Nature | Vol 577 | 2 January 2020 | 119To test this further, we resected the primary tumours to extend
mouse survival (see schematic in Extended Data Fig. 8d). Treatment
with AZD3965 before primary tumour resection, when cells were spon-
taneously metastasizing, significantly reduced metastatic tumour
burden (Extended Data Fig. 8e). By contrast, treatment with AZD3965
only after primary tumour resection—after metastatic tumours were
established—did not reduce metastatic tumour burden (Extended
Data Fig. 8e). Melanoma cells are, therefore, particularly dependent
on MCT1 during metastasis.
Analyses of clinical data^27 and TCGA data showed that higher expres-
sion of MCT1 is associated with significantly worse overall survival
(Extended Data Fig. 9a). Differences in MCT2 (also known as SLC16A7)
or MCT4 (SLC16A3) expression did not significantly affect survival
(Extended Data Fig. 9b, c). Consistent with the correlation between
CD147 and MCT1 expression^26 , higher CD147 expression was also associ-
ated with significantly worse survival (Extended Data Fig. 9d).
Inhibition of MCT1 induces oxidative stress
Inhibition of MCT1 or MCT4 in cancer cells in culture promotes oxida-
tive stress by inhibiting lactate export, leading to reduced glycolysis^21 ,^32.
AZD3965 treatment increased levels of reactive oxygen species (ROS)
in all three melanomas (Fig. 3a–c; see Extended Data Fig. 9e, f for the
gating strategy to identify melanoma cells), as did deletion of MCT1
from YUMM cells (Extended Data Fig. 7d). AZD3965 did not increase
ROS levels in melanomas after shRNA-mediated knockdown of MCT1,
which suggests an on-target effect (Extended Data Fig. 6i). AZD3965 also
reduced the ratios of GSH to oxidized glutathione (GSSG) (Extended
Data Fig. 10a) and levels of NADPH (Extended Data Fig. 10b). Moreover,
treatment with the antioxidant N-acetyl cysteine (NAC) rescued the
effects of AZD3965 on circulating melanoma cells and metastatic
disease burden (Fig. 3d–f). MCT1 inhibition thus impairs metastasis at
least partly by increasing oxidative stress.
To test whether MCT1 inhibition affected the pentose phosphate
pathway (PPP), we infused [1,2-^13 C]glucose into xenografted mice and
compared the relative flux of labelled glucose through glycolysis versus
the oxidative PPP by comparing the ratio of m + 1 lactate (derived from
the oxidative PPP) to m + 2 lactate (derived from glycolysis)^33 (Extended
Data Fig. 10c). We observed a trend towards increased glucose enrich-
ment in tumours treated with AZD3965 (Fig. 4a). We consistently
observed a lower m + 1 lactate/m + 2 lactate ratio in AZD3965-treated
as compared to control tumours for all three melanomas (Fig. 4b,
Extended Data Fig. 10d). This suggests that MCT1 inhibition reduced
flux through the oxidative PPP relative to glycolysis.
After infusion of [U-^13 C]glucose into xenografted mice (Extended
Data Fig. 10e), AZD3965 treatment did not alter isotope enrichment in
glucose or glycolytic intermediates (Fig. 4e–h), but reduced isotope
enrichment in the oxidative PPP (Fig. 4i, j). AZD3965 treatment did not
generally reduce the levels of glycolytic intermediates (Extended Data
Fig. 10f, h) but did reduce the levels of oxidative PPP intermediates
(Extended Data Fig. 10g, i). Therefore, the effect of MCT1 inhibition in
melanoma cells in vivo (inhibition of lactate import, favouring glyco-
lysis over the PPP) was quite different from MCT1 inhibition in culture
(inhibition of lactate export, reducing glycolysis^21 ,^32 ). In lung cancer,
MCT1 deletion also reduced lactate export and glycolysis in culture,
but reduced lactate uptake and enhanced glucose metabolism in vivo^17.
Lactate import can alter intracellular pH and the NAD+/NADH ratio
because lactate is co-transported with a proton and converted to pyru-
vate intracellularly, converting NAD+ to NADH^19. Consistent with this, in
all three melanomas, AZD3965 treatment significantly increased intra-
cellular pH (Fig. 4c), strongly suggesting substantial MCT1-dependent
lactate and proton import in these tumours. The increase in pH after
MCT1 inhibition could reduce flux through the PPP relative to glycoly-
sis as increased pH activates the activity of phosphofructokinase and
suppresses the activity of glucose-6-phosphate dehydrogenase^34 ,^35 —
rate-limiting enzymes in glycolysis and the PPP, respectively. AZD3965
treatment also significantly increased the NAD+/NADH ratios (Fig. 4d),
which has the potential to enhance glycolysis at the expense of the PPP.Heterogeneity in MCT1 expression
Flow cytometry revealed a more prominent MCT1high cell popula-
tion among melanoma cells in the blood (see arrows in Fig. 5b, d) as
compared with subcutaneous tumours in the same mice (Fig. 5a, c).f1001021041061081010MCT1:Total flux
(photons s–1)–/LowHigh1001021041061081010M405 M481
eTotal flux
(photons s–1)EfficientaMCT1-Alexa Fluor647M405
Isotype
controlb1001021041061081010UT10Total flux
(photons s–1)Isotype
controlM405EfficientMCT1-Alexa Fluor647MCT1MCT1EfficientcMCT1-Alexa Fluor647M481
Isotype
controld
Isotype
controlM481EfficientMCT1-Alexa Fluor647MCT1
MCT1100M405M481UT10M405M481UT10M405M481UT10M405M481UT10020406080100Mice forming tumours (%)MCT1−/low
MCT1highCell dose1,000 100
Cell dose1,000P = 0.0465 P = 0.005
P = 0.0003 P = 0.01020 16 15 15 910205 5 15 151017–/LowHigh –/LowHigh10101010
10
2020Fig. 5 | Heterogeneous MCT1 expression among melanoma cells from the
same tumour. a–d, Flow cytometric analysis of anti-MCT1 staining in
melanoma cells from subcutaneous tumours (a, c) or circulating melanoma
cells (b, d) from the same mice xenografted with M405 (a, b) or M481 (c, d) (the
gating strategies to identify human melanoma cells is in Extended Data Fig. 9e,
f; data are representative of two experiments). e, Flow cytometrically isolated
MCT1high or MCT1−/ low melanoma cells were intravenously transplanted into NSG
mice, using 100 or 1,000 cells per injection. The percentage of injections that
formed metastatic tumours is shown (one or two experiments per melanoma).
The number of mice analysed per treatment is indicated. f, Metastatic disease
burden in the visceral organs of mice that survived to end point after injection
with 100 (M405 and M481) or 1,000 (UT10) cells based on bioluminescence
signal intensity. Data are mean ± s.d. Statistical significance was assessed using
multiple linear regression (e) or Mann–Whitney tests (f).