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2007;67:2469-2479. Cancer Res
Susmita Esufali, George S. Charames, Vaijayanti V. Pethe, et al.
Decreased Adhesion of Colorectal Cancer Cells
Dishevelled Promotes Canonical Wnt Signaling and
Activation of Tumor-Specific Splice Variant Rac1b by
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Activation of Tumor-Specific Splice Variant Rac1b by Dishevelled
Promotes Canonical Wnt Signaling and Decreased Adhesion of
Colorectal Cancer Cells
Susmita Esufali,1,2,3 George S. Charames,1,2,3 Vaijayanti V. Pethe,1,2 Pinella Buongiorno,1,2,3
and Bharati Bapat1,2,3
1Samuel Lunenfeld Research Institute and 2Department of Pathology and Laboratory Medicine, Mount Sinai Hospital; 3Department of
Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
Abstract
Rac1b is a tumor-specific splice variant of the Rac1 GTPase
that displays limited functional similarities to Rac1. We have
shown previously a novel cross-talk between Rac1 and B-
catenin, which induces canonical Wnt pathway activation in
colorectal cancer cells. This prompted us to investigate if
Rac1b, frequently overexpressed in colon tumors, contributes
to Wnt pathway dysregulation. We show that Rac1b over-
expression stimulates Tcf-mediated gene transcription, where-
as depletion of Rac1b results in decreased expression of the
Wnt target gene cyclin D1. Reconstitution experiments
revealed an important difference between Rac1 and Rac1b
such that Rac1b was capable of functionally interacting
with Dishevelled-3 (Dvl-3) but not B-catenin to mediate
synergistic induction of Wnt target genes. In agreement, Dvl-
3 but not B-catenin caused increased activation of Rac1b
levels, which may explain the functional cooperativity dis-
played in transcription assays. Furthermore, we show that
Rac1b negatively regulates E-cadherin expression and results
in decreased adhesion of colorectal cancer cells. RNA
interference–mediated suppression of Rac1b resulted in
reduced expression of Slug, a specific transcriptional repres-
sor of E-cadherin, and a concomitant increase in E-cadherin
transcript levels was observed. Intriguingly, mutation of the
polybasic region of Rac1b resulted in complete loss of Rac1b
stimulatory effects on transcription and suppressive effects
on adhesion, indicating the importance of nuclear and
membrane localization of Rac1b. Our results suggest that
Rac1b overexpression may facilitate tumor progression by
enhancing Dvl-3–mediated Wnt pathway signaling and induc-
tion of Wnt target genes specifically involved in decreasing the
adhesive properties of colorectal cancer cells. [Cancer Res
2007;67(6):2469–79]
Introduction
Rac1 is one of the most extensively studied members of the Rho
family of small GTP binding proteins. It regulates diverse cellular
processes, which include actin cytoskeleton organization, mem-
brane trafficking, proliferation, and gene expression (1–4). Similar
to all members of the Ras superfamily proteins, the GTP binding/
GTP hydrolysis state of Rac1 is tightly controlled by guanine
nucleotide exchange factors (GEF) and GTPase-activating proteins
(GAP), respectively. An additional level of regulation exists for Rho
GTPases, whereby Rho-GDP dissociation inhibitors (Rho-GDI)
sequester GDP-bound forms in the cytoplasm, preventing their
activation. Exhaustive characterization of this GTPase has revealed
the pivotal role it plays in the genesis of many cancers. A recently
discovered splice variant of Rac1, designated as Rac1b, has also
received much attention for its plausible role in tumorigenesis of
colon and breast tissues. However, deciphering the functional
consequences of its expression and contribution to tumorigenesis
has been challenging because Rac1b shows limited similarities to
known Rac1 functions.
Overexpression of Rac1, as well as altered function of Rac1-
specific regulators (GEFs, GAPs, and GDIs) or downstream effec-
tors, have been found in several cancers (5). Studies using
activated point mutants of Rac1 (G12V or Q61L) indicate that
aberrant activation of Rac1 can alter many cellular processes
important for cancer progression. Rac1 is essential for cell cycle
progression and activates several pathways important for cellular
proliferation, such as serum response factor, cyclin D1, and E2F
(6, 7). Rac1 also promotes cell survival by activating the nuclear
factor-nB (NF-nB) pathway and by preventing anoikis and apop-
tosis (8–10). Rac1 can induce cellular transformation in rodent
fibroblast models and is required for Ras-induced transformation
(11–14). Rac1 also regulates cell-cell and cell-matrix adhesion as
well as stimulates motility and invasion by modulating the actin
cytoskeleton, activities important for tumor metastasis (15).
Rac1b is a naturally occurring splice variant, preferentially
expressed in colon and breast tumors (16, 17). It is created by
alternative splicing of an additional exon, resulting in a 19–amino
acid insertion between codon 75 and 76 of Rac1, directly C-
terminus to the switch II region. This structural modification
enables it to behave as a constitutively activated GTPase, largely
because of an accelerated GEF-independent guanine nucleotide
exchange rate, a decreased intrinsic GTPase activity, and an
inability to interact with Rho-GDI (18, 19). It has been postulated
that the insertion may create a novel effector binding site, which
may enable Rac1b to participate in signaling pathways related to
neoplastic growth (19). Most studies to date indicate impaired
effector signaling activity of Rac1b. Unlike Rac1, Rac1b does not
activate the protein kinases c-Jun NH2-terminal kinase (JNK) and
p21-activated kinase 1 and is not involved in lamellipodia
formation. However, similar to Rac1, Rac1b can promote growth
transformation of NIH3T3 cells as well as a loss of density-
dependent and anchorage-dependent growth and stimulates AKT
serine/threonine kinase, hence promoting cell survival (20).
Furthermore, a constitutively activated Q61L Rac1b mutant, but
Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
Requests for reprints: Bharati Bapat, Samuel Lunenfeld Research Institute, Mount
Sinai Hospital, Room L6-304B, 60 Murray Street, Toronto, Ontario, Canada M5T 3L9.
Phone: 416-586-4800, ext. 5175; Fax: 416-586-8869; E-mail: bapat@mshri.on.ca.
