The role of sphingosine kinase 1 (SphK1) in primary glioblastomas

描述(申请人提供)：多形性胶质母细胞瘤(GBM)是最常见的恶性胶质瘤，具有极具侵袭性的表型。胶质母细胞瘤细胞向健康组织扩散的能力是目前治疗的主要障碍，包括手术和放射治疗。多年来，已建立的人脑胶质母细胞瘤细胞系被用作研究侵袭的分子机制的模型；然而，由于已建立的细胞系在体内不具侵袭性，这些机制仍然难以捉摸。与已建立的细胞系相比，原发的未建立的胶质母细胞瘤，包括GBM6和GBM12细胞，在体内是侵袭性的，因此可以成功地用于研究生长、运动和侵袭。最近，神经鞘氨醇激酶1(SphK1)的高表达与GBM患者的不良预后有关。S1P是一种对肿瘤生长、存活、迁移和侵袭具有重要作用的强效脂质介质。在大脑中，S1P以高浓度存在，并已被证明在体外刺激已建立的胶质母细胞瘤系的迁移和侵袭。SphK1的活性可以被各种生长因子和细胞因子快速而短暂地刺激，然而，SphK1基因的转录调控机制尚不清楚，促进GBM患者SphK1表达的因素也尚未确定。最近，炎症与许多癌症的发生和发展有关，炎症细胞因子参与了这些过程。IL-1是脑内炎症的主要调节因子之一，在体外可以诱导其他促炎细胞因子的分泌，并促进胶质母细胞瘤细胞的增殖。更重要的是，胶质母细胞瘤细胞分泌大量的IL-1。在初步研究中，我们发现IL-1和SphK1在基底膜中共表达，外源性IL-1增加SphK1的表达，IL-1和S1P都促进胶质母细胞瘤细胞的侵袭和增殖。更重要的是，中和抗IL-1抗体在体外抑制了胶质母细胞瘤细胞的增殖，而一种新的SphK1抑制剂(SKI)则减少了小鼠胶质母细胞瘤移植瘤的生长。我们的假设是，IL-1介导的SphK1表达刺激导致S1P的产生，S1P进而激活基底膜的增殖、运动和侵袭。我们建议研究SphK1在原代GBM细胞中的功能及其调控。此外，在动物模型中有效和特异的SphK1抑制剂的开发可能为治疗GBM的新疗法铺平道路。本研究的具体目的如下：1.研究SphK1的表达及其抑制对原代未建立的小鼠肾小管上皮细胞和裸鼠脑内移植瘤的增殖、运动和侵袭的影响。目的2.探讨IL-1对内源性SphK1表达的调控机制。公共卫生相关性：多形性胶质母细胞瘤(GBM)具有极具侵袭性的表型，这是目前治疗的主要障碍。我们的假设是，IL-1介导的SphK1(鞘氨醇激酶1)表达的刺激在GBM的生长和侵袭性中起着重要作用，并可能成为潜在的治疗靶点。因此，我们建议研究SphK1在原代未建立的GBM细胞中的功能及其调控。此外，在动物模型中有效和特异的SphK1抑制剂的开发可能为治疗GBM的新疗法铺平道路。

DESCRIPTION (provided by applicant): Glioblastoma multiforme (GBM) is the most common malignant glioma and has an extremely invasive phenotype. The ability of glioblastoma cells to diffusely infiltrate healthy tissue is the major obstacle for current treatments, including surgery and radiotherapy. Over the years, established human glioblastoma cell lines were used as models to study molecular mechanisms of invasion; however, these mechanisms remain elusive since established lines are not invasive in vivo. In contrast to established lines, primary non-established glioblastomas, including GBM6 and GBM12 cells, are invasive in vivo, and thus can be successfully used to study growth, motility, and invasion. Recently, an elevated expression of sphingosine kinase 1 (SphK1), an enzyme that generates sphingosine 1-phosphate (S1P), was correlated with poor prognosis of patients with GBM. S1P is a potent lipid mediator important for cancer growth, survival, migration, and invasion. In the brain, S1P is present at high concentrations, and has been shown to stimulate migration and invasion of established glioblastoma lines in vitro. The activity of SphK1 is rapidly and transiently stimulated by various growth factors and cytokines; however, mechanisms of the transcriptional regulation of the sphk1 gene are not understood and the factors enhancing SphK1 expression in GBM patients have not been identified. Recently, inflammation has been linked to the development and progression of many cancers with inflammatory cytokines involved in these processes. IL-1 is one of the major regulators of inflammation in the brain, which can induce the secretion of other proinflammatory cytokines and also promote proliferation of glioblastoma cells in vitro. More importantly, glioblastoma cells secrete significant amounts of IL-1. In preliminary studies, we show that IL-1 is coexpressed with SphK1 in GBMs, exogenous IL-1 increases expression of SphK1, and both IL-1 and S1P enhance invasion and proliferation of glioblastoma cells. More importantly, neutralizing anti-IL-1 antibodies inhibit proliferation of glioblastoma cells in vitro, while a novel SphK1 inhibitor (SKI) reduces xenografted glioblastoma tumor growth in mice. It is our hypothesis that the IL-1-mediated stimulation of SphK1 expression leads to the production of S1P, which in turn activates proliferation, motility, and invasion of GBMs. We propose to study SphK1 functions and its regulation in primary GBM cells. Moreover, development of potent and specific SphK1 inhibitors that are active in animal models could pave the way for new therapeutics for treatment of GBM. Our specific aims are as follows: Aim 1. To determine the role of SphK1 expression and the effect of its inhibition on proliferation, motility, and invasion of primary non-established GBMs in vitro, and xenografts implanted in the brains of athymic mice. Aim 2. To identify the mechanisms that control endogenous SphK1 expression in response to IL-1. PUBLIC HEALTH RELEVANCE: Glioblastoma multiforme (GBM) has an extremely invasive phenotype, which is the major obstacle for current treatments. It is our hypothesis that the IL-1-mediated stimulation of SphK1 (sphingosine kinase 1) expression plays an important role in GBM growth and invasiveness, and may be a potential therapeutic target. Therefore, we propose to study SphK1 functions and its regulation in primary non-established GBM cells. Moreover, development of potent and specific SphK1 inhibitors that are active in animal models could pave the way for new therapeutics for treatment of GBM.