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The ZEB1/miR-200 feedback loop controls Notch
signalling in cancer cells
Simone Brabletz
1
, Karolina Bajdak
1,2
,
Simone Meidhof
1,2,3
, Ulrike Burk
1
,
Gabriele Niedermann
4
, Elke Firat
4
,
Ulrich Wellner
1
, Arno Dimmler
5
,
Gerhard Faller
5
,Jo¨ rg Schubert
1
and Thomas Brabletz
1,6,
*
1
Department of Visceral Surgery, University of Freiburg, Freiburg,
Germany,
2
Faculty of Biology, University of Freiburg, Freiburg,
Germany,
3
Spemann Graduate School of Biology and Medicine (SGBM),
University of Freiburg, Freiburg, Germany,
4
Department of
Radiotherapy, University of Freiburg, Freiburg, Germany,
5
Department
of Pathology, Karlsruhe, Germany and
6
Comprehensive Cancer Center
Freiburg, University of Freiburg, Freiburg, Germany
Notch signalling is important for development and tissue
homeostasis and activated in many human cancers.
Nevertheless, mutations in Notch pathway components
are rare in solid tumours. ZEB1 is an activator of an
epithelial–mesenchymal transition (EMT) and has crucial
roles in tumour progression towards metastasis. ZEB1 and
miR-200 family members repress expression of each
other in a reciprocal feedback loop. Since miR-200 mem-
bers target stem cell factors, ZEB1 indirectly induces
stemness maintenance and associated drug resistance.
Here, we link ZEB1 and its cancer promoting properties
to Notch activation. We show that miR-200 members target
Notch pathway components, such as Jagged1 (Jag1) and
the mastermind-like coactivators Maml2 and Maml3,
thereby mediating enhanced Notch activation by ZEB1.
We further detected a coordinated upregulation of Jag1
and ZEB1, associated with reduced miR-200 expression in
two aggressive types of human cancer, pancreatic adeno-
carcinoma and basal type of breast cancer. These findings
explain increased Notch signalling in some types of
cancers, where mutations in Notch pathway genes are
rare. Moreover, they indicate an additional way how
ZEB1 exerts its tumour progressing functions.
The EMBO Journal (2011) 30, 770–782. doi:10.1038/
emboj.2010.349; Published online 11 January 2011
Subject Categories: RNA; molecular biology of disease
Keywords: EMT; miR-200; Notch; stemness; ZEB1
Introduction
Notch signalling is important for embryonic development and
adult tissue homeostasis and if aberrantly activated has a
crucial role in the development of many human cancers. The
Notch pathway is activated by binding of Notch ligands
(Jag1, Jag2, DLL1, DLL3, DLL4) to Notch receptors, which
are subsequently cleaved extracellularly by ADAM-type pro-
teases, and intracellularly by g-secretase. The resulting Notch
intracellular domain (NICD) translocates to the nucleus,
where it builds up a transcription activator complex together
with the transcription factor CSL and coactivators, such as
mastermind-like factors (Maml 1,2,3). This complex activates
the transcription of typical Notch target genes like Hes and
Hey1 (Koch and Radtke, 2007). The Notch pathway controls
central cellular processes including stemness, differentiation,
proliferation and survival (Hurlbut et al, 2007; Roy et al,
2007). This may explain why aberrant Notch pathway activa-
tion is described for many human cancer types, including
lung, colorectal and breast cancer, as well as leukaemias (Koch
and Radtke, 2007; Rizzo et al, 2008). Its relevance was also
shown in mouse models for pancreatic cancer, where inhibi-
tion of Notch signalling by a g-secretase inhibitor (GSI)
completely blocked tumour formation (Plentz et al, 2009).
Epithelial–mesenchymal transition (EMT) is a reversible
embryonic program, which allows partial or complete transi-
tion between an epithelial and a mesenchymal phenotype.
EMT is essential for embryonic processes like gastrulation,
and if aberrantly activated is a trigger of tumour progression
and metastasis. EMT is activated by key signalling pathways,
including the TGFband FGF pathway, converging in the
stimulation of EMT activators, a group of transcription factors
repressing epithelial gene expression. These include mem-
bers of the snail family, of the bHLH family and of the ZFH
family (ZEB1 and ZEB2) (Thiery et al, 2009). It is now known
that EMT activators not only activate cellular motility, but are
also associated with the maintenance of stem cell properties
and cell survival (Mani et al, 2008; Morel et al, 2008). We and
others have shown that thereby the miR-200 family of
microRNAs has a crucial role (Shimono et al, 2009; Wellner
et al, 2009). ZEB1 suppresses the expression of all miR-200
family members (miR-141, -200a,b,c and -429), which in turn
inhibit translation of ZEB1 mRNA, resulting in the double-
negative ZEB/miR-200 feedback loop (Burk et al, 2008;
Gregory et al, 2008; Korpal et al, 2008; Park et al, 2008;
Brabletz and Brabletz, 2010). Since stem cell factors, such as
Bmi1, are additional targets of miR-200, an overexpression of
ZEB1 in tumour cells indirectly, through inhibition of miR-
200 expression, leads to maintenance of stem cell properties,
as shown for breast and pancreatic cancer (Shimono et al,
2009; Wellner et al, 2009).
Thus, it emerged that EMTand Notch signalling have many
functional overlaps by controlling central processes such as
stemness and cell survival. An obvious link is indicated by
the finding that Notch signalling can also induce EMT in cell
culture (Leong et al, 2007; Sahlgren et al, 2008). However,
albeit the importance of Notch signalling for tumour forma-
tion and malignant progression, mutations in Notch pathway
components are rarely detected in solid cancers, including
human pancreatic adenocarcinomas, as analysed in a recent
Received: 3 August 2010; accepted: 7 December 2010; published
online: 11 January 2011
*Corresponding author. Department of Visceral Surgery, University of
Freiburg, Hugstetterstr. 55, 79106 Freiburg, Germany.
Tel.: þ49 761 270 2577; Fax: þ49 761 270 2579;
E-mail: thomas.brabletz@uniklinik-freiburg.de
The EMBO Journal (2011) 30, 770–782 |
&
2011 European Molecular Biology Organization |All Rights Reserved 0261-4189/11
www.embojournal.org
The EMBO Journal VOL 30 |NO 4 |2011 &2011 European Molecular Biology Organization
EMBO
THE
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comprehensive survey of multiple genomes (Jones et al,
2008). Thus, other ways of aberrant Notch signalling activa-
tion must exist in cancer. We investigated if EMT activation
can stimulate the Notch pathway and show here that the EMT
inducer ZEB1 can trigger Notch signalling in cancer cells by
stabilizing the expression of Notch pathway components,
such as Jag1, Maml2 and Maml3, through inhibition of
miR-200 expression.
