer (500 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 mM EDTA, 0.5% NP-40, 1%
Triton X-100), LiCl wash buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 mM
EDTA, 300mM LiCl, 0.5% NP-40, 1% Triton X-100) and ChIP buffer. The
chromatin (Beads-Ab-chromatin) were eluted with freshly prepared elution buffer
(1% SDS and 50 mM NaHCO3), followed by reverse cross-linking with 0.3M NaCl
at 67°C overnight. Samples were incubated with 20 μg proteinase K in proteinase K
buffer (10mM Tris pH 7.5, 5mM EDTA and 0.25% SDS) at 55°C for 4 hours to
degrade protein. DNA was then recovered by phenol-chloroform extraction and
ethanol precipitation. Recovered DNA from ChIP was analyzed by quantitative
PCR using primers specific for corresponding ISREs as shown in the following.
ISRE of IFIT2: Forward,5’-GCTTCAGTTTCACTTTCCAG-3’
Reverse,3’-TTCTCTCGTCTCTCAGTTC-5’
ISRE of MDA5: Forward,5’-ACCAAAGTCCTCACCTAAC-3’
Reverse,3’-CACCCACCTTCCGTTAT-5’
Exon5 of MDA5: Forward,5’-TTATTATCTGCCTCCCCAC-3’
Reverse,3’-CTGTTCTTCTTCGTCCGTA-5’
Chapter 4 Results
4.1 WP1066 enhances IFN-α4-mediated expression of ISGs
It has been previously reported that STAT3 could negatively regulate type I
IFN-mediated antiviral response (Wang et al., 2011). Therefore, we hypothesize
that inhibition of STAT3 by small molecule inhibitors may mimic
STAT3-deficiency to enhance type I IFN-mediated antiviral response. Furthermore,
this strategy may be applied to clinical therapy against severe viral infections.
Type I IFNs mediate their antiviral effect through the induction of
antiviral-associated genes, such as PKR, IRF-7, IP-10, RNaseL, RIG-I and MDA5.
To evaluate the ability of STAT3 inhibitors on enhancing antiviral response, WT
MEFs were pretreated with several STAT3 inhibitors, followed by IFN-α4
stimulation. STAT3 inhibitors, including WP1066, LLL12, FLLL32, Cpd188 and
Stattic were used. As expected, the expression of ISGs was induced by IFN-α4
alone. Interestingly, pretreatment of FLLL32 enhanced the induction of PKR and
IRF7 by 2-fold at 5 μM (Fig. 1). However, IP-10 and RIG-I expression were
reduced under the same conditions. At high concentration of FLLL32 exerted an
adverse effect on gene induction, suggesting that this compound might have
nonspecific toxicity at this dose. On the other hand, LLL12, Cpd188 and Stattic did
Interestingly, WP1066 enhanced the expression of all ISG genes tested,
including PKR, IRF-7, IP-10, RIG-I, RNaseL and MDA5 to 2-3 folds as cpmpared
with IFN-α4 treatment alone, and the effect was in a dose-dependent manner (Fig.
5). To investigate if the effect of WP1066 on IFN response is depend on STAT3,
STAT3KO MEFs were subjected to the same treatment. As shown in Fig. 6,
increased gene expression by WP1066 was abolished in STAT3KO MEFs.
Macrophages are essential components of the innate immunity and a critical
linker between the adaptive and innate immunity. During viral infections,
macrophages establish an antiviral state to clear virus (Gendelman et al., 1990). To
further confirm the enhancement of type I IFN-mediated response, BMMs were
used. Primary BMMs were first prepared from WT and STAT3KO mice and then
subjected to the similar treatment as MEFs. As shown in Fig. 7, compared to cells
treated with IFN-α4 alone, WP1066 enhanced gene expression of PKR, IRF-7,
RIG-I, IFIT1, IFIT2 and IFIT3, but the phenomena was abrogated in STAT3KO
BMMs (Fig. 8). Taken together, these results suggest that WP1066 efficiently
enhances IFN-α4-mediated expression of antiviral-associated genes in the WT
MEFs and primary macrophages and the effect is STAT3-dependent.
