Preferential inhibition of Wnt/β-catenin signaling by novel benzimidazole compounds in triple-negative breast cancer

Article abstract of DOI:10.3390/ijms19051524

Wnt/β-catenin signaling is upregulated in triple-negative breast cancer (TNBC) compared to other breast cancer subtypes and normal tissues. Current Wnt/β-catenin inhibitors, such as niclosamide, target the pathway nonspecifically and exhibit poor pharmacokinetics/ pharmacodynamics in vivo. Niclosamide targets other pathways, including mTOR, STAT3 and Notch. Novel benzimidazoles have been developed to inhibit Wnt/β-catenin signaling with greater specificity. The compounds SRI33576 and SRI35889 were discovered to produce more cytotoxicity in TNBC cell lines than in noncancerous cells. The agents also downregulated Wnt/β-catenin signaling mediators LRP6, cyclin D1, survivin and nuclear active β-catenin. In addition, SRI33576 did not affect mTOR, STAT3 and Notch signaling in TNBC and noncancerous cells. SRI35889 inhibited mTOR signaling less in noncancerous than in cancerous cells, while not affecting STAT3 and Notch pathways. Compounds SRI32529, SRI35357 and SRI35361 were not selectively cytotoxic against TNBC cell lines compared to MCF10A cells. While SRI32529 inhibited Wnt/β-catenin signaling, the compound also mitigated mTOR, STAT3 and Notch signaling. SRI33576 and SRI35889 were identified as cytotoxic and selective inhibitors of Wnt/β-catenin signaling with therapeutic potential to treat TNBC in vivo.

Full text of DOI:10.3390/ijms19051524

International Journal of
Molecular Sciences
Article
Preferential Inhibition of Wnt/β-Catenin Signaling
by Novel Benzimidazole Compounds in Triple-
Negative Breast Cancer
Abhishek Gangrade 1, Vibha Pathak 2 ID , Corinne E. Augelli-Szafran 2, Han-Xun Wei 2,
Patsy Oliver 3, Mark Suto 2 and Donald J. Buchsbaum 1,
*
ID
1
Department of Radiation Oncology, University of Alabama at Birmingham, Birmingham, AL 35294, USA;
abhigan8@gmail.com
2
Department of Chemistry, Drug Discovery Division, Southern Research, Birmingham, AL 35205, USA;
vpathak@southernresearch.org (V.P.); caugelli-szafran@southernresearch.org (C.E.A.-S.);
hweihml@hotmail.com (H.-X.W.); msuto@southernresearch.org (M.S.)
Division of Pulmonary, Allergy and Critical Care, University of Alabama at Birmingham, Birmingham,
AL 35233, USA; poliver@uab.edu
3
*
Correspondence: djb@uab.edu; Tel.: +1-205-934-7077
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 8 May 2018; Accepted: 15 May 2018; Published: 20 May 2018
Abstract: Wnt/
β-catenin signaling is upregulated in triple-negative breast cancer (TNBC)
compared to other breast cancer subtypes and normal tissues. Current Wnt/
β-catenin inhibitors,
such as niclosamide, target the pathway nonspecifically and exhibit poor pharmacokinetics/
pharmacodynamics in vivo. Niclosamide targets other pathways, including mTOR, STAT3 and
Notch. Novel benzimidazoles have been developed to inhibit Wnt/
specificity. The compounds SRI33576 and SRI35889 were discovered to produce more cytotoxicity in
TNBC cell lines than in noncancerous cells. The agents also downregulated Wnt/ -catenin signaling
mediators LRP6, cyclin D1, survivin and nuclear active -catenin. In addition, SRI33576 did not
β-catenin signaling with greater
β
β
affect mTOR, STAT3 and Notch signaling in TNBC and noncancerous cells. SRI35889 inhibited mTOR
signaling less in noncancerous than in cancerous cells, while not affecting STAT3 and Notch pathways.
Compounds SRI32529, SRI35357 and SRI35361 were not selectively cytotoxic against TNBC cell lines
compared to MCF10A cells. While SRI32529 inhibited Wnt/
mitigated mTOR, STAT3 and Notch signaling. SRI33576 and SRI35889 were identified as cytotoxic
and selective inhibitors of Wnt/ -catenin signaling with therapeutic potential to treat TNBC in vivo
β-catenin signaling, the compound also
β
.
Keywords: receptor-targeted therapy; LRP6; triple-negative breast cancer; Wnt/β-catenin
signaling; benzimidazoles
1. Introduction
Of all cancers, breast cancer affects women the most with an incidence of about 12%.
High morbidity of this disease is associated with triple-negative breast cancer (TNBC), which accounts
for 10–20% of all breast cancers [
estrogen and HER2 receptors, commonly targeted by chemotherapy, hormone therapy and HER2
antibody. As a result, these cancers exhibit drug resistance [ ]. Known as the most aggressive breast
1–3] The moniker of the disease derives from lack of progesterone,
4
cancer subtype, TNBC requires new targeted therapies to overcome resistance and lessen probability
of relapse.
Underlying signaling characteristics of TNBC may be exploited for developing novel therapies.
Wnt/β-catenin signaling drives tumorigenesis in a variety of cancers and contributes to progression of
TNBC compared to that of other breast cancer subtypes [
5
–
8]. Wnt/
β-catenin signaling is activated
Int. J. Mol. Sci. 2018, 19, 1524; doi:10.3390/ijms19051524
www.mdpi.com/journal/ijms

