Tumor Biol. (2016) 37:11947–11957
DOI 10.1007/s13277-016-5039-5
ORIGINAL ARTICLE
Dishevelled proteins are significantly upregulated in chronic
lymphocytic leukaemia
Abdul Salam Khan 1 & Mohammad Hojjat-Farsangi 1 & Amir Hossein Daneshmanesh 1 &
Lotta Hansson 1,2 & Parviz Kokhaei 1,3 & Anders Österborg 1,2 & Håkan Mellstedt 1,4 &
Ali Moshfegh 1
Received: 18 December 2015 / Accepted: 28 March 2016 / Published online: 16 April 2016
# International Society of Oncology and BioMarkers (ISOBM) 2016
Abstract Dishevelled (DVL) proteins are components of the
Wnt signalling pathways, and increased expression is associated with various malignancies. Information on DVLs in
chronic lymphatic leukaemia (CLL) is limited. The aim of
the present study was to investigate the role of DVLs in
CLL cells and association with Wnt pathways downstream
of ROR1. DVL1, 2 and 3 were exclusively expressed in
CLL cells as compared to normal peripheral blood mononuclear cells (PBMCs). The expression of DVL1 and DVL3
proteins was significantly more pronounced in progressive
than in non-progressive disease (p < 0.01), whereas the level
of DVL2 was significantly higher in non-progressive as compared to progressive disease (p < 0.001). Treatment of CLL
cells with anti-ROR1 specific monoclonal antibodies induced
dephosphorylation of ROR1 as well as of tyrosine and serine
residues of both DVL2 and DVL3. However, gene silencing
of DVLs in the CLL cell line (EHEB) did not induce detectable apoptosis. Non-progressive CLL patients had a different
protein activity pattern with regard to Wnt signalling pathway
proteins as GSK-3β, β-catenin and AKT as compared to progressive disease. The DVL2 protein may play a role in the
* Håkan Mellstedt
[email protected]
1
Department of Oncology-Pathology, Immune and Gene Therapy
Lab, Cancer Center Karolinska (CCK), Karolinska University
Hospital Solna and Karolinska Institutet, Stockholm, Sweden
2
Department of Hematology, Karolinska University Hospital Solna,
Stockholm, Sweden
3
Cancer Research Center and Department of Immunology, Semnan
University of Medical Sciences, Semnan, Iran
4
Cancer Centre Karolinska, Department of Oncology, Karolinska
University Hospital Solna, SE-171 76 Stockholm, Sweden
activation of signalling pathways in CLL during early stages
of the disease, while DVL1 and 3 may have a role in later
phases of the leukaemia.
Keywords CLL . DVL . ROR1 . Wnt
Introduction
Chronic lymphocytic leukaemia (CLL) is characterized by the
accumulation of mature B cells with the phenotype CD19+/
CD5+/CD23+, which have an increased proliferative activity
and a defect in apoptotic machinery [1]. The clinical course is
highly variable. Advanced clinical stage and high-risk molecular prognostic characteristics (e.g. cytogenetic abnormalities
such as p53 abnormalities or complex karyotype, unmutated
IgVH, expression ZAP70 and CD38) are associated with disease progression and short survival [2].
Recent studies have demonstrated overexpression of the
receptor tyrosine kinase-like orphan receptor ROR1 in CLL
[3–5]. ROR1 is a transmembrane protein of the receptor tyrosine kinase (RTK) family and related to the Trk–RTK, musclespecific kinase (MuSK) and neurotrophic tyrosine kinase
(NTRTK) receptor families [6]. ROR1 is normally expressed
during the embryonic development and plays a key role in
skeletal, lung and neural organogenesis, but is repressed in
adult tissues [7]. ROR1 is of importance for cell proliferation,
survival, differentiation, metabolism and polarity [8] and for
tumour-like behaviour as migration and invasiveness [9]. In
haematological malignancies, ROR1 is upregulated in e.g.
chronic lymphocytic leukaemia [3–5], acute lymphocytic leukaemia [10] and mantle cell lymphoma [11].
Proteins of the wingless-type MMTV integration site family (Wnt) have been suggested to be ligands for ROR receptors
[8, 12]. Wnt signalling pathways play pivotal roles during
11948
embryogenesis, but are progressively turned off in differentiated cells [13]. Wnt signalling pathways seem to be stepwise involved in tumourigenesis [14]. Multiple Wnts
(Wnt-3, Wnt-5b, Wnt-6, Wnt-14, Wnt-16) and Frizzled
(Fzd) receptors (Fzd-3) as well as the lymphoid enhancerbinding factor 1 (LEF1) messenger RNAs (mRNAs) have
been shown to be elevated in CLL cells compared to normal B cells [15].
