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Received: 17 May 2019 Revised: 2 December 2019 Accepted: 22 December 2019
DOI: 10.1111/bph.14985

RESEARCH PAPER

Overcoming tamoxifen resistance in oestrogen receptor-

positive breast cancer using the novel thiosemicarbazone anti-
cancer agent, DpC

Sundus N. Maqbool1,2 | Syer C. Lim1 | Kyung Chan Park1 | Rumeza Hanif2 |

Des R. Richardson1 | Patric J. Jansson1 | Zaklina Kovacevic1

1
Molecular Pathology and Pharmacology
Program, Department of Pathology and Bosch
Institute, University of Sydney, Sydney, NSW,
Australia
2
Atta-ur Rahman School of Applied
Biosciences, National University of Sciences
and Technology, Islamabad, Pakistan
Correspondence
Zaklina Kovacevic, Patric J. Jansson, and Des
R. Richardson, Molecular Pharmacology and
Pathology Program, Department of Pathology
and Bosch Institute, University of Sydney,
Sydney, NSW 2006, Australia.
Email: zaklina.kovacevic@sydney.edu.au;
patric.jansson@sydney.edu.au; d.richardson@
med.usyd.edu.au
Funding information
Cancer Institute New South Wales, Grant/
Award Numbers: CDF141147 and
CDF171126; National Health and Medical
Research Council, Grant/Award Numbers:
1062607, 1159596 and APP1140447;
National Breast Cancer Foundation, Grant/
Award Number: 1146599; Cancer Australia;
Cure Cancer Australia Foundation, Grant/
Award Number: 1086449
Background and Purpose: Breast cancer is the leading cause of death in women
worldwide, with resistance to current therapeutic strategies, including tamoxifen,
causing major clinical challenges and leading to more aggressive and metastatic dis-
ease. To address this, novel strategies that can inhibit the mechanisms responsible
for tamoxifen resistance need to be assessed.
Experimental Approach: We examined the effect of the novel, clinically-trialled,
thiosemicarbazone anti-cancer agent, DpC, and its potential as a combination therapy
with the clinically used estrogen receptor (ER) antagonist, tamoxifen, using both
tamoxifen-resistant and -sensitive, human breast cancer cells (MDA-MB-453, MDA-
MB-231 and MCF-7) in 2D and 3D cell-culture. Synergy was assessed using the
Chou-Talalay method. The molecular and anti-proliferative effects of these agents
and their combination was examined via Western blot, immunofluorescence and col-
ony formation assays.
Key Results: Combinations of tamoxifen with DpC were highly synergistic, leading to
potent inhibition of cell proliferation, colony formation, and ER-α transcriptional
activity. The combination also more efficiently reduced major molecular drivers of
proliferation of tamoxifen-resistant cells, including c-Myc, cyclin D1, and p-AKT,
while up-regulating the cell cycle inhibitor, p27, and inhibiting oncogenic phosphory-
lation of ER-α at Ser167. Assessing these effects using 3D cell culture further con-
firmed the greater effects of DpC combined with tamoxifen in reducing ER-α
expression, and that of the proliferation marker, Ki-67, in both tamoxifen-sensitive
and -resistant MCF-7 spheroids.
Conclusions and Implications: These studies demonstrate that the synergistic combi-
nation of DpC with tamoxifen could be a promising new therapeutic strategy to over-
come tamoxifen resistance in ER-positive breast cancer.

Abbreviations: 4-OHT, 4-hydroxy-tamoxifen; CI, combination index; DpC, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone; EGFR, EGF receptor; ER, oestrogen receptor; EREs,
oestrogen response elements; IGF1R, insulin-like growth factor 1 receptor.

Br J Pharmacol. 2020;177:2365–2380. wileyonlinelibrary.com/journal/bph © 2020 The British Pharmacological Society 2365

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2366 MAQBOOL ET AL.

