Journal of Pathology

J Pathol 2015; 237: 152–165 ORIGINAL PAPER

Published online 4 June 2015 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/path.4562

Functional screening identifies MCT4 as a key regulator of breast

cancer cell metabolism and survival
Franziska Baenke,1# Sébastien Dubuis,2 Charlene Brault,3 Britta Weigelt,4 Beatrice Dankworth,3 Beatrice Griffiths,1
Ming Jiang,5 Alan Mackay,6 Becky Saunders,5 Bradley Spencer-Dene,7 Susana Ros,1 Gordon Stamp,7 Jorge S
Reis-Filho,4 Michael Howell,5 Nicola Zamboni3 and Almut Schulze1,3,8*
1 Gene Expression Analysis Laboratory, Cancer Research UK London Research Institute, UK
2 Institute of Molecular Systems Biology, ETH Zurich, Switzerland
3 Department of Biochemistry and Molecular Biology, Theodor-Boveri-Institute, Biocentre Am Hubland, Würzburg, Germany
4 Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA
5 High Throughput Screening Facility, Cancer Research UK London Research Institute, UK
6
Divisions of Molecular Pathology and Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey, UK
7 Experimental Histopathology, Cancer Research UK London Research Institute, UK
8 Comprehensive Cancer Centre Mainfranken, Würzburg, Germany

*Correspondence to: A Schulze, Department of Biochemistry and Molecular Biology, Theodor-Boveri-Institute, Biocenre, Am Hubland, 97074
Würzburg, Germany. E-mail: almut.schulze@uni-wuerzburg.de
#
Current address: Molecular Oncology Laboratory, Cancer Research UK Manchester Institute, Wilmslow Road, Manchester M20 4BX, UK.

Abstract
Metabolic reprogramming in cancer enhances macromolecule biosynthesis and supports cell survival. Oncogenic
drivers affect metabolism by altering distinct metabolic processes and render cancer cells sensitive to perturbations
of the metabolic network. This study aimed to identify selective metabolic dependencies in breast cancer by
investigating 17 breast cancer cells lines representative of the genetic diversity of the disease. Using a functional
screen, we demonstrate here that monocarboxylate transporter 4 (MCT4) is an important regulator of breast cancer
cell survival. MCT4 supports pH maintenance, lactate secretion and non-oxidative glucose metabolism in breast
cancer cells. Moreover, MCT4 depletion caused an increased dependence of cancer cells on mitochondrial respiration
and glutamine metabolism. MCT4 depletion reduced the ability of breast cancer cells to grow in a three-dimensional
(3D) matrix or as multilayered spheroids. Moreover, MCT4 expression is regulated by the PI3K–Akt signalling
pathway and highly expressed in HER2-positive breast cancers. These results suggest that MCT4 is a potential
therapeutic target in defined breast cancer subtypes and reveal novel avenues for combination treatment.
Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keywords: breast cancer; metabolism; MCT4; lactate; PI3K–Akt pathway; HER2

Received 12 August 2014; Revised 23 March 2015; Accepted 4 May 2015

No conflicts of interest were declared.

