1. AbstractAbstract
Triple negative breast cancer (TNBC) represents an aggressive and deadly form of breast cancer and it accounts
for 15-25% of all breast cancer cases. Unlike other subgroup of breast cancer, currently there are no molecular
and therapeutic targets for TNBC in that it lacks the expression of estrogen receptor (ER), progesterone receptor
(PR) and human epidermal growth factor receptor type 2 (HER2). Recently, it has been reported that MCT1
(the lactate transporter) is significantly elevated in human breast cancer tumors, compared with normal breast
tissue. Moreover, high expression of MCT1 and its ancillary protein CD-147 were found be associated with the
poor prognostic factors of basal-like subtype and high grade tumors. Recently, our laboratory reported mRNA
expression of MCT1 in a murine breast cancer cell lines (4T1) which has been shown to very closely mimic
human TNBC. Based on these findings, our goal was to characterize MCT1 and CD-147 membrane protein
expression and evaluate the impact of a novel MCT1 inhibitor, AR-15585 on L-lactate uptake in 4T1 cells.
Immunoblotting analyses showed that MCT1 and CD-147 proteins are expressed on the plasma membrane of
4T1 cells. The concentration dependent uptake of L-lactate in 4T1 cells was best fitted to a Michaelis-Menten
equation plus a diffusional uptake clearance (Km of 5.3 mM, Vmax of 31.4 nmol/mg/min and diffusional uptake
clearance of 2.69 uL/mg/min). AR-C15585 (200 and 2000 nM) significantly inhibited L-lactate uptake in 4T1
cells at pH 6.0 and 7.4. Our hypothesis, to be tested, is that MCT1 inhibition will result in cell death in TNBC
mediated by its effects on L-lactate transport and represents a new therapeutic treatment strategy in TNBC.
ObjectivesObjectives
 To Characterize MCT1 and CD-147 membrane protein expression in murine breast cancer 4T1 cells.
 To assess and evaluate uptake of L-lactate in 4T1 cells.
 To evaluate the impact of the MCT1 inhibitor AR-155858 on L-lactate uptake in 4T1 cells.
MethodsMethods
ResultsResults
ConclusionsConclusions
AcknowledgementsAcknowledgements
Characterizing MCT 1 and CD-147 in the Murine Breast Cancer 4T1 Cell line
Andy Lalka , Xiaowen Guan , Melanie Felmlee, Marilyn E. Morris
(1) Zhao S et al., Med Oncol. 2013 Mar;30(1):335.
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(7) Zhang Y et al., J Pharmacol Exp Ther. 2004
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(8) Roiko SA, et al., Drug Metab Dispos. 2012
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• MCT1 and CD-147 proteins are expressed on
the plasma membrane of 4T1 cells
• L-Lactate uptake in 4T1 cells exhibits pH and
concentration dependence and can be inhibited
by a specific MCT1 inhibitor, indicating the
importance of MCT1 in its uptake. The Km is
similar to that reported for MCT1-mediated
uptake of L-lactate in other cell systems.
• AR-C15585, a novel MCT1 inhibitor,
significantly inhibited L-lactate uptake in 4T1
cell at pH 6.0 and 7.4.
• Further studies are needed to investigate the
potential therapeutic role of MCT1 inhibition in
TNBC.
TNBC represents an aggressive and deadly form of breast cancer and it accounts for 15-25% of all breast
cancer cases (1) with higher incidence in African American women than other ethnic groups (2).The high
aggressive entity of TNBC is related to metastasis, poor prognosis, high recurrence and high mortality rates
observed clinically as compared to non-triple negative (non-TN) cancers (3). Unlike other subgroups of
breast cancer, currently there are no molecular and therapeutic targets for TNBC in that it lacks the
expression of ER, PR and HER2. Therefore, there remains a hurdle in finding and characterizing new
therapeutic targets in effort to improve clinical outcome for patients with this invasive form of breast tumor.
It is widely known that
tumor cells are highly glycolic and displace a phenomenon
Known as “Warburg effect“. The large production of
lactate from the high demand for glucose consumption
and glycolysis requires involvement of monocarboxylate
transporters (MCTs) for the removal of lactate from the
tumor cells. MCTs are SLC16A family of transporter that
comprises 14 members and of which only the first four
(MCT1-4) are shown to exhibit proton-linked transport
of many of the important endogenous monocarboxylates
such as lactate, pyruvate and ketone bodies (4). Functional
transport by MCT1, 3 and 4 require an ancillary protein,
CD-147 (5). Recently, it has been reported that MCT1