1 | I N T RO DU CT I O N
What is already known
Breast cancer is a leading cause of cancer-related deaths, affecting 2.1
• Tamoxifen resistance is a major clinical problem, leading
million women each year (Ali et al., 2016). The oestrogen receptor-α
to development of aggressive, metastatic breast cancers.
(ER-α) is expressed in 75% of breast cancer cases (Zhang, Man, Zhao,
• Intrinsic and ligand-independent mechanisms of ER-α
Dong, & Ma, 2014) and is a ligand-responsive transcription factor that
activation drive development of tamoxifen resistance.
plays a pivotal role in driving breast cancer progression (Hayashi et al.,
2003). The orthodox genomic ER-α pathway entails oestradiol (E2)
What this study adds
binding to ER-α, resulting in dimerization and nuclear localization,
• The novel clinically trialled anti-cancer agent DpC inhibits
where it binds to oestrogen response elements (EREs) to promote
major pathways that drive ligand-independent ER-α
transcription responsible for growth, proliferation, and metastasis
activation.
(Acconcia & Kumar, 2006; Klinge, 2001).
• Combining DpC with tamoxifen demonstrates potent
Therapies for ER-positive breast cancer include selective ER
synergy and overcomes tamoxifen resistance in breast
modulators, with tamoxifen being the standard of care for younger
cancer.
women with breast cancer (Osborne, 1998). Tamoxifen binds ER-α,
blocking its ability to bind EREs (Obrero, Yu, & Shapiro, 2002). How-
What is the clinical significance
ever, 20% of all ER-positive breast cancer develop resistance to
• Combining DpC with tamoxifen may overcome tamoxifen
tamoxifen, leading to more aggressive neoplasms (Ali et al., 2016;
resistance, preventing onset of more aggressive breast
Ring & Dowsett, 2004). Several mechanisms drive tamoxifen resis-
cancer.
tance, including the interaction of ER-α with growth factors, such as
• These findings are highly significant, as 20% of ER-
EGF and/or the over-expression of human EGF receptors (i.e., EGFR
positive breast cancer patients develop tamoxifen
and HER2), activating downstream MAPK and PI3K/AKT pathways
resistance.
(Campbell et al., 2001; Fan, Wang, Santen, & Yue, 2007; Ghayad
et al., 2010; Osborne, Shou, Massarweh, & Schiff, 2005; Riggins,
Schrecengost, Guerrero, & Bouton, 2007). This enables ligand-
independent phosphorylation of ER-α at Ser118 and Ser167
(Campbell et al., 2001; Ghayad et al., 2010; Kato et al., 1995; Sun Considering this, we examined the role of DpC and its potential
et al., 2001), promoting c-Myc and cyclin D1 expression (Ali et al., for synergy with tamoxifen in ER-positive breast cancer. We have
2016; Green et al., 2016; Kilker, Hartl, Rutherford, & Planas-Silva, demonstrated that DpC combined with the active tamoxifen metabo-
2004; Santen et al., 2009). In fact, over-expression or activation of lite, 4-hydroxy-tamoxifen (4-OHT), down-regulates key oncogenes,
these latter proteins is extensively linked to tamoxifen resistance including p-AKT, c-Myc, and cyclin D1, while potently inhibiting prolif-
(Santen et al., 2009), and it is critical to develop novel strategies to eration and colony formation of tamoxifen-resistant and -sensitive
inhibit or prevent resistance. MCF-7 cells. Hence, the combination of DpC with tamoxifen has the
We synthesized the novel thiosemicarbazone, di-2-pyridylketone potential to overcome tamoxifen resistance and prevent the progres-
4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), which has potent sion of breast cancer.
and selective anti-cancer activity against multiple neoplasms in vitro
and in vivo (Guo et al., 2016; Kovacevic, Chikhani, Lovejoy, & Richard-
son, 2011; Lovejoy et al., 2012). DpC binds iron and copper to form 2 | METHODS
redox-active complexes that are cytotoxic to cancer cells (Lane,
Saletta, Suryo Rahmanto, Kovacevic, & Richardson, 2013) and up- 2.1 | Cell culture
regulates the metastasis suppressor, N-myc downstream regulated
gene-1 (Kovacevic et al., 2011). Following extensive pharmacokinetic The human breast cancer cell lines, MCF-7 (RRID:CVCL_0031),
and toxicity studies (Kovacevic et al., 2011; Lovejoy et al., 2012; T47D (RRID:CVCL_0553), MDA-MB-453 (RRID:CVCL_0418), and
Sestak et al., 2015), DpC entered Phase I clinical trials to assess its MDA-MB-231 (RRID:CVCL_0062), were purchased from the Ameri-
efficacy in advanced cancers (Jansson et al., 2015). can Type Culture Collection (Rockville, MD). Cells were grown in
Importantly, DpC decreases the expression of key EGF receptors MEM complete media (ThermoFisher Scientific, MA, USA) sup-
including EGFR, HER2, and HER3 (Kovacevic et al., 2011), while plemented with 10% FCS (ThermoFisher Scientific). Tamoxifen-
inhibiting downstream PI3K/AKT and MAPK signalling (Kovacevic resistant MCF-7 cells (resistant) and their sensitive vehicle control
et al., 2011; Kovacevic et al., 2016; Lui et al., 2015; Xi et al., 2017). As counterparts (sensitive) were generated (Knowlden et al., 2003)
these pathways play major roles in tamoxifen resistance (Butt, McNeil, and generously provided by Dr. Elizabeth Caldon (Garvan Institute
Musgrove, & Sutherland, 2005; Ghayad et al., 2010; Kato et al., 1995; of Medical Research, Sydney). The resistant cells were generated
Riggins et al., 2007; Sun et al., 2001), DpC may overcome this major by continuous incubation with the active metabolite of tamoxifen,
clinical problem. namely, 4-OHT, for 6 months (Knowlden et al., 2003). All cells

