AMP-activated protein kinase is dispensable for maintaining ATP levels and for survival following inhibition of glycolysis, but promotes tumour engraftment of Ras-transformed fibroblasts.

Lactic acid generated by highly glycolytic tumours is exported by the MonoCarboxylate Transporters, MCT1 and MCT4, to maintain pHi and energy homeostasis. We report that MCT1 inhibition combined with Mct4 gene disruption severely reduced glycolysis and tumour growth without affecting ATP levels. Because of the key role of the 5′-AMP-activated protein kinase (AMPK) in energy homeostasis, we hypothesized that targeting glycolysis (MCT-blockade) in AMPK-null (Ampk−/−) cells should kill tumour cells from ‘ATP crisis’. We show that Ampk−/−-Ras-transformed mouse embryonic fibroblasts (MEFs) maintained ATP levels and viability when glycolysis was inhibited. In MCT-inhibited MEFs treated with OXPHOS inhibitors the ATP level and viability collapsed in both Ampk+/+ and Ampk−/− cells. We therefore propose that the intracellular acidification resulting from lactic acid sequestration mimicks AMPK by blocking mTORC1, a major component of an ATP consuming pathway, thereby preventing ‘ATP crisis’. Finally we showed that genetic disruption of Mct4 and/or Ampk dramatically reduced tumourigenicity in a xenograft mouse model suggesting a crucialrolefor these two actors in establishment of tumours in a nutrient-deprived environment. These findings demonstrated that blockade of lactate transport is an efficient anti-cancer strategy that highlights the potential in targeting Mct4 in a context of impaired AMPK activity.

Clonogenicity growth assay 5x10 3 cells were seeded onto 60mm dishes. 24h later, medium was replaced with regular medium, for 8 days of growth in the presence or not of MCTi (300nM), Phenformin (50M), or both under normoxic or hypoxic (1% O2) conditions. Dishes were then stained with Giemsa (Fluka).
Proliferation Assay 5x10 4 cells were seeded onto 35mm dishes. 24h after seeding, medium was replaced with medium containing the indicated compounds. Cells were detached and counted with Coulter Z1 (Beckman) every 24h during 4 to 5 days. Proliferation units were calculated by dividing the cell number obtained for each day by the one obtained 24h after seeding. Cell counting was done on two independent dishes at each time point and the experiment was repeated at least three times.
Cell Survival Assay 5x10 4 cells were seeded onto 35mm dishes. At the indicated time, the rate of mortality was assessed using Trypan blue exclusion staining (Sigma, St. Louis, MO).

Immunoblot Analysis
Cells were lysed in 1.5x sodium dodecyl sulfate (SDS)-buffer, and protein concentration was determined using the bicinchoninic acid assay. 40µg of each whole-cell extract were resolved by electrophoresis on SDS-polyacrylamide gels and transferred onto a polyvinylidene difluoride membrane (Millipore). Membranes were blotted with monoclonal antibodies to Phospho-AMPK (Thr172), Phospho-Acetyl-CoA Carboxylase (Ser79) and to total AMPK proteins (F6) purchased from Cell Signaling. Rabbit polyclonal antibodies to MCT4 (H-90) and MCT1 (M-45) were purchased from Santa Cruz Biotechnology. The rabbit polyclonal anti-HIF-1 antibody (antiserum 2087) was produced and characterized in our laboratory, and has been reported previously 66 . Antibody to Hsp90 (Abcam) was used as a loading control. The rabbit polyclonal antibody to arrest-defective-1 protein (ARD1) (previously produced and characterised in our laboratory 67 ) was also used as loading control.
Bands were detected with a horseradish peroxidase antimouse or anti-rabbit antibody (Promega) by ECL (Amersham Biosciences or Millipore).

