A novel small molecule STAT3 inhibitor SLSI-1216 suppresses proliferation and tumor growth of triple-negative breast cancer cells through apoptotic induction

Article abstract of DOI:10.1016/j.bcp.2020.114053

Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer, characterized by the lack of expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Owing to the absence of molecular targets, there are limited treatment options, and TNBC patients exhibit high mortality rates. Signal transducer and activator of transcription 3 (STAT3) is overexpressed and aberrantly activated in TNBC cells. Therefore, inhibition of STAT3-mediated signaling provides a potential strategy for the treatment of TNBC. In this study, A series of synthetic derivatives of SLSI-1 (a STAT3 inhibitor) were designed and evaluated for antitumor activity in TNBC cells. A novel derivative (SLSI-1216) exhibited the most potent anti-proliferative activity. SLSI-1216 effectively inhibited STAT3 activity and activation of STAT3, leading to the downregulation of AXL, a downstream target of STAT3 and epithelial-mesenchymal transition (EMT) progression. The inhibition of EMT by SLSI-1216 was associated with modulation of E-cadherin and N-cadherin. Furthermore, SLSI-1216 induced apoptosis by targeting STAT3 and effectively inhibited tumor growth in vivo. These findings suggest that SLSI-1216, as a potential inhibitor of STAT3, may be a promising therapeutic agent for TNBC.

Full text of DOI:10.1016/j.bcp.2020.114053

Journal Pre-proofs
A novel small molecule STAT3 inhibitor SLSI-1216 suppresses proliferation
and tumor growth of triple-negative breast cancer cells through apoptotic in‐
duction
Soo Kyung Park, Woong Sub Byun, Seungbeom Lee, Young Taek Han, Yoo-
Seong Jeong, Kyungkuk Jang, Suk-Jae Chung, Jeeyeon Lee, Young-Ger Suh,
Sang Kook Lee
PII:
DOI:
Reference:
S0006-2952(20)30287-2
https://doi.org/10.1016/j.bcp.2020.114053
BCP 114053
To appear in:
Biochemical Pharmacology
Received Date:
Accepted Date:
28 February 2020
21 May 2020
Please cite this article as: S. Kyung Park, W. Sub Byun, S. Lee, Y. Taek Han, Y-S. Jeong, K. Jang, S-J. Chung, J.
Lee, Y-G. Suh, S. Kook Lee, A novel small molecule STAT3 inhibitor SLSI-1216 suppresses proliferation and
tumor growth of triple-negative breast cancer cells through apoptotic induction, Biochemical Pharmacology
(2020), doi: https://doi.org/10.1016/j.bcp.2020.114053
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A novel small molecule STAT3 inhibitor SLSI-1216 suppresses proliferation and tumor
growth of triple-negative breast cancer cells through apoptotic induction
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Soo Kyung Parka,1, Woong Sub Byuna,1, Seungbeom Leeb,1, Young Taek Hanb, Yoo-Seong
Jeongc, Kyungkuk Jangc, Suk-Jae Chungc, Jeeyeon Leec, Young-Ger Suhb,*, Sang Kook Leea,*
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aCollege of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul
08826, Republic of Korea
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bCollege of Pharmacy, CHA University, Gyeonggi-do 11160, Republic of Korea
cCollege of Pharmacy, Research Institute of Pharmaceutical Sciences, Seoul National
University, Seoul 08826, Republic of Korea
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*Corresponding author:
College of Pharmacy, Natural Products Research Institute, Seoul National University, 1
Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea (S.K. Lee), and College of Pharmacy,
CHA University, Gyeonggi-do 11160, Republic of Korea (Y. Suh).
E-mail address: sklee61@snu.ac.kr (S.K. Lee), ygsuh@snu.ac.kr (Y. Suh).
1These authors contributed equally to this work.
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ABSTRACT
Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer,
characterized by the lack of expression of estrogen receptor, progesterone receptor, and human
epidermal growth factor receptor 2. Owing to the absence of molecular targets, there are limited
treatment options, and TNBC patients exhibit high mortality rates. Signal transducer and
activator of transcription 3 (STAT3) is overexpressed and aberrantly activated in TNBC cells.
Therefore, inhibition of STAT3-mediated signaling provides a potential strategy for the
treatment of TNBC. In this study, A series of synthetic derivatives of SLSI-1 (a STAT3
inhibitor) were designed and evaluated for antitumor activity in TNBC cells. A novel derivative
(SLSI-1216) exhibited the most potent anti-proliferative activity. SLSI-1216 effectively
inhibited STAT3 activity and activation of STAT3, leading to the downregulation of AXL, a
downstream target of STAT3 and epithelial-mesenchymal transition (EMT) progression. The
inhibition of EMT by SLSI-1216 was associated with modulation of E-cadherin and N-
cadherin. Furthermore, SLSI-1216 induced apoptosis by targeting STAT3 and effectively
inhibited tumor growth in vivo. These findings suggest that SLSI-1216, as a potential inhibitor
of STAT3, may be a promising therapeutic agent for TNBC.
Keywords: SLSI-1216, triple-negative breast cancer, STAT3, antitumor activity, AXL
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1. Introduction
Breast cancer is the main cause of cancer-associated death in women worldwide [1, 2].
Breast cancer is typically classified into subtypes based on the expression of three surface
receptors, namely the estrogen receptor (ER), progesterone receptor (PR), and human
epidermal growth factor receptor 2 (HER2) [3, 4]. Therefore, these receptors are plausible
molecular targets for the development of specific therapeutics against breast cancer. Triple-
negative breast cancer (TNBC), which is characterized by the lack of ER, PR, and HER2
expression, is the most aggressive type of breast cancer and accounts for approximately 15–20%
of breast cancer cases [5]. TNBC is more frequently found in women aged <40 years and
exhibits markedly higher rates of recurrence and metastasis versus other types of breast cancer
[6]. Owing to a lack of specific molecular targets and an inherent clinical aggressiveness,
TNBC patients are linked to a relatively poorer clinical outcome compared with that reported
in patients with other subtypes of breast cancers [7]. Conventional chemotherapy (i.e.,
anthracyclines, taxanes, and platinum agents) and radiation therapy are mainly used as standard
therapeutic approaches for TNBC patients at the early and advanced stages of the disease [8,
9]. However, these chemotherapeutic drugs for TNBC patients have exhibited relatively low
selectivity for cancer cells compared with that reported for normal cells. In addition, TNBC
patients develop drug resistance after repeated exposure to these agents. Consequently, there is
a significant challenge for the cure of patients with TNBC. Currently, there is an ongoing major
effort to discover potential molecular targets for the treatment of patients with this disease [10,
11]. Accumulating evidence has demonstrated the critical role of the transcription factor signal
transducer and activator of transcription 3 (STAT3) in TNBC [12, 13]. STAT3 is overexpressed
and aberrantly activated in TNBC cells. It regulates cellular signaling pathways associated with
cancer cell proliferation, migration, anti-apoptosis, invasion, and chemoresistance [14-17]. A
body of evidence suggests that STAT3 may be a promising molecular target for TNBC cells.