Results
Knockdown of ZEB1 expression reduces Notch pathway
activity
We have previously shown that, in addition to classical EMT-
associated properties, the EMT activator ZEB1 also controls
other central cellular processes and states, such as stemness
and survival, by inhibiting expression of miR-200 family
members (Wellner et al, 2009). Since the Notch signalling
pathway is known to control these processes as well, we
investigated if the EMT inducer ZEB1 can influence Notch
signalling. Stable knockdown of ZEB1 decreased the endo-
genous activity of a Notch reporter construct (4xCSL-Luc)
in the undifferentiated pancreatic cancer cell line Panc1
(Figure 1A). A mutated control reporter was not significantly
affected by ZEB1. Also a transient, siRNA-mediated knock-
down of ZEB1 led to a reduced Notch reporter activity
(Supplementary Figure S1A). We re-analysed whole-genome
gene expression arrays of stable ZEB1 knockdown clones
performed earlier in our laboratory (Spaderna et al, 2008) for
Notch pathway-related genes affected by ZEB1. Decrease of
ZEB1 expression led to a downregulation of mRNAs for the
Notch ligand Jag1, the Notch transcriptional coactivators
Maml2 and Maml3, as well as the Notch target factor Hey1
in pancreatic and breast cancer cell lines (Figure 1B). Other
Notch pathway components were not affected by ZEB1 in the
investigated cell lines (not shown). We initially focused on
Jag1, because it was already shown to be overexpressed in
many cancer types, such as the basal type of breast cancer
(Reedijk et al, 2008). Downregulation of Jag1 after both
stable and transient knockdown of ZEB1 was confirmed by
quantitative real-time PCR (Figure 1C–E). Moreover, also the
expression of the Notch target Hey1 was reduced (Figure 1C
and D). These data indicate that ZEB1 is enhancing Notch
pathway activation, at least partially, by increasing the
expression of the ligand Jag1.
Jag1 is a target of miR-200 family members
ZEB1-dependent expression of Notch pathway components is
likely not due to direct activation by ZEB1, since it is mainly
described as a transcriptional repressor (Vandewalle et al,
2009). Like previously shown for other genes indirectly
activated by ZEB1, we also hypothesized that Jag1 is a miR-
200 target and upregulated upon inhibition of miR-200 genes
by ZEB1. We first analysed if miR-200 family members can
affect Notch signalling. The miR-200 family members can be
subdivided into two subgroups according to their seed se-
quences (subgroup I: miR-141 and miR-200a; subgroup II:
miR-200b, c and miR-429), which indicate slight differences
in their target gene sets (Peter, 2009). Both stable and
transient overexpression of miR-141 and miR-200c, belonging
to subgroups I and II, respectively, reduced the activity of the
Notch reporter, but not of the control reporter (Figure 1F;
Supplementary Figure S1A). Moreover, reduced Notch repor-
ter activity after ZEB1 knockdown could be partially restored
by inhibiting miR-141 and miR-200c using specific antagomirs
(Figure 1G). By using the microRNA target prediction server
mirecords (http://mirecords.biolead.org), we found that Jag1
is a predicted target of miR-200 family members (Figure 1H).
Stable overexpression of miR-141 and miR-200c in the
undifferentiated pancreatic cancer cell line Panc1, lacking
endogenous miR-200 expression, led to a slightly reduced
expression of Jag1 at mRNA level (Figure 2A). Accordingly, a
transient overexpression of miR-141 and miR-200c in Panc1
cells slightly reduced Jag1 mRNA expression (Supplementary
Figure S1B). Using immunoblots, we could show that knock-
down of ZEB1 and overexpression of both miR-141 and miR-
200c led to a reduction of Jag1 protein expression (Figure 2B).
Vice versa, we used the differentiated pancreatic cancer
cell line HPAF2 and breast cancer cell line MCF7, which
endogenously express miR-200 members. Inhibition of their
function by specific antagomirs led to an increase in Jag1
mRNA and in particular protein level (Figure 2C and D). We
further constructed a luciferase reporter vector under the
control of the Jag1 30UTR to show direct effects of miR-200
members. Both miR-141 and miR-200c inhibited the Jag1
30UTR driven reporter activity. The ZEB1 30UTR reporter
construct served as a known positive control, in particular
for miR-200c (Figure 2E). Again, we used the differentiated
line HPAF2 to confirm the inhibitory effect of endogenous
miR-200 expression on the Jag1 30UTR. Subsequent muta-
tions of predicted miR-200 binding sites 1 to 5 steadily
increased Jag1 30UTR reporter activity, indicating that all
five detected binding sites can confer to the inhibitory
function of miR-200 (Figure 2F). Additionally, antagomirs
against both miR-200c and miR-141 increased the activity of
the Jag1 30UTR reporter in HPAFII cells, and thus further
confirmed that Jag1 is a direct target of miR-200 (Figure 2G).
Notch activity and Jag1 expression are important
for essential properties of cancer cells
Next, we wanted to analyse if Notch signalling, and in
particular expression of the Notch ligand Jag1, are important
for crucial properties of cancer cells. Notch signalling was
blocked by treatment of tumour cells with a g-secretase in-
hibitor (GSI), and Jag1 expression was transiently knocked
down by two different, specific siRNAs (Supplementary
Figure S1B). By applying Notch reporter assays and by detec-
tion of activated (cleaved) Notch2, we could demonstrate
that both GSI treatment and the knockdown of Jag1 inhibited
Notch signalling (Figure 3A and B). Inhibition of Notch
signalling by Jag1 knockdown was also confirmed by a reduced
expression of the Notch target gene Hey1 (Supplementary
Figure S1C and D). Moreover, we detected a reduced expression
of ZEB1 after knockdown of Jag1, which confirms published
data of ZEB1 expression being activated by Notch signalling
(Wang et al, 2009). Both GSI treatment and knockdown of
Jag1 also reduced the proliferative capacity of cancer cells
(Figure 3C; Supplementary Figure S1E) and increased apop-
tosis after irradiation of cancer cells with 5 Gy (Figure 3D).