The Wnt signalling cascade constitutes two major pathways: the β-catenin-dependent (canonical) and -independent
(non-canonical) pathways. In the β-catenin-dependent signalling, Wnt ligands bind to Fzd receptors in the presence of the
low-density lipoprotein receptor-related protein 6 (LRP6),
inhibiting β-catenin degradation by glycogen synthase kinase
3β (GSK-3β) through dishevelled proteins (DVL1, 2, 3). βcatenin then remains stable, accumulates in the cytoplasm and
translocates to the nucleus, where β-catenin activates the target genes through interaction with the transcription factors Tcell factor (TCF) and LEF1 respectively. Accumulation of βcatenin by inhibiting GSK-3β or LEF1 increased the survival
of CLL cells in vitro, whereas inhibition of Wnt signalling
induced apoptosis of CLL cells, indicating that this pathway
might play a role in the survival of CLL cells [16].
DVLs are upstream mediators of both Wnt signalling pathways [17]. DVLs were originally identified based on the phenotype of disorientation in the body and wing hairs of
Drosophila [18]. Three DVL homologues (DVL1, 2, 3) have
been identified in humans and mice. The proteins consist of
about 750 amino acids and display a high sequence homology.
Genes of DVL1, DVL2 and DVL3 are expressed early during
mouse embryogenesis [19]. DVL knockout mice (DVL2−/−
and DVL3−/−) died prenatally [20, 21].
Wnt/Fzd interactions activate several signalling pathways
[22]. In the β-catenin-dependent pathway, DVLs act as
scaffold proteins bridging together the receptor and downstream signalling molecules [23]. Phosphorylated LRP5/6
and activated DVLs are important for recruiting axin to
the cell membrane and to the cytosolic stabilization of βcatenin [16]. In the β-catenin-independent pathway, DVLs
play a role in the downstream signalling of planar cell
polarity (PCP) as well as the Wnt/calcium pathway [24].
Recruitment of DVLs induced activation of both ROCK
(Rho-associated protein kinase) and protein kinase C
(PKC), which triggered the activation of Ca2+-calmodulindependent calcineurin and CAMKII (Ca2+-calmodulin-dependent kinase II) signalling [25].
ROR1 has been shown to be of importance for the survival
of CLL cells [4, 5, 26]. Assuming that DVLs play a role in the
downstream signalling of ROR1-associated Wnt pathways,
we addressed the question of whether DVLs might be involved in the survival of CLL cells. The protein expression
pattern of DVL1, 2 and 3 was studied in progressive and nonprogressive CLL patients and CLL cell lines.
Tumor Biol. (2016) 37:11947–11957
Materials and methods
Patients and controls
Eighteen CLL patients (nine with progressive and nine with
non-progressive disease) (Table 1) and nine healthy agematched donors were included. Approval by the regional
ethics committee (www.epn.se) was obtained as well as oral
and written informed consent from the donors in accordance
with the Helsinki Declaration. The diagnosis of CLL was
established as previously described [27]. Progressive (P) disease was defined as progression during the preceding 3 months
in anaemia (haemoglobin <100 g/l), thrombocytopenia
(<100 × 109) and size of spleen/liver/lymph nodes or in more
than twice the lymphocyte count. Patients that did not meet
these criteria were considered to have non-progressive (NP)
disease [28], in which all patients were untreated before the
bloods were taken. FISH analyses and p53 mutation tests of
CLL cells were done at the Department of Clinical Genetics at
the Karolinska University Hospital, Stockholm (Table 1).
Peripheral blood mononuclear cells (PBMCs) were isolated
using Ficoll-Hypaque (GE Healthcare, Uppsala, Sweden)
density centrifugation according to manufacturer’s instructions. B cells were purified from healthy donors using a magnetic bead cell separation system (MiniMACS) (Miltenyi
Biotec, Bergisch Gladbach, Germany). Briefly, PBMCs were
isolated by Ficoll-Hypaque. B cells were enriched using
immunomagnetic MACS beads coated with anti-CD19 according to manufacturer’s instructions. B cell purity was
>95 % (flow-cytometry).
Cell lines
The CLL-derived cell lines, EHEB, CII, I83-E95 and 232-B4,
were used as well as the Lukas (Burkitt lymphoma-derived)
and the Jurkat (T cell lymphoma) cell lines. All cell lines were
obtained from the American Type Culture Collection and cultured in RPMI 1640 (Invitrogen, CA, USA) with 10 % heatinactivated FBS (Invitrogen) and penicillin/streptomycin at
37 °C in humidified air with 5 % CO2.