Thus, the use of STAT3 inhibitors may be appropriate for the suppression of tumor growth and
metastasis of TNBC cells. The activation of STAT3 signaling is generally mediated by the
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phosphorylation of a specific tyrosine residue (Tyr705) through upstream growth factor
receptor-associated tyrosine kinases and cytokine receptor-associated cytoplasmic kinases. The
phosphorylated STAT3 (p-STAT3) undergoes dimerization through interaction between the
phosphorylated Tyr705 residue and SH2 domain; subsequently, the STAT3 translocates to the
nucleus to activate the target gene transcription associated with the regulation of the
aggressiveness of various types of cancer cells [18, 19]. Several STAT3 inhibitors are currently
under investigation in clinical trials; however, there is limited information available regarding
the use of STAT3 inhibitors as anticancer drugs in the clinical setting [20-23].
Recently, we have been attempting to identify novel STAT3 inhibitors from our in-house
chemical library with a variety of chemical scaffolds. In our previous study, we found that
SLSI-1, a B,C-ring-truncated deguelin derivative, exhibited potential STAT3 inhibition,
accompanied by a strong growth inhibition of TNBC cells [24]. In particular, SLSI-1 showed
selective cytotoxicity against cancer cells with high expression levels of p-STAT3 (e.g., MBA-
MB-231 TNBC cells) compared with that observed for normal breast epithelial cells and breast
cancer cells with lower levels of p-STAT3 expression [24].
In the present study, a series of SLSI-1 derivatives were designed, synthesized, and
subsequently evaluated to determine their anti-proliferative activity in MBA-MB-231 TNBC
cells. The novel derivative SLSI-1216 was identified as the enhanced growth inhibitor of
TNBC cells in vitro and in vivo, with potential inhibition of the STAT3-mediated signaling
pathway in TNBC cells.
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2. Materials and Methods
2.1. Synthesis of SLSI-1216
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Sodium bicarbonate (3 equiv.) and Dess-Martin periodinane (1.5 equiv.) were added to a
solution of alcohol 1 (1 equiv.) in dichloromethane (CH2Cl2) at 0 °C. The reaction mixture was
stirred for 1 h at room temperature, quenched with 10% sodium thiosulfate solution, and
extracted with CH2Cl2. The organic layer was washed with brine, dried over magnesium sulfate
(MgSO4), and concentrated under reduced pressure. The residue was then purified using flash
column chromatography on silica gel to afford the corresponding carbonyl products. To a
solution of aryl bromide 5 (1.8 equiv.) in tetrahydrofuran was added dropwise n-butyllithium
solution (1.6 M solution in n-hexane, 1.7 equiv.) at −78 °C. The resulting solution was stirred
for 30 min at −78 °C and aldehyde 2 (1.0 equiv.) in tetrahydrofuran was added. The reaction
mixture was stirred for 1h at −78 °C, washed with saturated ammonium chloride solution, and
extracted with ethyl acetate (EtOAc). The organic layer was washed with brine, dried over
MgSO4, and concentrated under reduced pressure. The residue was directly used for the next
oxidation reaction without further purification via the standard procedure for the Dess-Martin
oxidation reaction. Following the completion of the reaction (monitored using thin-layer
chromatography), the reaction mixture was diluted with CH2Cl2. The mixture was washed with
10% sodium sulfite solution, brine, and saturated copper (II) sulfate solution. The organic layer
was dried over MgSO4 and concentrated under reduced pressure. The residue was purified
using flash column chromatography on a silica gel (EtOAc/n-hexane = 1:4) to afford ketone 3
(38% for three steps). K2CO3 (2 equiv.) and paraformaldehyde (3 equiv.) were added to a
solution of ketone 3 (1 equiv.) in dry dimethylformamide at room temperature under Argon
atmosphere. After completion of the reaction, the resultant solution was diluted with EtOAc
and brine. The combined organic layer was concentrated under reduced pressure and purified
via flash column chromatography on a silica gel (EtOAc/n-hexane = 1:15) to afford olefin 4
(72% yield) (Scheme 1).
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2.2. Cell culture and chemicals
MDA-MB-231 and HCC38 cells (human TNBC cell lines), HEK293 (human embryonic
kidney cell line), and MCF-10A (human breast epithelial cell line) were provided by the
American Type Culture Collection (Manassas, VA, USA). Cells were maintained in medium
[(MDA-MB-231and HCC38 cells in RPMI 1640 medium; HEK293 in Dulbecco's Modified
Eagle Medium (DMEM) with 10% fetal bovine serum and antibiotic-antimycotics (i.e.,
penicillin G sodium: 100 units/mL; streptomycin: 100 μg/mL; amphotericin B: 250 ng/mL)] at
37 °C in a humidified incubator with 5% carbon dioxide. MCF-10A cells were cultured in
DMEM/F12 containing 5% donor horse serum, 100 ng/mL cholera toxin, 10 µg/mL human
insulin, 20 ng/mL epidermal growth factor, 0.5 µg/mL hydrocortisone, and antibiotics. All
reagents used for cell culture, including culture medium, fetal bovine serum, trypsin-EDTA
solution (1×) and antibiotic-antimycotic solution (100×), were purchased from Gibco (Grand
Island, NY, USA). All cell lines were validated by short tandem repeat analysis and tested
mycoplasma-free in 2018. Laemmli sample buffer (2×) and 2-mercaptoethanol were purchased
from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Dimethyl sulfoxide (DMSO),
bicinchoninic acid, copper (II) sulfate solution, bovine serum albumin (BSA), trichloroacetic
acid, sulforhodamine B (SRB), and etoposide were purchased from Sigma–Aldrich (St. Louis,
MO, USA). Lipofectamine 3000 and Lipofectamine RNAiMAX were purchased from
Invitrogen (Carlsbad, CA, USA).
