Cancer Letters 325 (2012) 42–53

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Cancer Letters
journal homepage: www.elsevier.com/locate/canlet

Activation of canonical WNT/b-catenin signaling enhances in vitro motility of

glioblastoma cells by activation of ZEB1 and other activators of
epithelial-to-mesenchymal transition
Ulf D. Kahlert a,h, Donata Maciaczyk a, Soroush Doostkam b, Brent A. Orr c, Brian Simons c, Tomasz Bogiel a,
Thomas Reithmeier a, Marco Prinz b,f, Jörg Schubert d,1, Gabriele Niedermann g, Thomas Brabletz d,
Charles G. Eberhart c, Guido Nikkhah a, Jaroslaw Maciaczyk e,⇑
a
Division of Stereotactic Neurosurgery, Department of General Neurosurgery, University Medical Center Freiburg, Breisacher Straße 64, 79106 Freiburg, Germany
b
Department of Neuropathology, University Medical Center Freiburg, Breisacher Straße 64, 79106 Freiburg, Germany
c
Department of Pathology, Johns Hopkins Medical Institutions, Smith Building, 400 North Broadway, Baltimore, 21287 MD, USA
d
Department of General- and Visceral Surgery, University Medical Center Freiburg, Hugstetter Straße 55, 79106 Freiburg, Germany
e
Department of General Neurosurgery, University Medical Center Freiburg, Breisacher Straße 64, 79106 Freiburg, Germany
f
BIOSS Center for Biological Signaling Studies, University of Freiburg, 79106 Freiburg, Germany
g
Department of Radiation Oncology, University Medical Center Freiburg, 79106 Freiburg, Germany
h
Faculty of Biology, Albert-Ludwigs University of Freiburg, Schaenzlestr 1, 79194 Freiburg, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Here we show that activation of the canonical WNT/b-catenin pathway increases the expression of stem
Received 15 March 2012 cell genes and promotes the migratory and invasive capacity of glioblastoma. Modulation of WNT signal-
Received in revised form 15 May 2012 ing alters the expression of epithelial-to-mesenchymal transition activators, suggesting a role of this pro-
Accepted 22 May 2012
cess in the regulation of glioma motility.
Using immunohistochemistry in patient-derived glioblastoma samples we showed higher numbers of
cells with intranuclear signal for b-catenin in the infiltrating edge of tumor compared to central tumor
Keywords:
parenchyma. These findings suggest that canonical WNT/b-catenin pathway is a critical regulator of
Glioblastoma
b-Catenin
GBM invasion and may represent a potential therapeutic target.
ZEB1 Ó 2012 Elsevier Ireland Ltd. All rights reserved.
Epithelial-to-mesenchymal transition (EMT)
Invasion

1. Introduction capacity of BTSC appears to be regulated by developmental path-

Glioblastoma multiforme (GBM), the most malignant primary
brain tumor is characterized by dismal prognosis with median sur-
vival of approximately 16–19 months despite multimodal treat-
ment including surgical resection followed by combined radio-
chemotherapy [53,54]. In spite of enormous efforts towards under-
standing the molecular basis of the disease [2] and the develop-
ment of novel therapeutic strategies only limited advances have
been achieved. However, the identification of brain tumor stem-
like cells (BTSCs [19,51]), in both adult and pediatric human malig-
nant gliomas, have provided new insights in the biology of malig-
nant glial tumors rendering these cells a potentially viable
therapeutic target. BTSC share many characteristics of normal neu-
ral stem cells. Similar to normal neural stem cells the proliferative
⇑ Corresponding author. Tel.: +49 761 270 63570; fax: +49 761 270 63580.
E-mail address: jaroslaw.maciaczyk@uniklinik-freiburg.de (J. Maciaczyk).
1
Present address: Novartis Pharma GmbH, Roonstr. 25, 90429 Nürnberg, Germany.
ways including Sonic Hedgehog [5], Notch [16] and possibly Hippo
[40] signaling. Interestingly, while WNT/b-catenin signaling has
been implicated in the proliferation of the neural stem cells and
the regulation of stem-like cell population in a number of solid tu-
mors (for review see [10,39]), only recently has it been discussed in
the context of GBM [3,24,43,62,64].
Mutations of pathway members, including adenomatous polyp-
osis coli (APC) or b-catenin itself result in aberrant activation of the
target genes, including those encoding for activators of epithelial-
to-mesenchymal transition (EMT). EMT has been shown to pro-
mote mesenchymal differentiation and migration in a number of
human tumors including, i.e. carcinomas of the colon [65], prostate
[36] and breast [13].
Integrated genomic analysis enabled the molecular classifica-
tion of GBM into neural, proneural, classical and mesenchymal
subtypes [56]. Compared to the more favorable proneural subtype,
the mesenchymal signature is associated with poor response to
therapy and worse prognosis [41]. Interestingly, evaluation of

