Published OnlineFirst January 21, 2013; DOI: 10.1158/0008-5472.CAN-12-1752
Cancer
Research
Microenvironment and Immunology
The Planar Cell Polarity Pathway Drives Pathogenesis of
Chronic Lymphocytic Leukemia by the Regulation of
B-Lymphocyte Migration
ta Kaucka
1, Karla Plevova
2,4, Ša
rka Pavlova
2,4, Pavlína Janovska
1, Archana Mishra1, Jan Verner2,4,
Marke
1
1,5,6
2,4
í
, Petra Ovesna
3, Boris Tich
4,
zkova
, Pavel Krejc
, Jana Kotaškova
y2,4, Yvona Brychtova
Jirina Procha
2,4
1,5
2,4
2,4
1,5
rka Pospíšilova
, and Víte
zslav Bryja
Michael Doubek , Alois Kozubík , Jirí Mayer , Ša
Abstract
The planar cell polarity (PCP) pathway is a conserved pathway that regulates cell migration and polarity in
various contexts. Here we show that key PCP pathway components such as Vangl2, Celsr1, Prickle1, FZD3, FZD7,
Dvl2, Dvl3, and casein kinase 1 (CK1)-e are upregulated in B lymphocytes of patients with chronic lymphocytic
leukemia (CLL). Elevated levels of PCP proteins accumulate in advanced stages of the disease. Here, we show that
PCP pathway is required for the migration and transendothelial invasion of CLL cells and that patients with high
expression of PCP genes, FZD3, FZD7, and PRICKLE1, have a less favorable clinical prognosis. Our findings
establish that the PCP pathway acts as an important regulator of CLL cell migration and invasion. PCP proteins
represent an important class of molecules regulating pathogenic interaction of CLL cells with their microenvironment. Cancer Res; 73(5); 1491–501. 2012 AACR.
Introduction
Wnt signaling pathways are crucial for cell-to-cell communication, differentiation, and morphogenesis in embryonic
development. Dysfunction or deregulation of Wnt signaling
accounts for a number of developmental defects, inherited
diseases, and many types of cancer (1). The best-known Wnt
pathway—canonical or Wnt/b-catenin pathway—induces
b-catenin–dependent activation of T-cell factor/lymphoid
enhancer factor (TCF/LEF)-mediated transcription. However,
Wnt ligands can activate other so-called noncanonical Wnt
pathways, which are b-catenin–independent and biochemically distinct from the canonical Wnt pathway (2). Among
those, the pathway that regulates planar cell polarity (Wnt/
PCP pathway) is the best known (3). The core components of
the Wnt/PCP pathway (referred here as PCP or polarity
Authors' Affiliations: 1Institute of Experimental Biology, Faculty of Science, 2CEITEC - Central European Institute of Technology, 3Institute of
Biostatistics and Analyses, Masaryk University; 4Center of Molecular
Biology and Gene Therapy, Department of Internal Medicine–Hematology
and Oncology, University Hospital Brno and Medical Faculty MU; 5Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the
Czech Republic, Brno, Czech Republic; and 6Medical Genetics Institute,
Cedars-Sinai Medical Center, Los Angeles, California
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
and Š. Pavlova
contributed equally to this work.
K. Plevova
Corresponding Author: Vitezslav Bryja, Institute of Biophysics v.v.i.,
Academy of Sciences of the Czech Republic, Kralovopolska 135, Brno
CZ-612 65, Czech Republic. Phone: 420-549493291; Fax: 420541211214; E-mail: [email protected]
doi: 10.1158/0008-5472.CAN-12-1752
2012 American Association for Cancer Research.
proteins) are conserved in evolution and include Vang-like
protein 2 (Vangl2), cadherin EGF LAG 7-pass transmembrane
G-type receptor (Celsr1), Frizzleds (FZD, in mammals mainly
FZD3, FZD6, and FZD7), which act as receptors, and cytoplasmic components such as Disheveled (Dvl1-3 in mammals),
Prickle-like proteins (mainly Prickle1), casein kinase 1e (CK1e),
and the small GTPases Rho/Rac and downstream kinases
ROCK/JNK. PCP proteins regulate cell polarity (3), convergent
extension movements during gastrulation and neurulation,
and polarity of the hair cells in the inner ear (3, 4).