2.3. Western blotting analysis
Total cells lysates were prepared in 2× sample loading buffer (i.e., 250 mM Tris-hydrochoride:
pH 6.8, 4% sodium dodecyl sulfate, 10% glycerol, 0.006% bromophenol blue, 2% β-
mercaptoethanol, 50 mM sodium fluoride, and 5 mM sodium orthovanadate). The samples with
cell lysates were boiled for 10–20 min at 100 °C. The concentrations of proteins in the cell
lysates were quantified using the bicinchoninic acid method. Equal amounts of protein were
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subjected to 8–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred
to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA) activated with 100%
methanol. The membranes were blocked using 5% BSA in a mixture of Tris-buffered saline
and Tween 20 (1×), and subsequently probed with the following antibodies: anti-phospho-
STAT3 (Tyr705), p-STAT3 (S727), p-STAT1 (Y701), STAT1, anti-Activating transcription
factor 2 (ATF2), anti-c-Jun, and anti-cleaved-caspase8 (Asp391) (Cell Signaling Technology,
Beverly, MA, USA), anti-STAT3, anti-AXL, anti-c-fos, anti-Cyclin-dependent kinase 4, anti-
Cyclin B1, anti-survivin, anti-bcl-xL, anti-β-actin-horseradish peroxidase (Santa Cruz
Biotechnology, Dallas, TX, USA), or anti-cleaved-Poly (ADP-ribose) polymerase (PARP),
anti-Cyclin D1, anti-c-Myc, anti-N-cadherin, anti-E-cadherin (BD Biosciences, Franklin Lakes,
NJ, USA). The blots were visualized using an enhanced chemiluminescence detection kit
(Intron Biotechnology, Sungnam, Korea) and ImageQuant LAS-4000 Imager (Fujifilm Corp.,
Tokyo, Japan) [25].
2.4. Transfection and luciferase reporter gene assays
Transient transfections were conducted using Lipofectamine 3000 according to the instructions
provided by the manufacturer. The cells were seeded in 48-well plates and incubated for 24 h.
After incubation, the cells were transfected with 100–200 ng of a p-STAT3-Luc reporter vector
(Firefly) and 0.4–4 ng of the Renilla luciferase vector for normalization. For MDA-MB-231,
the cells were treated with test compounds at 24 h after transfection. For HEK293 cells, the
activation of STAT3 was induced using interleukin-6 (IL-6; 40 ng/mL) at 24 h after
transfection, and test compounds were subsequently administered at 4 h after induction. After
24 h, the MDA-MB-231 and HEK293 cells were lysed and subjected to a dual luciferase
activity assay using the Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA)
according to the instructions provided by the manufacturer. All experiments were performed
in triplicate. The levels of luciferase activity were normalized according to the levels of Renilla
luciferase activity, and the data are presented as values relative to those obtained in the control
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group.
2.5. RNA interference
RNA interference for STAT3 was performed using siRNA duplexes purchased from Bioneer
Corporation (Daejeon, Korea). The coding strand for STAT3 was designed as follows: #1-
sense CUC CAA CAU CUG UCA GAU G and antisense CAU CUG ACA GAU GUU GGA
G; #2-sense UGU UCU CUG AGA CCC AUG A and antisense UCA UGG GUC UCA GAG
AAC A; #3-sense CUA UCU AAG CCC UAG GUU U and antisense AAA CCU AGG GCU
UAG AUA G. MDA-MB-231 cells were transfected with 10 nM siRNA duplexes using
Lipofectamine RNAiMAX (Invitrogen, Grand Island, NY, USA) according to the
recommendations provided by the manufacturer. The cells were treated with the test
compounds for 24 h. Cells transfected with a control non-specific siRNA duplex were used as
controls for direct comparison [26].
2.6. Cell proliferation assay
Cell proliferation was measured using the SRB assay [27]. Briefly, cells were seeded in 96-
well plates with test compounds and incubated for 72 h at 37 °C in an incubator with 5% carbon
dioxide. After incubation, the cells were fixed with 10% trichloroacetic acid solution at 4 °C
for 30 min and air-dried overnight. Fixed cells were stained with 0.4% SRB dissolved in 1%
acetic acid at room temperature for 1 h. Unbound SRB dye was washed, and the stained cells
were resuspended in 10 mM Tris buffer (pH 10.0). The absorbance was measured at 515 nm
and cell viability was determined as a percentage, relative to the control (treated with solvent).
The half maximal inhibitory concentration (IC50) in samples in of treated cells was calculated
through non-linear regression analysis using the TableCurve 2D v5.01 software (Systat
Software Inc., San Jose, CA, USA).
2.7. RNA extraction and real-time polymerase chain reaction (PCR)
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Total RNA was extracted from cells using the TRI reagent (Invitrogen), and reverse-transcribed
using the ReverTra Ace® qPCR RT Kit & Master Mix (Toyobo, Osaka, Japan) according to
the instructions provided by the manufacturer. Real-time PCR was performed using the iQTM
SYBR® Green Super mix (Bio-Rad Laboratories, Hercules, CA, USA) according to the
instructions provided by the manufacturer. The threshold cycle (CT) value was determined
using the Bio-Rad CFX manager 3.1 software. The relative quantification between compounds
and the untreated controls was normalized according to the levels of β-actin mRNA and
calculated using the comparative CT method [28]. The following primers were used for real-
time PCR: ATF2, 5’- AGA TTT ATT AAT TTT TCT GTG CTC AA -3’ and 5’- ACA CCC
CCA TTT ATT AAA ACA CC -3’; c-Jun, 5’- CCA AAG GAT AGT GCG ATG TTT -3’ and
5’- CTG TCC CTC TCC ACT GCA AC -3’; AXL, 5’- CGT AAC CTC CAC CTG GTC TC -
3’ and 5’- TCC CAT CGT CTG ACA GCA -3’; β-Actin, 5′-AGC ACA ATG AAG ATC AAG
AT-3′ and 5′-TGT AAC GCA ACT AAG TCA TA-3′ (Bioneer, Daejeon, Korea).
2.8. Wound Healing Assay
MDA-MB-231 human TNBC cells were grown to 90% confluence in a 6-well plate.
