Ceramide signaling in the regulation of cellular response to folate stress

 描述（由申请方提供）：本提案的主要目的是了解鞘脂途径在介导抗叶酸剂细胞毒性作用中的作用，并研究抗叶酸剂和基于神经酰胺的药物联合治疗小鼠实验性肿瘤的治疗潜力。作为潜在的机制，我们将探讨叶酸和鞘脂代谢之间的功能联系，重点是神经酰胺合成酶6（CerS 6）的调节，以建立神经酰胺途径在细胞对膳食叶酸的反应中的作用。虽然膳食叶酸缺乏或抗叶酸剂治疗的直接代谢后果已被详细研究，但缺乏有关下游机制的知识。我们的初步研究已经证明，叶酸撤出或甲氨蝶呤治疗作为促死亡刺激后，激活癌细胞中神经酰胺的产生。具体来说，我们已经确定CerS 6和C16-神经酰胺作为叶酸状态的递质，通过神经酰胺信号传导进入细胞反应。此外，我们已经证明，p53肿瘤抑制因子是叶酸胁迫后CerS 6的上游调节因子。我们推测，通过CerS 6诱导激活神经酰胺代谢是细胞适应膳食叶酸状态的机制，p53是CerS 6在该途径中的主要调节因子。我们进一步假设CerS 6本身通过反馈回路放大p53反应。提出了以下机制：CerS 6产生的C16-神经酰胺直接与p53结合，并将其从与MDM 2的复合物中释放出来;这导致p53积累，这是由于对MDM 2依赖性泛素化的保护。测试我们假设的具体目标是：目标1。确定叶酸应激反应中CerS 6调节和神经酰胺生成的机制。目标二。区分导致神经酰胺生成的叶酸代谢途径。目标3。在动物模型中评估神经酰胺途径对膳食叶酸/抗叶酸剂应激的反应。这项提议将把两个基本的蜂窝网络 本研究旨在探讨神经通路、鞘脂和叶酸在癌症疾病中的作用，并将评估作为癌症治疗中膳食叶酸调控和抗叶酸治疗靶点的潜在机制。了解这些机制也将有助于在未来评估未能响应叶酸状态的神经酰胺途径作为叶酸代谢的开关，从肿瘤发生保护肿瘤发生启动模式的调整。考虑到同时应用抗叶酸剂和神经酰胺类药物的联合治疗，该项目将测试它们在未来转化研究中的治疗潜力。

 DESCRIPTION (provided by applicant): 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.