A novel aminothiazole KY-05009 with potential to inhibit Traf2- And Nck-Interacting Kinase (TNIK) attenuates TGF-β1-mediated epithelial-to-mesenchymal transition in human lung adenocarcinoma A549 cells

Article abstract of DOI:10.1371/journal.pone.0110180

Transforming growth factor (TGF)-β triggers the epithelial-to-mesenchymal transition (EMT) of cancer cells via wellorchestrated crosstalk between Smad and non-Smad signaling pathways, including Wnt/β-catenin. Since EMT-induced motility and invasion play a critical role in cancer metastasis, EMT-related molecules are emerging as novel targets of anticancer therapies. Traf2- and Nck-interacting kinase (TNIK) has recently been considered as a first-in-class anti-cancer target molecule to regulate Wnt signaling pathway, but pharmacologic inhibition of its EMT activity has not yet been studied. Here, using 5-(4-methylbenzamido)-2-(phenylamino)thiazole-4-carboxamide (KY-05009) with TNIK-inhibitory activity, its efficacy to inhibit EMT in cancer cells was validated. The molecular docking/binding study revealed the binding of KY-05009 in the hinge region of TNIK, and the inhibitory activity of KY-05009 against TNIK was confirmed by an ATP competition assay (K_i, 100 nM). In A549 cells, KY-05009 significantly and strongly inhibited the TGF-β-activated EMT through the attenuation of Smad and non-Smad signaling pathways, including the Wnt, NF-κB, FAK-Src-paxillin-related focal adhesion, and MAP kinases (ERK and JNK) signaling pathways. Continuing efforts to identify and validate potential therapeutic targets associated with EMT, such as TNIK, provide new and improved therapies for treating and/or preventing EMT-based disorders, such as cancer metastasis and fibrosis.

Full text of DOI:10.1371/journal.pone.0110180

A Novel Aminothiazole KY-05009 with Potential to
Inhibit Traf2- and Nck-Interacting Kinase (TNIK)
Attenuates TGF-b1-Mediated Epithelial-to-Mesenchymal
Transition in Human Lung Adenocarcinoma A549 Cells
1
2
3
4
4
2
Jiyeon Kim *, Seong-Hee Moon , Bum Tae Kim , Chong Hak Chae , Joo Yun Lee , Seong Hwan Kim *
1
Department of Biomedical Laboratory Science, School of Medicine, Eulji University, Jung-gu, Daejeon, Republic of Korea, 2 Laboratory of Translational Therapeutics,
Korea Research Institute of Chemical Technology, Yuseong-gu, Daejeon, Republic of Korea, 3 Division of Drug Discovery Research, Korea Research Institute of Chemical
Technology, Yuseong-gu, Daejeon, Republic of Korea, 4 Drug Discovery Platform Technology Team, Korea Research Institute of Chemical Technology, Yuseong-gu,
Daejeon, Republic of Korea
Abstract
Transforming growth factor (TGF)-b triggers the epithelial-to-mesenchymal transition (EMT) of cancer cells via well-
orchestrated crosstalk between Smad and non-Smad signaling pathways, including Wnt/b-catenin. Since EMT-induced
motility and invasion play a critical role in cancer metastasis, EMT-related molecules are emerging as novel targets of anti-
cancer therapies. Traf2- and Nck-interacting kinase (TNIK) has recently been considered as a first-in-class anti-cancer target
molecule to regulate Wnt signaling pathway, but pharmacologic inhibition of its EMT activity has not yet been studied.
Here, using 5-(4-methylbenzamido)-2-(phenylamino)thiazole-4-carboxamide (KY-05009) with TNIK-inhibitory activity, its
efficacy to inhibit EMT in cancer cells was validated. The molecular docking/binding study revealed the binding of KY-05009
in the hinge region of TNIK, and the inhibitory activity of KY-05009 against TNIK was confirmed by an ATP competition assay
i
(K , 100 nM). In A549 cells, KY-05009 significantly and strongly inhibited the TGF-b-activated EMT through the attenuation of
Smad and non-Smad signaling pathways, including the Wnt, NF-kB, FAK-Src-paxillin-related focal adhesion, and MAP
kinases (ERK and JNK) signaling pathways. Continuing efforts to identify and validate potential therapeutic targets
associated with EMT, such as TNIK, provide new and improved therapies for treating and/or preventing EMT-based
disorders, such as cancer metastasis and fibrosis.
Citation: Kim J, Moon S-H, Kim BT, Chae CH, Lee JY, et al. (2014) A Novel Aminothiazole KY-05009 with Potential to Inhibit Traf2- and Nck-Interacting Kinase
(TNIK) Attenuates TGF-b1-Mediated Epithelial-to-Mesenchymal Transition in Human Lung Adenocarcinoma A549 Cells. PLoS ONE 9(10): e110180. doi:10.1371/
journal.pone.0110180
Editor: Neil A. Hotchin, University of Birmingham, United Kingdom
Received June 9, 2014; Accepted September 8, 2014; Published October 22, 2014
Copyright: ß 2014 Kim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: This work was supported by KRICT’s project, SI-1304 funded by the Ministry of Knowledge Economy of Korea and Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2014002349). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
*
Email: yeon@eulji.ac.kr (JK); hwan@krict.re.kr (SHK)
receptors, TGF-b type I (TbR-I) and type II (TbR-II), transpho-
sphorylates TbR-I, and subsequently phosphorylates the down-
stream molecules, Smad2 and Smad3. The phosphorylated
Smad2/3 complex recruits Smad4, then this trimeric complex
further translocates into the nucleus, where it binds to transcrip-
tion factors, such as Snail/Slug and Twist, to activate TGF-b-
responsive genes [7–12].
Introduction
Epithelial-to-mesenchymal transition (EMT) is the complicated
process of change that epithelial cells undergo to acquire the
characteristics of mesenchymal cells during embryogenesis,
development, wound healing, organ fibrosis, and cancer metastasis
[1,2]. As they undergo EMT, the polarized and closely packed
epithelial cells become more motile and invasive, which are the
main characteristics of spindle-shaped mesenchymal cells. In
cancer cells, particularly, EMT-induced motility and invasion play
critical roles in the process of metastasis. Since metastasis is the
major cause of death in cancer patients, the signaling molecules
involved in the process of EMT are emerging as important, new
therapeutic targets for inhibition.
TGF-b-activated, non-Smad signaling pathways, including the
Wnt/b-catenin signaling pathway, are also required for EMT
induction in cancer cells [13–15]. The canonical Wnt signaling
pathway is mediated by the DNA-binding, HMG box transcrip-
tion factors, lymphoid enhancer-binding factor 1 and T cell-
specific factor (LEF1/TCF), and their coactivator, b-catenin.
Aberrant Wnt signaling is well known for its involvement in cancer
progression and metastasis [16,17]. The interdependence between
Smad and Wnt/b-catenin signaling pathways has been reported in
several studies; the Snail family promotes formation of a b-catenin-
Transforming growth factor (TGF)-b is the major cytokine that
triggers EMT during cancer progression and metastasis [3–6]. The
binding complex of TGF-b with its transmembrane Ser/Thr
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TNIK Inhibition to Block EMT in Cancer
TCF4 transcription complex to regulate the expression of TGF-b3
15]; and TGF-b-dependent activation of LEF1/TCF target genes
[
requires both Smad and LEF1/TCF DNA-binding sites [18].