Subsequently, each cell monolayer was artificially wounded using a Scratcher (SPL Life
Sciences, Pocheon, Republic of Korea), and the detached cells were removed by washing with
phosphate-buffered saline (PBS, Invitrogen Corp.). The wounded cultures were then incubated
with medium containing 1% FBS and various concentrations of SLSI-1216 for 24 h. The
wounds were photographed at 0 and 24 h under an inverted microscope (Olympus, Tokyo,
Japan). The wound areas ere quantified using ImageJ software (National Institutes of Health)
and presented as the percent wound healing (%) relative to the wound area at 0 h
2.9. Transwell Cell Invasion Assay
Twenty-four-well Transwell membrane inserts (diameter: 6.5 mm, pore size: 8 μm; Corning,
Tewksbury, MA, USA) were each coated with 10 μl of type I collagen (0.5 mg/ml, BD
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Biosciences, San Diego, CA, USA) and 20 μl of a 1:20 mixture of Matrigel (BD Biosciences)
in PBS. After treatment with the indicated compounds for 24 h, MDA-MB-231 human TNBC
cells were harvested, resuspended in serum-free medium, and plated (2 × 105 cells/chamber) in
the upper chambers of the Matrigel-coated Transwell inserts. Medium containing 30% FBS
was used as a chemoattractant in the lower chambers. After a 24-h incubation, the cells that
had migrated to the outer surfaces of the lower chambers were fixed and stained using the Diff-
Quik Staining Kit (Sysmex, Kobe, Japan) and imaged using a Vectra 3.0 Automated
Quantitative Pathology Imaging System (Perkin Elmer, Waltham, MA, USA). Representative
images from three separate experiments are shown, and the numbers of invaded cells were
counted in 5 randomly selected microscopic fields (200× magnification)
2.10. Cell cycle analysis
Cell cycle dynamics were measured through flow cytometry. MDA-MB-231 cells were treated
with either vehicle or test compounds for 24 h in complete medium. After incubation, the cells
were harvested, washed twice with phosphate-buffered saline (PBS), and subsequently fixed
in 70% cold ethanol overnight at -20 °C. The fixed cells were washed with cold PBS and
resuspended in 100 μg/mL of RNase A in a shaker for 30 min. The cells were then stained with
50 μg/mL of propidium iodide (PI) in the dark. The fluorescence-activated cells were sorted
and the cellular DNA content was analyzed using flow cytometry (FACSCalibur flow
cytometer; BD Biosciences). The data were calculated using the CELLQuest 3.0.1 software
(BD Biosciences) and the content of DNA is presented as histograms [29].
2.11. Annexin V-fluorescein isothiocyanate (V-FITC)/PI double staining
MDA-MB-231 cells were treated with test compounds for 48 h in complete medium and then
stained with Annexin V-FITC and PI using an annexin V-FITC apoptosis detection kit (BD
Biosciences) according to the recommendations provided by the manufacturer. Briefly, the
incubated cells were harvested and washed twice in cold PBS. The cells were resuspended in
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1× binding buffer and treated with Annexin V-FITC and PI in the dark for 15 min. The stained
cells were diluted with 1× binding buffer and immediately analyzed using a flow cytometer
[30].
2.12. In vivo tumor xenograft model
All animal experiments were conducted following the guidelines approved by the Seoul
National University Institutional Animal Care and Use Committee (permission number: SNU-
180129-4). Female nude mice (BALB/c-nu) (age: 4–5 weeks, 18 g) were purchased from the
Central Laboratory Animal, Inc. (Seoul, Korea), and housed under pathogen-free conditions
with a 12 h light-dark schedule. MDA-MB-231 cells were subcutaneously injected into the
flanks of the mice (4 × 106 cells in 200 μL of 50:50 DMEM/Matrigel), and tumors were allowed
to grow for 10 days until the tumor volume reached approximately 100 mm3. The mice were
randomized into vehicle control and treatment groups (n = 5). Vehicle control
(DMSO/cremophor/normal saline = 10:10:80), SLSI-1216 (5 or 10 mg/kg body weight), SLSI-
1 (10 mg/kg), or paclitaxel (5 mg/kg) was administered intraperitoneally thrice per week for
28 days; mice were euthanized 1 week later. Tumors were excised, weighed, and frozen for
further biochemical analysis or fixed for immunohistochemistry. Tumor volume was measured
using a caliper according to the following formula: tumor volume (mm3) = 3.14 × length ×
width × height/6. Toxicity was assessed based on body weight loss [31].
2.13. Ex vivo immunohistochemistry and biochemical analyses of tumors
Tumor tissues fixed in 4% paraformaldehyde were embedded in paraffin, sectioned, mounted
to slides, deparaffinized with xylene, rehydrated with ethanol series, and subjected to antigen
retrieval. The slides were incubated with the indicated antibodies, which were detected using
the LSAB+ System-HRP kit (Dako, Glostrup, Denmark). Stained sections were observed and
imaged using the Vectra 3.0 Automated Quantitative Pathology Imaging System (Perkin Elmer,
Waltham, MA, USA). A portion of the frozen tumors excised from nude mice was
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homogenized using a hand-held homogenizer in complete lysis buffer (Active Motif, Carlsbad,
CA, USA). Aliquots were stored at −80 °C and the levels of protein expression in the tumor
lysates were determined. The protein concentrations in the tumor lysates were measured using
the Bradford assay [32].
2.14. Evaluation of solubility
SLSI-1 and SLSI-1216 prepared in an organic solvent (i.e., final concentration of 10 mM in
DMSO) were added to an aqueous buffer (Dulbecco’s PBS) at pH 7.4, providing a theoretical
concentration of 200 μM with the final DMSO concentration of 2%, to determine their kinetic
solubility. The mixture was subsequently agitated for 24 h at room temperature. After
centrifugation of the mixture at 4 °C for 5 min at 16,100 g (Eppendorf 5415R; Eppendorf,
Hamburg, Germany), the supernatant was filtered with a filter (0.2 μm pore size syringe filter,
Minisart RC15; Sartorius Stedim Biotech, Goettingen, Germany) and analyzed using a liquid
chromatography-tandem mass spectrometry (LC-MS/MS) system (Applied Biosystems 3200
Qtrap MS/MS system with Alliance Waters e2695 LC system) to determine their concentration
in the media.