In the present study, we investigated the role of PCP proteins
in chronic lymphocytic leukemia (CLL). CLL is characterized
by clonal expansion and apoptosis dysregulation of CD5þ B
lymphocytes. Neoplastic CLL cells accumulate in blood, bone
marrow, and lymphoid tissue. It is recognized that cell migration and recirculation between peripheral blood and lymphoid
niches in bone marrow and lymphoid tissue are important
factors contributing to CLL biology (5–7) and that alterations
in survival and proliferation of CLL are affected by their
interaction with the microenvironment. Here, we show for the
first time that the core cassette of the PCP pathway composed
of Celsr1, Prickle1, Vangl2, Dvl2, Dvl3, FZD3, and FZD7 is
upregulated in B cells of patients with CLLs. We show that
PCP proteins, which are conserved regulators of the cell–cell
interaction and cell migration, are important mediators of CLL
chemotactic responses and homing.
Materials and Methods
Cell isolation, purification, and cell treatments
Primary B cells from previously untreated patients with
CLLs or healthy volunteers were separated using gradient
centrifugation followed by non–B-cell depletion (RosetteSep
B Cell Enrichment Kit and Human CD3þ Depletion Kit;
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StemCell Technologies; or MACS B cell Isolation Kit II; Miltenyi
Biotec) according to the manufacturer's instructions. Flow
cytometry was conducted for evaluation of CD5 and CD19
expression on purified cells (tricolor panel: CD45-TRI-COLOR,
MHCD45065, Invitrogen, CD5-FITC, A08932, CD19-PE, A07769,
Beckman Coulter). For further analyses, samples with the
purity higher than 95% B cells were used. The cell line MEC1
was obtained from DSMZ, cell line was tested by DSMZ using
molecular genetic methods; MEC1 cells were cultured in RPMI1640 supplemented with 10% FBS and antibiotics at 37o C and
5% CO2.
Transwell assay
The chemotaxis assay was conducted in HTS Transwell-96
well plates (Corning Incorporated) with 5.0-mm pore size
polycarbonate membranes following the manufacturer's
instructions. A total of 0.5 106 cells (primary CLL or MEC1
cells) were seeded in the Transwell upper insert, which was
either nontreated or coated with a human umbilical vein
endothelial cells monolayer (HUVEC). Cells in the insert were
treated with antibodies or inhibitors as described in Supplementary Material. Cells were incubated overnight in full medium (including 10% fetal calf serum) at 37o C and 5% CO2, and
after 18 hours the migration toward the chemokine was
analyzed by a Coulter Counter (model FN, Coulter
Electronics; Figs. 2A–C, 3A–D, and 4A–C; Supplementary Figs.
S2B and S4E–S4H) or by C6 flow cytometer (Accuri; Figs. 2E–
G, 3E–G, and 4D; Supplementary Fig. S2E). The migration index
was calculated as the number of cells (treated or untreated)
migrating in response to the chemokine divided by the number
of cells migrating toward the control medium only. In case of
very variable migration indexes (in primary CLL cells), cell
numbers were normalized to the chemokine-only condition
and expressed as "relative migration."
In vivo experiments
Nonirradiated mice (8- to 16-week-old) nonobese diabetic/
severe combined immunodeficient (NOD/SCID) IL2Rg-null
(NSG) mice were used for transplantation experiments. Freshly
isolated human CLL lymphocytes (25 106 per condition)
were pretreated as indicated for 20 hours and stained with
Calcein AM. Mice underwent transplantation by intraperitoneal injection of 20 106 of pretreated and calcein AM–stained
human CLL lymphocytes in 120 mL of sterile PBS. Recipient
mice were analyzed 24 hours after the injection by fluorescence-activated cell-sorting (FACS) analysis (for details, see
Supplementary Material).
Further methods including information on patient samples,
quantitative real-time (qRT)-PCR, Western blotting, flow cytometry, nucleofection, and statistics are provided in the Supplementary Material.
Results
Core PCP pathway components are upregulated in CLL
patients
Our earlier microarray expression analysis (8) suggested that
components of Wnt/PCP signaling are frequently upregulated
in CLLs and expressed differently in prognostically distinct
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subsets of patients with CLLs. To confirm that the levels of
PCP components are indeed altered in CLLs, we collected
RNA samples from purified peripheral blood B cells (CD19þ
cell population purity 95%) of control individuals (n ¼ 6)
and patients with CLL (n ¼ 93). qRT-PCR analysis showed
statistically increased mRNA levels of 3 PCP genes, CELSR1,
PRICKLE1, and FZD7, in CLL samples and identified that 2
other genes VANGL2 and FZD3 have high levels in a subset
of patients with CLLs [Fig. 1A—normalized to the expression
of b2-microglobulin; Supplementary Fig. S1A—normalized to
b-actin].