Additionally, the functional blockade of Smad4 leads to decreased
b-catenin levels [19]. Taken together, these data suggest that
TGF-b triggers EMT through the cross-talk between Smad and
Wnt/b-catenin signaling pathways. Thus, we hypothesize that the
molecules, such as kinases, which directly control both signaling
pathways, could effectively regulate the process of EMT.
Recent reports have proposed the Traf2- and Nck-interacting
kinase (TNIK) as a first-in-class anti-cancer target molecule [20].
A functional study revealed that its kinase activity is essential for
the maintenance of colorectal cancer growth [21]. Through the
interaction with TCF4 and b-catenin, TNIK phosphorylates
TCF4 at serine 154, and mediates the activation of Wnt target
genes that are involved in cancer cell growth [20,21]. Additionally,
TNIK has been identified as one of the kinases responsible for a-
helix 1 phosphorylation of Smad, thus, causing its inhibition [22].
Although only a few biological activities of TNIK are involved in
controlling the Wnt and Smad signaling pathways, we believe that
it might be valuable in the early stage of drug discovery to show
that the pharmacologic inhibition of TNIK has an anti-EMT
effect in cancer cells; this effort could improve the druggability of
TNIK, particularly regarding target validation. Therefore, in this
study, we evaluated the effect of a novel aminothiazole inhibitor of
TNIK, 5-(4-methylbenzamido)-2-(phenylamino)thiazole-4-carbox-
amide (hereinafter referred to as our database code number, KY-
0
5009; Fig. 1A), on TGF-b-mediated EMT in human lung
adenocarcinoma A549 cells [23]. The kinase selectivity assay
revealed that KY-05009 also inhibited Mixed lineage kinase 1
(MLK1) as well as TNIK [23], but we here focused on its cellular
activity relevant to TNIK.
Materials and Methods
Molecular Docking
All computational calculations for molecular docking were
performed using the Schr o¨ dinger suite [24]. The Nck-interacting
kinase structure (PDB ID 2X7F) [25] was revised in Protein
Preparation Wizard, and KY-05009 was generated in LigPrep.
The docking study was carried out with a grid box of
3
˚
3
0630630 A , centered on the corresponding ligand using the
Standard Precision (SP) protocol in Glide. The backbone carbonyl
group of Glu106, the backbone nitrogen, and the carbonyl groups
of Cys108 were selected as H-bond constraints.
KY-05009 synthesis and determination of its binding
constant (K ) for TNIK
i
KY-05009 was synthesized as described previously, with
modifications (Figure S1) [23]. The inhibition activity of ATP
binding to TNIK by KY-05009 was determined using an
d
orthogonal ATP competition assay (K ELECT, KINOMEscan,
DiscoveRx, USA).
i
Figure 1. Binding mode and K of KY-05009 for TNIK. (A)
Cell Culture
Chemical structure of KY-05009. (B) Binding mode of KY-05009 for TNIK.
KY-05009 has two H-bond interactions with Cys108 (red dotted lines) in
the hinge region, and CH/p interactions with Val31, Gly111, and Leu160.
The yellow ball represents the CH/p interactions among Val31, ligand,
A549 human lung epithelial adenocarcinoma cell line was
purchased from ATCC (USA) and maintained in Dulbecco’s
modified Eagle’s medium (DMEM; Hyclone, UT), containing
1
0% heat-inactivated fetal bovine serum (FBS) and 1% antibiotics
i
and Gly111. (C) The binding constant, K , of KY-05009 for TNIK was
determined using an ATP competition assay.
doi:10.1371/journal.pone.0110180.g001
(100 U/mL penicillin and 100 mg/mL streptomycin), in a
humidified atmosphere of 5% CO at 37uC.
2
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TNIK Inhibition to Block EMT in Cancer
Cell viability assay
A549 cells (5610 cells/well) were seeded in a 96-well plate, and
carried out overnight at 4uC with an anti-TCF4 antibody in
combination with 30 mL of PureProteome Protein A magnetic
beads. TCF4-bound proteins were subjected to Western blot
TM
3
incubated for 24 h. After serum starvation for 24 h, cells were
treated with 5 ng/mL human recombinant TGF-b1 (R&D
systems) and KY-05009 for 48 h. Cell viability was measured
using the Cell Counting Kit-8 (Dojindo Molecular Technologies,
Japan), according to the manufacturer’s instructions. Absorbance
was measured using the Wallac EnVision microplate reader
analysis.
Immunocytochemistry
5
A549 cells (1610 cells/mL) were incubated on a 1% gelatin-
coated slide glass for 24 h. After serum starvation for 24 h, cells
were treated with TGF-b1 and KY-05009 for 48 h. All cells were
fixed with 2% formaldehyde, permeabilized with 0.1% Triton X-
(PerkinElmer, Finland). All experiments were performed in
triplicate.
1
00 in PBS, and blocked with 3% BSA in PBS. Expression of p-
Smad2, E-cadherin, and vimentin was detected using each
respective primary antibody, and visualized with Alexa Fluor
TOP/FOPflash and NF-kB reporter luciferase assays
A549 cells were trasfected using TOPflash TCF reporter
plasmid (wild type TCF binding site, Millipore), FOPflash plasmid
4
88-conjugated secondary antibodies (Invitrogen). Nuclei were
2
000
counterstained with Hoechst 33258. All images were observed by
DeltaVision RT wide-field epifluorescence microscope imaging
system and softWoRxs image analysis program (Applied Precision,
NW, USA).
(
mutant TCF binding site, Millipore) and Lipofectamine
Invitrogen) in an antibiotics-free medium. FOPflash-normalized
(
TOPflash luciferase activity was represented to the relative TCF/
LEF luciferase activity. A549 cells were also transfected using the
5
NF-kB reporter (5610 TU; SABioscience), in an antibiotic-free
medium. After 48 h, the medium was changed to culture medium
containing 10% FBS, and transduced cells were selected in culture
medium with 30 mg/mL of puromycin (Sigma, MO). After
Migration assay
A549 cells (1.5610 cells/well) were incubated for 24 h in a
bottom line-marked, 96-well ImageLock Microplate (ESSEN
4
4
puromycin selection, cells (1610 cells/well) were incubated in a
BioScience). After serum starvation for 24 h, wounds were made
TM
using a 96-well WoundMaker
were treated with TGF-b1 and KY-05009. The continuous kinetic
9
6-well plate for 24 h. After TOP/FOPflash or NF-kB reporter
(ESSEN BioScience), and cells
transfection, serum deprived cells were treated with TGF-b1 and
KY-05009 for 48 h. Cells were washed with PBS, then lysed using
passive lysis buffer (Promega). Luciferase activity was evaluated
using the luciferase reporter assay (Promega). All experiments were
performed in triplicate.
TM
output of migrated cells was analyzed by IncuCyte
software,
every 12 h. The relative wound density (RWD) was calculated in
comparison with a similar cell population that was treated with
TGF-b1 alone. All experiments were performed in triplicate.
