Structure-based discovery of a novel inhibitor targeting the β-catenin/Tcf4 interaction.

Abstract

Overactivation or overexpression of β-catenin in the Wnt (wingless) signaling pathway plays an important role in tumorigenesis. Interaction of β-catenin with T-cell factor (Tcf) DNA binding proteins is a key step in the activation of the proliferative genes in response to upstream signals of this Wnt/β-catenin pathway. Recently, we identified a new small molecule inhibitor, named BC21 (C(32)H(36)Cl(2)Cu(2)N(2)O(2)), which effectively inhibits the binding of β-catenin with Tcf4-derived peptide and suppresses β-catenin/Tcf4 driven reporter gene activity. This inhibitor decreases the viability of β-catenin overexpressing HCT116 colon cancer cells that harbor the β-catenin mutation, and more significantly, it inhibits the clonogenic activity of these cells. Down-regulation of c-Myc and cyclin D1 expression, the two important effectors of the Wnt/β-catenin signaling, is confirmed by treating HCT116 cells with BC21. This compound represents a new and modifiable potential anticancer candidate that targets β-catenin/Tcf-4 interaction.

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Structure-Based Discovery of a Novel Inhibitor Targeting the
β-Catenin/Tcf4 Interaction
Wang Tian,†,‡Xiaofeng Han,†,‡Maocai Yan,§Yan Xu,†,‡Srinivas Duggineni,†,‡Nan Lin,†,‡Guifen Luo,†,‡
Yan Michael Li,⊥Xiaobing Han,†,‡Ziwei Huang,†,‡and Jing An*,†,‡
†Department of Pharmacology, State University of New York, Upstate Medical University, Syracuse, New York 13210, United States
‡Upstate Cancer Research Institute, Syracuse, New York 13210, United States
§Department of Molecular Biology, University of Southern California, Los Angeles, California 90089, United States
⊥Department of Neurosurgery, State University of New York, Upstate Medical University, Syracuse, New York 13210, United States
ABSTRACT: Overactivation or overexpression of β-catenin in the Wnt
(wingless) signaling pathway plays an important role in tumorigenesis.
Interaction of β-catenin with T-cell factor (Tcf) DNA binding proteins is
a key step in the activation of the proliferative genes in response to
upstream signals of this Wnt/β-catenin pathway. Recently, we identified a
new small molecule inhibitor, named BC21 (C32H36Cl2Cu2N2O2), which
effectively inhibits the binding of β-catenin with Tcf4-derived peptide and
suppresses β-catenin/Tcf4 driven reporter gene activity. This inhibitor
decreases the viability of β-catenin overexpressing HCT116 colon cancer
cells that harbor the β-catenin mutation, and more significantly, it inhibits the clonogenic activity of these cells. Down-regulation
of c-Myc and cyclin D1 expression, the two important effectors of the Wnt/β-catenin signaling, is confirmed by treating HCT116
cells with BC21. This compound represents a new and modifiable potential anticancer candidate that targets β-catenin/Tcf-4
interaction.
T
tion of the Wnt/β-catenin pathway, resulting from mutations of
proteins in this pathway, is common in a wide range of human
cancers.2−4For example, an abnormally high level of β-catenin
has been found in many types of cancers, and this is necessary
for tumorigenesis.2The overactivation or overexpression of
β-catenin is associated with resistance to cancer treatment
through enhancement of cancer cell renewal and prevention of
apoptosis.3,4Inhibitors that specifically target the key regulators
in this pathway therefore not only represent a generation of
new medicine for treating cancers but also can serve as valuable
chemical probes for studying the molecular mechanism under-
lying the control of cancer cell fate by Wnt/β-catenin signaling.
