The main objective of this proposal is to understand the role of sphingolipid pathways in mediating cytotoxic effects of antifolates and to investigate the therapeutic potential of the treatment of experimental tumors in mice with combinations of antifolate and ceramide-based drugs. As the underlying mechanism, we will explore the functional connection between folate and sphingolipid metabolism, with the focus on the regulation of ceramide synthase 6 (CerS6), to establish the role of ceramide pathways in cellular response to dietary folate. While the immediate metabolic consequences of dietary folate deficiency or antifolate treatments have been studied in some detail, there is a lack of knowledge regarding the engaged downstream mechanisms. Our pilot study has demonstrated the activation of ceramide generation in cancer cells upon folate withdrawal or methotrexate treatment as a pro-death stimulus. Specifically, we have identified CerS6 and C16-ceramide as transmitters of folate status into cellular responses through ceramide signaling. Furthermore, we have demonstrated that p53 tumor suppressor is an upstream regulator of CerS6 upon folate stress. We hypothesize that the activation of ceramide metabolism, through CerS6 induction, is a mechanism of cellular adaptation to dietary folate status with p53 being the main regulator of CerS6 in this pathway. We further hypothesize that CerS6 itself, through a feedback loop, amplifies p53 response. The following mechanism is proposed: CerS6-generated C16-ceramide directly binds to p53 and releases it from the complex with MDM2; this results in the p53 accumulation due to protection from MDM2-dependent ubiquitinylation. The specific aims to test our hypotheses are: Aim 1. Determine the mechanisms of CerS6 regulation and ceramide generation in folate stress response. Aim 2. Differentiate metabolic pathways of folate leading to ceramide generation. Aim 3. Evaluate the activation of ceramide pathways in response to dietary folate/antifolate stress in animal models. This proposal will link two fundamental cellular pathways, sphingolipid and folate, with regard to their role in cancer diseases and will evaluate underlying mechanisms as targets for the dietary folate manipulation and antifolate treatment in cancer therapy. Understanding these mechanisms will also help in the future to evaluate the failure to respond to folate status by the adjustment of ceramide pathways as a switch of folate metabolism from tumorigenesis protective to tumorigenesis-initiating mode. Keeping in mind the combinational therapy with simultaneous application of antifolate and ceramide- based drugs, this project will test their therapeutic potential for the future translational research.