is significantly elevated in human breast cancer tumors,
compared with normal breast tissue. Moreover, high expression of MCT1 and its ancillary protein CD-147
were found be associated with the poor prognostic factors basal-like subtype and high grade tumors (6).
Furthermore an analogue of a novel MCT1 inhibitor AR-C155858 (AZD3965) is being used in a Phase I
study in small cell lung cancer in a UK/AstraZeneca study. In the present study, our aim is to characterize
MCT1 and CD-147 membrane protein expression and to evaluate the impact of MCT1 inhibition on L-
lactate uptake in 4T1 cells.
Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, SUNY
Figure 4. Inhibition of L-lactate uptake in 4T1 cells by AR-
155858. AR-C15585 (200 and 2000 nM) significantly
inhibited L-lactate uptake in 4T1 cells at pH 6.0 and 7.4. All
data represent mean ± SEM. ANOVA followed by a
Dunnett’s post hoc test were used for analyzing statistical
significance among means of different groups and P<0.05
was considered statistically significant.
Figure 2. (A.) Plasma membrane
expression of MCT1 and CD-147 in 4T1
cells. 20 ug per lane. KNRK cells were
used as the positive control.
(B.) PCR analysis of MCT1 mRNA
expression in 4T1 cells. Mouse kidney was
used as the positive control.
Figure 3. (A). Time dependent L-lactate uptake at pH 6.0 in 4T1 cells. (B). Concentration dependent uptake of L-
lactate at pH 6.0 in 4T1 cells was best fitted to a Michaelis-Menten equation with a diffusional uptake clearance
component.
Cell Culture: Murine breast cancer, 4T1 cells were cultured in RPMI media (standard medium) supplemented with 10%
fetal bovine serum and 1% penicillin/streptomycin at 37°C in a humidified atmosphere with 5%CO 2/95% air.
Western blot: Total plasma membrane protein was isolated using ultracentrifugation followed by plasma membrane
enrichment according to the protocol by Zhang Y et al. 2004 (7). 20 µ g of membrane protein per lane were ran in 10%
SDS PAGE gel and transferred electrophoretically onto nitrocellulose membranes. Membranes were blocked with 5%
(w/v) nonfat milk in Tris-buffer saline containing 0.05% (v/v) Tween 20 (TBST) overnight at 4° C. Selective antibody
against MCT1 and CD147 were used to blot the membranes followed by incubation with secondary antibody coupled to
HRP for 1 hr. Membranes were incubated with ECL substrates for 5 min followed with visualization in ChemiDocTM
XRS + system (Bio Rad, Hercules, CA).
Uptake study: 4T1 cells were plated in 35 mm (diameter) culture dishes at a density of 200,000 cells/ml two days before
the uptake studies. Uptake studies of [3H]-L-lactate were performed according to the protocol detailed by Roiko et al.
(2011)(8). In brief, growth medium was removed from the culture dishes and cells were washed three times followed by
equilibration in uptake buffer for 20 min at 37°C. All uptake studies were performed at room temperature. One milliliter
of uptake buffer containing [3H]-L-lactate was added to the dishes to initiate the uptake. The uptake was terminated by
aspirating out the uptake buffer and the cells were washed three times with ice-cold buffer immediately. The cells were
lysed and the radioactivity was determined using liquid scintillation counter (1,900 CA, Tri-carb liquid scintillation
analyzer). Protein concentrations were determined using Bicinchoninic Acid assay. All the data points were triplicated.
The overall L-lactate accumulation in the cells was normalized with total protein content.

For time-dependent uptake, cells were incubated with uptake buffer (pH6.0) containing 1 µ Ci of [3 H]-L-lactate for
0.5, 1, 5, 10, 15, 30, 60 and 120 min at room temperature.

For concentration dependent uptake of L-lactate, L-lactate concentration ranged from 0 to 30 mM.

For assessing the impact of AR-155858 on L-lactate uptake in 4T1 cells, cells were pre-incubated with AR-C-155858
(0, 20, 200 and 2000 nM) for 30 mins followed by L-lactate (0.5M) uptake reaction.
Statistical Analysis: All data represent mean ± SEM. ANOVA followed by a Dunnett’s post hoc test were used for
analyzing statistical significance among means of different groups and P<0.05 was considered statistically significant.
BackgroundBackground
ResultsResults
A. B.
B.A.
Parameter Estimates %CV
Vmax (nmol/mg/min) 31.4 39.84
Km (mM) 5.33 37.70
P (uL/mg/min) 2.69 16.95
ReferencesReferences
Table 1: L-lactate uptake parameter estimates in 4T1 cells.
Finding from the National Institute of Drug Abuse grant DA-
023223