Introduction Breast cancer comprises a heterogeneous group of

Metabolic transformation in cancer supports macro-
molecule biosynthesis and is crucial for cancer cell sur-
vival and tumour formation [1,2]. Many solid tumours
exhibit increased glucose uptake and lactate secre-
tion, a feature known as the Warburg effect. Most
metabolic alterations observed in cancer are the conse-
quence of oncogene activation or inactivation of tumour
suppressors [3], but genetic alterations in metabolic
enzymes have also been observed. Cancer metabolism
is a highly dynamic network that adapts to changes in
environmental conditions. However, metabolic repro-
gramming produces selective dependencies in cancer
cells [4] and metabolic enzymes may provide suitable
targets for the development of novel treatment strate-
gies [5,6].
Copyright © 2015 Pathological Society of Great Britain and Ireland.
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
tumours. Clinically, it is classified by expression of
oestrogen receptor (ER), progesterone receptor (PR)
and human epidermal growth factor receptor 2 (HER2).
Comprehensive genomic and transcriptomic analyses
revealed that breast cancers can be divided into several
subgroups with distinct molecular and clinical features
[7–12]. These analyses have revealed the extent of the
diversity of the constellation of somatic genetic aberra-
tions found in breast cancer; only a few genes are recur-
rently mutated, many genes are altered in a small number
of cases, and each breast cancer may harbour a distinct
repertoire of somatic genetic changes [10,11,13,14].
One common feature within the complex genetic land-
scape of breast cancer is the activation of the PI3K–Akt
signalling pathway [9–11]. Akt is one of the main
drivers of anabolic metabolism and activates glucose
J Pathol 2015; 237: 152–165
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MCT4 supports breast cancer cell survival
uptake, glycolysis and lipid synthesis [15]. In addi-
tion to genetic heterogeneity, breast cancers also show
environmental complexity with regions of high and
low delivery of nutrients and oxygen. The presence of
low-oxygen niches in solid tumours (hypoxia) is asso-
ciated with poor clinical outcome and can promote ded-
ifferentiation, chemoresistance, enhanced migration and
metastasis formation [16,17]. Identifying the molecular
dependencies of hypoxic cancer cells is integral to suc-
cessful treatment.
In this study, we sought to identify specific metabolic
dependencies of genetically diverse breast cancer cell
lines under normoxic and hypoxic conditions. Using an
unbiased functional RNA interference (RNAi) screen,
we found that the proton-linked monocarboxylate
transporter 4 (MCT4) is an important regulator of
breast cancer cell survival in vitro and in vivo. We
found that MCT4 supports metabolite transport, pH
homeostasis and redox balance in breast cancer cells.
Detailed metabolic characterization of MCT4-depleted
cells revealed an increased dependence on oxida-
tive metabolism and glutaminolysis. MCT4 depletion
blocked the ability of breast cancer cells to grow in 3D
culture systems and form xenograft tumours. Moreover,
we found that MCT4 is regulated by the PI3K–Akt
signalling axis and over-expressed in human breast
cancer tissue. The results of this study confirm MCT4
as a potential drug target in breast cancer and suggest
strategies for patient stratification and combination
treatment.
Materials and methods
Additional methods not included in this section are pro-
vided in the supplement (see supplementary material).
Cell culture, antibodies and reagents
Cell lines were obtained from the American Type
Culture Collection (ATCC) and LRI Cell Services.
Cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM)/F12 (1:1), with 2 mM L-glutamine
and penicillin–streptomycin. The medium was sup-
plemented with 10% fetal calf serum (FCS; Gibco)
for the cancer cell lines and 5% horse serum, 20 ng/ml
epithelial growth factor (EGF), 5 μg/ml hydrocorti-
sone, 10 μg/ml insulin and 100 ng/ml cholera toxin for
the non-malignant cell lines. MCT4 antibodies were
obtained from Santa Cruz (cat. no. sc-50329), BSG from
Novus (cat. no. NBP1-19677) and MCT1 from Millipore
(cat. no. AB3538P). Horseradish peroxidase-conjugated
anti-β-actin and secondary antibodies were from
Abcam and Sigma-Aldrich, respectively. All other
antibodies were from Cell Signaling. Hydrocortisone,
EGF, PI-103 and metformin were from Calbiochem;
PD98059, UO126 and LY294002 from New England
Biolabs; and lapatinib was from LC Laboratories.
Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl
sulfide (BPTES), carbonyl cyanide 4-(trifluoromethoxy)
Copyright © 2015 Pathological Society of Great Britain and Ireland.
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
153
phenylhydrazone (FCCP) and oligomycin were from
Sigma-Aldrich.
siRNA screen and data analysis
Cells were reverse-transfected with 37.5 nM Dharmacon
siRNA SMARTpools of 231 genes in 96-well plates,
using Lullaby reagent (Oz Biosciences). After 24 h, the
culture medium was topped up with fresh medium; 96 h
post-transfection, cells were fixed with 80% ice-cold
ethanol, stained with 4′ ,6-diamidino-2-phenylindole
(DAPI) and cell numbers quantified by Acumen
Explorer (TPP Labtech) (see supplementary material).
Intracellular pH, apoptosis and ROS levels
Intracellular pH [pH(i)] was determined with 10 mM
SNARF-4 F 5-(and-6)-carboxylic acid (Molecular
Probes). The cells were trypsinized, washed and
incubated with SNARF-4 F for 30 min. Changes in
fluorescence were measured using a Fortessa cytometer
(BD Biosciences). Cell death was determined 96 h
post-transfection, using the Apo-ONE assay (Promega).
For ROS detection, cells were incubated with 5 μM
CM-H2 DCFDA (Molecular Probes) for 30 min at 37 ∘ C,
trypsinized, washed twice with phosphate-buffered
saline (PBS), stained with DAPI and analysed on a
LSRII-SORP flow cytometer (Becton Dickinson).
Glycolytic rate and respiration
Glycolytic rate was determined by monitoring the con-
version of 5-3 H-glucose to 3 H2 O. Cells were incubated
in glucose-free DMEM for 30 min before the addition
of 10 mM glucose spiked with 10 mCi 5-3 H-glucose for
1 h at 37 ∘ C. The reaction was quenched with 0.2 N
HCl and 3 H2 O was captured by diffusion. Respiration
experiments were performed in a 96-well format, using a
Seahorse Bioscience XF96 Extracellular Flux Analyser.
Cells were incubated in glucose- and glutamine-free
DMEM (phenol red-free) for 2 h. The oxygen consump-
tion rate (OCR) was measured after injection of 10 mM
glucose and normalized to total protein content.
3D cultures and spheroid assays
Cells were transfected with siRNA and grown as a
monolayer, ie in two-dimensional (2D) culture. After
24 h, the cells were trypsinized and seeded in an on-top
Matrigel assay [18]. For spheroid formation, cells were
mixed with 2% Matrigel in culture medium and placed in
96-well ultra-low attachment plates (Costar). Spheroid
formation was initiated by centrifugation at 650 × g and
the cultures were incubated for 7–14 days. At least
six spheroids/group were photographed and size was
calculated using Photoshop (CS5.1).
Co-culture experiments
MDA-MB-468 cells were plated together with red flu-
orescent protein-expressing murine cancer-associated
fibroblasts (mCherry-CAFs; a gift from E. Sahai, CRUK
J Pathol 2015; 237: 152–165
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154
LRI), at a 5:1 ratio, and reverse-transfected with siRNA
targeting human MCT4 (37.5 nM Dharmacon siGenome
MCT4 5). After 3 days, the cells were stained with
Annexin V–Pacific blue and propidium iodide (PI) and
apoptotic cells in the mCherry-negative population were
determined by FACS analysis.
Xenograft experiments
MDA-MB-468 cells (1.5 × 106 cells) expressing
inducible shRNA targeting MCT4 (Tet-PLKO-shMCT4
88) were mixed with 10% Matrigel and injected into
the mammary fat pad of nude mice (nu/nu). After
tumour initiation (day 10), the mice were divided into
two cohorts (n = 5 each). One cohort was treated with
doxycycline (0.2 g/kg food pellet; Harlan D.98186)
and tumour growth was followed over 65 days. Animal
experiments were performed according to UK Home
Office guidelines.
Statistical analysis
Graphs were generated using GraphPad Prism 5.0
(GraphPad software). All experiments were performed
independently at least twice, with at least three biologi-
cal replicates; p values were generated using two-tailed
unpaired Student’s t-test or Mann–Whitney U-test, as
indicated.
Results
Functional screen identifies metabolic enzymes
required for breast cancer cell survival
To identify metabolic liabilities in breast cancer, we per-
formed an unbiased RNA interference (RNAi) screen,
targeting 231 enzymes, transporters and regulators
involved in central carbon metabolism (Figure 1A).
The screen was performed under normoxic and hypoxic
conditions in 17 breast epithelial cell lines representa-
tive of different disease subtypes (see supplementary
material, Figure S1). Cell numbers were determined
after 4 days of silencing and used to calculate z-scores
(see supplementary material, Table S1). For 12 genes
we confirmed the effect on cell number with at least
two individual siRNA sequences (see supplementary
material, Table S2). The gene with the strongest selec-
tivity for cancer cells was SLC16A3. Silencing of
SLC16A3 in normoxia caused a substantial reduction
in cell number in six of 14 cancer cell lines, while
not affecting non-malignant cells (Figure 1B). This
was even more pronounced under hypoxic conditions,
with eight of 14 cancer cell lines showing a substantial
response (Figure 1C; see also supplementary material,
Table S1). A comparison of different disease subtypes
revealed that the effect of SLC16A3 silencing was more
pronounced in HER2+ cells under normoxic conditions.
Under hypoxic conditions, most breast cancer cell
lines were sensitive to ablation of SLC16A3, which
Copyright © 2015 Pathological Society of Great Britain and Ireland.
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
F Baenke et al
is consistent with an induction of SLC16A3 expres-
sion in most breast cancer cell lines under hypoxia
(Figure 1D, E).
To confirm the differential sensitivity of breast cancer
cells towards SLC16A3 depletion, three representative
cell lines were chosen. Silencing of SLC16A3 caused
a substantial reduction in cell number in HCC1954
and MDA-MB-468 cells, while non-malignant
MCF10A cells were insensitive to SLC16A3 deple-
tion (Figure 1 F). Loss of viability in MDA-MB-468
cells was further validated using four different siRNA
sequences and efficient depletion of SLC16A3 mRNA
was confirmed (see supplementary material, Figure
S2A). Non-malignant MCF10A cells were insensitive
to SLC16A3 silencing despite efficient down-regulation
of mRNA and protein by three of four siRNA sequences
(see supplementary material, Figure S2B).
Next, we investigated the effect of long-term silenc-
ing of SLC16A3 on viability of MCF10A cells and
MDA-MB-468 cells. Inducible shRNA expression
resulted in efficient down-regulation of SLC16A3
mRNA and protein expression in both cell lines (see
supplementary material, Figure S2C, D), but only
reduced the viability of MDA-MB-468 cells (see sup-
plementary material, Figure S2C, D). Moreover, growth
factors used for culture of MCF10A cells did not alter
the sensitivity of either cell line towards SCL16A3
ablation (see supplementary material, Figure S2E),
while expression of an RNAi-insensitive version of
SLC16A3 (MCT4) rescued the observed reduction in
cell number caused by silencing of endogenous MCT4
(see supplementary material, Figure S2F).
Interestingly, meta-analysis of human gene expres-
sion data revealed that SLC16A3 mRNA levels are
elevated in invasive breast cancer compared to nor-
mal breast tissue (Figure 1G). Increased expression of
SLC16A3 was also associated with tumour recurrence
(Figure 1H).
Expression MCT1 and MCT4 in breast cancer cells
SLC16A3 encodes the monocarboxylate transporter 4
(MCT4), a lactate–proton symporter that regulates intra-
cellular pH in cancer cells [19,20]. We investigated the
expression levels of MCT4, the related monocarboxy-
late transporter 1 (MCT1) and the ancillary glycoprotein
basigin (BSG) across the cell line panel. Expression of
MCT4 and MCT1 mRNA was higher in non-malignant
cells than in cancer cells, while BSG showed little vari-
ation (Figure 2A). Interestingly, HER2+ breast cancer
cell lines showed higher levels of MCT4 mRNA expres-
sion than the other subtypes (Figure 2B). Expression of
MCT4 and MCT1 protein was variable, with high lev-
els of MCT1 protein found in some ER− HER2− cell
lines (Figure 2C), consistent with a previous report [21].
However, ablation of MCT1 did not affect the viability
of the breast cancer cell lines used here, despite efficient
depletion of the protein (see supplementary material,
Table S1, Figure S2G).
J Pathol 2015; 237: 152–165
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MCT4 supports breast cancer cell survival 155

A B Normoxia C Hypoxia
non- non-
cancer malign. cancer malign.