The cellular location of β-catenin typically is predominantly
at cell−cell junctions and at very low levels in both the cyto-
plasm and nucleus. Excess β-catenin is usually subjected to
GSK-3β/APC/axin complex-mediated phosphorylation and
subsequent proteasome-mediated degradation.2Binding of
Wnt ligands to Frizzled (Fzd), a cell surface receptor, activates
Disheveled (Dsh), which leads to accumulation of cytoplasmic
β-catenin. This accumulation can also be seen in presence of
mutations in APC, Axin, and also the β-catenin gene.5The ele-
vation of the cytoplasmic β-catenin level results in its tran-
slocation to the nucleus and binding to members of the T cell
factor/lymphoid enhancer factor (Tcf/Lef) family, stimulating
transcription of Wnt target genes, including c-Myc and cyclin
D1, that are critical for cell growth, proliferation, and differen-
tiation.6,7About 50% of colon cancers contain activating β-catenin
he Wnt/β-catenin signaling pathway plays a critical role in
cancer cell self-renewal and proliferation.1,2Overactiva-
mutations at putative GSK-3β phosphorylation sites, which render
the β-catenin protein resistant to proteasome-mediated destruc-
tion.8Recent data indicate that enhanced clonogenic capacity and
poor prognosis correlate positively with the constitutively active
β-catenin overexpression of β-catenin by acute chronic myelo-
genous leukemia (AML), chronic myelogenous leukemia
(CML), and many solid tumors.9−12Thus, disruption of the
interaction between β-catenin and Tcfs would be considered as
a good strategy for treating cancer cells, particularly CSCs.
The crystalline structures of β-catenin complexed with Tcf3
and Tcf4 have been resolved.13,14Both Tcf3 and Tcf4 have
similar structures when complexed with β-catenin; both consist
of a long irregular head and an α-helix tail. The irregular head
forms dense hydrogen bonding contacts with β-catenin, while
the α-helix tail binds to β-catenin primarily by hydrophobic
interactions. In this study, we used the crystal structure of
β-catenin, alone and in complexes with Tcf3/Tcf4, in docking
studies and screened a compound library. A small molecule
compound was identified that showed potential inhibition of
β-catenin/Tcf4 interaction and cancer cell proliferation.
■EXPERIMENTAL PROCEDURES
Virtual Screening by Molecular Docking. The diversity
set of National Cancer Institute (NCI) open database,15consisting
Received:
Revised:
Published: December 23, 2011
September 14, 2011
December 22, 2011
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© 2011 American Chemical Society
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of 1990 small molecules, was selected for the virtual screening by
using Autodock4.16Receptor structure solved at 2.5 Å resolution
was extracted from crystal structure of the β-catenin/Tcf4 complex
(PDB entry 1JPW, chain A) and the N-terminal moiety which are
far from site A (Arg151−Asn387) was truncated to facilitate the
preprocessing of receptor. The receptor file was converted to
PDBQT file, and a 64 × 62 × 54 grid box with a grid spacing of
0.375 Å was defined to cover the whole groove in which site
A locates. Side chains of high polar residues around site A
(such as Lys435, Arg469, Lys508, Arg515, and Glu571) were
set flexible during the docking. Grid maps were then generated
by Autogrid416for Autodock simulations. The whole
preparation process of receptor files was accomplished with
AutoDockTools-1.5.2.16Preparation of ligands and docking
parameter files were accomplished using scripts of Molecular
Graphics Laboratory of Scripps Institute, with all parameters
set to default values.
Reagents. Anti-c-Myc (#9402), anticyclin D1 (DCS6, #2926),
and anti-β-actin (#4967) antibodies were purchased from Cell
Signaling, Inc. Quercetin was purchased from Sigma-Aldrich.
pCMV-SPORT-FL-Tcf4 (Catalog #MHS1010-7508380) was
purchased from Open Biosystem. Primers used for amplifying
Tcf4 (N-terminal 1−54 amino acids) were synthesized from
Invitrogen. Restriction endonuclease and T4 ligase were
purchased from New England Lab. Compounds identified by
virtual screening were requested from NCI and dissolved in
DMSO to 10 mM stock and stored at −20 °C.
