Ceramide signaling in the regulation of cellular response to folate stress

 描述(由申请人提供)：本提案的主要目的是了解鞘磷脂通路在抗叶酸的细胞毒性效应中的作用，并研究抗叶酸和神经酰胺类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，我们将探索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(CerS6)的调节，以确定神经酰胺途径在细胞对叶酸的反应中的作用。虽然饮食中叶酸缺乏或抗叶酸治疗的直接代谢后果已经有了一些详细的研究，但对参与其中的下游机制缺乏了解。我们的初步研究已经证明，在停用叶酸或甲氨蝶呤治疗时，癌细胞中神经酰胺的生成被激活，这是一种亲死亡的刺激。具体地说，我们已经确定CerS6和C16-神经酰胺是通过神经酰胺信号将叶酸状态传递到细胞反应中的。此外，我们还证明了P53肿瘤抑制因子是叶酸应激时CerS6的上游调节因子。我们推测神经酰胺代谢的激活，通过CerS6的诱导，是细胞对膳食叶酸状态的一种适应机制，而P53是这一途径中CerS6的主要调节因子。我们进一步假设CerS6本身通过反馈环放大P53反应。提出了以下机制：CerS6生成的C16-神经酰胺直接与P53结合，并将其从与MDM2的复合体中释放出来；这导致了P53的积聚，这是由于保护了MDM2依赖的泛素化。验证我们的假设的具体目的是：目的1.确定CerS6在叶酸应激反应中的调节和神经酰胺产生的机制。目的2.区分导致神经酰胺生成的叶酸代谢途径。目的3.在动物模型上评价神经酰胺通路在膳食叶酸/抗叶酸应激中的激活情况。这项提案将把两个基本的蜂窝 我们还将研究神经鞘脂脂和叶酸途径在癌症疾病中的作用，并将评估其作为饮食叶酸操纵和抗叶酸治疗癌症治疗的靶标的潜在机制。了解这些机制也将有助于未来通过调节神经酰胺通路来评估叶酸状态的失败，因为神经酰胺通路是叶酸代谢从肿瘤发生保护模式向肿瘤发生启动模式的切换。考虑到同时应用抗叶酸和神经酰胺类药物的联合治疗，该项目将为未来的翻译研究测试它们的治疗潜力。

 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.