Western blot
PBMCs were lysed in buffer containing 1 % Triton X-100,
150 mM NaCl, 50 mM Tris–HCl, 5 mM EDTA, 1 % protease
inhibitor cocktail (Sigma-Aldrich Corp., Saint Louis, MO,
USA) and phosphatase inhibitors (Roche Ltd, Basel,
Switzerland) on ice for 30 min and centrifuged at 13,
000 rpm. Supernatants were collected and protein concentration measured by Thermo Scientific BCA Protein Assay Kit
(Thermo Scientific, Rockford, IL, USA). About 15 μg of the
lysate was loaded onto 10 % Bis-Tris SDS-PAGE gel
(Invitrogen, Carlsbad, CA, USA) and run at 135 V for 2 h.
Tumor Biol. (2016) 37:11947–11957
Table 1 Clinical characteristics
of patients
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Hb (g/l)
PLC
(× 109/l)
Gender
Age
Karyotype
Rai
stage
No.
Disease
progression
WBC
(× 109/l)
CLL1
P
353.6
53
68
M
45
13q
III
CLL2
CLL3
P
P
46.4
38.5
81
73
79
75
M
M
76
78
Trisomy 12
13q−, 11q−
III
III
CLL4
P
184
59
55
F
71
13q−
I
CLL5
CLL6
P
P
16
157
68
81
51
72
M
F
63
71
Normal
ND
I
III
CLL7
P
390
61
88
M
77
13q−
III
CLL8
CLL9
P
P
60
88.4
92
75
50
57
M
M
77
69
13q
Normal
I
0
CLL10
NP
43
139
185
M
65
ND
I
CLL11
NP
20
161
172
M
83
ND
0
CLL12
CLL13
NP
NP
13
100
125
155
220
159
M
F
65
72
Trisomy 12
ND
II
0
CLL14
CLL15
NP
NP
33.4
150
119
129
211
189
M
F
80
48
ND
ND
III
I
CLL16
CLL17
CLL18
NP
NP
NP
50
350
95
141
134
157
191
149
234
F
M
M
71
73
50
ND
Trisomy 12
13q
I
II
0
P progressive disease, NP non-progressive, ND not done, M male, F female, Hb hemoglobin, PLC platlet
concetration
Protein was transferred to immobilon-PVDF membranes
(Millipore Corporation, Billerica, MA, USA) and blotted at
35 V for 1.5 h in transblot cell (Invitrogen) at room temperature. Membranes were blocked in 5 % bovine serum albumin
(BSA) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in
TBS with 0.1 % Tween 20 (TBS-T) at room temperature for
2 h. Membranes were probed with the respective primary
antibodies overnight at 4 °C and washed five times for
10 min in TBS-T at room temperature and then with the secondary antibody conjugated with peroxidase (Dako
Cytomation, Glostrup, Denmark) for 1.5 h in 5 % non-fat milk
in 0.1 % TBS-T at room temperature. The membranes were
washed five times and developed using the ECL chemiluminescence detection system (GE Healthcare, Uppsala,
Sweden). The following antibodies were used: DVL2,
DVL3, pPKC, PKC, pan pAKT, AKT, ERK, pGSK-3α,
GSK-3α, pan pGSK-3β, GSK-3β and β-catenin (Cell
Signaling Technology, Danvers, MA, USA), DVL1, DVL2
and DVL3 (Santa Cruz Biotechnology) as well as antiphospho-tyrosine mAb (clone 4G10) and anti-phosphoserine mAb (clone 4A4) (Millipore Corporation, Bedford,
MA, USA). For loading control, membranes were probed
for β-actin (Sigma-Aldrich Corp., Saint Louis, MO, USA).
Immunoprecipitation
Immunoprecipitation (IP) was done with DVL1, DVL2 and
DVL3 antibodies respectively using Dynabeads protein A and
G (Invitrogen Dynal, Oslo, Norway) according to the manufacturer’s instructions. Immunoprecipitated proteins were subjected to blotting under reducing conditions. Membranes were
probed with anti-phospho-tyrosine and anti-phospho-serine
antibodies (Millipore).