Cell Culture. The colon cancer cell line HCT116 was
obtained from American Type Culture Collection (ATCC) and
cultured in RPMI 1640 (Hyclone, Thermal Scientific)
supplemented with 10% fetal bovine serum, 2 mM glutamine,
100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were
maintained in a humidified 5% CO2atmosphere at 37 °C.
Vector Construction. The expression plasmid containing
Tcf4 β-catenin binding domain (CBD, N-terminal 1−54 amino
acids) was constructed by amplifying the corresponding DNA
fragment by PCR using pCMV-SPORT6-FL-Tcf4 as template
and the primers (forward: 5′-CGCGGATCCATGCCGCAG-
CTGAACG-3′; reverse: 5′-CGCGAATTCTTACGTTTCT-
GATTCATTG-3′; BamHI and EcoRI sites are underlined,
respectively). Purified PCR fragment was digested with BamHI and
EcoRI and inserted into pGEX-6p-1 vector treated with the same
enzymes. The ligation product was transformed into competent
DH5α, and single colonies were picked up for characterization.
Protein Expression and Purification. Plasmid pET28a-
FL-hβ-catenin (kindly provided by Dr. Wenqing Xu, University
of Washington) was used to transform BL21(DE3)star
(Invitrogen). Bacteria were induced with 0.5 mM IPTG and
then harvested and lysed. The supernatant was loaded onto an
affinity column containing Ni-NTA beads for His-tag fusion
protein (QIAGEN). Purified protein was concentrated to
6 mg/mL in buffer containing 20 mM Tris-HCl, 100 mM
NaCl, 2 mM DTT, and 20% glycerol and stored at −80 °C.
The GST-Tcf4-1-54 fusion protein was purified by similar
protocol, except that glutathione Sepharoase 4B (GE Health-
care) was used to affinity chromatography and the protein was
concentrated to 1 mg/mL.
Luciferase Reporter Gene Assay. HCT116 cells at 1 ×
104cells/well were transfected with TOP-FLASH, containing
Tcf4 binding sites, or FOP-FLASH harboring mutant Tcf4
binding sites. At 4 h post-transfection, candidate compounds
were added to cells. Luciferase activities were determined 24 h
after compounds treatment using the Firefly Luciferase Assay
Kit (Biotium, Inc. Hayward, CA). Cell viabilities were also
determined at this time point as described in the Cell Viability
Assay section. The luciferase activities were normalized with
cell viability to rule out the nonspecific cytotoxicity. For HEK293
cell, pCDNA3.1-β-catenin was cotransfected into cells with TOP
(or FOP) vectors. At 24 h post-transfection, compounds were
added to cells. The luciferase activities and cell viabilities were
determined as described above. The relative luciferase activity
was expressed as follows: for TOP, (TOP-luciferase activity)treatment/
(TOP-luciferase activity)control× 100; for FOP, (FOP-luciferase
activity)treatment/(TOP-luciferase activity)control× 100.
Cell Viability Assay. Human colon cancer 116 (HCT116),
human embryonic kidney 293 (HEK293), and human umbilical
vein endothelial (HUVE) cells were plated at 1 × 104/well in
96-well plate and incubated overnight at 37 °C. Cells were
treated with different concentrations of selected candidate
compounds and incubated for 72 h. Cell viability was measured
by a CellTiter-Blue reagent-based assay (Promega).
Clonogenicity Assay. HCT116 cells were plated at 300/
well in 6-well plates and incubated overnight at 37 °C. Cells were
treated with various concentrations of selected candidate com-
pounds and incubated for 7 days. Colonies containing more than
50 cells were scored. Individual assays were performed with dupli-
cate wells. Relative colony number was calculated as [(colony
number)treatment/(colony number)control] × 100%.