0304-3835/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.canlet.2012.05.024
 U.D. Kahlert et al. / Cancer Letters 325 (2012) 42–53
morphometric details (i.e. nuclear staining) from the TCGA data-
base revealed that high levels of WNT/b-catenin activity is associ-
ated with shortest overall patient survival [11]. This may be due to
increased cell motility leading to early tumor recurrence, processes
that may be dependent on EMT [33,60].
In this study, we present evidence of canonical WNT/b-catenin
pathway activation predominantly within the invasive tumor front
in GBM samples. Activation of the pathway in GBM-derived cul-
tures induced expression of multiple EMT activators including
ZEB1 [57], Twist [33] and Snail [60] and augmented in vitro cell
migration/invasion. Conversely, inhibition of the pathway using
RNA-interference, led to down regulation of EMT-inducing tran-
scription factors and decrease of in vitro cell motility.
Our findings support a novel role of the canonical WNT pathway
in regulating GBM invasion by inducing a mesenchymal develop-
mental program and suggest that WNT/b-catenin pathway may
represent a potential therapeutic target.
2. Materials and methods
2.1. Clinical data and tissue collection
All procedures described were approved by the local ethical committee of the
University of Freiburg and were in accordance with the German law. For immuno-
histochemical analysis, tumor tissue from 30 patients diagnosed with glioblastoma
in accordance with World Health Organization established guidelines [31] was used
for this study.
Primary tumor-derived cell cultures (KK, FF, AH and GB) were established
according to procedure developed for human fetal neural stem cells adopted from
Maciaczyk et al. [32].
Cell lines NCH421k, NCH644 were kindly provided by C. Herold-Mende, Depart-
ment of Neurosurgery, Medical Center Heidelberg, Germany.
Cell line HSR-GBM1 was gratefully provided by A. Vescovi, Stemgen S.p.A., Mi-
lan, Italy, whereas cell line 276 was generously provided by A. Quinones-Hinojosa,
Department Neurosurgery, Johns Hopkins Medical Institutions, Baltimore, USA.
HEK293T cells were provided by M. Bredel, Department General Neurosurgery,
Medical Center Freiburg, Germany.
U87 was graciously provided by A. Weyerbrock, Department General Neurosur-
gery, Medical Center Freiburg, Germany.
30 GBM samples for immunohistochemical evaluation were obtained from
Department of General Neurosurgery, Medical Center Freiburg (head Prof. J.
Zentner).
The adult cerebral cortex from a non-tumor patient was gratefully provided by
C. Haas, Experimental Epilepsy, Department of General Neurosurgery, Medical Cen-
ter Freiburg, Germany.
2.2. Analysis of expression of WNT target genes
Subgroup specific mRNA expression data (z-normalized) from the TCGA [56]
was downloaded from the cbio genomic portal on February 2012 (http://www.cbio-
portal.org). Box plots were generated applying Interactive Box plot tool provided by
the shodor portal (http://www.shodor.org).
2.3. Cell culture
Tumors spheres (KK, HSR-GBM1, NCH421k, GB, FF, NCH644 and 276) were cul-
tured as described previously [32] in Dulbecco’s modified Eagle’s medium (DMEM)/
F12 (3:1) medium containing 1  B27 (Gibco) supplements. The media was supple-
mented with 20 ng/ml recombinant human basic fibroblast growth factor (bFGF,
R&D Systems), 20 ng/ml recombinant human epidermal growth factor (EGF, R&D),
penicillin/streptomycin (Invitrogen), L-glutamine (Invitrogen), and 5 lg/ml heparin
(Sigma–Aldrich).
Twice per week the spheres were passaged via mechanic dissociation (low vol-
ume tituration up to 150 times with polished 200 ll pipettes) combined with enzy-
matic digestion with Trypsin Like Enzyme (TrypLE, Gibco) at 37 °C.
The HEK 293T cells as well as U87 were grown in DMEM (Gibco) supplemented
with 10% fetal calf serum (FCS) and 1 antibiotics and antimycotics (Gibco).
2.4. Generation of lentiviral constructs
For stable transfection we used the 3rd generation of lentiviral packaging sys-
tem (VSV-G; RRE, REV, original distributed by Trono et al. [15]). To overactivate
the canonical WNT/b-catenin pathway we used a mutant form of b-catenin, bearing
a Cytosine to Adenine exchange at position 33 (originally generated by Morin et al.
[34]). This transversion gives an altered amino acid sequence for Serine to Tyrosine
43
(S33Y) and therefore missing the phosphorylation site subsequently leading to
aberrant turn-over of the protein with arrested ubiquitination, cytoplasmatic accu-
mulation and high nuclear transfer. We cloned the mutant b-catenin under the con-
trol of an EF1alpha promoter into the cFUGW lentiviral backbone, containing a GFP-
sequence as a selection marker, driven by human ubiquitin promoter. This dual pro-
moter system appears to be constitutively on in all tissues and rarely undergoes
silencing events [30].
For overexpression of Dickkopf1 (DKK1) we cloned the sequence from pCS
hDKK1 (Addgene #15494) coupled with a CMVpromoter into the lentiviral back-
bone of pCDH-CMV-MCS-EF1copGFP (System Biosciences). Here the selection mar-
ker is a GFP-sequence from Pontellina plumata (copGFP).
In case of b-catenin-knock down experiments we applied short hairpin (sh) RNA
transfection method. To block the intrinsic b-catenin signaling we applied the
pLKO.1.sh.beta-catenin plasmid [17] (Addgene plasmid #1248) and its control
pLKO.1.sh.scramble plasmid [46] (Addgene plasmid #1864) both obtained from
Addgene Cambridge, MA.
HEK 293T cells were cultured to 80% confluence in 10 cm petri dishes. In anti-
biotics free environment, the cells were transiently transfected with 16 lg plasmid
DNA using Lipofectamine 2000 (Invitrogen). The transduction of >90% of the cells
was checked under fluorescence microscope. The virus was collected 48, 72 and
96 h after transfection and concentrated via ultracentrifugation using ultra-speed
centrifuge (Beckman Coulter) at 22.500 rpm/min. Each virus production from a
10 cm2 dish was concentrated in 100 ll PBS, aliquoted and stored at 80 °C. The
virus titer was determined by quantitative PCR according the protocol described
by Sastry et al. [48].
2.5. Lentiviral infection of target cells and positive selection for infected cells
To infect the GBM cultures (suspension as well as adherent) we plated
3  105 cells in a volume of 1 ml culture media in one well of a six well dish. The
cells were infected with a multiplicity of infection of 10. After an initial infection
time span of 5–6 h at 37 °C 2 ml of growth media was added. For some cell lines
we used two consecutive rounds of infections for S33Y mutation. To obtain a pure
population of infected cells with the S33Y-, cFUGW-plasmids as well as for DKK1
overexpression and its control we used FACS sorting (100 lm nozzle, MoFlow Sys-
tem, Dako Cytomation) against high FITC-intensity.
In case of sh-RNA transfections, the Puromycin resistance gene enabled an anti-
biotic selection of the transfected cell population starting 72 h post infection.
2.6. Immunocytochemistry
For immuncytological studies, the cells were plated on pre-coated glass cover-
slips in a 24 well plate. Cells were plated as drop culture to enhance their attach-
ment for 3 h. After incubation the cells were fixed in 4% paraformaldehyde,
blocked with 4% goat serum and stained with the corresponding primary antibod-
ies. The primary antibody incubation was performed overnight at 4 °C. Afterwards,
following several washes, secondary Alexa 488 (Invitrogen, anti-mouse, A11001
1:250) was applied for 1 h at room temperature. The nuclei were counterstained
with Dapi (Sigma–Aldrich). (For detailed information on antibodies applied see
Supplementary Fig. S1).
2.7. Immunohistochemistry
Specimens of surgical tumor resections (n = 30) from patients with GBM, of
which two exhibited additional sarcomatous components, were immunohisto-
chemically assessed for presence of intranuclear b-catenin (#610153, Becton Dick-
inson Biosciences, 1:200). In brief, the formalin fixed and paraffin embedded
samples were sectioned to 3 lm thickness. Following deparaffinization, the tissue
sections were subjected to water steam for 40 min at pH6 for antigen retrieval
and stained with anti-b-catenin primary antibody at 4 °C overnight. To distinguish
tumor cells from endothelium, which often shows abundant intranuclear b-catenin
signal, specimens were stained with anti-CD31 (#JC70a, Dako,) at room tempera-
ture. Subsequently, the slices were incubated with secondary antibody (#E464, bio-
tinylated polyclonal rabbit anti-mouse, Dako, 1:200) for 60 min at room
temperature. b-catenin and CD31 were detected, following incubation with horse
radish peroxidase (HRP)-conjugated-Avidin (1:1000, 45 min, room temperature),
using DAB solution (10 min, room temperature, Merck). Counterstaining was per-
formed with Haematoxylin for 30 s.
Each specimen was evaluated for nuclear b-catenin expression, indicating WNT
pathway activation. Sections were divided in central tumor parenchyma (vital tu-
mor center), infiltration/margin zone (progression of tumor core to non-neoplastic