SLC16A3 SLC16A3
OXCT2 MT−ND4
ODC1 CHPT1
GPI GNPNAT1
GPX4 COX4I1
SOD1 ODC1
CHPT1 MT−ND2
OLAH PPAP2A
GPX2 IDH1
C12orf5 MT−ND6
HSD17B7 GGPS1
DLST PPAP2B
Glycolysis & galactose metabolism DDO RPE
CHKA OXCT2
TCA cycle
MVD GFPT2
Pentose phosphate pathway PGM3
Aminoacid metabolism LOC441996
PFKL
PHGDH ≤-2.0
Hexosamine metabolism GLUD2
PGAM4
Oxphos & ROS metabolism COX4I2
DHCR7 ACSS1
Fatty acid metabolism SREBF1 FDFT1
Cholesterol synthesis PDP2 PDP2
Metabolic regulation PMVK DHCR7
BT-20
BT-549
HCC1806
HCC1954
HCC38
HS-578T
MCF7
MDA-MB-231
MDA-MB-436
MDA-MB-468
SK-BR-3
T-47D
ZR-75-1
MCF10A
MCF12A
184B5
AU-565

Others MCAT
Total=231 siRNAs PMVK 0

BT-20
BT-549
HCC1806
HCC1954
HCC38
HS-578T
MCF7
MDA-MB-231
MDA-MB-436
MDA-MB-468
SK-BR-3
T-47D
ZR-75-1

184B5
MCF10A
MCF12A
AU-565
D Normoxia Hypoxia E
** ** ER+/HER2- ER-/HER2-
3 **** 3 *** HER2+ non-malign.
*** ****
2 2 20 20% O2
relative SLC16A3 mRNA
SLC16A3 z-score

0.5% O2
SLC16A3 z-score

1 1
15
0 0
-1 -1
10
-2 -2
-3 -3 5
-4 -4
-5 -5 0
2+

2+
n.
2-

2-
2-

2-
n.

184B5
BT-549
HCC1954

HCC1806
AU-565

HS-578T
BT-20
SK-BR-3

MCF10A
MCF12A
MDA-MB-468

MDA-MB-231
MDA-MB-436
ZR-75-1

T-47D

HCC38
MCF7
ig
ER

ER
ig

ER

ER
ER

ER
al
al

m
H

H
-/H

-/H
H

H
m

+/

+/
n-
n-

ER

ER
ER

ER
no
no

F HCC1954 MDA-MB-468 MCF10A

20 30 50 n.s.
*** ****
cell number (x1000)
cell number (x1000)

cell number (x1000)

40
15
20
**** ** 30
10 n.s.
20
10
5
10

0 0 0
M rl

M rl

M rl

M rl
T4

T4

T4

T4

T4
M rl

M rl
T4
si iCt

si iCt

si iCt

si iCt

si iCt

si iCt
C

C
C
s

20% O2 0.5% O2 20% O2 0.5% O2 20% O2 0.5% O2

G H
TCGA Zhao Richardson Finak
(p<0.001) (p<0.001) (p<0.001) (p<0.003)
2 1 6 2
SLC16A3 expression

SLC16A3 expression

SLC16A3 expression
SLC16A3 expression

0 4 1
0
-1 2 0
-2
-2 0 -1
-4 -3 -2 -2
-6 -4 -4 -3
a l
ar
a l

om ta

re yeance
st

om la
om ta
st

a l

ar e
ar

as
va nomcta
va nomas

om la

s
ye c
in c

at cur rs
ea

in u
ea

ca ive a

in c

3 ren
re
in u

rc du
rc lob
rc du
i e

3 rre
rc du

rc lob

br
br

rc br

at cu
al
al
al

ca e

ca ve
i
v

rm
m

re
rm

ca
ca
si

ca
s

si

no
va

no
no
no

in
in

in

Figure 1. Legend on next page.

Copyright © 2015 Pathological Society of Great Britain and Ireland. J Pathol 2015; 237: 152–165
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com
156 F Baenke et al

MCT4 supports pH regulation, redox balance
and non-oxidative glucose metabolism in breast
cancer cells
We next examined whether loss of viability observed
after MCT4 silencing relates to changes in cellular pH
homeostasis. We assayed the intracellular pH [pH(i)]
of viable cells after silencing of MCT4. HCC1954 and
MDA-MB-468 cells showed a significant reduction
in pH(i) upon MCT4 depletion, while no change was
observed in MCF10A cells (Figure 3A). Intracellu-
lar acidification can cause the formation of reactive
oxygen species (ROS), potentially by increasing the
NADH:NAD+ ratio and inhibiting complex I of the res-
piratory chain [22,23]. HCC1954 and MDA-MB-468
cells showed increased ROS levels after MCT4 ablation.
This was not observed in MCF10A cells (Figure 3B),
suggesting that these cells do not depend on MCT4 to
maintain pH(i) and prevent ROS formation.
Lactate production and secretion is important to main-
tain non-oxidative glucose metabolism in cancer cells
[24]. We therefore assayed the effect of MCT1 or
MCT4 ablation on glycolytic flux in MDA-MB-468
cancer cells. Depletion of MCT4 resulted in a 50%
reduction in glycolytic flux, while depletion of MCT1
had a smaller effect (Figure 3C). We also used stable
isotope labelling to follow changes in metabolite dis-
tribution. Depletion of MCT4 increased the levels of
M + 2 citrate and M + 2 glutamate in cells labelled with
U-13 C-glucose (Figure 3D), confirming the enhanced
entry of glucose-derived pyruvate into the TCA cycle.
In contrast, levels of M + 3 aspartate, M + 5 citrate
and M + 4 glutamate were decreased (see supplemen-
tary material, Figure S3A), suggesting that the direct
conversion of pyruvate into oxaloacetate by pyruvate
carboxylase (PC) was reduced. We next determined
glucose-dependent respiration using the Seahorse Bio-
analyser. We observed a reduction in the oxygen con-
sumption rate (OCR) of MDA-MB-468 cells upon addi-
tion of glucose (Figure 3E), suggesting that cells switch
from oxidative to non-oxidative glucose metabolism as
glucose becomes available, a phenotype known as the
’Crabtree-effect’ [25]. Silencing of MCT4 reduced the
ability of cancer cells to lower their OCR in response
to glucose, most likely due to impaired glycolytic flux
(Figure 3E), indicating that MCT4 is required to main-
tain the non-oxidative use of glucose. We also treated
MCT4-depleted MDA-MB-468 cells with the complex
I inhibitor metformin. Interestingly, MCT4-depleted
cells showed increased sensitivity towards this drug
(Figure 3F; see also supplementary material, Figure
S3B), indicating that depletion of MCT4 increases the
dependence of cancer cells on respiration.
As expected, silencing of MCT4 caused a reduction in
lactate secretion (Figure 3G, H; see also supplementary
material, Table S3). Instead, we noticed an increased
uptake of several amino acids, particularly glutamine,
accompanied by the secretion of glutamate and trypto-
phan (Figure 3G, H). This implies that cells increase
catabolic use of some amino acids, particularly glu-
tamine, when MCT4 function is impaired. This was also
confirmed by increased sensitivity of MCT4-depleted
cells towards BPTES, a selective inhibitor of glutami-
nase, which is essential for the utilization of glutamine
by the TCA cycle (Figure 3I; see also supplementary
material, Figure S3C).
MCT4 supports spheroid formation, 3D growth
and tumour formation in breast cancer cells
The regulation of pH(i) by carbonic anhydrase IX (CA9)
can promote the ability of tumour cells to grow as
three-dimensional (3D) spheroids [26]. These culture
conditions recapitulate the oxygen, nutrient and pH
gradients experienced by cancer cells within tumours
[27]. Analysis of histological sections of spheroids from
MDA-MB-468 cells showed that MCT4 is expressed
mainly in a ring surrounding the necrotic core, which
also stained positive for cleaved caspase 3 (Figure 4A).
Silencing of MCT4 caused a significant reduction in
spheroid size in MDA-MB-468 and HCC1954 cells
(Figure 4B), indicating that MCT4 is required for effi-
cient cell growth under these conditions.
Silencing of MCT4 significantly reduced the
growth of MDA-MB-468 and HCC1954 cells in a
3D Matrigel-based model (Figure 4C). In contrast,
acinar formation of MCF10A cells induced during 14
days of 3D culture was insensitive to shRNA-mediated
depletion of MCT4 (Figure 4D). Breast epithelial