Western Blots. HCT116 cells at 1 × 106/mL were treated
with different concentrations of selected compounds. Cell
lysates were prepared after 24 h treatment and subjected to
SDS-PAGE and Western blotting with anti-c-Myc or anticyclin
D1 antibody (Cell Signaling). The intensities of bands were
quantified by Image J. In order to exclude the differences in
loading levels, the intensity of c-myc or cyclin D1 was divided
by the corresponding intensity of β-actin. The relative intensity
expressed as follows: (intensity)treatment/(intensity)control× 100.
Real-Time PCR. HCT116 cells at 1 × 106/mL were treated
with different concentrations of BC21 for 24 or 5 h. Total RNA
was isolated using a RNeasy Kit (Qiagen). The cDNA was
synthesized with a high-capacity cDNA reverse transcription
Kit (Invitrogen). Real-time PCR was performed on an Applied
Biosystems Stepone Plus system using SYBR-green Mastermix
(SABiosciences). The threshold cycle (CT) values were nor-
malized to β-actin internal reference. The primers used in real-
time PCR were as follows: β-actin, forward 5′-AGAAAATCT-
GGCACCACACC-3′; reverse 5′-AGAGGCGTACAGGGATAG-
CA-3′; c-myc, forward 5′-TCAAGAGGCGAACACACAAC-3′;
reverse 5′-GGCCTTTTCATTGTTTTCCA-3′; cyclin D1, for-
ward 5′-AACTACCTGGACCGCTTCCT-3′; reverse 5′-GGGGA-
TGGTCTCCTTCATCT-3′.
Fluorescence Polarization (FP)-Based Competitive Bind-
ing Assay. The effect of candidate compounds on β-catenin/
Tcf4 binding was tested using a FP-based assay developed for
that purpose (unpublished data). In the competitive binding
experiments, 20 nM tracer (FITC labeled Tcf4 peptide,
sequence is not shown) and 1.5 μM β-catenin were mixed
and incubated with/without competitor in 1 × PBS (containing
0.01% Triton X-100) to produce a final volume of 100 μL. The
reaction was incubated at room temperature for 3 h to reach
equilibrium.17Polarization values were read using the “synergy
2” reader (BioTek) at excitation and emission wavelengths of
485/20 and 528/20 nm, respectively. The relative binding was
calculated as (mPT− mPf)/(mPC− mPf) × 100, where mPC=
control (without inhibitor), mPf= free tracer (without β-catenin
and inhibitor), and mPT= treatment (treated with inhibitors).
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Statistical Analysis. The statistical analysis was performed
using the paired Student’s t-test. Two-sided P values of <0.05
were considered statistically significant. Average values were
expressed as mean ± SD.
■RESULTS
Identify β-Catenin and Tcf4 Interaction-Targeted
Lead Inhibitor BC21 by Virtual Screen. We analyzed the
interactions of β-catenin and Tcf4 in the crystalline structures
(Figure 1) based on the proposed hot spots by Fasolini et al.18
Three “hot spots” (sites A, B, and C) on the β-catenin were
critical for the binding of Tcf4. We chose site A as our
pharmacophore for virtual screening because it is sufficiently
large and deep for a small molecule to bind. This hot spot
consists of a number of polar residues, including Lys435,
Arg469, Lys508, Arg515, and Glu571. These polar residues are
critical for binding of β-catenin with Tcf4 as well as for
β-catenin-mediated proliferative signaling. Asp16 of Tcf4
formed a salt bridge with Lys435 with a distance of 2.5 Å
and three hydrogen bonds with Asn430 on site A,13,19while
Glu17 of Tcf4, which was adjacent to Asp16, formed a salt
bridge with Lys508 on site A. In addition, the Ile19 and Phe21
of Tcf4 side chain packed together into a cleft, which is lined by
Cys466 and Pro463 residues and the Arg386 aliphatic portion
Figure 1. Complex of β-catenin with Tcfs and predicted binding model for BC21. (I) Tcf3 (cyan) and Tcf4 (magenta) are structurally similar when
complexed with β-catenin; both consist of a long, irregular head and α-helix tail. Three “hot spots” (A, B, and C) on β-catenin were critical for Tcf4
binding. Site A is sufficiently large and deep for a small molecule to bind and consisted of a number of polar residues (Lys435, Arg469, Lys508,
Arg515, Glu571, etc.). In the present study, site A was chosen as a binding pocket for the identification of small molecules that could disrupt
β-catenin/Tcfs interaction. (II) The detailed interaction of β-catenin/Tcf4 at site A. Key residues for the interaction in Tcf4 and β-catenin are
labeled in red or black, respectively. (III) The predicted binding position for BC21. The Tcf4 peptide is shown in yellow. BC21 is shown as a ball−
stick model. Gray balls represent carbon atoms, blue balls represent nitrogen atoms, red balls represent oxygen atoms, green balls represent chloride
atoms, and brown balls represent copper atoms. The pictures were generated by Autodock and PyMol.