Subsequent analysis of the protein level by Western blotting
confirmed increased protein expression of the PCP proteins
Vangl2, Dvl2, Dvl3, Prickle1, and CK1e (Fig. 1B, quantified
in Fig. 1C) in patients with CLLs. The level of b-catenin did
not differ between CLLs and healthy cells (Fig. 1B), and we
failed to detect activated (dephosphorylated) b-catenin (not
shown), which suggest that the Wnt/b-catenin pathway is
not active in CLL cells. All CLL samples tested had high expression of LEF1 and Ror1 (Fig. 1B), which were previously
identified as abundantly and consistently expressed in CLL
cells (9, 10). Flow cytometry detection (Fig. 1D) further confirmed increased cell surface levels of Ror1, FZD3, Celsr1, and
Vangl2 in comparison to healthy B cells.
Next, we asked whether the levels of PCP proteins are
stable or they change during the disease course. When we
compared samples of 6 patients with CLLs collected at 2
different time points (t1 and t2; Fig. 1E), we were able to
detect changes in the levels of PCP proteins in CLL cells from
individual time points. Specifically, we observed a clear
increase in Dvl2, Dvl3, and Prickle1 levels in patients followed in time, which is quantified in Supplementary Fig.
S1B. The most prominent changes were observed mainly in
patients, who progressed from Rai stages (11) 0/I/II to the
stage III or IV (see patients C, H, P). The levels of LEF1 and
b-catenin do not change (Fig. 1E).
In summary, using several methods, we provide evidence
that a host of PCP proteins are overexpressed in B cells of
patients with CLLs, with some accumulating to a greater extent
in advanced stages of the disease.
Wnt5a, a PCP ligand, increases migration of primary CLL
cells in a chemokine gradient via a Wnt/PCP pathway
It is becoming increasingly evident that the role of microenvironment in the pathogenesis of CLLs is of a crucial
importance (7, 12). Chemokines and their receptors, mainly
CXCL12-CXCR4, CXCL9/10/11-CXCR3, and CCL19/21-CCR7,
have been shown to mediate invasiveness and transendothelial
migration of CLL cells (13–16). In that line, it has been recently
shown that Wnt-5a, which is the major ligand for Ror1/2
receptors and a crucial component of the Wnt/PCP pathway
(17–19), promotes cell polarization and directional movement
of melanoma cells and T cells in a chemokine (CXCL12)
gradient (20, 21).
We thus hypothesized that Wnt5a, which is also expressed in
CLL cells (Supplementary Fig. S2A) together with receptor PCP
protein complexes, may contribute to the pathogenesis of CLLs
by the effects on the polarized migration of CLL cells toward
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PCP Proteins Drive Pathogenesis of CLL
Figure 1. PCP genes and proteins are upregulated in patients with CLL. A, expression of the indicated genes from CD19þ cells of healthy individuals
(control; n ¼ 6) and patients with CLLs (n ¼ 93) was analyzed by qRT-PCR. Data represent the fold difference relative to the reference gene 2DCt b2microglobulin. B, the amounts of the indicated PCP proteins were analyzed by Western blotting in B cells of healthy controls and patients with CLLs. One
lane represents one individual. C, densitometric quantification of data in B. D, the cell surface expression of the PCP proteins Ror1, FZD3, Celsr1,
and Vangl2 in CD19þ B cells of healthy individual and patients with CLLs were analyzed by flow cytometry. E, Western blotting of Dvl2, Dvl3, Prickle1,
LEF1, Ror1, and b-catenin in B cells from 6 patients, where each patient has been taken and analyzed at 2 different time points (t1 and t2). b-Actin served
as the loading control. The Rai clinical stages of each patient at t1 and t2, and the time between t1 and t2 are indicated. Mean timespan between t1 and t2 is 1,308
days. , P < 0.001; , P < 0.05. n.s., not significant (Student t test).
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Figure 2. PCP proteins regulate
migration of primary CLL cells in a
chemokine gradient. A, migration
of primary CLL cells (expressed as
migration index) in the presence of
CXCL12, Wnt5a, and CXCL12/
Wnt5a was analyzed using
Transwell migration chambers
(n ¼ 6). The observed increase is
dependent on the activity of CK1
(n ¼ 3; B) and on presence of the
Ror1 cell surface receptor (aRor1,
Ror1-blocking antibody; n ¼ 4; C).
