Discovering alkylamide derivatives of bexarotene as new therapeutic agents against triple-negative breast cancer

Article abstract of DOI:10.1016/j.bmcl.2017.12.033

Triple-negative breast cancer (TNBC) has been reported to be correlated with high expression of proliferation markers as well as constitutive activation of metastasis-relevant signaling pathways. For many years, breast cancer researchers have been investi

Full text of DOI:10.1016/j.bmcl.2017.12.033

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovering alkylamide derivatives of bexarotene as new therapeutic
agents against triple-negative breast cancer
⇑
Luxi Chen, Chao Long, Jennifer Nguyen, Dhiraj Kumar, Jiyong Lee
Department of Chemistry and Biochemistry, The University of Texas at Dallas, 800 W. Campbell Rd., Richardson, TX 75080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Triple-negative breast cancer (TNBC) has been reported to be correlated with high expression of prolifer-
ation markers as well as constitutive activation of metastasis-relevant signaling pathways. For many
years, breast cancer researchers have been investigating specific and effective methods to treat or to con-
trol the development of TNBC, but promising therapeutic options remain elusive. In this study, we have
demonstrated that alkylamide derivatives of bexarotene DK-1–150 and DK-1–166 induce apoptotic cell
death in TNBC cell lines without causing cytotoxicity in the normal mammary epithelial cell line.
Furthermore, the bexarotene derivatives also showed significant effects in inhibiting TNBC cell prolifer-
ation and migration, modulating cancer stem cell markers expressions, as well as limiting the epithelial-
mesenchymal transition (EMT) activities of TNBC cell lines in terms of downregulating EMT marker and
blocking nuclear translocation of b-catenin. Therefore, we propose the alkylamide derivatives of bexaro-
tene as potential candidates for novel anticancer therapeutics against TNBC.
Received 25 August 2017
Revised 25 November 2017
Accepted 13 December 2017
Available online xxxx
Keywords:
Triple-negative breast cancer
Apoptosis
Cancer stem cell
Epithelial-mesenchymal transition
Anti-cancer therapeutics
Ó 2017 Elsevier Ltd. All rights reserved.
Triple-negative breast cancer (TNBC), characterized by mam-
mary tumors that lack detectable expressions of estrogen receptor
(ER), progesterone receptor (PR) and human epithermal growth
factor receptor 2 (HER2),1 contributes to 15–20% of all breast can-
cer cases diagnosed.2,3 This breast cancer subtype is associated
with high expression of proliferation markers such as Ki-67,4 and
activation of the b-catenin pathway.5 TNBC is further classified
into 6 subtypes based on the gene expression profiles, namely
basal-like 1 (BL1), basal-like 2 (BL2), immunomodulatory (IM),
mesenchymal (M), mesenchymal stem-like (MSL), and luminal
androgen receptor (LAR) subtype. Among these TNBC subgroups,
mesenchymal (M) and mesenchymal stem-like (MSL) subtypes
are enriched in genes associated with the epithelial-mesenchymal
transition (EMT) and growth factor signaling pathways.6 In con-
trast to other subtypes of breast cancer, TNBC is more aggressive
and invasive, more resistant to chemotherapies, and also possesses
elevated EMT activity and a higher percentage of cancer stem cell
(CSC) population. Such properties of TNBC tumors give rise to
resistance to conventional anti-cancer therapies, metastasis and
tumor relapse.
effective treatments, most TNBC patients with advanced diseases
experienced relapse shortly after neoadjuvant chemotherapy,
frequently with visceral metastases and a short life expectancy.10
Particularly, advanced TNBC has a higher rate of early recurrence
and distant metastasis to the brain and lungs, as compared to other
breast cancer subtypes.11–13 Thus, identifying effective, tailored
and less toxic therapeutic options for TNBC is a pressing need. In
this study, we are demonstrating the alkylamide derivatives of
bexarotene as a new class of therapeutic agents targeting TNBC,
imposing anti-cancer activities through induction of apoptotic cell
death, suppression of cell proliferation and migration, as well as
limiting EMT and CSC properties of TNBC cells.
