cancer cell line MDA-MB231. Consistent with a recent
report(Smith et al., 2009), we observed TGF-dependent induction
ofSNAIL in MDA-MB231 cells. Similarly, we detected that
TGFactivates expression of SLUG, PAI-1 and SMAD7 in an
IKK-dependent fashion. Furthermore, TGF-induced SMAD binding toDNA
is controlled by IKK. Because TGF signaling in MDA-MB231 cells is
implicated in diverse processes like invasion (Farinaet al., 1998;
Matsuura et al., 2010), metastasis (Yin et al., 1999),angiogenesis
(Safina et al., 2007) or autophagy (Kiyono et al.,2009) our
observations might argue that the described TGF–IKK–SMAD signaling
controls molecular functions beyond EMT.However, this hypothesis
needs further experimental validation.
In summary, we demonstrate that TGF–IKK–SMAD signalingdrives
SNAIL and SLUG expression during EMT of Panc1 cells,pointing to a
novel tumor-promoting pathway controlled by IKK.
Materials and MethodsCell culture, transfection, luciferase
assays, siRNAs, plasmids and reagentsThe pancreatic cancer cell
lines Panc1 was cultivated in DMEM supplemented with10% fetal calf
serum (FCS) and 1% (w/v) penicillin/streptomycin (Invitrogen,
Karlsruhe,Germany) as recently described (Fritsche et al., 2009;
Schild et al., 2009). MDA-MB231 cells were a kind gift from Oliver
Krämer (Friedrich-Schiller University, Jena,Germany) and cultivated
in DMEM supplemented with 10% FCS and 1%
(w/v)penicillin/streptomycin. TGF1 was purchased from PeproTech
(Hamburg, Germany).Untreated controls received vehicle alone.
Double-stranded siRNAs were transfectedat a final concentration of
50 nM using oligofectamine (Invitrogen, Karlsruhe,
Germany)according to the manufacturer’s protocol. siRNAs were
purchased from Eurofins(Ebersberg, Germany). Target sequences of
the used siRNAs were: control siRNA, 5�-CAGTCGCGTTTGCGACTGGdtdt-3�;
IKK siRNA, 5�-GTCTTGTCGC -CTAGAGCTAdtdt-3�; IKK siRNA,
5�-ATGTCATCCGATGGCACAAdtdt-3�;RelA/p65 siRNA, 5�-GATCAATG
GCTACACAGGAdtdt-3�; RelB siRNA, 5�-GACTGCACCGACGGCATCTdtdt-3�;
c-Rel siRNA, 5�-GAACG -CAGACCTTTGTTTTdtdt-3�; SMAD2 siRNA,
5�-GTCCCATGA AAAGA CT -TAAdtdt-3�; SMAD3 siRNA,
5�-GGAGAAATGGTGCGAGAAGdtdt-3�; and SMAD4siRNA,
5�-GGTCTTTGATTTGCGTCAGdtdt-3�. The 3xNFB luciferase reporterwas as
described (Häussler et al., 2005). Stable transfection of Panc1
cells was carriedout as described (Schneider et al., 2006). The
SureSilencing shRNA plasmid waspurchased from SuperArray Bioscience
(Frederick, MD). The shRNA target sequenceswere: control shRNA,
5�-GGAATCTCATTCGATGCATAC-3� and IKK shRNA,
5�-AGCGTGCCATTGATCTATATA-3�. The SMAD luciferase reporter
(pSMAD-Luc)contains the 5�-AGTATGTCTAGACTGAAGTATG TCTAGACTGAAG TATG
-TCTAGACTGA-3� SMAD-binding site between the NheI and BglII
restriction sitesof pTL-Luc (Panomics, Santa Clara, CA) and was
purchased from BioCat (Heidelberg,Germany). pGL3control and phRG-B
vectors were purchased from Promega(Mannheim, Germany). The 5�
regulatory region of the murine E-cadherin gene wasamplified by PCR
using the following primers: E-cadherin-FW, 5�-TAGGAAGCTGGGAAG-3�
and E-cadherin-RV 5�-CTCGGGTGCGGTCG-3�. Thefragment corresponds to
–174 to +94 of the murine E-cadherin promoter, containingthe two
proximal E-boxes. The resulting fragment was blunt-end cloned into
SmaIopened phRG-B (Promega). All plasmids were verified by
sequencing. Transfectionsof the luciferase reporter genes (500 ng
per well) were performed using FuGene6(Roche Applied Science,
Mannheim, Germany) according to the manufacturer’sprotocol in
12-well plates. After stimulation with TGF1 for indicated time
points,cells were incubated in lysis buffer (Promega), harvested,
and cleared by centrifugation.Lysates were normalized for protein
content. Luciferase activity was determined in aLB 9501 luminometer
(Berthold, Bad Wildbad, Germany) using a renilla luciferaseassay
system (Promega).