4.2 WP1066 enhances IFN-α4-mediated antiviral response to EMCV
In previous results, WP1066 showed a greater ability and selectivity to
enhance the expression of IFN-α4-stimulated ISGs. To further investigate whether
the antiviral activity of IFN-α4 is also enhanced by WP1066, EMCV was used to
infect WT MEFs. WT MEFs pretearted with WP1066, followed by treatment with
low dose of IFN-α4 (1 IU/ml) and infected with EMCV at an MOI of 1. EMCV
gene 2A-2B, as indicative of viral replication, was reduced in a dose-dependent
manner (Fig. 9A), and the inhibition effect could be enhanced as high as 3-fold by
WP1066 at 3.75 μM (Fig. 9C). WT BMMs were also infected with EMCV at an
MOI of 10 after WP1066 and IFN-α4 treatment. EMCV 2A-2B gene was reduced
by 7.5 μM of WP1066 pretreatment (Fig. 9B), and the inhibition effect also could
be enhanced up to 3-fold (Fig. 9D).
To further confirm if the ability of WP1066 to promote antiviral activity is
dependent on STAT3, STAT3KO MEFs that have been treated WP1066 and
IFN-α4 were infected with EMCV at an MOI of 1. The enhancement of antiviral
response to EMCV infection by WP1066 was abrogated in the absence of STAT3,
suggesting that the effect is STAT3 dependent (Fig. 11). Nevertheless, the
suppressive effect on viral replication under 3.75 μM treatment in STAT3KO
MEFs appeared to be an off-target effect because there was a sudden drop of
2A-2B mRNA at this dose.
4.3 WP1066 promotes ISG gene induction independent of alteration of phosphorylation of STAT1 and STAT2
To understand whether the STAT3 inhibitor enhancing ISG genes expression
is due to upregulation of STAT1 and STAT2 phosphorylation, Western blot
analysis was performed. Since STAT family members, especially STAT1 and
STAT3, share structurally conserved domains, the specificity of STAT3 inhibitors
was examined. Total cell extracts from WT MEFs or BMMs pretreated with
STAT3 inhibitors and IFN-α4 were subjected to immunoblotting. As shown in Fig.
9, WP1066 did not promote phosphorylation of STAT1 and STAT2 at the dose
enhanced ISG gene expression in WT MEFs and BMMs, suggesting that the
underlying mechanism for promoting IFN-α4 response is likely independent of
enhancing STAT phosphorylation.
4.4 WP1066 does not affect nuclear translocation of STAT1 and STAT2
We next examined whether WP1066 altered nuclear translocation of STATs.
After the treatment of the indicated doses of WP1066 and IFN-α4, cytoplasmic and
nuclear extracts were prepared from WT MEFs, followed by immunoblotting. As
shown in Fig. 12, IFN-α4 treatment stimulated STAT phosphorylation and
promoted it translocate into nuclear. Although WP1066 slightly reduced the level of
STATs phosphorylation, it did not affect the nuclear level of STAT protein. WT
BMMs also showed a similar result (Fig. 13). These results suggest that WP1066
does not alter nuclear translocations of STATs to enhance gene induction.
4.5 WP1066 affects the binding ability of ISGF3 to ISRE
Since type I IFN-mediated gene transcription requires the binding of ISGF3
complex to ISRE in the promoter region, we next investigated whether WP1066
alters ISGF3 binding ability to ISRE by ChIP assay. As shown in Fig. 14A-B, ISRE
of IFIT1 and MDA5 promoter were greatly enriched by STAT1-specific antiserum
in WT BMMs stimulated by IFN-α4 alone, while exon of MDA5 was not. These
result suggesting that the recruitment of ISGF3 to ISRE was induced by IFN-α4.
Furthermore, the binding ability of ISGF3 to ISRE was further enhanced in cells
treated with WP1066. We also used gel electrophoresis analysis to confirm this
finding (Fig.14C-E). The results were consistent with quantitative PCR that
WP1066 increased the abundance of STAT1 containing ISGF3 on ISRE of IFIT1
and MDA5.
4.6 WP1066 blocks STAT3 NTD-mediated suppression on IFN-α-induced gene expression and antiviral responses
We have previously shown that NTD (1-134 a.a.) of STAT3 was sufficient to
suppress IFN-αsignaling (Wang et al., 2011). To further address whether WP1066
could reverse the suppression effect. STAT3KO MEF was transfected with empty
vector (EV), full length (FL) and NTD of STAT3. As shown in Fig. 15, both FL
and NTD of STAT3 exhibited inhibitory effect on IFN-α-mediated expression of
PKR, IRF-7, IFIT1, IFIT2 and IFIT3. The treatment of WP1066 reversed the
inhibition effect of FL and NTD of STAT3 in a dose-dependent manner, but did not
affect the gene induction of EV-transfected cells.