Both CK1 inhibition and Ror1
blocking antibody clearly interfere
with Wnt/PCP pathway as shown
by their ability to reduce the Wntinduced mobility shift of Dvl3 (full
arrowhead, open arrowhead—
non-shifted Dvl3; D). E–G, the
effects of the inhibitors of the
Rho-associated kinase (Y27632;
E), Rac1 (F), and porcupine
(IWP-2; G) on the chemotaxis
driven by Wnt5a/CXCL12 were
tested by Transwell assays. Values
represent mean SEM. Statistical
differences were tested by oneway ANOVA and Tukey post hoc
test ( , P < 0.05; , P < 0.01;
, P < 0.001). n.s., not significant.
chemokines. To test this prediction, we studied primary cells
from patients with CLLs for their ability to respond to CXCL12
in the presence and absence of recombinant Wnt5a (200
ng/mL) using Transwell migration chambers (16, 22). As we
show in Fig. 2A, CXCL12 alone increased migration of CLL cells,
whereas Wnt5a alone had no effect. In combination with
CXCL12, Wnt5a significantly increased migration of CLLs cells
compared with all other experimental conditions. A similar
effect was seen for CXCL10, CXCL11, and CCL21 but not for
CXCL9 and CCL19 (Supplementary Fig. S2B). Importantly, the
Wnt5a-induced synergism was dependent on the activity of
CK1, which phosphorylates a key PCP protein Dvl and reg-
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ulates its activity in the PCP pathway (19, 23–25). Treatment
with the CK1 inhibitor D4476 (CK1 inh. I; Fig. 2B; ref. 26)
significantly reduced the positive effects of Wnt5a on the
chemotaxis toward CXCL12. To exclude that these effects are
due to the activation of the Wnt/b-catenin pathway, we tested
in primary CLL cells the ability of Wnts to activate sensitive
TCF/LEF (TopFlash) luciferase reporter (27) and analyzed the
expression of bona fide Wnt/b-catenin target genes AXIN2 and
DKK1 by quantitative PCR. As we show in Supplementary Fig.
S2C and S2D, no experimental treatments, including treatment
with Wnt3a, a typical ligand of the Wnt/b-catenin pathway,
trigger the expression from TCF/LEF-responsive promoters.
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PCP Proteins Drive Pathogenesis of CLL
Figure 3. Regulation of MEC1 cell migration by PCP proteins. A, migration of MEC1 cells was analyzed in presence of CXCL12, CCL19, and CCL21 (200 ng/mL)
with or without Wnt5a using Transwell migration assays. Wnt5a does not further promote migration driven by CXCL12, CCL19, and CCL21. B–G,
MEC1 migration triggered by CCL21 and/or CCL19 chemokines is blocked by CK1 inhibitor D4476 (CK1 inh. I; 100 mmol/L; B), PF670462 (CK1 inh. II;
50 mmol/L; C), knockdown of Dvl2, the efficiency of knockdown was determined by Western blot analysis and quantified by densitometry (Dvl2/actin; D),
inhibition of the Rho-associated kinase (Y27632; E), and the inhibitor of endogenous Wnt production (inhibitor of porcupine IWP-2; G). The inhibitor of
Rac1 (F) does not have an effect. Values represent mean SEM. Statistical differences were tested by one-way ANOVA and Tukey post hoc test ( , P < 0.05;
, P < 0.01; , P < 0.001; n.s., not significant). Each experiment has been carried out in at least 3 independent replicates.
This strongly suggests that Wnt/b-catenin pathway is not
involved in the observed phenotypes.
The effects similar to the inhibition of CK1 have been
observed, when we blocked the putative Wnt5a receptor in
the PCP complex, Ror1, with the goat Ror1 blocking antibody
(aRor1) but not with the control goat IgG (Fig. 2C). The
efficiency of this antibody, which binds Ror1 and promotes
its internalization, has been shown earlier (28). Both D4476
(CK1 inh. I) and aRor1 antibody clearly inhibited activation of
PCP pathway in primary CLL cells as shown by their ability to
block phosphorylation-dependent shift of endogenous Dvl3
(PS-Dvl3), which is a hallmark of Dvl3 activation in the noncanonical Wnt pathway (Fig. 2D; ref. 19).
In the Wnt/PCP pathway, Dvl mediates the activation of
small GTPases RhoA and Rac1, which trigger subsequent
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cytoskeletal rearrangements and cell migration. As we show
in Fig. 2E, the inhibition of RhoA-driven signaling by Y27632,
an inhibitor of Rho-associated ROCK protein kinase (29) but
not the inhibition of Rac1 by a Rac1 inhibitor NSC23766
(Fig. 2F; ref. 30), blocked the migration of primary CLL cells.
No inhibitors used in this section affect the viability of primary CLL cells, as assessed by WST1 test, and the effects on
chemotaxis thus do not reflect differences in the condition of
CLL cells (Supplementary Fig. S3).