I2007 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-06-2843
www.aacrjournals.org 2469 Cancer Res 2007; 67: (6). March 15, 2007
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not wild-type (WT) Rac1b, can stimulate the transcription factor
NF-nB and cyclin D1 expression (19, 20). Rac1b has been shown to
induce epithelial-mesenchymal transition (EMT) in mouse mam-
mary carcinoma cells via reactive oxygen species–dependent
activation of the transcription factor Snail (21).
Although these studies implicate a role for Rac1b in cellular
transformation, downstream signaling pathways by which this
tumor-associated splice variant may contribute to colorectal
tumorigenesis remain to be identified. The canonical h-catenin–
mediated Wnt signaling pathway is aberrantly activated in the vast
majority of colorectal cancers (22, 23). A hallmark feature of Wnt
pathway activation is nuclear translocation of h-catenin, where it
complexes with Tcf4 and inappropriately activates transcription of
target genes important for tumorigenesis (24, 25). We have shown
previously that Rac1 can cooperate with h-catenin to augment
canonical Wnt pathway activation by promoting its nuclear
accumulation and amplifying its transcription coactivator function
(26). This prompted us to investigate if Rac1b, frequently overex-
pressed in colon tumors, contributes to Wnt pathway dysregula-
tion, which is characteristically activated in these tumors. We
describe that Rac1b is a downstream target of Dishevelled-3 (Dvl-3)
and amplifies Dvl-3–initiated Wnt pathway activation leading to
increased h-catenin/Tcf–dependent transcription. Furthermore, we
show that Rac1b negatively regulates E-cadherin expression and
results in decreased adhesion of colorectal cancer cells. Thus, our
results contribute to the understanding of Rac1b as a putative
accelerator of tumor progression by positively regulating the
expression of proliferation-promoting genes and decreasing the
adhesive properties of colorectal cancer cells.
Materials and Methods
Cell culture and transfection. HEK293, SW480, and MCF-7 cells were
maintained in DMEM. HCT116 and HT29 cells were cultured in McCoy’s 5A.
SW48 and MDA-MB-231 cells were cultured in Leibovitz’s L-15 medium. All
media were supplemented with 10% (v/v) fetal bovine serum (Invitrogen,
Carlsbad, CA) and cultured at 37jC in a humidified atmosphere of 5% CO
2
.
Cells were transfected as described previously (26). For endogenous gene
knockdown experiments, the following small interfering RNAs (siRNA) were
used: Rac1 siRNA was a SMARTpool reagent (Dharmacon, Lafayette, CO),
whereas Rac1b knockdown was achieved via an equal mixture of two
siRNAs against the target sequences 5¶-GAAACGUACGGUAAGGAUA-3¶and
5¶-GGCAAAGACAAGCCGAUUG-3¶. Gene expression data were normalized
against transfections with a siCONTROL nontargeting siRNA (Dharmacon).
Plasmids. Dvl-3 construct was purchased from American Type Culture
Collection (Manassas, VA). Activator protein (AP-1)–luc and serum
response element (SRE)–luc luciferase reporter constructs were purchased
from Stratagene (La Jolla, CA). To generate the Rac1b polybasic region
(PBR) mutants, site-directed mutagenesis was done using the QuikChange
Site-Directed Mutagenesis kit (Stratagene). Using a WT FLAG-tagged Rac1b
cDNA construct as template, the KKRKRK sequence at amino acids 202 to
207 of Rac1b was replaced sequentially by pairs of glutamines to create Q2,
Q4, and Q6 mutants as depicted in Fig. 4A.
Reverse transcription-PCR. cDNA synthesis was done using 5 Ag total
RNA, random primer p(dN
6
), and SuperScript II reverse transcriptase. Rac1
and Rac1b mRNA levels were analyzed using the following primers specific
for both Rac1 and Rac1b: 5¶-ATGCAGGCCATCAAGTGTGTG-3¶( forward)
and 5¶-CAACAGCAGGCATTTTCTCTT-3¶(reverse). The PCR conditions
used were as follows: 1 cycle at 94jC for 4 min; 35 cycles at 94jC for 30 s,
55jC for 45 s, and 72jC for 30 s; and 1 cycle at 72jC for 5 min. PCR products
were separated on 1.5% agarose gel stained with ethidium bromide.
Quantitative real-time reverse transcription-PCR. First-strand cDNA
was synthesized from 5 Ag RNA using SuperScript III reverse transcriptase
(Invitrogen). A mixture of 2SYBR Green (15 AL/well), 10 Amol/L of
forward and reverse primer (0.6 AL each/well), 1 AL cDNA, and DNase-free
water to bring the reaction to 30 AL was added to each well. Samples were
analyzed in triplicate and carried out in an Applied Biosystems 7500 Real-
time PCR System (Foster City, CA). Real-time results were collected and
analyzed (standard curve method) using the ABI 7500 System software
according to the manufacturer’s protocol. Expression values were
normalized with the b-actin gene expression values. Primer sequences
used to amplify cDNA are described in Supplementary Table S1.
Luciferase reporter gene assays. Cells were plated at a seeding density
of 2 10
5
per well of a 24-well dish 24 h before transfection. Luciferase
reporter constructs (0.1 Ag) were added to each well together with 0.03 Ag
pCMV h-galactosidase construct for normalization. For coexpression of
additional proteins, FLAG-tagged WT or PBR-mutated Rac1b, WT h-
catenin, or Dvl-3 constructs were added as indicated in figures. Cells were
harvested after 24 h in 100 AL reporter lysis buffer (Promega, Madison, WI).
Assays of luciferase and h-galactosidase activity were done as described
previously (26).
Cell fractionation, coimmunoprecipitation, and Western blotting.
Whole-cell lysates as well as cytoplasmic and nuclear extracts were
prepared and detected as described (26). Coimmunoprecipitation experi-
ments were done using both whole-cell lysates and fractionated cytoplasmic
and nuclear lysates. The antibodies used in this study were horseradish
peroxidase–conjugated anti-FLAG (Sigma, St. Louis, MO), monoclonal anti–
h-catenin and monoclonal anti-paxillin (Transduction Laboratories, BD
Biosciences, Franklin Lakes, NJ), monoclonal anti–topoisomerase II
(Oncogene Research Products, San Diego, CA), monoclonal Dvl-3 (Santa
Cruz Biotechnology, Santa Cruz, CA), and anti–h-actin (Ambion, Austin,
TX) as a loading control.