Next, we analysed the effects on stemness-associated proper-
ties. We used the stem cell sphere assay to quantify the
numbers of potential cancer stem cells in culture: if tumour
cells are cultivated in suspension in a serum-free selection
medium, only cancer cells with stem cell properties are able
ZEB1 activates Notch signalling
S Brabletz et al
&2011 European Molecular Biology Organization The EMBO Journal VOL 30 |NO 4 |2011 771
DC
50
100
% mRNA expression level
Jag1
ZEB1 Hey1
shZEB1shctrl
MDA-MB231
50
100
% mRNA expression level
shZEB1
shctrl
Panc1
50
100
% mRNA expression level
siZEB1
sictrl
Panc1
Jag1ZEB1 Hey1
1 18001000 1200 1400 1600800600400200
JAG1 3′UTR
H
B
ctrl shZEB1 Ratio % ctrl shZEB1 Ratio %
ZEB1 1057 63 5.9 4634 872 18.8
Jag1 18009 6351 36.2 7204 2011 27.9
Maml2 1376 359 26.1 2330 598 25.7
Maml3 NE NE —1737 516 29.7
Hey1 447 248 55.4 6586 282 4.3
MDA-MB231 Panc1
Panc1 sh
ctrl
sh
ZEB1
sh
ctrl
sh
ZEB1
100
% Relat. luciferase activity
50
A
WT
mut
Notch reporter
wt
sictrl
siZEB1
Jag1
Actin
E
Panc1
50
100
Relative reporter activity
mut
Notch reporter
sh
ZEB1
sh
ZEB1
sh
ZEB1
sh
ZEB1
sh
ZEB1
ctrl shctrlshctrl
Clones Clones
F
miR-141 miR-200c
B2 B3 E2 A4 B3
WT
Panc1
50
100
Relative reporter activity
Antagomir ctrl ctrl miR141
miR200c
ctrl ctrl miR141
miR200c
GmutNotch reporter WT
MDA-MB231 Panc1
′
′′
′
′′
′
′′
′
′
′′
′
Figure 1 ZEB1 and miR-200 family members affect Notch signalling. (A) Stable knockdown of ZEB1 results in reduced Notch reporter activity
(WT). There was no significant effect on a mutated (mut) reporter construct. Shown are mean values of each two independent clones. Control
clones were set to 100%. (B) Expression values of ZEB1 and the indicated Notch pathway components deduced from a whole-genome
expression screen comparing ZEB1 knockdown and control knockdown clones (NE ¼not expressed). (C,D) Quantitative RT–PCR after stable
(C) and transient (D) knockdown of ZEB1. (E) Immunoblot showing reduced expression of Jag1 after transient knockdown of ZEB1. (F) Notch-
luciferase reporter assay showing reduced activity in different clones after stable knockdown of ZEB1 or stable overexpression of miR-141 and
miR-200c. (G) Antagomirs against miR-141 and miR-200c partially rescue Notch reporter inhibition by shZEB1. (H) Scheme showing five
predicted target sites of miR-200 family members in the 30UTR of Jag1.
ZEB1 activates Notch signalling
S Brabletz et al
The EMBO Journal VOL 30 |NO 4 |2011 &2011 European Molecular Biology Organization772
to grow out and form spheres, whereas all other cells undergo
anoikis (Gou et al, 2007). The number of spheres formed per
1000 cultivated cells reflects the number of potential cancer
stem cells. We have previously demonstrated that knock-
down of ZEB1 or overexpression of miR-200c affects the
sphere-formation capacity in breast and pancreatic cancer
cells (Wellner et al, 2009). Here, we show that inhibition
of Notch signalling by GSI treatment strongly inhibited
the sphere-forming capacity of cancer cells (Figure 4A).
This indicated that stem cell associated properties depend
on Notch signalling, although the reduced sphere-forming
capacity after GSI treatment might be an additive effect
of reduced cancer stem cell numbers and reduced growth
capacities. A transient knockdown of Jag1 had no effect on
sphere formation in the first generation, however, led to a
reduction of the sphere-forming capacity in the second-sphere
generation (Figure 4B; Supplementary Figure S1F). This in-
dicated that Jag1 expression is necessary for maintenance of
stem cell properties, in particular for a self-renewal capacity.
We further analysed in vivo expression of ZEB1, miR-200
family members and Jag1 in orthotopic xenograft tumours
derived from pancreatic cancer cells. We have previously
Fold luciferase activity
5
miR-
200c
miR-
141
miR-200c
miR-141
ctrlAntagomir
G
Jag1 3′UTR
ZEB1 3′UTR
F
% Luciferase activity
Jag1 WT mut mut mut mut mut
3′UTR 5 4–5 3–5 2–5 1–5
100
200
HPAF2
X-fold miRNA
expression
1 17 / 15 1944 75 1021 1651 1108
50
100
% mRNA expression level
Jag1
ZEB1
shZEB1ctrlTransfectand
A
miR-141 miR-200c
B2 B3 E2 A4 B3Clone no.
Panc1
ZEB1
Jag1
Actin
Control GD4
shZEB1 ZB4
miR-141 B2
miR-141 B3
miR-141 E2
miR-200c A4
miR-200c B3
Wild type
B
Transfectand
*
20
40
60
80
100
E
% Luciferase activity
miRNA ctrl 141 200c
Jag1 3′UTR
ZEB1 3′UTR
Antagomir ctrl
Antagomir 200c, a
Antagomir 200c, b
Actin
Jag1
D
MCF7
5
Fold mRNA expression level
Jag1
ZEB1
miR-200c ctrl miR-200c
12 12
ctrlAntagomir
C
HPAF2 MCF7
miR-141
Figure 2 Jag1 is a target of miR-200 family members. (A,B) Quantitative RT–PCR (A) and immunoblot (B) for ZEB1 and Jag1 after stable
knockdown of ZEB1 or overexpression of miR-141 and 200c in Panc1 cells (* is a degradation product of ZEB1). Also shown is the relative
expression of the indicated microRNAs in the different clones. (C,D) Inhibition of endogenous miRs by the indicated specific antagomirs
increase the mRNA levels of ZEB1 and Jag1 (C) and the protein level of Jag1 (D) in differentiated cancer cell lines. (E) Transient overexpression
of miR-141 or miR-200c reduced the activity of Jag1 30UTR reporter and a ZEB1 30UTR control reporter in Panc1 cells. (F) Subsequent mutations
of predicted miR-200 binding sites in the Jag1 30UTR-luciferase construct increased reporter activity in HPAFII cells, endogenously expressing
miR-200 family members. (G) Inhibition of endogenous miRs in HPAFII cells by the indicated specific antagomirs (antagomir miR-200c (b) was
used) increased the activity of the Jag1 30UTR reporter and a ZEB1 30UTR control reporter.
ZEB1 activates Notch signalling
S Brabletz et al
&2011 European Molecular Biology Organization The EMBO Journal VOL 30 |NO 4 |2011 773
shown that stable ZEB1 knockdown in the pancreatic cancer
cell lines Panc1 and MiaPaCa2 affected important aspects of
tumour formation. After ZEB1 knockdown, the cancer cells
showed strong reduction in tumourigenicity and the few
grown tumours were much smaller and completely lost
their invasive and metastatic capacity (Wellner et al, 2009).