tissue, with significant decreased cell density compared to tumor core) and adjacent
non-neoplastic brain tissue. In each tumor the percentage of b-catenin positive nu-
clei was assessed. Afterwards, a semi quantitative scoring system was applied
which distinguished 0 – ‘‘no signal’’ indicating a lack of nuclear b-catenin staining,
1 – ‘‘mild activity’’ corresponding to less than 1% stained nuclei, 2 – ‘‘moderate
activity’’ for staining present in 1–5% of nuclei and 3 – ‘‘substantial activity’’ corre-
sponding to more than 5% of stained nuclei for each of three tested regions per sam-
44 U.D. Kahlert et al. / Cancer Letters 325 (2012) 42–53
ple. Colon carcinoma samples were used as a positive control, whereas non-cancer-
ous adult brain served as negative control. (For complete table with pathological
scoring see Supplementary Fig. S2).
2.8. RNA extraction and reverse transcription
Total RNA was isolated using the RNeasy Mini kit (Qiagen) according to the
manufacturer’s instructions. Contamination with genomic DNA was minimized by
applying DNA digestion step with DNAse easy set (Qiagen). Total RNA extracts were
photometrical quantified in a UV cuvette in a Biophotometer (Eppendorf). Reverse
transcription (RT) was performed according to manufacturer’s protocol using the
SuperScriptÒ III Reverse Transcriptase kit (Invitrogen). Translation of messenger
RNA only was achieved by applying oligo-dT primers. Copy-DNA was diluted to a
10 ng/ll working solution. RNA stocks were stored at 80 °C and cDNA at 20 °C.
2.9. Real-time quantitative RT-PCR
Abundance of transcripts was determined by real-time quantitative PCR on a
MyiQ Real-Time PCR Detection System (Bio-Rad Laboratories) via SYBR Green incor-
poration (Applied Biosystems) for each gene (performed in triplets). Primers were
used at a concentration of 10 pmol/primer. PCR conditions were as follows: 35 cy-
cles, 15 min at 95 °C, followed by 45 cycles of 15 s at 95 °C and 1 min at 60 °C each.
Melting curves of the amplified products were generated to control for specificity of
the amplification reaction. All relative quantifications were normalized to endoge-
nous housekeeping control (b-Actin). In case of down regulated expression a value
of 1 was set as reference level.
Amplification values were automatically determined by supplied software of
the MyiQ5 Real-Time PCR Detection System (Bio-Rad). (For primer sequences see
Supplementary Fig. S3).
2.10. Western blot
Protein analysis was performed with standard semi-dry Western blot method
under established protocol settings. Samples were analyzed with a loading of
40 lg protein/lane. The primary anti-body staining was done via overnight incuba-
tion at 4 °C after blocking with 5% milk powder solution or 2% SlimFastÓ solution. A
list of primary antibodies applied can be found in Supplementary Fig. S1. Secondary
HRP-conjugated antibodies (anti-mouse: #474-1802, anti-rabbit: #474-1506, both
KPL) were diluted 1:10,000 in blocking solution and incubated for 1 h at room tem-
perature. Signal detection was performed on a film based system by applying Im-
mun-Star Western C Kit (Bio-Rad) or Super Signal West Pico Chemiluminescent
Substrate (Thermo Scientific).
2.11. Cell proliferation assay
Mitochondrial activity for determining biological active cell mass was measured
using CellTiter 96Ò AQueous One Solution Cell Proliferation Assay according to the
instructions of the manufacturer (Promega GmbH). The individual values were re-
trieved trough Elisa-based (Tecan) absorption measurements at 490 nm. The forma-
zan dye generated by the metabolizing cells directly corresponds to viable cell mass
in culture. In each proliferation experiment we initially plated 20,000 cells per 24-
well in 1 ml culture media. In order to plate as exact as possible the cells were pas-
saged as described above directly prior plating.
2.12. In vitro motility assays
Invasion and migration of GBM-derived cells was analyzed using a Boyden
Chamber assay. A 24-well transwell system (Corning Inc.) was used, with each well
containing a permeable transwell insert containing a 6.4 mm polycarbonate mem-
brane with 8 lm pores. To mimic three-dimensional extracellular environment and
investigate infiltrative potential of the cells, the inserts were coated with growth
factor reduced Matrigel (BD) and incubated for 1 h in at 37 °C. Migratory potential
of the cells were analyzed on 1% gelatine-coated inserts. Subsequently,
7.5  105 GBM cells solved in 500 ll DMEM were placed on top of each insert mem-
brane. The bottom was filled with 500 ll DMEM media containing 10% FCS. After
incubation for 24–36 h, depending on the cell line. The upper side of the membrane
was then wiped carefully with a cotton swab to remove the rest of the plated cells.
The membrane was then fixed in 4% formaldehyde for 40 min and stained with
Haematoxylin. The migration/invasion of the GBM cells was evaluated by counting
the cell nuclei on the lower side of the membrane under light microscope and
20  magnification (6 view fields).
Cell numbers counted in the control condition (infection with cFUGW vector for
the WNT overexpression, sh scramble for the knock down experiments, copGFP/
control for the DKK1 overexpression) were set as 100%. In relationship to that,
counted cells with modulated WNT activity (S33Y, sh b-catenin, DKK1-overexpres-
sion) were normalized to 100%.
2.13. Statistical analysis
A statistical analysis was carried out with Statistica v5.1 (StatSoft Inc., USA). A
one way ANOVA test was used to compare the different groups. In case of statisti-
cally significant differences, the Newman–Keuls post hoc analysis was used to iden-
tify the nature of the differences.
For q-PCR results, means based on the CP value–raw data for each gene and con-
dition were compared with Student t-Test using Statview 5.0 (SAS Institute Inc.,
USA).
If not mentioned otherwise, the level of significance was set at p < 0.05 for all
tests. Results are expressed as means ± standard derivation.
3. Results
3.1. Multiple WNT/b-catenin targets are overexpressed in the
mesenchymal subgroup of GMBs
The Cancer Genome Atlas (TCGA) recently completed a compre-
hensive genomic analysis of GBM which included the subdivision
of tumors into molecular subtypes including the proneural, neural,
classical, and mesenchymal subgroup ([38], [56]). To determine if
genes related to the WNT/b-catenin pathway were differentially
expressed in specific subgroups of GBMs we interrogated the TCGA
dataset using the caBio portal (www.cbioportal.org). Besides CD44,
a cell surface glycoprotein implicated in Apc/Tcf- mediated tumor
formation [58], Runx2 a transcription factor responsible for mis-
regulated differentiation in WNT-mutant chondrocytes and osteo-
blasts [14], the WNT-ligand receptor Frizzled-1 [29], as well as
others were investigated. Surprisingly, we discovered significant
higher expression of Dickkopf-1 (DKK1), one of main regulators
of the canonical WNT/b-catenin pathway, exclusively in the
aggressive mesenchymal subgroup of GBM (Box plots for TCGA
dataset expression values see Fig. 1). Following this analysis we
investigated the role of canonical WNT/b-catenin network in an
in vitro model of GBM-derived cultures using both gain-of-function
as well as pathway inhibition model.
3.2. Constitutive activation of the canonical WNT/b-catenin signaling
pathway with lentiviral construct encoding for mutant b-catenin
First, we examined our tumor cultures for intrinsic WNT/b-cate-
nin pathway activity using quantitative PCR-based assessment of
Axin2, DKK1 and CD44 expression compared to non-neoplastic hu-
man adult cortical tissue as control. This revealed variations in
WNT pathway activity among the cell lines, with cells showing
low expression levels (i.e. U87: Axin2 0.05 and DKK1 1.7) as
well as many neurosphere cultures clearly over-expressing the
investigated markers including, i.e. FF (Axin2 340, DKK1 24
and CD44 12) or NCH421k (Axin2 24, DKK1 1211 and CD44
8) as shown in Fig. 2A.
To investigate the effects of WNT activation, we created glioma
lines expressing the activating mutant form of b-catenin (S33Y
[27]). The presence of reporter gene GFP enabled the FACS-based
purification for cells overactivating the canonical b-catenin
pathway.
The activity of the pathway was measured using quantitative
PCR-based evaluation of expression of previously reported tar-
gets/negative feedback regulators of canonical WNT pathway,
DKK1 [8], and AXIN2 [23]. Axin2 was upregulated in all GBM-cul-
tures expressing mutant form of b-catenin compared to control
(Fig. 2B) highlighted by strongest upregulation the case of U87 cell
line. This corresponded to intranuclear b-catenin localization in
immunostaining of cells transfected with mutant b-catenin
(Fig. 2D).
Overactivation of canonical WNT/b-catenin signaling led also to
increase of DKK1 expression compared to control group (U87:
Axin2 501 and DKK1 7.8, NCH421k: Axin2 39.2 and DKK1
 U.D. Kahlert et al. / Cancer Letters 325 (2012) 42–53 45