2.15. Evaluation of liver microsomal stability
In this study, the time-dependent metabolic stability of SLSI-1 and SLSI-1216 in rat liver
microsomes was determined. The reaction mixture (total volume: 0.5 mL) consisted of
nicotinamide adenine dinucleotide phosphate (NADPH)-regenerating solution containing 1.3
mM NADP+, 3.3 mM glucose-6-phosphate, 0.4 U/mL glucose-6-phosphate dehydrogenase,
and 3.3 mM magnesium dichloride, according to the protocol provided by the manufacturer
(Corning Gentest, Woburn, MA, USA). An aqueous solution (100 mM potassium phosphate
buffer) containing SLSI-1 or SLSI-1216 (final concentration: 10 μM in the reaction mixture)
was also added. The concentration of organic solvent (i.e., DMSO) in the incubation mixture
was maintained at 0.1% to limit its influence on microsomal enzymes. After preincubation (37
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°C for 10 min), the reaction was initiated by the addition of rat liver microsomes (25 μL; the
final microsomal protein concentration: 0.5 mg/mL). Using the results from preliminary
experiments performed to determine the incubation time for the given concentration for a linear
rate of disappearance, samples (50 μL) were collected at various predetermined time points.
Subsequently, ice-cold methanol (200 μL) was added to terminate the reaction. After mixing
by vortexing and subsequent centrifugation at 4 °C for 5 min at 16,100 g (Eppendorf 5415R),
the clear supernatant was collected and analyzed using an LC-MS/MS system for the
quantification of SLSI-1 and SLSI-1216.
2.16. Evaluation of binding titration experiments
Fluorescence-based equilibrium binding experiments were performed to examine the binding
affinity of SLSI-1 and SLSI-1216 for STAT3. All titration experiments were conducted at
20 °C using a Jasco FP 6500 spectrofluorometer (Easton, MD, USA). Recombinant human
STAT3 protein (BSA and azide free) was equilibrated with various concentrations of ligands
prior to measuring the fluorescence emission. Ligand stock solutions were titrated into a protein
sample dissolved in phosphate buffer (pH 7.4). The protein was excited at 285 nm and the
decrease in fluorescence emission at 305 nm after ligand binding was measured as a function
of ligand concentration. All titration data were fit to a hyperbolic binding equation to obtain
the KD values.
2.17. Molecular docking analysis
Modeling and initial docking calculations were performed using the Interaction Simulation
Package of Discovery Studio (by in Silico co.). The default parameters were used. Constrained
energy minimization of SLSI-1216 and STAT3 protein was then performed via Ligand and
Protein Preparation Package of Discovery Studio.
2.18. Statistical analysis
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Data are presented as the mean values ± standard deviation for the indicated number of
independently performed experiments. All data are representative of the results of at least three
independent experiments. Statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001) was
assessed using Student’s t-test or one-way analysis of variance coupled with Dunnett’s t-test.
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3. Results
3.1. SLSI-1216 inhibited the transcriptional activity of STAT3 in MDA-MB-231 cells and IL6-
stimulated HEK293 cells
In our previous study, we identified SLSI-1 (Figure 1A) as a novel STAT3 inhibitor with
a selective growth inhibitory activity against human breast cancer cells compared with that
observed against normal cells [24]. In addition, SLSI-1 exhibited more profound growth
inhibitory activity against MDA-MB-231 TNBC cells (high expression levels of p-STAT3)
than that reported against non-TNBC MCF7 cells (lower expression levels of p-STAT3). Based
on these findings, a series of SLSI-1 derivatives were newly designed and synthesized.
Primarily, we evaluated the anti-proliferative activity of the synthetic compounds in MBA-
MB-231 cells (data not shown). We found that the novel derivative SLSI-1216 (Figure 1A)
showed the most potent growth inhibitory activity against MBA-MB-231 TNBC cells.
Therefore, we selected the SLSI-1216 and attempted to elucidate the mechanisms of action of
SLSI-1216 in MBA-MB-231 cells.
Initially, we analyzed the clinical significance of STAT3 expression in patients with
breast cancer in terms of relapse-free survival (RFS) rate using the Kaplan−Meier method. The
auto-select best cutoff method was applied to classify patients with breast cancer. Patients were
further classified according to their levels of STAT3 expression and RFS probability. The
classification showed that breast cancer patients with high expression of STAT3 were
associated with a decreased probability for RFS versus those with low expression of STAT3
(Figure 1B). In addition, following the classification of patients with TNBC (mesenchymal
subtypes only), the probability of RFS in patients with a high expression of STAT3 was
significantly decreased compared with that reported in patients with low expression of STAT3
(Figure 1B). These data suggest that the expression of STAT3 is inversely correlated with RFS
in patients with TNBC and, thus, that STAT3 may be a therapeutic biomarker for TNBC cells.
The expression levels of AXL and STAT3 are highly correlated with the progression and
metastasis of breast cancer [12]. Therefore, the expression levels of AXL and STAT3 were
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evaluated in a normal breast epithelial cell line (MCF-10A) and two TNBC cell lines (MDA-
MB-231 and HCC38). The TNBC cells showed higher expression levels of AXL and p-STAT3
(activated STAT3) compared with the normal breast epithelial cells. Moreover, MDA-MB-231
cells showed relatively high expression levels of both markers compared with HCC38 cells
(Figure 1C).
The transcriptional activity of STAT3 was determined using a reporter gene assay in
MDA-MB-231 cells and HEK293 cells to primarily investigate the compound SLSI-1216 as
an inhibitor of STAT3. MDA-MB-231 cells, which overexpress STAT3, were treated with
SLSI-1216 for 24 h, and the STAT3-mediated luciferase reporter gene assay was subsequently
performed. SLSI-1216 effectively inhibited the transcriptional activity of STAT3 in a
concentration-dependent manner (Figure 1D). We also determined the inhibitory effect of
SLSI-1216 on the transcriptional activity of STAT3 in IL-6-induced HEK293 cells. HEK293
cells, which are normal kidney cells with lower endogenous expression of STAT3, were
induced with IL-6 (40 ng/mL) and subsequently treated with SLSI-1216. Following the
treatment of HEK293 cells with IL-6, the transcriptional activity of STAT3 was induced by
approximately 2.5-fold. However, the compound SLSI-1216 significantly inhibited the IL-6-
induced transcriptional activity of STAT3 in HEK293 cells (Figure 1E). These data suggest
that SLSI-1216 effectively suppressed the transcriptional activity of STAT3 in either
overexpressing TNBC cells or IL-6-induced normal cells. In addition, the inhibitory effect of
SLSI-1216 on the transcriptional activity of STAT3 was more pronounced versus that observed
with SLSI-1 (a previously reported inhibitor of STAT3) at the same concentration (2 μM) in
both cell lines. To further verify the involvement of STAT3 in the anti-proliferative activity of
SLSI-1216 against TNBC cells, MDA-MB-231 cells were transfected with STAT3 siRNA for
48 h, and subsequently treated with SLSI-1216 for 72 h. Scrambled siRNA was used as a
negative control. As shown in Figure 1F, SLSI-1216 exhibited anti-proliferative activity
against MDA-MB-231 cells with scrambled siRNA. However, the anti-proliferative activity of
SLSI-1216 in cancer cells was abolished by co-treatment with STAT3 siRNA. These data
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suggest that SLSI-1216 is able to inhibit the proliferative activity of STAT3-overexpressing
cancer cells. Furthermore, the anti-proliferative activity of SLSI-1216 is partly associated with
the involvement of STAT3 in TNBC cells.