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upon binding of secreted cysteine-rich Wnt glycoproteins to LRP5/6 and Frizzled co-receptors,
leading to inactivation of -catenin-phosphorylating protein GSK3 . Lack of phosphorylation enables
-catenin to avoid ubiquitination and degradation, thus allowing the protein to translocate to the
β
β
β
nucleus, bind to TCF/LEF transcription factors and promote transcription of target genes, such as
cyclin D1 and survivin, that drive tumorigenesis and cell proliferation [9–12].
Unlike colorectal cancers, of which 80–90% contain APC mutations, TNBC generally lacks
mutations in Wnt/
activation of -catenin-mediated transcription [
mediators such as Frizzled [14 15] and LRP6 [16]. Both autocrine and paracrine Wnt/
signaling are implicated in TNBC progression [17 19]. LRP6 surface receptor is overexpressed in
β
-catenin pathway related proteins. Instead, pathway dysregulation is mediated by
13] and high expression of Wnt/ -catenin pathway
-catenin
β
7,
β
,
β
–
triple-negative, ER-negative and HER2-negative breast cancers. At the genetic level, LRP6 expression
is more than 6-fold higher in 6 of 7 TNBC cell lines compared to noncancerous MCF10A cells [16].
At the protein level, LRP6 was overexpressed in 4 of 7 TNBC cell lines compared to MCF10A cells.
Little overlap was observed between tumor samples displaying higher LRP6 transcript and those
with higher HER2 transcript, signifying that Wnt/β-catenin signaling is possibly an independent
diagnostic indicator [16]. Inhibition of LRP6 expression and/or activity with Mesd, an LRP6 antagonist,
in MDA-MB-231 cells decreased cell viability, proliferation and colony formation [16]. In addition,
inhibition of LRP6 activity led to decrease in migration and invasion of TNBC cells. Treatment with
Mesd also decreased expression of S100A4, a Wnt/β-catenin signaling target and a contributor to
cancer metastasis [20]. As a result, targeting LRP6 represents an appealing therapeutic strategy for
TNBC. Blood of mice dosed with 5, 10, 20 mg/kg Mesd had the highest concentration after 2 h. Tissue
distribution of Mesd was rapid and widespread as the bioavailability averaged about 60% [16].
Novel small molecule inhibitors targeting Wnt/
developed [21]. Appealing targets have included CREB-binding protein (CBP), a coactivator involved
in -catenin-mediated transcription by binding either -catenin or TCF [22] and porcupine, a Wnt-
β-catenin signaling in cancers have been recently
β
β
modifying enzyme [23]. Currently, agents such as PRI-724, a CBP suppressor and WNT974, a porcupine
inhibitor, are undergoing investigation for use as sole agents in phase I clinical trials in TNBC
patients [24]. Small molecule inhibitor CWP232228 suppressed
TNBC by mitigating -catenin/TCF interaction, inhibiting stem cell proliferation and lowering tumor
bulk [25]. The agent displayed minimal toxicity and no obvious clinical signs in animals. CWP232228,
administered IV at a dose of 200 mg/kg yielded a blood concentration of 0.8 g/mL for 7 h [25].
β-catenin-mediated transcription in
β
µ
Inhibitors such as XAV939 [26] and IWP-2 [27] have been shown to target Wnt/β-catenin activity via
in vitro studies, although they exhibit suboptimal pharmacokinetic and pharmacodynamic (PK/PD)
characteristics. As a result, Wnt/β-catenin pathway inhibitors with improved PK/PD activities are
required for more promising therapies in vivo.
Maintenance of cancer stem cells (CSCs), a subpopulation of cells involved in self-renewal and
regeneration of tumors, has been associated with Wnt/
by CD44+/CD24 markers, have the capacity to generate tumors [28
correlates with the CD44+/CD24 phenotype [30]. In addition, suppression of WNT1 changes the
phenotype and lessens tumor formation and cell migration [31]. Attenuation of Wnt/ -catenin
β-catenin signaling. Breast CSCs, identified
−
,
29]. High -catenin expression
β
−
β
signaling due to inhibition of protein kinase D1 (PKD1) decreased CSC characteristics in breast
cancer cells [32].
High-throughput screening of more than 4000 clinically approved compounds revealed the
anthelmintic agent niclosamide as a potent Wnt/
activity [33]. Currently undergoing clinical trials in colorectal and prostate cancers, niclosamide
inhibits proliferation of various cancers at concentrations that also inhibit Wnt/ -catenin signaling
activities [34 35]. Niclosamide inhibited Wnt/ -catenin signaling in TNBC by inducing LRP6
degradation, enabling Frizzled1 internalization and mitigating interaction between -catenin and
TCF [34 37]. Anthelmintic activities of niclosamide have been shown to influence ATP homeostasis and
uncoupling of oxidative phosphorylation [38 39]. Potentially, niclosamide may inhibit Wnt/ -catenin
β-catenin signaling inhibitor with anticancer
β
,
β
β
–
,
β