Figure 1. Identification of metabolic enzymes required for breast cancer cell survival: 17 breast epithelial cell lines were reverse-transfected
with Dharmacon SMARTpool siRNAs targeting 231 different metabolic genes; the cells were placed under normoxic (20% O2 ) or hypoxic
(0.5% O2 ) conditions for 96 h; cell number was used to calculate z-scores for each gene in the different cell lines/conditions. (A) Pie chart
of the metabolic processes represented by the genes targeted by the siGenome siRNAs used in the screen. (B) Supervised cluster analysis of
z-scores derived from cells grown in normoxia; non-malign., non-malignant; genes that showed significant differences in z-score between
the two groups are displayed; p ≤ 0.05. (C) Supervised cluster analysis of z-scores derived from cells grown in hypoxia. (D) Comparison
of z-scores for SLC16A3 (MCT4) between breast cancer subtypes in normoxia and hypoxia. (E) SLC16A3 (MCT4) mRNA expression in
cells grown under normoxic and hypoxic conditions for 24 h; mean ± SD of three biologically independent replicates. (F) Effect of SLC16A3
(MCT4) silencing on cell number in HCC1954, MDA-MB-468 or MCF10A cells under normoxic (black bars) and hypoxic (red bars) conditions;
mean ± SD of two independent experiments with three replicates each; subtypes were compared using Mann–Whitney U-test, *p ≤ 0.05,
**p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; n.s., not significant. (G) Differential expression of SLC16A3 (MCT4) in three breast cancer datasets:
significant comparisons between cancerous and normal tissue are shown; p ≤ 0.001; data are publicly available in Oncomine and references
are provided in Supplementary materials and methods (see supplementary material). (H) Differential expression of SLC16A3 (MCT4) in a
study comparing breast cancers that show no recurrence to those that show recurrence at 3 years: statistical comparisons were performed
using Student’s t-test, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; n.s., not significant

Copyright © 2015 Pathological Society of Great Britain and Ireland. J Pathol 2015; 237: 152–165
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com
MCT4 supports breast cancer cell survival 157

A
SLC16A3 SLC16A1 BSG
15
(normalized to population mean)

(normalized to population mean)

3

(normalized to population mean)

ER+/HER2- ER+/HER2- ER+/HER2-
HER2+ HER2+ HER2+
SLC16A3 mRNA levels

SLC16A1 mRNA levels

ER-/HER2- ER-/HER2- ER-/HER2-
non-malign.

BSG mRNA levels

6 non-malign. non-malign.
10 2

5 1
2

0 0 0

184B5

184B5
BT-549

BT-549
HCC1954

HCC1806

HCC1954

HCC1806
AU-565

AU-565
HS-578T

HS-578T
BT-20

BT-20
184B5

SK-BR-3

SK-BR-3
MCF10A
MCF12A

MCF10A
MCF12A
MDA-MB-468

MDA-MB-231
MDA-MB-436

MDA-MB-468

MDA-MB-231
MDA-MB-436
ZR-75-1

ZR-75-1
T-47D

HCC38

T-47D

HCC38
BT-549
HCC1954

HCC1806

MCF7

MCF7
AU-565

HS-578T
BT-20
SK-BR-3

MCF10A
MCF12A
MDA-MB-468

MDA-MB-231
MDA-MB-436
ZR-75-1

T-47D

HCC38
MCF7

-5 B- 1
6
B C

68

BT M 3
H 49 43
****

A- -2
-2 B-4

C 6
54
n.s.
(normalized to population mean)

D B
D T
- 3
(normalized to population mean)

C A
S A

C 0
15

M 5-1

M 578
M -M
H 565
AU R-

H 18
BT -M

R 38
R C19
10

M 10
R 2
****

M 5
SK D
T- F7

18 0

F1
-B

4B

A
-7

C
A

F
47

S-
****
SLC16A1 mRNA levels

C
C
SLC16A3 mRNA levels

ZR

S
S
M

H
****
8
*** 10 MCT4
**
6
MCT1
4
5

2
BSG
0
β-actin
0
ER .
H n.

+/ lign

ER+/HER2-
H 2-
-/H 2+
2-
ER ER -
-/H 2+

ER-/HER2-
2-
H 2
ig

ER
ER

ER

ER ER
al

HER2+ non-malign.
ER -ma
m

H
n-

+/

n
no
no
ER

Figure 2. Expression of SLC16A3 and SLC16A1 in breast cancer cells. (A) mRNA levels of SLC16A3 (MCT4), SLC16A1 (MCT1) and BSG in
breast epithelial cell line panel. (B) Comparison of mRNA levels of SLC16A3 (MCT4) and SLC16A1 (MCT1) between different breast cancer
subtypes; non-malign., non-malignant. (C) Protein levels of MCT4, MCT1 and BSG across breast epithelial cell line panel: β-actin is shown
as loading control; a reference sample (RS) was loaded to allow comparison of signal intensities across multiple gels; Student’s t-test,
**p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; n.s., not significant