Western blot analysis
Invasion assay
5
Cytoplasmic or nuclear protein fractions of A549 cell lysates
were prepared using the RIPA buffer (Cell Signaling Technology,
Inc.) or the NucBuster Protein Extraction kit (Novagen, Germany).
After protein quantification, cytoplasmic or nuclear protein
A549 cells (1610 cells/well) were incubated in a 24-well plate
for 24 h. After serum starvation for 24 h, cells were treated with
TGF-b1 and KY-05009 for 48 h. Cells were collected using
trypsin-EDTA, and resuspended in serum-free medium for
counting. Culture medium (30 mL) containing 10% FBS was
added to the bottom of a Boyden chamber. After placing over the
gelatin-coated membrane filter, the silicone gasket, and the top
(40 mg) was loaded on 8–15% polyacrylamide gels, and separated
proteins were transferred to PVDF membranes. Proteins were
detected using specific primary antibodies against: TNIK, TCF4,
b-catenin, vimentin, Twist, actin, and histone H3, purchased from
Santa Cruz Biotechnology, Inc. (TX, USA); phospho (p)-ERK1/2,
ERK1/2, p-Smad2 (S465/467), Smad2/3, Snail, p-FAK (Y925),
FAK, p-Src (Y416), Src, p-paxillin (Y118), paxillin, p-JNK (Y183/
Y185), and JNK, purchased from Cell Signaling Technology, Inc.;
p-Ser, a-smooth muscle actin (a-SMA), and IgG, purchased from
Abcam; and E-cadherin and N-cadherin, purchased from BD
Biosciences and Millipore. After incubation with HRP-conjugated
secondary antibodies, membranes were developed using Super-
Signal West Femto Maximum Sensitivity Substrate (Pierce) and
the LAS-3000 luminescent image analyzer (Fuji Photo Film Co.,
Ltd., Japan). Each analysis was repeated three times in order to
check the reproducibility of result and ImageJ (NIH, USA)
software-based quantification of the detected band was carried out
in the images represented in each figure. The relative, normalized
ratio between phosphorylated protein and the protein itself or the
loading control was presented in each image of figures.
4
chamber, the cell suspension (2610 cells/50 mL) was added to the
top chamber, followed by incubation at 37uC in 5% CO for 6 h.
2
The membrane filter was collected, fixed, and stained using the
Diff-Quick staining kit (Dade Behring), according to the manu-
facturer’s instructions. After the filter was dried and stabilized on a
glass slide using 30% glycerol solution, the migrated cells were
counted in three randomly selected fields at 4006 magnification.
All experiments were performed in triplicate.
Quantitative Real Time-PCR
5
A549 cells (1610 cells/mL) were incubated in a 6-well plate for
4 h. After serum starvation for 24 h, cells were treated with TGF-
2
b1 and KY-05009 for 72 h. Total RNA was isolated using the
TRIzol reagent (Life Technologies), and cDNA was synthesized
using the Omniscript Reverse Transcriptase Kit (Qiagen),
according to the manufacturer’s instructions. Quantitative reverse
transcriptase (RT)-PCR was performed using Brilliant SYBR
Green Master Mix (Stratagene) and the Mx3000P Real-Time
PCR system (Stratagene). Primer sequences used in this study were
designed as the following: MMP-2, forward, 59-TTG ACG GTA
AGG ACG GAC TC-39, reverse, 59-ACT TGC AGT ACT CCC
CAT CG-39; MMP-9, forward, 59- TTG ACA GCG ACA AGA
AGT GG-39, reverse, 59-GCC ATT CAC GTC GTC CTT AT-
39; and GAPDH, forward, 59-GAG TCA ACG GAT TTG GTC
GT-39, reverse, 59-GATCTCGCTCCTGGAAGATG-39. All
Immunoprecipitation
A549 cells were seeded at a density of 1610 cells/mL, and
cultured for 24 h. After serum starvation for 24 h, cells were
5
treated with TGF-b1 and KY-05009 for 48 h. After protein
quantification, total lysates were pre-cleared with normal control
TM
IgG and PureProteome Protein A magnetic beads (Millipore).
Immunoprecipitation of endogenous protein complexes was
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TNIK Inhibition to Block EMT in Cancer
reactions were run in triplicate, and data were analyzed using the
2
In the process of EMT, TGF-b1-induced activation of TCF4-
mediated transcription is dependent on the protein level of b-
catenin, and/or its nuclear translocation. As shown in Fig. 2B,
TGF-b1 induced the nuclear translocation of b-catenin in A549
cells, while 10 mM KY-05009 completely inhibited this induction.
KY-05009 also reduced b-catenin protein levels at 3–10 mM.
The binding of b-catenin to TCF4, and subsequent phosphor-
ylation of TCF4, is necessary for TNIK to activate Wnt signaling
[20,21]. Here, TGF-b1 induced the phosphorylation of TCF4,
and this induction was strongly inhibited by KY-05009 (Fig. 2C).
Although KY-05009 did not alter the TGF-b1-induced increase in
nuclear TNIK protein levels (Fig. 2B), it inhibited TNIK binding
to TCF4 (Fig. 2C).
DDC
2
T
method [26]. GAPDH was used as an internal standard.
Statistical significances were determined using the Student’s t-test
2
DDC
with GAPDH-normalized 2
values.
T
Gelatin zymography
To analyze MMP-2 and MMP-9 activity, A549 cells (1610
5
cells/well) were incubated in a 24-well plate for 24 h. After serum
starvation for 24 h, cells were treated with TGF-b1 (5 ng/mL) and
KY-050095 for 48 h. The supernatants were collected, centrifuged
at 3,0006g for 10 min, concentrated using Amicon Ultra
Centrifugal Filter Units (Millipore), and quantified using the
BCA protein assay kit (Pierce). Proteins (40 mg) were loaded onto a
gelatin-containing acrylamide gel (8% acrylamide; 1.5 mg/mL
gelatin), and separated by electrophoresis. Next, the gel was
washed with 2.5% Tween-20 solution, developed overnight at
KY-05009 inhibits TGF-b1-induced activation of Smad
signaling
3
and 5 mM CaCl ), stained with 0.25% Coomassie blue R250
7uC in Zymogram incubation buffer (50 mM Tris-HCl, pH 7.6;
To investigate the effect of KY-05009 on TGF-b1-induced
activation of Smad signaling, protein levels of p-Smad2, Smad2/3,
and the transcription factors, Snail and Twist, were evaluated. As
shown in Fig. 3A, TGF-b1 strongly induced the phosphorylation
of Smad2 and nuclear translocation of p-Smad and Smad2/3,
while KY-05009 inhibited these inductions. The inhibitory effect
of KY-05009 on TGF-b1-induced phosphorylation of Smad2 was
also confirmed by immunocytochemistry analysis (Fig. 3B).
Additionally, TGF-b1 strongly induced the protein expression of
Snail and Twist, and KY-05009 also dramatically inhibited these
inductions.
2
solution, and destained with a solution of 50% methanol and 10%
acetic acid until the part of membrane degraded by MMP-2 or
MMP-9 became clear.
