1. RESEARCH POSTER PRESENTATION DESIGN © 2012
www.PosterPresentations.com
High-grade serous ovarian cancer (HGS-OvCa) accounts for two-thirds of
deaths of OvCa patients. Enhanced capacity to invade surrounding tissues
and metastasize is a major characteristic of advanced HGS-OvCa, although
molecular pathways that drive OvCa recurrence and metastasis are largely
unknown. Several studies have provided significant evidence that TAK1 holds
a critical role in tumor invasion, and metastasis of breast cancer. TGF-β-
activated kinase 1 (TAK1) is a member of several signaling pathways that are
stimulated by cytokines. Our previous studies have indicated that TAK1
controls production of metalloproteinase-9 (MMP-9) that plays an essential
role in the invasive and metastatic mechanism. We have reason to believe
that TAK1-MMP9 axis maintains a similar role in ovarian cancer since they
share similar genetic abnormalities. This study will explore TAK1’s role in
ovarian cancer invasion and metastatic potential. Cytotoxicity assays with
inhibitors of the TAK1-IKK signaling axis, (5Z)-7-oxozeaenol, CAY10657, and
BMS-345541, showed that ovarian cancer cell lines OVCAR-3, OVCAR-4,
OVCAR-5, OVCAR-8, and IGROV-1 are growth inhibited by these compounds;
this result indicates that growth of these ovarian cancer cell lines is
dependent on the TAK1-IKK pathway. Effects of the inhibitors will be further
assessed by Cell Cycle Analysis and Anchorage Independent Growth Assays.
This should indicate whether TAK1-IKK axis contributes to survival and/or cell-
cycle progression. Biochemical analysis of TAK1 signaling pathways and
secreted MMP-9 will be carried out using Western Blot assays and whole-cell
lysates or conditioned media from ovarian cancer cells lines treated with the
TAK1-IKK inhibitors. These assays will provide biochemical proofs and
biomarkers of the TAK1 function in ovarian cancer cells. Zymography will be
used to assess the activity of MMP-9 in conditioned media. Together these
studies will provide molecular evidence for the possible role of TAK1 in the
invasive and metastatic potential of OvCa cells.
Abstract
Introduction
Cell Culture:
Ovarian cancer cell lines—IGROV1, OVCAR3, OVCAR4, OVCAR5, OVCAR8—
were grown in RPMI, 10% FBS, 1% Penicillin/Streptomycin media in a
humidified environment at 37 oC with 5% CO2.
Cytotoxicity:
Cells were seeded at 10,000 cells per well in 96 well plates. The next day,
serial dilutions of inhibitors were prepared and added to the wells using a
multichannel pipette. Cells were incubated for 48 hours, fixed and stained
with 1% Methylene Blue, in 50%Methanol (MeOH), 50% H2O. Wells were
washed thoroughly and air-dried overnight. Cells were solubilized in 1% SDS
in PBS and absorbance at 650 nm was measured in a Spectramax M2®
microplate reader. Data were analyzed in Microsoft Excel and SigmaPlot™
where IC50s were determined.
Western Blot and Zymography Sample Preparation:
Cells were seeded at 300,000 cells per well in 6-well plates. The next day,
cells were washed 3X with PBS, and 600 µL serum-free media alone or with
inhibitor was added to each well. Cells were incubated for 48 hours .
Conditioned media was collected and cell debris was removed by
centrifugation. Conditioned media samples were used for In-Gel Zymography
and immunoblotting. Whole-cell lysates were prepared from cells for
immunoblot analysis of GAPDH.
Western Blot (Immunoblotting):
Western blot samples were loaded onto 12.5% SDS-PAGE gels and run at 40
mA until loading buffer ran off. Proteins were transferred to nitrocellulose
membranes at 100 V. Protein transfer was validated by Ponceau S staining.
Membranes were blocked for 1 h with 5% milk in TBS-Tween and incubated
overnight at 4°C with primary antibodies. Membranes were washed with
TBS-Tween, incubated with secondary antibodies at room temperature for 1
h then washed with TBS-Tween and TBS. Immune complexes were visualized
using the chemiluminescent ECL substrate and exposed to X-Ray film.
Zymography:
Conditioned media samples were loaded onto 12.5% SDS-PAGE gels
containing 2% gelatin and run at 120 V until loading buffer ran off. Gels were
washed with 2.5% TritonX-100 then incubated in Development Buffer for 18 h
in a 37°C water bath with agitation. Gels were stained with Coomassie Blue
staining solution for 2 h and distained with Methanol/Acetic Acid solution.
Wound Closure:
Cells were seeded at 200,000 cells per well in 12-well plates and grown to
complete confluency. Wounds were created in each well with a P200 pipet tip
then washed with PBS. RPMI media containing 1% FBS was added to the
wells then cytokines and inhibitors were added as necessary. Inhibitors were
added to the appropriate wells 1 h prior to cytokines. Pictures of the wound
were taken with Spot Software immediately after scratching (0 h) and every
24 h after until the control wounds have closed.