3.2. SLSI-1216 inhibited the cell viability of TNBC cells
The anti-proliferative potential of SLSI-1216 for 72 h was determined in two TNBC cells and
a normal breast epithelial cell to evaluate its effect on the growth of TNBC cells (Table 1). As
shown in Table 1, SLSI-1216 effectively inhibited the growth of MDA-MB-231 and HCC38
TNBC cells. The growth inhibitory activity of SLSI-1216 was also relatively higher than that
reported for the STAT3 inhibitor SLSI-1 in both cell lines. Moreover, the growth inhibitory
activity of SLSI-1216 against normal human breast MCF-10A cells was found to be
approximately 10-fold lower (IC50 value: 19.92 μM for MCF-10A vs. 1.86 μM for MDA-MB-
231). This finding indicates that SLSI-1216 selectively inhibits the proliferation of TNBC cells
versus normal cells. In addition, based on the sensitivity of the MDA-MB-231 cells to SLSI-
1216, the plausible mechanisms of action of SLSI-1216 were further elucidated using these
cells.
3.3. SLSI-1216 suppresses the protein expression of p-STAT3 and AXL, and regulates
epithelial-mesenchymal transition (EMT) biomarkers in MDA-MB-231 cells
Since SLSI-1216 inhibited the transcriptional activity of STAT3, we further determined
its effect on STAT3-mediated signaling using western blotting. As shown in Figure 2A, in
MDA-MB-231 cells, the levels of the activated STAT3 protein p-STAT3 (Tyr705) were
suppressed by SLSI-1216 in a concentration-dependent manner. In addition, it is well
established that AXL is overexpressed in TNBC cells and highly associated with metastasis
[33, 34]. Therefore, we also evaluated the effect of SLSI-1216 on the expression of AXL in
MDA-MB-231 cells. The high levels of AXL in the TNBC cells were also effectively
suppressed by SLSI-1216 (Figure 2A). The suppressive potential of SLSI-1216 on the
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expression of AXL was shown to be higher than that reported for SLSI-1 at the same
concentration (4 μM). In addition, the treatment of SLSI-1216 for 24 h effectively suppressed
the phosphorylation of STAT3 (both tyrosine 705 and serine 727 residue) in a concentration-
dependent manner, but the phosphorylation of STAT1 (tyrosine 701) was not affected by SLSI-
1216, indicating the relatively selective suppression of STAT3 activity by SLSI-1216 (Figure
2B). Further study was performed to elucidate the mechanisms of actions involved in the
suppression of STAT3 activation and AXL expression by SLSI-1216. STAT3 acts as a
transcription factor for the components of the AP-1 protein complex, which consists of ATF2,
c-Jun, and c-Fos [35]. The AP-1 protein complex also plays a role as a transcription factor for
AXL [36]. Interestingly, the protein levels of ATF2 and c-Jun were downregulated by SLSI-
1216 (Figure 2C). In addition, the mRNA expression levels of ATF2, c-Jun, and AXL were
also found to be downregulated by SLSI-1216 (Figure 2D). These data suggest that the
inhibitory activity of SLSI-1216 on the activation of STAT3 led to the downregulation of the
AP-1 complex and suppressed the expression of the AXL protein.
3.4. SLSI-1216 Inhibits Cell Invasion and Migration by Regulating EMT Gene Expression in
Human TNBC Cell
AXL is related to cell invasiveness, EMT, and metastasis [37, 38]. The overexpression of
AXL regulates the expression of EMT biomarkers, such as E-cadherin and N-cadherin [39].
The inactivation of AXL is able to induce the expression of E-cadherin (an epithelial marker)
and decrease the expression of N-cadherin (a mesenchymal marker), suppressing the invasive
potential of cells [37]. Since cancer metastasis is multistep processes including cell invasion
and migration, we evaluated whether SLSI-1216 is able to regulate the metastatic potential of
human TNBC cells via the inhibition of cancer cell invasion and migration. Notably, SLSI-
1216 treatment for 24 h effectively inhibited cell invasion in a Transwell-invasion assay (Figure
3A) and wound closure in a wound healing assay (cell migration, Figure 3B) in MDA-MB-231
cells. To further elucidate the molecular mechanism underlying the inhibition of cancer cell
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invasion and migration by SLSI-1216, the effects of SLSI-1216 on the expression levels of
EMT-associated genes were determined by western blotting in MDA-MB-231 cells. The
expression of E-cadherin was upregulated, whereas that of N-cadherin was downregulated
following treatment with SLSI-1216 (Figure 3C). This finding suggests that SLSI-1216 is able
to suppress cancer cell invasion and migration through the AXL-mediated downregulation of
EMT progression in MDA-MB-231 cells.