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signaling and elicit anthelmintic effects via different mechanisms [40]. In addition, the drug has also
been found to impact mTOR, STAT3 and Notch signaling in various cancers [41 46]. Our laboratory
has previously shown niclosamide affects both Wnt/ -catenin and STAT3 signaling pathways in
–
β
bulk and stem TNBC cells [47]. Niclosamide has shown some efficacy after systemic administration
against various cancers in animal studies but the agent is not well absorbed from the gastrointestinal
tract following oral administration as used for treatment of tapeworm, attributed to its weak
metabolic stability (t1/2 = 29 min in rat liver microsomes) and solubility (1.6
the pharmacokinetic t1/2 should be at least 60 min, requiring twice daily dosing.
Benzimidazole compounds (agents featuring fused benzene and imidazole rings) have been
evaluated for anticancer effects [40 48 50]. Due to the poor bioavailability of niclosamide, several
µM, pH 7.4). Ideally,
,
–
benzimidazole compounds were developed by modifying the structure of niclosamide to improve
in vivo potential. The amide group of niclosamide was constrained to generate the five-membered
ring scaffold for benzimidazole analogs used in this study. A class of compounds, known as
2,5-disubstituted phenyl benzimidazoles, produced cytotoxic and Wnt/β-catenin-inhibiting effects.
These compounds are characterized by substituents located in the 2 and 5 positions of the phenyl ring
in benzimidazoles. The NO2 group in niclosamide, known to elicit toxic effects, was removed from
most of the inhibitors. Since niclosamide was postulated to affect Wnt/
from ATP homeostasis, Mook et al. (Duke University) developed novel compounds to improve
Wnt/ -catenin selectivity. The structure of a selective compound evaluated in this study, SRI33576,
was first identified by Mook et al. to have specificity for Wnt/ -catenin signaling over ATP homeostasis
β-catenin signaling separately
β
β
in colorectal cancer cells [40]. SRI32529 was first developed in a screen of thirty 6-nitrobenzimidazoles
for phosphodiesterase inhibitors. SRI32529 was not selected as one of the effective inhibitors [51].
In addition, SRI32529 was found to have less potency in inhibiting Wnt/
SRI33576 [40]. Neither compound has been evaluated for antiproliferative effects in cancer cells. Also,
the effects of SRI33576 have not been distinguished from those of niclosamide on non-Wnt/ -catenin
β-catenin signaling than
β
signaling pathways, such as mTOR, STAT3 and Notch. Benzimidazole compounds developed at
Southern Research (SR) were screened for effects on cell viability and apoptosis in TNBC and selectivity
for Wnt/β-catenin signaling. SRI33576 and SRI35889 were discovered to have more cytotoxic efficacy
and specificity against Wnt/β-catenin signaling in TNBC than in noncancerous cells.
In the current study, modification of niclosamide has led to the identification of novel
benzimidazole compounds that inhibit Wnt/β-catenin signaling. SRI33576 and SRI35889 are effective
and selective Wnt/ -catenin inhibitors which do not affect mTOR, STAT3 and Notch signaling.
β
We propose these compounds are potentially effective therapeutic agents for cancers dependent on
inhibition of Wnt/β-catenin signaling.
2. Results
2.1. SRI33576 and SRI35889 are More Cytotoxic to TNBC Than to Noncancerous MCF10A Cells
Five of the 2,5-disubstituted phenyl benzimidazoles were tested for cytotoxicity and inhibition of
Wnt/β-catenin signaling (Table 1). First, it was important to compare efficacies of the SR compounds
in inhibiting proliferation of TNBC and noncancerous cells. Both SRI33576 and SRI35889 exhibit more
cytotoxicity in TNBC cell lines than in noncancerous MCF10A cells. IC50 values for SUM149, SUM159,
MDA-MB-231 and MDA-MB-468 TNBC cell lines treated with SRI33576 ranged from 1.9 to 3.2
µM,
compared to 5.0
from 1.1 to 2.4
µ
M for MCF10A cells. IC50 values for TNBC cell lines treated with SRI35889 ranged
µ
M, compared to 5.4
µ
M for treated MCF10A cells (Figure 1). IC50 values for TNBC cell
M,
M. Thus, compared to SR agents, niclosamide exhibits
less selectivity in eliciting cytotoxicity in TNBC cells versus noncancerous cells.
lines treated with niclosamide, an inhibitor of multiple signaling pathways, ranged from 0.3 to 1.0
while the IC50 value for MCF10A cells was 1.0
µ
µ