Statistical analysis
Data are presented as mean 6 SD. Statistical significance of
independent three experiments was determined using the
Student’s t-test, and differences were considered significant, as
#
#
###
follows:
p,0.01,
p,0.001 (versus the control); * p,0.05,
** p,0.01, *** p,0.001 (versus the cell population treated with
TGF-b1 alone).
KY-05009 inhibits TGF-b1-mediated modulation of EMT
markers
Results
To verify EMT inhibition by KY-05009, the effect of KY-05009
on the expression of epithelial and mesenchymal markers was
evaluated. As shown in Fig. 4A, TGF-b1 reduced the expression
of the epithelial marker, E-cadherin, but increased the expression
of mesenchymal markers, N-cadherin and vimentin. KY-05009
treatment blocked these effects of TGF-b1. This inhibitory effect
of KY-05009 on the TGF-b1-mediated change of EMT markers
was also confirmed by immunocytochemistry analysis (Fig. 4B);
both the TGF-b1-mediated decrease of E-cadherin and increase of
vimentin were strongly attenuated by KY-05009.
Molecular docking and binding mode of KY-05009 to
TNIK
Before evaluating the effect of KY-05009 on TGF-b-mediated
EMT in human lung adenocarcinoma A549 cells, we investigated
the binding mode and main interactions of KY-05009 with TNIK.
As shown in Fig. 1B, there are two hydrogren bonds between KY-
05009 and the backbone of Cys108 in the hinge region that play
crucial roles in the mechanism by which KY-05009 inhibits
TNIK. Leu160 and Val170 undergo CH/p interactions with the
˚
thiazole and benzamide of KY-05009 at a distance of 3.34 A and
˚
.99 A, respectively. Additionally, located between Val31 and
KY-05009 inhibits TGF-b1-induced migration and
invasion
3
Gly111, the benzene ring of KY-05009 stabilizes the binding
mode with CH/p interactions.
Next, the effects of KY-05009 on TGF-b1-induced migration
and invasion were investigated in A549 cells. As shown in Fig. 5A,
the TGF-b1-induced migration of A549 cells was significantly
inhibited by KY-05009. KY-05009 also significantly inhibited
TGF-b1-induced invasion of A549 cells across the gelatin-coated
membrane (Fig. 5B). Quantitative RT-PCR and gelatin zymo-
graphy were used to further investigate the effect of KY-05009 on
the TGF-b1-induced activation of the gelatinases, matrix metallo-
proteinase (MMP)-2 and MMP-9. TGF-b1 induced the mRNA
expression levels and gelatinase activities of both MMP-2 and
MMP-9, while KY-05009 inhibited these inductions (Fig. 5C and
Inhibitory binding constant (K ) of KY-05009 for TNIK
i
The inhibitory binding constant, K , of KY-05009 for TNIK
i
was calculated from triplicate, 11-point dose-response curves, as
shown in Fig. 1C. KY-05009 inhibited the binding of ATP to
TNIK in a dose-dependent manner (K = 100 nM).
i
KY-05009 inhibits TGF-b1-induced activation of Wnt
signaling
To confirm the inhibitory effect of KY-05009 on TCF4-
mediated transcription, and determine the concentration of KY-
5
D). Furthermore, TGF-b1 induced the transcriptional activity of
NF-kB, which is a transcription factor that regulates MMP-2 and
MMP-9 expression, and is well-known to play a role cancer
metastasis; KY-05009 also attenuated this effect in a dose-
dependent manner (Fig. 5E).
0
5009 used in this study, we conducted cell viability and TOP/
FOPflash luciferase activity assays in parallel. As shown in Fig. 2A,
KY-05009 did not cause significant cytotoxicity up to 10 mM, but
it significantly inhibited the TGF-b1-mediated induction of TCF4-
mediated transcription at 1–10 mM. Therefore, a concentration
range of 3–10 mM was used for KY-05009 in the following
experiments.
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TNIK Inhibition to Block EMT in Cancer
Figure 2. KY-05009 inhibits TGF-b1-induced Wnt signaling. (A) Effects of KY-05009 on cell viability and TCF4 transcriptional activity. Serum-
deprived A549 cells were treated with TGF-b1 or its combination with KY-05009 for 48 h, and then cell viability and TOPflash luciferase activity were
measured. FOPflash-normalized TOPflash luciferase activity was represented to the relative TCF/LEF luciferase activity. The expression of TNIK and b-
catenin in cytosolic and nuclear fractions (B), and the protein levels of TCF4-interacting proteins c were measured by Western blot analysis and
immunoprecipitation assay, respectively. Actin, histone H3, and IgG were used as loading controls. The expression of cytosol and nucleus proteins
###
was normalized by actin and histone H3, respectively. Reported results are representatives of triplicate experiments.
control’); ** p,0.01, *** p,0.001 (versus ‘the group treated with TGF-b1 only’).
doi:10.1371/journal.pone.0110180.g002
p,0.001 (versus ‘the
[
30,31]. Therefore, the inhibitory effects of KY-05009 on TGF-
b1-induced activation of ERKs and JNKs were investigated. KY-
5009 inhibited the TGF-b1-induced phosphorylation of ERK1/2
KY-05009 inhibits TGF-b1-induced activation of focal
adhesion and other non-Smad signaling pathways
Phosphorylation of focal adhesion kinase (FAK) at Y925 has
been observed in different invasive tumors, and is associated with
integrin adhesion dynamics and E-cadherin deregulation during
EMT [27,28]. Therefore, we further investigated the effect of KY-
0
and JNK1/2, as well as the TGF-b1-induced increase in JNK1
protein levels (Fig. 6).
Discussion
05009 on the activation of TGF-b1-induced focal adhesion and
invasion-related signaling molecules, FAK, Src, and paxillin [29].
As shown in Fig. 6, TGF-b1 induced the phosphorylation of FAK
TNIK was first identified in a yeast two-hybrid screen, and it
was named for its interaction with both Traf2 and Nck [32]. It
belongs to a subgroup of the Ste20 family of kinases, the germinal
center kinase (GCK) family, which all have an N-terminal kinase
domain and a C-terminal regulatory region. TNIK has been
shown to regulate several signaling molecules and events, but the
focus of this study was on its involvement in TGF-b1-induced
(Y925), Src (Y416), and paxillin (Y118), and these phosphorylation
events were inhibited by KY-05009.
In addition to its canonical, Smad-dependent pathway, TGF-b
can also control EMT through non-Smad signaling pathways,
including those of the MAP kinases, MEK/ERK and JNK
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TNIK Inhibition to Block EMT in Cancer
Figure 3. KY-05009 inhibits TGF-b1-induced Smad signaling. (A) Effects of KY-05009 on TGF-b1-activated Smad signaling. Serum-deprived
A549 cells were treated with TGF-b1 or its combination with KY-05009 for 48 h, and then the levels of p-Smad2 and endogenous Smad2/3 in cytosolic
and nuclear fractions were evaluated by Western blot analysis. The nuclear expression levels of Snail and Twist were also evaluated. The expression of
cytosol p-Smad2 was normalized by endogenous Smad2/3 and all nucleus proteins were normalized by histone H3. (B) Immunocytochemical
confirmation of KY-05009 inhibition of TGF-b1-activated p-Smad2. Nuclei were counterstained with Hoechst 33342. All scale bars represent 50 mm.