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of β-catenin.20We used structure-based virtual screening to
identify a new class of small molecule BC21 among 200 top-
ranked compounds in a 1990 compound library. In the case of
BC21, hydrophobic interactions contributed to blocking of the
binding of Tcf4 and β-catenin. The estimated binding free
energy between BC21 and β-catenin given by Autodock 4 is
−7.07 kcal/mol, and predicted Ki= 6.55 μM. Half of the BC21
molecule located at the same position where the Ile19 and
Phe21 residues of Tcf4 were found and showed very good
hydrophobic interactions with the β-catenin hydrophobic cleft,
especially with Pro463 ring. The other half of the BC21 mole-
cule occupied the position at Asp16, and this also contributed
to blocking of the β-catenin/Tcf4 interaction.
BC21 Inhibits the β-Catenin/Tcf4 Transcriptional
Activity. We investigated the biological activity of 100 lead
compounds using a cell-based TOP/FOP assay for testing the
β-catenin/Tcf4 signaling. HCT116 cells were transfected with a
reporter gene (luciferase) harboring the Tcf4 binding site (TOP)
or a mutant Tcf4 binding site (FOP). At 4 h post-transfec-
tion, candidate compounds (20 μM) were added to the cells.
Luciferase activities were determined 24 h post-transfection.
Compounds capable of passing through the cell membrane and
specifically inhibiting the Wnt/β-catenin signaling would
reduce TOP-luciferase activity, without reducing FOP-lucifer-
ase activity. Figure 2 shows that the lead compound, BC21
(C32H36Cl2Cu2N2O2, MW = 679, NSC109268), at concen-
tration of 10 μM, effectively reduced the β-catenin/Tcf4 driven
TOP-luciferase activity in HCT116 cells, without significantly
affecting FOP-luciferase activity (Figure 2A,B). BC21 also exhibited
an inhibitory effect on TOP-luciferase activity in HEK293 cells that
transiently expressed high levels of β-catenin (Figure 2C,D)
Quercetin is a flavonoid that has recently been shown to have
ability to inhibit β-catenin/Tcf4 pathway;21it was used here as
a positive control.
BC21 Decreases the Viability and Reduces the Colony
Forming Activity of β-Catenin Overexpressing Colon
Cancer Carcinoma Cells. We examined the cell death and
clonogenic cell death effects of this compound on HCT116
cells using CellTiter-Blue Cell Viability and colony-formation
assays. BC21 effectively induced HCT116 cell death in a dose-
dependent manner; over 80% of the cells died after a 72 h
treatment with 30 μM BC21 (Figure 3). At concentrations of
5 and 10 μM, BC21 was significantly blocked more than 80 and
100% of the colony-forming activity of HCT116 cells, respec-
tively (Figure 4). However, at 3−15 μM, BC21 exhibited no
cytotoxic effects on normal HEK293 and HUVEC cells. These
effects on cell viability and clonogenic cancer cell death were
stronger for BC21 than for quercetin, p < 0.05. These results
indicated that the interruption of β-catenin/Tcf4-mediated
Figure 2. BC21 can inhibit the transcriptional activity of β-catenin/Tcf4 in HCT116 cells and HEK293 cells. (A) HCT116 cells were transfected
with a reporter gene (luciferase) harboring the Tcf4 binding site (TOP) or a mutant Tcf4 binding site (FOP). At 4 h post-transfection, candidate
compounds were added to the cells. Luciferase activities were determined 24 h post-transfection. BC21 can effectively reduce the TOP-luciferase
activity at a concentration of 10 μM, without significantly affecting FOP-luciferase activity. Quercetin was used as a positive control. (B) HEK293
cells were co-transfected with TOP (or FOP) and pcDNA3.1-β-catenin. 24 h post-transfection, BC21 were added to cells. Luciferase activities were
determined 24 h after drug treatment.