Active Rac pull-down experiments. Active GTP-bound Rac1 and
Rac1b levels were determined using a nonradioactive Rac1 activation
assay kit according to the manufacturer’s protocol (Pierce Biotechnology,
Rockford, IL). Following transfection, cells were washed in cold PBS and
lysed on ice in 600 AL lysis buffer. Total lysates were cleared by
centrifugation at 16,000 gfor 15 min, and 100 AL lysate was kept for
protein quantitation of total Rac1/Rac1b protein. The remaining lysate
was incubated for 30 min at 4jC with 20 Ag PAK binding domain (PBD)
agarose beads. Precipitated complexes were washed thrice with excess
lysis buffer. After the final wash, the supernatant was discarded and 40 AL
of 2Laemmli sample buffer were added to the beads. Total lysates and
precipitates were then analyzed by Western blot.
Fluorescence microscopy. HCT116 cells cultured on glass coverslips
were transfected with indicated constructs and harvested after 24 h by
fixing in formaldehyde (3.7%, 30 min, room temperature) and permeabiliz-
ing in Triton X-100 (0.2%, 10 min, room temperature). Cells were incubated
in blocking buffer (1% goat serum, 1 h, room temperature) followed by
incubation with appropriate primary and fluorescently labeled secondary
antibodies. FLAG-tagged constructs were detected with rabbit polyclonal
anti-FLAG antibody (Sigma) followed by Texas red–conjugated anti-
rabbit polyclonal antibody (Molecular Probes, Eugene, OR). Endogenous
E-cadherin was visualized using mouse monoclonal anti–E-cadherin
(Transduction Laboratories) and antimouse FITC-conjugated secondary
antibody (Molecular Probes). Actin was stained using an actin-phalloidin
conjugate (Molecular Probes). The level of nonspecific background
immunostaining was established by omitting primary antibodies. Coverslips
were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and
examined using the Olympus (Markham, Ontario, Canada) 1X-70 inverted
deconvolution microscope (100 lens magnification).
Adhesion assay. HCT116 colorectal cancer cells were seeded at a cell
density of 2 10
6
per 60-mm tissue culture dish the day before transfection.
Cells were transfected with indicated constructs, and DNA concentration
was held constant at 8 Ag per dish. Cells were harvested 24 h post-
transfection by trypsinization, resuspended in serum-free McCoy’s 5A
medium at cell density of 100,000 and 400,000 cells/mL, and used in the
Innocyte Adhesion Assay from Calbiochem (San Diego, CA). Briefly, 100 AL
of prepared cell suspension were added, in triplicate, to wells of 96-well
dish, coated with poly-L-lysine (general attachment) or bovine serum
albumin (negative control), and incubated 2 h at 37jC. Wells were gently
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washed twice with PBS to remove nonadherent cells and then incubated
with 100 AL alcein-AM green fluorescent dye solution for an additional 1 h at
37jC. Adherent cells were quantitated by measuring fluorescence of the
samples using a fluorescent plate reader at excitation wavelength 485 nm
and emission wavelength 520 nm.
Results
Rac1b induces transcriptional activation of Wnt-responsive
promoters in colorectal cancer cells. The splice variant Rac1b
exhibits selective expression, largely confined to colonic mucosa
and crypt epithelial cells, and is overexpressed specifically in colon
and breast tumors (16, 17). To assess the role of Rac1b in Wnt
pathway regulation, we first determined its endogenous expression
profile in a panel of colon cancer cell lines harboring aberrant Wnt
pathway activation. Using reverse transcription-PCR (RT-PCR)
analysis, we observed expression of the Rac1b transcript in SW480
and HT29 colorectal cancer cells as well as MCF-7 breast cancer
cells and very low levels in HCT116 and SW48 colorectal cancer
cells (Fig. 1A, top). Concordantly, cells expressing Rac1b transcript
also showed Rac1b protein expression (Fig. 1A, bottom). The level of
activation of Rac1b, assessed by PBD pull down-assay, was
comparable with Rac1 in SW480 and MCF-7 cells, although
examination of corresponding whole-cell lysates indicated far
more Rac1 versus Rac1b total protein in these cells. This
emphasizes that Rac1b is more efficiently activated than Rac1,
which agrees with previously published reports (19, 20).