The expression of p-Smad2 was represented by the relative intensity of green fluorescence (n = 30), and reported images are representatives of
##
triplicate experiments.
doi:10.1371/journal.pone.0110180.g003
p,0.01 (versus ‘the control’), ** p,0.01 (versus ‘the group treated with TGF-b1 only’).
EMT, as hypothesized from its ability to control both the Wnt and
Smad signaling pathways, which are both important for EMT
study revealed that KY-05009 has two H-bond interactions with
Cys108 in the hinge region of TNIK, and CH/p interactions with
Val31, Gly111, and Leu160. Furthermore, the inhibitory activity
of KY-05009 against TNIK was confirmed by an ATP compe-
[20–22]. In order to validate TNIK as a druggable, anti-cancer
target molecule, it is essential to verify the pharmacologic
relevance of targeting TNIK. Thus, to address this relevance, we
performed this study to investigate the effects of KY-05009 with
the potential to inhibit TNIK activity, on TGF-b-induced EMT in
A549 cells.
tition assay (K , 100 nM).
i
KY-05009 inhibition of TGF-b1-induced activation of TCF4-
mediated transcription in A549 cells is consistent with a previous
study, which showed similar results in colorectal cancer cells [23].
One possible mechanism for this effect could be through KY-
05009 inhibition of ATP binding to TNIK, which is downstream
of TGF-b1 signaling, thus blocking the phosphorylation and
activation of TCF4. In this study, TGF-b1 strongly induced the
nuclear translocation of TNIK protein, but KY-05009 did not
Recently, KY-05009 was shown to be a potent inhibitor of
TNIK (IC50, 9 nM in a kinase assay), attenuating b-catenin/
TCF4-mediated transcription [23]. It also inhibited MLK1 (IC50,
8 nM in a kinase assay), but we focused on its cellular activity
1
relevant to TNIK. Reported here, the molecular docking/binding
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TNIK Inhibition to Block EMT in Cancer
Figure 4. KY-05009 inhibits TGF-b1-mediated modulation of EMT markers. The effect of KY-05009 on TGF-b1-mediated modulation of EMT
markers was evaluated by (A) Western blot analysis and (B) immunofluorescence microscopy. Serum-deprived A549 cells were treated with TGF-b1 or
its combination with KY-05009 for 48 h. Actin was used as a loading control. Nuclei were counterstained with Hoechst 33342, and all scale bars
represent 20 mm. The expressions of E-cadherin and vimentin were represented by the relative intensity of green fluorescence (n = 30), and reported
##
images are representatives of triplicate experiments.
doi:10.1371/journal.pone.0110180.g004
p,0.01,* p,0.05 (versus ‘the group treated with TGF-b1 only’).
change this increased nuclear protein level. However, KY-05009
inhibited serine phosphorylation of TCF4, and attenuated the
formation and activity of the TGF-b1-induced TNIK-TCF4-b-
catenin complex, which culminates in canonical Wnt signaling.
These results provide evidence that KY-05009 inhibits TGF-b1-
mediated Wnt signaling through its inhibition of the kinase activity
of TNIK, which phosphorylates TCF4. In further support, TNIK
has been shown to interact with TCF4 through amino acids 1–
signaling directly induces the expression of Snail [34]. Here, we
showed that KY-05009 strongly inhibited TGF-b1-induced
phosphorylation and nuclear translocation of Smad2 and the
expression of Snail and Twist. KY-05009 also inhibited the TGF-
b1-induced nuclear translocation of Smad2/3, but at the highest
concentration tested (10 mM), it appeared to reduce the expression
or stability of cytosolic Smad2/3. From these results, we
hypothesized that the pharmacologic inhibition of TNIK by
KY-05009 could inhibit TGF-b1-induced EMT through the
attenuation of both Smad activation and expression of its
transcriptional cofactors, such as b-catenin and Snail.
E-cadherin, N-cadherin, and vimentin have all been shown to
be regulated by the Snail family and Twist transcription factors
[35,36], and b-catenin-Smad2/3 complexes have been reported to
transcriptionally activate expression of a-SMA [33]. The translo-
cation of b-catenin from the membrane into the nucleus has also
been observed during the loss of E-cadherin [37], suggesting that
EMT-related gene expression could be regulated by the compli-
cated crosstalk between Wnt and Smad signaling pathways. Here,
we showed that TGF-b1 caused a decrease in E-cadherin and
2
89, which includes the kinase domain [21]. KY-05009 also
caused a decrease in b-catenin protein levels, providing another
possible mechanism through which it inhibits TGF-b1-induced
activation of Wnt signaling.
In its canonical pathway, TGF-b1 binding to its receptors leads
to phosphorylation of Smad2/3, which then associates with
Smad4, and translocates into the nucleus, where Smad complexes
regulate specific target gene expression by interacting with
transcriptional cofactors, such as b-catenin, Snail, and Twist.
Increased expression of these cofactors can sensitize cells to TGF-
b-induced EMT. Additionally, b-catenin-Smad2/3 complexes are
rapidly formed during TGF-b-induced EMT [33], and Wnt
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TNIK Inhibition to Block EMT in Cancer
Figure 5. KY-05009 inhibits TGF-b1-induced migration and invasion. The effect of KY-05009 on TGF-b1-induced migration and invasion of
A549 cells was evaluated using (A) IncuCyte software and (B) Boyden chambers, respectively. The red and white dashed lines a represent the
wounded area and the edge of migrated cells, respectively. Values (% RWD; Relative Wound Density) represent mean 6 SD of triplicate samples, and
reported images are representatives of triplicate experiments. Numbers of invaded cells were represented by an average number of cells per
randomly selected three high-power field (HPF). Effects of KY-05009 on TGF-b1-induced expression and activation of MMP-2 and MMP-9 were
measured by (C) quantitative RT-PCR and (D) gelatin zymography, respectively. (E) The effect of KY-05009 on NF-kB transcriptional activity was
#
#
###
determined by reporter assay.
b1 only’).
doi:10.1371/journal.pone.0110180.g005
p,0.01,
p,0.001 (versus ‘the control’); * p,0.05, ** p,0.01, *** p,0.001 (versus ‘the group treated with TGF-
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TNIK Inhibition to Block EMT in Cancer
tissues, and positively correlates with increased EMT [45].
Additionally, enhanced paxillin phosphorylation has been ob-
served in breast cancer cells derived from mouse lung metastases
[46]. Here, we showed that TGF-b1 strongly induced the
phosphorylation of FAK, Src, and paxillin, while KY-05009
inhibited these inductions.