Fig. 1. Box plots representing TCGA expression data for WNT/b-catenin targets DKK1, DKK2, RUNX2, CD44, LEF1 and FZD1 in glioblastoma multiforme (GBM). Analysis of
different subgroups of GBM samples according to Verhaak et al. [56] (proneural (P), neural (N), classical (C) and mesenchymal (M)) showed WNT-target overexpression in the
mesenchymal subtype,  = dataset outliers, Student t-Test, # = p-value 6 0.002.

2.8, HSR-GBM1: Axin2 2 and DKK1 900, KK: Axin2 & DKK1
about 2) (Fig. 2B). Furthermore, the mutant cells were character-
ized by moderate increased expression (2–3) of CD44, another
marker regulated by the WNT network [58]. Effective physiological
modulation of WNT targets Axin2 and DKK1 could be confirmed
with Western blot-based protein analysis (Fig. 2C).
Upon WNT-overactivation, substantial induction of DKK1 in
case of HSR-GBM1 and Axin2 in U87 could presumably be ex-
plained by their explicit low expression levels in wild type (WT)
conditions. Vice versa, high basal levels of CD44 in U87 might ex-
plain why we could not detect its significant induction.
Moreover, inhibition of intrinsic WNT/b-catenin pathway was
achieved by lentiviral transfection with short hairpin (sh) RNAs
against b-catenin, leading to a decrease of Axin2 expression in all
lines tested (Fig. 2E KK 1.3, FF 1.5, HSR-GBM1 over 2, GB
2.8, Western blot exemplary shown in Fig. 2F for NCH421k and
U87). Surprisingly, in this loss-of-function model we could not de-
tect any DKK1 transcripts any longer, except for U87, were we
46 U.D. Kahlert et al. / Cancer Letters 325 (2012) 42–53