Dephosphorylation of protein
We have previously shown that our anti-CRD ROR1 mouse
monoclonal antibody could induce dephosphorylation of
ROR1 [29] as well as of ROR1 associated downstream signalling molecules as PI3K/AKT/mTOR/CREB [29]. PBMCs
from CLL patients were treated with the anti-ROR1 antibody
(CRD-1D8) (IgG2A) (10 μg/ml) in Aim V medium (Life
Technologies, Carlsbad, CA, USA) at 37 °C in humidified
air with 5 % CO2. After 30 min of incubation, cells were lysed
in a buffer containing 1 % Triton X-100, 150 mM NaCl,
50 mM Tris–HCl, 5 mM EDTA, 1 % protease inhibitor cocktail (Sigma-Aldrich Corp., Saint Louis, MO) and phosphatase
inhibitors (Roche Ltd, Basel, Switzerland) on ice for 30 min
and centrifuged at 13,000 rpm. Lysed cells were collected and
Western blot was performed. A rabbit polyclonal pRor-1 antibody against the phosphorylated TK domain of ROR1 was
used to detect phosphorylated ROR1 [30].
Lysates were also immunoprecipitated using DVL2 and
DVL3 antibodies and subjected to immunoblotting under reducing conditions. The membranes were probed with antiphospho-tyrosine and anti-phospho-serine antibodies
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(Millipore). Immunoprecipitation of DVL1 probed with anti
phospho-tyrosine and anti-phospho-serine antibodies in the
dephosphorylation experiment could technically not be performed (data not shown).
Tumor Biol. (2016) 37:11947–11957
After incubation, 20 μl of MTT reagent (5 mg/ml) was added.
The plate was left for 4 h at 37 °C. The reactions were stopped
with MTT solvent (10 % SDS in 0.01 N HCl). Optical density
(OD) was read in a Multiskan FC microplate reader (Thermo
Scientific) at 595 nm.
Quantification of total and phospho-proteins
Statistics
The level of phosphorylation was measured by analysing the
intensity of phosphorylated proteins in relation to the respective total protein using the ImageJ 1.44p software (National
Institutes of Health, USA). The expression of nonphosphorylated proteins was related to β-actin [29].
siRNA transfection
Gene silencing of DVL1, 2 and 3 in EHEB cells and PBMCs
from CLL patients was done using Accell smart pool small interfering RNA (siRNA; mixture of 4 siRNA duplexes; Thermo
Fisher Scientific, Pittsburgh, PA, USA) against DVL1 (cat. no. E004068-00), DVL2 (cat. no. E-004069-00), DVL3 (cat. no. E004070-00), GAPDH (cat. no. D-001930-01-20) and nontargeting siRNA (cat. no. D-001910-01-20). In siRNA delivery
medium, 1 × 106 EHEB cells or 106 CLL PBMCs/ml were
transfected with 1 μM siRNA (Accell; Thermo Fisher
Scientific) for 48 h at 37 °C. After incubation, expression of
DVL genes was determined by qPCR analysis. siRNA
transfected cells were also used in the MTT assay (see below).
qPCR analysis
Total RNA (2 μg) was used to synthesize single-strand complementary DNA (cDNA) by the Superscript II reverse transcriptase system and Oligo(dT)12–18 according to the manufacturer’s instructions (Life Technologies). TaqMan gene expression assays were performed by the FAM dye labelling
system according to the manufacturer’s instructions using
Hs00182896_m1 (DVL1), Hs00182901_m1 (DVL2),
Hs00610263_m1 (DVL3) and the endogenous control gene
G A P D H ( H s 0 2 7 5 8 9 9 1 _ g 1 ) ( L i f e Te c h n o l o g i e s ) .
Quantitative PCR (qPCR) was performed in a total volume
of 10 μl in a 384-well plate thermal cycler ABI 7900 (Life
Technologies), and the relative expression of genes (CT
values) was determined.
Statistical analysis was done by using GraphPad Prism version
5 (GraphPad Software, Inc., La Jolla, CA, USA). Statistical
calculation of normalized intensity values for each protein
was done by one-way analysis of variance (ANOVA) test
followed by post-hoc Tukey’s honest significant difference test
(HSD). The cut-off for significance was set to p < 0.05.
Results
Expression of DVL proteins
DVL1, DVL2 and DVL3 were highly expressed in CLL cells,
but not detectable in B cells of healthy donors (Fig. 1a, b). The
relative expression of DVL1 and DVL3 was significantly
higher in progressive compared to non-progressive disease
(p < 0.01), while DVL2 was higher expressed in nonprogressive compared to progressive disease (p < 0.001). The
CLL cell lines EHEB, CII, I83-E95 and 232-B4 as well as the
Lukas and Jurkat cell lines expressed DVL2 and DVL3, while
DVL1 was only weakly expressed (data not shown).