Activation of MAP kinases is also involved in TGF-b1-induced
EMT. The activation of ERK is required for TGF-b1-induced
EMT in vitro [47]. Consistent with a previous study [48], TGF-b1
strongly induced phosphorylation of ERK1/2, while KY-05009
completely inhibited this induction. Several studies have shown
that TGF-b1 can induce JNK phosphorylation, and constitutively
active JNK can cause cancer cell migration and invasion, and
induces the expression of mesenchymal-specific markers, vimentin
and fibronectin [31,49]. JNK signaling has also been shown to
regulate cancer cell migration through phosphorylation of paxillin
[50]. JNK1 is also able to promote TGF-b1-induced EMT
through its interaction with Smad3 [51], and its down-regulation
blunts TGF-b1-mediated EMT in epithelial cells [52]. Further-
more, inhibiting JNK1 activation attenuates TGF-b1-mediated
EMT-related modulation of E-cadherin and a-SMA expression
[53]. Interestingly, TNIK can promote the activity of JNK2, and
activation of both JNK1 and JNK2 can be blocked by TNIK
knockdown, suggesting that TNIK is required for the activation of
the JNK signaling pathway [32,54,55]. In this study, TGF-b1
strongly induced the expression of JNK1 and the phosphorylation
of JNK1/2 in A549 cells, while KY-05009 completely inhibited
these effects.
Cross-talk between several signaling pathways plays a role in the
acquisition of EMT in cancer cells. Therefore, targeting
molecule(s), which are involved in the process of EMT, might be
sufficient to control cancer metastasis. Although the possible
involvement of MLK1 on the anti-EMT activity of KY-05009
could not be excluded, we suggested in this study that the
pharmacologic inhibition of TNIK by KY-05009 could suppress
TGF-b-activated EMT in A549 cells through the attenuation of
downstream Smad and non-Smad signaling pathways, including
the Wnt, NF-kB, FAK-Src-paxillin-related focal adhesion, and
MAP kinase (ERK and JNK) pathways. These findings imply that
pharmacologic inhibition of TNIK is a possible new method to
control EMT, which contributes to metastatic processes in cancers.
The next step is to identify more potent inhibitors of TNIK, and to
perform functional studies to solidify the druggability of TNIK as
an anti-EMT target, using siRNA and/or expression of a
dominant negative form. This study substantiates the continuous
efforts to identify and validate potential therapeutic targets
associated with EMT, such as TNIK, that may provide new and
improved therapies for treating and/or preventing EMT-based
disorders, such as cancer metastasis and fibrosis.
Figure 6. KY-05009 inhibits TGF-b1-induced activation of focal
adhesion and non-Smad signaling pathways. Serum-deprived
A549 cells were treated with TGF-b1 or its combination with KY-05009
for 48 h. Effects of KY-05009 on TGF-b1-induced activation of focal
adhesion-related and ERK and JNK MAP kinase signaling molecules was
evaluated by Western blot analysis. The ERK1 and 2 (p44 and p42,
respectively) or JNK1 and 2 (p46 and p54, respectively) isoforms could
be separated and differentially evaluated by gel electrophoresis,
according to their molecular weight differences. The expression of p-
FAK, p-Src, p-Paxillin, p-ERK1/2, and p-JNK2/1 was normalized by
endogenous FAK, Src, Paxillin, ERK1/2, and JNK2/1, respectively. Actin
was used as a loading control. Reported results are representatives of
triplicate experiments.
doi:10.1371/journal.pone.0110180.g006
increases in N-cadherin, vimentin, and a-SMA levels, and KY-
0
5009 blocked these effects. Our results suggest that KY-05009
blocks these effects through its inhibitory actions on both the Wnt
and Smad signaling pathways.
Through loss of epithelial and acquisition of mesenchymal
characteristics, tumor cells undergoing EMT become more motile
and invasive, allowing for metastatic spread. Here, we showed that
TGF-b1 strongly induced the migration and invasion of A549
cells, and KY-05009 significantly inhibited these inductions. One
possible mechanism through which KY-05009 inhibits invasion
could be through its attenuation of TGF-b1-induced expression
and activation of MMP-2 and MMP-9 via NF-kB, which is a well-
known transcription factor that controls cancer metastasis [38].
The functionally interdependent protein kinases (i.e., FAK and
Src) and focal adhesion molecules (i.e., paxillin) are also involved
in TGF-b1-induced EMT [39–41]. Hyperactivation of FAK has
been reported in cancer cell lines and cancer metastasis [42], and
Src-mediated regulation of E-cadherin and vimentin has also been
reported in several cancers [43,44]. Paxillin is an oncogene that is
involved in cell migration; it is highly expressed in lung cancer
Supporting Information
Figure S1 Synthesis of KY-05009. Step 1: Preparation of
ethyl 2-cyano-2-(hydroxyimino)acetate. Acetic acid (6.9 g,
1
8
15 mmol) was added to a suspension of ethyl cyanoacetate (10 g,
8 mmol) and sodium nitrite (7.3 g, 106 mmol) in water (40 mL)
at 0–5uC over a period of 1 h. The temperature was slowly raised
to room temperature, and the reaction mixture was stirred for 1 h
at that temperature. After the complete consumption of ethyl
cyanoacetate (monitored by TLC), the reaction mixture was
extracted with ethyl acetate (56150 mL). The combined organic
layer was successively washed with 10% sodium bicarbonate
(26150 mL) and brine solution (125 mL), and dried over sodium
sulfate. The solvent was removed under reduced pressure. The
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9
October 2014 | Volume 9 | Issue 10 | e110180

TNIK Inhibition to Block EMT in Cancer
resulting solid was stirred with n-hexane (300 mL) for 30 minutes
at room temperature, then filtered and dried under vacuum to
mino)thiazole-4-carboxylate (0.66 g, 2.5 mmol) in pyridine
(12 mL), and the mixture was heated at 60uC for 12 h. The
solvent was evaporated, and the residue was purified by silica gel
column chromatography, eluted with 5% MeOH in DCM to
1
afford 11 g (88% yield) of title compound. H-NMR (300 MHz,
CDCl
7
3
) d (ppm) 9.02 (br, 1H), 4.46 (q, 2H, J = 7.1 Hz), 1.42 (t, 3H,
1
.4 Hz). Step 2: Preparation of ethyl 2-amino-2-cyanoa-
cetate. The H-cube system was charged with a Pt/C CatCart
column and was heated to 25uC. The hydrogen pressure was set to
produce 0.4 g (56% yield) of the titled compound. H-NMR
(300 MHz, CDCl ) d (ppm) 11.58 (s, 1H), 7.89 (d, 2H, J = 8.4 Hz),
3
7.40-7.30 (m, 5H), 7.18 (s, 1H), 7-13-7.08 (m, 1H), 4.49 (q, 2H,
J = 7.1 Hz), 1.47 (t, 3H, J = 7.3 Hz). Step 5: Prepara-
tion of 5-(4-methylbenzamido)-2-(phenylamino)thiazole-
1
0 bars. Ethyl 2-cyano-2-(hydroxyimino)acetate (7.3 g, 52 mmol)
was dissolved in EtOH (250 mL), and the solution was pumped
through the H-Cube system with a flow rate of 1 mL/min for 2
days. The collected solution was concentrated under reduced
pressure to produce a yellowish oil. The solution was evaporated
4
-carboxamide. 7M NH
solution of ethyl 5-(4-methylbenzamido)-2-(phenylamino)thiazole-
-carboxylate (0.13 g, 0.33 mmol) in THF (1 mL), and the
3
in MeOH (3 mL) was added to a
4
under reduced pressure to produce 6.4 g (96% yield) of the title
1
compound as a pale yellowish oil. H-NMR (300 MHz, CDCl
solution was heated in a sealed tube at 80uC for 12 h. The
solvent evaporated, and the resulting solid was collected. The
solids were washed with ether and dried to produce 50 mg (43%
3
) d
ppm) 4.43 (s, 1H), 4.34 (q, 2H, J = 7.1 Hz), 1.95 (br, 2H), 1.36 (t,
(
3
(
H, 7.2 Hz). Step 3: Preparation of ethyl 5-amino-2-
phenylamino)thiazole-4-carboxylate. Phenyl isothiocya-
1
yield) of the title compound. H-NMR (300 MHz, CDCl
3
) d (ppm)
2.09 (s, 1H), 7.89 (d, 2H, J = 8.5 Hz), 7.41-7.36 (m, 3H), 7.33-
1
7
5
nate (1.5 g, 9.0 mmol) was added to a suspension of ethyl 2-
amino-2-cyanoacetate (1.0 g, 8.2 mmol) in THF (17 mL), and the
mixture was refluxed for 3 h. The solvent was evaporated, and the
residue was purified by silica gel column chromatography, eluted
.30 (m, 2H), 7.09 (t, 1H, J = 6.9 Hz), 6.90 (m, 1H), 6.80 (s, 1H),
.49 (s, 2H), 2.44 (s, 3H).
