Use of 18 F-FDG uptake as a surrogate marker of therapeutic response requires the recognition of biologic factors that influence cancer cell glucose metabolism. Estrogen is a potent stimulator of breast cancer proliferation, a process that requires sufficient energy, which is likely met by increased glycolysis. We thus explored the effect of estrogen on 18 F-FDG uptake in responsive breast cancer cells and investigated the mediating molecular mechanisms. Methods: T47D breast cancer cells were stimulated with 17b-estradiol (E 2 ) or bovine serum albumin (BSA)-E 2 and measured for 18 F-FDG uptake, lactate release, and mitochondrial hexokinase activity. The effects of antiestrogens, cycloheximide, and major protein kinase inhibitors were investigated. Immunoblots were performed for membrane glucose transporter type 1, phosphorylated phosphatidylinositol 3-kinase (PI3K), and Akt. Results: E 2 augmented T47D cell 18 F-FDG uptake in a dose-and time-dependent manner that preceded and surpassed its proliferative effect. With exposure to 10 nM E 2 , protein content-corrected 18 F-FDG uptake reached 172.7% 6 6.6% and 294.4% 6 9.5% of controls by 24 and 48 h, respectively. Lactate release reached 110.9% 6 7.3% and 145.2% 6 10.5% of controls at 24 and 48 h, and mitochondrial hexokinase activity increased to 187.1% 6 31.6% at 24 h. Membrane glucose transporter type 1 expression was unaltered. The effect was absent in estrogen receptor (ER)-negative breast cancer cells and was abrogated by ICI182780, indicating ER dependence. The E 2 effect was not blocked by tamoxifen and was mimicked by membrane-impermeable BSA-E 2 , consistent with nongenomic membrane-initiated E 2 action. Inhibition by cycloheximide demonstrated the requirement of a new protein synthesis. Immunoblots displayed rapid phosphorylation of PI3K and Akt within minutes of E 2 treatment, and the specific PI3K inhibitors wortmannin and LY294002 abolished the ability of E 2 to elevate 18 F-FDG uptake. Conclusion: Estrogen augments breast cancer cell 18 F-FDG uptake by stimulating glycolysis and hexokinase activity via membrane-initiated E 2 action that activates the PI3K-Akt pathway. These findings yield important insight into our understanding of the biology of breast cancer metabolism and may have potential implications for 18 F-FDG uptake as a surrogate marker of therapeutic response.

Although the success of sodium/iodide symporter (NIS) genebased cancer therapy is critically dependent on the level of radioiodide accumulation attained, recent evidence indicates that successful therapy relies not solely on NIS amount but also crucially on its functional activity. In this study, we investigated the role of kinase-linked signaling on the regulation of NIS function in cancer cells. Methods: T47D human breast cancer and PC-12 rat pheochromocytoma cells were transduced with the human NIS genes via an adenoviral vector. Cells were measured for 125 I uptake, and the effects of activation or inhibition of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase pathways were evaluated. Membrane localization of NIS was evaluated by biotinylation-immunoblotting and confocal microscopy. 131 Imediated cancer cell killing was evaluated by clonogenic assays. Results: NIS function was acutely reduced by short stimulation with the PKC activator phorbol 12-myristate 13-acetate and increased by its inhibition with staurosporine or prolonged phorbol 12-myristate 13-acetate exposure. Surprisingly, epidermal growth factor (EGF) caused a strong dose-dependent augmentation of radioiodide transport, accompanied by extracellular signal-regulated kinase (ERK)-1/2 activation. Both effects were completely abrogated by specific MAP kinase kinase (MEK) inhibitors, which also reduced basal NIS function. Hence, radioiodide uptake levels could differ 24-fold, depending on ERK activity. Biotinylationimmunoblotting and confocal microscopy revealed that EGF increases plasma membrane-localized NIS without affecting total cellular levels. EGF stimulation was sufficient to enhance the killing effect of 131 I on the cancer cells. Conclusion: Thus, PKC and ERK signaling play important roles in the regulation of NIS function, and control of these signaling pathways may help enhance the efficacy of radioiodide cancer therapy.

There has been recent interest in the relationship between 18 F-FDG uptake and the angiogenic activity of endothelial cells (ECs). The angiogenic process is strongly dependent on the interaction of ECs with matrix fibronectin (FN), a key regulator of EC survival, migration, and proliferation. Therefore, we investigated how FN influences EC glucose uptake and elucidated the signaling pathways that mediate this effect. Methods: Human umbilical vein ECs were allowed to adhere to FN-coated plates and were compared with control cells for 18 F-FDG uptake, membrane GLUT1 levels, and hexokinase activity. The roles of focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K), and Akt were evaluated with Western blotting, small interfering RNA (siRNA), and specific inhibitors. Results: FN adhesion significantly enhanced the protein-corrected 18 F-FDG uptake in HUVEC, to 2.1-, 2.7-, and 4.3-fold that in control cells by 2, 3, and 5 d, respectively. This effect was mediated by the upregulation of both membrane GLUT1 expression and hexokinase activity and was accompanied by FAK activation. Silencing of FAK signaling by siRNA completely abrogated both FN-induced FAK phosphorylation and 18 F-FDG uptake. FN also activated PI3K and Akt, well-known angiogenesis mediators, and the inhibition of either pathway totally abolished the effect of FN on 18 F-FDG uptake. Nitric oxide, a downstream Akt effector that stimulates glucose uptake, was not involved in the metabolic effect of FN. Conclusion: The results of this study demonstrated that an EC-FN interaction induces strong enhancement of 18 F-FDG uptake through the upregulation of GLUT1 expression and hexokinase activity. The findings also showed that the response occurs through FAK-mediated activation of PI3K and Akt, indicating a role for this pathway in modulating EC glucose metabolism.

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