The bexarotene alkylamide derivatives (Fig. 1A), DK-1–150 and
DK-1–166 were synthesized by reacting acyl chloride of bexaro-
tene with 1-(2-aminoethyl)pyrrolidine or N,N-diethylethylenedi-
amine, respectively. Cellular toxicity of the alkylamide
derivatives of bexarotene was assessed in two of the invasive TNBC
cell lines, MDA-MB-231 (MSL subtype) and BT549 (M subtype), via
the evaluation of post-treatment cell viabilities (Fig. 1B). In addi-
tion to the TNBC cell lines, we also used normal mammary epithe-
lial cell line MCF10A and non-TNBC breast cancer cell MCF-7, in
order to examine if the cytotoxic effects induced by bexarotene
derivatives are selectively high in TNBC cells. MTT cell viability
assay results showed that 5 mM of bexarotene failed to cause signif-
icant reduction in cell viabilities in MCF10A, and in the three breast
cancer cell lines. However, 5 mM of DK-1–150 or DK-1–166 was
Although a variety of single agents and combination regimens
are available for breast cancer prevention and/or treatment, none
of them are recommended specifically for TNBC.7–9 Without
⇑
Corresponding author.
E-mail address: jiyong.lee@utdallas.edu (J. Lee).
https://doi.org/10.1016/j.bmcl.2017.12.033
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Chen L., et al. Bioorg. Med. Chem. Lett. (2017), https://doi.org/10.1016/j.bmcl.2017.12.033

2
L. Chen et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Fig. 1. Bexarotene derivatives display cytotoxicity against TNBC cell lines. A) Chemical structures of bexarotene and its alkylamide derivatives, DK-1–150 and DK-1–166. B)
Cytotoxicity evaluation by MTT assay comparing post-treatment cell viabilities among MCF10A and breast cancer cell lines. (*p < .05, **p < .005). C) Activation of caspase-3 of
TNBC cell lines by the bexarotene derivatives. b-Actin was used as endogenous loading control. Densitometry measurement for quantification was performed, and the results
were normalized to DMSO-treated samples. Numbers stated underneath indicate expression fold-change values from ImageJ analysis. See Supplementary Fig. 2 for the
activation of caspases-8 and -9.
able to induce about 40% reduction in MCF-7 cell viability, and
about 60% reduction in cell viability of MDA-MB-231 and BT549
cells. The EC50 of bexarotene on TNBC cells is estimated to be
higher than 56 mM while the EC50 of DK-1–150 and DK-1–166 on
TNBC cells are less than 10 mM (Fig. 1B; Supplementary Fig. 1).
Importantly, both derivatives showed minimal cytotoxicity on
MCF10A. These results suggest the two bexarotene derivatives
are more cytotoxic than bexarotene to breast cancer cells, but
may not be harmful to the normal breast epithelial cells.
To examine if the cytotoxicity of the derivatives is due to induc-
tion of apoptosis, the activation of caspases in TNBC cells was eval-
uated. Bexarotene and the two derivatives significantly induced
activation of caspase-8, caspase-9, and caspase-3 in TNBC cell lines
by at least 2 folds (Fig. 1C; Supplementary Fig. 2), suggesting that
the derivatives are able to initiate both intrinsic and extrinsic
apoptotic cascades, as well as induce both early and late apoptosis
in TNBC cells. In contrast, no significant activation of caspases was
observed in MCF10A and MCF-7 cell lines, upon treatments with
bexarotene or either of the derivatives (Supplementary Fig. 2).
Interestingly, BT549 cell line responds better to DK-1–150 or DK-
1–166 than to bexarotene of the same dosage (Fig. 1C; Supplemen-
tary Fig. 2). Furthermore, both MDA-MB-231 and BT549 cells trea-
ted with 5 mM DK-1–150 showed increased levels of cleaved PARP
(Fig. 4B), suggesting induction of DNA damage by the derivative
through apoptosis mechanism.