(TIF)
with 5% MeOH in DCM to produce 1.2 g (52% yield) of the titled
1
compound. H-NMR (300 MHz, CDCl
Author Contributions
3
) d (ppm) 9.56 (s, 1H),
.50 (d, 2H, J = 8.1 Hz), 7.28–7.23 (m, 1H), 6.70–6.85 (m, 3H),
.21 (t, 2H, J = 7.1 Hz), 1.27 (t, 3H, J = 7.02 Hz). Step 4:
7
4
Conceived and designed the experiments: JK SHK. Performed the
experiments: JK SHM BTK CHC JYL. Analyzed the data: JK SHK.
Contributed reagents/materials/analysis tools: SHK CHC BTK JYL.
Wrote the paper: JK SHK.
Preparation of ethyl 5-(4-methylbenzamido)-2-(phenyla-
mino)thiazole-4-carboxylate. p-toluoyl chloride (0.39 g,
2
.5 mmol) was added to a solution of ethyl 5-amino-2-(phenyla-
References
1
2
3
4
.
.
.
.
Kalluri R, Weinberg RA (2009) The basics of epithelial-mesenchmal transition.
J Clin Invest 119: 1420–1428.
19. Romero D, Iglesias M, Vary CP, Quintanilla M (2008) Functional blockade of
Smad4 leads to a decrease in beta-catenin levels and signaling activity in human
pancreatic carcinoma cells. Carcinogenesis 29: 1070–1076.
Thiery JP, Acloque H, Huang RY, Nieto MA (2009) Epithelial-mesenchymal
transitions in development and disease. Cell 139: 871–890.
20. Mahmoudi T, Li VS, Ng SS, Taouatas N, Vries RG, et al. (2009) The kinase
TNIK is an essential activator of Wnt target genes. EMBO J 28: 3329–3340.
21. Shitashige M, Satow R, Jigami T, Aoki K, Honda K, et al. (2010) Traf2- and
Nck-interacting kinase is essential for Wnt signaling and colorectal cancer
growth. Cancer Res 70: 5024–5033.
Drabsch Y, ten Dijke P (2012) TGF-b signalling and its role in cancer
progression and metastasis. Cancer Metastasis Rev 31: 553–568.
Behrens J, Mareel MM, Van Roy FM, Birchmeier W (1989) Dissecting tumor
cell invasion: epithelial cells acquire invasive properties after the loss of
uvomorulin-mediated cell-cell adhesion. J Cell Biol 108: 2435–2447.
Barcellos-Hoff MH, Akhurst RJ (2009) TGF-b in breast cancer: too much, too
late. Breast Cancer Res 11: 202.
22. Kaneko S, Chen X, Lu P, Yao X, Wright TG, et al. (2011) Smad inhibition by
the Ste20 kinase Misshapen. Proc Natl Acad Sci USA 108: 11127–11132.
23. Yamada T, Shitashige M, Yokota K, Sawa M, Morlyama H (2010) TNIK
inhibitor and its use. US2010/0216795
5.
6.
7.
8.
9.
Buck MB, Knabbe C (2006) TGF-b signaling in breast cancer. Ann NY Acad
Sci 1089: 119–126.
24. Schr o¨ dinger LLC (2009) New York, NY, USA.
Serra R, Crowley MR (2005) Mouse models of TGF-b impact in breast
development and cancer. Endocr Relat Cancer 12: 749–760.
Massague J, Seoane J, Wotton D (2005) Smad transcription factors. Genes Dev
25. Protein Data Bank (PDB) website. Available: www.rcsb.org.
26. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using
real-time quantitative PCR and the 2(2Delta Delta C(T)) Method. Methods 25:
402–408.
1
9: 2783–2810.
Heldin CH, Miyazono K, ten Dijke P (1997) TGF-beta signaling from cell
membrane to nucleus through SMAD proteins. Nature 390: 465–471.
27. Brunton VG, Avizienyte E, Fincham VJ, Serrels B, Metcalf CA III, et al. (2005)
Identification of Src-specific phosphorylation site on focal adhesion kinase:
dissection of the role of Src SH2 and catalytic functions and their consequences
for tumor cell behavior. Cancer Res 65: 1335–1342.
1
1
1
0. Massague J (1998) TGF-beta signal transduction. Annu Rev Biochem 67: 753–
91.
7
1. Peinado H, Portillo F, Cano A (2004) Transcriptional regulation of cadherins
during development and carcinogenesis. Int J Dev Biol 48: 365–375.
2. Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, et al. (2004) Twist
a master regulator of morphogenesis, plays an essential role in tumor metastasis.
Cell 117: 927–939.
28. Avizienyte E, Wyke AW, Jones RJ, McLean GW, Westhoff MA, et al. (2002)
Src-induced de-regulation of E-cadherin in colon cancer cells requires integrin
signalling. Nat Cell Biol 4: 632–638.
29. McLean GW, Carragher NO, Avizienyte E, Evans J, Brunton VG, et al. (2005)
The role of focal-adhesion kinase in cancer - a new therapeutic opportunity. Nat
Rev Cancer 5: 505–515.
1
3. Galliher AJ, Neil JR, Schiemann WP (2006) Role of TGF-b in cancer
progression. Future Oncol 2: 743–763.
30. Suzuki K, Wilkes MC, Garamszegi N, Edens M, Leof EB (2007) Transforming
growth factor beta signaling via Ras in mesenchymal cells requires p21-activated
kinase 2 for extracellular signal-regulated kinase-dependent transcriptional
1
4. Chen XF, Zhang HJ, Wang HB, Zhu J, Zhou WY, et al. (2012) Transforming
growth factor-b1 induces epithelial-to-mesenchymal transition in human lung
cancer cells via PI3K/Akt and MEK/Erk1/2 signaling pathways. Mol Biol Rep
responses. Cancer Res 67: 3673–3682.
