Therapeutics, Targets, and Chemical Biology MEK1/2 Inhibition Decreases Lactate in BRAF-Driven Human Cancer Cells Maria Falck Miniotis 1 , Vaitha Arunan 1 , Thomas R. Eykyn 1,3 , Richard Marais 4 , Paul Workman 2 , Martin O. Leach 1 , and Mounia Beloueche-Babari 1 Abstract The RAS/BRAF/MEK/ERK signaling pathway is a central driver in cancer with many BRAF and MEK inhibitors being evaluated in clinical trials. Identifying noninvasive biomarkers of early pharmacodynamic responses is important for development of these targeted drugs. As increased aerobic glycolysis is often observed in cancer, we hypothesized that MEK1/2 (MAP2K1/MAP2K2) inhibitors may reduce lactate levels as detected by magnetic resonance spectroscopy (MRS), as a metabolic biomarker for the pharmacodynamic response. MRS was used to monitor intracellular and extracellular levels of lactate in human cancer cells in vitro and in melanoma tumors ex vivo. In addition, we used 1 H MRS and a fluorescent glucose analog to evaluate the effect of MEK inhibition on glucose uptake. MEK1/2 signaling inhibition reduced extracellular lactate levels in BRAF-dependent cells but not BRAF-independent cells. The reduction in extracellular lactate in BRAF-driven melanoma cells was time- dependent and associated with reduced expression of hexokinase-II driven by c-Myc depletion. Taken together, these results reveal how MEK1/2 inhibition affects cancer cell metabolism in the context of BRAF oncogene addiction. Furthermore, they offer a preclinical proof-of-concept for the use of MRS to measure lactate as a noninvasive metabolic biomarker for pharmacodynamic response to MEK1/2 inhibition in BRAF-driven cancers. Cancer Res; 73(13); 4039–49. Ó2013 AACR. Introduction Hyperactivated extracellular signal–regulated kinase (ERK) 1/2 signaling occurs in many human cancers due to mutation or overexpression of components of the RAS/RAF/MEK1/2/ ERK1/2 pathway. For example, BRAF mutation, especially the V600E variant, has been reported in approximately 50% of malignant melanomas and 40% of thyroid cancers (www. sanger.ac.uk/genetics/CGP/cosmic/). Thus, inhibition of BRAF/MEK1/2 signaling constitutes a key strategy for molec- ularly targeted cancer treatment (1), and the recent regulatory approval of the BRAF inhibitor vemurafenib (PLX4032/ RG7204) for BRAF-driven melanoma shows the effectiveness of this approach. Many MEK1/2-selective inhibitors have also been described including PD184352 (CI-1040), PD325901, selumetinib (AZD6244/ARRY-142866), and trametinib (GSK1120212). PD325901 and selumetinib have shown potent antitumor activity in preclinical tumor models (2), and selu- metinib has recently undergone phase II clinical trials in non– small cell lung cancer and melanoma (3, 4). Trametinib has shown clinical activity in BRAF-mutant melanoma (5). Compared with cytotoxic therapy, RAF/MEK1/2 inhibitors are predominantly, but not exclusively, cytostatic. In BRAF- mutant cells, MEK inhibition can elicit both apoptotic and cytostatic effects (6). Tumor shrinkage may therefore not always be apparent in the early stages of treatment, indicating the need to identify early, and preferably, noninvasive, response biomarkers to assess target modulation and treat- ment efficacy. Magnetic resonance spectroscopy (MRS) provides a nonin- vasive approach for monitoring metabolism. MRS has shown different metabolic characteristics in tumors versus normal tissues (e.g., elevated signals from lipids, choline-containing metabolites, and glycolytic intermediates; ref. 7), which are altered with chemotherapy and various molecularly targeted anticancer agents (8, 9). The increased glycolytic activity of cancer cells under nor- moxic conditions, known as the "Warburg effect" is charac- terized by increased glucose uptake and lactate production (10). This allows fast-growing cancer cells to shunt glycolytic intermediates into anabolic reactions while the acid produced aids tumor invasion and stroma destruction (11). Increased tumor lactate correlates with poor prognosis in some cases of brain, breast, lung, and liver cancers (12). Furthermore, in human cervical cancer, high tumor lactate concentration is Authors' Affiliations: 1 Cancer Research UK and EPSRC Cancer Imaging Centre, Division of Radiotherapy and Imaging, 2 Cancer Research UK Cancer Therapeutics Unit, Division of Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey; 3 Division of Imaging Sciences and Biomedical Engineering, The Rayne Institute, St. Thomas' Hospital, Lon- don; and 4 Cancer Research UK Centre for Cell and Molecular Biology, Division of Cancer Biology, The Institute of Cancer Research, London, United Kingdom Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Martin O. Leach, Institute of Cancer Research and Royal Marsden NHS Foundation Trust, Downs Road, Sutton, Surrey SM2 5PT, United Kingdom. Phone: 44-2086613338; Fax: 44-2086610846; E-mail: martin.leach@icr.ac.uk doi: 10.1158/0008-5472.CAN-12-1969 Ó2013 American Association for Cancer Research. Cancer Research www.aacrjournals.org 4039 on December 9, 2021. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst May 2, 2013; DOI: 10.1158/0008-5472.CAN-12-1969