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Table 1. Scaffold and structures of 2,5-disubstituted phenyl benzimidazoles. Compounds SRI32529,
SRI33576, SRI35357, SRI35361 and SRI35889 were evaluated for cytotoxicity and inhibition of
Wnt/β-catenin signaling.
R1
H
H
Cl
F
R2
NO2
CF3
H
Cl
Cl
R3
H
H
Cl
H
H
R4
R7
Cl
Cl
F
Cl
Cl
SRI32529
SRI33576
SRI35357
SRI35361
SRI35889
OH
OH
OH
OH
OH
Cl
Figure 1. Effects of benzimidazole compounds on viability of triple negative breast cancer (TNBC) and
noncancerous MCF10A cells. Cells were treated with 0–5 M SRI32529 ( ), SRI33576 ( ), SRI35357 ( ),
SRI35361 (D), SRI35889 (E) and niclosamide (F) for 72 h. Assays were performed three times.
µ
A
B
C

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2.2. SRI33576 and SRI35889 Induce Apoptosis in TNBC
Apoptosis of TNBC cell lines was evaluated by Annexin V/PI assay following treatment with the
agents. Compounds SRI33576 and SRI35889 induced significantly more apoptosis in MDA-MB-231
and MDA-MB-468 cells treated with 5
not induce significantly more apoptosis than DMSO in either cell line (Figure 2A,B). In addition,
a higher percentage of MDA-MB-231 cells treated with 5 M SRI33576 (24% of total) and SRI35889
µM for 48 h. SRI35361 was the only compound which did
µ
(26.5%) resided in the late apoptotic phase as compared to vehicle control-treated cells (8.1%). This was
comparable to cells treated with niclosamide (29.5%) as shown in Figure 2C.
Figure 2. Apoptosis of (A) MDA-MB-231 and (B) MDA-MB-468 TNBC cells following treatment with
Southern Research (SR) compounds and niclosamide for 48 h. Apoptosis was measured by staining
cells with Annexin V and PI prior to analysis by flow cytometry. Apoptosis of cells treated with
compounds was compared to DMSO-treated cells. (
cells treated with DMSO, 5 M SRI33576, SRI35889 and niclosamide for 48 h. Assays were performed
three times. * p < 0.05, ** p < 0.01.
C) Representative scatterplots of MDA-MB-231
µ

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2.3. SRI33576 and SRI35889 Inhibit Wnt/
β
-Catenin Signaling in TNBC Cell Lines But Not Noncancerous Cells
We sought to determine specificity of the two compounds for inhibition of Wnt/
β-catenin
signaling in TNBC over noncancerous cells. As niclosamide is an inhibitor of several pathways,
we compared the effects of this agent with the SR benzimidazoles. Modulation of Wnt/
β
-catenin
-catenin
pathway-related proteins by the compounds was detected by Western blotting. Nuclear
β
promotes transcription of pro-tumorigenesis genes and cytoplasmic
prognosis in breast cancer patients [52]. To detect cytoplasmic/nuclear active (non-phosphorylated)
-catenin, we used a monoclonal antibody which recognizes non-phosphorylated sites Ser-37 and
Thr-41. The antibody has been shown to visualize production of active -catenin via the canonical
Wnt/ -catenin pathway during murine embryogenesis [53]. Interestingly, active -catenin was
observed in the cytoplasm of MDA-MB-468 cells but not MDA-MB-231 cells. SRI35889 decreased
cytoplasmic -catenin (Figure 3A). SRI33576 and SRI35889 decreased nuclear active -catenin
expression following 18 h treatment (Figure 3B). Immunocytochemistry also revealed a decrease
in nuclear active -catenin expression with SRI33576 and SRI35889 treatment in MDA-MB-231 and
β-catenin is associated with poor
β
β
β
β
β
β
β
MDA-MB-468 cells (Figure 4).
Because the receptor LRP6 is an initiator of Wnt/β-catenin signaling, we sought to assess
whether the compounds inhibited expression of the phosphorylated (active) form. SRI33576 decreased
phosphorylated LRP6 at 5 M in both TNBC cell lines but not MCF10A cells. SRI35889 decreased
phosphorylated LRP6 at 2.5 and 5 M in both TNBC cell lines but not MCF10A cells. Niclosamide
inhibited decreased phosphorylated LRP6 in MCF10A cells (Figure 5).
µ
µ
Figure 3. Effects of SR compounds and niclosamide on active
) Cytoplasmic and ( ) nuclear active -catenin expression was evaluated in the cells following 18 h
treatment with 5 M concentrations of the compounds and cytoplasmic-nuclear fractionation. Intensity
of nuclear active -catenin bands in ( ) were quantified by densitometry with ImageJ software and
normalized to corresponding band of lamin A/C. Western blotting was performed on lysates (40 ug
loaded). Active -catenin was not detected in cytoplasm of MDA-MB-231 cells. Tubulin and lamin
β-catenin expression in TNBC cells.
(
A
B
β
µ
β
B
β
A/C were detected to ensure proper separation of cytoplasmic and nuclear fractions. Western blotting
was performed two times.