To assess the effect of the bexarotene derivatives on breast can-
cer cell proliferation, we performed adherent colony-forming assay
and found that both DK-1–150 and DK-1–166 led to 80% reduction
of colony formation in MCF-7 cell line at 5 mM, while bexarotene
did not affect the colony-forming capacity of MCF-7 (Fig. 2A). To
further evaluate if the derivatives can inhibit anchorage-indepen-
dent proliferation, we conducted the soft-agar colony-forming
assay. Similar to the adherent colony-forming assay, soft-agar col-
ony-forming assay results reflected that both bexarotene deriva-
tives at 2 mM inhibited the formation of colonies by at least 90%,
while 10 mM bexarotene failed to inhibit colony formation in
MDA-MB-231 cells (Fig. 2B; Supplementary Fig. 3). TNBC is well
known for its high metastatic potential. To assess the in vitro
anti-metastatic activity of bexarotene derivatives on TNBC cell
lines, we performed wound-healing assay on MDA-MB-231 cells.
While DMSO- or bexarotene (5 mM)-treated MDA-MB-231 cells
were able to migrate, and eventually close the ‘‘wound gap” after
24 h incubation, cells treated with 2 mM DK-1–150 or 2 mM DK-
1–166 had significantly lowered capability of cell migration (Fig.
2C; Supplementary Fig. 4). Collectively, these data prove that the
bexarotene derivatives are able to suppress TNBC cell proliferation
and migration.
It has been proposed that CSCs, while existing as a minor pop-
ulation, exert resistance to the contemporary anti-cancer thera-
pies, and hence become a potential root of tumor recurrence or
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L. Chen et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
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Fig. 2. Suppression of proliferation and migration of breast cancer cells by the alkylamide derivatives of bexarotene. A) Adherent colony-forming assay performed on MCF-7
cells. Number of colonies was counted at the end of 7-day treatment, and the results were normalized with untreated samples (**p < 0.005). B) Soft-agar colony-forming assay
performed on MDA-MB-231 cells (see quantitative analysis in Supplementary Fig. 3). Scale bar indicates 200
lm. C) Wound-healing assay performed on MDA-MB-231 cells,
and the images taken at 24 h. See Supplementary Fig. 4 for the cells treated with higher dose (5 mM) of DK-1–150 or DK-1–166.
metastasis.14–16 Previous studies have reported that such CSC pop-
ulation is enriched in the tissues or cell lines of triple-negative
breast cancer than those of other breast cancer subtypes, and such
CSC population contributes to the aggressiveness and poor progno-
sis of TNBC.17–19 To assess the derivatives’ activities on modulating
CSC properties, we conducted the mammosphere formation
assay20,21 that evaluates the self-renewal capability of cancer cells.
The result showed that while DMSO-treated and bexarotene-trea-
ted MCF-7 cells still retained capability to form mammospheres,
cells treated with 2 mM DK-1–150 or 2 mM DK-1–166 failed to form
detectable mammospheres (Fig. 3A and B). We have also evaluated
the expression levels of CSC-related markers such as c-Myc and
KLF4, Nanog, Oct4A and Sox-2. These stemness-associated tran-
scription factors contribute to pluripotency and cannot be replaced
by other tumor-promoting oncogenes.22–24 Western blot analysis
on MCF-7 cells indicated that 5 mM DK-1–150 or 5 mM DK-1–166
downregulated protein expressions of c-Myc and KLF4 by about
10–30%; downregulated protein expression of Nanog by about
20%. Particularly, 5 mM DK-1–150 caused about 60% reduction in
Sox-2 expression, while 5 mM DK-1–166 induced approximately
50% decrease in Oct4A protein expression (Fig. 3C). Western blot
analysis on MDA-MB-231 cells revealed that 5 mM DK-1–150 or
5 mM DK-1–166 downregulated protein expressions of KLF4 by
about 50%, while no significant effect was observed in c-Myc
expressions in TNBC cells (Fig. 3D). Suppression of KLF4 by
DK-1–150 or DK-1–166 is significant as KLF4 has been known to
be required to maintain CSC properties and breast cancer cell
invasion and migration.25 There were no substantial changes in
both c-Myc and KLF4 expressions in bexarotene-treated TNBC
cells. These results suggest that both bexarotene derivatives
not only effectively suppress the self-renewal capacity of
stem cell-like breast cancer cells, but also selectively
downregulate stemness-associated transcription factors in TNBC
cell lines.
