Regulation of Sphingosine Kinase 1 by p53 and Caspase 2

描述(申请人提供)：癌症是一种疾病，在这种疾病中，异常细胞无法控制地分裂，并具有侵袭其他组织的能力。正常细胞以受控的方式生长和分裂，以产生更多的细胞，因为它们是保持身体健康所需的。为了确保这一过程顺利进行，有各种保护机制，以确保受损的DNA得到修复，而不是传递给下一代细胞。然而，由于许多原因，包括环境压力、遗传因素和年龄等，受损或突变的DNA无法消除或修复，并可能继续影响正常的细胞生长和分裂。因此，异常细胞的不受控制的分裂会导致癌症，这是美国第二大死亡原因。在消除受损或改变的DNA的过程中，关键分子之一是肿瘤抑制蛋白p53。功能性P53在保护人类健康和预防疾病方面发挥着重要作用。当P53状态受损时，细胞将无法管理DNA受损细胞的复制。不幸的是，所有癌症中有50%在p53内存在突变，导致活性受损(14)；因此，了解正常p53如何阻止肿瘤进展的确切机制对于确定能够有效恢复p53功能的化疗开发靶点是绝对关键的。我们实验室以前的结果表明，响应DNA损伤的P53信号可能涉及鞘磷脂。鞘磷脂是一类生物活性脂类，是细胞生长和死亡的重要调节因子(1，2)。当生物活性脂质神经酰胺和鞘氨醇作为促死亡分子时，鞘氨醇-1-磷酸(S1P)被证明可以刺激增殖和血管生成。调节这些促死亡和促生长生物活性分子之间平衡的主要酶是鞘氨醇激酶1(SK1)。这一角色强调了了解SK1如何被调控的重要性，因为它的活动可能在控制细胞命运方面发挥作用(3，4)。最近，我们实验室的研究建立了p53和SK1(11-13)之间深刻而新颖的联系。在这些研究中，已经证明了P53的诱导导致蛋白降解导致SK1的丢失，而这种P53诱导的SK1的丢失对于允许P53介导的胸腺淋巴瘤、骨肉瘤和其他癌症的体内抑制至关重要，这一点在使用P53/SK1联合敲除小鼠的研究中得到了证明。因此，生物活性鞘脂水平的调节可能是P53 DNA损伤反应的关键组成部分，这些途径之间的相互作用值得进一步研究。这个项目的目标是确定参与P53依赖的SK1降解的蛋白酶(S)。通过使用MCF7乳腺癌细胞以及Caspase2基因敲除的小鼠胚胎成纤维细胞进行的各种细胞研究，我们产生了强有力的初步数据，暗示Caspase2是负责P53依赖的SK1降解的蛋白酶。Caspase 2是Caspase家族中进化上最保守的成员，但尽管如此，Caspase 2的生理功能仍然是一个谜，也是有争议的[42]。混淆这个问题的是缺乏对Caspase 2真正底物的了解，以及到目前为止确定的少数底物还没有清楚地揭示该酶的功能的事实。有趣的是，最近的工作暗示了Caspase 2可能的肿瘤抑制作用，尽管确切的分子机制仍不清楚(25，33，44)。根据这些数据，本建议的目的是：1)确定Caspase 2是P53介导的SK1蛋白降解所必需的；2)从机制上确定Caspase 2在调节SK1/S1P途径中的作用；3)证明解除对P53/Caspase2/SK1/S1P的调控的生物学意义。这项提案中概述的研究将为P53和SK1之间令人兴奋的联系提供宝贵的机制洞察，同时也证明SK1是P53抑制肿瘤作用的关键下游靶点。此外，这项工作还旨在揭示Caspase 2在鞘磷脂调节中的新作用，并发现一个新的Caspase 2底物SK1，它可以解释这种神秘的Caspase的肿瘤抑制作用。

DESCRIPTION (provided by applicant): Cancer is a disease in which abnormal cells divide uncontrollably and possess the ability to invade other tissues. Normal cells grow and divide in a controlled way to produce more cells as they are needed to keep the body healthy. In order to ensure this process proceeds without mishap there are various protective mechanisms in place to ensure that damaged DNA is repaired and not passed on to subsequent generations of cells. However, due to a number of causes including, environmental stresses, genetic factors and age among other things, damaged or mutated DNA is not eliminated or repaired and can go on to affect normal cell growth and division. Consequently, uncontrolled division of abnormal cells leads to cancer, the second greatest cause of mortality in the United States. One of the key molecules involved in this process of eliminating damaged or changed DNA is the tumor suppressor protein p53. Functional p53 plays an important role in protecting human health and preventing disease. When p53 status is compromised, the cell is left unable to manage the replication of cells with damaged DNA. Unfortunately 50% of all cancers harbor mutations within p53, resulting in compromised activity (14); therefore understanding the exact mechanisms of how normal p53 prevents tumor progression is absolutely crucial to define targets for chemotherapeutic development that can effectively restore p53 function. Previous results in our lab suggest that p53 signaling, in response to DNA damage, may involve sphingolipids. Sphingolipids are a class of bioactive lipids that are important regulators of cell growth and death (1,2). While the bioactive lipids ceramide and sphingosine act as pro-death molecules, sphingosine-1- phosphate (S1P) has been shown to stimulate proliferation and angiogenesis. The primary enzyme regulating the balance between these pro-death and pro-growth bioactive molecules is sphingosine kinase 1 (SK1). This role highlights the importance of understanding how SK1 is regulated as its activity may play a role in controlling cell fate (3,4). Recently research from our lab established a profound and novel connection between p53 and SK1 (11-13). In these studies it was demonstrated that induction of p53 results in loss of SK1 through proteolysis and this p53-induced loss of SK1 is critical for allowing p53-mediated suppression of thymic lymphoma, osteosarcoma, and other cancers in vivo as evidenced in studies using the combined p53/SK1 knock out mice. Thus, regulation of bioactive sphingolipid levels may be a key component in the p53 DNA damage response and the interaction of these pathways warrants further investigation. The goal of this project is to define the protease(s) involved in p53-dependent SK1 degradation. Through various cell studies using MCF7 breast cancer cells as well as Caspase 2 knockout mouse embryonic fibroblasts we have generated strong preliminary data implicating Caspase 2 as the protease responsible for p53-dependent SK1 degradation. Caspase 2 is the most evolutionarily conserved member of the caspase family but despite this, the physiological function of Caspase 2 has remained enigmatic and controversial (42). Confounding this issue is the lack of knowledge of bona fide substrates of Caspase 2 and the fact that the few substrates identified thus far have not clearly revealed the enzyme's function. Interestingly, recent work has implicated a possible tumor suppressor role for Caspase 2 although the exact molecular mechanisms are still unknown (25,33,44). In light of these data this proposal aims to 1) Establish that Caspase 2 is required for p53-mediated proteolysis of SK1, 2) Define mechanistically the role of Caspase 2 in the regulation of the SK1/S1P pathway, 3) Demonstrate the biological significance of deregulation of the p53/Caspase2/SK1/S1P. The research outlined in this proposal will provide invaluable mechanistic insight into the exciting connection between p53 and SK1 while also demonstrating that SK1 is critical downstream target for the tumor suppressive action of p53. In addition this work also aims to uncover a novel role for Caspase 2 in the regulation of sphingolipids and identify a new Caspase 2 substrate, SK1 that could explain the characterized tumor suppressive roles of this enigmatic caspase.