Material Methods
Figure 3. (A) Graphical representations of cytotoxicity assays. The
indicated cell lines were treated with 0.01 µM -100 µM (5Z)-7-oxozeaenol
(oxo), 0.005 µM -50 µM CAY 10657 (CAY), or 0.005 µM -50 µM BMS
345541 (BMS). (B)Table shows the IC50 values of the indicated cell lines
treated with each inhibitor for 48 hours.
Results
• Ovarian cancer cell growth is markedly reduced by inhibitors of the
TAK1-IKK pathway. Given the increased expression of TAK1 target genes
is associated with OvCa recurrence and poor prognosis, this result
suggests that TAK1 signaling may drive ovarian cancer progression.
• Matrix Metalloproteinase-9 (MMP9) secretion is stimulated by TGF-β
and TNF-α in 3 out of 5 tested OvCa lines. MMP9-expressing cells may
have greater invasive, metastatic, and angiogenic potentials.
• Motility of OVCAR3 cells is increased in response to TNF-α, and
decreased by TAK1-IKK pathway inhibitors. This suggests that TAK1-IKK
signaling plays an important role in ovarian cancer cell invasion.
Acknowledgments
This summer research experience was supported in part by DRP-OvCa SPORE
grant (to AVB) and by funding from the National Cancer Institute of the
National Institutes of Health under award number: 3P30CA016056-37S3. The
content is solely the responsibility of the authors and does not necessarily
represent the official views of the National Institutes of Health.”
• Cytotoxicity (IC50): Is the growth of ovarian cancer cells TAK1
dependent?
• Western Blot: Is MMP9 production or secretion TAK1 dependent?
• Zymography: Do ovarian cancer cells secrete active MMP9 and can MMP9
secretion be enhanced by cytokine treatment? If so, does MMP9 secretion
depend on TAK1, TGFβ-Receptor, or p38 signaling? Can TNF and TGF-β
synergistically regulate MMP9 production?
• Cell Motility/Invasion: Is motility of ovarian cancer cells TAK1 dependent?
Do cytokines increase motility? (Wound Closure Assays)
1Canisius College, Buffalo, NY, 14208; 2Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263;
3Summer Research Experience Program in Cancer Science; 4University at Buffalo, SUNY, Buffalo, New York 14260
Korry Wirth1,2,3, Michelle Limoge MS2, Aleksandar Gruevski2,4, Justin Zonneville2,4, Andrei Bakin PhD2
Investigating the Role of TAK-1 in the Invasive and Metastatic Potential of Ovarian Cancer
Objectives
Future Directions
• Test in pre-clinical models whether TAK1-IKK inhibitors can improve
efficacy of current anti-OvCa therapeutics such as cisplatin, gemcitabine.
• Validate the role of TAK1 as a OvCa driver by knockdown (shRNA) or
knockout (CRISPR/Cas9) of TAK1 or IKKs in human OvCa cell lines.
• Assess the role of TAK1 in tumor dormancy and tumor-stroma
interactions.
Conclusions
IC50 (µM)
OXO CAY BMS
Cell Line Exp 1 Exp2 Exp 1 Exp 2 Exp 1 Exp 2
IGROV1 0.50 nd 12 4.2 7.4 nd
OVCAR3 1.7 1.6 0.058 1.2 13 18
OVCAR4 3.0 9.3 0.24 0.25 23 24
OVCAR5 1.7 1.8 nd 4.0 10 21
OVCAR8 0.88 1.2 nd 4.2 7.4 16
IGROV1+OXO
X Data
10-3 10-2 10-1 100 101 102 103
YData
0.0
0.2
0.4
0.6
0.8
1.0
1.2
IGROV1+CAY
X Data
10-3 10-2 10-1 100 101 102 103
YData
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
IGROV1+BMS
X Data
10-3 10-2 10-1 100 101 102 103
YData
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
IC50= 0.50 µM IC50= 4.23 µM IC50= 7.42 µM
OVCAR8+OXO
X Data
10-3 10-2 10-1 100 101 102 103
YData
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
OVCAR-5 + BMS
X Data
10-3 10-2 10-1 100 101 102 103
YData
0.0
0.2
0.4
0.6
0.8
1.0
1.2
OVCAR5+CAY
X Data
10-3 10-2 10-1 100 101 102 103
YData
0.0
0.2
0.4
0.6
0.8
1.0
1.2
OVCAR5+OXO(1)
X Data
10-3 10-2 10-1 100 101 102 103
YData
0.0
0.2
0.4
0.6
0.8
1.0
1.2
OVCAR-8 + BMS
X Data
10-3 10-2 10-1 100 101 102 103
YData
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
OVCAR8+CAY
X Data
10-3 10-2 10-1 100 101 102 103
YData
0.0
0.1
0.2
0.3
0.4
0.5
0.6
IC50= 1.65 µM IC50= 21.3 µM
IC50= 15.8 µM
IC50= 4.03 µM
IC50= 4.18 µMIC50= 1.18 µM
(5Z)-7-oxozeaenol
(TAK1 inhibitor)
BMS 345541
(IKKβ inhibitor)
CAY 10657
(TAK1 inhibitor)
IGROV1
OVCAR5
OVCAR8
A)
B)
Figure 4. Zymography gel (upper) and Western blots (lower) of 48-hrs
conditioned media. The indicated cell lines were treated with 10 ng/mL TNF-
α and 2ng/mL TGF-β.