EMT is a key developmental program that is often activated
during cancer invasion and metastasis. It is also suggested that
EMT is a key event by which cancer stem cells are generated.26
As the bexarotene derivatives show CSC-inhibitory activities, we
sought to examine if the derivatives can also affect EMT properties
of TNBC cell lines. Both DK-1–150 and DK-1–166 at concentration
of 5 mM were able to induce downregulation of mesenchymal
markers, vimentin and slug in TNBC cell lines at a similar or
slightly better effectiveness, as compared to 5 mM bexarotene.
Moreover, the two TNBC cell lines respond differently to the two
bexarotene derivatives, in terms of changes in the mesenchymal
markers expression. The suppression of slug by the derivatives
was observed in BT549, but not in MDA-MB-231. In addition,
MDA-MB-231 responded well to both derivatives, but BT549
Fig. 3. Modulation of cancer stem cell (CSC) properties of breast cancer cell lines by bexarotene derivatives. A) Mammosphere-formation assay performed on MCF-7 cells.
Scale bar indicates 200 mm. (B) Number of spheres larger than or equal to 60 mm was counted for quantitation. C, D) MCF-7 cells (C) and MDA-MB-231 cells (D) were subjected
to Western blot analysis on post-treatment expressions of selected CSC markers. b-Actin was used as endogenous loading control. The basal level expressions of Nanog, Oct4A
and Sox-2 in MDA-MB-231 cells were under the detection limit in Western blot analysis. Densitometry measurement with Image J software for quantification was performed,
and the results were normalized to DMSO-treated samples. Numbers stated underneath indicate expression fold-change values.
Please cite this article in press as: Chen L., et al. Bioorg. Med. Chem. Lett. (2017), https://doi.org/10.1016/j.bmcl.2017.12.033

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L. Chen et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Fig. 4. Epithelial-mesenchymal transition (EMT) activities of TNBC cell lines are affected by bexarotene derivatives. A) TNBC cell lines were subjected to Western blot analysis
on post-treatment expressions of selected epithelial and mesenchymal markers. B) Post-treatment TNBC cell lines were subjected to cytosolic and nuclear fractionation,
followed by Western Blot analysis on translocation of b-catenin. PARP was used as fraction purity indicators for nuclear fraction. b-Actin was used as endogenous loading
control. Densitometry measurement for quantification was performed, and the results were normalized to DMSO-treated samples. Numbers stated underneath indicate
expression fold-change values from ImageJ analysis.
responded better to DK-1–166 than to DK-1–150 by almost 2 folds
(Fig. 4A). Interestingly, both MDA-MB-231 and BT549 treated with
DK-1–150 (5 mM) showed increased level of epithelial marker, E-
Cadherin, while DK-1–166 (5 mM) displayed a slight increase in
E-Cadherin expressions in the TNBC cell lines. These results indi-
cate that the derivatives can induce the acquisition of epithelial
properties and the concurrent loss of mesenchymal properties.
Next, we examined the nuclear localization of b-catenin in the
presence of the derivatives. Translocation of b-catenin into the
nucleus facilitates the transcription of mesenchymal proteins,
while suppressing the expressions of epithelial proteins.27 We
observed that in both MDA-MB-231 and BT549, 5 mM DK-1–150
is more effective than 10 mM bexarotene in restraining b-catenin
in the cytosol and preventing its translocation into the nucleus
(Fig. 4B). Collectively, our results suggest that the bexarotene
derivatives are capable of limiting mesenchymal characteristics,
while promoting epithelial feature in TNBC cell lines.
In conclusion, the results of our study provide strong evidence
that the alkylamide derivatives of bexarotene effectively target
TNBC by inducing apoptotic cell death, suppressing proliferation
and migration, and inhibiting CSC and EMT properties of TNBC
cells. The overall results proved that both DK-1–150 and DK-1–
166 work more effectively than their parental molecule bexarotene
as anti-breast cancer therapeutic agents against TNBC cells. We
envision that detailed structure and activity relationship (SAR)
studies would result in more potent derivatives. Identification of
cellular target(s) of the derivatives are underway to elucidate their
mechanism of action.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2017.12.033.
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