Obesity, diabetes and insulin resistance are associated with increased risk and worse outcomes for endometrial cancer (EC). Metformin is a biguanide that is widely used in the treatment of type 2 diabetes. Epidemiological and preclinical data suggest that metformin may have anti-tumorigenic activity, due to its indirect effects within the metabolic milieu (↓insulin, ↓glucose) and direct effects on tumor cells through AMPK activation/mTOR inhibition and suppression of fatty acid/lipid biosynthesis. Metformin is dependent on cation-selective transporters for entry into cells, and the multi-drug and toxin extrusion transporters, MATE1 and 2, are expressed in human EC cell lines and tumors. Thus, metformin may break the link between obesity and EC, emerging as a metabolically targeted agent for this disease. Within The Cancer Genome Atlas database, endometrioid ECs arising in obese versus non-obese women have distinguishing patterns of gene expression, including upregulation of lipoprotein lipase and modulators of the insulin/insulin growth factor-1 (IGF-1) pathway. These findings suggest that ECs arising in obesity may have distinct metabolic vulnerabilities that could be targeted for treatment. In a phase 0 clinical trial of obese EC patients, short-term metformin treatment reduced proliferation and decreased expression of the IGF-1 receptor and targets of the mTOR pathway within the endometrial tumor tissues. Responders to metformin had higher pre-treatment levels of fatty acids/glycolipids in their serum and MATE2 in their ECs, suggesting that these biomarkers might predict metformin response. Lastly, in the LKB1fl/flp53fl/fl EC mouse model, diet-induced obesity led to a doubling of tumor size, accompanied by increases in energy metabolism and lipid biosynthesis. Importantly, metformin had increased efficacy against EC in obese versus lean mice and reversed the detrimental metabolic effects of obesity in the ECs, via shunting fatty acids to beta-oxidation as opposed to lipid production. The overall goal of this proposal is to assess the contribution of indirect effects (via downregulation of insulin/IGF-1 signaling) and direct effects (via transporter-dependent cell entry, activation of AMPK/inhibition of mTOR signaling, blunting of fatty acid/lipid biosynthesis) of metformin (+/- chemotherapy) to its overall anti-cancer efficacy in (i) a clinically relevant EC mouse (obese/lean) model and (ii) an ongoing randomized phase 2/3 clinical trial evaluating metformin versus placebo, in combination with standard of care paclitaxel/carboplatin for the treatment of EC [through the NRG Oncology Group]. Our central hypothesis is that predictors of metformin response (+/- chemotherapy) will include both molecular and metabolic biomarkers, specifically obesity, insulin resistance, upregulation of insulin/IGF1 signaling, heightened fatty acid/lipid biosynthesis and higher MATE 1/2 expression. The proposed research will rigorously test this hypothesis in parallel pre-clinical and clinical studies and support it with diverse measurements of metabolic and molecular markers associated with obesity and modulated by metformin treatment. This strategy should delineate the interplay of metformin’s indirect and direct effects on tumor growth, identify metabolic and molecular biomarkers predictive of response to metformin, and define the role of this agent in obesity-driven EC treatment.