Because Rac1b expression was minimally detected in HCT116
cells, they were used to evaluate the role of Rac1b overexpres-
sion in Wnt pathway regulation. A hallmark feature of canonical
Wnt pathway activation is h-catenin/Tcf–mediated transcription
of Wnt target genes. h-catenin/Tcf–dependent gene expression
was tested with the Wnt-responsive Tcf reporter TOPflash
together with the mutant reporter FOPflash as a negative
control. As shown in Fig. 1B(top), transient transfection of
Rac1b led up to a 7-fold activation of h-catenin/Tcf–mediated
transcription of TOPflash. Under similar conditions, WT Rac1
also stimulated TOPflash activity in HCT116 cells although much
less robustly compared with Rac1b, reaching only a maximal 2-
fold induction (Fig. 1B, bottom). To further elucidate the specific
contribution of Rac1b in Wnt pathway activation, we down-
regulated endogenous Rac1b in HT29 cells by transfection of
small interfering RNAs (siRNA) targeting the splice insertion
sequence within Rac1b. As shown in Fig. 1C(right), f70%
specific knockdown of Rac1b was achieved and did not affect
WT Rac1 transcript levels. We examined the effects of Rac1b
knockdown on the regulation of an endogenous Wnt target gene
Figure 1. Rac1b expression stimulates
transcription of Wnt-responsive promoter
TOPflash in HCT116 colorectal cancer
cells. A, top, endogenous Rac1b
expression was analyzed by RT-PCR in a
panel of human colorectal and breast
cancer cell lines: breast (MDA-MB-231 and
MCF-7) and colon (SW480, HT29,
HCT116, and SW48); bottom,
endogenous protein expression and
activation status of Rac1b was analyzed by
Western blotting of whole-cell lysates
(10 Ag) and PBD pull-down lysates,
respectively, in human embryonic kidney
HEK293 cells, HCT116 and SW480
colorectal cancer cells, and MCF-7 breast
cancer cells. B, pTOPflash (5)or
pFOPflash (n) reporter constructs were
transiently transfected with increasing
amount of Rac1b expression vector
(0.2, 0.4, and 0.9 Ag) or WT Rac1 (0.4 and
0.9 Ag) for 24 h in HCT116 cells.
Luciferase activity is expressed as total
relative light units (RLU ). Columns,
average of experiments carried out in
triplicate; bars, SE. Empty vector ()was
transfected to establish basal TOPflash/
FOPflash activity. C, quantification of
knockdown of endogenous Rac1b gene
expression. HT29 cells were transfected
with a scrambled control siRNA (negative
control), Rac1 siRNA (targets both Rac1
and Rac1b), or siRNA selectively targeting
Rac1b for 24 h and then subjected to
real-time RT-PCR to quantitate Rac1/
Rac1b gene expression. Data are triplicate
values of at least two independent
experiments. D, endogenous cyclin D1
transcript expression was analyzed
following specific Rac1b knockdown in
HT29 cells under serum-starved
conditions. Cells were transfected with
Rac1b siRNA and then changed to
low-serum conditions (0.1% fetal bovine
serum) 4 h later. RNA was harvested for
real-time RT-PCR after 20 h.
Dishevelled-Mediated Activation of Rac1b
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cyclin D1 and observed f40% reduction (Fig. 1D). These findings
suggest that Rac1b expression contributes to inappropriate
transcription of Wnt target genes in colorectal cancer cells.
Rac1b augments Dishevelled-mediated activation of Tcf/
Lef–dependent transcription. We have shown previously that
constitutively active V12Rac1 synergizes with stabilized h-catenin
to activate transcription of Wnt target genes in HEK293 cells (26).
To see if Rac1b acts analogously, Rac1b was cotransfected with the
TOPflash reporter into HEK293 cells, which have an intact and
therefore tightly regulated Wnt pathway. As shown in Fig. 2A,
Rac1b failed to activate TOPflash activity on its own compared
with 6-fold activation by h-catenin. Surprisingly, Rac1b did not
show functional synergy with h-catenin as shown previously for
V12Rac1. In agreement with our V12Rac1 studies, WT Rac1 showed
a remarkable synergy with h-catenin, increasing TOPflash activity
up to 30-fold (data not shown).
Failure to see a functional interaction between Rac1b and h-
catenin in HEK293 cells that could account for the transcriptional
activity observed in HCT116 cells prompted us to explore other
Wnt signaling components that lay upstream of h-catenin along
the Wnt signaling axis. Dishevelled is one such protein that is a
well-established positive regulator of Wnt signaling, functioning
very early in the cascade. It is believed that Dishevelled transmits
Wnt signals from Frizzled receptors by inhibiting glycogen
synthase kinase-3hactivity through an unknown mechanism,
enabling h-catenin to escape proteasomal degradation. Coexpres-
sion of Rac1b with Dvl-3 caused a striking transcriptional
activation of up to 11-fold of TOPflash compared with 6-fold
induction with Dvl-3 alone (Fig. 2B). Taken together, these data
suggest that the functional cross-talk between Rac1b and Wnt
pathway activation likely occurs downstream of Dvl-3 but
upstream of h-catenin.
Having established a role for Rac1b as an upstream activator of
transcription of Wnt target genes, we wished to compare its
transcription regulatory function with respect to other well-
established Rac1-specific transcriptional targets. Canonical Rac1
effector pathways that result in transcriptional regulation include
NF-nB, mitogen-activated protein kinase (MAPK), and JNK path-
ways (8, 27, 28). As depicted in Fig. 2C, Rac1b very modestly
activated NF-nB–dependent transcription in a dose-dependent
manner and exerted no effect on AP-1– or SRE-dependent gene
transcription, corresponding to MAPK and JNK pathway activation,
respectively. Furthermore, Rac1b showed a dose-dependent
activation of transcription of the cyclinD1 promoter (Fig. 2D),
which agrees with our observations of Rac1b suppression causing
reduced endogenous cyclin D1 expression (Fig. 1D). These
Figure 2. Rac1b cooperates with Dvl-3 to stimulate Wnt target gene transcription. Aand B, pTOPflash (5) or pFOPflash (n) reporter constructs were transiently
transfected with increasing amounts of Rac1b, h-catenin, or Dvl-3 expression vectors for 24 h in HEK293 cells. Amounts of expression constructs used are 0.2, 0.4, and
0.9 Ag. The lowest amount of h-catenin or Dvl-3 (0.2 Ag) was used in cotransfection experiments with Rac1b. Cand D, HEK293 cells were cotransfected with
0.9 Ag of empty vector () or 0.2 and 0.9 Ag of Rac1b expression vector and either NF-nB–, AP-1–, or SRE-responsive promoter luciferase reporter constructs (C)ora
full-length cyclin D1 promoter construct, CycD1-luc (D). Results are representative of at least three or more independent experiments. Luciferase activity is
expressed as total relative light units. Columns, average; bars, SE (A–D ).
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differential transcriptional responses reiterate the selective signal-
ing capability of Rac1b versus Rac1.
Dvl-3 activates Rac1b and together they synergistically
stimulate cyclin D1 transcription. Because Rac1b was unable
to activate TOPflash activity on its own but showed significant
functional synergy in the presence of Dvl-3 in HEK293 cells, we
speculated whether Rac1b activity was being modified by Dvl-3.
Using the PBD assay, we found that expression of h-catenin did not
change the activity of exogenously transfected Rac1b (Fig. 3A, top).
In contrast, cotransfection with Dvl-3 resulted in a significant
increase in Rac1b activation levels. These findings help to explain
why we observed a differential response in our TOPflash assay
(Fig. 2B). Rac1b is activated by Dvl-3 but not h-catenin and
consequently proceeds to stimulate transcription from the Wnt-
responsive promoter in a Dishevelled-dependent manner. Cyclin
D1 is a well-characterized target gene of both the Wnt and Rac1
signaling pathways (12, 29, 30). We observed cooperativity between
Dvl-3 and Rac1b in regulation of the cyclin D1 promoter (Fig. 3B).
Combined expression of Dvl-3 with Rac1b yielded >5-fold
activation of cyclin D1 promoter activity, which was greater than
their effects individually, suggesting a synergistic interaction.