We next used EMCV to infect the FL- or NTD-STAT3 restored STAT3KO
MEFs that had been treated with IFN-α4 and WP1066. As shown in Fig. 16, the
EMCV 2A-2B gene was induced after EMCV infection for 8 hours, which was
blocked by the treatment of IFN-α4. The introduction of FL- or NTD-STAT3 into
STAT3KO MEFs resulted in increased 2A-2B mRNA, probably was due to
suppressive effect of STAT3. Under the same conditions, WP1066 further
enhanced the antiviral ability of IFN-αin FL- and NTD-STAT3 transfected cells.
These results suggests that WP1066 would abrogate the inhibitory effects of FL-
and NTD-STAT3 on type I IFN-mediated antiviral function.
Chapter 5 Discussion
STAT3 inhibitors have been studied for years, but most of them were focused
on the therapeutic effect in cancer treatment though inhibition of STAT3 activation
(Lavecchia et al., 2011; Yu et al., 2007). It is well known that STAT3 not only
promotes cancer development but also suppresses anti-tumor immunity (Yu et al.,
2009). Here, we reported another effect of STAT3 inhibits, which is fortify type I
IFN response. One of the mechanism is reverse the negative effect of STAT3 on
IFN response. We have demonstrated that WP1066 could further enforce
IFN-α-mediated response, leading to decreased EMCV 2A-2B levels in the treated
cells, suggesting that WP1066 indeed enhances IFN-α-mediated gene induction
and antiviral response by targeting STAT3. Together, these results have prove the
principle that inhibition of STAT3 activity could promote type I IFN-mediated
antiviral response.
5.1 The inhibitory effect of WP1066
WP1066 is an analogues of AG490, a member of the tryphostin family of
tyrosine kinase inhibitors (Ferrajoli et al., 2007; Verstovsek et al., 2008). Although
the target of WP1066 is JAK2, it also affects type I IFN-mediated signaling
share structurally and functionally conserved domains, one possibility is that
WP1066 also targets on JAK1 or TYK2, which are involved in type I IFN-induced
pathway. It remains unclear that WP1066 inhibits STAT3 directly or indirectly
through the action on JAKs. Nevertheless, WP1066 enhances type I IFN signaling
is STAT3-dependent, because STAT3-deficiency inhibited the effect exerted by
WP1066 (Fig. 5 and Fig. 7).
5.2 How does WP1066 affect the activity of STAT3
As described previously, WP1066 enhances type I IFN signaling though a
STAT3-dependent manner. WP1066 also promotes the recruitment of ISGF3 to
ISRE, leading to enhancement of ISG expression (Fig. 14), but how the inhibitor
suppresses STAT3 activity is still unknown. We hypothesize that WP1066 inhibits
the post-translational modifications of STAT3, such as acetylation. A member of
our laboratory, has already found the acetylation of NTD of STAT3 is involved in
its suppressive effect (unpublished data). Mutation in two potential acetylation sites
K49 and K87 resulted in the loss of suppressive function of STAT3. These two site
are located in the N-terminal domain of STAT3. Interestingly, WP1066 also blocks
NTD of STAT3-mediated suppressive effect. Therefore, it is possible that WP1066
inhibits the acetylation of STAT3, leading to the blockage of its function. However
we cannot exclude other possibilities, such as blocking the recruitment of
co-repressors.
5.3 The clinical implication of WP1066
IFN-α is currently approved by Food and Drug Administration for patients with
HCV infection. In our results, the combination of IFN-α and WP1066 enhances the
antiviral ability of cells, it provides a possibility that a STAT3 inhibitor like
WP1066 can enforce IFN-mediated antiviral therapy (Fig. 9 and Fig. 10). However,
there are some problems in application of WP1066. We observe that the toxic
concentration of WP1066 is very close to the effective concentration. Therefore, the
therapeutic window for WP1066 is quite narrow. However, in research of cancer
therapy, normal cell lines are much more resistant to WP1066 than cancer cell lines
(Ferrajoli et al., 2007), and that the inhibitor has been used in animal models (Kong
et al., 2008). Since WP1066 has shown efficacy in vitro, we would further examine
the effect of WP1066 on IFN-mediated antiviral ability in animal models, hoping to
gain insight into its in vivo activity.
References
Alonzi, T., Maritano, D., Gorgoni, B., Rizzuto, G., Libert, C., and Poli, V. (2001).
Essential role of STAT3 in the control of the acute-phase response as revealed by
inducible gene inactivation [correction of activation] in the liver. Mol. Cell. Biol. 21,
1621-1632.
