The Role of Ceramidases in Cancer Chemotherapy

描述（由申请人提供）：我们的长期目标是通过关注人碱性神经酰胺酶2（ACER 2）及其脂质底物（神经酰胺）和产物（鞘氨醇），确定抗癌化疗药物（ACCA）诱导肿瘤细胞抗增殖反应（增殖抑制和程序性细胞死亡）的机制。许多ACCA诱导肿瘤细胞中生物活性鞘脂神经酰胺的产生，并且阻断神经酰胺的产生抑制药物的抗增殖作用，这表明神经酰胺是ACCA在这种抗增殖反应中的重要介质。神经酰胺可以通过人细胞中的神经酰胺酶水解成鞘氨醇，并且与神经酰胺一样，SPH可以在体外和动物模型中有效地诱导肿瘤细胞的抗增殖作用。中性神经酰胺酶ASAH 2在质膜上的表达增加也减弱了由TNF-1（一种已知的神经酰胺产生细胞因子）诱导的细胞凋亡。相反，我们的初步研究表明，增加神经酰胺转化为SPH的高尔基碱性神经酰胺酶2（ACER 2）显着增强阿霉素，一个已知的神经酰胺诱导ACCA，在HeLa细胞的细胞毒性，而敲低ACER 2有相反的效果。这一令人兴奋的发现使我们假设ACCA通过神经酰胺/ACER 2/SPH途径在癌细胞中发挥抗增殖反应，并且该途径的激活将增强癌症治疗中的化疗作用。为了检验这一假设，我们将通过确定1）AACA是否通过激活神经酰胺/ACER 2/SPH途径诱导癌细胞中的抗增殖反应; 2）如果ACER 2诱导剂增强而ACER 2抑制剂减弱ACCA诱导的癌细胞抗增殖作用（目的1）。在目的2中，我们将通过研究1）将ACER 2靶向内质网（ER）是否消除其在肿瘤细胞中介导ACCA诱导的抗增殖作用的能力来检验其高尔基体定位是ACER 2介导ACCA诱导的抗增殖反应所必需的假设;和2）如果将ASAH 2靶向高尔基复合体赋予在肿瘤细胞中介导ACCA诱导的抗增殖作用的能力。对于目标3，我们将检验以下假设：ACER 2/SPH通路的激活通过高尔基体片段化增强肿瘤细胞中ACCA的抗增殖应答。我们将确定1）ACCA是否通过激活神经酰胺/ACER 2/SPH途径诱导高尔基体断裂; 2）SPH是否在体外诱导高尔基体断裂并抑制高尔基体重组; 3）激活神经酰胺/ACER 2/SPH是否通过抑制GRASP 65的堆积功能抑制高尔基体组装（65 kD的高尔基体重组堆积蛋白）和GRASP 55;和4）如果神经酰胺/ACER 2/SPH途径的激活通过高尔基体片段化诱导有丝分裂停滞和凋亡。这些研究将为深入了解ACER 2及其产物SPH在介导癌细胞抗增殖反应中的作用和作用机制提供帮助。

DESCRIPTION (provided by applicant): Our long-term goal is to define the mechanisms by which anti-cancer chemotherapeutic agents (ACCAs) induce anti-proliferative responses (proliferation inhibition and programmed cell death) in tumor cells by focusing on human alkaline ceramidase 2 (ACER2) and its lipid substrate (ceramide) and product (sphingosine). Many ACCAs induce the generation of the bioactive sphingolipid ceramide in tumor cells, and blocking ceramide generation inhibits anti-proliferative effects of the drugs, suggesting that ceramide is an important mediator of ACCAs in this anti-proliferative response. Ceramide can be hydrolyzed into sphingosine by ceramidases in human cells and, like ceramide, SPH can potently induce anti-proliferative effects in tumor cells both in vitro and in animal models. Increasing expression of neutral ceramidase ASAH2 on the plasma membrane also attenuates cell apoptosis induced by TNF-1, a known ceramide-generating cytokine. In contrast, our preliminary studies show that increasing the conversion of ceramide to SPH by the Golgi alkaline ceramidase 2 (ACER2) markedly enhances the cytotoxicity of doxorubicin, a known ceramide-inducing ACCA, in HeLa cells, whereas knocking down ACER2 has the opposite effect. This exciting finding led us to the hypotheses that ACCAs exert anti-proliferative responses in cancer cells through the ceramide/ACER2/SPH pathway and that activation of this pathway will augment chemotherapy action in cancer treatment. To test this hypothesis, we will establish that the ceramide/ACER2/SPH pathway is an important mediator of the anti- proliferative responses of ACCAs in cancer cells by determining 1) if ACCAs induce the anti-proliferative responses in cancer cells by activating the ceramide/ACER2/SPH pathway; 2) if ACER2-inducing agents enhance whereas ACER2-inhibiting agents attenuate the ACCA-induced anti-proliferative effects in cancer cells (Aim 1). In Aim 2, we will test the hypothesis that its Golgi localization is required for ACER2 to mediate the ACCA-induced anti-proliferative responses in cancer cells by investigating 1) if targeting ACER2 to the endoplasmic reticulum (ER) abolishes its ability to mediate the ACCA-induced anti-proliferative effects in tumor cells; and 2) if targeting ASAH2 to the Golgi complex confers the ability to mediate ACCA-induced anti- proliferative effects in tumor cells. For Aim 3, we will test the hypothesis that the activation of the ACER2/SPH pathway enhances anti-proliferative responses of ACCAs in tumor cells through Golgi fragmentation. We will determine 1) if ACCAs induces Golgi fragmentation by activating the ceramide/ACER2/SPH pathway; 2) if SPH induces Golgi fragmentation and inhibits Golgi reassembly in vitro; 3) if activating the ceramide/ACER2/SPH inhibits Golgi assembly by inhibiting the stacking function of GRASP65 (Golgi reassembly stacking protein of 65 kD) and GRASP55; and 4) if activation of the ceramide/ACER2/SPH pathway induces mitotic arrest and apoptosis through Golgi fragmentation. These studies will provide insights into the role and mechanism of action of ACER2 and its product SPH in mediating anti-proliferative responses in cancer cells.