High glycolytic flux by cancer cells is believed to be essential for tumor growth, and in situ glucose uptake predicts tumor progression and overall patient mortality and morbidity.  The long-term objective of our laboratory is to understand the mechanisms responsible for high glycolytic flux during neoplastic transformation in order to develop metabolic strategies to treat cancer.  Fructose-2,6-bisphosphate (F2,6BP) is a potent activator of PFK-1, the rate-limiting enzyme of glycolysis, and is increased by neoplastic transformation.  The steady-state concentration of F2,6BP depends on the activity of the enzyme PFK-2.  We recently identified an inducible isoform of PFK-2 (iPFK-2) that is over-expressed by several human solid tumors and is required for the growth of K562 leukemia tumors in athymic mice.  Our central hypothesis is that the high glycolytic flux of cancer cells is activated by iPFK-2 catalyzed production of F2,6BP and is required for neoplastic growth. The rationale for our studies is that once knowledge of the mechanisms for increased glycolysis in cancer cells has been obtained, new therapeutic strategies to inhibit tumor growth can be developed.  We will test our central hypothesis by pursuing the following two specific aims:

1. ExaminetheeffectsofhumaniPFK-2over-expressionandunder-expressionontheglucosemetabolismandneoplasticphenotypeoftransformedandnon-transformedcells. Transformed and non-transformed cells will be transduced with retroviruses that under- or over-express iPFK-2 and the effects on glucose metabolism (by ¹³C-NMR) and their neoplastic phenotype (i.e. growth, invasiveness, resistance to apoptosis) will be examined in vitro and in vivo. We also will expose the cell lines to various substrate analogs of fructose-6-phosphate and putative small molecule inhibitors of iPFK-2 and examine the resultant effects on glycolytic flux and growth.
2. Examinetheneoplasticpotentialandmetabolic phenotype of cells derived from iPFK-2 knockout mice in vitro and in situ. We recently cloned the murine homolog to iPFK-2 and have generated iPFK-2 knockout mice.  We propose to compare, in iPFK-2 knockout and wild-type mice, the appearance and growth of cutaneous tumors that have been initiated and promoted by the carcinogens DMBA and TPA. We also will measure in situ tumor glucose uptake by positron emission tomography and expand cells from the tumor masses and analyze their neoplastic and metabolic phenotype.  Next, we will purify fibroblasts from iPFK-2^-/- and iPFK-2^+/+ mice and determine their potential to be transformed by SV40 large T antigen and ras.