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Figure 4. Cont.

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Figure 4. Immunocytochemistry (ICC) of active
IF of active β-catenin in (A) MDA-MB-231 and (B
β
-catenin following treatment with SR compounds.
) MDA-MB-468 cells treated with 0.05% DMSO,
M SRI33576, SRI35889 and niclosamide. Cells were counterstained with Hoechst 33342 (blue).
) Fluorescence intensities were quantified by ImageJ and means were compared to DMSO-treated
cells (n = 3). Scale bar = 100 µm.
5
µ
(
C
Figure 5. Cont.

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Figure 5. Effect of SR compounds on Wnt/
and (C) MCF10A cells treated with 2.5 or 5 µ
β
-catenin signaling in (
M of compounds for 18 h. Hes1, pSTAT3 (Y705), STAT3,
p4EBP1 (p65), 4EBP1, pLRP6, cyclin D1, survivin and tubulin in whole cell lysates were detected by
immunoblotting. ( ) Densitometry readings for select proteins Hes1, pSTAT3, p4EBP1, pLRP6, LRP6,
A) MDA-MB-231, (B) MDA-MB-468
D
cyclin D1 and survivin according to Western blots. Results are averages of two replicate experiments
and normalized to tubulin. 0.05% DMSO was used as vehicle control. Western blotting was performed
two times.
2.4. SRI33576 and SRI35889 Have Less Effect on mTOR, STAT3 and Notch Signaling Than Niclosamide
Niclosamide affects mTOR, STAT3 and Notch pathways, potentially conferring increased toxicity
in normal tissues. Specificity of benzimidazole compounds was evaluated by assessing their effects
on non-Wnt/β-catenin signaling pathways mTOR, STAT3 and Notch. Cell lines were treated with
the compounds and assayed by Western blotting for changes in protein levels associated with
these pathways. Niclosamide downregulated STAT3 phosphorylation in TNBC and MCF10A cells,
whereas SRI33576 and SRI35889 produced noticeably less of an effect (Figure 5). SRI33576 inhibited