Expression of Dvl-3 and Rac1b has been shown in both the
cytoplasm and the nucleus. A recent study has shown that nuclear
translocation of Dvl is essential for its ability to activate Wnt/h-
catenin signaling (31). In addition, we as well as others have
reported previously the expression of Rac1 in the nucleus (26, 32,
33). For this reason, we examined complex formation of Dvl-3 and
Rac1b in these specific cellular fractions. We observed complexes of
Rac1b and Dvl-3 in the cytoplasm as well as the nucleus (Fig. 3C).
Interestingly, Rac1b coimmunoprecipitated with Dvl-3 more effi-
ciently in the nuclear fraction. Similarly, endogenous h-catenin also
preferentially coimmunoprecipitated with Rac1b in the nuclear
fraction. Protein expression levels of Rac1b, Dvl-3, and h-catenin in
whole-cell lysates and cytoplasmic and nuclear fractions are
shown. Blots were stripped and reprobed for marker proteins for
cytoplasm and nucleus, paxillin and topoisomerase II, respectively.
Considered together, our data suggest that Dvl-3 and h-catenin can
both form complexes with Rac1b in the nucleus and cytoplasm.
PBR of Rac1b regulates its nuclear localization. The C-
terminal PBR of Rac1 has been shown to function as a nuclear
localization signal (NLS) and mutation of this region drastically
reduces its nuclear entry (34). To examine if the PBR of Rac1b was
involved in mediating its nuclear localization, we generated various
FLAG-tagged Rac1b PBR mutant constructs, by substituting the
basic amino acids of the PBR with the neutral amino acid
glutamine, depicted in Fig. 4A. We examined cellular distribution
of these mutants in fractionated lysates of HCT116 cells. As shown
in Fig. 4B, progressive mutation of the PBR resulted in a significant
progressive decrease in nuclear expression of Rac1b and was
accompanied by a corresponding progressive increase in cytoplas-
mic expression of Rac1b. The Rac1b (Q6) mutant, with all six basic
amino acids mutated, showed the greatest reduction in nuclear
expression.
Mutation of the PBR did not compromise Rac1b activation
levels. As shown in Fig. 4C, all PBR mutants (Q2, Q4, and Q6)
showed similar levels of activation to WT PBR-intact Rac1b. We
also examined the distribution of Rac1b and Rac1b (Q6) by
fluorescence microscopy in HCT116 cells (Fig. 4D). Whereas Rac1b
was prominently localized to the plasma membrane, and to a lesser
extent in the cytoplasm and nucleus, Rac1b (Q6) transfectants
showed complete loss of membrane staining, distinct exclusion
Figure 3. Dvl-3 stimulates activation of Rac1b and cooperatively induces
transcription of cyclin D1. A, FLAG-tagged Rac1b was transiently cotransfected
with increasing concentrations of either h-catenin or Dvl-3 (0, 1, and 4 Ag) to
determine activation status in transiently transfected HEK293 cells. Top, active
GTP-bound Rac1b was determined by glutathione S-transferase (GST)
pull-down assays using the PBD followed by immunoblot analysis using
anti-FLAG antibody to detect exogenous Rac1b; bottom, total expression
levels of exogenously transfected FLAG-tagged Rac1b were determined by
immunoblot analysis of whole-cell lysates (10 Ag). B, HEK293 cells were
transfected with cyclin D1 promoter-luciferase construct (CycD1-luc ) and Dvl-3
or Rac1b (0.4 Ag) alone or in combination. Luciferase activity is expressed as
total relative light units. Columns, average of experiment carried out in triplicate;
bars, SE. C, top, complex formation of Rac1b with Dvl-3 and h-catenin was
examined in HEK293 cells cotransfected with FLAG-tagged Rac1b and Dvl-3 for
24 h. Whole-cell lysates (WCL ), cytoplasmic extracts (CE ), or nuclear extracts
(NE) were immunoprecipitated (IP) with anti–Dvl-3 or anti–h-catenin antibody or
agarose beads alone (negative control). Immunoblotting was done with indicated
antibodies. FLAG-specific antibody was used to detect FLAG-tagged Rac1b.
Bottom, 10 Ag WCL, CE, and NE were immunoblotted with h-catenin, Dvl, or
FLAG antibodies to confirm expression of endogenous h-catenin and exogenous
Rac1b and Dvl-3 in different cellular fractions.
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from the nucleus, and diffuse cytoplasmic staining. The cells were
costained with actin to distinguish individual cells. Taken together,
the PBR of Rac1b controls both membrane localization and nuclear
accumulation of Rac1b. We used the Rac1b (Q6) mutant in
subsequent experiments because it exhibited the greatest reduction
in plasma membrane and nuclear localization.
Mutation of PBR compromises the transcription activator
function of Rac1b and the association of Dishevelled and B-
catenin with Rac1b. To gain insight into the role of subcellular
localization on Rac1b functions, we examined the transcription
activator potential of PBR-mutated Rac1b. As shown in Fig. 5A,
Rac1b (Q6) was unable to activate transcription of the TOPflash
promoter in HCT116 cells compared with WT Rac1b (Fig. 1B).
Consistent with these findings, the Rac1b (Q6) mutant also failed to
stimulate transcription from the NF-nB promoter (Fig. 5B) and
cyclin D1 promoter (Fig. 5C). These data show that the PBR of
Rac1b is important for mediating transcriptional activation.
Furthermore, disrupting plasma membrane and/or nuclear local-
ization of Rac1b interferes with this activity.