The experiments above suggest that PCP pathway is
capable to promote and is required for the efficient chemotactic migration of primary CLL cells. To further elaborate
on the mechanism of the interaction between Wnt/PCP and
the chemokine signaling, we tested functionally the role of
the CXCR4 (the receptor for CXCL12) and the role of
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Figure 4. Regulation of transendothelial migration of CLL cells by PCP pathway. A–D, transendothelial invasion of primary human CLL lymphocytes and MEC1
cells was tested in the Transwell assays coated with the HUVEC endothelial cell monolayer and is indicated as the migration index. The level of
invasion of human primary CLL lymphocytes (A) was analyzed in the presence of chemokine CCL19, Wnt5a, or a combination of CCL19 and Wnt5a. Wnt5a
strongly promotes CCL19-induced invasion of CLL cells through the endothelial monolayer. B–D, the positive effect of CCL19 on transendothelial
migration of MEC1 cells is fully blocked by the addition of the D4476 (CK1 inh. I; B). siRNA-mediated knockdown of Dvl2 (C) and the inhibition of
Rho-associated kinase (Y27632) and endogenous Wnt production (IWP-2; D). Statistical differences were tested by one-way ANOVA and Tukey post hoc
test ( , P < 0.05; , P < 0.01; , P < 0.001). Each experiment has been carried out in at least 3 independent replicates.
endogenous Wnts in CLL chemotaxis. Interestingly, CXCR4blocking antibody was unable to inhibit CLL migration
(Supplementary Fig. S2E), whereas the disruption of the
endogenous Wnt production by the porcupine inhibitor
IWP-2 (31) reduced CLL migration (Fig. 2G). Given the fact
that CXCL12-CXCR4 signaling does not regulate Wnt-5a
expression and Wnts do not regulate expression of CXCR4
in CLL cells (see Supplementary Fig. S2F), we conclude that
(i) the effects do not take place at the level of transcription,
(ii) that the autocrine stimulation by Wnts contributes to the
CLL chemotaxis. and (iii) that the chemotactic response is
not critically mediated by CXCR4.
PCP protein Dvl and its kinase CK1« are required for the
migration in the chemokine gradient
To study the role of Wnt/PCP pathway in CLL migration in
more detail and to overcome the natural variability among
patients and limitations in the experimental manipulation
with primary CLL cells, we carried out further experiments
in MEC1 cells (32). MEC1 is a well-defined cell line derived from
a patients with CLLs, which recapitulates many aspects of the
CLL biology and is used as the transplantation model of CLL
(33). Quantitative analysis of mRNA (WNT5A, CELSR1, PRICKLE1, FZD3, FZD7; Supplementary Fig. S4A) and protein levels
(Ror1, Vangl2, Dvl2, Dvl3, CK1e; Supplementary Fig. S4B)
showed that MEC1 cells express most PCP genes in high levels,
although the levels of Ror1 were relatively low (Supplementary
Fig. S4B).
MEC1 cells were unable to respond to CXCL12 but
responded clearly to CCL19 and CCL21 in a dose-dependent
manner (Fig. 3A; Supplementary Fig. S4C). Interestingly,
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treatment with Wnt5a did not further promote the promigratory effect of chemokines (Fig. 3A) and Wnt5a-blocking
antibody did not block the migration (Supplementary Fig.
S4E) despite the high endogenous expression of Wnt5a (see
Supplementary Fig. S4A and S4D) Inhibition of CK1e, a
kinase, which is required for PCP signaling due to its role
in the phosphorylation of Dvl, with 2 unrelated inhibitors
D4476 (CK1 inh. I) and PF670462 (CK1 inh. II; 50 mmol/L;
ref. 34) blocked the chemotactic response of MEC1 cells
toward CCL21 and CCL19 (Fig. 3B and C). It can be expected
that CK1 blocks CLL migration via phosphorylation of the
key PCP protein Dvl. Indeed, even partial Dvl2 knockdown
efficiently reduced the motility of MEC1 cells (Fig. 3D).
Similarly to primary CLL cells, even in MEC1, the chemotaxis
was blocked by the inhibitors of Rho-associated kinase
ROCK (Fig. 3E) and by the inhibitor of Wnt secretion
IWP-2 (Fig. 3F) but not by the inhibitor of Rac1 (Fig. 3G).
These data suggest that endogenous Wnts (but not uniquely
Wnt5a), Dvl2, and likely the whole PCP machinery activating
Rho and ROCK kinase is required for the polarized migration
of CLL cells.
