3/30/2012
Supplemental Information
TGF-β and NF-κB signal pathway crosstalk is mediated through
TAK1 and Smad7 in a subset of head and neck cancers
Christian Freudlsperger MD, DDS1,2, Yansong Bian MD, PhD1, Stephanie Contag
BS1,3, Jeffrey Burnett BS1, Jamie Coupar1, Xinping Yang MD1, Zhong Chen MD,
PhD1,*, Carter Van Waes MD, PhD1,*
1Tumor
Biology Section, Head and Neck Surgery Branch, National Institute on
Deafness and Other Communication Disorders, NIH, Bethesda, Maryland, USA.
2Department
of Oral and Maxillofacial Surgery, University Hospital Heidelberg,
Germany.
3Clinical
Research Training Program, a public-private partnership supported jointly by
the NIH and Pfizer Inc.
Supplemental Methods
Reagents
Recombinant TNFα and TGF-β1 were obtained from R&D Systems (Minneapolis,
MN). A 50-mM solution of celastrol (Cayman Chemicals, Ann Arbor, MI) was
prepared in 100% dimethyl sulfoxide (Sigma, St. Louis, MO) and stored as small
aliquots at -20°C. Aliquots were then diluted as needed in cell-culture medium.
Transfection
All transfections were performed using Lipofectamine 2000 according to the
manufacturer’s instructions (Invitrogen, Carlsbad, CA). Transient transfection was
performed using expression vectors and respective control vectors for: TAK1, pWZLNeo-Myr-Flag-MAP3K7 and pWZL-Neo-Myr-Flag-DEST control vector; SMAD7,
pCMV5-SMAD7-HA and pCMV5 control vector (all from Addgene Inc., Cambridge,
MA); TβRII, TβRII-pcDNA3 and pcDNA3 control vector. Validated On-Target siRNAs
from Dharmacon ThermoScientific (Chicago, IL) used included Control (Nontargeting Pool; D-001810-10-05), TAK1 (MAP3K7; L-003790-00-0005) and p65
(RELA; L-003533-00-0005).
Immunohistochemistry
Immunohistochemical staining (IHC) and quantifications of IHC slides were
performed using a previously published method (Bian et al 2009). Briefly, the tissue
slides were dewaxed in xylenes, hydrated through graded alcohols, and incubated in
3% hydrogen peroxide in phosphate-buffered saline (PBS) for 30 min to block the
endogenous peroxidase. After washing in distilled water, antigen retrieval was
performed using 10 mM citric acid buffer in a microwave for 2 minutes at 100%
power and then 18 minutes at 20% power for TGFBR2, TAK1, SMAD7 and control
immunostaining, or 10 mM citrate buffer with 0.01% tween20 in a microwave for 20
minutes at 80% power for other immunostains. . Slides were allowed to cool down to
room temperature, rinsed thoroughly with distilled water and PBS, then incubated in
blocking solution (2.5% BSA in PBS) for 30 min at room temperature. Excess
solution was discarded, and the sections were incubated overnight at 4 °C with the
primary antibody diluted in blocking solution. Antibodies used were TGFBR2 antibody
(sc400, Santa Cruz, Santa Cruz, CA); phospho-Smad2 (Ser465/467) antibody
(Millipore, Billerica, MA); p-Smad3 (S423+S425) antibody (abcam, Cambridge, MA)
at 1:200 dilution; TAK1 antibody (07-263, Upstate Cell Signaling Solutions, Lake
Placid, NY) at 1:500; phospho-NF-κB p65 (Ser276) antibody (Cell Signaling
Technology, Danvers, MA) at 1:100 dilution; and SMAD7 antibody (MAB2029 R&D
Systems, Minneapolis, MN). After washing with PBS, the slides were sequentially
incubated with the biotinylated secondary antibody (1:400; Vector, Burlingame, CA)
for 30 min, followed by the avidin-biotin complex method (Vector Stain Elite, PK-6100
Standard ABC kit; Vector, Burlingame, CA) for 30 min at room temperature. The
slides were washed and developed in 3’3’-diaminobenzidine (FASTDAB tablet;
Sigma, St. Louis, MO) under microscopic control. The reaction was stopped in tap
water, and the tissues were counterstained with hematoxylin, dehydrated, and
mounted. Whole slide images were acquired using an Aperio Scanscope at X20
magnification. Two staining patterns were distinguished relative to the numbers of
positive cells as well as the staining intensity of normal mucosa: 1) ++: no change in
expression compared with normal mucosa tissues, 2) +/-: reduced or absence of
expression compared with normal mucosa tissues.