Because PBR-mutated Rac1b (Q6) was unable to stimulate
TOPflash activity, we speculated that activation by Dvl-3 may also
be compromised. Comparison of the activation levels of WT Rac1b
and Rac1b (Q6) in the presence of Dvl-3 indicate that Dvl-3 is
unable to further stimulate the activation of Rac1b (Q6) (Fig. 5D,
left). Because we have shown that Rac1b can complex with Dvl-3
and h-catenin (Fig. 3C), we examined whether they were associated
with active Rac1b by reprobing the PBD blot with Dvl-3 and h-
catenin antibodies. As shown in Fig. 5D(left), Dvl-3 and h-catenin
were associated with active GTP-bound Rac1b. Interestingly, the
Rac1b (Q6) pull-down fractions contained considerably reduced
levels of Dvl-3 and h-catenin. The reduced binding to Rac1b (Q6)
was not due to lack of expression of Dvl-3 and h-catenin, which
was confirmed by Western blotting of whole-cell lysates (Fig. 5D,
right). Because Rac1b (Q6) was refractory to further activation by
Figure 4. The PBR of Rac1b regulates its nuclear and plasma membrane localization but not activity. A, schematic representation of the Rac1b protein sequence
and an enlarged view of the C-terminal PBR sequence of WT Rac1b and the substitutions that were made to alter PBR function (Q2, Q4, and Q6). B, the expression of
FLAG-tagged Rac1b and the various FLAG-tagged PBR mutants (Q2, Q4, and Q6) were determined by Western blotting of fractionated cytoplasmic and nuclear
lysates of transiently transfected HEK293 cells. Blots were stripped and reprobed with topoisomerase II, paxillin, or h-actin antibodies to assess purity of nuclear and
cytosolic fractions and to normalize for protein loading, respectively. Interestingly, a doublet was observed in the Rac1b (Q6)–transfected cells. The doublet
could also be detected by an anti-Rac1 antibody on Western blots (data not shown). In addition, RT-PCR analysis of Rac1b (Q6) plasmid DNA did not show an
additional band. We conclude that the doublet is specific for Rac1b and may result due to a posttranslational modification. C, the activation status of Rac1b and
PBR-mutated forms of Rac1b were assessed by PBD assay. Indicated FLAG-tagged constructs were transiently transfected into HEK293 cells for 24 h. Top, active
GTP-bound Rac1b expression levels determined by PBD-GST pull-down assays followed by immunoblot analysis using anti-FLAG antibody; bottom, total cellular
expression of transfected Rac1b constructs determined by immunoblot analysis of whole-cell lysates (10 Ag) with anti-FLAG antibody. D, the distributions of
FLAG-tagged Rac1b and Rac1b (Q6) in HCT116 cells were determined by fluorescence microscopy. Cells were stained with actin-phalloidin to demarcate individual
cells. Approximately 50 to 60 transfected cells were examined. Bar, 10 Am.
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Dvl-3 and we failed to see association of Dvl-3 or h-catenin with
Rac1b (Q6), these observations strongly support the role of the
PBR in mediating binding and activation of Rac1b by Dvl-3.
Rac1b reduces E-cadherin expression and cellular adhesion
of colorectal cancer cells. Transcriptional activation of Wnt
target genes requires h-catenin accumulation and translocation to
the nucleus. Rac1b does not likely modulate proteasomal
degradation of h-catenin because our observations were made in
HCT116 cells, which already contain stabilized h-catenin (35).
Recently, it was shown that loss of E-cadherin can lead to Wnt-
dependent transcription in colorectal cancer cells (36). Therefore,
we next explored whether Rac1b may mediate its positive effects
on transcription by altering E-cadherin expression in HCT116 cells.
We observed that the total amount of endogenous E-cadherin
protein was decreased in the presence of Rac1b (Fig. 6A).
Densitometry indicated a 40% reduction of endogenous E-cadherin
levels in the presence of Rac1b. In agreement, specific suppression
of Rac1b using Rac1b siRNA resulted in a modest increase in the
expression of E-cadherin (Supplementary Fig. S1). Interestingly,
E-cadherin levels gradually returned to basal (untransfected) levels
with progressive mutation of the PBR, such that Rac1b (Q6)
showed minimal effects on E-cadherin expression (Fig. 6A). This
shows that the PBR is required for Rac1b-mediated regulation of
E-cadherin. Because the PBR is important in regulating the tar-
geting of Rac1b, it is possible that the loss of E-cadherin regulation
is related to change in its cellular localization.
Next, we wished to see if the membrane pool of E-cadherin
reflected the same changes as total E-cadherin. Overexpression of
Rac1b changed the distribution of endogenous E-cadherin by
causing a marked reduction of E-cadherin staining at the plasma
membrane (Fig. 6B) compared with untransfected cells. In contrast,
PBR-mutated Rac1b (Q6) did not alter E-cadherin staining at the
plasma membrane, which remained intact at cell-cell junctions.
Changes in E-cadherin expression often translate to changes in
cell-cell adhesion. We wanted to assess whether expression of
Rac1b changed the adhesive properties of HCT116 cells. Expression
of Rac1b decreased the adhesion of HCT116 cells, whereas Rac1b
(Q6)–transfected cells showed no change (Fig. 6C, left). Interest-
ingly, V12Rac1 caused an even more robust reduction in cell
adhesion compared with Rac1b. Because V12Rac1 is a constitu-
tively active mutant, we predicted that the greater reduction in
adhesion was attributed to its greater level of activity. Because we
Figure 5. Mutation of Rac1b PBR disrupts Rac1b-mediated transcriptional activation and activation by Dvl-3. A, HCT116 cells were transfected with pTOPflash/
pFOPflash promoter luciferase constructs and increasing doses of PBR-mutated Rac1b (Q6; 0.2, 0.4, and 0.9 Ag). Band C, HEK293 cells were transfected with
indicated promoter-luciferase constructs and either WT Rac1b or Rac1b (Q6; 0.4 Ag). D, activation status of FLAG-tagged WT Rac1b or Rac1b (Q6) in the absence ()
or presence of Dvl-3 (1 or 4 Ag). Indicated constructs were transiently transfected into HEK293 cells for 24 h. Left, active GTP-bound Rac1b/Q6 expression levels
determined by PBD-GST pull-down assays followed by immunoblot analysis using anti-FLAG antibody. Blots were stripped and reprobed with Dvl-3 and h-catenin
antibodies. Right, total cellular expression of transfected Rac1b/Q6 constructs determined by immunoblot analysis of whole-cell lysates (10 Ag) with anti-FLAG
antibody. Blots were stripped and reprobed for Dvl-3, h-catenin, and h-actin. Luciferase activity is expressed as total relative light units. Columns, average of
experiments carried out in triplicate; bars, SE (A–C ). Data are representative of three independent experiments (A–D ).