By re-investigating these tumours, we detected a strongly
decreased expression of Jag1 and the mesenchymal marker
ctrl
GSI
2.5 μM
GSI
5μM
B
si ctrl
si Jag1 a
si Jag1
a+b
si Jag1 b
Activated
Notch2
Dapi
Panc1
A
50
% Relat. luciferase activity
Notch reporter WT WT WT WT mut mut mut mut
Treatment ctrl GSI si si ctrl GSI si si
Jag
1a
Jag
1b
Jag
1a
Jag
1b
100 Panc1
D
10.0%
16.6%
71.7%
1.6%
Annexin V
Propidium idodide
ctrl (26.6%)
11.8% 51.1%
19.3%
17.8%
GSI 2.5 μM (70.4%)
10.1% 55.9%
8.7% 25.2%
GSI 5 μM (81.1%)
7.8%
1.6%
77.5% 13.1%
si ctrl (20.9%)
13.7%
67.5% 16.4%
2.4%
si Jag1 a (30.1%)
2.2% 11.7%
71.5% 14.6%
si Jag1 b (26.3%)
6.0 *P<0.05 (ANOVA)
Error bars: +/– 95% CI
5.0
4.0
3.0
2.0
1.0
0.0
12
Time (days)
34
Time (days)
Relative MTT activity
C
DMSO
GSI 2.5 μM
GS1 5.0 μM
Panc1
*
*
*
1234
*P<0.05 (ANOVA)
Error bars: +/– 1 SE
Relative MTT activity
3.5
3.0
2.5
2.0
1.5
1.0
0.5
si ctrl
si Jag1 a
si Jag1 b
si Jag1 a+b
*
*
*
Figure 3 Notch signalling and Jag1 expression are important for cancer cell properties. (A) Treatment with 5 mM GSI or transient knockdown
of Jag1 inhibits Notch reporter activity. (B) Immunofluorescence showing reduced nuclear expression of activated Notch2 after GSI treatment
or transient Jag1 knockdown. (C) GSI treatment or transient knockdown of Jag1 inhibits proliferation measured in an MTT assay. Xaxis
indicates days after cell seeding. Asterisks indicate significance of control versus treated cells. (D) GSI treatment or transient knockdown of
Jag1 increases apoptosis of Panc1 cells irradiated with 5Gy. The percentage of apoptotic cells is indicated, resulting from the addition of the two
right, annexin V positive quadrants.
ZEB1 activates Notch signalling
S Brabletz et al
The EMBO Journal VOL 30 |NO 4 |2011 &2011 European Molecular Biology Organization774
vimentin, and increased expression of miR-141 and miR-200c
in tumours derived from ZEB1 knockdown clones. This
correlated with their reduced tumourigenicity and aggressive-
ness (Figure 4C and D; Supplementary Figure S2A).
Jag1 alone cannot explain the effects of ZEB1
and miR-200 on Notch signalling
Next, we wanted to determine whether Jag1 is the major
or only target mediating the inhibitory effect of ZEB1
knockdown or miR-200 overexpression on the Notch path-
way. Overexpression of a Jag1 expression construct lacking
the 30UTR increased the Notch reporter activity, which was
suppressed by knockdown of ZEB1 or overexpression of miR-
141 and miR-200c. However, it could not fully rescue the
suppressive effect (Figure 4E). Vice versa, inhibition of
endogenous miR-141 and miR-200c in differentiated cancer
cells by specific antagomirs increased Notch reporter activity,
which again could be only partially repressed to the control
level by siRNA-mediated knockdown of Jag1 (Figure 4F;
Supplementary Figure S2B). In the same setting, the en-
hanced sphere-forming capacity after inhibition of miR-141
and miR-200c in HPAF2 cells was fully reduced by Jag1
knockdown (Figure 4G). Interestingly, reduction of Jag1 did
not significantly affect the proliferative capacity of the differ-
entiated lines HPAF2 and MCF7 (Figure 4H; Supplementary
Figure S2C), in contrast to the undifferentiated lines Panc1
and MDA-MB231, which already expressed high endogenous
levels of Jag1 (Figure 3C; Supplementary Figure S1E). Further
work will address this difference. Altogether these data
indicate that Jag1 alone is not the only target mediating the
effects of ZEB1 and miR-200 on Notch signalling. To investi-
gate if downstream Notch signalling is also directly affected
by ZEB1 knockdown, we cotransfected an expression vector
for the cleaved Notch1 ICD, a constitutive Notch signalling
activator, which led to increased Notch reporter activation in
Panc1 cells. NICD-induced activation was also decreased
by stable knockdown of ZEB1 (Figure 4I), further indicating
that ZEB1 and/or miR-200 influence Notch pathway activity
not only by regulating the ligand Jag1 but also on down-
stream levels.
miR-200 targets the Notch coactivators Maml2
and Maml3
Besides Jag1, the only Notch pathway components detected
in expression arrays to be affected by ZEB1 were the intra-
cellular transcriptional coactivators Maml2 and Maml3
(Figure 1B), which could explain the reduced Notch reporter
activity by ZEB1 knockdown also after cotransfection of
NICD. Both coactivators are predicted miR-200 targets
(Figure 5A). Knockdown of ZEB1 or overexpression of miR-
200c and miR-141 inhibited expression of Maml2 and to a
lesser extend Maml3 (Figure 5B–D). Moreover, 30UTR repor-
ter constructs of both factors were also affected by miR-200c/
141 overexpression (Figure 5E). siRNA-mediated knockdown
of Maml2 and Maml3 demonstrated their crucial role for
Notch signalling, since it inhibited both Notch reporter
activity (Figure 5F) and expression of the Notch target gene
Hey1 (Figure 5G and H). Similar to a knockdown of Jag1 (see
Figure 4F and Supplementary Figure S2B), a knockdown
of Maml2 and Maml3 also led to a reduction of the sphere-
forming capacity in the second-sphere generation of Panc1
cells (Figure 5I).
ZEB1, miR-200 and Notch signalling in human cancer
Finally, we analysed the expression of ZEB1, miR-200 family
members and Notch pathway activation in human breast and
pancreatic cancers. Distinct subtypes of these tumours are
known to be associated with high Notch signalling activity.