Real-time QRT-PCR
cDNA products were subjected to real-time QRT-PCR using TaqMan Assays-onDemand probes (Applied Biosystems, Carlsbad, CA). PCR primer/probes included
p65:
Hs00153294_m1;
PAI1
(SERPINE1):
Hs00167155_m1;
and
SMAD7:
Hs00998193_m1. An 18S rRNA probe was used as an internal control. Each sample
was examined in triplicate. The relative mRNA expression levels were determined by
normalizing with the 18S transcripts, the values of which were calculated using the
comparative CT method. Amplification conditions for all QRT-PCR reactions were:
activation of enzymes for 2 min at 50°C and 10 min at 95°C, followed by 40 cycles at
15 s at 95°C and 1 min at 60°C. Thermal cycling and fluorescence detection were
done using an ABI Prism 7700 Sequence Detection System (Applied Biosystems,
Carlsbad, CA).
Luciferase Reporter Assays
A luciferase reporter plasmid with 5X NF-B binding consensus sequence in the
promoter was purchased from Stratagene (La Jolla, CA). Transfection and NF-kB
reporter assays were done as described previously (Bancroft et al 2002). Briefly, UMSCC cells were seeded in 24-well plates as triplicates and transiently transfected with
the indicated expression construct, reported plasmid, and β-galactosidase control
vector, and lysates were analyzed at the indicated time using the Dual-Light
Combined Reporter Gene Assay System (Tropix, Applied Biosystems, Carlsbad, CA)
in the Wallac VICTOR2 1420 Multilabel Counter (Perkin Elmer, Waltham, MA). Each
sample was assayed in triplicate and data were presented as the mean + standard
deviation (SD).
Western Blot analysis
Proteins were separated by electrophoresis on 4-12% gradient gels (Invitrogen),
transferred
to
nitrocellulose
membranes
and
subjected
to
immunoblotting.
Membranes were blocked in 5% non-fat milk in Tris Buffered Saline containing 0.1%
Tween-20. Primary antibodies against TβRII and Oct-1 were purchased from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA); antibodies against Smad, p-Smad2,
Smad3, p-Smad3, β-actin, TAK1, p-TAK1, p65, p-p65, p-IKK/β, p-IκB-, β-actin
were from Cell Signaling Technology, Inc. (Danvers, MA); and antibody against
Smad7 was from R&D Systems Inc. (Minneapolis, MN). Primary antibodies were
used at the indicated concentrations, and HRP-coupled secondary antibodies were
generally used at 1:2000. Immunoblots were developed using Super Signal West
Pico substrate (Pierce, Rockford, IL).
NF-B DNA binding assay
NF-B DNA binding activity was assessed using TransAM NF-B Family
Transcription Factor Assay Kit (Active Motif, Carlsbad, CA) following the
manufacturer’s protocol as described in Supplemental Methods. 10 μg of nuclear
extracts were tested in triplicate, with 5 μg Raji nuclear extract as the positive control
(Active Motif). Colorimetric results were read by a μQuant microplate reader under
absorbance at 570 nm (Bio-Tek Instruments, Winooski, VT). Data were calculated
from triplicates and presented as the mean + standard deviation (SD).
Cell Proliferation Assay
UM-SCC cells were initially plated in quadruplicate onto a 96-well plate at 5 x 103
cells per well in 50 μl of complete MEM. After 48h, cells were exposed to different
concentrations of celastrol and cell proliferation was analyzed at 1, 2 and 3 days
using a (sodium 3´-[1-(phenylaminocarbonyl)- 3,4-tetrazolium]-bis (4-methoxy-6-nitro)
benzene sulfonic acid hydrate (XTT) Cell Proliferation kit (Roche Diagnostics,
Indianapolis, IN). An aliquot of 50 ml of XTT solution was added for 5 h, and
absorbance was measured at a test wavelength of 450nm and a reference
wavelength of 630nm by a microplate reader.
Wound healing and Matrigel migration assays. For Wound assay, cells were
transfected with control or TAK1 siRNA (Dharmacon) for 24 hours. Wounds were
made through the confluent cell sheets using a 1ml pipette tip. Scratches were
monitored for % wound closure over the next 14 hours. Twelve measurements at
preset distances on the wound were measured and quantified. The statistical
analysis relative to the control siRNA was performed (t-test, * p<0.05). To further
confirm the effect of knocking down TAK1 on cell migration and invasion, Matrigel
invasion assay was performed using BD Biocoat Growth Factor Reduced Matrigel
Invasion Chamber (Cat# 354483, BD Bioscience). Briefly, cells were transfected with
siRNA at concentration 50nM for 4 hours in serum free media (Opti-MEM I, Reduced
Serum Medium, Invitrogen Cat# 31985). After 5hrs, medium was changed to MEM
with Serum/without Antibiotics and with TGF-beta 10ng/ml for 19hrs. Then the cells
were detached from the culture dishes by treating with 1X accutase according to
manufacturer’s protocol (Sigma, Cat#A6964). The cells were counted and plated at
5X104 cells/insert well of Matrigel plate in triplicates using DMEM serum free medium
supplemented with TGF-10ng/ml). The bottom well was plated with DMEM serum
free medium plus 10ng/ml EGF (R&D Systems) as chemoattractant.