Western blotting and immunoprecipitationWhole-cell lysates were
prepared and western blots and immunoprecipitations werecarried out
as recently described (Fritsche et al., 2009; Schneider et al.,
2010;Schneider et al., 2006). Nuclear extracts were prepared as
described (Häussler et al.,2005). The following antibodies were
used: HDAC1 (Millipore, Schwalbach,Germany), SMAD2/3 (FL-425,
sc-8332), SMAD4 (B-8, sc-7966), RelA/p65 (C-20,sc-372), -tubulin
(B-7, sc-5286), RelB (C-19, sc-226), c-Rel (C, sc-71),
vimentin(VI-REI1, sc-51721) (Santa Cruz Biotechnology, Santa Cruz,
CA), IKK (CellSignaling, Danvers, MA), E-cadherin and N-cadherin
(BD Biosciences, Heidelberg,Germany), IKK (clone 10AG2) (Biomol,
Hamburg, Germany) and -actin (Sigma-Aldrich, Munich, Germany).
Western blots were scanned and quantified usingOdyssey Infrared
Imaging System (LI-COR Biosciences, Bad Homburg, Germany).
Quantitative reverse-transcriptase PCRTotal RNA was isolated
from pancreatic carcinoma cell lines using the RNeasy kit(Qiagen,
Hilden, Germany) following the manufacturer’s instructions.
Quantitative
mRNA analyses were performed as previously described using
real-time PCRanalysis (TaqMan, PE Applied Biosystems, Norwalk CT)
(Fritsche et al., 2009;Schneider et al., 2006). Expression was
normalized to GAPDH expression levels.Primer sequences were:
SNAIL-FW, 5�-CCCAATCGGAAGCCTAACTA-3�;SNAIL-RV,
5�-CAGGACAGAGTCCCAGATGAG-3�; SLUG-FW, 5�-ATGCATATTCGGACCCACAC-3�;
SLUG-RV, 5�-GCAGATGAGCCC -TCAGATTT-3�; SMAD2-FW,
5�-TCAGTTCCGCCTCCAATC-3�; SMAD2-RV,5�-CAAGCCACGCTAGGAAAAC-3�;
SMAD3-FW, 5�-CCACGCAGAAC -GTCAACA-3�; SMAD3-RV,
5�-TTGAAGGCGAACTCACACAG-3�; SMAD4-FW,5�-ACAAGGTGGAGAGAGTGAAACA-3�;
SMAD4-RV, 5�-CTCCAGAGAC -GGGCATAGAT-3�; PAI-1-FW,
5�-AATCAGACGGCAGCACTGTCT-3�; PAI-1-RV,5�-GGCAGTTCCAGGATGTCGTAGT-3�;
Smad7-FW, 5�-TGCTCCCATCCT -GTGTGTTAAG-3�; Smad7-RV,
5�-TCAGCCTAGGATGGTACCTTGG-3�;GAPDH-FW, 5�-CGTGGAAGGACTCATGACCA-3�;
and GAPDH-RV, 5�-GCCATCACGCCACAGTTTC-3�.
Chromatin immunoprecipitation assaysChromatin
immunoprecipitation (ChIP) assays were performed as recently
described(Fritsche et al., 2009; Schneider et al., 2010; Schneider
et al., 2006). An equalamount of chromatin (50–100 g) was used for
each precipitation. The followingantibodies were used: SMAD2/3
(FL-425, sc-8332) and control IgG from SantaCruz Biotechnology
(Heidelberg, Germany) and IKK from BD Biosciences(Heidelberg,
Germany). One-twentieth of the precipitated chromatin was used
foreach PCR reaction. To ensure linearity, 28–38 cycles were
performed, and onerepresentative result is shown. Sequences of the
promoter specific primers were:GAPDH-FW,
5�-AGCTCAGGCCTCAAGACCTT-3�; GAPDH-RV, 5�-AAGAAGATGCGGCTGACTGT-3�;
SNAIL-FW, 5�-CGCTCCGTAAA CACT -GGATAA-3�; SNAIL-RV,
5�-GAAGCGAGGAAAGGGACAC-3�; SLUG-FW,5�-GCCTG CCTTTAGAGGGCTAC-3�; and
SLUG-RV, 5�-TGCGCTA -CTCAGGGCTTC-3�.