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signaling pathway caused by BC21 resulted in the induction of
HCT116 cell death, particularly clonogenic cell death.
BC21 Down-Regulates the Expression of Wnt/β-Catenin
Target Genes. Cyclin D1 and c-Myc are two important target
genes of the Wnt/β-catenin signaling pathway that are up-
regulated in many cancer cells including HCT116 carcinoma
cells. After a 24 h treatment with 20 and 40 μM BC21, the
expressions of c-myc (Figure 5A) and cyclin D1 (Figure 5B),
respectively, were significantly reduced to levels comparable to
those seen with 50 μM quercetin. We also examined the mRNA
levels for c-myc and cyclin D1 after treated with BC21. As
shown in Figure 6, after 5 h treatment at 40 μM, the cyclin D1
mRNA amount was significantly reduced. The c-myc mRNA
levels were decreased at both 20 and 40 μM treatments. Most
RNA were degraded after treating with 40 μM BC21 for 24 h
due to the high cytotoxicity. So the data at this time point and
concentration were not determined.
BC21 Interferes the Binding between β-Catenin and
Tcf4 Peptide. We determined the direct inhibitory effects of
Figure 3. Effect of BC21 on the viability in HCT116 cells. The
HCT116 cells were treated with the indicated concentration of each
compound. The cell viability was determined by CellTiter-Blue assays
(Promega) after a 72 h treatment. The IC50for quercetin and BC21
was 35 and 15 μM, respectively.
Figure 4. Effect of BC21 on HCT116 colony forming activity. The
HCT116 cells were treated with the indicated concentrations of BC21
or quercetin and incubated for 7 days at 37 °C in a humidified
atmosphere containing 5% CO2. Colonies consisting of more than
50 cells were scored (left panel). Relative colony number was
calculated as [(colony number)treatment/(colony number)control] × 100%
and is shown in the right panel.
Figure 5. Effect of BC21 on the expression of the Wnt/β-catenin
target gene. The HCT116 cells were treated with the indicated
concentrations of BC21 or quercetin. Cell lysates were prepared after
24 h treatment and subjected to Western blotting with anti-c-myc (A)
or with anticyclin D1 (B) antibody. Anti-β-actin antibody was used as
a loading control. The intensity of each band was quantified by Image J
and normalized against a nontreatment control. The result was repre-
sentative of three independent experiments.
Figure 6. Effect of BC21 on the mRNA level of the Wnt/β-catenin
target gene. The HCT116 cells were treated with the indicated
concentrations of BC21. Total RNA was prepared after 24 h (for the
20 μM treatment) or 5 h (for the 40 μM treatment). The cDNA was
synthesized and quantified by real-time PCR and normalized against a
nontreatment control.
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BC21 on interaction between β-catenin and Tcf4 by developing
a new cell-free FP-based competitive binding assay. BC21
was able to inhibit the binding between β-catenin and FITC-
labeled Tcf4 probe with an IC50value of 5 μM (Figure 7).
The β-catenin binding domain of Tcf4 (GST-Tcf4-1-54) was
used as a positive control, which has an IC50of 1 μM.