The
membranes with migrated cells were harvested, stained with crystal violet, and
mounted onto glass slides. The cell images were scanned and analyzed by
ScanScope Aperio Digital Pathology System (Aperio Technologies, Inc, Vista, CA).
The quantitation of migrated cells was performed using Aperio ImageScope and
presented as mean + standard deviation (SD). Statistical significance was tested by
Student t test, and * indicates p<0.05.
References
Bancroft CC, Chen Z, Yeh J, Sunwoo JB, Yeh NT, Jackson S et al (2002). Effects of
pharmacologic antagonists of epidermal growth factor receptor, PI3K and MEK signal
kinases on NF-kappaB and AP-1 activation and IL-8 and VEGF expression in human head
and neck squamous cell carcinoma lines. Int J Cancer 99: 538-548.
Bian Y, Terse A, Du J, Hall B, Molinolo A, Zhang P et al (2009). Progressive tumor
formation in mice with conditional deletion of TGF-beta signaling in head and neck epithelia
is associated with activation of the PI3K/Akt pathway. Cancer Res 69: 5918-26.
Supplemental Table 1
Tumor and Outcome Characteristics of Patients Providing Human HNSCC Cell Lines
Cell line
UM-SCC 1
Age
72
Sex
M
Stage
I
TNM
T1N0M0
Primary site
FOM
Specimen site
Local recur
UM-SCC 6
37
M
II
T2N0M0
Tongue
Pri bx
UM-SCC 9
UM-SCC 11A
UM-SCC 11B
UM-SCC 22A
UM-SCC 22B
UM-SCC 38
UM-SCC 46
72
65
F
M
II
V
T2N0M0
T2N2aM0
Tonsil/BOT
Hypopharynx
59
F
III
T2N1M0
Hypopharynx
60
57
M
F
IV
III
T2N2aM0
None given
Tonsil/BOT
Supraglottic larynx
Local recur
Pri bx
Pri resect
Pri bx
LN met
Pri
Local recur
Survival
15
LTF
15
14
10
11
6
The clinical information was kindly provided by Drs. Thomas E. Carey and Carol R. Bradford,
and some information was presented previously in the literature. ‘Age’ represents age at
diagnosis in years. ‘Primary site’ refers to the origin of the primary tumor. ‘Specimen site’
refers to origin of tissue used to establish cultures. ‘Survival’ represents time in months from
diagnosis to last follow up. BOT, base of tongue; bx, biopsy; F, female; FOM, floor of mouth;
LN, lymph node; LTF, lost to follow-up; M, male; met, metastasis; Pri, primary tumor site;
recur, recurrence; resect, surgical resection specimen; HNSCC, head and neck squamous
cell carcinoma; TNM, tumor-node-metastasis (staging system); UM-SCC, University of
Michigan series head and neck squamous cell carcinoma.
Normal
Mucosa
HNSCC (++)
Supplemental Figure 1. TGF- receptor II immunostaining for normal mucosa and
HNSCC, higher original magnification of 400X is shown. Anti-TGF- receptor II
antibody (Santa Cruz sc400) was used as described in supplemental methods.
H&E
TAK
1
p-p65S276
Normal
Mucosa
HNSCC (++)
HNSCC (+/-)
Supplemental Figure 2A. TAK1 and phospho-p65 immunostaining for normal mucosa
and HNSCC, original magnification (400X). Anti-TAK1 antibody (07-263, Upstate
Signaling Solutions) and anti-phospho-p65 (Cell Signaling Technology, Danvers, MA)
was used as described in supplemental methods.
no Tx
TGF-β1
30’
1h
2h
p-TAK1
p-p65S536
p65
β-Actin
Supplemental Figure 2B. UM-SCC 6 cells were treated with 10 ng/ml TGF-β1 for
different time intervals, and increases in p-TAK1, and p-p65 were observed in whole
cell protein extracts by Western blot.
Supplemental Figure 3A. Densitometry of nuclear p65 shown in Figure 3A. TNF-
and TGF- induced 2-3 fold increase in nuclear p65, which is partially attenuated by
TAK1 depletion.
Supplemental Figure 3B. left panel, TAK1 vector transfected UM-SCC 6 cells show
increased expression of TAK1 by quantitative RT-PCR. Right panel, co-transfection and
expression of TAK1 with NF-B reporter gene in UM-SCC 6 cells increased basal, TGFβ1 and TNFα-induced NF-κB reporter gene activity. Columns, mean of triplicate or
quadruplicate samples; bars, SD. **, p<0.01,***, p<0.001.