Fig. 2. Modulation of WNT/b-catenin signaling pathway. Different basal levels of WNT/b-catenin pathway activity in wildtype GBM-derived cell cultures has been observed
as assessed by Axin2, DKK1 and CD44 expression normalized to non-neoplastic adult cortex (CTX) (A); transfection with mutant form of b-catenin (S33Y)-led to stable
overexpression of the WNT-targets (B) and increase of corresponding protein levels as exemplary shown for U87, KK and NCH421k cell lines (C); immunocytochemistry
against b-catenin in transfected lines revealed stronger nuclear accumulation as compared to control (cFUGW) as shown for U87S33Y (D); in contrary, inhibition of WNT/b-
catenin signaling via shRNA interference against b-catenin decreased expression of relevant markers (E) as well as protein levels of Axin2 (F), (A,B,E Student t-Test, p-
value 6 0.05).

proofed an over 2 decrease of DKK1 transcript (Fig. 2E). All tested
cultures showed also lower CD44 expression (1–2).
Based on our experimental results we assume that DKK1
expression seen in the mesenchymal subgroup of GBMs correlates
with high WNT/b-catenin signaling and can be used as an indirect
marker for the canonical WNT pathway activity.
Taken together, we established robust systems enabling modu-
lation of the canonical WNT pathway and identified two targets,
Axin2 and DKK1, to monitor pathway activity in our subsequent
in vitro studies.
3.3. WNT/b-catenin level does not effect proliferation capacity but
directly correlates with in vitro cell motility
To assess the effect of WNT/b-catenin modulation on prolifera-
tion of GBM cultures, we tested their metabolic activity using MTS
following activation or inhibition of the pathway. In all tested cell
lines the analysis showed no significant differences in growth upon
genetic pathway modulation. In fact, cells with mutated b-catenin/
sh RNA-based b-catenin inhibition and empty virus controls dis-
played apparently similar growth dynamics (Fig. 3A and B).
Interestingly, further characterizations of the genetically modi-
fied cells revealed significantly changed in vitro migratory poten-
tial as tested in Boyden Chambers-based experiments. In our
investigations we used two different insert coatings- 1% gelatine
and 1  Matrigel to test migration or invasiveness, respectively.
In comparison to empty virus control, all tested cell lines (n = 4)
showed stronger invasion (Fig. 3A: NCH421k 2, U87 54.3, KK
5.2, 276 7.3) as well as migration (NCH421k 2.8, U87 6, 1
and 1.2 for KK and 276) when infected with mutant b-catenin.
No significant difference was detected between wild type cell lines
or those infected with empty viral vector (data not shown).
Conversely, cells with reduced WNT/b-catenin signaling
showed decreased numbers of invading and migrating cells as
compared to cells infected with empty virus control (Fig. 3B). In
fact, for KK only 50%, 80% for HSR-GBM1 and 48% for NCH421k
of the cells traversing the membrane, on both, gelainte- and Matri-
gel surfaces. There was no significant motility difference compar-
 U.D. Kahlert et al. / Cancer Letters 325 (2012) 42–53 47