PCP proteins regulate chemokine-driven
transendothelial invasion of CLL cells
In the human body, cells behave in a complex environment
and have to pass via extracellular matrix or endothelial barriers, which is a process known as cell invasion. We have tested
the role of PCP proteins in the invasion of CLL cells by the
analysis of their invasion through a layer of HUVEC. In this
experimental system, Wnt5a significantly promoted transendothelial migration of primary patient CLL cells in a gradient of
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PCP Proteins Drive Pathogenesis of CLL
CCL19 (Fig. 4A), to a lesser extent in the gradient of CXCL12
(Supplementary Fig. S4F) but showed no effect in the gradient
of CCL21 (Supplementary Fig. S4G). This suggests that cooperation between Wnt/PCP and chemokine signaling is not
limited to chemotaxis but takes place also in the process of
transendothelial invasion.
To check whether invasion is controlled by PCP signaling,
specifically via CK1e-Dvl2-Rho pathway (as shown in Fig. 3 for
chemotaxis), we have carried out another set of experiments in
MEC1 cells. O the 3 tested chemokines, only CCL19 was able to
induce transendothelial migration of MEC1 cells (Supplementary Fig. S4H). This response has not been promoted by Wnt5a
but has been almost completely abolished by CK1 inhibition
(Fig. 4B). Similar to the basal chemotaxis, the downregulation
of the key PCP protein Dvl2 by siRNA (Fig. 4C) as well as
inhibition of ROCK kinase (by Y27632) and porcupine (by IWP2; Fig. 4D) efficiently reduced also transendothelial invasion of
MEC1 cells. In summary, these results show that PCP proteins
mediate both migration/chemotaxis and chemokine-driven
invasion of CLL cells.
PCP proteins regulate CLL homing in vivo
To test the role of PCP proteins in CLL cells in vivo, we
transplanted primary CLL cells into NSG mice (Fig. 5A),
which lack mature T cells, B cells, and functional natural
killer cells (35). Primary CLL cells efficiently home in NSG
mice and can be detected mainly in spleen, liver, and bone
marrow. Interestingly, pretreatment with the casein kinase 1
inhibitor D4476 decreased homing to spleen, liver, and bone
marrow without any effect on the apoptosis of the cells (Fig.
5B and C; for typical dot plots for apoptosis assay, see
Supplementary Fig. S5A). This experiment suggests that
inhibition of CK1 diminishes the ability of CLL cells to
migrate and colonize murine tissues. Interference with the
Ror1- and FZD7-driven signaling by treatment with antibodies directed against Ror1 and FZD7 (for validation of
anti-FZD7 antibody, see Supplementary Fig. S5B) did not
affect the survival of CLL cells either (Fig. 5D). Importantly,
treatment with the blocking anti-Ror1 antibody (28) efficiently eliminated homing of CLL cells from most patients to
the spleen (Fig. 5E). The homing to bone marrow could not
be determined because treatment with any antibody (even
control IgG) for unknown reasons completely abolished
migration to bone marrow in vivo (data not shown). Liver
infiltration was diminished only for some patients (Fig. 5F).
Treatment with the anti-FZD7 antibody did not show any
effect on the CLL cells in liver (Fig. 5F); however, decreased
spleen infiltration by CLL cells occurred in a subset of
patients (Fig. 5E). Interestingly, the patient subset responding to anti-FZD7 antibody had mutated p53, a strong marker
of aggressive disease, whereas the patients not responding to
anti-FZD7 antibody treatment had wild-type (wt) p53.
In summary, these results show that CK1 activity and the
cell surface receptors Ror1 and FZD7 regulate biologic
properties of CLL cells in vivo. These observations are
consistent with the in vitro results and further strengthen
the conclusion that PCP proteins act as regulators of CLL
biology and pathogenesis.
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PCP-high patient cohorts show worse clinical
characteristics
The expression as well as functional data suggested that PCP
proteins are strongly associated with CLL cell behavior. In the
next step, we analyze the clinical significance of PCP protein
expression by analysis of samples from 93 previously untreated
patients (identical to patients analyzed in Fig. 1A). The normalized expression of FZD3, FZD7, CELSR1, VANGL2, and
PRICKLE1 assessed by qRT-PCR (see also Fig. 1A) was correlated to the clinical parameters.
Importantly, the increased expression of FZD3, FZD7,
VANGL2, and PRICKLE1 correlated positively with unmutated
immunoglobulin heavy chain (IGHV; Table 1), which is strongly associated with worse prognosis. In contrast, patients with
unfavorable chromosomal aberrations del11q22-23 (ATM) or
del17p13 (TP53) did not show significantly higher expression
of PCP genes. However, it should be noted that the number
of patients with del11q22-23 (n ¼ 17; 18%) or del17p13 (n ¼ 7;
7.5%) was rather low and insufficient for robust statistical
analysis.