Figure 5. (A) Wound Closure Assay. OVCAR3 cells were treated with 2 ng/mL
TGF-β, 10 ng/mL TNF-α, 2 ng/mL TGF-β/ 10 ng/mL TNF-α combination, 5
μM OXO, or 20 μM BMS. (B) Graphical representation of the cell-free space
relative to control at each time-point for each treatment.
0%
20%
40%
60%
80%
100%
120%
0 10 20 30 40 50 60 70 80 90
%Cell-FreeSpace
Hours
B)
0 hr 24 hr 48 hr 80 hr
Control
TGFβ
TNFα
TGFβ+TNFα
Oxo
BMS
A)Cytotoxicity Assay :
Wound Closure Assay :
Western Blotting and In-Gel Zymography:
Table 1. IC50 values of TAK1-IKK inhibitors for ovarian cancer cell lines
IGROV1
GAPDH
MMP9
37 -
50 -
100 -
100 -
75 -
MMP9
- + - + - + - + - + TGFβ+TNFα
3 4 5 8
OVCAR
kDa
Figure 1. TAK1 signaling in cytokine pathways and metastasis.
A B C
Basal-like Luminal A Luminal B HGS-OvCa
GENE 80 cases 209 cases 113 cases 316 cases
TP53 * 85.0 11.5 35.4 94.9
EGFR ** 20.0 0.0 0.9 6.7
RB1 *** 45.0 1.0 8.0 19.0
MYC ** 35.0 8.1 16.8 32.3
RELB ** 20.0 1.9 0.9 10.1
FOSL1 ** 17.5 1.4 1.8 5.7
BIRC2 ** 18.8 0.5 0.9 8.0
PTGS2 ** 27.5 4.3 4.4 7.3
VEGFA ** 23.8 2.4 0.0 6.7
IL8 ** 17.5 3.8 2.7 7.7
CCNE1 ** 42.5 1.4 39.8 23.3
IKBKB** 13.8 13.9 24.8 20.0
FADD** 12.5 18.2 25.7 15.7
* Mutations or deletions
** Amplifications or increase in expression
*** Mutations, deletions or decrease
Cases with Alterations
Cases without Alterations
Logrank Test P-value: 0.09
TAK1-targets
Months Disease Free
0 20 40 60 80 100 120 140 160 180
DiseaseFree
HG-Serous Ovarian Cancer (TCGA); Altered in 42 of 158 cases
Cases with Alterations
Cases without Alterations
Logrank Test P-value: 0.005
TAK1-targets
Survival
0 20 40 60 80 100 120 140 160 180
Months Survival
TAK1 targets: FRA1, JUN, MMP9, NFKB2, RELA
Figure 2. Genetics of ovarian and breast cancer. (A) Panel shows percentage
of alterations for each gene in Invasive Breast Carcinoma cases (TCGA, Nature,
2012) and Serous OvCa (TCGA, Nature 474, 2011, 609–615). Basal tumors have
a higher presentation of alterations in TP53, RB1, and increased expression of
TAK1-dependent targets. (B-C) Disease-free and survival data are from TCGA:
http://www.cbioportal.org.
TAB1
TARGET GENES
COX2, IL8, VEGFA, FRA1, JUN, cIAPs,
MMP9
TNF-αTGF-β IL1-β
TAB2
P
AP1
METASTASES
Invasion
Angiogenesis
Survival
P P
NFκB
MAPK
(ERK, p38, JNK)
IKKα-IKKβ
P
TAK1
TRAF6
TRAF2,5
cIAP
Nemo
TRAF4,6 RIP
TAK1-Inh
(5Z)-7-Oxozeaenol
CAY-compound
IKKβ-Inh
BMS-345541
MEK-Inh
(U0126)
The prodomain, catalytic domain, fibronectin domains, and
hemopexin domain are shown in red, blue, green, and yellow,
respectively. Zn2+ ions are indicated in red, and Ca2+ ions are
magenta. Asterisk indicates the cleavage site.
Cleavage site for u/t-PA; MMP-3; MMP-2; MMP-13.
Electrophoresis
(protein separation)
Incubation in Zn/Ca-supplemented
reaction buffer; Coomassie Staining
Conditioned Media
(Serum-free media)
CTR
BMS
OXO
TGFβ
TNF+TGFβ
TNF
I would like to express my gratitude towards the Roswell Park Summer Internship Program, Department of Cancer
Genetics, and Andrei Bakin for providing me with this invaluable opportunity. Many thanks go to Michelle Limoge for
helping and supporting me throughout this project. Special Acknowledgments go to my colleagues Aleksandar
Gruevski, and Justin Zonneville for additional contributions made to this project.