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phosphorylation of 4EBP1 at 5
µM in both TNBC cell lines but it did not yield such a change in MCF10A
cells. SRI35889 inhibited 4EBP1 phosphorylation at 2.5 and 5
less of a change in MCF10A cells at 2.5 M. Neither of the compounds downregulated phosphorylation
of 4EBP1 as much as niclosamide (Figure 5). Expression of the pSTAT3 and total STAT3 isoform
µM in both TNBC cell lines but it produced
µ
β
β
(lower band), associated with tumor-suppressor function [54], was markedly decreased by niclosamide
but not by SRI33576 or SRI35889 in MDA-MB-468 and MCF10A cells. SRI33576 and SRI35889 decreased
expression of Wnt/
β-catenin pathway proteins cyclin D1 and survivin in TNBC cells at 2.5 and 5
µM
concentrations. At 2.5
µM concentration, SRI35889 did not affect cyclin D1 or survivin expression as
much as niclosamide in MCF10A cells (Figure 5).
3. Discussion
Despite the fact Wnt/β-catenin signaling is not driven by mutations encoding pathway-related
proteins, Wnt/ -catenin signaling represents an appealing target for inhibitors due to higher activity
β
in TNBC than in normal tissues. Niclosamide has been established as an effective inhibitor of
Wnt/β-catenin signaling in several cancers, including colon, breast and myeloma [36,55–58]. Studies
indicated niclosamide exhibits no carcinogenic effects in animals [38]. However, several studies
have shown the agent elicits genotoxic effects [59
Wnt/ -catenin signaling in colorectal cancer cells by decreasing cytosolic axin2 and
overall c-myc, survivin and cyclin D1 levels [40]. However, it was not known whether nuclear
the major driver of canonical Wnt/ -catenin signaling, was abrogated by the agent in colorectal cancer
–
61]. SRI33576 was previously found to inhibit
-catenin and
-catenin,
β
β
β
β
cells. To our knowledge, this is the first study evaluating the effects of 2,5-disubstituted phenyl
benzimidazoles on TNBC cell lines.
Benzimidazoles SRI33576 and SRI35889 were identified with improved specificity for Wnt/
β
-catenin signaling in TNBC cell lines than in noncancerous cells. Compared to niclosamide,
both agents preferentially inhibit Wnt/ -catenin signaling, indicated by lower expression of Wnt/
-catenin pathway proteins survivin and cyclin D1. A member of the inhibitor of apoptosis (IAP)
family, survivin controls cell division, mitigates apoptosis and suppresses active caspases [62 66].
Overexpression of cyclin D1, a driver of G1/S transition during cell cycle progression, has been
observed in more than half of breast cancers [67]. Inhibition of Wnt/ -catenin signaling in TNBC
is associated with promotion of apoptosis [ ]. Both agents increased apoptotic levels in TNBC cells,
potentially due to their effects on Wnt/ -catenin signaling. In addition, it appears that compounds
SRI33576 and SRI35889 decrease active -catenin expression via targeting of LRP6 in TNBC cells over
noncancerous MCF10A cells. As TNBC is characterized by high LRP6 expression [16 20], targeting of
β
β
–
β
5
β
β
,
this receptor by the compounds underscores their therapeutic relevancy. In addition, the inhibitors
exhibited less pronounced effects on mTOR signaling in TNBC cell lines than in noncancerous cells.
Neither SRI33576 nor SRI35889 affected Notch or STAT3 signaling activities in TNBC cell lines.
It is known autocrine Wnt/
β-catenin signaling drives TNBC cell proliferation [17]. Both SRI33576
and SRI35889 inhibited autocrine Wnt/β-catenin signaling using established human cancer cell lines.
However, paracrine signaling via activity of ligands such as Wnt3a may also play a role in TNBC
growth [19] and it remains to be elucidated whether SRI33576 and SRI35889 can inhibit this pathway.
We previously evaluated other novel analogs of niclosamide that inhibited Wnt/
mTOR signaling [68 69]. SRI35889 and to a lesser extent, SRI33576, were found to inhibit mTOR
signaling in addition to Wnt/ -catenin activity, which may confer increased cytotoxic potency in TNBC.
Recently, a study supported the use of dual inhibition of both PI3K/AKT/mTOR and Wnt/ -catenin
pathways for synergistic effects in TNBC [70]. Interestingly, treatment of TNBC with the pan-PI3K
inhibitor buparlisib increased Wnt/ -catenin activity via increased expression of Wnt/ -catenin
β-catenin and
,
β
β
β
β
pathway mediators such as FZDs, Wnt ligands, LRP4/6 and porcupine. Unfortunately, the inhibitor
was found to elicit toxic effects, such as anxiety, pneumonititis and liver toxicity, in humans.
An appealing target of new therapies, CSCs participate in tumor recurrence, metastasis and
resistance to chemotherapies in breast cancer. Evidence indicates β-catenin expression correlates