MAQBOOL ET AL.
were authenticated based on viability, recovery, growth, morphol-
ogy, and cytogenetic analysis (i.e., antigen expression, DNA profile,
and iso-enzymology) by the provider.
The cell lines were maintained in phenol red-free RPMI media
(ThermoFisher Scientific), supplemented with 5% (v/v) charcoal-
stripped serum (csFBS; Sigma-Aldrich; MO, USA) and 100 μg
ml−1
penicillin/streptomycin/glutamine (ThermoFisher Scientific). Resistant
cells were grown in 10 μM 4-OHT (re-suspended in tetrahydrofuran)
along with 10 pM β-E2 (re-suspended in ethanol). Sensitive cells were
maintained in base media with 10 μM of tetrahydrofuran and 10 pM
of ethanol. Cells were grown in an incubator at 37 C in a humidified
atmosphere of 5% CO2 and 95% air. For studies assessing the effects
of E2 and EGF, cells were incubated with phenol-red free media con-
taining 5% csFBC for 3 days prior to treatment with E2 and EGF at
−1
concentrations of 10 nM and 100 ng
ml , respectively, for
10 min/37 C.
2.2 | MTT assay
Both tamoxifen-sensitive and -resistant MCF-7 cells were seeded in
96-well plates and allowed to attach overnight. Cells were then
treated with DpC or 4-OHT at a range of concentrations (0–1.25 μM
for DpC and 0–40 μM for 4-OHT) and incubated for a further 72 hr.
Proliferation was examined using the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium (MTT; Sigma-Aldrich) assay, using standard
methods (Richardson, Tran, & Ponka, 1995). As shown previously,
MTT colour formation was directly proportional to viable cell number
(Richardson et al., 1995).
2.3 | Chou–Talalay combination analysis
For the combination studies with DpC and 4-OHT, the Chou–Talalay
method was used (Chou, 2006; Chou, 2010). The resulting combina-
tion index (CI) offers a quantitative definition for an additive effect,
where synergism is evident when CI < 1 and antagonism when CI > 1
in drug combinations (Chou, 2010). Briefly, to calculate the CI and
dose reduction index, cells were incubated with DpC and 4-OHT in a
range that was eight times below, and eight times above, their respec-
tive IC50 values after a 72 hr/37 C incubation (i.e., 0.125, 0.25, 0.5,
1, 2, 4, and 8 × IC50). The concentrations of DpC and 4-OHT used
were cell type dependent, as each cell line had its own IC50 values for
these agents. The resulting dose–response curves were then analysed
using Chou–Talalay methodology (Chou, 2006; Chou, 2010).
2.4 | Protein extraction, Western blot, and
antibodies
The antibody-based procedures used in this study comply with the
recommendations made by the British Journal of Pharmacology. Fol-
lowing treatment with either control, DpC (5 μM), 4-OHT (5 μM), or
14765381, 2020, 10, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.14985 by CAPES, Wiley Online Library on [24/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
2367
their combination (5 μM each) for 24 hr/37 C, cells were then
harvested and protein extracted using standard methods (Chen et al.,
2012). Western blot analysis was performed via established methods
(Gao & Richardson, 2001). The primary antibodies used (diluted at
1:1,000) included ER-α (RRID:AB_2632959), p-ER-α (Ser167; RRID:
AB_2102074), EGFR (RRID:AB_2262057), p-EGFR (Tyr1068; RRID:
AB_1903957), AKT (RRID:AB_329827), p-AKT (Ser473; RRID:
AB_329824), c-Myc (RRID:AB_2151827), p-c-Myc (Ser62; RRID:
AB_2151830), cyclin D1 (RRID:AB_2228523), p27 (RRID:
AB_10693314), SRC3 (RRID:AB_823642), NF-κB-p65 (RRID:
AB_2341215), and p-NF-κB-p65 (Ser536; RRID:AB_331281; Cell Sig-
naling Technology, MA); β-actin (diluted at 1:10,000, RRID:
AB_476692) was purchased from Sigma-Aldrich. The secondary anti-
bodies implemented (diluted 1:10,000) include anti-rabbit and anti-
mouse antibodies (Sigma-Aldrich; RRID:AB_2336059, RRID:
AB_2336060).
2.5 | Spheroid generation and
immunofluorescence
Spheroids were prepared by seeding 35,000 cells per well in phenol
red-free RPMI supplemented with 5% csFBS and 100 μg
ml−1
penicillin/streptomycin/glutamine in 96-well plates coated with
0.75% (w/v) agarose. The plates were then incubated for
3–4 days/37 C in 5% CO2. Once established, spheroids were treated
with either 5-μM DpC, 5-μM 4-OHT, or these agents combined at
5 μM each (combination) for a further 24 hr/37 C. Spheroids were
then fixed in 4% paraformaldehyde for 3 hr/20 C. Following fixation,
spheroids were washed three times with PBS, permeabilized with
0.1% Triton X-100, and blocked using 5% BSA in PBS overnight at
4 C. They were then incubated with either mouse Ki-67 (RRID:
AB_2797703) or rabbit ER-α monoclonal antibodies (Cell Signaling
Technology) overnight at 4 C. After washing 3 × 10 min with PBS,
secondary antibodies (anti-mouse IgG Alexa Fluor 488 [RRID:
AB_10694704] and anti-rabbit IgG Alexa Fluor 594 [RRID:
AB_2716249]; Cell Signaling Technology) were added at 1:200 dilu-
tion in BSA buffer for 1 hr/20 C. Nuclei were stained with DAPI (Life
Technologies), and spheroids imaged and photographed using a META
confocal microscope (Carl Zeiss, Germany). Intensity analysis was per-
formed using Fiji ImageJ software (NIH, Baltimore, MD; RRID:
SCR_003070).
2.6 | Colony formation assay
MCF-7-sensitive and -resistant cells were harvested and plated
(70 cells per plate) in 100-mm Petri dishes for 21 days with media
containing either DpC (0.125 μM), 4-OHT (0.125 μM), or their combi-
nation at 0.125 μM each. Colonies were then rinsed with 10-ml PBS,
fixed (acetic acid/methanol [1:7 v/v]), and stained with 0.5% crystal
violet. After staining, colonies were visualized and examined
microscopically.

2368
2.7 | Dual-luciferase assay for ER-α
The ERE Luciferase Reporter Assay Kit (Cignal Reporter Assay Kit;
Qiagen, Netherlands) was used following the manufacturer's proto-
col. Briefly, the MCF-7-sensitive and -resistant cells were trans-
fected with the ERE Luciferase Reporter, as well as the relevant
positive and negative controls for 48 hr. Cells were then incubated
with either DpC (5 μM), 4-OHT (5 μM), or their combination (5 μM
each), in the presence of E2 for a further 24 hr. Luciferase activity
was then assessed implementing the Dual-Luciferase Reporter
Assay kit (Cat. No. 1910; Promega) using a luminometer (FLUOstar;
BMG Labtech, Germany).
2.8 | Densitometry
Densitometry was performed using Quantity One software (Bio-Rad;
CA; RRID:SCR_014280) and normalized using the β-actin loading
control.
2.9 | Data and statistical analysis
Data are presented as mean ± SD. The group data subjected to
statistical analysis have a minimum of n = 5 independent samples
per group. Statistical comparisons between two groups were evalu-
ated by the unpaired Student's t test and were considered signifi-
cant when P < .05. The data and statistical analysis comply with
the recommendations of the British Journal of Pharmacology on
experimental design and analysis in pharmacology (Curtis et al.,
2018). To provide blinding in our experiments, analysis of the
experimental data was performed by different people to those that
completed the experiment. All studies were designed to provide
randomization, with cells being randomly assigned to different
treatment groups.
2.10 | Materials
DpC was synthesized as described previously (Lovejoy et al., 2012).
4-OHT was supplied by Sigma-Aldrich (MO, USA) and EGF was sup-
plied by Cell Signaling Technology (MA, USA).
2.11 | Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to
corresponding entries in http://www.guidetopharmacology.org, the
common portal for data from the IUPHAR/BPS Guide to PHARMA-
COLOGY (RRID:SCR_013077; Harding et al., 2018), and are perma-
nently archived in the Concise Guide to PHARMACOLOGY
2019/20 (Alexander, Cidlowski et al., 2019; Alexander, Fabbro
et al., 2019a, 2019b).
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MAQBOOL ET AL.
3 | RE SU LT S
3.1 | Assessing the synergy between DpC and
4-OHT in breast cancer cells
The novel anti-cancer agent, DpC, and its potential to synergize
with the active metabolite of tamoxifen, 4-OHT, was
assessed using well-characterized ER-a positive (MCF-7 and T47D)
and ER-a negative (MDA-MB-231 and MDA-MB-453) breast
cancer cells (Miller & Katzenellenbogen, 1983; Vranic, Gatalica, &
Wang, 2011).
DpC substantially inhibited proliferation of all breast cancer cells
examined, with IC50 values (72 hr) ranging from 0.006 to 0.024 μM
(Figure 1a). ER-positive breast cancer cells (T47D and MCF-7) were
most sensitive, while triple-negative MDA-MB-231 cells were least
sensitive to DpC (Figure 1a). 4-OHT alone displayed much higher IC50
values than DpC, ranging from 7 to 14 μM (72 hr; Figure 1b). As
expected, ER-positive cells were two to three times more sensitive to
4-OHT compared to ER-negative cells (MDA-MB-453 and MDA-MB-
231; Figure 1b).
Next, we examined the potential synergy between DpC and
4-OHT using the Chou–Talalay method (Chou, 2010). For ER-
positive MCF-7 and T47D cells, the combination of DpC with
4-OHT was highly synergistic (Figure 1c). In contrast, the ER-
negative MDA-MB-231 and MDA-MB-453 cells had CI values close
to or >1, indicating no synergism (Figure 1c). These results suggest
that the combination of DpC and 4-OHT was synergistic only in ER-
positive breast cancer cells.
3.2 | Assessing synergy between DpC and 4-OHT
in tamoxifen-sensitive and -resistant cells
We next investigated the potential of combining DpC with 4-OHT in
overcoming tamoxifen resistance in ER-positive cells using
tamoxifen-resistant MCF-7 (resistant) cells and their sensitive coun-
terparts (sensitive; Knowlden et al., 2003). MTT studies were con-
ducted to assess the IC50 values of each agent in these cells under
different conditions, including (a) no ligands, (b) E2 alone (10 nM),
(c) EGF alone (100 ng
ml−1), or (d) E2 (10 nM) and EGF (100 ng
ml−1)
together, for 72 hr. This was done as E2 or EGF can activate ER-α,
with EGF also promoting tamoxifen resistance (Ghayad et al., 2010;
Knowlden et al., 2003).
Tamoxifen-sensitive MCF-7 cells were highly sensitive to DpC
under all conditions assessed, with IC50 values <0.02 μM
(Figure 1d). Tamoxifen-resistant cells had significantly higher IC50
values for DpC in the absence of ligands, although sensitivity was
restored upon addition of E2, EGF, or their combination
(Figure 1d). The IC50 values for 4-OHT in the sensitive cells
(Figure 1e) were up to 100-fold higher compared to DpC
(Figure 1d). As expected, the IC50 values for 4-OHT in the resistant
cells were significantly higher, or slightly higher (i.e., for E2), when
compared to their sensitive counterparts (Figure 1e). However, in
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MAQBOOL ET AL. 2369