3
9: 3549–3556.
31. Wang J, Kuiatse I, Lee AV, Pan J, Giuliano A, et al. (2010) Sustained c-Jun-
NH2-kinase activity promotes epithelial-mesenchymal transition, invasion, and
survival of breast cancer cells by regulating extracellular signal-regulated kinase
activation. Mol Cancer Res 8: 266–277.
1
5. Medici D, Hay ED, Olsen BR (2008) Snail and Slug promote epithelial-
mesenchymal transition through beta-catenin-T-cell factor-4-dependent expres-
sion of transforming growth factor-beta3. Mol Biol Cell 19: 4875–4887.
6. Polakis P (2000) Wnt signaling and cancer. Genes Dev 14: 1837–1851.
7. Behrens J, von Kries JP, Kuhl M, Bruhn L, Wedlich D, et al. (1996) Functional
interaction of beta-catenin with the transcription factor LEF-1. Nature 382:
1
1
32. Fu CA, Shen M, Huang BC, Lasaga J, Payan DG, et al. (1999) TNIK, a novel
member of the germinal center kinase family that activates the c-Jun N-terminal
kinase pathway and regulates the cytoskeleton. J Biol Chem 274: 30729–30737.
33. Kim Y, Kugler MC, Wei Y, Kim KK, Li X, et al. (2009) Integrin alpha3beta1-
dependent beta-catenin phosphorylation links epithelial Smad signaling to cell
contacts. J Cell Biol 184: 309–322.
6
38–642.
1
8. Labbe E, Letamendia A, Attisano L (2000) Associateion of Smads with lymphoid
enhancer binding factor 1/T cell-specific factor mediates cooperative signaling
by the transforming growth factor-beta and wnt pathways. Proc Natl Acad Sci
USA 97: 8358–8363.
34. Yook JI, Li XY, Ota I, Fearon ER, Weiss SJ (2005) Wnt-dependent regulation of
the E-cadherin repressor snail. J Biol Chem 280: 11740–11748.
PLOS ONE | www.plosone.org
10
October 2014 | Volume 9 | Issue 10 | e110180

TNIK Inhibition to Block EMT in Cancer
3
3
3
3
3
5. Xu J, Lamouille S, Derynck
mesenchymal transition. Cell Res 19: 156–172.
R
(2009) TGF-beta-induced epithelial to
46. Chen J, Gallo KA (2012) MLK3 regulates paxillin phosphorylation in
chemokine-mediated breast cancer cell migration and invasion to drive
metastasis. Cancer Res 72: 4130–4140.
47. Xie L, Law BK, Chytil AM, Brown KA, Aakre ME, et al. (2004) Activation of
the Erk pathway is required for TGF-beta1-induced EMT in vitro. Neoplasia 6:
603–610.
6. Ivaska J (2011) Vimentin: Central hub in EMT induction? Small GTPases 2: 51–
3.
5
7. Barker N, Clevers H (2001) Tumor environment: a potent driving force in
colorectal cancer? Trends Mol Med 7: 535–537.
4
4
5
5
8. Kim J, Kim SH (2013) CK2 inhibitor CX-4945 blocks TGF-b1-induced
epithelial-to-mesenchymal transition in A549 human lung adenocarcinoma cells.
PLoS One 8: e74342.
9. Santiba n˜ ez JF (2006) JNK mediates TGF-beta1-induced epithelial mesenchymal
transdifferentiation of mouse transformed keratinocytes. FEBS Lett 580: 5385–
8. Wu Y, Zhou BP (2010) TNF-alpha/NF-kappaB/Snail pathway in cancer cell
migration and invasion. Br J Cancer 102: 639–644.
9. Tumbarello DA, Brown MC, Hetey SE, Turner CE (2005) Regulation of
paxillin family members during epithelial-mesenchymal transformation:
putative role for paxillin delta. J Cell Sci 118: 4849–4863.
a
5
391.
4
4
4
0. Galliher AJ, Schiemann WP (2006) Beta3 integrin and Src facilitate
transforming growth factor-beta mediated induction of epithelial-mesenchymal
transition in mammary epithelial cells. Breast Cancer Res 8: R42.
1. Cicchini C, Laudadio I, Citarella F, Corazzari M, Steindler C, et al. (2008)
TGFbeta-induced EMT requires focal adhesion kinase (FAK) signaling. Exp
Cell Res 314: 143–152.
0. Wei W, Li H, Li N, Sun H, Li Q, et al. (2013) WNT5A/JNK signaling regulates
pancreatic cancer cells migration by phosphorylating paxillin. Pancreatology 13:
3
84–392.
1. Velden JL, Alcorn JF, Guala AS, Badura EC, Janssen-Heininger YM (2011) c-
Jun N-terminal kinase 1 promotes transforming growth factor-b1-induced
epithelial-to-mesenchymal transition via control of linker phosphorylation and
transcriptional activity of Smad3. Am J Respir Cell Mol Biol 44: 571–581.
2. Alcorn JF, Guala AS, van der Velden J, McElhinney B, Irvin CG, et al. (2008)
Jun N-terminal kinase 1 regulates epithelial-to-mesenchymal transition induced
by TGF-beta1. J Cell Sci 121: 1036–1045.
3. Liu Q, Zhang Y, Mao H, Chen W, Luo N, et al. (2012) A crosstalk between the
Smad and JNK signaling in the TGF-b-induced epithelial-mesenchymal
transition in rat peritoneal mesothelial cells. PLoS One 7: e32009.
2. Cance WG, Harris JE, Iacocca MV, Roche E, Yang X, et al. (2000)
Immunohistochemical analyses of focal adhesion kinase expression in benign
and malignant human breast and colon tissues: correlation with preinvasive and
invasive phenotypes. Clin Cancer Res 6: 2417–2423.
5
5
4
3. Wei J, Xu G, Wu M, Zhang Y, Li Q, et al. (2008) Overexpression of vimentin
contributes to prostate cancer invasion and metastasis via src regulation.
Anticancer Res 28: 327–334.
4
4
4. Nagathihalli NS, Merchant NB (2012) Src-mediated regulation of E-cadherin
and EMT in pancreatic cancer. Front Biosci 17: 2059–2069.
54. Gui J, Yang B, Wu J, Zhou X (2011) Enormous influence of TNIK knockdown
on intracellular signals and cell survival. Hum Cell 24: 121–126.
55. Shkoda A, Town JA, Griese J, Romio M, Sarioglu H, et al. (2012) The germinal
center kinase TNIK is required for canonical NF-kB and JNK signaling in B-
cells by the EBV oncoprotein LMP1 and the CD40 receptor. PLoS Biol 10:
e1001376.
5. Jagadeeswaran R, Surawska H, Krishnaswamy S, Janamanchi V, Mackinnon
AC, et al. (2008) Paxillin is a target for somatic mutations in lung cancer:
implications for cell growth and invasion. Cancer Res 68: 132–142.
PLOS ONE | www.plosone.org
11
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