Phosphorylation of DVL proteins
DVL1, DVL2 and DVL3 proteins were immunoprecipitated
using the respective antibodies and immunoblotted for
phospho-tyrosine and phospho-serine. DVL1, DVL2 and
DVL3 were phosphorylated at both serine and tyrosine residues (Fig. 2). Incubation of CLL cells with an anti-ROR1
monoclonal antibody directed against the CRD region induced apoptosis of CLL cells preceded by dephosphorylation
of ROR1 [30]. The anti-ROR1 antibody in addition to dephosphorylating ROR1 also dephosphorylated tyrosine and serine
residues of DVL2 and DVL3 (Fig. 3).
Expression of GSK-3β and β-catenin
MTT assay
siRNA-treated EHEB cells were analysed by the colorimetric
MTT (3-[[4,5]-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide) assay (Sigma-Aldrich). The MTT assay is a cell
proliferation/apoptosis assay. Briefly, EHEB cells were cultured in 96-well flat-bottomed tissue culture plates (104
cells/well) for 48 h under standard culture conditions with or
without siRNA (DVL1, DVL2, DVL3 and pool of DVLs).
GSK-3β and β-catenin are key proteins in the Wnt canonical
pathway. Phosphorylated GSK-3β was detected in CLL cells
of all patients as well as in PBMCs of healthy donors (Fig. 4a).
Phosphorylation intensity of GSK-3β was significantly higher
in progressive than in non-progressive disease (p < 0.01) as
well as compared to healthy controls (p < 0.001) (Fig. 4b).
The expression of β-catenin varied between donors
(Fig. 4c), but in general, the expression intensity was
Tumor Biol. (2016) 37:11947–11957
11951
on
on
on
tro
tro
tro
l3
l2
,B
,B
lls
lls
kDa
ce
ce
P
P
P
lls
,N
,N
,N
,P
,P
,P
ce
22
21
20
19
18
17
,B
LL
LL
LL
LL
LL
LL
l1
C
C
C
C
C
C
A
C
C
C
Fig. 1 a Representative Western
blots of DVL1, DVL2 and DVL3
in CLL cells of progressive (P)
and non-progressive (NP) patients
as well as B cells of healthy
control donors (control 1, 2, 3 B
cells). b Relative expression
(mean ± SEM) of DVL proteins
compared to β-actin. Significance
levels are shown at the top.
*p < 0.05, **p < 0.01 and
***p < 0.001
DVL-1
85
DVL-2
95
DVL-3
93
Actin
B
DVL/B Actin
1
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
***
***
**
***
***
***
***
P
(n=9)
NP
(n=9)
DVL-1
significantly higher in CLL patients compared to healthy donors and more pronounced in non-progressive compared to
progressive disease (Fig. 4d).
contr
(n=9)
P
(n=9)
NP
(n=9)
IP DVL1
IP DVL2
kDa
CLL 16, NP
CLL 9, P
kDa
pTyr
85
pTyr
95
pSer
85
pSer
95
DVL1
85
DVL2
95
IP DVL3
CLL 16, NP
CLL 9, P
kDa
pTyr
93
pSer
93
DVL3
93
Fig. 2 Immunoprecipitated (IP) DVL1, DVL2 and DVL3 probed with
phospho-tyrosine (ptyr) and phospho-serine (pser) antibodies in
progressive (P) and non-progressive (NP) CLL patients and as well as
healthy control donors (contr). Direct blotting for DVL1, DVL2 and
DVL3 is shown at the bottom lanes. One representative experiment out
of three
P
(n=9)
NP
(n=9)
contr
(n=9)
DVL-3
patients or in comparison to control donors. However, phosphorylation of AKT was significantly higher in CLL patients
with non-progressive compared to progressive disease
(p < 0.01) (Fig. 4i, j).