F I G U R E 1 The combination of DpC and 4-OHT is synergistic in ER-positive breast cancer cells. IC50 values for (a) DpC and (b) 4-OHT
in MCF-7, T47D, MDA-MB-231, and MDA-MB-453 breast cancer cells. (c) Combination index (CI) values of DpC in combination with
4-OHT in MCF-7, T47D, MDA-MB-231, and MDA-MB-453 breast cancer cells. IC50 values for (d) DpC and (e) 4-OHT in MCF-7-sensitive
and -resistant cells in the presence of control media or media containing E2, EGF, or both. (f) CI values of DpC combined with 4-OHT in
MCF-7-sensitive and -resistant cells in control media or media containing E2, EGF, or both. Data represent the mean ± SD of five
independent experiments. *P < .05, significantly different from the respective control; #P < .05, significantly different from the
corresponding treatment in MCF-7-sensitive cells
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2370 MAQBOOL ET AL.

the presence of E2, EGF, or their combination, the resistant
cells also became significantly more sensitive to 4-OHT compared
with the control (Figure 1e).
For both sensitive and resistant cells, a synergistic effect was
observed when DpC and 4-OHT were combined at the same dose
ratios, with CI values <1 under all conditions (Figure 1f). Notably,
synergy was more pronounced in control and E2-treated resistant cells
when compared with their sensitive counterparts. Assessing CI values
using different doses of DpC and 4-OHT (Tables S1 and S2) further
confirmed that these agents were synergistic in the majority of combi-
nations. Further, dose reduction index values (Tables S1 and S2) indi-
cated the doses of DpC and 4-OHT could be reduced to maintain
efficacy, while potentially reducing toxicity, which would be highly
beneficial in clinical use. Overall, these studies demonstrate that DpC
in combination with 4-OHT exhibited a synergistic effect in ER-
α-positive cell types.

F I G U R E 2 The effect of DpC, 4-OHT, and their combination on ER-α levels and transcriptional activity. Western blot analysis of total
ER-α and its phosphorylation at Ser167 in (a) sensitive and (b) tamoxifen-resistant MCF-7 cells treated with either control media or media
containing DpC (5 μM), 4-OHT (5 μM), or their combination (5 μM each) in the presence or absence of E2. β-actin was used as a protein
loading control. Data represent the mean ± SD of five independent experiments. *P < .05, significantly different from the respective
controls. Luciferase activity of ERE-containing constructs in MCF-7 (c) sensitive and (d) resistant cells treated with either control media or
media containing DpC, 4-OHT, or their combination. Data represent the mean ± SD of five independent experiments. *P < .05, significantly
different from the respective control