Bowie, A.G., and Unterholzner, L. (2008). Viral evasion and subversion of
pattern-recognition receptor signalling. Nat Rev Immunol 8, 911-922.
Fensterl, V., and Sen, G.C. (2011). The ISG56/IFIT1 gene family. J. Interferon Cytokine
Res. 31, 71-78.
Fensterl, V., White, C.L., Yamashita, M., and Sen, G.C. (2008). Novel characteristics of
the function and induction of murine p56 family proteins. J. Virol. 82, 11045-11053.
Ferrajoli, A., Faderl, S., Van, Q., Koch, P., Harris, D., Liu, Z., Hazan-Halevy, I., Wang,
Y., Kantarjian, H.M., Priebe, W., and Estrov, Z. (2007). WP1066 disrupts Janus kinase-2
and induces caspase-dependent apoptosis in acute myelogenous leukemia cells. Cancer
Res. 67, 11291-11299.
Fletcher, S., Turkson, J., and Gunning, P.T. (2008). Molecular approaches towards the
inhibition of the signal transducer and activator of transcription 3 (Stat3) protein.
ChemMedChem 3, 1159-1168.
Fossey, S.L., Bear, M.D., Lin, J., Li, C., Schwartz, E.B., Li, P.K., Fuchs, J.R., Fenger, J.,
Kisseberth, W.C., and London, C.A. (2011). The novel curcumin analog FLLL32
decreases STAT3 DNA binding activity and expression, and induces apoptosis in
osteosarcoma cell lines. BMC Cancer 11, 112.
Gao, Q., Hua, J., Kimura, R., Headd, J.J., Fu, X.Y., and Chin, Y.E. (2004). Identification
of the linker-SH2 domain of STAT as the origin of the SH2 domain using
two-dimensional structural alignment. Mol Cell Proteomics 3, 704-714.
Gendelman, H.E., Baca, L.M., Turpin, J.A., Kalter, D.C., Dieffenbach, C., Friedman,
R.M., and Meltzer, M.S. (1990). Interactions between interferon and the human
immunodeficiency virus. J. Exp. Pathol. 5, 53-67.
Giordanetto, F., and Kroemer, R.T. (2002). Prediction of the structure of human Janus
kinase 2 (JAK2) comprising JAK homology domains 1 through 7. Protein Eng. 15,
727-737.
Haftchenary, S., Avadisian, M., and Gunning, P.T. (2011). Inhibiting aberrant Stat3
function with molecular therapeutics: a progress report. Anticancer. Drugs 22, 115-127.
Ho, H.H., and Ivashkiv, L.B. (2006). Role of STAT3 in type I interferon responses.
Negative regulation of STAT1-dependent inflammatory gene activation. J. Biol. Chem.
281, 14111-14118.
Horiguchi, A., Asano, T., Kuroda, K., Sato, A., Asakuma, J., Ito, K., Hayakawa, M., and
Sumitomo, M. (2010). STAT3 inhibitor WP1066 as a novel therapeutic agent for renal
cell carcinoma. Br. J. Cancer 102, 1592-1599.
Iversen, M.B., and Paludan, S.R. (2010). Mechanisms of type III interferon expression.
J. Interferon Cytokine Res. 30, 573-578.
Iwamaru, A., Szymanski, S., Iwado, E., Aoki, H., Yokoyama, T., Fokt, I., Hess, K.,
Conrad, C., Madden, T., Sawaya, R., et al. (2007). A novel inhibitor of the STAT3
pathway induces apoptosis in malignant glioma cells both in vitro and in vivo.
Oncogene 26, 2435-2444.
Kasembeli, M.M., Xu, X., and Tweardy, D.J. (2009). SH2 domain binding to
phosphopeptide ligands: potential for drug targeting. Front. Biosci. 14, 1010-1022.
Katze, M.G., He, Y., and Gale, M., Jr. (2002). Viruses and interferon: a fight for
supremacy. Nat Rev Immunol 2, 675-687.
Kisseleva, T., Bhattacharya, S., Braunstein, J., and Schindler, C.W. (2002). Signaling
through the JAK/STAT pathway, recent advances and future challenges. Gene 285, 1-24.
Kong, L.Y., Abou-Ghazal, M.K., Wei, J., Chakraborty, A., Sun, W., Qiao, W., Fuller,
G.N., Fokt, I., Grimm, E.A., Schmittling, R.J., et al. (2008). A novel inhibitor of signal
transducers and activators of transcription 3 activation is efficacious against established
central nervous system melanoma and inhibits regulatory T cells. Clin. Cancer Res. 14,
5759-5768.