Next, we analyzed the association of PCP gene expression
with the treatment-free survival (TFS) using Kaplan–Meier
survival curves. Patients were sorted into 2 groups (high vs.
low) based on the mRNA levels (as determined by qRT-PCR)
of individual PCP genes (FZD3, FZD7, CELSR1, VANGL2, and
PRICKLE1). The cutoff for each gene was set up as the value
corresponding to 75% percentile for the expression levels in
normal B cells from the periphery (see Fig. 1A and below).
TFS analysis was conducted in both groups of patients.
Importantly, FZD3-, FZD7-, and PRICKLE1-high patient
cohorts had significantly (Breslow test, P < 0.05) worse
prognosis than the group of patients with low expression
of these genes (Fig. 6). Specifically, the median TFS of FZD3low (cutoff 1.2) patients was 40 months compared with 17
months in FZD3-high group, 54 versus 25 months for FZD7
low versus high (cutoff 2.0), and 54 versus 31 months for
PRICKLE1-low versus PRICKLE1-high (cutoff 1.0) patient
cohorts. Very similar data were obtained when we conducted the analysis with the data normalized to b-actin
(Supplementary Fig. S6).
These findings suggest that mainly more aggressive CLL
cells with unmutated IGHV use PCP pathway to regulate their
chemotaxis toward chemokines. Indeed, when we looked at
unmutated IGHV patient subset only, we have not observed
any statistically significant difference in survival curves
between PCP-high and -low patient cohorts (data not shown).
In summary, the expression analyses in patients with CLLs
showed that (i) the expression of key PCP genes correlates with
clinically important mutational status of IGHV and (ii) on the
clinical sample show the relevance of processes controlled by
PCP pathway for disease pathogenesis.
Discussion
In the present study we, for the first time, show that (i) core
PCP components such as Vangl2, Prickle1, CK1e, Dvl, and
Celsr1 are upregulated in CLLs, (ii) PCP pathway components
help regulate chemotaxis and transendothelial migration in
the chemokine gradient and in vivo homing of CLL cells, and
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Figure 5. PCP proteins affect engraftment of CLL cells in vivo. A, scheme of the experiment. Typical FACS dot blot is shown (see gate G1 for transplanted cells.).
B, the level of apoptosis in primary CLL cells after dimethyl sulfoxide (DMSO)/D4476 (CK1 inh. I) was determined by flow cytometric analysis of the
mitochondrial membrane potential using tetramethylrhodamine ethyl ester dye in combination with the green calcein staining. C, proportion of CLL cells (number of
positive cells/number of total cells in the organ) treated with DMSO or D4476 (CK1 inh. I) that were recovered from the spleen (Ci), liver (Cii), and bone marrow
(Ciii) of transplanted mice. D, treatment with the aRor1 and aFZD7 antibodies does not induce apoptosis as determined by tetramethylrhodamine ethyl ester
analysis. E and F, proportion of CLL cells, when treated with a nonspecific antibody (IgG) or antibodies directed against Ror1 and FZD7, were recovered from the
mouse spleen (E) or liver (F). Please note that samples responding to aFZD7 come from patients with mutated p53 (p53 MUT). B and D, graphs show mean SEM.
C and E, one symbol on the graph represents one patient. , P < 0.05, Wilcoxon paired t test (C) or one-way ANOVA and Tukey post hoc test (E).
(iii) PCP-high patient cohorts show worse clinical parameters
such as TFS. Our findings suggest that PCP proteins affect CLL
pathogenesis via regulation of chemokine-driven migration
and as such influence disease progression.
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Our data suggest that PCP pathway contributes to the
CLL pathogenesis mainly via regulation of chemotactic
responses to chemokines, which are the leading mediators
of the interaction between CLL cells and their microenvi-
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Published OnlineFirst January 21, 2013; DOI: 10.1158/0008-5472.CAN-12-1752
PCP Proteins Drive Pathogenesis of CLL
Table 1. The expression of PCP genes determined by qRT-PCR in the patient cohorts with mutated (IGHV
MUT) and unmutated (IGHV UNMUT) immunoglobulin heavy chain
Gene name
IGHV MUT
(n ¼ 39)
IGHV UNMUT
(n ¼ 54)
P (Mann–Whitney test)
FZD7
FZD3
VANGL2
PRICKLE1
2.697 (0.9081)
0.7684 (0.07373)
4.937 (2.137)
1.942 (0.2322)
3.674 (0.6834)
1.217 (0.1130)
11.12 ( 6.119)
2.978 (0.2950)
0.0075
0.0054
0.007
0.0113
NOTE: Numbers indicate the mean, SEM; n, number of patients; P, statistical significance of the difference between both cohorts
determined by Mann-Whitney test.
ronment (for review, see ref. 36). Components of the PCP
machinery thus become an interesting target for the novel
therapies, which interfere with the communication between
CLL cells and their environment, similarly to the inhibitors
of chemokine receptor or B-cell receptor (BCR) signaling
pathways (37–39). In agreement with these data, our observations show that PCP-controlled migration is mainly relevant for the CLL cells with unmutated IGHV, which were
shown to be more dependent on chemokine and BCR
signaling.