MAQBOOL ET AL.
3.3 | DpC alone or in combination with 4-OHT
decreases ER-α expression
To elucidate the mechanisms underlying the synergy between DpC
and 4-OHT, we next examined the effect of DpC alone or in combina-
tion with 4-OHT on ER-α in tamoxifen-sensitive and -resistant MCF-7
cells (Figure 2a,b). Both cell types were incubated for 24 hr with either
DpC (5 μM), 4-OHT (5 μM), or these agents combined (5 μM each),
followed by a 10-min incubation with or without the ER-α activating
ligand, E2 (10 nM). These doses were selected based on our studies
using DpC in vitro (Kovacevic et al., 2011) and studies by others using
4-OHT (Lee et al., 2018). The levels of total ER-α or its phosphoryla-
tion at Ser167 (p-ER-α), which contributes to tamoxifen resistance
(Bostner et al., 2013; Campbell et al., 2001), were then assessed via
Western blot.
In sensitive cells, DpC significantly down-regulated ER-α and p-
ER-α (Figure 2a), while 4-OHT markedly increased ER-α and p-ER-α
under all conditions (Figure 2a). This was expected, as 4-OHT binds to
ER-α, leading to its stabilization and the inhibition of downstream sig-
nalling (Jaiyesimi, Buzdar, Decker, & Hortobagyi, 1995). Assessing the
ratio of p-ER-α to total ER-α for 4-OHT demonstrated a significant
increase, compared with the control, suggesting that 4-OHT facili-
tated ER-α phosphorylation (Figure 2a). In contrast, this ratio did not
significantly alter for DpC relative to the control, suggesting that a
decrease in ER-α phosphorylation can be attributed to decreased total
ER-α. When DpC was combined with 4-OHT (combination), total ER-
α and p-ER-α remained similar to the untreated controls under all
conditions.
In resistant cells, DpC again markedly reduced total ER-α, while
4-OHT had no significant effect (Figure 2b). In contrast to sensitive
cells (Figure 2a), DpC significantly increased p-ER-α in the absence of
E2, while reducing its levels in the presence of E2 in resistant cells
(Figure 2b). The combination of DpC with 4-OHT in resistant cells
gave similar results to DpC alone (Figure 2b).
3.4 | DpC and 4-OHT inhibit ER-α transcriptional
activity in sensitive and resistant MCF-7 cells
Further studies assessed ER-α transcriptional activity, as activated ER-
α can directly or indirectly bind to target gene EREs to promote breast
cancer progression (Levin & Pietras, 2008). To achieve this, ERE dual-
luciferase reporter assays were performed, which was transfected into
tamoxifen-sensitive and -resistant MCF-7 cells followed by incubation
with DpC (5 μM), 4-OHT (5 μM), or their combination for 24 hr.
A significant decrease in luciferase activity was observed in sensi-
tive (Figure 2c) and resistant (Figure 2d) cells upon treatment with
DpC or 4-OHT. The combination of DpC and 4-OHT also led to a pro-
nounced down-regulation of luciferase activity in sensitive and resis-
tant cells, with the combination being most effective in resistant cells
(Figure 2d).
Considering reduced ER-α transcriptional activity, we further
examined two major co-activators of ER-α, namely, SRC3 and NF-κB-
14765381, 2020, 10, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.14985 by CAPES, Wiley Online Library on [24/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
2371
p65 (Gionet, Jansson, Mader, & Pratt, 2009; Osborne et al., 2003).
SRC3 was reduced by DpC and the combination in sensitive cells,
while being reduced by DpC, 4-OHT, and their combination in resis-
tant cells (Figure S1). While total NF-κB-p65 was not affected by
these agents in sensitive cells, DpC, 4-OHT, and their combination
reduced NF-κB-p65 in resistant cells. Notably, in the presence of E2,
the combination treatment was most effective at reducing SRC3 and
NF-κB-p65 levels in the resistant cells (Figure S1), demonstrating the
synergy between DpC and 4-OHT. The activating phosphorylation of
NF-κB-p65 (Ser536) was also most reduced by the combination in
sensitive cells. However, its expression in the resistant cells was
increased by DpC or 4-OHT (Figure S1), which may account for the
lower sensitivity of these cells to these agents alone (Figure 1d,e).
Importantly, this adverse effect was prevented when resistant cells
were incubated with DpC and 4-OHT as a combination (Figure S1),
further highlighting their synergy.
3.5 | DpC down-regulates AKT activation in
tamoxifen-sensitive and -resistant MCF-7 cells
To determine whether the synergy between DpC and 4-OHT was due
to inhibition of EGFR signalling, we assessed total EGFR and its acti-
vating phosphorylation at Tyr1068 in tamoxifen-sensitive and
-resistant MCF-7 cells.
Considering its ability to down-regulate EGFR in other cell types
(Kovacevic et al., 2016), it was unexpected that DpC alone or in com-
bination with 4-OHT up-regulated EGFR in sensitive and resistant
cells, regardless of E2 treatment (Figure 3a,b). Further, DpC and the
combination of DpC with 4-OHT also distinctly up-regulated p-EGFR,
although this was only evident in the presence of E2 in sensitive cells
(Figure 3a). In resistant cells, DpC also significantly increased p-EGFR
when E2 was present (Figure 3b), although this effect was far less pro-
nounced than in sensitive cells (Figure 3a).
Conversely, 4-OHT had no significant effect on total or phos-
phorylated EGFR in the sensitive cells (Figure 3a), while a slight but
significant reduction in total EGFR was observed in resistant cells in
the absence of E2 (Figure 3b). The ratio of p-EGFR/EGFR was signifi-
cantly increased for DpC and the combination in sensitive cells in
the presence of E2 (Figure 3a), while a significant decrease in this
ratio was observed under these conditions in resistant cells
(Figure 3b). Overall, these results suggest that DpC promotes EGFR
phosphorylation in sensitive MCF-7 cells to a much greater extent
than in resistant cells.
To further investigate this, we examined these cells in the pres-
ence of E2 and EGF together, as addition of EGF results in more
robust EGFR activation (Stabile et al., 2005). Under control conditions,
endogenous EGFR was very low in sensitive cells, with E2 + EGF hav-
ing no substantial effect on EGFR (Figure S2A). In response to DpC
alone, total and p-EGFR levels were markedly increased in sensitive
cells (Figure S2A), as observed with E2 alone (Figure 3a). However, the
stimulatory effect of EGF + E2 on p-EGFR was inhibited when DpC
was combined with 4-OHT (Figure S2A).
 14765381, 2020, 10, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.14985 by CAPES, Wiley Online Library on [24/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
2372 MAQBOOL ET AL.

F I G U R E 3 DpC inhibits downstream AKT activation in tamoxifen-sensitive and -resistant cells. Western blot analysis of total EGFR and its
phosphorylation at Tyr1068, along with total AKT and its phosphorylation at Ser473 in (a) tamoxifen-sensitive and (b) tamoxifen-resistant MCF-7
cells treated with either control media or media containing DpC (5 μM), 4-OHT (5 μM), or their combination (5 μM each) in the presence or
absence of E2. β-actin was used as a protein loading control. Data represent the mean ± SD of five independent experiments. *P < .05,
significantly different from the respective control

The resistant cells had much higher endogenous EGFR and to (Figure S2A). However, this increase of p-EGFR in resistant cells
a lesser extent, p-EGFR, than sensitive cells (Figure S2A). Further, incubated with E2 and EGF was not observed with the combina-
resistant cells responded strongly to E2 + EGF treatment, with a tion treatment (Figure S2A). Hence, in the presence of E2 + EGF,
pronounced increase in p-EGFR observed after control, DpC, and the DpC and 4-OHT combination potently inhibited EGFR
4-OHT treatment, compared with the control without E2 + EGF activation.
 14765381, 2020, 10, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.14985 by CAPES, Wiley Online Library on [24/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
MAQBOOL ET AL. 2373

To determine whether other major receptors were affected by
these chemotherapeutic agents, we also assessed HER2 and insulin-
like growth factor 1 receptor (IGF1R) in sensitive and resistant cells,
as these receptors contribute to tamoxifen resistance (Osborne et al.,
2003; Osborne et al., 2005; Riggins et al., 2007). While HER2 was not
markedly affected by DpC or 4-OHT in sensitive cells, this receptor
was reduced by DpC and the combination in resistant cells, regardless
of E2 (Figure S2B). Further, the combination of DpC with 4-OHT
decreased IGF1R levels in sensitive cells, while having no distinct
effect in resistant cells (Figure S2B).