-
PKC, AKT and GSK-3α are regulators of the Wnt noncanonical (β-catenin-independent) pathway. Phosphorylation
of PKC (Fig. 4e, f) and GSK-3α (Fig. 4g, h) varied, and there
was no statistically significant difference between CLL
CLL 9, P
contr
(n=9)
DVL-2
Expression of PKC, AKT and GSK-3α
CLL 16, NP
***
**
+
kDa
ROR1
130
pROR1
130
DVL2
95
DVL2, p-tyrosine
95
DVL2, p-serine
95
DVL3
93
DVL3, p-tyrosine
93
DVL3, p-serine
93
Fig. 3 CLL cells were incubated with an anti-ROR1 CRD Mab (+) and
an isotype control Mab (-) for 30 min and probed for p (phosphorylated)
ROR1. Immunoprecipitated DVL2 and DVL3 were probed for phosphotyrosine and phospho-serine. One representative experiment out of three
11952
r,
nt
Co
r,
nt
Co
5
6
7
pGSK-3β
46
tGSK-3β
46
B
Relative expression of
pGSK-3β/tGSK-3β
r,
nt
Co
kDa
P
P
,N
17
L
CL
,N
16
L
CL
P
,N
15
L
CL
,P
L8
CL
,P
L7
CL
P
6,
L
CL
A
Tumor Biol. (2016) 37:11947–11957
**
1.2
1
0.8
0.6
0.4
0.2
0
P
r,
nt
Co
r,
nt
Co
6
7
8
D
92
β-Catenin
β-Actin
42
Relative expression of
β-Catenin/β-Actin
r,
nt
Co
P
P
,N
17
L
CL
,N
16
L
CL
P
,N
15
L
CL
,P
L3
CL
,P
L2
CL
P
1,
L
CL
C
2
**
1.5
1
0.5
0
P
(n=9)
7
8
F
pPKC
78
tPKC
78
Relative expression of
pPKC/tPKC
r,
nt
Co
NP
NP
r,
nt
Co
4,
3,
6
L1
L1
NP
r,
nt
Co
CL
CL
2,
L1
CL
,P
L8
CL
,P
L7
CL
,P
L6
CL
E
r,
nt
Co
5
6
7
tGSK-α
51
1.4
1.2
tAKT
60
Relative expression of
pKT/tAKT
NP
NP
NP
r, 6
nt
Co
r, 5
nt
Co
r, 4
nt
Co
7,
6,
L1
CL
5,
L1
CL
L1
CL
,P
L3
CL
,P
L2
CL
,P
L1
CL
60
Contr
0.2
P
(n=9)
(n=9)
1
0.8
0.6
0.4
0.2
P
(n=9)
pAKT
NP
0.4
H
J
Contr
0.6
0
I
NP
(n=9)
0.8
Relative expression of
pGSK-3α/tGSK-3α
r,
nt
Co
NP
NP
NP
r,
nt
Co
6,
5,
L1
CL
L1
CL
4,
L1
CL
,P
,P
L4
CL
L3
CL
,P
L2
CL
51
Contr
1
(n=9)
pGSK-α
NP
(n=9)
1.2
0
G
(n=9)
(n=9)
*
2.5
Contr
NP
(n=9)
**
3
(n=9)
(n=9)
**
* **
2.5
2
1.5
1
0.5
0
P
(n=9)
NP
(n=9)
Contr
(n=9)
Tumor Biol. (2016) 37:11947–11957
11953
ƒFig. 4
Western blots and column bar graphs showing protein expression
in progressive (P) and non-progressive (NP) CLL patients as well as in
healthy control donors (contr). a Western blots showing total (t) GSK-3β
and pGSK-3β, b intensity of pGSK-3β relative to tGSK-3β, c Western
blots for β-catenin and d intensity of β-catenin in relation to β-actin. e
Western blots showing total (t) PKC and phosphorylated (p) PKC and f
intensity of pPKC relative to tPKC. g Western blots showing total (t)
GSK-3α and phosphorylated (p) GSK-3α, h intensity of pGSK-3α
relative to tGSK-3α, i Western blots showing total (t) AKT and
phosphorylated (p) AKT and j intensity of pAKT relative to tAKT.
Intensity values are shown as mean ± SEM. Significance levels are
shown at the top. *p < 0.05, **p < 0.01 and ***p < 0.001
Silencing of DVL in the CLL-derived cell line EHEB
The apoptotic effect of DVL gene silencing was analysed
using the CLL-derived EHEB cell line. siRNA transfection
with DVL1, DVL2 and DVL3 was successful (Fig. 5), but no
effect on cell survival was noted (data not shown).
Discussion
DVL1, DVL2 and DVL3 proteins were highly expressed in
CLL cells compared to normal PBMCs. The expression of
DVL1 and DVL3 was more pronounced in progressive than
in non-progressive disease, whereas the level of DVL2 was
higher in non-progressive compared to progressive CLL. All
three DVLs were expressed in the four CLL cell lines as well
as in the Jurkat and Lukas cell lines. DVL1, 2 and 3 proteins
were phosphorylated at both serine and tyrosine residues.