Fig. 3. Modulation of WNT/b-catenin signaling influences in vitro motility of GBM-derived cultures. Activation of WNT/b-catenin pathway did not alter proliferation of
cultivated GBM tissue as assessed by MTS assay. WNT-pathway overactivation directly correlated with increased in vitro cell motility based on Boyden Chamber experiments
with gelatine and Matrigel coatings. There was no significant difference in motility or proliferation between WT and control lines (data not shown) (A). In contrast, WNT/b-
catenin inhibition resulted in significantly decreased cell motility (B), (A, B Student t-Test for qPCR, ANOVA with Newman–Keuls post hoc analysis for motility assays, p-
value 6 0.05).

ing WT cells and empty control cells except for KK showing 26%
less motility in both surfaces upon virus infection. Again, metabolic
activity assessed by MTS was not altered upon WNT/b-catenin
inhibition (Fig. 3B).
3.4. WNT activation induces expression of EMT activators
Increased invasive potential of cancer cells in solid tumors has
been associated with EMT of the migrating tumor cell population
[55,57]. To determine if induction of EMT in GBM-derived cell cul-
tures may explain their increased invasion following WNT path-
way activation, we analyzed mRNA expression levels of EMT-
related transcription factors ZEB1, Twist, Snail and Slug. Indeed,
increased levels of ZEB1 and Twist (both 2–3.5) could be de-
tected. KK showed the strongest induction of Snail (3.7). These
alterations could be proved also on protein level as shown in
Fig. 4A. Another important feature of EMT is an increased ratio
of N-Cadherin to E-Cadherin. The expression of E-Cadherin could
not be observed in any of the GBM cultures. Nevertheless N-Cad-
herin transcript levels were clearly enhanced in cells with acti-
vated WNT/b signaling (up to 3 for NCH421k and over 2 for
U87, Fig. 4A).
48 U.D. Kahlert et al. / Cancer Letters 325 (2012) 42–53

Fig. 4. Activation of WNT/b-catenin signaling promotes the expression of EMT activators and CD133. S33Y transfected cell cultures show induction of epithelial-to-
mesenchymal (EMT) activators ZEB1, Twist, Snail as well as N-Cadherin as showed at mRNA and protein levels (A), whereas the inhibition of intrinsic WNT/b-catenin
signaling led to their decreased transcription and translation (B). Also a putative brain tumor stem cell marker CD133 appeared to up- and down regulated in response to
WNT/b-catenin pathway modulation with the highest transcript and protein level following WNT activation (C,D) (A,B Student t-Test, p-value 6 0.05).

Consistent with this finding, shRNA interference against b-cate-
nin reduced the mRNA levels of EMT factors (2 decrease for Twist
in GB, 3 ZEB1 and Twist for FF and up to 2.7 for N-Cadherin in
HSR-GBM1; Fig. 4B). Furthermore, clear reduction of ZEB1 and
Twist by Western blot could be demonstrated.
of the canonical WNT/b-catenin cascade, we observed overexpres-
sion of Prominin1 mRNA (3 for NCH421k, 2 for NCH644, KK and
HSR-GBM1) as well as increase of corresponding protein (Fig. 4C).
Similarly, following inhibition of WNT/b-catenin pathway we
consistently observed decreased signals for CD133 (1.5–3 for
U87, HSR-GBM1 and KK, Fig. 4D).

3.5. Activation of WNT/b-catenin pathway leads to the increase of

mRNA and protein levels of putative brain tumor stem cell marker
CD133 3.6. Overexpression of DKK1 did not enhance migratory behavior

Next, we evaluated the relationship between levels of WNT DKK1 has been previously reported to act in a feedback loop for
pathway activity, expression of mesenchymal activators and brain canonical WNT/b-catenin pathway activation [8]. Based on the
tumor stem cell marker CD133 (Prominin1). Following activation overexpression of DKK1 in the mesenchymal subgroup of GBMs
 U.D. Kahlert et al. / Cancer Letters 325 (2012) 42–53 49

