Statin-induced metabolic reprogramming in head and neck cancer: a biomarker for targeting monocarboxylate transporters

Prognosis of HPV negative head and neck squamous cell carcinoma (HNSCC) patients remains poor despite surgical and medical advances and inadequacy of predictive and prognostic biomarkers in this type of cancer highlights one of the challenges to successful therapy. Statins, widely used for the treatment of hyperlipidaemia, have been shown to possess anti-tumour effects which were partly attributed to their ability to interfere with metabolic pathways essential in the survival of cancer cells. Here, we have investigated the effect of statins on the metabolic modulation of HNSCC cancers with a vision to predict a personalised anticancer therapy. Although, treatment of tumour-bearing mice with simvastatin did not affect tumour growth, pre-treatment for 2 weeks prior to tumour injection, inhibited tumour growth resulting in strongly increased survival. This was associated with increased expression of the monocarboxylate transporter 1 (MCT1) and a significant reduction in tumour lactate content, suggesting a possible reliance of these tumours on oxidative phosphorylation for survival. Since MCT1 is responsible for the uptake of mitochondrial fuels into the cells, we reasoned that inhibiting it would be beneficial. Interestingly, combination of simvastatin with AZD3965 (MCT1 inhibitor) led to further tumour growth delay as compared to monotherapies, without signs of toxicity. In clinical biopsies, prediagnostic statin therapy was associated with a significantly higher MCT1 expression and was not of prognostic value following conventional chemo-radiotherapy. These findings provide a rationale to investigate the clinical effectiveness of MCT1 inhibition in patients with HNSCC who have been taking lipophilic statins prior to diagnosis.


Measurement of ATP levels and mitochondrial mass
ATP levels were measured using the ATPlite™ Luminescence Assay System (Perkin Elmer) according to manufacturer's instructions. Cells were seeded and exposed to 100 µl of medium with and without simvastatin for 96 hours. At endpoint, lysis solution was added to stop the reaction followed by substrate solution and luminescence was measured.
Mitochondrial mass was measured by staining live cells with 10-Nonyl acridine orange dye (Molecular probes, Invitrogen). Cells were grown to 70% confluency in a 6-well dish. The dye was diluted in RPMI media (final concentration: NAO = 20 nM) and exposed to the cells for 30 minutes at 37°C. Cells were then washed with PBS, collected by scraping and analysed using LSRFortessa flow cytometer (BD Biosciences).

Biotinylation of cell surface MCTs/ Cell surface protein isolation
Cells were washed with ice-cold PBS followed by incubation with 0.25 mg/mL Sulfo-NHS-SS-Biotin in 48 mL ice-cold PBS per flask on a rocking platform for 30 minutes at 4ºC. The biotinylation reaction was quenched by adding 500 μL of the provided Quenching Solution. Cells were harvested by gentle scraping, pelleted by centrifugation and lysed using the provided Lysis Buffer containing a protease inhibitor cocktail for 30 minutes on ice with intermittent vortexing. The lysates were then centrifuged and the clarified supernatant was used for purification of biotinylated proteins on NeutrAvidin Agarose. The captured surface proteins were eluted from the biotin-NeutrAvidin Agarose by incubation with dithiothreitol (DTT) in PBS containing 62.5 mM Tris-HCl for 1 h at room temperature. The eluted proteins, representing the cell surface proteins, were collected by column centrifugation at 1,000 x g for 2 minutes. For all cell lines, three biological replicates were obtained. Protein concentrations were quantified using the BCA protein Assay Kit (Pierce) and the lysates were stored at -20ºC until use.

Analysis of IHC staining
IHC staining was scored by two independent researchers and validated by a c l i n i c a l pathologist blinded to all clinico-pathological variables. Protein expression was scored for membrane M C T1 a n d M C T 4 a s both proteins are functional at the plasma membrane. Areas of necrosis, stroma, normal epithelium and distinct edge effects were ignored.
Immunoreactivity was scored for both the intensity and the proportion of cells stained; intensity was given scores of 0-4 (0, no staining; 1, very weak staining; 2, weak staining; 3, moderate staining; 4, strong staining), as shown in Supplementary Figure 7. The two scores were multiplied to obtain the final score within the range of 0-400. MCT score ≥200 and <200 were described as high and low, respectively.

Induced metabolic bioluminescence imaging
Snap-frozen tumours were cut into serial cryosections for structural hematoxylin and eosin (H&E) staining and metabolic measurements. The spatial concentrations of ATP, glucose, and lactate in cryosections of tumours were obtained using the method of metabolic imaging with induced bioluminescence (imBI), as previously described (20,21). For measurement, metabolites are enzymatically linked to the light reaction of bioluminescence enzymes, leading to light emission with the intensity being proportional to the tissue content of each metabolite.
Light emission was induced in a temperature-stabilized reaction chamber, which was placed under a microscope (Axiophot, Zeiss, Oberkochen, Germany) connected to a 16bit CCD camera with an imaging photon counting system (iXon EM + DU-888, Andor Technology PLC, Belfast, Northern Ireland). Light intensities were calibrated using appropriate standards. Metabolite content was calculated in µmol/g tumour tissue and images were displayed in colors coding for tissue concentration of metabolites in units of µmol/g.
Computerised image analysis allowed for separate data assessment in selected histological areas of xenograft tumours. Five tumours of vehicle-treated and simvastatin-treated were analysed using three sections from each tumour. Pixel values were summarised for individual tumours into one distribution histogram. From this histogram, mean values(± SEM) and additional statistical parameters were calculated. All metabolite concentrations shown here were acquired exclusively from vital tumour regions.