Typical human breast cancers are of the ductal invasive type,
which express steroid hormone receptors and show a more
differentiated phenotype. In contrast, the basal type of breast
cancer is undifferentiated, does not express steroid hormone
receptors (triple negative), and has a highly aggressive phe-
notype with poor clinical prognosis. The basal type of breast
cancer is characterized by a Notch activation signature (Lee
et al, 2008), and strikingly it was shown that this type is
associated with a high expression of Jag1 (Reedijk et al,
2008). We observed that a high expression level of ZEB1
correlated with significantly lower miR-141 and miR-200c
levels in the basal type, compared with the common ductal
invasive type of breast cancer (Figure 6A). ZEB1 expression
in basal type of breast cancers was heterogeneous within
individual tumours. High ZEB1 expression levels, particularly
in invasive tumour regions, correlated with expression of
Jag1 and activation of Notch signalling, as indicated by
expression of activated Notch1 and Notch2 (Figure 6B;
Supplementary Figure S2D). Pancreatic cancer is also asso-
ciated with increased Notch signalling activity (Miyamoto
et al, 2003). In differentiated pancreatic adenocarcinomas
only few tumour cells expressed ZEB1 (not shown). In
contrast, a high proportion of the investigated undifferen-
tiated pancreatic adenocarcinomas (classified with gradings
G3 and G4) showed areas with strong expression of ZEB1 in
tumour cells, again particularly in invasive regions. These
tumour cells have undergone an EMT, as demonstrated by the
expression of the mesenchymal marker vimentin. ZEB1 ex-
pression and EMT were strongly associated with expression
of Jag1, mainly in invasive tumour regions (Figure 6C;
Supplementary Figure S2D). Moreover, also undifferentiated,
ZEB1 expressing pancreatic cancers expressed significantly
lower miR-141 and miR-200c levels compared with differen-
tiated cases (Figure 6A). These correlative data indicate
that the experimentally validated link of ZEB1 expression,
reduction of miR-200 family members and expression of
Notch pathway components might be causal to increased
Notch signalling in some types of human cancer.
Discussion
We have shown that the EMT activator ZEB1 increases
Notch signalling in cancer cells via repression of miR-200
family members. Members of this microRNA family inhibit
expression of Notch pathway components, as demonstrated
for the Notch ligand Jag1 and the intracellular Notch coacti-
vators Maml2 and Maml3. Notch signalling is known to be
important for key cancer cell properties, including prolifera-
tion, survival and stemness (Hurlbut et al, 2007; Koch and
Radtke, 2007; Rizzo et al, 2008), which could be confirmed in
this study, in particular for Jag1. A correlated overexpression
of ZEB1 and Jag1, associated with reduced miR-200
expression was detected in poorly differentiated pancreatic
adenocarcinomas as well as in the highly aggressive basal
type of breast cancer, particularly in invasive tumour cells.
Although the importance of Notch signalling for tumour
formation and malignant progression is documented for
ZEB1 activates Notch signalling
S Brabletz et al
&2011 European Molecular Biology Organization The EMBO Journal VOL 30 |NO 4 |2011 775
many cancer types, mutations in Notch pathway components
are detected very rarely in solid tumours. Our results describe
an additional way to activate and stabilize Notch signalling
by linking EMT and aberrant Notch signalling in cancer cells.
This is of particular relevance since many of the properties
already known to be regulated by the Notch pathway were
recently shown to be also controlled by EMT activators. For
instance, it was shown that factors like Snail, Twist and in
GSI – 2.5 5 – 2.5
50
No. of spheres per 1000 cells
A
100
Panc1 MDA-MB231 B
Treatment si si si si si si
ctrl Jag
1a
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1. Generation 2. Generation
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Notch reporter WT
F
Relative reporter activity
ctrl miR141 miR141
miR200c miR200c
Antagomir
HPAF2
100
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siRNA ctrl ctrl Jag1
G
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No. of spheres per 1000 cells
100 HPAF2
ctrl miR141 miR141
miR200c miR200c
Antagomir
siRNA ctrl ctrl Jag1
WT
sh/miRNA ctrl shZEB1 miR-141 miR-200c
E
mut
Notch reporter
Panc1
50
100
Relative reporter activity
Jag1 –+ –+ –+ –+
T
T
TT
T
Panc1
Overview ZEB1 ZEB1 Jag1 Vim
shctrl
shZEB1
P
S
C
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P
Du
M
M
T
T
C
M
M
T
T
C
M
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T
T
C
DmiR-141
ZEB1
X-fold miRNA expression level
50
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miR-200cJag1
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sh ZEB1
Panc1 sh ctrl
Notch ICD – – + + – – + +
I
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mut
Notch reporter
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% Relat. reporter activity
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shRNA ctrl ZEB1 ctrl ZEB1 ctrl ZEB1 ctrl ZEB1
Panc1
Anta ctrl
si ctrl
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si ctrl
Anta 141/200c
si Jag1
HPAF2
H
4.0
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0.0
Time (da
y
s)
12
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Error bars: 95% CI
34
ZEB1 activates Notch signalling
S Brabletz et al
The EMBO Journal VOL 30 |NO 4 |2011 &2011 European Molecular Biology Organization776
particular ZEB1 not only control EMT-associated processes
like cell motility, but also stemness and survival (Thiery et al,
2009). A link of EMT and Notch activation at molecular level
can explain such overlaps in the control of crucial cellular
processes. Moreover, a reverse link was already described
recently, by showing that Notch signalling can induce EMT
in cell culture (Leong et al, 2007; Sahlgren et al, 2008).
For instance, an inhibition of Notch signalling resulted
in the reversal of EMT and the associated drug resistance in
pancreatic cancer cell lines by decreasing expression of
ZEB1, Snail1 and NFkB (Wang et al, 2010). This is supported
by our data, showing that a knockdown of Jag1 also affected
the expression of ZEB1 in cancer cells (see Supplementary
Figure S1B).
Our results indicate the existence of reciprocal activation of
Notch signalling and EMT in the sense of a positive feedback
loop. We therefore would like to propose the following model
(Figure 7): aberrant expression of ZEB1, for example induced
by environmental signals and hypoxia at the tumour invasion
front, was shown to induce stemness and EMT in cancers
cells through suppression of stemness-inhibiting microRNAs,
including miR-200 family members (Shimono et al, 2009;
Wellner et al, 2009). Targets of miR-200 are not only ZEB1
itself and stem cell factors, such as Bmi1 (Brabletz and
Brabletz, 2010), but also Notch pathway components like
Jag1, Maml2 and Maml3. Accordingly, indirect stabilization
of Notch pathway components by aberrant ZEB1 re-enforces
a Notch signalling loop, with all its known effects. In
addition, it was already shown (Wang et al, 2009) and also
confirmed in this study (see Supplementary Figure S1B), that
Notch signalling stimulates ZEB1 expression, which might
further stabilize motility, survival and a stemness phenotype
in cancer cells, particularly at the invasive front. We have
validated our experimental findings by analysing the expres-
sion of ZEB1 and Jag1 and the activation of Notch signalling
in subtypes of human pancreatic and breast cancers, which
were selected, because of their strong association with
aberrant Notch signalling. In particular, the highly aggressive
basal type of breast cancer is characterized by high expres-
sion of Jag1 (Reedijk et al, 2008). Here, we found an
associated expression of Jag1 and ZEB1, as well as Notch
activation in tumour cells of this type of breast cancer,
supporting the proposed link of high ZEB1, low miR-200
and Notch activation at molecular level. However, in the
more differentiated ductal invasive type of breast cancer, we
rarely found ZEB1 expressing tumour cells, although Jag1
was expressed in some tumour areas (not shown). This
indicates that additional ways of aberrant Notch activation
might exist in different cancer subtypes. A second example is
pancreatic cancer, which is also characterized by abundant
activation of Notch signalling (Miyamoto et al, 2003). We
already described a high frequency of aberrant ZEB1 expres-
sion in poorly differentiated pancreatic adenocarcinomas, in
particular in invasive regions (Wellner et al, 2009). As
described for basal type of breast cancers (Burk et al, 2008;
Gregory et al, 2008), we show here that also undifferentiated
pancreatic cancers express significantly lower levels of miR-
200 family members compared with differentiated cases. We
further detected a correlated expression of ZEB1, the me-
senchymal marker vimentin and Jag1 expression in invasive
regions of such cancers, supporting the existence of the
proposed molecular link also in pancreatic adenocarcinomas.