Dishevelled-Mediated Activation of Rac1b
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have shown that Dvl-3 can increase the activation level of Rac1b,
we speculated whether Rac1b would decrease adhesion to a greater
extent in the presence of Dvl-3. Indeed, cotransfection of Dvl-3 with
Rac1b caused f80% reduction in adhesion of HCT116 cells
compared with Rac1b or Dvl-3 alone, which individually caused a
50% reduction in adhesion (Fig. 6C, right). A primary mechanism of
E-cadherin down-regulation in tumors is a result of the action of
transcriptional repressors (e.g., proteins of the Snail/Slug family),
which bind to E-box elements within the promoter region of E-
cadherin (37). This prompted us to investigate whether such
underlying molecular mechanisms were involved in mediating the
Rac1b-associated decrease in E-cadherin expression. Suppression
of endogenous Rac1b in HT29 cells resulted in increased E-
cadherin transcript expression and a concomitant decrease in Slug
Figure 6. Rac1b decreases the expression of E-cadherin and cellular adhesion in HCT116 cells. A, top, Western blot analysis of the total amount of E-cadherin after
24 h of transfection with Rac1b or PBR mutants of Rac1b. FLAG-tagged Rac1b and PBR-mutant expression was detected by probing blots with FLAG antibody.
Expression of actin was used as a loading control. Bottom, the band of densitometry was normalized with actin to allow quantification. Data show a significant decrease
in E-cadherin expression mediated by Rac1b, which is abolished on mutation of the PBR region. Data are representative of three independent experiments.
B, distribution of endogenous E-cadherin (red fluorescence) was compared in HCT116 cells transfected with FLAG-tagged Rac1b versus FLAG-tagged Rac1b (Q6),
or untransfected cells by fluorescence microscopy. Rac1b and Rac1b (Q6) transfectants were identified by green fluorescence. Bar, 10 Am. C, HCT116 cells were
transfected for 24 h with indicated constructs followed by assessment of adhesion on poly-L-lysine–coated surface. Results are the percentage of adhesion compared
with empty vector–transfected cells. Representative data obtained from triplicate values. Data are representative of at least two independent experiments.
D, E-cadherin (top) and Slug (bottom ) transcript levels in response to RNA interference–mediated inhibition of Rac1b expression in HT29 cells.
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(Snai2) transcript levels (Fig. 6D). We did not observe any change in
Snail transcript levels under similar conditions (data not shown).
These data agree of a repressive role for Rac1b in cellular adhesion
by inhibition of E-cadherin transcription.
Discussion
Because its discovery as a splice variant specifically overex-
pressed in colon and breast tumors, there has been considerable
interest in characterizing the functions of Rac1b to understand
how it may contribute to tumorigenesis in these tissues (16, 17).
Our data show functionality of naturally occurring Rac1b in
modulating Wnt signaling by augmenting Dishevelled-mediated
effects on Tcf-dependent target gene transcription. In addition, we
show that Rac1b decreases membrane E-cadherin expression and
reduces adhesion of colorectal cancer cells and this is likely via up-
regulation of Slug, the E-cadherin-specific transcriptional repressor.
The contribution of Rac1b to Wnt pathway activation may
represent an important mechanism by which it promotes colon
tumorigenesis. We have shown previously that V12Rac1 requires
the presence of stabilized h-catenin to mediate its stimulatory
effects on transcription. In agreement with these data, coexpres-
sion of WT Rac1 with h-catenin also resulted in synergistic
induction of TOPflash promoter activity in HEK293 cells (data not
shown). Unlike Rac1, Rac1b was unable to stimulate Tcf-dependent
transcription in HEK293 cells, despite coexpression of h-catenin.
However, when exogenous Dvl-3 was coexpressed with Rac1b in
these cells, we observed striking synergistic activation of TOPflash.
Interestingly, Dvl proteins have been noted to form cytoplasmic
puncta when overexpressed in cells, and their formation has been
correlated with their ability to induce Wnt pathway activation
(38, 39). In this regard, we observed a striking increase in the size
and number of Dvl-3 punctae in HCT116 cells cotransfected with
Dvl-3 and either Rac1b or L61Rac1 compared with Dvl-3 alone
(Supplementary Fig. S2). Interestingly, we also observed Rac1b and
L61Rac1 colocalize with Dvl-3 within these punctae. Understanding
how these vesicles form and correlate with Wnt signaling activity
may uncover new roles for Dvl and Rac1b/Rac1 proteins in
regulating the canonical arm of Wnt signaling.
It is tempting to speculate that Rac1b and Rac1 may act at
different stages of tumor progression to further exacerbate Wnt
signaling in colonic tissues. Our data show that Rac1b and Rac1
likely require different cellular cues, at which they can participate
in Wnt signaling. In contrast to Rac1, which can synergize with
h-catenin to augment Wnt signaling, Rac1b requires a signal
further upstream, likely stemming from Dishevelled proteins. This
implies that expression of Rac1b in colorectal tumor cells that
already harbor inherent Dishevelled-dependent activation of Wnt
pathway may increase the intensity of Wnt signaling within those
cells. This may be a critical step in tumor progression, assisting
tumor cells to metastasize to distal sites. Clinical evidence suggests
that Wnt signaling acts differently at different stages of tumor
progression (40). This is best exemplified by nuclear staining for h-
catenin, a hallmark of Wnt pathway activation, in colorectal
carcinomas, which often show a heterogeneous pattern, with
strongest nuclear enrichment at the invasion front. The molecular
basis for this differential distribution of h-catenin is not known, but
it has been speculated that signals from the mesenchymal tissue,
which surrounds the invasive tumor cells, might superactivate the
pathway by unknown ways. Matrix metalloproteinases (MMP) are
important microenvironmental factors present in mesenchymal
tissue, implicated in tumor initiation and progression. Interestingly,
some MMPs are Wnt target genes (41). Recently, Rac1b was shown
to be the key mediator of MMP-3–induced malignant transforma-
tion of mouse mammary epithelial cells (21). Specifically, MMP-3
treatment induced Rac1b expression, which was responsible for the
EMT of the cells. It is tempting to speculate that a positive feedback
loop exists, in which initial Wnt pathway activation induces
expression of a subset of gene products, such as MMPs, which
feedback in an autocrine or paracrine manner, on Wnt-dysregu-
lated cells to induce Rac1b expression. In turn, as our data suggest,
Rac1b expression would further augment nuclear Wnt signaling in
the cancer-initiated cells, promoting tumor progression by aiding
tumor cells in invasion and metastasis. Whether such a feedback
loop exists warrants future investigation.