Lavecchia, A., Di Giovanni, C., and Novellino, E. (2011). STAT-3 inhibitors: state of the
art and new horizons for cancer treatment. Curr. Med. Chem. 18, 2359-2375.
Levy, D.E., and Garcia-Sastre, A. (2001). The virus battles: IFN induction of the
antiviral state and mechanisms of viral evasion. Cytokine Growth Factor Rev. 12,
143-156.
Levy, D.E., and Lee, C.K. (2002). What does Stat3 do? J. Clin. Invest. 109, 1143-1148.
Lim, C.P., and Cao, X. (2006). Structure, function, and regulation of STAT proteins.
Mol Biosyst 2, 536-550.
Lin, L., Benson, D.M., Jr., Deangelis, S., Bakan, C.E., Li, P.K., Li, C., and Lin, J. (2011).
A small molecule, LLL12 inhibits constitutive STAT3 and IL-6-induced STAT3
signaling and exhibits potent growth suppressive activity in human multiple myeloma
cells. Int. J. Cancer.
Lin, L., Deangelis, S., Foust, E., Fuchs, J., Li, C., Li, P.K., Schwartz, E.B., Lesinski,
G.B., Benson, D., Lu, J., et al. (2010a). A novel small molecule inhibits STAT3
phosphorylation and DNA binding activity and exhibits potent growth suppressive
activity in human cancer cells. Mol Cancer 9, 217.
Lin, L., Hutzen, B., Li, P.K., Ball, S., Zuo, M., DeAngelis, S., Foust, E., Sobo, M.,
Friedman, L., Bhasin, D., et al. (2010b). A novel small molecule, LLL12, inhibits
STAT3 phosphorylation and activities and exhibits potent growth-suppressive activity in
human cancer cells. Neoplasia 12, 39-50.
Lin, R., Genin, P., Mamane, Y., and Hiscott, J. (2000). Selective DNA binding and
association with the CREB binding protein coactivator contribute to differential
activation of alpha/beta interferon genes by interferon regulatory factors 3 and 7. Mol.
Cell. Biol. 20, 6342-6353.
Marie, I., Durbin, J.E., and Levy, D.E. (1998). Differential viral induction of distinct
interferon-alpha genes by positive feedback through interferon regulatory factor-7.
EMBO J. 17, 6660-6669.
McMurray, J.S. (2006). A new small-molecule Stat3 inhibitor. Chem. Biol. 13,
1123-1124.
Nelson, J.D., Denisenko, O., and Bomsztyk, K. (2006). Protocol for the fast chromatin
immunoprecipitation (ChIP) method. Nat Protoc 1, 179-185.
Page, B.D., Ball, D.P., and Gunning, P.T. (2011). Signal transducer and activator of
transcription 3 inhibitors: a patent review. Expert Opin Ther Pat 21, 65-83.
Park, I.H., and Li, C. (2011). Characterization of molecular recognition of STAT3 SH2
domain inhibitors through molecular simulation. J. Mol. Recognit. 24, 254-265.
Pestka, S., Krause, C.D., and Walter, M.R. (2004). Interferons, interferon-like cytokines,
and their receptors. Immunol. Rev. 202, 8-32.
Sadler, A.J., and Williams, B.R. (2008). Interferon-inducible antiviral effectors. Nat Rev
Immunol 8, 559-568.
Sato, M., Hata, N., Asagiri, M., Nakaya, T., Taniguchi, T., and Tanaka, N. (1998).
Positive feedback regulation of type I IFN genes by the IFN-inducible transcription
factor IRF-7. FEBS Lett. 441, 106-110.
Schust, J., Sperl, B., Hollis, A., Mayer, T.U., and Berg, T. (2006). Stattic: a
small-molecule inhibitor of STAT3 activation and dimerization. Chem. Biol. 13,
1235-1242.
Takeda, K., Kaisho, T., Yoshida, N., Takeda, J., Kishimoto, T., and Akira, S. (1998).
Stat3 activation is responsible for IL-6-dependent T cell proliferation through
preventing apoptosis: generation and characterization of T cell-specific Stat3-deficient
mice. J. Immunol. 161, 4652-4660.
Takeda, K., Noguchi, K., Shi, W., Tanaka, T., Matsumoto, M., Yoshida, N., Kishimoto,
T., and Akira, S. (1997). Targeted disruption of the mouse Stat3 gene leads to early
embryonic lethality. Proc. Natl. Acad. Sci. U. S. A. 94, 3801-3804.