Metabolic measurements
Glycolytic rates and mitochondrial respiration were assessed by monitoring extracellular acidification and oxygen consumption rates simultaneously in realtime using the Seahorse Biosciences Extracellular Flux Analyzer 68 . Cells were plated at an optimum density (1.5 to 2x10 5 cells/well) to achieve a confluent layer for the fluorescent probes to detect changes. The experimental procedure involved monitoring cells for oxygen consumption and lactic acid production (extracellular acidification) while injecting metabolic compounds into the medium (glucose 10mM, oligomycin 1M, FCCP 1M, rotenone/antimycinA 1M each, MCTi 300nM). Seahorse Biosciences Assay medium was used during experimentation. This media is an unbuffered DMEM without glucose, pyruvate, and bicarbonate. pHe of the media was adjusted before each use. Data are presented as extracellular acidification rate (ECAR; mpH/min/g protein) for glycolysis and oxygen consumption rate (OCR; pMolesO 2 /min/g protein). ECAR and OCR were determined at 3min measurement intervals. Protein samples were taken at the end of each experiment to standardize the results. Each assay was performed in quadruplicate at 37°C and representative data from at least three independent experiments are shown.
Determination of intracellular lactate concentration 5x10 5 cells were washed twice with PBS and maintained in DMEM without glucose, pyruvate, and serum for 30min with either DMSO (0.1% vol) or MCTi (300nM). At the indicated times (0-6h) after glucose (25mM) and serum addition, cells were washed twice with cold PBS, once with cold water and lysed in cold water (200μL). Lactate concentration was determined by an enzyme based assay using 900μM β-NAD (BioChemika), 175μg/mL lactate dehydrogenase (BioChemika), and 100μg/mL glutamate pyruvate transaminase (Roche) diluted in a sodium carbonate (620mM)-L-glutamate (79mM) buffer adjusted to pH10. Lithium lactate was used as a standard. Measurements were done with a microplate reader after incubation for 30min at 37 °C. Determinations were done in triplicate, and the experiment was repeated at least three times.
Determination of glucose consumption and lactate production 5x10 5 cells were seeded onto 60mm dishes and following cell attachment, medium was replaced with 5 ml of medium with DMSO or MCTi (300nM) and cells were incubated in normoxia or hypoxia (1%O2). 48h later, medium was replaced with a fresh one still containing DMSO/MCTi. Supernatant was collected 1h and 6h later. Then, cells were lysed and protein concentration was determined to normalize lactate production/glucose consumption to protein content. Glucose and lactate concentrations were measured in the supernatant by the Ysi 2300 STAT Plus analyzer (YSI Life Sciences). Rate of lactate production was determined by substracting the amount of lactate in the medium after 6h to the value obtained 1h after addition of the fresh medium. Similarly, rate of glucose consumption was determined by substracting the amount of glucose in the medium after 1h to the value obtained 6h after addition of the fresh medium. Determinations were done in triplicate, and the experiment was repeated two times.

ATP assays
To determine cellular ATP content we utilized a luminescence assay (CellTiter-Glo, Promega). Briefly, 1,000 cells were plated per well in 200l on a 96 well plate and following cell attachment medium was replaced with 200μl of medium with the indicated compounds.
After 48h incubation, the relative ATP content was determined following the manufacturers instructions. An identical plate was always prepared to standardize ATP results to protein content (lysis in 0.1M NaOH, BCA protein assay). For the short lasting experiments (1min to 3h), 5,000 cells were plated per well in 200l on a 96 well plate. 24h later, cells were washed twice with PBS, and 100l of medium with the indicated concentration of glucose was added.
45min later, 100l of medium with the indicated concentration of glucose and two times concentrated oligomycin (final concentration of 1g/l) or 2DG (final concentration of 10mM) was added. Solution provided by the kit, lysing instantaneously the cells was added at the indicated time after addition of oligomycin/2DG.

Nude mice tumorigenicity and immunohistochemistry
1x10 6 cells suspended in 300 μL of serum-free DMEM supplemented with insulintransferrin-selenium (Gibco) were s.c. injected into the backs of 6wk-old female athymic nude mice (Harlan). The mice were housed under strict pathogen-free conditions and given sterile food and water. Animals were housed 5 per cage. When the tumor reached 70mm 3 , mice were treated twice a day by gavage for ten days with vehicle (methyl cellulose 5g/l, Tween 1g/l) or with MCT1/2 inhibitor (200mg/kg/day, dissolved in the vehicle). The tumor volume was determined by using the formula: 0.52 x L x W x H, where L represents length, W the width, and H the height. Tumors were collected for immunohistochemical analysis. Tumor sections were incubated with antibodies to P-AMPK or P-ACC for 1.5h followed by incubation with antirabbit IgG-HRP antibodies. Analysis was performed with a Leica microscope (20x objective).