F I G U R E 4 Combining DpC with 4-OHT inhibits c-Myc and cyclin D1, while promoting p27 expression. Western blot analysis of c-Myc and
its phosphorylation site at Ser62, along with cyclin D1 and p27 in (a) sensitive- and (b) tamoxifen-resistant MCF-7 cells treated with either control
media or media containing DpC (5 μM), 4-OHT (5 μM), or their combination (5 μM each) in the presence or absence of E2. β-actin was used as a
protein loading control. Data represent the mean ± SD of five independent experiments. *P < .05, significantly different from the respective
control

2374
As EGFR, HER2, and IGF1R signalling pathways converge on sim-
ilar downstream targets, we investigated their major target, AKT, and
its activating phosphorylation at Ser473 (Levin & Pietras, 2008), in
sensitive and resistant MCF-7 cells (Figure 3a,b). Once activated via
phosphorylation, AKT directly phosphorylates ER-α at Ser167, which
is a major contributor to tamoxifen resistance (Campbell et al., 2001).
In sensitive cells, total AKT was significantly reduced by the combina-
tion in the absence of E2, while no distinct effect was observed under
other conditions (Figure 3a). Interestingly, 4-OHT markedly increased
p-AKT in the absence of E2, while p-AKT was significantly reduced by
DpC alone or the combination in the presence of E2 (Figure 3a). A
similar effect was observed in resistant cells, with DpC and the combi-
nation also significantly decreasing p-AKT under all conditions
(Figure 3b).
Taken together, our results showed that AKT activation was
substantially inhibited in resistant cells, suggesting a mechanism
by which DpC and its combination with 4-OHT overcome
resistance.
3.6 | DpC combined with 4-OHT attenuates c-Myc
and cyclin D1, while promoting p27 expression
We next assessed the major downstream targets of ER-α and AKT,
including c-Myc and cyclin D1, both of which play oncogenic roles in
breast cancer and are associated with tamoxifen resistance (Butt
et al., 2005; Green et al., 2016; Sears, 2004). We also examined the
tumour suppressor, p27, a major cyclin D1 inhibitor that attenuates
proliferation (Chiarle, Pagano, & Inghirami, 2001).
In sensitive cells, c-Myc was significantly down-regulated under
all conditions, with the combination having the most potent effect
(Figure 4a). In these cells, c-Myc was detected as two bands (~50 and
60 kDa), which may reflect different isoforms (Benassayag et al.,
2005), or post-translational modification (Hann, 2006), with quantita-
tion assessing both bands as a total. Next, we examined phosphory-
lated c-Myc (Ser62), as this phosphorylation is associated with
increased c-Myc stability and oncogenic activity (Sears, 2004; Yeh
et al., 2004). Both DpC and the combination significantly decreased p-
c-Myc under all conditions (Figure 4a). 4-OHT also potently reduced
p-c-Myc, but only in the presence of E2 in sensitive cells. A similar
trend was observed for cyclin D1, with DpC and the combination sig-
nificantly decreasing cyclin D1 under all conditions (Figure 4a). p27
was significantly increased by 4-OHT in sensitive cells, while DpC had
no significant effect (Figure 4a).
In resistant cells, c-Myc was predominantly expressed as the
upper band (Figure 4b). DpC alone again significantly reduced total c-
Myc, p-c-Myc, and cyclin D1, while having no effect on p27
(Figure 4b). However, in direct contrast to its effects in sensitive cells
(Figure 4a), 4-OHT failed to decrease total c-Myc or cyclin D1, while
significantly reducing p27 in resistant cells (Figure 4b). This is in agree-
ment with these cells being tamoxifen resistant (Kilker & Planas-Silva,
2006). The combination was most effective in resistant cells, signifi-
cantly decreasing c-Myc phosphorylation and cyclin D1 expression
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MAQBOOL ET AL.
and significantly increasing the cyclin-dependent kinase inhibitor, p27
(Figure 4b).
While the above studies were conducted with DpC at 5 μM,
lower doses of DpC (i.e., 0.1, 0.5, and 1 μM) alone or combined with
5-μM 4-OHT induced similar, dose-dependent effects on ER-α, p-ER-
α, p-AKT, p-c-Myc, and cyclin D1 in both sensitive and resistant cells
(Figure S3).
3.7 | Combining DpC with 4-OHT decreases
proliferation and inhibits colony formation
To further assess the synergy between DpC and 4-OHT using more
pathologically relevant tumour models, we examined these agents
using 3D cell culture, where MCF-7-sensitive and -resistant cells were
grown as spheroids. Spheroids better represent the in vivo setting and
are more resistant to chemotherapy (Edmondson, Broglie, Adcock, &
Yang, 2014; Nyga, Cheema, & Loizidou, 2011). Once established,
spheroids were treated with DpC (5 μM), 4-OHT (5 μM), or their com-
bination for 24 hr and examined for ER-α and Ki-67 (marker of prolif-
eration; Yerushalmi, Woods, Ravdin, Hayes, & Gelmon, 2010) via
immunofluorescence.
A significant down-regulation of ER-α and Ki-67 was observed
in sensitive and resistant spheroids treated with DpC alone or in
combination with 4-OHT (Figure 5a). Notably, 4-OHT alone did
not affect ER-α and Ki-67 under these conditions. These results
support our current findings that down-regulation of c-Myc and
cyclin D1 by DpC and the combination decreases proliferation. We
also performed colony formation assays to determine the ability of
sensitive and resistant cells to undergo unlimited division. While
control cells formed large colonies, no colony formation was
observed with DpC or the combination (Figure 5b,c). Further,
4-OHT reduced colony formation in sensitive cells, but not in
resistant cells where colonies were similar to the control
(Figure 5b,c), which further confirms that these cells are resistant
to tamoxifen.
4 | DI SCU SSION
Various pathways contribute to tamoxifen resistance, with ER-α over-
expression and its crosstalk with multiple oncogenic pathways such as
EGFR and its downstream targets (i.e., AKT, c-Myc, and cyclin D1)
being major players (Butt et al., 2005; Campbell et al., 2001; Chang,
2012; Fan et al., 2007; Ghayad et al., 2010; Knowlden et al., 2003).
We demonstrated that the novel, clinically tested anti-cancer agent,
DpC, inhibits multiple oncogenic pathways, including EGFR, AKT, c-
Myc, and cyclin D1, in pancreatic and prostate cancer cells (Kovacevic
et al., 2011; Kovacevic et al., 2016; Xi et al., 2017), leading to potent
anti-tumour activity in vitro and in vivo (Kovacevic et al., 2011;
Lovejoy et al., 2012). These studies prompted us to examine the effi-
cacy of DpC against breast cancer and its potential to overcome
tamoxifen resistance.
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MAQBOOL ET AL. 2375

