Targeting MCT1 Blocks Tumor Angiogenesis Figure 7. MCT1 inhibition blocks lactate-induced endothelial tube formation. HUVECs were seeded on growth factor-reduced Matrigel containing 10 mM lactate or not and monitored for their ability to generate vascular tubes. (A) Pictures of the upper panel show endothelial network formation over time (bar = 500 mm), which was quantified in the lower graph. n = 4. Error bars reflect mean 6 SEM and are sometimes smaller than symbols. (B) The tube formation assay was performed in the presence of 10 mM SU5402 (targeting VEGF and bFGF receptors), 10 nM echinomycin (targeting the transcriptional activity of HIF-1), or 2 mM of CHC (targeting MCT1). Pictures were captured after 8-h of culture in the Matrigel matrix. Bar = 200 mm. doi:10.1371/journal.pone.0033418.g007 VEGF protein secretion despite increased VEGF-A gene transcrip- tion. Because VEGFR2 is the main transducer of the pro- angiogenic effects of VEGF [27], one can logically propose that, in complex tumor tissues, lactate exquisitely stimulates VEGF signaling by acting on both the growth factor and its cognate receptor. This paradigm is further supported by the influence of lactate on stromal cells. Lactate indeed promotes VEGF production by fibroblasts and macrophages [18,19], and enhances VEGF activity through inhibition of ADP-ribosylation in several cell types [18–20]. Of note, the existence of a HIF-1a-driven, VEGF-mediated autocrine loop in ECs could further reinforce the anti-angiogenic effects of MCT1 inhibition in ECs [37,38]. Finally, it should be emphasized that although the lactate induction of the VEGF/VEGFR2 signaling pathway is indepen- dent of hypoxia in the target cells, lactate may arise from anaerobic glycolysis [24] as well as aerobic glycolysis (the Warburg effect) [39], or through the malate shuttle from the oxidative metabolism of glucose and glutamine [40,41]. Lactate flux rate is likely to be different between ‘‘target’’ cells signaling through lactate vs cells consuming or producing lactate. For instance, while lactate-fueled respiration of tumor cells requires high-rate lactate uptake and high MCT1 expression [26], lower MCT1 expression in ECs could efficiently support the low-rate lactate uptake accounting for lactate signaling. Different levels of MCT1 expression could explain why Koukourakis et al. [42] failed to detect MCT1 in the vascular structures of colorectal adenocarci- nomas using immunohistochemistry. Although we focused on the paradigm that HIF-1a-driven VEGFR2 receptor expression is a target of MCT1 inhibition to account for the observed anti-angiogenic effects, inhibition of other pro-angiogenic HIF-1a targets is very likely to support the functional impact of blocking lactate transport in ECs. For instance, we found that bFGF release was increased in response to lactate (Figure 2C) and thus that in the absence of increased VEGF, the inhibition of lactate-induced tubulogenesis on Matrigel with SU4502, a mixed inhibitor of VEGFR2 and fibroblast growth factor receptor 1 (FGFR1) (Figure 7B), strongly suggests that bFGF participates to the pro-angiogenic effects of lactate. Of interest is also the ability of bFGF to stimulate the activity of aminophospholipid translocase in ECs [43], an enzyme required for the maintenance of lipid asymmetry of the membrane, proper membrane anchorage of proteins, endocytosis and membrane recycling [44,45]. In erythro- cytes, GLUT-dependent arabinose transport and MCT-dependent lactate transport were shown to be inhibited by cleavage of either phosphatidylcholine or sphingomyelin [46], stressing out that membrane fluidity and asymmetry are important for the activity of these transporters. Whether lactate-induced bFGF production facilitates the membrane translocation of transporters (such as MCT1) and receptors (such as bFGFR and VEGFR2) involved in the angiogenic phenotype of ECs warrants further investigation. More generally, previous work indicates that targeting MCT1 offers the possibility to simultaneously inhibit several major pro- angiogenic pathways with a single therapeutic molecule. First, MCT1 inhibition may interfere with the viability of angiogenic- factor-producing-cells including cancer cells, macrophages and fibroblasts; all these cells express MCT1 [26,42]. Note that the impact may be indirect since MCT1 inhibition in oxidative tumor cells may favor the death of hypoxic glycolytic cells [26], and alteration in the fibroblast capacity to take up lactate and export pyruvate (both through MCT1) in tumors may reduce tumor cell PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e33418