3.5. SLSI-1216 induces apoptotic cell death in MDA-MB-231 cells
It is well established that STAT3 is associated with cell cycle regulation and apoptotic
cell death in cancer cells [40, 41]. Since SLSI-1216 inhibits STAT3-mediated cell signaling in
its anti-proliferative activity against cancer cells, the role of SLSI-1216 in cell cycle regulation
was investigated in MDA-MB-231 cells. The cells were treated with SLSI-1216 for 24 h, and
the distribution of cell cycle was then analyzed using flow cytometry. As shown in Figure 4A,
the population of cells in the sub-G1 phase increased to 15.95%, 23.24%, and 38.96% after 24
h of treatment with 0.25 μM, 1 μM, and 4 μM SLSI-1216, respectively. These data suggest that
SLSI-1216 may induce apoptosis in MDA-MB-231 cells. To further confirm the induction of
apoptosis by SLSI-1216, a flow cytometric analysis using Annexin V-FITC and PI double
staining was performed after treatment with SLSI-1216 for 48 h in MDA-MB-231 cells. As
shown in Figure 4B, the early apoptotic (Annexin V+/PI-) cell population increased to 5.08%,
18.84%, and 36.02% after treatment with 1 μM, 2 μM, and 4 μM SLSI-1216, respectively. The
late apoptotic (Annexin V+/PI+) cell population also increased to 3.92%, 7.46%, and 9.24%
after treatment with 1 μM, 2 μM, and 4 μM SLSI-1216, respectively. In addition, the rate of
apoptotic cell death induced by SLSI-1216 was shown to be higher than that reported for SLSI-
1 at the same concentration (4 μM). These data suggest that the longer treatment with SLSI-
1216 is able to induce apoptotic cell death via the inhibition of STAT3 in MDA-MB-231 cells.
The expression of apoptosis-related proteins was determined to further explore the molecular
mechanisms associated with the SLSLI-1216-induced apoptosis in MDA-MB-231 cells. It is
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known that STAT3 regulates numerous anti-apoptotic or pro-apoptotic proteins and members
of the Bcl-2 family [42, 43]. As shown in Figure 4C, the expression of anti-apoptotic proteins,
such as c-Myc and Survivin, was downregulated. However, the activation of caspase 8
(cleaved-caspase 8) was upregulated by SLSI-1216 in a concentration-dependent manner. The
activation of caspase 8 by SLSI-1216 also led to the induction of PARP cleavage in MDA-
MB-231 cells. In addition, the expression of Bcl-xL, an anti-apoptotic Bcl-2 family, was
downregulated by SLSI-1216. These findings indicated that the induction of apoptosis by
SLSI-1216 might be partly associated with the modulation of apoptotic regulatory proteins
through the activation of the caspase 8 cascade.
3.5. SLSI-1216 exhibits antitumor activity in a xenograft mouse model
A tumor xenograft model in nude mice implanted with MDA-MB-231 cells (4 × 106
cells/mouse) was employed to further evaluate the in-vivo efficacy of SLSI-1216. When the
tumor volume reached approximately 100 mm3, SLSI-1216 (5 or 10 mg/kg), SLSI-1 (10
mg/kg), or paclitaxel (5 mg/kg, as a positive control) was administered intraperitoneally thrice
a week for 28 days. Compared with the vehicle-treated control group, the tumor volumes in the
groups treated with 5 and 10 mg/kg SLSI-1216 were significantly reduced by 42.6% and 56.9%,
respectively, as measured at the termination of the experiment (Figure 5A). Under the same
experimental conditions, the tumor volume in the group treated with 10 mg/kg SLSI-1 was
reduced by 46.9% (Figure 5A). There was no overt toxicity or change in body weight observed
in the groups treated with SLSI-1216 compared with the vehicle control group (Figure 5B).
In addition, as determined on the final day of experiment, tumor weight inthegroupstreatedwith
5 and 10 mg/kg SLSI-1216 was reduced by 48.8% and 60.2%, respectively, compared with the
vehicle control group (Figure 5C). These data suggest that SLSI-1216 effectively inhibits the
growth of human breast cancer cells MDA-MB-231 in vivo.
Consistent with the in-vitro results, additional biochemical analyses of the tumor tissues
revealed that SLSI-1216 significantly suppressed the activation of STAT3 (p-STAT3 [Tyr705]
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and the expression of AXL protein) (Figure 5D). Immunohistochemical analysis also
demonstrated that SLSI-1216 effectively suppressed the expression levels of p-STAT3
(Tyr705), AXL, and Ki-67 (a proliferation biomarker) in tumor tissues (Figure 5E). These
results confirmed that the antitumor activity of SLSI-1216 in MDA-MB-231 cells might be
partly associated with the downregulation of p-STAT3 and AXL.
3.6. Physicochemical properties of SLSI-1216
The solubility and microsomal stability of SLSI-1 and SLSI-1216 were determined to
obtain preliminary data for in vitro ADME. We expected that the solubility of SLSI-1216 might
decrease owing to its lipophilic tert-butyl substituent in lieu of the 3,4-dimethoxy group of
SLSI-1. However, the solubility of SLSI-1216 was significantly higher compared with that
measured for SLSI-1, as depicted in Figure 6A. This result may be attributed to the change in
molecular 3D conformation since SLSI-1216 contains a bulky tert-butyl group, which renders
it more amorphous than SLSI-1. As shown in Figure 6B, SLSI-1216 exhibited increased
microsomal stability compared with SLSI-1. This finding was supported by the observation of
an increase in the remaining tested compound in rat liver microsomes.
In a previous study, we reported that SLSI-1 directly binds to STAT3 and may form a
covalent bond between the thiol moiety of STAT3 cysteine and Michael acceptor of SLSI-1,
without determining the KD value for the SLSI-1 binding to STAT3 [24]. In this study, we
determined the KD values of SLSI-1 and SLSI-1216 binding to STAT3 through a fluorescence-
based binding titration analysis. At 305 nm of a maximum emission wavelength of STAT3,
SLSI-1 and SLSI-1216 exerted a moderate concentration-dependent inhibitory activity, which
decreased the fluorescence intensities of STAT3 with a KD value of 43.13 μM and 32.77 μM,
respectively (Figure 6C and 6D). This result implied that SLSI-1216 exhibits higher affinity
for STAT3 than SLSI-1. Collectively, these physicochemical data suggest that SLSI-1216 may
serve as a useful tool to further study the role of STAT3 pathways in vivo, with improved
parameters compared with SLSI-1.
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To understand the binding interactions between SLSI-1216 and STAT3, molecular
docking studies were carried out using the X-ray crystal structure of STAT3 protein (PDB ID:
1BG1). As shown in Figure 6E and 6F, SLSI-1216 binds to SH2 domain of STAT3 protein. In
addition, Michael acceptor of SLSI-1216 and Michael donor of Cys259 of STAT3 protein are
very closely located. These results imply that SLSI-1216 has potential to covalently bind to
STAT3 protein via Michael addition reaction between β-carbon of SLSI-1216 and thiol group
of Cys259 of STAT3 protein. Since previous study reported that the interaction between
Cys259 of STAT3 and chemicals leading to the inhibition of phosphorylation of STAT3 and
dimerization of STAT3, we assumed that the direct binding between SLSI-1216 and SH2
domain of STAT3 evokes the suppression of formation of phosphorylated STAT3 dimer.
