Neutral Ceramidase in Colon Cancer

描述(申请人提供)：这个项目的长期目标是了解中性神经酰胺酶(NCDase)在结肠癌发病机制、化学预防和治疗中的作用。NCDase是调节重要生物活性分子相互转化的关键酶，但研究较少，这些生物活性分子包括具有整体抗肿瘤特性的神经酰胺和具有促癌特性的1-磷酸鞘氨醇。本实验室首次对哺乳动物nCDase进行了鉴定、纯化和克隆，并对其生化性质进行了研究。在最近的研究中，我们发现nCDase在肠上皮细胞中丰富，并且正在进行的初步结果显示，在AOM(偶氮甲烷)诱导的小鼠结肠腺癌模型中，nCDase的缺失对结肠癌的发生有显著的影响。最近的更多研究开始将这种酶与ss-catenin途径联系起来。在此基础上，我们提出了nCDase是转化的结肠细胞，特别是那些由ss-catenin途径异常激活所定义的细胞的“合成致死”靶点的假设。我们将利用化学、生化、分子和体内研究来评估这一假说，并将nCDase作为一个新的靶点。我们将集中于以下特定目标：1)通过a)确定nCDase在结肠癌发生发展中的体内作用；b)确定nCDase在人类结肠癌中的表达；以及c)确定nCDase在体内和细胞中调节结肠癌细胞生长和应激反应的作用，从而确定NCDase在调节结肠癌进展中的作用。2)确定通过干扰nCDase来阻止结肠癌进展的机制。我们将集中于a)确定nCDase在体内和细胞培养模型中调节ss-catenin途径的作用；b)确定nCDase作用的候选生物活性脂质介质和下游靶标的作用；以及c)确定nCDase调节ss-catenin途径的机制。3)开发NCDase作为肿瘤治疗的新靶点。在这里，我们将a)开发基于初始HITS的nCDase小分子抑制剂；b)评估这些抑制剂抑制细胞内nCDase的能力和功能后果；c)开展PK/PD研究；以及d)确定这些抑制剂在体内的作用。这些结果可能允许我们定义和建立nCDase作为一种新的、迄今未被认识的结肠癌进展的主要调节因子。NCDase可能成为合理化疗的新的有效靶点。

DESCRIPTION (provided by applicant): The long-term aims of this project are to understand the role of neutral ceramidase (nCDase) in colon cancer pathogenesis, chemoprevention, and therapeutics. nCDase is a key, yet poorly studied, enzyme in regulating the interconversion of important bioactive molecules that include ceramides with an overall anti- tumor properties and sphingosine 1-phosphate with tumor-promoting properties. Our laboratory first identified, purified, and cloned the mammalian nCDase and studied its biochemical properties. In recent studies, we find that nCDase is enriched in the intestinal epithelium, and ongoing preliminary results show dramatic effects of deletion of nCDase on development of colon cancer in a mouse model of AOM (azoxymethane)-induced colonic adenocarcinoma. Additional recent studies are beginning to implicate this enzyme in the ss-catenin pathway. Based on the above, we propose the hypothesis that that nCDase is a 'synthetically lethal' target for transformed colon cells, especially those defined by abnormal activation of the ss-catenin pathway. We will employ chemical, biochemical, molecular, and in vivo studies to evaluate this hypothesis and to advance nCDase as a novel target. We will focus on the following specific aims: 1) Establish and define the role of NCDase in regulating colon cancer progression, by a) defining the in vivo role of nCDase in the development of colon cancer using the knock out mouse; b) determine the expression of nCDase in human colon cancer; and c) define the in vivo and cellular roles of nCDase in regulating cell growth and stress responses in colon cancer. 2) Determine the mechanisms underlying the prevention of colon cancer progression by disruption of nCDase. We will focus on a) determining the role of nCDase in regulating the ss-catenin pathway of colon cancer development in vivo and in cell culture models; b) defining the candidate bioactive lipid mediators of action of nCDase and roles of downstream targets; and c) defining mechanisms by which nCDase regulates the ss-catenin pathway. 3) Develop NCDase as a novel target for cancer therapy. Here we will a) develop small molecule inhibitors of nCDase based on initial hits; b) evaluate these inhibitors for their ability to inhibit nCDase in cells and the functional consequences; c) develop PK/PD studies; and d) Define the effects of these inhibitors in vivo. These results may allow us to define and establish nCDase as a novel and heretofore unappreciated major regulator of colon cancer progression. nCDase may emerge as a novel and effective target for rationally-based chemotherapy.