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with chemoresistance of TNBC as
β-catenin knockdown sensitized TNBC cell lines to doxorubicin-
or cisplatin-mediated cell death [13]. Wnt/
and salinomycin were found to effectively target breast and ovarian CSCs [47
Wnt/ -catenin signaling mediates self-renewal and migration of CSCs, SRI33576 and SRI35889 may
represent clinically useful therapeutic agents.
β-catenin pathway inhibitors niclosamide, its analogs
,
69 71 75]. Because
, –
β
This study supports SRI33576 and SRI35889 for further investigation in animal studies.
These compounds must be further evaluated for their PK/PD properties to elucidate their potential
as cancer-targeting agents. In addition, both compounds may be combined with chemotherapy or
immunotherapeutic agents to further enhance their therapeutic effects.
4. Materials and Methods
4.1. Compound Synthesis
Synthesis of compounds SRI32529 and SRI33576 have been previously described [40,51], Figure 6.
Figure 6. Synthesis of compounds SRI32529 and SRI33576. Method A. NaHSO3, DMSO, 210 ◦C, 1 h;
◦
Method B: Na2S2O5. DMF, 170 C, 15 min.
Method A utilized the condensation of diamines and aldehydes in presence of sodium sulfite and
DMSO at 210 ◦C for 1 h. An improved synthetic method (Method B) was determined which utilized
microwave conditions in the reaction of the substituted diamines and aryl rings in the presence of
sodium metabisulfite and dry DMF. Method B was preferred versus Method A due to shorter reaction
times (15 min) and higher yields.
4.1.1. Method A
A mixture of an appropriate 1,2-phenylenediamine (1.0 eq), appropriate aldehyde (1.0 eq) and
sodium bisulfite (1.0 eq) in 10 mL of DMSO was heated at 210 ◦C for 1 h. The reaction mixture was
filtered, concentrated in vacuo and purified on a purification system using 10–50% hexanes and EtOAc.
4.1.2. Method B
A mixture of an appropriate 1,2-phenylenediamine (1.0 eq), appropriate aldehyde (1.0 eq) and
sodium metabisulfite (1.0 eq) in 8 mL of DMF was heated in a CEM microwave at 170 ◦C for 15 min.
The reaction mixture was filtered, concentrated in vacuo and purified on a purification system using
10–50% hexanes and EtOAc.
4.1.3. 2-(5,7-Dichloro-3H-benzo[d]imidazol-2-yl)-4-fluorophenol (35357)
This compound was prepared from 3,5-dichlorobenzene-1,2-diamine and 5-fluoro-2-
1
hydroxybenzaldehyde using Method A. Yield 21%. TLC Rf = 0.40 (Hexanes–EtOAc, 2:1). H NMR
(400 MHz, DMSO-d6)
δ 13.40 (s, 1H), 7.95 (s, 1H), 7.71 (s, 1H), 7.48 (s, 2H), 7.30 (ddd, J = 9.2, 8.1, 3.1 Hz,
1H), 7.09 (dd, J = 9.1, 4.7 Hz, 1H). HRMS m/z calcd. for C13H7Cl2FN2O [M + H]+: 296.9992, found:
296.9993.
4.1.4. 2-(5-Chloro-6-fluoro-1H-benzo[d]imidazol-2-yl)-4-fluorophenol (35361).
This compound was prepared from 4-chloro-5-fluorobenzene-1,2-diamine and 5-fluoro-2-
1
hydroxybenzaldehyde using Method A. Yield 64%. TLC Rf = 0.45 (Hexanes–EtOAc, 2:1). H NMR

Int. J. Mol. Sci. 2018, 19, 1524
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(400 MHz, DMSO-d6)
δ
12.60 (s, 1H), 8.54 (s, 1H), 8.22–8.11 (m, 2H), 7.82 (d, J = 8.9 Hz, 1H), 7.24–7.09
(m, 1H). HRMS m/z calcd. for C13H7ClF2N2O [M + H]+: 280.0215, found: 280.0215.
4.1.5. 4-Chloro-2-(5,6-dichloro-1H-benzo[d]imidazol-2-yl)phenol (35889)
This compound was prepared from 4,5-dichlorobenzene-1,2-diamine and 5-chloro-2-
1
hydroxybenzaldehyde using Method B. Yield 92%. TLC Rf = 0.30 (Hexanes–EtOAc, 1:1). H NMR
(400 MHz, DMSO-d6)
δ 12.99 (s, 1H), 8.24 (s, 1H), 7.95 (s, 1H), 7.70 (q, J = 1.8 Hz, 1H), 7.45–7.40 (m, 2H),
7.08 (dd, J = 8.9, 2.4 Hz, 1H). HRMS m/z calcd. for C13H7Cl3N2O [M + H]+: 312.9697, found: 312.9696.
HPLC: 100% (tR = 3.05 min).
4.2. Materials
All experimental compounds were synthesized at Southern Research (Birmingham, AL, USA)
and dissolved in DMSO at stock concentrations of 10 mM. Antibodies purchased from Cell Signaling
Technologies (Danvers, MA, USA) include phospho-LRP6 (#2568), LRP6 (#2560), cyclin D1 (#2978),
survivin (#2808), phospho-4EBP1 (#9451), 4EBP1 (#9452), phospho-STAT3 (Tyr705) (#9145), STAT3
(#4904), Hes1 (#11988), α-tubulin (#2144) and lamin A/C (#2032). Tissue culture media were obtained
from ThermoFisher (Waltham, MA, USA) and FBS was obtained from Atlanta Biologicals (Flowery
Branch, GA, USA). Niclosamide, insulin, cholera toxin, hydrocortisone, protease and phosphatase
inhibitor cocktails were purchased from Sigma-Aldrich (St. Louis, MO, USA). Epidermal growth factor
was obtained from PeproTech (Rocky Hill, NJ, USA).
4.3. Cell Culture
Nonmalignant mammary cell line MCF10A and TNBC cell lines MDA-MB-231 and MDA-MB-468
were purchased from ATCC (Manassas, VA, USA). MCF10A cells were cultured in DMEM/F-12
(1:1) supplemented with 5% horse serum, 10 µg/mL insulin, 0.5 µg/mL hydrocortisone, 0.02 µg/mL
epidermal growth factor, 0.1 µg/mL cholera toxin. MDA-MB-231 and MDA-MB-468 cell lines were
cultured in DMEM with 10% FBS. SUM149 and SUM159 cell lines were purchased from Asterand
(Detroit, MI, USA) and cultured in DMEM/F12 with 5% FBS, hydrocortisone and insulin. All cell lines
were maintained in an incubator at 37 ◦C and 5% CO2.
4.4. In Vitro Cytotoxicity
MCF10A and TNBC cells (2 × 103) were seeded with appropriate growth media containing 10%
FBS in each well of tissue culture-treated 96-well black plates (Corning #3904) (Corning, NY, USA)
16 h prior to treatment. Cells were treated with SR compounds for 3 days at concentrations of 0–5 µM.
Cell viability was then evaluated with Cell Titer Glo (Promega, Madison, WI, USA) and luminescence
was measured using a TOPCount NXT plate reader (PerkinElmer, Waltham, MA, USA). Curves were
fit according to nonlinear regression and IC50 values were calculated using GraphPad Prism 7. Cells
were treated in quadruplicates and experiments were performed three times.
4.5. Apoptosis Assay
Cells were seeded in 6-well tissue-culture treated plates (Corning) for overnight incubation.
Culture media was replaced with growth media containing 1% FBS. Cells were treated with compounds
for 48 h prior to harvest. Apoptosis/necrosis was detected by the FITC Annexin V Apoptosis Detection
Kit I from Becton Dickinson (San Jose, CA, USA). Cells positive for Annexin V, propidium iodide (PI)
or both were all designated as apoptotic.
4.6. Western Blot Analysis
Cells were seeded in 6-well tissue-culture treated plates for 48 h until 80% confluency prior to
treatment. Whole cell extracts were prepared with Laemmli buffer (5% SDS, 10% glycerol, 0.5 M Tris-Cl)