Taniguchi, T. (1995). Cytokine signaling through nonreceptor protein tyrosine kinases.
Science 268, 251-255.
Taniguchi, T., and Takaoka, A. (2002). The interferon-alpha/beta system in antiviral
responses: a multimodal machinery of gene regulation by the IRF family of
transcription factors. Curr. Opin. Immunol. 14, 111-116.
Turkson, J. (2004). STAT proteins as novel targets for cancer drug discovery. Expert
Opin Ther Targets 8, 409-422.
Uddin, S., and Platanias, L.C. (2004). Mechanisms of type-I interferon signal
transduction. J Biochem Mol Biol 37, 635-641.
Verstovsek, S., Manshouri, T., Quintas-Cardama, A., Harris, D., Cortes, J., Giles, F.J.,
Kantarjian, H., Priebe, W., and Estrov, Z. (2008). WP1066, a novel JAK2 inhibitor,
suppresses proliferation and induces apoptosis in erythroid human cells carrying the
JAK2 V617F mutation. Clin. Cancer Res. 14, 788-796.
Wang, W.B., Levy, D.E., and Lee, C.K. (2011). STAT3 negatively regulates type I
IFN-mediated antiviral response. J. Immunol. 187, 2578-2585.
Xu, X., Kasembeli, M.M., Jiang, X., Tweardy, B.J., and Tweardy, D.J. (2009). Chemical
probes that competitively and selectively inhibit Stat3 activation. PLoS One 4, e4783.
Yamaoka, K., Saharinen, P., Pesu, M., Holt, V.E., 3rd, Silvennoinen, O., and O'Shea, J.J.
(2004). The Janus kinases (Jaks). Genome Biol 5, 253.
Yu, H., Kortylewski, M., and Pardoll, D. (2007). Crosstalk between cancer and immune
cells: role of STAT3 in the tumour microenvironment. Nat Rev Immunol 7, 41-51.
Yu, H., Pardoll, D., and Jove, R. (2009). STATs in cancer inflammation and immunity: a
leading role for STAT3. Nat Rev Cancer 9, 798-809.
Figures
Figures 1. The effect of FLLL32 on ISG gene
pretreated with the indicated dose
IFN-α4 (1000 IU/ml) for 6 hours.
using primers to PKR (A), IRF-7
was normalized to β-actin level.
The effect of FLLL32 on ISG gene induction by IFN-α4. WT MEFs were
pretreated with the indicated doses of FLLL32 for 1 hour, followed by treatment of
IU/ml) for 6 hours. Total RNA was prepared for quantitative RT
7 (B), IP-10 (C), RIG-I (D) and β-actin. Relative mRNA WT MEFs were
followed by treatment of
quantitative RT-PCR
Relative mRNA
Figures 2. The effect of LLL12 on ISG gene
MEFs were pretreated with the indicated dose
treatment of IFN-α4 (1000 IU/ml) for 6 hours. Total
RT-PCR using primers to PKR (A),
Relative mRNA was normalized to
The effect of LLL12 on ISG gene induction by IFN-α4 stimulation.
MEFs were pretreated with the indicated doses of LLL12 for 1 hour, followed by
IU/ml) for 6 hours. Total RNA was prepared for quantitative
PKR (A), IRF-7 (B), IP-10 (C), RIG-I (D) and
Relative mRNA was normalized to β-actin level.
4 stimulation. WT
hour, followed by
quantitative
and β-actin.
Figures 3. The effect of Cpd188 on ISG gene
MEFs were pretreated with the in
treatment of IFN-α4 (1000 IU/ml) for 6 hours. Total
RT-PCR using primers to PKR (A),
Relative mRNA was normalized to
The effect of Cpd188 on ISG gene induction by IFN-α4 stimulation.
MEFs were pretreated with the indicated doses of Cpd188 for 1 hour, followed by
IU/ml) for 6 hours. Total RNA was prepared for quantitative
PKR (A), IRF-7 (B), IP-10 (C), RIG-I (D) and
Relative mRNA was normalized to β-actin level
.
4 stimulation. WT
hour, followed by
quantitative
and β-actin.
Figures 4. The effect of Stattic
MEFs were pretreated with the in
treatment of IFN-α4 (1000 IU/ml) for 6 hours. Total
RT-PCR using primers to PKR (A),
Relative mRNA was normalized to
Stattic on ISG gene induction by IFN-α4 stimulation.
MEFs were pretreated with the indicated doses of Stattic for 1 hour, followed by
IU/ml) for 6 hours. Total RNA was prepared for quantitative
PKR (A), IRF-7 (B), IP-10 (C), RIG-I (D) and
Relative mRNA was normalized to β-actin level
.