F I G U R E 5 DpC and 4-OHT inhibit 3D growth and colony formation of tamoxifen-resistant and -sensitive MCF-7 cells. (a) Representative
images of ER-α (red), Ki-67 (green), and DAPI (blue) staining in multicellular 3D tumour spheroids of MCF-7-sensitive and -resistant cells treated
with either control, DpC (5 μM), 4-OHT (5 μM), or their combination (5 μM each) for 24 hr. The scale bar in the bottom right-hand corner of the
first image in each set represents 2 μm and is the same across all images. Quantification of pixel intensity for ER-α and Ki-67 was performed
using ImageJ software and expressed as mean ± SD (five experiments). *P < .05, significantly different from the respective control. (b) Colony
formation was analysed in tamoxifen-sensitive and -resistant MCF-7 cells in the presence of control media or media containing DpC (0.125 μM),
4-OHT (0.125 μM), or their combination (0.125 μM each) for 21 days. Higher magnification (10×) images are shown in (c). Scale bar in the bottom
right-hand corner of the first image in (c) represents 2 μm and is the same across all images. Data represent five independent experiments

2376
Here, we have demonstrated that DpC has potent anti-
proliferative activity against ER-positive, HER2 over-expressing, and
triple-negative breast cancer cells. DpC was least effective against
MDA-MB-231 triple-negative cells, and this may be due to the
absence of both ER-α and HER2 (Holliday & Speirs, 2011), which
we demonstrate are major DpC targets. DpC was most effective
against ER-positive cells, potently inhibiting proliferation and colony
formation for tamoxifen-sensitive and -resistant MCF-7 cells. Fur-
ther, Chou–Talalay analysis demonstrated that the combination of
DpC and 4-OHT was synergistic in tamoxifen-sensitive and
-resistant cells.
To elucidate the mechanisms of synergy between DpC and
4-OHT, major molecular effectors in the development of tamoxifen
resistance were examined. ER-α was potently decreased by DpC in
sensitive and resistant MCF-7 cells using 2D and 3D culture. Further,
the combination of DpC with 4-OHT also decreased ER-α and
inhibited its transcriptional activity. These agents also decreased two
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MAQBOOL ET AL.
major ER-α co-activators, namely, SRC3 and NF-κB-p65, further con-
tributing to the reduced ER-α transcriptional activity. In fact, NF-κB-
p65 and SRC3 both contribute to tamoxifen resistance in breast can-
cer, and their inhibition may be a vital aspect of the synergy between
DpC and 4-OHT in resistant cells (Biswas, Singh, Shi, Pardee, &
Iglehart, 2005; Osborne et al., 2003).
The effect of DpC on reducing cyclin D1 and c-Myc may further
contribute to the synergy between DpC and 4-OHT, as cyclin D1 and
c-Myc are necessary for growth of tamoxifen-resistant cells (Butt
et al., 2005; Kilker et al., 2004; Kilker et al., 2006). Notably, cyclin D1
and c-Myc are up-regulated by p-AKT, a major effector in the
PI3K/AKT pathway (Butt et al., 2005; Kilker et al., 2006). We found
that DpC alone, or in combination with 4-OHT, strongly inhibited AKT
activation in sensitive and resistant MCF-7 cells. In fact, this inhibitory
effect on AKT may be responsible for the decreased cyclin D1 and c-
Myc observed and could be central to the synergism between these
two agents (Figure 6a,b).
F I G U R E 6 Diagram of the effects of
4-OHT alone or in combination with DpC
in MCF-7 tamoxifen-sensitive and
-resistant cells. (b) In the sensitive cells, E2
and 4-OHT compete to bind to the ER-α
ligand binding site. Binding of 4-OHT
results in conformational change of the
receptor, inhibiting its ability to promote
the transcription of genes containing
oestrogen response elements (EREs).
4-OHT also up-regulates p27, which
further inhibits the cell cycle. When
combined with 4-OHT, DpC inhibits p-
AKT, leading to decreased c-Myc and
cyclin D1 levels, which further decrease
tumour cell proliferation. (b) In tamoxifen-
resistant cells, increased activation of
non-genomic pathways enables enhanced
AKT activation, which leads to
(a) phosphorylation of the ER-α receptor
at Ser167, which enables its
transcriptional activity, despite 4-OHT
binding (Likhite et al., 2006); (b) activation
of downstream c-Myc and cyclin D1; and
(c) inhibition of p27 levels and activity
(Kilker et al., 2006; Motti et al., 2004).
Together, these effects drive proliferation
and form the major mechanisms of
resistance to 4-OHT. Combining DpC
with 4-OHT in these cells inhibits p-AKT,
leading to reduced phosphorylation of ER-
α receptor at Ser167, as well as
decreasing c-Myc and cyclin D1 levels,
consequently reducing tumour cell
proliferation. By inhibiting p-AKT, DpC
also enables increased expression of p27
to further inhibit cell cycle progression

