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（nCD酶）在结肠癌发病机制、化学预防和治疗中的作用。nCD酶是调节重要生物活性分子相互转化的关键酶，但研究较少，所述生物活性分子包括具有总体抗肿瘤特性的神经酰胺和具有肿瘤促进特性的1-磷酸鞘氨醇。本实验室首先鉴定、纯化和克隆了哺乳动物nCD酶，并研究了其生化特性。在最近的研究中，我们发现nCD酶在肠上皮中富集，并且正在进行的初步结果显示，在AOM（氧化偶氮甲烷）诱导的结肠腺癌的小鼠模型中，nCD酶的缺失对结肠癌的发展具有显著影响。最近的其他研究开始暗示这种酶在β-连环蛋白途径。基于上述情况，我们提出了这样的假设，即nCD酶是转化结肠细胞的“合成致死”靶标，特别是那些由β-连环蛋白途径的异常激活定义的细胞。我们将采用化学，生物化学，分子和体内研究来评估这一假设，并推进nCD酶作为一种新的目标。我们将专注于以下具体目标：1）通过a）使用敲除小鼠确定nCD酶在结肠癌发展中的体内作用; B）确定nCD酶在人结肠癌中的表达;以及c）确定nCD酶在结肠癌中调节细胞生长和应激反应中的体内和细胞作用，建立和确定NCD酶在调节结肠癌进展中的作用。2)确定通过破坏nCD酶预防结肠癌进展的潜在机制。我们将重点关注a）确定nCD酶在体内和细胞培养模型中调节结肠癌发展的β-连环蛋白途径中的作用; B）定义nCD酶作用的候选生物活性脂质介质和下游靶点的作用;以及c）定义nCD酶调节β-连环蛋白途径的机制。3)开发NCDase作为癌症治疗的新靶点。在此，我们将a）基于初始命中开发nCD酶的小分子抑制剂; B）评价这些抑制剂抑制细胞中nCD酶的能力和功能后果; c）开发PK/PD研究; d）确定这些抑制剂的体内作用。这些结果可能使我们能够定义和建立nCD酶作为一种新的和迄今为止未被认识到的结肠癌进展的主要调节因子。nCD酶可能成为一个新的和有效的目标，合理的化疗。

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.