Future work will show if this link between ZEB1, miR-200
and Notch activation is also active in other cancer types
and is controlling physiological processes in embryonic
development and adult tissue homeostasis as well.
In summary, we here described that ZEB1 can indirectly
activate Notch signalling by inhibiting miR-200 expression,
leading to a stabilization of Notch pathway components and
Notch signalling. Thus, activation of Notch signalling is an
additional way how ZEB1 exerts its established tumour
progressing function. This link could explain how aberrant
EMT activation is molecularly connected to crucial properties
driving cancer initiation and progression, such as mainte-
nance of cancer stem cells, as well as proliferation and drug
resistance of cancer cells. It could further explain how Notch
signalling is amplified in some types of poorly differentiated
cancers, where mutations in Notch pathway genes are rarely
detected.
Materials and methods
DNA constructs
For construction of miR-200c and miR-141 expression constructs,
mature sequences of miR-141 and miR-200c were cut out of pUC57
miR-141 or pUC57 miR-200c (GenScript, Piscataway, NJ) and cloned
in pRetroSuper (Brummelkamp et al, 2002). For construction of the
Jagged1 30UTR reporter plasmid, nucleotides þ4164 to þ5966 of
human Jagged 1 cDNA, for construction of the Maml2 and Maml3
30UTR reporter plasmids, nucleotides þ4759 to þ5419 of human
Maml2 cDNA and nucleotides þ5564 to þ7064 of human Maml2
cDNA were amplified and cloned into the multiple cloning site
downstream of the luciferase gene in the pMIR-REPORT vector
(Ambion, Austin, TX). For construction of the Jag1 expression
Figure 4 Jag1 alone is not sufficient for the effects of ZEB1 and miR-200 on Notch signalling. (A) GSI treatment inhibits the sphere-forming
capacity. (B) Transient knockdown of Jag1 inhibits the sphere-forming capacity in the second, but not in the first generation of spheres in Panc1
cells. A representative picture of spheres from the second generation is shown. (C) Representative immunohistochemistry showing a huge
control tumour (T) invading the duodenal wall (Du) and small, encapsulated, non-invasive tumour nodules of shZEB1 cells surrounded by
pancreas (P) and spleen (S) tissue (overview). Squares indicate magnified areas showing a coordinated reduction of ZEB1, Jag1 and vimentin
expression in tumours derived from ZEB1 knockdown cells, encapsulated by ZEB1 expressing fibroblasts (c) and high expression in control
tumour cells invading the duodenal muscle layers (M). Size bar is 200 and 20 mm. (D) Quantitative RT–PCR after microdissection of orthotopic
xenograft tumours showing decrease of Jag1 and increase of miR-141 and miR-200c after knockdown of ZEB1. Shown are the mean values
of all grown tumours (six mice for control and four mice for ZEB1 knockdown Panc1 cell clones), control knockdown was set to 100% or 1.
(E) Coexpression of Jag1 lacking the 30UTR can only partially rescue Notch reporter inhibition by shZEB1 or miR-141 and miR-200c.
(F) Inhibition of endogenous miR-141 and miR-200c in differentiated HPAF2 cells increases Notch reporter activity, which is only partially
reversed by siRNA-mediated knockdown of Jag1. (G) Inhibition of endogenous miR-141 and miR-200c increases the second-generation sphere-
forming capacity, which is reversed by siRNA-mediated knockdown of Jag1. (H) Proliferation of differentiated HPAF2 cells is not affected by
antagomirs and Jag1 knockdown. (I) Cotransfection of the Notch ICD increases the Notch reporter activity in control clones, but not in stable
ZEB1 knockdown clones. There was no significant effect on a mutated (mut) reporter construct. Shown are mean values of each two
independent clones. Control clones were set to 100%.
ZEB1 activates Notch signalling
S Brabletz et al
&2011 European Molecular Biology Organization The EMBO Journal VOL 30 |NO 4 |2011 777
1600400200
MAML2 3′UTR MAML3 3′UTR
2800800 1200 1600 2000 2400
400
1
A
B
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% mRNA expression level
Maml3
Maml2
Transfection
Transient
Panc1
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40
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Jag1 3′UTR
Maml2 3′UTR
Maml3 3′UTR
MDA-MB231
Panc1
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Relative reporter activity
F
mut
Notch reporter WT
Panc1
G
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100
% mRNA expression level
siMaml2sictrl
Panc1 Maml3
Maml2
Hey1
H
50
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% mRNA expression level
siMaml2
sictrl
MDA-MB231
Maml2
Hey1
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Transfection
C
miRNA 141/200c
ctrl
Jag1
Maml2
Maml3
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MDA-MB231
Maml2
I
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No. of spheres per 1000 cells
100 Panc1
sictrl
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shctrl
DPanc1
Maml3
Maml2
β-Actin
β-Actin
β-Actin
′
′
′′
′
′′
miR-ctrl
ctrl siZEB1 141 200c shZEB1 141 200c
miR-200c
miR-141
shZEB1
ctrl siZEB1 141 200c ctrl ctrl 141/200c
siRNA
siMaml3
siMaml2
siMaml3
Stable
Figure 5 The Notch coactivators Maml2 and Maml3 are targets of miR-200. (A) Scheme showing predicted target sites of miR-200 family
members in the 30UTR of Maml2 and Maml3. (B–D) Knockdown of ZEB1 or overexpression of miR-141 and miR-200c decrease the expression of
Maml2 and Maml3 on mRNA (B,C) and protein (D) level. Overexpression of miR-200 in Panc1 was done by lentiviral transduction, shown in
(D) are each three independent lines. (E) Overexpression of miR-141 and miR-200c decreases activity of the indicated 30UTR reporter
constructs. (F) Knockdown of Jag1, Maml2 or Maml3 decreases Notch reporter activity. (G,H) Knockdown of Maml2 or Maml3 decreases
expression of the Notch target gene Hey1 in Panc1 (G) and MDA-MB231 (H). Maml3 is not expressed in MDA-MB231 (see Figure 1B).