(TCGA data), we tested weather a DKK1 overexpression has any di-
rect effect on in vitro motility.
We established a lentiviral system to overexpress DKK1 and
validated its efficiency using qPCR and Western blot (Fig. 5A). Next,
we evaluated the effect of DKK1 overexpression of migration and
invasion of glioblastoma using the Boyden Chamber assay. Inter-
estingly, these assays revealed a diminished motility of cells over-
expressing DKK1 in both conditions (percentages traversing cells
upon DKK1 overexpression comparing to control, on gelatine:
U87 2%, 276 13%; on Matrigel: U87 10%, 276 39%, no difference be-
tween WT and control [data not shown], Fig. 5C).
3.7. Nuclear localization of b-catenin is predominantly found in the
infiltration zone in patient-derived glioblastoma specimens
Immunohistochemical assessment of GBM specimens, subdi-
vided in tumor parenchyma and infiltration zone (Fig. 6A) upon
exclusion of non-tumorous CD31 positive endothelial cells
(Fig. 6B), revealed cells with strong intranuclear b-catenin signal
in all 30 observed cases (Fig. 6C). The quantity ranged from few
cells to a substantial fraction of positive nuclei (over 5%) in 14 from
30 samples (Fig. S2). Interestingly, in 11 GBM-derived samples we
observed higher numbers of cells with intranuclear b-catenin sig-
nal in peripheral infiltration zone of the tumor as compared to
its central parenchyma (for evaluation of average pathological
scoring of the 30 samples see Fig. 6D). In 4 tumors WNT activation,
as evidenced by nuclear b-catenin, was seen exclusively in the
infiltration zone (Fig. S2).
A high frequency of co-localization of the endothelial marker
CD31 and nuclear b-catenin (data not published) was also ob-
served, consistent with previous reports [61]. Aside from the vas-
cular staining, intranuclear b-catenin signal was significantly
lower in non-cancerous sections compared to the tumor tissue
(Fig. 6D).
4. Discussion
Aberrant activation of developmental signaling pathways
important in stem cells are hallmarks of neoplastic transformation
in the brain and various other tissues (i.e. Hedgehog [5], Hippo [12]
and Notch [16]). Our current work aims at deciphering the role of
the canonical WNT/b-catenin pathway in the biology of adult glial
tumors. We present clinical data suggesting an important role for
the canonical WNT/b-catenin signaling network in a subset of hu-
man-derived GBM samples, as well as an in vitro experimental
model of pathway modulation supporting its relevance especially
in the regulation of tumor cell migration and invasion.
The analysis of the TCGA-derived gene expression data of over
200 tumor samples enabled the identification of several subclasses
of glioblastomas [38]. Interestingly, the gene expression data pub-
lished as a supplementary material by Verhaak et al. [56] showed
up to 6 up-regulation of Dickkopf1 (DKK1), 3 for Frizzled1
(FZD1) and Runx2 almost exclusively in the mesenchymal group
of GBMs, characterized by frequent multi-focal growth and early
tumor recurrence leading to fast clinical deterioration and nega-
tively influencing of patient overall survival. These findings at-
tracted our attention to a potential role of canonical WNT
signaling in adult gliomas. Since DKK1, a direct target of b-catenin
[37], playing a pivotal role in the negative feedback-based control
of pathway activity [8], showed the highest expression in mesen-
chymal GBMs and in our in vitro experiments was not able to in-
duce the motility of cultivated GBM cells, we hypothesize, that
elevated DKK1 expression levels indirectly indicate WNT pathway
overactivation, responsible for observed phenotypic changes. In
fact, secreted soluble frizzled receptors 1 and 2 were predomi-
nantly inactivated in primary GBMs [20].
Immunohistochemical assessment of 30 patient-derived GBM
samples, consisting of tumor parenchyma, infiltration zone and
non-cancerous adjacent brain tissue showed significantly more

Fig. 5. Biological effects of WNT inhibitor overexpression – DKK1. Using 3rd generation lentiviral transfection system DKK1 was stably overexpressed in GBM-derived
cultures as shown with transcript and corresponding protein (A). It resulted in diminished in vitro cell motility on gelatine- and Matrigel coated Boyden Chambers (B) (A,
Student t-Test, B, ANOVA with Newman-Keuls post hoc analysis for motility assays, p-value 6 0.05).
50 U.D. Kahlert et al. / Cancer Letters 325 (2012) 42–53

Fig. 6. Presence of cells with intranuclear b-catenin signal in IHC assessment of patient-derived GBM samples. In 11 of 30 GBM samples increased number of b-catenin
positive nuclei at the tumors infiltration zone as compared to tumor parenchyma (examples shown by red arrows) could be observed. Overall averaged scoring revealed
significant increased numbers of cells with nuclear b-catenin in the infiltration zone compared to the parenchyma. The lowest levels of intranuclear b-catenin signal were
observed in the adjacent neuropil (D, ANOVA p-value 6 0.05); H&E (A), endothelial staining CD31 (B) and intranuclear b-catenin (C) of patient-derived GBM tissue. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

