Abstract Most proliferating and tumor cells generate the energy required to support rapid cell division by aerobic glycolysis instead of oxidative phosphorylation (Warburg effect). By extension, in order to meet the increased requirements of proliferation, they often display essential changes in energy metabolism and nutrient uptake pathways. Several oncogenes, including Myc HIF-1α, and AKT, drive this switch directly, by targeting Hexokinase II, Lactate Dehydrogenase A, Pyruvate Kinase M2, and Pyruvate Dehydrogenase Kinase 1. The AMP-activated protein kinase (AMPK) is a highly conserved protein kinase complex that plays a central role in the regulation of cellular energy homeostasis. The fact that AMPK is an evolutionarily conserved energy sensor and plays a role both downstream and upstream of defined tumor suppressors, suggests that it may function as a tumor suppressor; however, the exact contribution of AMPK to tumor progression is unclear. We have generated a Pten−/- mouse model in which the PI3K pathway is selectively activated in the thyroid epithelial cells, leading to hyperplasia which progresses to follicular carcinoma. The early hyperplastic lesions exhibit a dramatic down-regulation of the expression levels of many tricarboxylic acid cycle proteins due to reduced mRNA expression, which results in enhanced compensatory glycolysis (over 3-fold increase in lactate production in mutants vs. wt). In addition, the expression of most mitochondrially-encoded genes is heavily down-regulated, and none of the genes and proteins classically associated with the Warburg effect is found deregulated in the mutant thyroid cells. In this context, while AMPK is found to be activated in wt thyroids through phosphorylation on Thr172, AMPK is inactivated in mutant thyroids through PI3K-dependent phosphorylation on Ser485. Pten−/− mice treated with 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR, IP 500 mg/kg/day for 4 weeks) display a restoration of the metabolic and mitochondrially-encoded gene expression, as well as a significant decrease in the lactate production. Pharmacological PI3K inhibition in human tumor-derived thyroid cancer cell lines increases the expression levels of both TCA cycle and OXPHOS genes. Moreover, it also causes AMPK reactivation through Thr172 phosphorylation with a concomitant reduction in pAMPK Ser485. Based on these compelling data, we propose a novel mechanism leading to a Warburg-like effect in preneoplastic lesions: PI3K activation initiates through AMPK inactivation a coordinated rearrangement of the expression of metabolic genes, which favors aerobic glycolysis at the expense of TCA/OXPHOS. Further elucidation of this process will lead to innovative targeted approaches to selectively disrupt tumor progression, while preserving normal metabolism in non mutated cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5141. doi:1538-7445.AM2012-5141