Our data provide to our best knowledge (i) the first
evidence for the increased levels of the conserved proteins
unique for the PCP pathway—Prickle1, Vangl2, Celsr1—in
leukemia and (ii) the first implication for the role of Celsr1 in
human cancer. PCP signaling has been, however, functionally implicated earlier in the metastatic process of various
solid tumors with the best defined role in melanoma, breast,
and gastric cancer (40–42). It has been convincingly shown,
mainly in melanoma, that PCP pathway increases the capacity of tumor cells to migrate and invade into surrounding
tissue (43). Our data support the possibility that PCP pathway mediates physical aspects of cell migration via its effects
on cytoskeleton and that chemokine signaling can serve as
the navigator of invading cells into their final destination.
Interesting prediction of this hypothesis is that PCP pathway
is dynamically and temporarily used by migrating immune
Figure 6. Expression of PCP genes defines CLL progression. Patients were sorted into 2 groups (high vs. low expression; cutoff indicated by the line in
the upper graph) based on the mRNA levels of individual PCP genes (FZD3, FZD7, and PRICKLE1), and Kaplan–Meier survival curves (TFS) were
plotted and survival analysis was conducted in both groups of patients. PRICKLE1- (A), FZD3- (B), and FZD7-high (C) patient cohorts had a significantly
(Breslow test, P < 0.05) worse prognosis than the group of patients with low expression of these genes.
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Kaucka
cells during the immune response. This interesting topic
clearly exceeds the current study and has to be tested in
future.
In summary, our findings provide strong evidence that core
components of the Wnt/PCP pathway play an important role
in CLL pathogenesis via regulation of CLL migration. We for
the first time show the biologic importance of the crosstalk
between chemokine and PCP signaling, which might have
implication for understanding of the biology of invasiveness
in other tumors. Given the fact that Wnt/PCP pathway is an
evolutionary conserved regulator of cell polarity and migration, our data further emphasize the role of cell migration in the
development of CLLs.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: M. Kaucka, P. Krejcí, Š. Pospíšlova, V. Bryja
Development of methodology: M. Kaucka, P. Krejcí
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): M. Kaucka, K. Plevova, S. Pavlova, P. Janovska,
A. Mishra, J. Verner, J. Prochazkova, J. Kotaškova, B. Tichy , Y. Brychtova,
M. Doubek, J. Mayer
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): M. Kaucka, S. Pavlova, A. Mishra, J. Prochazkova,
P. Krejcí, J. Kotaškova, P. Ovesna, B. Tichy , V. Bryja
Writing, review, and/or revision of the manuscript: M. Kaucka, K. Plevova,
S. Pavlova, A. Mishra, A. Kozubík, J. Mayer, Š. Pospíšlova, V. Bryja
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K. Plevova, J. Kotaškova, A. Kozubík,
J. Mayer
Study supervision: P. Krejcí, Š. Pospíšlova, V. Bryja
Grant Support
This work was supported by grants from the Czech Science Foundation (204/
09/H058, 301/11/0747), Ministry of Health of the Czech Republic (NT11217-5/
2010, NT 13493-4/2012), Ministry of Education, Youth and Sports of the Czech
Republic (MSM0021622430), Masaryk University Rector's programme to support
MU student's creative work (MUNI/E/0128/2009, MUNI/A/0784/2011), European Regional Development Fund (CZ.1.07/2.3.00/20.0180, CZ.1.05/1.1.00/02.0068,
CZ.1.07/2.3.00/20.0045), and by an EMBO Installation Grant. P. Krejcí is supported by the grants Kontakt LH12004 (Ministry of Education) and P305/11/0752
(Czech Science Foundation).
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received May 7, 2012; revised November 29, 2012; accepted December 12, 2012;
published OnlineFirst January 21, 2013.
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The Planar Cell Polarity Pathway Drives Pathogenesis of Chronic
Lymphocytic Leukemia by the Regulation of B-Lymphocyte
Migration
Markéta Kaucká, Karla Plevová, Sárka Pavlová, et al.
Cancer Res 2013;73:1491-1501. Published OnlineFirst January 21, 2013.
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