Neutral Ceramidase in Colon Cancer

描述（由申请人提供）：本项目的长期目标是了解中性神经酰胺酶（ncase）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（包括具有整体抗肿瘤特性的神经酰胺和具有促肿瘤特性的鞘氨醇1-磷酸）相互转化的关键酶，但研究很少。本实验室首次鉴定、纯化、克隆了哺乳动物nCDase，并对其生化特性进行了研究。在最近的研究中，我们发现nCDase在肠上皮中富集，并且正在进行的初步研究结果表明，在AOM（偶氮甲烷）诱导的结肠腺癌小鼠模型中，nCDase的缺失对结肠癌的发展有显著影响。最近的其他研究开始暗示这种酶与ss-连环蛋白途径有关。基于上述，我们提出了一个假设，即nCDase是转化结肠细胞的“合成致死”靶标，特别是那些由ss-catenin通路异常激活定义的细胞。我们将采用化学、生化、分子和体内研究来评估这一假设，并推进nCDase作为一个新的靶点。我们将重点关注以下具体目标：1)建立和确定NCDase在调节结肠癌进展中的作用，a)使用敲除小鼠确定NCDase在结肠癌发展中的体内作用；b)测定nCDase在人结肠癌中的表达；c)明确ncase在调节结肠癌细胞生长和应激反应中的体内和细胞作用。2)确定通过破坏nCDase预防结肠癌进展的机制。我们将重点关注a)在体内和细胞培养模型中确定ncase在调节结肠癌发展的ss-catenin通路中的作用；b)确定nCDase的候选生物活性脂质介质及其下游靶点的作用；c)确定nCDase调节ss-catenin通路的机制。3)开发NCDase作为癌症治疗的新靶点。在这里，我们将a)开发基于初始命中的nCDase小分子抑制剂；b)评估这些抑制剂在细胞中抑制nCDase的能力和功能后果；c)开展PK/PD研究；d)确定这些抑制剂在体内的作用。这些结果可能使我们能够定义和确立nCDase作为一种新的、迄今为止未被认识到的结肠癌进展的主要调节因子。nCDase可能成为合理化疗的一个新的有效靶点。

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.