4 stimulation. WT
hour, followed by
quantitative
and β-actin.
Figures 5. WP1066 enhances ISG gene induction by IFN-α4 in a dose-dependent manner in WT MEFs. WT MEFs were pretreated with the indicated doses of WP1066
for 1 hour, followed by treatment with or without IFN-α4 (1000 IU/ml) for 6 hours.
Total RNA was prepared for quantitative RT-PCR using primers to PKR (A), IRF-7 (B),
IP-10 (C), RIG-I (D), RNaseL (E), MDA5 (F), and β-actin. Relative mRNA was
normalized to β-actin level.
Figures 6. Enhanced ISG gene expression by WP1066 is abrogated in STAT3KO MEFs. STAT3KO MEFs were pretreated with the indicated doses of WP1066 for 1
hour, followed by treatment with or without IFN-α4 (1000 IU/ml) for 6 hours. Total
RNA was prepared for quantitative RT-PCR using primers to PKR (A), IRF-7 (B),
IP-10 (C), RIG-I (D), RNaseL (E), MDA5 (F) and β-actin. Relative mRNA was
normalized to β-actin level.
Figures 7. WP1066 enhances ISG gene induction by IFN-α4 in a dose-dependent manner in WT BMMs. WT BMMs were pretreated with the indicated dose of
WP1066 for 1 hour, followed by treatment with or without IFN-α4 (1000 IU/ml) for 4
hours. Total RNA was prepared for quantitative RT-PCR using primers for PKR (A) ,
IRF-7 (B), RIG-I (C), IFIT1 (D), IFIT2 (E), IFIT3 (F) and β-actin. Relative mRNA was
normalized to β-actin level.
Figures 8. Enhanced IFN-α4-stimulated ISG gene expression by WP1066 is abrogated in STAT3KO BMMs. STAT3KO BMMs were pretreated with WP1066 for
1 hour, followed by stimulation with or without IFN-α4 (1000 IU/ml) for 4 hours. Total
RNA of treated cells was subjected to quantitative RT-PCR using primers for PKR (A) ,
IRF-7 (B), RIG-I (C), IFIT1 (D), IFIT2 (E) IFIT3 (F) and β-actin. Relative mRNA was
normalized to β-actin level.
Figures 9. WP1066 enhances IFN-α4-mediated antiviral response to EMCV in WT MEFs and BMMs. WT MEFs (A) and BMMs (B) were pretreated with the indicated
doses of WP1066 for 1 hour, followed by stimulation with or without IFN-α4 (1 or 10
IU/ml) for 16 hours. The treated MEFs and BMMs were infected with EMCV at an
MOI of 1 or 10 , respectively for 4 or 3 hours. Total RNA of the infected cells were
subjected to quantitative RT-PCR using primers for EMCV (2A-2B) and β-actin.
Relative mRNA was normalized to β-actin level. Inhibition percentage was cauculated
according to the following formula in (C) and (D) .
Percent inhibition = 1 – 2A-2B related mRNA+IFN±WP x 100%
2A-2B related mRNA-IFN
Figures 10. Enhancement of
WP1066 is abrogated in STAT3KO
the indicated doses of WP1066 for 1 hour, followed by s
IFN-α4 (1 IU/ml) for 16 hours. The treated cells were
of 1 for 4 hours. Total RNA of the infected cells were subjected to quantitative RT
using primers for EMCV 2A-2B (
(B) Percent inhibition was calculated
Percent inhibition = 1 – 2A-2B related mRNA 2A
Enhancement of IFN-α4-mediated antiviral response to EMCV s abrogated in STAT3KO MEFs. STAT3KO MEFs were pretreated
of WP1066 for 1 hour, followed by stimulation with or
IU/ml) for 16 hours. The treated cells were infected with EMCV at
of 1 for 4 hours. Total RNA of the infected cells were subjected to quantitative RT
2B (A). Relative mRNA was normalized to β-actin lev
calculated according to the following formula:
x 100%
2B related mRNA+IFN±WP 2A-2B related mRNA-IFN
to EMCV by
pretreated with
ion with or without
with EMCV at an MOI
of 1 for 4 hours. Total RNA of the infected cells were subjected to quantitative RT-PCR
actin level.