Avidin-biotin-complex DNA assayA total of 1000 g of untreated or
TGF-treated Panc1 whole-cell extract wasincubated with 3 g
biotinylated double-stranded oligonucleotides for 4 hours
withconstant rotation at 4°C in a total volume of 400 l
immunoprecipitation (IP) buffer(50 mM HEPES, 150 mM NaCl, 1 mM
EDTA, 0.5% NP-40, 10% glycerol, 1 mMdithiothreitol, 1 mM
phenylmethysulfonylfluoride and 5 mM NaF) as described(Schild et
al., 2009). After addition of 50 l equilibrated streptavidin
agarose beads(Invitrogen), incubation was continued overnight at
4°C on a rotator. Beads werecollected by centrifugation and washed
repeatedly with IP buffer. Afterwards, theprecipitated beads were
boiled in Laemmli sample buffer and proteins were separatedvia
SDS-PAGE. SMAD3 and SMAD4 were detected by western blot. The
following3� biotinylated oligonucleotides were used: SMAD-s,
5�-TCGA GAGCCAG -ACAAAAAGCCAGACATTTAGCCAGACAC-3� and SMAD-as,
5�-GTGTCT -GGCTAAATGTCTGGCTTTTTGTCTGGCTCTCGA-3�.
Boyden chamber assayCell migration was determined using a Boyden
chamber assay. HTS 24-MultiwellInsert Systems were purchased from
BD Biosciences (Bedford, VA). Panc1 cellswere transfected with
control or IKK-specific siRNAs and stimulated with TGF(10 ng/ml)
for 48 hours. Afterwards, 2.5�104 cells were seeded in
serum-freeDMEM in the upper wells of the multiwell insert system.
The lower wells were filledwith 500 l DMEM supplemented with 10%
fetal bovine serum. After 6 hours in anincubator (5% CO2, 37°C)
cells on the upper surface of the filter were removed witha
cotton-tipped swab. Migrated cells on the lower side of the
membrane were fixedwith methanol, stained with Giemsa solution
(Merck, Darmstadt, Germany) andcounted.
ImmunocytochemistryCells were washed twice with PBS and fixed
for fluorescence microscopy byincubation for 10 minutes at 37°C in
PBS containing 3.7% formaldehyde. Fordetection using specific
antibodies, cells were permeabilized with 0.5% Triton X-100 in PBS
for 5 minutes at room temperature. After washing the cells twice
withPBS, unspecific antibody binding sites were blocked by
incubation of cells in 3%BSA (in PBS) for 30 minutes at room
temperature. The following antibodies wereused: SMAD2/3 (Fl-425,
sc-8332), SMAD4 (B-8, sc-7966) antibody (Santa CruzBiotechnology)
and Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) as
secondaryantibody. For counterstaining of the nucleus, fixed cells
were incubated for 2 minutesin medium containing 600 nM
DAPI-solution (Biomol). Cells were washed twice inPBS, mounted on
slides with VectaShield mounting medium (Vector Labs,
Grünberg,Germany) and examined with an Axiophot epifluorescence
microscope (Zeiss, Jena,Germany) using an 40� oil immersion
objective. Optical data were collected withAxioVision Rel. 4.6
(Zeiss, Jena, Germany).
Statistical methodsAll data were obtained from at least three
independent experiments performed intriplicate, and the results
presented as mean and standard error of the mean (s.e.m.).To
demonstrate statistical significance, a two-tailed Student’s t-test
was used.* denotes a P-value of at least 0.05.
4238 Journal of Cell Science 123 (24)
Jour
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We thank Kerstin Hoffmann, Tatjana Netz and Birgit
Kohnke-Ertelfor excellent technical support and Oliver Krämer for
providing MDA-MB231 cells. This work was founded by DFG SFB456 (to
G.S.) andDeutsche Krebshilfe (to D.S.).
Supplementary material available online
athttp://jcs.biologists.org/cgi/content/full/123/24/4231/DC1
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4239IKK and EMT
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SummaryKey words: EMT, IKK, NFkB, Pancreatic cancer, SMAD,
SNAIL, SLUG,IntroductionResultsIKKa controls downregulation of
E-cadherin and EMT in TGFb-treated Panc1IKKa controls TGFb-induced
migration of Panc1 cellsIKKa controls SMAD signalingTGFb-induced
nuclear translocation of SMADs is not controlled by
IKKaTGFb-induced IKKa-SMAD signaling controls SNAIL and SLUG
expressionIKKa controls TGFb-induced binding of SMADs to the SNAIL
and
Fig. 1.Fig. 2.Fig. 3.DiscussionFig. 4.Fig. 5.Fig. 6.Fig.
7.Materials and MethodsCell culture, transfection, luciferase
assays, siRNAs, plasmids and reagentsWestern blotting and
immunoprecipitationQuantitative reverse-transcriptase PCRChromatin
immunoprecipitation assaysAvidin-biotin-complex DNA assayBoyden
chamber assayImmunocytochemistryStatistical methods
Supplementary materialReferences2747.pdfFig 1