■DISCUSSION
Current cancer treatments, such as chemo- and radiotherapies,
are successful at destroying bulk cancer cells but are ineffective
at eliminating self-renewing cancer stem cells (CSCs). The
Wnt/β-catenin pathway is one of the important signaling path-
ways that are critical for CSC renewal and proliferation. Tar-
geted disruption of the interaction between β-catenin and Tcf
DNA binding proteins, which is a key step involved in Wnt/
β-catenin-mediated proliferative signaling,18,22would be a valu-
able strategy as a tumor therapy. In the present studies, we dis-
covered a new class of small molecule inhibitor of β-catenin−
Tcf4 interaction. It not only effectively inhibits the binding
between β-catenin and Tcf4 derived peptide as well as the
β-catenin-Tcf4 reporter TOP gene activity but also causes
apoptosis and clonogenic cell death for β-catenin overexpres-
sing HCT116 colon cancer cells.
β-Catenin has a superhelix structure and bears a long and
shallow groove, while Tcf3 and Tcf4 bind to this groove as a
linear structure. The binding interfaces of β-catenin and Tcfs
are very large which create difficulty in finding a small molecule
that can compete with Tcfs by occupying the whole bind-
ing pocket. However, “hot spots” exist on the protein surface;
that is, small regions that are essential for maintenance of the
binding affinity of protein−protein interactions.23,24Fasolini et al.
defined the hot spots for Tcf4 in β-catenin-binding by per-
forming an alanine scanning of all Tcf4 residues on the binding
interface.18They found that, in addition to Asp16, which is the
most important residue for binding, alanine replace-
ment of Asp11 and three hydrophobic residues on the α-helix
(Leu41, Val44, and Leu48) also results in significant decrease in
the binding constants. Another research group showed that
Lys435 and Lys508 were also critical for β-catenin binding to
Tcf4.20Therefore, blocking a hot spot using a small molecule
resulted in disruption of the protein−protein interactions.
In fact, some small molecules have been reported to show
inhibitory effects on this interaction.21,25−27However, most of
these compounds are natural products extracted from plant or
fungi, and their chemical modification is difficult; this limits the
capacity for improving their potency and selectivity. BC21, the
small molecule we identified in this study, is a potent candidate
inhibitor for β-catenin/Tcf4 interaction. This small molecule is
an organic compound and, as far as we know, is different from
other published inhibitors in its chemical structure. Previous
studies showed that the hydrophobic interactions between Tcf4
Ile19 and Phe21 with β-catenin had ∼60% functional
contributions.20This means that blocking the Tcf4 Ile19−
Phe21 interaction with β-catenin is critical. BC21 is located at
the β-catenin hydrophobic cleft just where it supposedly would
interact with Tcf4 Ile19 and Phe21. In addition, BC21 occupied
another important binding site and blocked the Tcf4 Asp16
from forming a hydrogen bond with β-catenin Lys435, which is
a key Tcf4/β-catenin interaction. One naphthalene ring of
BC21 undergoes hydrophobic interactions with the side chains
of Pro463 and Cys466, and the other naphthalene ring also
undergoes hydrophobic interactions with the fatty carbon chains
of Lys508 and Arg469. Therefore, although BC21 binds part of
the Tcf4 binding pocket of the β-catenin, it is capable of
hindering the Tcf4 binding, thereby disrupting the β-catenin/
Tcf4 interactions. Our biological data demonstrated this
inhibitory activity of BC21.
