Introduction Increased glucose metabolism in neoplastic cells is orchestrated by the oncogenic proteins ras and c-myc. Ectopic expression of oncogenic ras rapidly increases glucose metabolism by activating the synthesis of fructose-2,6-bisphosphate (F2,6BP), an allosteric activator of 6-phosphofructo-1-kinase (PFK-1), the rate-limiting step of glycolysis. The steady-state concentration of F2,6BP depends on the activity of 6-phosphofructo-2-kinase (PFK-2), and an inducible isoform of PFK-2 (iPFK) was recently identified to be overexpressed by human solid tumors relative to adjacent normal tissues. We hypothesize that increased F2,6BP, arising from activation of iPFK-2 by oncogenic ras, is required for the increased flux of glucose carbons into anabolic pathways and is necessary for the anchorage-independent growth of neoplastic cells.
Methods/Results We isolated lung fibroblasts from iPFK-2+/+ and iPFK-2+/- mice and transduced the cells with retroviruses expressing SV40 large T-antigen (LT) and human H-rasV12 mutant. Introduction of LT and activated H-rasV12 into wild-type lung fibroblasts conferred the ability for anchorage-independent growth as soft agar colonies, a hallmark of neoplastic transformation (143.3 ± 29.9 colonies [n = 3], 9 days of culture). In contrast, introduction of LT and HrasV12 into iPFK-2+/- lung fibroblasts was unable to transform the cells and permit anchorage-independent growth (0 colonies [n = 3], 14 days of culture). Ras-transformed fibroblasts isolated from iPFK-2+/- mice were found to secrete significantly more protons than their iPFK-2+/+ counterparts (48 hrs exponential growth: iPFK-2+/-, pH 7.0 vs iPFK-2+/+, pH 7.5). We reasoned that such media acidification could be caused by a perturbation of mitochondrial respiration leading to a proton efflux and/or a compensatory increase in glycolytic flux to lactate. Although we found no significant difference in lactate secretion, we did observe a fully dissipated mitochondrial membrane potential in the ras-transformed fibroblasts isolated from iPFK2+/- mice.
Conclusions The observation that heterozygote genomic deletion of iPFK-2 causes a loss of the mitochondrial membrane potential indicates that iPFK-2 may serve as an essential coupler of glycolysis with respiration. That this metabolic phenotype is not compatible with anchorage-independent growth supports the rationale for molecular targeting of iPFK-2 as an antineoplastic agent. Given the recent observation that iPFK-2+/- mice display no overt pathology, these studies suggest that pharmacologic inhibition of iPFK-2 may yield antineoplastic effects without causing significant toxicity.
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