There are two major Wnt-signalling pathways in mammalians: the canonical and non-canonical pathways. The canonical Wnt signalling pathway has been shown to be activated in
embryonic and adult stem cells, but is progressively turned off
in differentiated cells. However, in mature cells the canonical
Wnt signalling pathway is activated during tumourigenesis
and of importance for tumour cell proliferation and migration
29
3
,2,
L1
NA
DV
DV
L2
NT
28
NA
28
25
siR
siR
40
siR
26
40
Un
t
Ct Value
30
40
Un
t
Un
t
3
,2,
L1
L1
DV
siR
NA
NA
siR
31
31
45
40
35
30
25
20
15
10
5
0
NT
siR
NA
DV
siR
L2
NA
DV
L1
,2,
3
40
40
27
DV
27
DVL2 Expression
NA NT
siR
DV
NA
L1
DV
L1
,2,
3
40
NT
45
40
35
30
25
20
15
10
5
0
Un
t
Ct Value
DVL1 Expression
DVL3 Expression
45
40
40
40
36
Ct Value
35
30
25
24
27
24
28
28
20
15
10
5
Fig. 5 Gene expression levels of DVL1, DVL2 and DVL3 in the EHEB
cell line (filled columns) and PBMCs of a CLL patient (open columns)
after 48 h of transfection with siRNA DVL1, siRNA DVL2, siRNA
DVL3, pool siRNA (DVL1, 2 and 3), non-target siRNA (NT) and
NT
siR
NA
siR
DV
NA
L3
DV
L1
,2,
3
Un
t
,3
1,2
L3
DV
L
DV
NA
siR
siR
NA
NT
Un
t
0
untransfected (Unt) cells as control. Gene expression was calculated in
CT values. The lower the CT values, the higher the expression. Gene
expression was always higher in control cells than in siRNA-transfected
cells
11954
Tumor Biol. (2016) 37:11947–11957
Normal B cells
Leukemic B cells in
non-progressive CLL
Leukemic B cells in
progressive CLL
Ror-1
Ror-1
DVL1
DVL2
DVL3
Axin
APC
DVL1
DVL2
DVL3
Axin
Axin
GSK3-ß
GSK3-ß
APC
GSK3-ß
Wnt/PCP pathway
AKT
Wnt/PCP pathway
APC
AKT
ß-catenin
ß-catenin
Degradation
Nucleus
Accumulation
TCF
Nucleus
ß-catenin
Accumulation
LEF-1
Survival
Proliferation
Migration
TCF
Nucleus
LEF-1
Survival
Proliferation
Migration
Fig. 6 In the absence of ROR1 and DVLs in normal B cells, the
destruction complex comprising APC, axin-1 and GSK-3β controls the
cellular level of beta-catenin by ubiquitination of β-catenin. In nonprogressive CLL, phosphorylated ROR1 contributes to phosphorylation
of the highly expressed DVL2 as well as AKT, which inactivates GSK3β and subsequently disrupts the destruction complex. β-catenin
accumulates in the nucleus, regulating gene expression by binding to
the TCF/LEF-1 DNA-binding proteins and promoting preferentially cell
survival. In contrast to progressive CLL, phosphorylated ROR1 activates
DVL1 and 3, disrupting the destruction complex and activating the Wnt/
PCP pathway, promoting preferentially proliferation and migration of
CLL cells [49, 50]. Bold marks indicate a relatively higher expression
of phosphorylated proteins and signalling activity comparing nonprogressive and progressive CLL
[31]. Activation is induced by binding of a Wnt ligand to a
Frizzled receptor inducing phosphorylation of DVLs [32, 33],
which may activate downstream signalling proteins of significance for survival and proliferation [23]. In our study silencing of DVLs of the EHEB cell line (a CLL-derived cell line)
and PBMCs of CLL patients did, however, not induce apoptosis, which may suggest that these proteins might not be of
major importance for survival of CLL cells.
We could also show the expression of other activated proteins of importance both for the canonical and non-canonical
Ror-1/Wnt signalling pathways in CLL. Inhibition of ROR1
dephosphorylated DVL2 and 3 at both Ser/Thr and tyrosine
residues. Grumolato et al. [34] showed that the canonical
Wnt3a and non-canonical Wnt5a ligands specifically triggered the completely unrelated endogenous coreceptors
LRP5/6 and ROR1/2 respectively through a common mechanism that involved their Wnt-dependent coupling binding to
the Frizzled (Fzd) coreceptor and recruitment of shared components including dishevelled (DVL), axin and glycogen synthase kinase 3 (GSK3) proteins. Furthermore, AKT seems to
be one of the key players in the non-canonical Wnt pathway
[35]. Activation may promote survival of CLL cells [36–39].
The present study also showed that AKT was activated in both
progressive and non-progressive disease compared to healthy
donors. Surprisingly, patients with non-progressive disease
showed a significantly higher phosphorylation level of
AKT as compared to patients with progressive disease,
but phosphorylated PKC upstream of AKT did not differ
significantly between CLL patients and healthy donors.
The results may be consistent with those of others suggesting that Wnt5a triggers AKT phosphorylation via
PI3K, but not via ERK or PKC [40].