cells alter their metabolic activity in response to loss material, Figure S1A). Moreover, analysis of the TCGA
of extracellular matrix (ECM) attachment [28] and dataset showed that SLC16A3 (MCT4) amplifications,
MCT4 seems to be required for this adaptation. We next which occur in 6.4% of breast cancers, have a tendency
investigated whether co-culture with cancer-associated towards co-occurrence with HER2/ERBB2 amplifica-
fibroblasts (CAFs) alters the sensitivity of breast can- tion (p = 0.000292) (MSKCC cBioPortal). Most breast
cer cells towards ablation of MCT4. Transfection of cancers harbouring these genetic alterations display
species-specific siRNA targeting human MCT4 caused high levels of Akt phosphorylation [10].
a substantial loss of viability (Figure 4E) and induc- To investigate whether MCT4 dependence is asso-
tion of apoptosis (Figure 4F) in MDA-MB-468 cells ciated with activation of the PI3K–Akt pathway, we
cultured either alone or in the presence of CAFs. This established levels of growth factor-independent Akt
demonstrates that MCT4 has essential functions in phosphorylation in all cell lines. High levels of Akt ser-
cancer cells, even under conditions that allow the shut- ine 473 phosphorylation were detected in ER− HER2−
tling of metabolites between cancer and stromal cells. breast cancer cells (see supplementary material, Figure
Moreover, inducible silencing of MCT4 substantially S4) and most cancer cells maintained high Akt phospho-
reduced the growth of MDA-MB-468 cells as orthotopic rylation in the absence of growth factors (Figure 5A;
tumours (Figure 4G, H), confirming the importance of see also supplementary material, Figure S4). We next
MCT4 for breast cancer cells under physiological compared levels of growth factor-independent Akt phos-
growth conditions. phorylation across the breast cancer cell line panel with
their sensitivity towards MCT4 ablation. Cell lines that
MCT4 is regulated by the PI3K–Akt signalling axis retained high levels of Akt phosphorylation, such as
in breast cancer MDA-MB-468 or HCC1954, displayed high sensitivity
Having established that MCT4 is essential for the towards MCT4 silencing (ie low z-scores; Figure 5B),
growth and survival of breast cancer cell lines, we suggesting a functional link between MCT4 and the
investigated whether it is linked to specific genetic PI3K–Akt signalling axis.
alterations found in the human disease. Sensitivity We next investigated whether activation of the
towards MCT4 depletion was most pronounced in PI3K–Akt signalling pathway regulates MCT4 expres-
the HER2+ subset or those cell lines that harbour sion. We chose two breast cancer cell lines that show
mutations in PTEN or INPP4B (see supplementary amplifications within EGFR (MDA-MB-468) or HER2
Copyright © 2015 Pathological Society of Great Britain and Ireland. J Pathol 2015; 237: 152–165
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com
158 F Baenke et al

A B
HCC1954 MDA-MB-468 MCF10A HCC1954 MDA-MB-468 MCF10A
** 2.5
8.5 8.5 2.5 6
8.5

H2DCFDA fluorescence
H2DCFDA fluorescence

H2DCFDA fluorescence
2.0

(relative to siCtrl)
**** ** 2.0

(relative to siCtrl)

(relative to siCtrl)
8.0 n.s.
8.0 8.0 n.s.
4 1.5
1.5 ****

pH(i)
pH(i)

pH(i)

7.5 7.5 7.5

1.0 1.0
2
7.0 7.0 7.0 0.5
0.5

6.5 0.0 0 0.0

6.5 6.5

T4
2
+ 4
si trl
2
T4
s trl

si rl
2O

2
2O

T
T4
T4

t
T4

trl

2O
C

C
trl
trl

C
C
si iMC

H
si

M
C

H
C

si

si
C

M
C
C

H
si
si
si

M
M
M

+
trl
si
si
si

trl

trl
C
C

C
si

si
C D U-13C-Glucose Citrate (M+2) Glutamate (M+2)
MDA-MB-468 - Dox 25 p=0.0052
- Dox
1.5 p=0.00027
+ Dox
+ Dox
relative 3H2O production

% of total pool
*** 30

% of total pool
Pyruvate 20
(relative to siCtrl)

**
15
1.0 20
Acetyl-CoA
10
Citrate 10
Oxaloacetate 5
0.5
0 0

8
R
8
R

#8
Glutamate

#8

SC
SC

0.0 Malate α-Ketoglutarate

T4
T4

sh
sh

C
T1

T4
trl

M
C

M
C

C
si

sh
M

sh
si

si

E shMCT4 #74 shMCT4 #88 F shMCT4 #88

shSCR
110 glucose 110 glucose 110 glucose
***
OCR [pM/min]/cell mass
OCR [pM/min]/cell mass

OCR [pM/min]/cell mass

- Dox - Dox - Dox

absorbance (560 nm) - Dox
100 100 100 (normalized to -Dox) 1.0 + Dox
+ Dox + Dox + Dox
90 90 90
80 ***
80 **** 80
***
70 70 70 0.5
** ***
60 60 60
***
50 50 50
40 40 0.0
40
8 14 21 28 35 43
in

8 14 21 28 35 43
in
8 14 21 28 35 43
t

rm
en

rm

time [min] time [min] time [min]

fo
lv

fo
so

et

et
m

G H
M

M
1m

5m

Lactate Glutamine
15 2
Lactate siCtrl
nM/(cell mass unit × h)

nM/(cell mass unit × h)

Malate siMCT4
1
Fumarate
10
Succinate
α-Ketoglutarate
0
I shMCT4 #88
Citrate -1 ***
5 - Dox
absorbance (560 nm)
(normalized to -Dox)

Alanine 1.0 + Dox

Asparagine -2
***
Aspartate ***
0 -3
Glutamate ***
trl
T4

T4
M rl
t
C

Glutamine 0.5
C
C

C
si

si
M

Glycine
si

si

Isoleucine
Glutamate Tryptophan
Leucine
0.25 1.5
Methionine 0.0
nM/(cell mass unit × h)

nM/(cell mass unit × h)

Phenylalanine
t

S
en

0.20
TE

TE
lv

Proline
BP

BP
so

1.0
Serine 0.15
M

μM
5μ

Succinate
10

Tryptophan 0.10
0.5
Tyrosine
0.05
Valine
-1.0 -0.5 0.0 0.5 1.0 10 15 20 0.00 0.0
-3 5
-2 0
-2 5
-1 0
.5
.
.
.
.
-3

trl
T4

nM/(cell mass unit × h)

trl
T4

C
C

C
si
C
si

M
M

si
si

Figure 3. Legend on the next page

Copyright © 2015 Pathological Society of Great Britain and Ireland. J Pathol 2015; 237: 152–165
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com
MCT4 supports breast cancer cell survival 159