MAQBOOL ET AL.
p-AKT also promotes ER-α activation by Ser167 phosphorylation,
increasing ER-α binding to DNA and promoting its transcriptional
activity (Campbell et al., 2001; Likhite, Stossi, Kim,
Katzenellenbogen, & Katzenellenbogen, 2006). However, it is impor-
tant to note that while high levels of p-ER-α (Ser167) can correlate to
good patient outcomes (Motomura et al., 2010), AKT is associated
with poor outcomes and tamoxifen resistance in breast cancer
(Campbell et al., 2001). Here, DpC alone, or its combination with
4-OHT, markedly decreased p-ER-α (Ser167) in resistant MCF-7 cells.
This may contribute to the synergy between DpC and 4-OHT, as the
resistant cells can no longer activate ER-α via an intrinsic AKT-
mediated mechanism, leaving them more vulnerable to 4-OHT. Alter-
natively, the ability of DpC to inhibit other major AKT targets
(i.e., cyclin D1 and c-Myc) and ER-α co-activators (i.e., SRC3 and NF-
κB-p65) may be responsible for overcoming resistance to 4-OHT.
Another protein that mediates breast cancer endocrine resistance
is the cyclin-dependent kinase inhibitor p27, with low levels being
associated with breast cancer relapse (Arteaga, 2004). Restoration of
p27 in endocrine-resistant breast cancer cells re-sensitizes them to
anti-oestrogen therapies (Arteaga, 2004). We showed that, in sensi-
tive cells, 4-OHT up-regulated p27, which is in accordance with its
anti-proliferative activity (Kilker et al., 2006; Figure 6a). However, in
resistant cells, p27 was decreased by 4-OHT, demonstrating signifi-
cant molecular alterations that confer tamoxifen resistance. While
DpC alone did not affect p27, its combination with 4-OHT induced a
significant increase in p27 in resistant cells. This synergy may be medi-
ated by the ability of DpC to decrease p-AKT levels, as p-AKT pro-
motes degradation of p27 (Kilker et al., 2006; Motti, De Marco,
Califano, Fusco, & Viglietto, 2004).
The expression and activation of EGFR is another key difference
between tamoxifen-sensitive and -resistant cells, with this protein
being a major driver of tamoxifen resistance (Knowlden et al., 2003).
In agreement with this, we demonstrate that the resistant cells had
higher endogenous EGFR levels and responded robustly to E2 + EGF
treatment by increasing activating phosphorylation of EGFR, which
was in contrast to the sensitive cells. While DpC or its combination
with 4-OHT increased EGFR expression and activation in the sensitive
cells, the activation of EGFR was reduced by DpC, and in particular its
combination with 4-OHT, in the resistant cells in the presence of
E2 + EGF.
Importantly, downstream targets of EGFR signalling, namely, AKT,
c-Myc, and cyclin D1, were all decreased by DpC and its combination
with 4-OHT in both sensitive and resistant cells. This suggests that
increased EGFR levels/activation observed under some conditions
may be a compensatory response to counteract the inhibition of
downstream signalling by DpC. Further, the inhibitory effect of these
agents on downstream AKT signalling may also be mediated by other
receptors, such as IGF1R and HER2, which were decreased by DpC
alone and in combination with 4-OHT in sensitive and resistant cells,
respectively.
Another recently identified mechanism of anti-oestrogen resis-
tance involves the tumour micro-environment (TME), with FGF2 being
produced by the TME and acting on breast cancer cells to promote
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2377
resistance (Shee et al., 2018). FGF2 mediates its effects via the activa-
tion of MEK/ERK signalling to promote cyclin D1, which is vital for
FGF2-mediated resistance (Shee et al., 2018). Notably, we demon-
strate here that DpC and particularly its combination with 4-OHT
potently inhibited cyclin D1 expression. Hence, this therapeutic
approach may potentially also inhibit TME-mediated mechanisms of
resistance. However, further studies utilizing humanized in vivo
models that incorporate the TME are required to assess this.
Overall, combining DpC with 4-OHT was more effective than
DpC alone against tamoxifen-resistant cells, as shown by (a) the
Chou–Talalay method demonstrating strong synergy between DpC
and 4-OHT (Figure 1c,f); (b) the combination treatment being most
effective at inhibiting ER-α transcriptional activity in resistant cells
(Figure 2c); (c) the combination treatment most potently reducing ER-
α co-activators SRC3 and NF-κB-p65 in resistant cells (Figure S1); and
(d) only the combination treatment up-regulated the tumour suppres-
sor, p27, in resistant cells (Figure 4b). These results indicate that com-
bining DpC with tamoxifen may be a promising therapeutic approach
for ER-positive breast cancer to overcome endocrine resistance.
AC KNOW LEDG EME NT S
This project was supported by (a) Priority-driven Collaborative Cancer
Research Scheme (PdCCRS) Young Investigator Grant (1086449)
awarded to Z.K., which was co-funded by Cure Cancer Australia
Foundation and Cancer Australia, and (b) PdCCRS Project Grant
(1146599) awarded to D.R.R. and Z.K. and co-funded by the National
Breast Cancer Foundation and Cancer Australia. Z.K. is grateful for a
University of Sydney Bridging Fellowship, a National Health and Med-
ical Research Council RD Wright Fellowship (APP1140447), and a
Cancer Institute New South Wales (CINSW) Career Development Fel-
lowship (CDF171126). D.R.R. appreciates an NHMRC Senior Principal
Research Fellowship (1062607 and 1159596). P.J.J. was supported by
a CINSW Career Development Fellowship (CDF141147). S.N.M
acknowledges funding provided by IRSIP-HEC (Pakistan) and the
Molecular Pathology and Pharmacology Program (USYD). We would
also like to thank Dr. Elizabeth Caldon (Garvan Institute of Medical
Research, Sydney) for providing the tamoxifen-sensitive and -resistant
MCF-7 cells and valuable experimental advice.
AUTHOR CONTRIBU TIONS
S.N.M. designed and performed experiments, analysed data, and
wrote the manuscript. S.C.L. performed experiments and analysed
data. R.H. analysed data. Z.K., P.J.J., and D.R.R. designed experiments,
analysed data, and wrote the manuscript.
CONFLIC T OF INT ER E ST
The authors declare no conflicts of interest.
DECLARATION OF T RANSPARENCY AND SCI ENTIFIC
RIGOUR
This Declaration acknowledges that this paper adheres to the princi-
ples for transparent reporting and scientific rigour of preclinical
research as stated in the BJP guidelines for Design & Analysis, and

2378
Immunoblotting and Immunochemistry, and as recommended by
funding agencies, publishers, and other organizations engaged with
supporting research.
ORCID
Zaklina Kovacevic https://orcid.org/0000-0002-4742-4690
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SUPPORTING INF ORMATION
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How to cite this article: Maqbool SN, Lim SC, Park KC, et al.
Overcoming tamoxifen resistance in oestrogen receptor-
positive breast cancer using the novel thiosemicarbazone anti-
cancer agent, DpC. Br J Pharmacol. 2020;177:2365–2380.
https://doi.org/10.1111/bph.14985