(I) Knockdown of Maml2 or Maml3 decreases the sphere-forming capacity of Panc1 in the second-sphere generation.
ZEB1 activates Notch signalling
S Brabletz et al
The EMBO Journal VOL 30 |NO 4 |2011 &2011 European Molecular Biology Organization778
% Rel. miRNA
expression
100
50
Undiff Diff
Undiff
Diff
No
miR-141
miR-200c
++ ++ ++ + + – – – – – – –
Pancreatic cancer
P<0.001
P<0.005
% Rel. miRNA
expression
100
50
Ductal invasive
Basal
Dic
No
Basal type
miR-141
miR-200c
++ ++ ++ + + + (+) (+) – – – – – – – – –
12345678 9101112
12345 6789101112
13 14 15 16 17 Average
Average
ZEB1
ZEB1
Case no.
Case no.
Type
Type
Breast cancer
A
P<0.02
P<0.001
ZEB1 Jag1 aNotch1 aNotch2
inv
cen
B
ZEB1 Jag1 Vim
inv
cen
C
Figure 6 Correlation of ZEB1 and Notch activity in human cancers. (A) qPCR for miR-141 and miR-200c after microdissection of tumour areas
from the indicated types of human primary tumours, showing a significant reduction in basal versus ductal invasive type (dic) of breast
cancers, as well as in undifferentiated versus differentiated pancreatic adenocarcinomas. The statistical significance for the mean values of all
included cases is shown. miRNA expression in normal (no) breast or pancreatic epithelial tissue was set to 100%. Shown are also the ZEB1
expression levels in tumour cells scored by immunohistochemistry. (B) Immunohistochemistry of a typical basal type of breast cancer showing
correlated expression of ZEB1, Jag1, activated Notch1 and activated Notch2 in cancer cells of invasive (inv) tumour areas. In central tumour
areas (cen), a correlated reduction was often detectable. (Inserts show magnifications, specific staining in red, nuclear counter staining in
blue). Size bar is 20 and 12 mm for inserts. (C) Immunohistochemistry of an undifferentiated pancreatic adenocarcinoma showing correlated
expression of ZEB1, Jag1 and the mesenchymal marker Vimentin in cancer cells of invasive (inv) tumour areas. In central tumour areas (cen)
often a correlated reduction was detectable (squares show magnifications). Arrows show positive staining of fibroblasts for ZEB1 and
Vimentin, and blood vessels for Jag1. T ¼tumour cells. Size bar is 20 and 12 mm for inserts.
ZEB1 activates Notch signalling
S Brabletz et al
&2011 European Molecular Biology Organization The EMBO Journal VOL 30 |NO 4 |2011 779
vector lacking the 30UTR nucleotides þ51 7 t o þ4174 of human
Jag1 cDNA were amplified and cloned into pCI-neo (Promega,
Madison, WI). The Notch-CSL-1 reporters, 4xwt-CSL-Luc, which
contains four tandem repeats of the consensus CSL DNA-binding
sequence, GTGGGAA and the mutant (4xmt-CSL-luc) were gifts
from Diane Hayward (Johns Hopkins University School of
Medicine, Baltimore, MD). The plasmid pcDNA-FLAG-Notch-IC,
expressing the Notch1 ICD, was a gift from Tilman Borggrefe (MPI
for Immunobiology, Freiburg, Germany). For mutagenesis of the
indicated miRNA seed sequence binding sites for mir-141, mir-200a
and mir-200c in the Jagged1 30UTR, PCRs were performed using the
Pfu Ultra Hotstart 2 Master Mix (Stratagene, Santa Clara)
followed by DpnI (Fermentas, St Leon-Rot, Germany) digestion
for 1 h at 371C. Mutagenesis was verified by sequencing.
Cell culture and various assays
All cell lines were purchased from ATCC. Stable knockdown clones
for ZEB1 and control clones were constructed as previously
described (Spaderna et al, 2008). For generation of stable over-
expression Panc1 clones for miRNA-141 or miRNA-200c, cells were
transfected with sequence verified pRetroSuper-miRNA constructs.
Cell lines were selected and cultivated under standard conditions in
DMEM þ10% fetal bovine serum þ2mg/ml puromycin. Transient
transfections, reporter assays, immunoblots and transient siRNA-
mediated knockdown were done as previously described (Spaderna
et al, 2006, 2008). All experiments were done at least three times.
For reporter assays, the firefly luciferase values were normalized
against the values of a cotransfected pCMV-Renilla-Luciferase
construct, in order to correct differences in transfection efficiencies.
RNA was isolated using RNeasy Plus Mini Kit (Qiagen), mirVana
TM
miRNA Isolation Kit (Ambion) for microRNAs and RecoverAll
TM
RNA Isolation Kit for formalin-fixed, paraffin-embedded tissues
(Ambion). mRNA expression values were measured in triplicates on
a Roche LightCycler 480 and normalized to b-actin expression as
control. Specific quantitative real-time PCR experiments for
miRNAs were carried out using TaqMan MicroRNA Assays (Applied
Biosystems). To block Notch signalling, cells were incubated for
24 h in the presence of GSI type I (Z-Leu-Leu-Nle-CHO; Calbiochem
#565750, San Diego, CA) in concentrations of 5 and 2.5 mM.
Lentiviral-based miRNA expression vectors were used for generat-
ing control GFP (pCDH-CMV-MCS-EF1-copGFP, System Biosciences,
Mountain View, CA), miR-141 þGFP (pMIRH141-PA-1, System
Biosciences) and miR-200c þGFP (pMIRH200c-PA-1, System
Biosciences) overexpressing cells. Lentiviral particles were pro-
duced by cotransfection of HEK293T cells with the appropriate
transfer and lentiviral helper plasmids (pCMVDR8.74 packaging
vector and pMD2.VSVG envelope vector) using Lipofectamine with
Plus reagent (Invitrogen). The medium was exchanged 3 h after
transfection and lentiviral supernatant was collected 36 and 72h
later. The lentiviral supernatants were concentrated by ultracen-
trifugation at 201C for 2.5 h at 19 500 r.p.m. Infection of Panc1 cells
was performed in 12-well plates in the presence of 6mg/ml
Polybrene (Hexadimethrinbromid, Sigma-Aldrich). Infection with
different amounts of viruses was tested on Panc1 cells and the
infection efficiency determined by GFP expression and quantitative
real-time PCR for microRNAs.