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4. Discussion
The oncogenic potential of STAT3 is highly associated with the constitutive
overexpression and aberrant activation of its signaling pathways in malignant cancers [44, 45].
The transcriptional activation of STAT3 regulates its downstream target genes involved in the
proliferation, apoptosis, metastasis, and angiogenesis of cancer cells [14, 40, 46]. Recently,
accumulating clinical evidence has suggested that STAT3 plays a pivotal role in TNBC. Thus,
inhibitors of STAT3 are considered promising candidates for the development of anticancer
agents against TNBC [12, 44]. Currently, there are no specific molecular targets for TNBC
(owing to the lack of ER, PR, and HER2 expression), and only limited therapeutic options for
these patients. Therefore, the inhibition of STAT3 activation may be a strategy for the treatment
of TNBC.
In our continuous efforts to procure chemical entities for anticancer agents against breast
cancers, we previously developed SLSI-1 as an inhibitor of STAT3 with a potential growth
inhibition of TNBC cells [24]. In the present study, we synthesized a series of SLSI-1 analogs
for the development of enhanced anti-proliferative activity against TNBC cells. Among them,
SLSI-1216 exhibited the most potent and an approximately three-fold higher growth inhibitory
activity compared with that reported for the parent SLSI-1 in MBA-MB-231 TNBC cells.
Therefore, we selected SLSI-1216 to further evaluate the antitumor activity and underlying
mechanisms of action in TNBC cells.
SLSI-1216 exhibited a relatively selective growth inhibitory activity against TNBC cells
(MBA-MB-231 and HCC38 cells) compared with normal epithelial breast cells (MCF-10A)
(i.e., 7–10-fold higher sensitivity for these cancer cells). A fluorescence-based binding study
also revealed that SLSI-1216 is able to more tightly bind to STAT3 protein than the parent
compound SLSI-1, leading to the inhibition of STAT3 phosphorylation and the suppression of
downstream target gene expression. Consequently, SLSI-1216 effectively inhibited the
transcriptional activity of STAT3 in MDA-MB-231 cells, which constitutively overexpress
STAT3, and in IL-6-induced HEK293 cells. These data suggest that SLSI-1216 inhibits the
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transcriptional activity of STAT3 in STAT3-overexpressing cells and exogenously STAT3-
induced cells. The STAT3-dependency of SLSI-1216 in terms of cytotoxicity was also
confirmed using siSTAT3 in MBA-MB-231 cells. The cell viability of SLSI-1216 was
abolished by co-treatment with siSTAT3 in MBA-MB-231 cells. As a STAT3 inhibitor, SLSI-
1216 effectively suppressed the expression of constitutive phosphorylation of STAT3 at
tyrosine 705 (activated form of STAT3, p-STAT3). SLSI-1216 also downregulated the
expression of AXL, which is associated with metastasis and the EMT process. The mechanism
involved in the regulation of STAT3 and AXL expression was identified based on the AP-1
complex-mediated responses via suppression of the gene and protein expression of ATF2 and
c-Jun. Inhibition of the EMT process by SLSI-1216 was also partly involved in the upregulation
of E-cadherin and downregulation of N-cadherin in MBA-MB-231 cells. Moreover, SLSI-1216
downregulated the STAT3 downstream target genes, such as Bcl-xL, Survivin, and c-Myc,
which are related to cancer cell survival. SLSI-1216 inhibits cell proliferation and activates the
caspase-dependent apoptotic pathways via caspase 8 activation and PARP cleavage in MBA-
MB-231 cells. In addition, SLSI-1216 exhibited a significant antitumor activity in an in-vivo
xenograft nude mouse model implanted with MDA-MB-231 cells.
In view of the suitability of druggable candidates, the metabolic stability and solubility
of chemical compounds are important parameters for possessing favorable pharmacokinetic
properties [47]. Cytochromes P450 (CYPs) are the major enzymes involved in drug metabolism
[48]. We found that SLSI-1216 showed higher metabolic stability versus SLSI-1 in liver
microsomes. In terms of chemical structures, SLSI-1 has two methoxy groups in the phenyl
ring A; however, SLSI-1216 has one tertiary butyl group at that site of one methoxy group.
The methoxy group is a metabolic site, which can be oxidized by CYPs in the drug metabolism
in the human body [49, 50]. Since SLSI-1216 does not have two methoxy groups in the phenyl
ring A, the degree of oxidation may be decreased, and the tertiary butyl group in the phenyl
ring part of SLSI-1216 may also protect the compound from oxidation by CYPs. These
differences in chemical properties may enhance the microsomal metabolic stability of SLSI-
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1216.
In summary, the findings of this study report SLSI-1216 as a novel inhibitor of STAT3,
with marked antitumor activity against TNBC cells. The underlying mechanism of action for
the antitumor activity of SLSI-1216 in MBA-MB-231 TNBC cells involves the STAT3-
medated signaling and apoptosis-induction pathways (Figure 7). Therefore, this evidence may
offer a basis for the development of effective inhibitors of STAT3 as therapeutic agents against
TNBC.
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Conflict of Interests
The authors declare no conflicts of interest.
Acknowledgments
This study was supported by the National Research Foundation of Korea (NRF) Grant funded
by the Korean Government [grant number NRF-2016M3A9B6903499].
Author contributions
S.K.P., W.S.B., and S.K.L. designed experiments and analyzed the data. W.S.B. and S.K.L.
wrote the manuscript. S.K.P. and W.S.B. performed most of the experiments. Y.-S.J., K.J., S.-
J.C., and J.L. contributed to the analysis of data. S.L., Y.T.H., and Y.-G.S. designed and
contributed deguelin analogues. All authors read and approved the final manuscript.
Abbreviations
STAT3, signal transducer and activator of transcription-3; TNBC, triple-negative breast cancer;
ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor
receptor 2; EMT, epithelial-mesenchymal transition; FBS, fetal bovine serum; IL-6, interleukin
6; ATF2, activating transcription factor 2; PCR, polymerase chain reaction; PBS, phosphate-
buffered saline.
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