Cell Density (fold increase)
Cell Density (fold increase)
Supplemental Figure 3C. MTT assay, proliferation upon knockdown with TAK1,
SMAD2, or both siRNAs, in FBS alone, or with added TGF-. UMSCC6 cells
(5x103/well in 6 replicates/condition) were cultured in medium containing 10% Fetal
bovine serum (FBS, upper panel), or with added TGF- (10ng/ml, lower panel).
Proliferation was examined upon knockdown with TAK1, SMAD2, or both siRNAs,
*p<0.05.
Expression (arbitrary units)
Supplemental Figure 3D. Confirmation of siRNA knockdown of TAK1, SMAD or
both, under conditions used in Suppl Fig. 3C by quantitative RT-PCR. Three
replicates for each experimental gene and two replicates for 18S were used for
normalization. All knockdowns were significant, (p<0.05).
Cell Density (fold increase)
Supplemental Figure 3E. MTT assay. UMSCC6 cell density in medium containing
serum is partially inhibited by anti-TGF antibody (** = significantly different from control
diluent and no tx absorbance). Addition of recombinant TGF- partially overcame the
inhibitory effect of anti-TGF- antibody on proliferation, but did not further increase
proliferation above that attributable to TGF- in 10%FBS (* = significantly different from
control diluent absorbance; + = not significantly different from control absorbance). UMSCC 6 (2x103 cells/well/6 replicates each) were cultured in medium with 10%FBS
without or with added TGF-1, 10ng/ml, and -/+ anti-TGF antibody (30 g/ml).
Supplemental Fig. 3F. Wound healing scratch assay performed 24 h after
transfection with control or TAK1 siRNA reveals decreased closure with TAK1 siRNA
14h post-scratch. Twelve measurements at preset distances on the wound were
measured and quantified. The statistical analysis relative to the control siRNA was
performed (t-test, * p<0.0001).
Supplemental Fig. 4A. Densitometry of western blot shown in Fig. 4A. UMSCC-6 and
22B cells were pre-treated with different concentrations of Celastrol for 1h and then
treated with 10 ng/ml TGF-β1 for 1h. p-TAK1, TAK1 and p-p65 were examined in
whole cell extract by Western blot. Celastrol suppressed TGF-β1 induced
phosphorylation of TAK1 and p65 in a dose dependent manner.
UMSCC6
1h
Celastrol (2.5µM)
TGF-β1 (10ng/ml)
-
+
-
+
2h
+
+
+
+
+
TAK1
p-TAK1Thr184/187
p-p65S536
p65
β-Actin
Supplemental Figure 4B. Celastrol, inhibits TAK1 and NF-B signal phosphorylation
in HNSCC cells. UMSCC-6 cells were pre-treated with 2.5 M of Celastrol for 1h and
then treated with 10 ng/ml TGF-β1 for 1 or 2h. TAK1, pTAK1 and p-p65 were
examined in whole cell extract by Western blot. Celastrol suppressed TGF-β1
induced phosphorylation of TAK1 and p65 in a time dependent manner.
Supplemental Figure 4C. DNA and Annexin V/Propidium Iodide fluorescence
analysis in UM-SCC 22B without and with exposure to TAK1 inhibitor Celastrol
2.5M for 12 and 24 hours. Left panels, DNA histograms reveal increase in G2/M
and sub-G0 DNA 12 and 24 hours after exposure to celastrol, versus no treatment/24
hours. Right panels, Two-color fluorescence X-axis, Annexin V, Y-axis, propidium
iodide shows increased Annexin V/PI double positive cells (right upper quadrant)
from 12- 24hours.
H&E
Smad7
p-p65S276
Normal
Mucosa
HNSCC (++)
HNSCC (+/-)
Supplemental Figure 5. A. NF-B and SMAD7 immunostaining of normal mucosa
and HNSCC. Histology at original magnification of 400X is shown. Anti-NF-B (Cell
Signaling Technology, Danvers, MA) and SMAD7 antibody (MAB2029, R&D
Systems) was used as described in supplemental methods.
(B) TGFβ-1 treatment for indicated time periods induced SMAD7 mRNA expression
UM-SCC 6 and 22B cells. Columns, mean of quadruplicate samples; bars, SD. ***,
p<0.001.
Supplemental Figure 5B. TGFβ-1 treatment for indicated time periods induced
SMAD7 mRNA expression UM-SCC 6 and 22B cells. Columns, mean of
quadruplicate samples; bars, SD. ***, p<0.001.
Vector
SMAD7
Control
no Tx
TGF-β1
+
+
+
+
(Cytoplasmic protein)
no Tx
β1
+
TGF-
+
+
+
(Nuclear protein)
Oct-1
β-Tubulin
Supplemental Fig. 5C. Demonstration of cytoplasmic/nuclear fractionation of extracts
from Fig. 5D by probing for cytoplasmic protein -tubulin and nuclear Oct-1