ROR1, Fzd receptors, multiple Wnts and LEF1 mRNAs
have been found to be increased in CLL cells compared to
healthy B cells [41]. Binding of Wnt ligands to Fzd receptors
in the presence of the LRP6 coreceptor inhibited degradation
of β-catenin by GSK-3β [35]. Accumulation of β-catenin in
the cytoplasm and subsequent translocation to the nucleus
may activate target genes through interaction with the transcription factors TCF and LEF1 regulating numerous target
genes involved in cell cycle progression, adhesion and
differentiation.
The non-canonical Wnt signalling may also be activated by
ROR1 and/or other Frizzled receptors activating downstream
signalling proteins, regulating e.g. cytoskeleton formation and
cell adhesion. One of the downstream signalling pathways is
PCP. Cellular polarization is essential for the function of many
tissues. PCP signalling consists of two major groups of
Tumor Biol. (2016) 37:11947–11957
proteins: the core PCP and the global module[42]. The core
PCP genes encode functions of importance for molecular
asymmetry within and between cells. These proteins are required for organization of multicellular structures, tissue remodelling, cellular polarization and movements. The global
module includes the atypical cadherins, Ft and Ds, which interact heterophilically across membranes, as well as the Golgi
protein four-jointed (Fj) modulating affinity by phosphorylation. It is not known whether the Ft/Ds module has a role
upstream of the core PCP module or represents an independent system [43]. Many proteins downstream of PCP as the
Rho family of GTPases or the JNK/p38 MAPK module are
important effector molecules involved in e.g. adhesion and
migration and thus are of significance in tumour invasion
and metastasis [44].
Deregulated PCP signalling is implicated in tumour metastasis. A recent study in non-small cell lung cancer (NSCLC)
(dishevelled-1 and dishevelled-3 affect cell invasion mainly
through canonical and non-canonical Wnt pathway respectively and associate with poor prognosis in non-small cell lung
cancer [45]) showed that the expression of DVL3 was significantly higher in nodal metastases than in the primary tumours. Correlation between DVL3 and β-catenin was noted
neither in primary tumours nor in metastases.
DVLs were not expressed in normal leucocytes. AKT was
slightly phosphorylated as well as GSK-3β, which may regulate ubiquitination of β-catenin. In CLL, cell DVL2 was
expressed. Based on the current and previous studies, we suggest a model for the interactions between DVLs, AKT, GSK3β and β-catenin in CLL (Fig. 6). In non-progressive CLL,
AKT and DVL2 were highly phosphorylated, which may prevent GSK-3β to be phosphorylated. Inhibition of activation of
GSK-3β by phosphorylated AKT and DVL2 may promote the
accumulation of β-catenin and activation of TCF/LEFmediated transcription of genes, which might preferentially
prolong survival of CLL cells [46, 47]. DVL1 and DVL3 were
highly phosphorylated in progressive CLL, while AKT was
slightly phosphorylated, which might prevent GSK-3β to be
fully phosphorylated preferentially to support proliferation
and migration. This is in agreement with Kaucka et al. [48].
Upregulation of the Wnt/PCP pathway has been shown to be
significantly phosphorylated in progressive as compared to
non-progressive CLL patients. Activation of both the canonical and non-canonical pathways in progressive CLL may not
only prolong cell survival but also support proliferation and
migration [49, 50]. The reason for the differential expression
of DVL1, 2 and 3 in non-progressive and progressive is not
clear [51].
In conclusion, DVL proteins were highly overexpressed in
CLL patients. Non-progressive CLL had a different activity
pattern of the Wnt signalling pathways compared to progressive disease. The different DVL proteins’ expression pattern
might contribute to the altered signalling pathway profile
11955
during tumour progression. However, future studies are needed to understand the mechanism for and relevance of differential DVL expression and the association with Wnt signalling in the pathobiology of CLL.
Acknowledgments This study was supported by grants from CLL
Global Research Foundation, the Cancer and Allergy Foundation
(149351, 149746, 150288), the Swedish Research Council (K201364X-21464-04-3), the Swedish Cancer Society (CAN 2009/852), the
Cancer Society in Stockholm (121332), the King Gustav Vth Jubilee
Fund (124272) and the Stockholm County Council (20120051). The
secretarial help from Leila Relander is highly appreciated.
Authors’ contributions ASK and AM designed the study, performed
experiments, interpreted data and wrote the manuscript; MHF performed
experiments and read the manuscript; AHDM read the manuscript; HM,
AÖ and LH provided clinical samples and all read the manuscript; and
HM supervised the study.
Compliance with ethical standards Approval by the regional ethics
committee (www.epn.se) was obtained as well as oral and written
informed consent from the donors in accordance with the Helsinki
Declaration.
Conflicts of interest None
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