Int. J. Mol. Sci. 2018, 19, 1524
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supplemented with protease and phosphatase inhibitors and equivalent amounts of proteins were
separated by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride (PVDF) membrane
(Bio-Rad, Hercules, CA, USA) and blocked with 5% BSA in tris-buffered saline (TBS) supplemented
with 0.1% Tween-20 for 1 h. Membranes were incubated with primary antibody overnight on a shaker
at 4 ºC. They were then washed and incubated with secondary antibody for 1 h. Bands were visualized
with the Enhanced Chemiluminescence Detection reagents (PerkinElmer).
Cells were lysed using the CERI and CERII reagents of the cytoplasmic-nuclear extraction kit
(ThermoFisher), to obtain cytoplasmic fractions following treatment. Nuclear fractions were isolated
by using 1
×
Laemmli buffer and sonication for a few seconds. Membrane was incubated overnight
with active β-catenin (MilliporeSigma, Burlington, MA, USA), lamin A/C and tubulin antibodies.
4.7. Immunocytochemistry
Cells were seeded on coverslips for 48 h prior to treatment with DMSO and SR compounds for
18 h. Cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min and permeabilized
with 0.5% Triton X-100 for 20 min. Following blocking with 3% BSA for 30 min, cells were incubated
◦
overnight with active
β
-catenin antibody (MilliporeSigma) at 4 C. Cells were incubated with Alexa 488
goat anti-mouse secondary antibody (1:1000) at room temperature for 1 h. To counterstain, cells were
incubated with 1:5000 Hoechst 33342 for 20 min. Coverslips were mounted with Prolong Gold
Antifade Reagent (ThermoFisher) and cells were observed with Nikon A1R confocal microscope
(Nikon Instruments, Melville, NY, USA). Images were obtained at 20× magnification.
4.8. Statistical Analysis
Statistical analyses were performed by the unpaired Student t test. p < 0.05 values were considered
significantly different. Results are shown as mean ± SD.
Author Contributions: A.G. conceived, designed, performed the experiments, analyzed the data and wrote the
manuscript; V.P., H.-X.W., C.E.A.-S., M.S. were involved in the design, synthesis and scale-up of the benzimidazole
compounds; M.S. and D.J.B. contributed reagents/materials/analysis tools. D.J.B. and P.O. edited the manuscript.
Acknowledgments: Supported by Breast Cancer Research Foundation of Alabama and UAB Comprehensive
Cancer Center support grant, NCI P30CA013148. Funds were received for covering the costs to publish in
open access.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
BSA
Bovine Serum Albumin
CSC
Cancer Stem Cell
FBS
Fetal Bovine Serum
DMSO
ECL
ICC
Dimethyl Sulfoxide
Enhanced Chemiluminescence
Immunocytochemistry
LRP6
Low-density Lipoprotein Receptor-Related Protein 6
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
T-cell factor/Lef-1 Family of Transcription Factors
Triple-Negative Breast Cancer
SDS-PAGE
TCF/LEF
TNBC
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