A high level of β-catenin has been found in many cancers,
especially colon cancer.28,29We investigated the biological
activities of our identified compound using a human colon
cancer cell line HCT116, which expresses elevated mutant
β-catenin. In addition, in order to determine the specific effects
of inhibitors on Wnt/β-catenin pathway, in addition to the
primary HCT116 colon cancer cell line, we employed HEK293
cells by transfecting them with plasmid pcDNA3.1-β-catenin
(wild-type). This caused them to express β-catenin and specifi-
cally activate the Wnt/β-catenin signaling pathway for the
measurement of the TOP/FOP luciferase activity of the re-
porter gene in this pathway. BC21 showed dose-dependent
inhibitory effects on the β-catenin/Tcf4 driven luciferase acti-
vity in both primary HCT116 and pcDNA3.1-β-catenin
transfecred HEK293 cell lines (Figure 2). The less inhibitory
effect of BC21 on TOP luciferase activity in HEK293 cells
compared to the effect seen in HCT116 cells may be caused by
higher transient expression level of β-catenin in HEK293 cells.
When we next examined the effects of BC21 on cell viability
and clonogenic activity of HCT116 cells, we found that BC21
effectively decreased the HCT116 cell viability and colony
forming activities (Figures 3 and 4). At 5 μM, BC21 was able to
block more than 80% of the colony-forming activity of HCT116
cells (Figure 4). However, 5 μM BC21 exhibited no cytotoxic
effects on normal HEK293 and HUVEC cells. This indicates that
BC21 represents a new anticolon cancer candidate which posses-
ses potential β-catenin/Tcf4 specific antiproliferative activity. We
further examined its effects on c-Myc and cyclin D1, which are
Wnt/β-catenin signal target genes,2and as expected, BC21
reduced the expression of these two important genes.4The
5 μM IC50value of BC21 in the colony forming assay (Figure 4)
was lower than the 15 μM IC50value of BC21 in the cell viability
assay (Figure 3) but was consistent with the concentration in the
competitive binding assay (Figure 7) and luciferase reporter
assay in HCT116 cells (Figure 1A). Possibly, BC21 at lower
concentrations exerts its antiproliferation activity mainly by
blocking Wnt/β-catenin pathway, whereas at higher concen-
trations, other pathways might be affected which result in
Figure 7. Competitive binding assay for GST-Tcf4-1-54 and BC21. A
mixture of 1.5 μM β-catenin and 20 nM tracer (FITC labeled Tcf4
peptide) was incubated in the absence or presence of indicated
concentrations of competitor. Fluorescence polarization values were
recorded after 3 h. The relative binding was calculated as (mPT− mPf)/
(mPC− mPf) × 100.
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cytotoxicity. For example, at concentrations above 50 μM,
BC21 has been reported to inhibit protein phosphatase 2C
and proteasome and to induce apoptosis in human cancer
cells.30,31
In summary, we demonstrated that BC21 represents a new
class of small molecule inhibitors of β-catenin/Tcf4 interaction
and signaling. It interrupts the direct binding of β-catenin to
Tcf4 and down-regulates the expression and activity of Wnt/
β-catenin target genes and gene products. This results in the
induction of HCT116 cell death, particularly clonogenic cell
death. Further modification and optimization of the BC21
chemical structure would be worth pursuing in order to gener-
ate new analogues with improved potency and selectivity against
β-catenin/Tcf4 interactions.
■AUTHOR INFORMATION
Corresponding Author
*Tel: 315-464-7952. E-mail: anji@upstate.edu.
Funding
This study was supported by grants from Connolly Endow-
ment/Hendricks Fund and LUNGevity Foundation and Carol
M. Baldwin Breast Cancer Research Fund.
■ACKNOWLEDGMENTS
This work was supported in part by grants from the Carol M.
Baldwin Breast Cancer Research Fund, the Connolly Endow-
ment/Hendricks Fund, and the LUNGevity Foundation.
■ABBREVIATIONS
Wnt, wingless; Tcf/Lef, T cell factor/lymphoid enhancer
factor; GSK-3β, glycogen synthase kinase-3β; APC, axin/
adenomatous polyposis coli tumor suppressor protein; Fzd,
frizzled; Dsh, disheveled; CSC, cancer stem cell; AML, acute
myelogenous leukemia; CML, chronic myelogenous leukemia;
FP, fluorescence polarization; FITC, fluorescein isothiocyanate.
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