(HCC1954) and have high levels of Akt activation Discussion

(Figure 5A). EGF increased MCT4 mRNA levels in
both cell lines (Figure 5C). Moreover, treatment with the
HER2/EGFR inhibitor lapatinib or inhibitors of PI3K
(PI-103 or LY294002) attenuated MCT4 induction,
while inhibitors of MEK–ERK signalling (PD98059
or UO126) were less effective (Figure 5C). Finally,
induction of MCT4 expression by hypoxia in MCF10A
cells was dependent on the presence of growth factors,
including insulin and EGF (Figure 5D).
MCT4 expression is induced in breast cancer
The MMTC–PyMT mouse model of breast cancer reca-
pitulates the different stages of breast cancer progres-
sion [30] and is highly dependent on Akt activity
[31]. We therefore investigated expression of MCT4
in tissues from these mice at different disease stages.
MCT4 expression was low in normal murine mam-
mary glands but increased with neoplastic transfor-
mation (Figure 6A). In the carcinoma stage, strong
staining was localized to cancer cells in areas surround-
ing necrotic tissue, suggesting that MCT4 is induced by
tumour hypoxia and/or oxidative stress.
We next analysed MCT4 expression in a tissue
microarray of 75 human breast specimens. MCT4
staining was low in normal breast (Figure 6B) but
enhanced in cancerous tissues from all clinical subtypes
(Figure 6C, E). Moreover, some HER2+ and ER+ breast
cancers showed high-intensity MCT4 staining localized
to the cancer compartment (Figure 6C, E). In contrast,
stromal expression of MCT4 was moderate and uniform
across all subtypes (Figure 6D, F). A meta-analysis of
expression data showed that high levels of SCL16A3
are indicative of poor overall survival in HER2+ breast
cancer (Figure 6G). Collectively, these results indicate
that MCT4 is regulated by the PI3K–Akt signalling
axis in human breast cancer and promotes the survival
of breast cancer cells in which this pathway is activated.
Metabolic reprogramming is a hallmark of cancer and
may offer novel treatment opportunities [4]. Here we
demonstrate the role of the monocarboxylate transporter
MCT4 in supporting cancer cell survival in a panel of
genetically diverse breast cancer cell lines. We found
that MCT4 is required for metabolite transport, pH
homeostasis and redox balance, and that it supports the
viability of breast cancer cells under physiologically rel-
evant growth conditions in vitro and in a breast cancer
xenograft model. Moreover, expression of MCT4 is reg-
ulated by the PI3K–Akt signalling axis and correlates
with disease progression in a mouse model of breast can-
cer. High levels of MCT4 are found in human breast
cancer tissue and are predictive of poor survival.
There is substantial evidence that proton-linked
monocarboxylate transporters perform important roles
in cancer cells [32]. The low affinity of MCT4 to pyru-
vate facilitates the conversion of pyruvate into lactate
in hypoxic cancer cells, thereby allowing the regener-
ation of cytosolic NAD+ [33]. In contrast, MCT1 has
high affinity for both lactate and pyruvate and may be
more important for lactate uptake in oxidative cancer
cells. However, it is now clear that the expression of
MCT1 and MCT4 can differ substantially, not only
between different cancer types but also between dif-
ferent intra-tumoural compartments and cell types.
Functional studies are therefore needed to unravel the
relative contributions of different lactate transporters.
It has been shown that combined inhibition of the
lactate transporters MCT4 and MCT1 blocks viability
and tumour growth in RAS-transformed fibroblasts and
LS174T colon cancer cells [19]. However, we found
that ablation of MCT4 alone was sufficient to impair
viability of human breast cancer cell lines. Sensitivity
towards MCT4 ablation did not correlate with expres-
sion of either MCT4 or MCT1, making it unlikely

Figure 3. MCT4 is required for pH maintenance, redox balance and non-oxidative glucose metabolism in breast cancer cells. (A) HCC1954,
MDA-MB-468 and MCF10A cells were transfected with siRNA targeting MCT4 (siMCT4) or controls (siCtrl): 72 h post-transfection,
intracellular pH [pH(i)] was analysed; mean ± SD of two independent experiments with two replicates each. (B) ROS levels in cells treated as
in (A): H2 O2 treatment was used as positive control for ROS induction; mean ± SEM of three independent experiments with two replicates
each. (C) Effect of MCT1 or MCT4 ablation on glycolytic activity in MDA-MB-468 cells; mean ± SEM of two independent experiments
with four replicates each. (D) MDA-MB-468 cells expressing inducible shRNAs targeting MCT4 (shMCT4 88) or scrambled controls (shSCR)
were grown in the presence or absence of doxycycline (± Dox) for 7 days: cells were labelled with U-13 C-glucose for 24 h before lysis and
metabolite levels were determined by mass spectrometry; increased levels of labelled citrate (M + 2) and glutamate (M + 2) are indicative
of enhanced entry of pyruvate into the TCA cycle. (E) MDA-MB-468 cells expressing inducible shRNAs targeting MCT4 (shMCT4 74 or
shMCT4 88) or scrambled controls (shSCR) were grown in the presence or absence of doxycycline (± Dox) for 6 days: the cells were starved
of glucose and glutamine for 2 h and oxygen consumption rate (OCR) was assessed before and after injection of 10 mM glucose, using a
Seahorse Bioanalyser; mean ± SD of two independent experiments with six replicates each. (F) MDA-MB-468 cells expressing inducible
shRNAs targeting MCT4 (shMCT4 88) were grown in the presence or absence of doxycycline (± Dox) for 8 days: the indicated doses of
metformin were added during the last 3 days; cell viability was determined by crystal violet staining; mean ± SEM of two independent
experiments with three replicates each. (G) Effect of MCT4 ablation on metabolite uptake and secretion in MDA-MB-468 cells under
normoxia: cells were transfected with siRNA targeting MCT4 (siMCT4) or controls (siCtrl); 72 h post-transfection, cells were placed in fresh
medium and samples of the medium were taken over a period of 24 h; metabolite levels in the medium were determined using GC–MS;
data represent six biological replicates. (H) Uptake and secretion rates of lactate, glutamine, glutamate and tryptophan in MDA-MB-468
cells, as in (G). (I) MDA-MB-468 cells expressing inducible shRNAs targeting MCT4 (shMCT4 88) were grown in the presence or absence of
doxycycline (± Dox) for 8 days: the indicated doses of BPTES were added during the last 3 days; cell viability was determined by crystal violet
staining; mean ± SEM of two independent experiments with three replicates each; Student’s t-test, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001;
n.s., not significant

Copyright © 2015 Pathological Society of Great Britain and Ireland. J Pathol 2015; 237: 152–165
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com
160 F Baenke et al

A B MDA-MB-468 HCC1954
MCT4 cleaved caspase 3
siCtrl 2000 siCtrl 1500

spheroid area [pixelx1000]

spheroid area [pixelx1000]
**** ****
1500
1000

100 μm
1000 100 μm
100 μm 100 μm
siMCT4 siMCT4
500
500

0 0

trl

T4
100 μm 100 μm

T4
trl

C
C

si

M
si

si
si
100 μm

H&E
C D
MDA-MB-468 HCC1954
siCtrl siCtrl MCF10A shSCR

1.5 1.5 1.5

(normalized to siCtrl)
(normalized to siCtrl)

(normalized to siCtrl)
****
****

cell viability
cell viability

1.0
cell viability

1.0 1.0 + Dox

siMCT4 siMCT4 shMCT4 #88

0.5 0.5 0.5

0.0 0.0 0.0 +Dox

siCtrl siMCT4 siCtrl siMCT4 shSCR shMCT4
(#88)

MDA-MB-468 siCtrl MDA-MB-468 + CAFs siCtrl

E MDA-MB-468 MDA-MB-468 + CAFs F 5 0.39% 1.68% 0.86% 3.12%
10 105
0.39%
4
10 104

PI-A
PI-A

siCtrl 10 3 103

10 2 102

0 0
94.1% 3.38% 90.5% 5.51%
0 10
2
10
3 4
10 10
5 0 102 103 104 105
Annexin V Pacific Blue Annexin V Pacific Blue
MDA-MB-468 siMCT4 MDA-MB-468 + CAFs siMCT4
0.7% 1.31% 105
0.96% 1.58%
105
siMCT4

4
104 10
PI-A

PI-A

3
103 10

G MDA-MB-468 shMCT4 #88 102 102

150 - Dox 0
0
+ Dox 74.6% 23.4% 75.6% 21.8%
tumour volume (mm )
3

2 3 4 5 2 3 4 5
0 10 10 10 10 0 10 10 10 10
Annexin V Pacific Blue Annexin V Pacific Blue
100
H - Dox + Dox