Figures 11. WP1066 does not alter IFN-α4-stimulated activation of STAT1, STAT2 and STAT3. WT MEFs (A) and BMMs (B) were pretreated with the indicated dose of
WP1066 for 1hr. Followed by treatment with or without IFN-α4 1000 IU/ml for 30
minutes. Total cell lysates were subjected to immunoblotting using antibodies to
pSTAT1, pSTAT2, pSTAT3, STAT1, STAT2, STAT3 and α-tubulin.
Figures 12. WP1066 does not STAT1, STAT2 and STAT3
indicated doses of WP1066 for 1 hour, followed by treatment with or without IFN
1000 IU/ml for 30 minutes. C
immunoblotting using antibodies to pSTAT1, pSTAT2, pSTAT3, STAT1, STAT2,
STAT3, α-tubulin and lamin B.
WP1066 does not affect nuclear translocation of IFN-α4 activated in WT MEFs. WT MEFs were pretreated with the
of WP1066 for 1 hour, followed by treatment with or without IFN
Cytoplasmic and nuclear extracts were subjected to
immunoblotting using antibodies to pSTAT1, pSTAT2, pSTAT3, STAT1, STAT2, activated
WT MEFs were pretreated with the
of WP1066 for 1 hour, followed by treatment with or without IFN-α4
were subjected to
immunoblotting using antibodies to pSTAT1, pSTAT2, pSTAT3, STAT1, STAT2,
Figures 13. WP1066 does not affect n STATs in WT BMMs. WT BMMs
WP1066 for 1 hour, followed by treatment with or without IFN
minutes. Cytoplasmic and nuclear extracts
antibodies to pSTAT1, pSTAT2, pSTAT3, STAT1, STAT2, STAT3,
lamin B.
WP1066 does not affect nuclear translocation of IFN-α4 activated
WT BMMs were pretreated with the indicated dose
WP1066 for 1 hour, followed by treatment with or without IFN-α4 1000 IU/ml for 30
ytoplasmic and nuclear extracts were subjected to immunoblotting
antibodies to pSTAT1, pSTAT2, pSTAT3, STAT1, STAT2, STAT3, α-tubulin and activated
were pretreated with the indicated doses of
IU/ml for 30
were subjected to immunoblotting using
tubulin and
Figures 14. WP1066 enhances the recruitment of transcription complex ISGF3 to ISRE of ISGs. WT BMMs were pretreated with the indicated doses of WP1066 for 1
hour, followed by treating with or without IFN-α4 1000 IU/ml for 15 mins. ChIP was
performed using antibody to STAT1, and QPCR using primers specific to the ISRE
region of IFIT1 (A) and ISRE or exon region of MDA5 (B). Relative abundance was
normalized to the values of enriched to that of regions of input control. (C) Gel
electrophoresis analysis of enriched chromatins were amplified by PCR with indicated
primers. (D) and (E) Quantitative analysis of PCR signals in (C). The results were
expressed as the percentage of immunoprecipitated DNA over total input DNA.
Figures 15. WP1066 reverses the suppression effect of full length and N-terminal domain of STAT3 on ISG induction. STAT3KO MEFs transfected with empty vector
(EV), wild type STAT3 (ST3-FL) and NTD of STAT3 (ST3-NTD) were pretreated with
the indicated doses of WP1066 for 1 hour, followed by treatment with or without
IFN-α4 100 IU/ml for 6 hours. Total RNA were subjected to quantitative RT-PCR using
primers for PKR (A), IRF-7 (B), IFIT1 (C), IFIT2 (D), IFIT3 (E) andβ-actin. Relative
mRNA was normalized to β-actin level.
Figures 16. WP1066 reverses domain of STAT3 on type I IFN
transfected with empty vector (EV), wild type STAT3 (ST3
(ST3-NTD) were pretreated with the indicated dose of WP1066 for 1 hour, followed by
treatment with or without IFN-α
with EMCV at an MOI of 1 for 8
to quantitative RT-PCR using primers for
normalized to β-actin level.
the repression effect of full length and N-terminal ype I IFN-inducing antiviral function. STAT3KO MEFs
vector (EV), wild type STAT3 (ST3-FL) and NTD of STAT3
NTD) were pretreated with the indicated dose of WP1066 for 1 hour, followed by
α4 1IU/ml for 16 hours. The treated cells were
MOI of 1 for 8 hours. Total RNA of the infected cells were subjected
PCR using primers for EMCV 2A-2B. Relative mRNA was terminal
STAT3KO MEFs
FL) and NTD of STAT3
NTD) were pretreated with the indicated dose of WP1066 for 1 hour, followed by
The treated cells were infected
hours. Total RNA of the infected cells were subjected
. Relative mRNA was
