Role of Sphingosine Kinase in p53 Cancer Biology

这个项目的长期目标是确定鞘氨醇介导的新的脂类通路的作用。 激酶(SK)及其活性产物鞘氨醇-1-磷酸(S1P)在癌症中的作用，并建立 作为潜在的癌症治疗新靶点的这个途径的组成部分。来自先前资助的研究 这一时期将我们带入了一个新的令人兴奋的方向，即SK/S1P在癌症中的作用和调控。S1P是 由鞘磷脂神经酰胺分解为鞘氨醇产生，然后被SK磷酸化， 然后，它调节/调节几个重要的生物活动，包括内源发育、细胞存活和 血管生成。在前一个资助期，我们发现SK1在 几种癌症，并在缺氧的情况下上调。此外，我们发现了一种新的调节机制 通过蛋白降解以依赖于P53的方式响应遗传毒性应激，并表明SK1的丢失 SK1导致癌细胞生长停滞和凋亡。这些数据使我们提出了以下建议 假设：P53的激活导致SK1的丢失，这种SK1蛋白分解介导了P53肿瘤 抑制子功能，以及SK1/S1P通路介导空/突变型P53诱导的癌症。为了测试这一点 假设我们提出以下具体目标：1)建立p53调节SK1/S1P通路 并通过以下方式破译所涉及的机制：A)证明SK1被蛋白分解以响应P53 激活剂、紫外线辐射和DMA破坏细胞内的化疗药物。B)确定P53是否 细胞内SK1蛋白降解所必需的。C)检测SK1在p53缺失和突变组织中的表达 并与匹配的P53缺失/突变动物模型的正常组织和人类肿瘤组织进行比较。d) 确定P53激活导致SK1蛋白降解的机制，以及切割位点。 E)评估诱导P53和SK1蛋白分解对细胞鞘脂谱的影响。 2)为了确定SK1/S1P通路的持续性是否对于介导P53缺失/突变的肿瘤是必要的 通过A)确定SK1/S1P通路在调节细胞和体内的进展中的作用 P53诱导细胞生长反应。B)通过合成开发更有效和更具体的抑制剂 SK1.C)评估这些化合物诱导细胞凋亡/衰老样生长的能力 抑制细胞中P53的零/突变生长。D)确定SK1/S1P在空/突变型p53介导中的作用 用SK1 K/O小鼠与P53缺失或突变小鼠杂交进行体内肿瘤检测 防止肿瘤的形成。这些研究可能提供P53和P53之间的一种新的分子联系 神经鞘磷脂途径调节细胞应激，这两个途径都在调节生长，细胞凋亡， 和衰老。这项研究的先导化合物可能为特效治疗铺平道路 开发目标为SK1

The long-term goal of this project is to define the role of the novel lipid pathway mediated by sphingosine kinase (SK) and its bioactive product sphingosine-1-phosphate (S1P) in cancer, and to establish components of this pathway as potential novel targets for cancer therapy. Studies from the previous funding period have led us into a novel exciting direction on the role and regulation of SK/S1P in cancer. S1P is generated by breakdown of the sphingolipid ceramide to sphingosine, followed by phosphorylation by SK, and it then mediates/modulates several important biologic activities, including mrtogenesis, cell survival, and angiogenesis. During the previous funding period, we established that SK1 is significantly overexpressed in several cancers and is upregulated by hypoxia. Moreover, we discovered a novel mechanism of regulation of SK1 by proteolysis in response to genotoxic stress in a p53-dependent manner, and showed that loss of SK1 led to growth arrest and apoptosis of cancer cells. These data have led us to propose the following hypothesis: that activation of p53 causes the loss of SK1, that this SK1 proteolysis mediates p53 tumor suppressor function, and that the SK1/S1P pathway mediates null/mutant p53-induced cancer. To test this hypothesis we propose the following specific aims: 1} To establish that p53 regulates the SK1/S1P pathway and decipher the mechanisms involved by A) Demonstrating that SK1 is proteolyzed in response to the p53 activators, UV radiation and DMA damaging chemotherapeutic agents in cells. B) Determining if p53 is required for SK1 proteolysis in cells. C) Determining the expression of SK1 in p53 null and mutant tissues and compare to matched normal tissues from p53 null/mutant animal models and human cancer tissues. D) Determining the mechanism by which activation of p53 leads to SK1 proteolysis, and the sites of cleavage. E) Evaluating the consequences of induction of p53 and SK1 proteolysis on the cellular sphingolipid profile. 2) To determine if the persistence of the SK1/S1P pathway is necessary to mediate null/mutant p53 tumor progression pathways in cells and in vivo by A) determining the role of the SK1/S1P pathway in mediating p53 induced growth responses in cells. B) To develop through synthesis more potent and specific inhibitors of SK1. C) To evaluate these compounds as to their ability to induce apoptosis/senescence-like growth arrest in null/mutant p53 growth in cells. D) To determine the role of SK1/S1P in null/mutant p53 mediated cancer in vivo using SK1 K/O mice crossed with p53 null or mutant mice and determining if they are protected from tumor formation. These studies may provide a novel molecular link between the p53 and the sphingolipid pathways of cell stress regulation, both of which play key roles in regulating growth, apoptosis, and senescence. Lead compounds from this study may pave the way towards specific therapeutic development targeting SK1