Abstract 5083: Inhibition of MEK1/2 signaling in human <i>BRAF</i>V600E melanoma cells reduces glucose uptake and lactate dehydrogenase activity resulting in a time-dependent decrease in lactate production
Authors: Maria Falck Miniotis, Thomas R. Eykyn, Paul Workman, Martin O. Leach, Mounia Beloueche-Babari
Journal: Cancer Research (2010)

Abstract Background: Oncogenic transformation is associated with changes in cell metabolism. The MEK1/2 activator BRAF is mutated in ∼70% of malignant melanoma. This provides a significant focus for advancing mechanism-based therapy and several inhibitors of the pathway are now undergoing clinical development. Our aim was to investigate whether MEK1/2 signaling inhibition in BRAFV600E human melanoma cells results in magnetic resonance spectroscopy (MRS) detectable changes in glycolysis that could serve as non-invasive biomarkers of target suppression. For this we assessed lactate production, glucose uptake and lactate dehydrogenase (LDH) activity in response to treatment. Methods: WM266.4 melanoma cells (BRAFV600E) were treated with 1 µM of the MEK1/2 inhibitor CI-1040 and lactate production was monitored using 1H MRS at 30 min and 2, 6, 16, 24, 48 h on a 11.7 T Bruker Avance spectrometer. LDH activity was measured using a dynamic nuclear polarization assay by suspending 107 control or treated cells (24h) in a neutralized solution of hyperpolarized 1-13C pyruvic acid and unpolarized lactate followed by serial 13C MRS acquisitions. Glucose uptake at 24 h was evaluated by adding 10 µM of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) to control and treated cells for the last 2 h of treatment. Median fluorescence intensities of 2-NBDG uptake were obtained using a BD FACSAriaTM Flow Cytometer. Statistical analysis was performed using Student's t-test with p ≤ 0.05 considered to be significant. Results: 1H MRS analysis indicated that lactate levels were unchanged at 30 min to 6 h but decreased significantly at 16 h (79±3%), 24 h (76±4%) and 48 h (80±6%) as compared to controls (n=3, p ≤ 0.006). LDH activity in control and treated cells was 0.83±0.26 and 0.48±0.08 nmol/s/106 cells, respectively (n=5, p=0.02) i.e. reduced by ∼40% following MEK1/2 inhibition. Similarly, CI-1040 treatment led to a reduction in 2-NBDG uptake to 88±2% relative to the control (n=3, p=0.003). Conclusions: Our findings demonstrate that MEK1/2 signaling inhibition leads to a time-dependent decrease in lactate production through modulation of both LDH activity and glucose uptake. These results show lactate as a potential non-invasive biomarker of response to MEK1/2 targeted therapeutics in human BRAFV600E melanoma cells. Further studies are required to establish the molecular processes linking MEK1/2 inhibition to decreased lactate production. Funding: Marie Curie Action: Early Stage Training (grant MEST-CT-2005-020718) and CRUK (grants C309/A8274, C1060/A5117 and C1060/A6916). We also acknowledge the support received from the CRUK and EPSRC Cancer Imaging Centre in association with the MRC and Department of Health (England) grant C1060/A10334 and NHS funding to the NIHR Biomedical Research Centre. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5083.