50 Dox added *

0
0 20 40 60 days

Figure 4. MCT4 depletion reduces 3D growth and tumour formation in breast cancer cells. (A) MDA-MB-468 cells were grown as spheroids
for 7 days; spheroids were sectioned and stained for MCT4 or cleaved caspase 3; H&E staining is shown as control. (B) MDA-MB-468 and
HCC1954 cells were transfected with siRNA targeting MCT4 (siMCT4) or controls (siCtrl): 12 h post-transfection, cells were trypsinized,
seeded as spheroids and incubated for 7 days; spheroid size was determined and compared to siCtrl-transfected cells; Student’s t-test,
****p ≤ 0.0001. (C) MDA-MB-468 and HCC1954 cells were transfected with siRNA targeting MCT4 (siMCT4) or controls (siCtrl): 12 h
post-transfection, cells were trypsinized and embedded in Matrigel; after 7 days, representative images were taken and viable cell number
determined with CellTiter-Blue, normalized to siCtrl-transfected cells; mean ± SEM of three independent experiments with two replicates
each; Student’s t-test, ****p ≤ 0.0001. (D) MCF10A cells expressing shMCT4 88 or scrambled controls (shSCR) were embedded in Matrigel
and treated with 1 μg/ml doxycycline for 14 days: representative images were taken and viable cell number determined with CellTiter-Blue;
mean ± SEM of three independent replicates. (E) Representative images of co-cultures of MDA-MB-468 with mCherry-labelled murine
cancer-associated fibroblasts (CAFs), 3 days after transfecting with siRNA targeting MCT4 (siMCT4) or control (siCtrl); arrows, apoptotic
MDA-MB-468 cells. (F) Apoptosis in the mCherry-negative populations (= MDA-MB-468 cells), determined by Annexin V–PI staining and
FACS. (G) MDA-MB-468 cells expressing shMCT4 88 were injected into the mammary fat pad of 8 week-old immunocompromised mice
(nu/nu): after tumour initiation on day 10, the mice were randomized into two cohorts (n = 5); one cohort was kept on a standard diet
and the other on a doxycycline-supplemented diet (0.2 g/kg doxycycline in food pellet); tumour size was measured twice weekly; statistical
comparisons of sizes at day 65 were performed using Student’s t-test, *p ≤ 0.05. (H) Representative images of tumour tissue

Copyright © 2015 Pathological Society of Great Britain and Ireland. J Pathol 2015; 237: 152–165
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com
MCT4 supports breast cancer cell survival 161

A 5% HS + GF
2.5 5% HS - GF
phospho-AKT/pan-AKT levels

10% FCS
(normalized to β-actin)

0.5% BSA
2.0

1.5

1.0

0.5

non-malign.
0.0 ER+/HER2-
HER2+

38
8
5

M 1

BT 6
AU -3
M 0A

ZR 2A

M CC 5
M 4

H 6
C 49
H -20

M MB T
F7

SK 7D
1

ER-/HER2-
46
4B

A- -23

43
6

0
5-

C
R

5
D 19

18
5
D -57
C
F1

F1

C
B-

BT
-7

-B

B-
-

-
18

T-
M

C
C

S
M

A-

A-
H

H
D
M
B 2 r = -0.598 C
184B5 p = 0.012 HCC1954 MDA-MB-468
M12A 2.5 ** 5
M-231
SLC16A3 mRNA levels

SLC16A3 mRNA levels

(normalized to control)

(normalized to control)
0 BT-20 ****
ZR-75-1
SLC16A3 z-score

M10A HS-578T 2.0

**
4
MCF7 **
M-436 H38
-2
AU-565 T-47D 1.5 *** ** 3 **
BT-549 M-468 ***
SK-BR-3 H1806 *** ***
1.0 2
-4 H1954
0.5 1

-6 0.0 0
0.0 0.5 1.0 1.5 2.0 2.5
F a

EG 103

LY

F a

EG 103

LY
EG F D

EG F D
l

l
+ F

+ F
O

O
ro

ro
EG ap

EG ap
EG

EG
EG + P

EG + P
F +U

F +U
nt

nt
+

+
L

-
pAKT in 0.5% BSA
co

co
PI

PI
F

F
+

+
F

F
(normalized to β-actin)
EG

EG
D MCF10A
2.5 - GF
(normalized to 20% O2 + GF)

**
+ GF
SLC16A3 mRNA levels

2.0

1.5
**
1.0

0.5

0.0
20% O2 0.5% O2

Figure 5. MCT4 expression is regulated by EGFR–HER2 signalling and increases during breast cancer progression. (A) Cells were cultured
in medium containing 10% FCS or 0.5% bovine serum albumin (BSA); non-malignant cells were cultured in medium with 5% horse serum
(HS), with or without growth factor supplements (GF): Akt phosphorylation levels were determined by immunoblotting (see supplementary
material, Figure S4); signal intensities were analysed using ImageJ to determine the phospho-AKT:pan-AKT ratio. (B) Akt phosphorylation
levels in medium with 0.5% BSA were compared to z-scores determined after MCT4 silencing, using the Spearman correlation. (C) HCC1954
and MDA-MB-468 cells were serum-starved for 16 h and stimulated with 20 ng/ml EGF for 6 h, with or without 1 h pretreatment with the
HER2/EGFR inhibitor lapatinib (Lapa; 0.5 μM), the MEK inhibitors PD98059 (PD; 10 μM) or UO126 (UO; 10 μM), the dual PI3K–mTOR inhibitor
PI-103 (1 μM) or the PI3K inhibitor LY294002 (LY; 20 μM). SLC16A3 (MCT4) mRNA levels were determined by qPCR; mean ± SEM of two
independent experiments with two replicates each. (D) MCF10A cells cultured in medium with (+GF) or without (–GF) growth factors
under normoxia (20% O2 ) or hypoxia (0.5% O2 ) for 24 h: expression of SLC16A3 (MCT4) mRNA was determined by qPCR; mean ± SD of two
replicates

that MCT1 compensates for MCT4 in breast cancer. We found that reduced viability after MCT4 deple-
Another study reported that 7-aminocarboxycoumarin tion was associated with intracellular acidification and
derivatives, which inhibit lactate uptake but not secre- oxidative stress, indicating that MCT4 is required for
tion, block xenograft growth of several cancer cell lines, the maintenance of pH homeostasis and redox bal-
including ER+ MCF7 cells [34]. Here ER+ breast cancer ance. MCT4 depletion reduced glycolytic flux in cancer
cells showed low sensitivity towards MCT4 depletion cells but increased the entry of glucose-derived pyru-
compared to other subtypes, suggesting that lactate vate into the TCA cycle. Consistent with the function
metabolism differs substantially between breast cancer of MCT4 as a lactate–proton symporter, we observed
cell lines. a reduction in lactate secretion after MCT4 depletion.
Copyright © 2015 Pathological Society of Great Britain and Ireland. J Pathol 2015; 237: 152–165
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com
162 F Baenke et al

adenoma
A normal hyperplasia early carcinoma carcinoma

100μm 100μm 100μm 100μm

B normal mammary gland C cancer D stromal MCT4

intensity 0 intensity 1 intensity 1

100μm 100μm 100μm 100μm

intensity 2 intensity 3 intensity 2

100μm 100μm 100μm

E F G
cancer HER2-positive breast cancer
staining stroma staining 100
100 intensity intensity
100
0 1 80
80
overall survival (%)

