Mounia Beloueche-Babari1, Slawomir Wantuch1, Harold G Parkes1, Markella Koniordou1, Vaitha Arunan1, Thomas R Eykyn1, Paul D Smith2, and Martin O Leach1
1CRUK Cancer Imaging Centre, The Institute of Cancer Research & Royal Marsden NHS Foundation Trust, London, United Kingdom, 2AstraZeneca, Cambridge, United Kingdom
Synopsis
The
monocarboxylate transporter 1 (MCT1), which mediates the bidirectional movement
of molecules such as lactate and pyruvate, is upregulated in cancer and constitutes
a promising drug target. Using MRS we investigate the impact of the MCT1
inhibitor AZD3965, currently in phase-I clinical trials, on human cancer cell
metabolism to assess potential for pharmacodynamic biomarker discovery. We show
that AZD3965 inhibits lactate efflux and blocks hyperpolarised 13C-pyruvate-lactate
exchange in human cancer cells in an MCT4-dependent manner. Thus, intracellular
lactate and hyperpolarized 13C-pyruvate-lactate exchange are promising
metabolic imaging biomarkers for monitoring the action of AZD3965 and
potentially other MCT1 inhibitors.Purpose
Monocarboxylate
transporters (MCTs) are transmembrane proteins that mediate the bi-directional
transport of monocarboxylic acids such as lactate, pyruvate and ketones. MCT1
and MCT4 are key modulators of lactate homeostasis in tumours and represent
promising targets for molecular cancer therapeutics. The MCT1 inhibitor AZD3965
is now in early phase clinical trials, thus understanding the impact of this
drug on cancer cell metabolism may enable the discovery of pharmacodynamic
biomarkers of target inhibition that will support the clinical development of
such agents. Since MCT1 mediates the flux of lactate and pyruvate in and out of cells, here we use magnetic
resonance spectroscopy (MRS) to investigate the effect of AZD3965 on a) intracellular
(Lactate-I) and extracellular lactate (lactate-E) levels
and b) hyperpolarised
13C-pyruvate-lactate exchange, as potential biomarkers
for MCT1 inhibition in human cancer cells with varying MCT4 expression levels,
a predictor of resistance to AZD3965.
1Methods
Human Raji (MCT4-) and Hut78 (MCT4 low (+))
lymphoma as well as HT29 (MCT4 high (+++))
colon carcinoma cells were treated with either 5nM or 25nM AZD3965 for
24h and levels of lactate in cell
extracts (Lactate-I)
or growth media (lactate-E) determined by
1H MRS on a 500MHz Bruker spectrometer. Spectra were
processed on MestRe-C v4.9 (University of Santiago de Compostela) and lactate
levels normalized to an internal standard and to cell number. For
13C-pyruvate-lactate exchange
studies, Raji, Hut78 and HT29 cells were treated as above for 24h or 1h (Raji
cells only). Cells were then incubated at 37°C in FBS-free medium and dynamic
13C MR
spectra acquired for 4 minutes with 2s intervals immediately after the addition
of 10mM hyperpolarised [1-
13C]pyruvic acid and 10mM unlabelled
lactate. The ratio of the area under the curve for the summed lactate and
pyruvate signals (Lactate
AUC/Pyruvate
AUC)
was determined, corrected for cell number and used to estimate pyruvate-lactate
exchange, as
previously shown.
2 Data
represent mean±SE.
Results
24h exposure to AZD3965 led
to increased Lactate-I in MCT4- Raji to 2.65-fold and 12-fold
relative to controls following exposure to 5nM and 25nM AZD3965, respectively
(p<0.05). Similar effects were observed in MCT4+ Hut78 human lymphoma cells
with Lactate-I increasing to ~10-fold with 5nM and maintaining this
level at 25nM AZD3965 (p=≤0.02);
however, in MCT4+++ HT29 human colon carcinoma cells Lactate-I showed
only a trend towards an increase which did not reach statistical significance
(167±30% to 204±34%, p≥0.056). AZD3965 treatment also led to a fall in Lactate-E in MCT4- Raji cells to ~60% and ~50% with 5nM and 25nM AZD3965,
respectively (p≤0.05) but its effects on Lactate-E levels at both concentrations were insignificant in Hut78 (92±8% to104±12%
of controls, p≥0.34) and HT29 cells (111±12% to126±17% of controls, p≥0.2), in line with the presence of an alternative transporter for lactate
efflux, MCT4.
Hyperpolarised
13C MRS
analyses showed a significant decrease in
Lactate
AUC/Pyruvate
AUC to 31±6% in 5nM and 19±2% in 25nM
AZD3965-treated Raji cells relative to controls at 24h (p<0.001), an effect
that was also observed at 1h following treatment with 25nM AZD396 (down to
27±7% of controls, p=0.002) indicating that drug-induced inhibition of
13C-pyruvate-lactate
exchange is immediate. Reduced Lactate
AUC/Pyruvate
AUC was
also observed in MCT4+ Hut78 cells and MCT4+++ HT29 cells treated with AZD3965,
however these effects appeared to be MCT4-dependent with
MCT4- Raji displaying the greatest fall in pyruvate-lactate exchange at 5nM
AZD3965 and MCT4+++ HT29 showing the lowest reduction at 25nM AZD3965 with
MCT4+ Hut78 falling in the middle (Figure
1). These results are consistent with MCT1-mediated transport of
13C-pyruvate
being a rate limiting step in the
13C MRS-observed
pyruvate-lactate exchange,
3 although additional factors appear
to be implicated.
Discussion
Our data show that AZD3965
triggers inhibition of lactate efflux leading to a MRS-detectable increase in
Lactate-I in human cancer cells with modest or no MCT4 expression. Further,
we show that AZD3965 inhibits
13C-pyruvate-lactate exchange (via
blockade of
13C-pyruvate uptake) in human cancer cells with MCT4-
cells showing the largest effect.
Conclusions
Lactate-I and hyperpolarised
13C-pyruvate-lactate
exchange are therefore promising non-invasive metabolic imaging biomarkers for monitoring
the action of AZD3965 and potentially other MCT1 inhibitors.
Acknowledgements
We Acknowledge CRUK support to the Cancer Imaging Centre
at ICR and RMH in association with MRC & Department of Health C1060/A10334,
C1060/A16464 and NHS funding to the NIHR Biomedical Research Centre and the
Clinical Research Facility in Imaging. MOL is an NIHR Senior Investigator.References
1) Polansky et al, Clin Cancer Res 2013.
2) Hill et al, PlosOne 2013.
3) Harris et al, Proc Natl Acad Sci U
S A. 2009.