Cell proliferation assay
Cells were seeded in 96-well plates at 3000 cells/well. After 24 h,
cells were transfected with siRNAs or treated with GSI. At indicated
time points, MTT (methylthiazolyldiphenyl-tetrazolium bromide;
Sigma #M5655) was added (5 mg/ml) to the medium. After 4 h
incubation, the medium was removed and the crystals were diluted
in 200 ml acidified isopropanol (0.04 N HCl). Absorption was
measured at 570nm with 650 nm as a reference wavelength
using a Tecan Infinite M200 Reader to determine MTT activity,
which is indicated in relation to activity measured at day 1 after
seeding (set to 1).
Cancer stem cell spheroid assay
To induce sphere formation, cells were dissociated to single cells by
0.05% trypsin-EDTA solution (Invitrogen) and plated at
20 000 cells/ml in serum-free medium (SFM) into Poly(2-hydro-
xyethyl metacrylate) (Polyhema, Sigma)-coated six-well plates to
prevent the cells from attaching to the surface. The SFM consists of
DMEM-F12 (Invitrogen) supplemented with 20 ng/ml epidermal
growth factor (R&D Systems), 0.4% bovine serum albumin (Sigma).
For pancreatic cell lines, it additionally contains 10 ng/ml fibroblast
growth factor (BD Bioscience) and 10 mg/ml Insulin, 10 mg/ml
Transferrin, 10 ng/ml Sodium-Selenite Mix (Sigma), whereas the
SFM for breast cancer cells is further supplemented with B27
Supplement 1:50 (Invitrogen) and 4 mg/ml Insulin (Invitrogen). For
in vitro propagation, the spheres were dissociated to single cells
after 5 days and again seeded as described to form the next
generation of spheres. For quantification of the sphere-formation
capacity, 1000 cells/well of the dissociated single cells were seeded
in SFM containing 1% methylcellulose (Sigma) into polyhema-
coated 96-well plates. After 7 days, Panc1 cells colony numbers
475 mm were counted, for MDA-MB231 cell line all colonies 45
cells were counted. For the treatment of HPAFII cells with
antagomirs against miR-141 and miR-200c and subsequent siRNA-
mediated knockdown of Jag1, tumour cells were first transfected
with siRNAs against Jag1 using Oligofectamine (Invitrogen). At 4 h
after transfection, medium was changed and 500nM antagomirs
added to the cell culture medium. After 24 h, cells were trypsinized
and seeded for the spheroid assay in the presence of 500 nM
antagomirs.
Apoptosis assay
Exponentially growing cells were transfected with the indicated
siRNAs or treated with GSI. At 8 h after transfection or 3 h after GSI
treatment, cells were irradiated with 5 Gy using a Gammacell 40
137
Cs laboratory irradiator. After 3 days, cells were stained for
Annexin V and with propidium iodide using an Annexin V-FITC Kit
from Milteniy Biotec. Apoptosis was measured by flow cytometry
on a Cytomics FC 500 instrument from Beckman Coulter.
miRNA target gene search
For identification of potential mircoRNA target genes, the miR-
ecords website, which integrates different microRNA target
prediction tools, was used (http://mirecords.biolead.org).
MicroRNA overexpression
In all, 3 10
4
cells/well were seeded in 12-well plates. After 24h,
cells were transfected with 30 pmol miRNA oligonucleotides for
hsa-miR-141 or hsa-miR-200c (Ambion). The Ambion Pre-miRTM
miRNA Precursor-Negative Control #1 and #2 was used as control.
Oligofectamine
TM
Reagent (Invitrogen) was used as transfection
reagent. After 3 to 6 days of incubation, cells were used for several
assays.
Specific inhibition of miRNAs using antagomirs
Antagomirs (Dharmacon) were designed as described (Krutzfeldt
et al, 2007). In all, 500nM of antagomirs were added to the normal
cell culture medium right after seeding in 12-well plates. In all, 4–5
days after seeding, the cells were harvested for specific assays.
ZEB1 miR-200
EMT
stemness
survival
Epithelial
differentiation
Bmi1
Jag1
Maml2
Maml3
Environment
Figure 7 Proposed model for a molecular link between ZEB1,
miR-200 and Notch signalling (explanation see Discussion section).
ZEB1 activates Notch signalling
S Brabletz et al
The EMBO Journal VOL 30 |NO 4 |2011 &2011 European Molecular Biology Organization780
Tissue specimens and immunohistochemistry
Immunohistochemistry on formalin-fixed, paraffin-embedded sam-
ples of breast and pancreatic carcinomas from patients who
underwent surgery or of nude mice xenografts was done as
previously described (Brabletz et al, 2004; Wellner et al, 2009).
For quantification of miRNA expression by qPCR, tumour areas
were separated by microdissection under microscope, total RNAs
including small RNAs were isolated by RecoverAll RNA Isolation Kit
for formalin-fixed, paraffin-embedded tissues (Ambion).
Statistics
For comparison of rational variables, the ANOVA algorithm of SPSS
17.0 (SPSS Inc., Chicago) with the significance level set to P¼0.05
was used. Relative MTT absorption levels were plotted with error
bars as indicated (confidence interval, standard error). Association
of dichotomous variables was analyzed using two-sided Fisher’s
exact test of SPSS Version 17.0 (SPSS Inc., Chicago), with a
significance level of Po0.05.
Supplementary data
Supplementary data are available at The EMBO Journal Online
(http://www.embojournal.org).
Acknowledgements
For expert technical assistance we thank Bettina Schuler, Jessica
Pfannstiel, Kerstin Meyer, Anja Schmitt and Stephanie Mewes. For
helpful discussions, we are grateful to Tilman Borggrefe (MPI for
Immunobiology, Freiburg, Germany). For generous gift of reagents,
we thank Tilman Borggrefe, Maria Dominguez (Instituto de
Neurosciencas, Alicante, Spain) and Diane Hayward (Johns
Hopkins University School of Medicine, Baltimore, MD). This
work was supported by grants to TB from the EU (MCSC contract
no. 037297), and the DFG (no. BR 1399/6-1 and the SFB 850, B2),
and to GN from the Clotten Foundation.
Author contributions: SB designed the study, performed the
experiments and analysed the data; KB, SM, UB, GN and EF
performed the experiments and analysed the data; UW performed
statistical analyses; AD and GF investigated and analysed human
cancers; JS designed the parts of the study, performed the experi-
ments and analysed the data; TB designed the study, analysed the
data and wrote the manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
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