1 80 2
p= 0.0314
percentage

2
percentage

60
60 60
3
40
40 p = 0.03 40
20 low MCT4
20 20
med MCT4
0 high MCT4
0 0
0 2 4 6 8 10
)
)

)
)
31
30

35
24

)
1)

24

35
30

Time (years)
=
=

=
=

=
=
=
(n
(n

(n
(n

(n

(n
(n
(n
+

3+
BC

2+

2+

3+
BC
ER

ER
2
TN

2
TN
ER
ER

ER

ER
H
H

Figure 6. MCT4 is expressed in human breast cancer and predicts poor survival in HER2-positive disease. (A) Breast tissues derived from
MMTV–PyMT mice at different stages of tumour development were stained for MCT4: expression of MCT4 in breast tissue was analysed
using a tissue microarray; representative images are shown. (B) Normal mammary gland. (C) Different staining intensities used for scoring
cancer cell positivity. (D) Different staining intensities used for scoring stromal cell positivity. (E) Percentage of tissue cores with different
staining intensities for MCT4 in cancer cells within the different subgroups, according to receptor status: staining intensities for HER2 and
ER according to the manufacturer’s information; HER2 2+ = equivocal, HER2 3+ = HER2 amplified; p value reflects c2 statistical significance
in the correlation analysis. (F) Percentage of tissue cores with different staining intensities for MCT4 in stromal cells, according to receptor
status. (G) Kaplan–Meier curves showing the overall survival of patients with HER2-positive breast cancers, showing low (grey line), medium
(black line) or high (red line) levels of SLC16A3 (MCT4) mRNA expression

Instead, cells increased the uptake of amino acids, partic-
ularly glutamine, suggesting an induction of anaplerosis
and use of amino acids for energy generation. Inter-
estingly, MCT4-depleted cells showed enhanced sen-
sitivity towards inhibitors of mitochondrial respiration
Copyright © 2015 Pathological Society of Great Britain and Ireland.
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
and glutaminolysis, indicating that cells adapt to MCT4
inhibition by increasing oxidative metabolism. Cancer
cells that are unable to convert to oxidative metabolism
should therefore be particularly sensitive to MCT4 inhi-
bition. Similar observations were also recently reported
J Pathol 2015; 237: 152–165
www.thejournalofpathology.com
MCT4 supports breast cancer cell survival
for a glycolytic subtype of pancreatic cancer [35], sug-
gesting that combination strategies targeting glutaminol-
ysis or mitochondrial respiration, together with MCT4
inhibitors, could be of clinical benefit.
As expected, sensitivity of breast cancer cells towards
MCT4 depletion was more pronounced when cells were
grown in hypoxia. MCT4 is induced by hypoxia in a
HIF-1a-dependent manner [29]. It is likely that ablation
of MCT4 lowers the ability of breast cancer cells to adapt
their metabolism to hypoxia. Consequently, MCT4 was
also required to support the growth of breast cancer cells
as 3D spheroids, which recapitulate oxygen, nutrient and
pH gradients experienced by cancer cells within growing
tumours [27].
Several studies have shown that metabolic reprogram-
ming contributes to the morphological transformation
of breast cancer cells [28,36,37]. Cancer phenotypes
such as loss of cell polarization, defective morphogen-
esis and enhanced invasion can be investigated in 3D
collagen cultures, and alter the sensitivity of cancer
cells towards inhibition of signalling molecules [38]. We
found that depletion of MCT4 reduced the 3D growth
of breast cancer cells but not non-malignant cells, sug-
gesting that MCT4 supports the metabolic adaptation of
cancer cells to these conditions. It has been suggested
that MCT4 participates in the shuttling of lactate from
glycolytic stromal cells to support oxidative metabolism
in cancer cells [39]. We found that co-culture with
cancer-associated fibroblasts (CAFs) did not alter the
sensitivity of cancer cells towards MCT4 depletion.
Instead, we found that MCT4 is essential for orthotopic
tumour formation, confirming that MCT4 is required for
cancer cell growth under physiological growth condi-
tions.
Our study also revealed a regulatory connection
between MCT4 and PI3K–Akt signalling in breast
cancer. Signalling through the PI3K–Akt axis also
results in enhanced HIF-1 activity in normoxic cancer
cells [41]. It is therefore possible that the induction of
MCT4 expression in response to EGF involves activa-
tion of HIF-1. Akt increases glucose uptake and lactate
secretion in cancer cells [40] and impairment of lactate
transport could be highly detrimental to cells with active
Akt. The connection between MCT4 and PI3K–Akt
was confirmed by the observation that MCT4 levels
increase with disease progression in the MMTV–PyMT
breast cancer model, which shows enhanced HER2
expression and Akt phosphorylation during progression
to the carcinoma stage [42]. Consistent with a role
of MCT4 in supporting the metabolism of hypoxic
cancer cells [43], staining was mainly localized to areas
surrounding necrotic tissue.
MCT4 staining shows substantial heterogeneity in
different tumour compartments, including stromal cells
[21,39,44–46]. MCT4 expression in cancer cells is
found in triple-negative breast cancer [47] and corre-
lates with high-grade disease, while stromal expression
was mainly restricted to lower grades [44]. Another
study found that stromal MCT4 expression predicts poor
disease outcome in triple-negative breast cancer [48].
Copyright © 2015 Pathological Society of Great Britain and Ireland.
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
163
We found that a subset of breast cancers showed mod-
erate stromal MCT4 staining, independent of receptor
status. In contrast, strong positivity for MCT4 in can-
cer cells was found in a proportion of tumours, with
the strongest staining detected in HER2+ and ER+ sub-
types. It is likely that the regulation of MCT4 expression
in breast cancer is highly dynamic. Cancer cells need
to respond to different metabolic niches determined by
the tumour microenvironment. Differential expression
of MCTs allows cancer cells to modify their metabolic
activity according to the nutrient and oxygen conditions
and their metabolic demand. Understanding the depen-
dencies of cancer cells within the context of this dynamic
regulation is essential to develop strategies to target lac-
tate transport for the treatment of cancer.
Acknowledgements
The authors thank Eric Sahai (LRI) for material, Tamara
Bunting for tissue analysis and LRI Research Services
for technical support. This study was funded by Cancer
Research UK and the Comprehensive Cancer Centre
Mainfranken.
Author contributions
FB, SD, CB, BD, BG, MJ and SR designed and carried
out experiments; BW, JRF, MH, NZ and AS conceived
the study; FB and AS wrote the manuscript; AM and
BS performed statistical analysis; and BSD and GS
performed histological analysis.
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MCT4 supports breast cancer cell survival 165

SUPPLEMENTARY MATERIAL ON THE INTERNET

The following supplementary material may be found in the online version of this article:
Supplementary materials and methods
Figure S1 (related to Figure 1). Panel of breast epithelial cell lines
Figure S2 (related to Figure 1). Validation of MCT4 as a differential target in breast cancer
Figure S3 (related to Figure 3). Metabolic characterization of MCT4-depleted breast cancer cells
Figure S4 (related to Figure 5). Analysis of Akt phosphorylation in breast epithelial cell lines
Figure S5 (related to supplementary methods). siRNA screen quality control and reproducibility
Table S1 (related to Figure 1). Results of siRNA screen targeting 231 metabolic genes
Table S2 (related to Figure 1). Validation of screen results
Table S3 (related to Figure 3). Metabolite uptake and secretion rates after MCT4 depletion

Copyright © 2015 Pathological Society of Great Britain and Ireland. J Pathol 2015; 237: 152–165
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com