cells displaying intranuclear b-catenin signal within the tumor
parenchyma and infiltration zone as compared to adjacent brain.
Interestingly, the strongest signal was present within tumor infil-
tration zones adjacent to healthy tissue (Fig. 6C + D). These find-
ings suggest that, similar to colorectal carcinoma [7], in a
subgroup of GBMs the activity of the canonical WNT/b-catenin sig-
naling pathway is directly associated with increased motility of the
tumor cells leading to expansion of the malignant process. Further
characterization of cells with intranuclear b-catenin signal within
tumor infiltration zone is compulsory.
The possible role of canonical WNT signaling in malignant glial
tumors has already been reported by Pulvirenti et al., who showed
an overexpression of Dishevelled-2 (DVL2), a key component of the
WNT pathway in patient-derived samples of GBM [43]. Interest-
ingly, RNA-interference mediated inhibition of Dvl2 expression
blocked in vitro proliferation of tumor-derived cells, promoting
their differentiation as well as inhibiting tumor formation capacity
upon intracranial implantation. Additionally, evaluation of clinical
outcome of patients with GBM revealed b-catenin as a negative
prognostic factor for overall survival [45].
In the current study we found that transduction of a mutant
form of b-catenin (S33Y) leading to constitutive activity of the
pathway significantly increased in vitro invasion and migration in
all studied cell lines with no effect on their growth kinetic. Inter-
 U.D. Kahlert et al. / Cancer Letters 325 (2012) 42–53
estingly, previously reported knock down experiments against
WNT2 and b-catenin showed significant impairment of the prolif-
eration capacity of GBM cultures both in vitro and in vivo [42]. Con-
cordantly, blocking the secretion of multiple WNTs by silencing of
Evi resulted in diminished proliferation capacity of GBM cultures
[3]. These observations could not be reproduced in our RNA-inter-
ference mediated loss-of-function model. In fact, inhibition of the
intrinsic canonical WNT pathway activity had no pronounced ef-
fect on the growth as determined by MTS assay. This might be
due to the significance of non-canonical WNT signaling mediated
by both Evi [1] and Dvl2 [43] on the glioma cell division, not mod-
ulated in our experiments.
Transfection with the lentiviral construct encoding mutated b-
catenin promoted the induction of Jag1 mRNA expression (Supple-
mentary Fig. S4), possibly linking WNT to the Notch pathway. Sim-
ilar interactions have been reported in the developing human brain
[28]. Furthermore, cross-talk between Notch and Sonic Hedgehog
pathways in GBM cultures have also recently been described
[50]. However, further studies addressing the involvement of
Notch-signaling upon WNT pathway activation are necessary.
The WNT network is also involved in the regulation of stem cell
pool during development as shown by numerous reports (for re-
view see [10]). It has also been reported as one of the regulatory
mechanism of stem-like cell population in a number of malignan-
cies including, i.e. colon and breast cancer or acute myeloid lym-
phoma [22,44]. Here we show that manipulation of WNT activity
influenced the expression of Prominin-1 indicating its possible role
in the regulation of brain tumor stem-like cells (BTSCs). Similar to
our findings, an increase of CD133 + cells in glioma cultures medi-
ated by the direct activator of the WNT/b-catenin pathway PLAGL2
has already been reported [64].
In our experiments, an over-activation of the canonical WNT/b-
catenin signaling constantly led to an increased in vitro motility. In
other solid tumors (i.e. bladder [59], breast [47], pancreas [25]) in-
creased invasiveness is often mediated by complete genetic repro-
gramming of the cancer cell- a process termed epithelial-to-
mesenchymal transition (EMT). Furthermore, this phenomenon
plays a pivotal role not only in tumor dissemination but also medi-
ates the resistance to treatment modalities, escape from the im-
mune surveillance, as well as stem cell maintenance.
Predominantly occurring within the tumor infiltration zone, EMT
can be induced by several factors including cytokines, hypoxia or
cellular and extracellular matrix [55,57]. Interestingly, highly con-
served canonical WNT/b-catenin pathway is one of the known
inducers of EMT in both physiological and pathological conditions
(i.e. [26,49]). Similar to these reports, over-activation of the WNT
pathway in GBM cultures induced the expression of EMT activators
ZEB1, Snail, Twist, Slug as well as N-Cadherin being responsible for
the alteration of the cell-cell contact and induction of increased cell
motility observed in the current study. Interestingly Snail has al-
ready been shown to directly modulate in vitro motility of GBM-
derived cells [21]. Furthermore, similar to silencing of the intrinsic
WNT/b-catenin activity via RNA-interference, overexpression of
the WNT pathway inhibitor DKK1 led to decreased mRNA expres-
sion of EMT-related transcription factors (Supplementary Fig. S5)
and abrogation of the in vitro migratory/invasive capacity of the
cells. Similarly, in prostate cancer, inhibition of WNT signaling
leads to the reversal of EMT [63]. The presence and possible role
of the EMT-like process in glioblastoma is currently very contro-
versial. Very recent report by Cheng et al. [9] proved the overex-
pression of EMT activators in a subgroup of GBMs showing
unequivocal negative prognostic value of the mesenchymal transi-
tion metagen for patient survival. In contrast, Nagaishi et al. [35]
reported on the presence of EMT activators in gliosarcomas but
not in glioblastomas based on immunohistochemical analysis of
patient-derived tumor specimens. However, due to methodological
51
differences these interesting data cannot be directly compared
with our observations. Here we show that the overexpression of
EMT activators in GBM-derived cell lines occurs following the
induction of WNT/b-catenin pathway activity. Amongst the pa-
tient-derived samples the intranuclear b-catenin staining could
be rarely observed in P5% of all tumor cells. Therefore the thresh-
old set by Nagaishi et al. at P10% of total cells may led to under-
estimation of the possible GBM samples showing WNT-
dependent overexpression of EMT activators. Furthermore, the lack
of b-catenin staining an exclusive assessment of the tumor paren-
chyma without infiltration zone precludes any direct comparison
with our study and does not exclude the presence of subpopula-
tions of cells undergoing EMT-like changes in some tumor
compartments.
EMT has also been associated with increased resistance to radi-
ation and chemotherapy in several solid tumors [52]. Furthermore,
WNT/b-catenin signaling has also been implicated in promoting
resistance against classical treatment modalities, attributed to tu-
mor stem-like cell population in various cancer types including
GBM [4]. Interestingly, following WNT activation, GBM cells
showed increased mRNA transcripts of ATP-binding cassette
(ABC) transporter ABCG2, involved in resistance against chemo-
therapy in GBMs [6] (Supplementary Fig. S6).
Taken together, our results point out for the first time, the
unequivocal significance of WNT/b-catenin-mediated EMT induc-
tion in regulation of the invasiveness of human malignant gliomas
and increases the signal for CD133 on mRNA and protein level sug-
gesting its involvement in regulating BTSC-pool, addressed par-
tially by recent reports [24,33]. Moreover, the spatial distribution
of the nuclear b-catenin signal in patient-derived samples, found
predominantly within the invasive front of the tumor, suggests
the pivotal role of this pathway in regulation of in vivo malignant
cell motility. These findings may contribute to the development
of an alternative therapeutic strategy based on the interference
with the canonical WNT/b-catenin pathway using numerous effi-
cient small molecules (i.e. [18]) specifically targeting the migrating
cell population and thus preventing tumor recurrence.
Acknowledgements
We gratefully thank K. Geiger, Core Facility 1, Department of
Internal Medicine, University Medical Center Freiburg, Germany
for the flow-cytometry sorting and C. Haas, Experimental Epilepsy,
Medical Center Freiburg for the generous opportunity to use the
qPCR system in her lab.
This work was supported by the Comprehensive Cancer Center
Freiburg Seeding Grant (Deutsche Krebshilfe) awarded to J. Mac-
iaczyk and G. Nikkhah.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/
j.canlet.2012.05.024.
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