Another important point worth noting is that overexpression of
the naturally occurring WT form of Rac1b was sufficient to induce
changes in Wnt signaling. Unlike the widely used ‘artificial’
constitutively active mutants (G12V or Q61L), WT forms of Rho
proteins are amenable to regulation and so their use can facilitate
identification of upstream or downstream signals that may be
critical in modulating expression and activity. In this context, our
findings are significant because overexpression of WT Rac1b had a
profound stimulatory effect, cooperating with Dvl-3 to increase
nuclear Wnt signaling events. Furthermore, we show that
expression of Dvl-3, but not h-catenin, leads to increased activation
of Rac1b. This is likely the reason why Rac1b augmented Tcf-
mediated transcription in the presence of Dvl-3 but not h-catenin.
Perhaps association of Dvl-3 with Rac1b not only further activates
Rac1b by modulating GEF/GAP activity but also may recruit other
proteins, such as h-catenin, to form functional signaling com-
plexes. Our findings that Dvl-3 and h-catenin were predominantly
present in the active GTP-bound fractions of Rac1b, in Dvl-
dependent manner, support this idea. Mechanisms of how Dvl-3
may increase activity of Rac1b are currently under investigation in
our laboratory. Aberrant expression of Dishevelled has been
observed in multiple cancers and has been linked to activation of
Wnt/h-catenin signaling and cell growth in these cancers. It would
be interesting to examine Rac1b expression and activity and
turnover in these tumors (42–44).
Because we observed nuclear expression of Rac1b, we wondered
if the putative NLS in the PBR was responsible. Substitution of all
six basic amino acids of the Rac1b PBR with neutral glutamines
resulted in almost complete exclusion of Rac1b from the nucleus,
as well as the plasma membrane. PBR-mutated Rac1b was unable
to activate transcription from TOPflash, cyclin D1, and NF-nB
promoters. It is tempting to speculate that the nuclear pool of
Rac1b is likely critical for transcription of Wnt target genes.
Interestingly, Dvl-3 was unable to augment activity of PBR-mutated
Rac1b, suggesting that it may mediate these effects within the
nucleus. In this regard, nuclear expression of many Rac1-specific
proteins, including GEFs and downstream effectors, further
supports the notion of Rac1 and Rac1b participating in nuclear
signaling pathways (33, 45). In agreement with this hypothesis, we
observed Dvl-3 coimmunoprecipitating with Rac1b mostly in the
nuclear fraction of cells, despite these proteins being present in the
cytoplasm, which lends further support for these effects occurring
in the nucleus.
In the colon, E-cadherin–mediated cell adhesion is critical in the
transition from adenoma to carcinoma, and reexpression of WT
E-cadherin in cancer cell lines reduces their invasiveness (46, 47).
Here, we show that expression of Rac1b in HCT116 colorectal
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cancer cells reduces endogenous E-cadherin expression and
cellular adhesion. Intriguingly, coexpression of Dvl-3 with Rac1b
resulted in a striking reduction of adhesion compared with effects
seen by either protein alone. Furthermore, we show that both
of these effects disappear on mutation of the PBR of Rac1b.
Because nuclear localization of Rac1b is disrupted by mutation of
the PBR, it is possible that Rac1b functions in the nucleus to alter
E-cadherin expression. We observed decreased Slug expression and
increased E-cadherin transcript expression on suppression of
Rac1b in HT29 cells. These data support an important nuclear
role for Rac1b, whereby it positively regulates Slug transcription,
which consequently represses E-cadherin expression leading to
diminished cellular adhesion. Interestingly, Slug has been reported
to be a Wnt target gene and shown to have Tcf-binding elements
within its promoter region (48). We propose that Rac1b expression
contributes to tumor progression by synergizing with Wnt
components to modulate Slug expression and therefore the
adhesive properties of cells. Future studies aimed at elucidating
how Rac1b regulates Slug and the discovery of other Rac1b/Wnt
target genes will further our understanding of the role of Rac1b in
tumor progression.
It is tempting to speculate that because Rac1b, being a tumor-
specific protein, seems to share only a selective repertoire of Rac1
functions, it is likely that those pathways retained by Rac1b are
critical for tumorigenesis, by perhaps imparting a growth advantage
and/or increased capacity for invasion and metastasis. In this regard,
further activation of Wnt signaling in colon cancer cells by Rac1b
may accelerate tumor progression in this tissue. Tumorigenesis is an
evolutionary process, where successive genetic changes transform a
normal cell into a self-sufficient, resilient ‘‘super’’ cancer cell capable
of metastasis. Mounting evidence suggests that deregulation of Rac1
signaling, through overexpression of itself or its regulators, has dire
consequences on cell physiology, changing signaling circuitry and
contributing to cellular transformation. Changes in Rac1 splicing to
create Rac1b, a much more efficient and active GTPase, likely
represents a further evolutionary advantage for cancer development.
Future studies aimed at identifying the mechanisms involved in
activation of Rac1b expression as well as clinical studies correlating
Rac1b expression with extent of colorectal cancer will increase our
understanding of Rac1b in tumorigenesis and may also be of
prognostic value or provide novel therapeutic targets.
Acknowledgments
Received 8/1/2006; revised 12/21/2006; accepted 1/11/2007.
Grant support: Canadian Institutes for Health Research MOP 64225 (B. Bapat), the
Ontario Graduate Scholarship (S. Esufali and P. Buongiorno), the Samuel Lunenfeld
Research Institute Ontario Student Opportunity Trust Funds Fellowship (S. Esufali and
P. Buongiorno), and the University of Toronto Open Fellowship (S. Esufali, G.S.
Charames, and P. Buongiorno).
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.
We thank the following colleagues for their kind gifts of cDNA constructs: FLAG-
Rac1b (Dr. M. Ahmadian, Max-Planck Institute, Heidelberg, Germany); GFP-Rac1b (Dr.
C. Der, University of North Carolina, Chapel Hill, NC); W T h-catenin and FLAG–Dvl-3
(Dr. L. Attisano, University of Toronto, Ontario, Canada); CycD1-luc (Dr. T. Akiyama,
University of Tokyo, Tokyo, Japan); NF-nB–luc (Dr. A. Bosserhoff, University of
Regensburg, Regensburg, Germany); and pTOPFLASH and pFOPFLASH (Dr. B. Alman,
Hospital for Sick Children, Toronto, Ontario, Canada).
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