Heparan Sulfate--a Novel Target for Cancer Therapeutics

硫酸乙酰肝素——癌症治疗的新靶点

• 批准号: 6334303
• 负责人: Jeffrey D Esko
• 金额: $ 43.64万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6334303
• 关键词: CHO cells; Drosophilidae; RNA; antineoplastics; antisense nucleic acid; carbohydrate biosynthesis; cooperative study; gene mutation; genetically modified animals; heparan sulfate; high throughput technology; isomerase; laboratory mouse; lung neoplasms; metastasis; neoplasm /cancer chemotherapy; neoplastic growth; nonhuman therapy evaluation; ovary neoplasms; prostate neoplasms; site directed mutagenesis; tumor suppressor genes

Tumor growth depends on a variety of secreted growth and angiogenic factors. Cellular responses to many of these factors rely on the presence of cell surface heparan sulfate proteoglycans, which serve as co-receptors for several signaling pathways. Mounting evidence suggests that inhibition of heparan sulfate biosynthesis could bock the ability of tumor cells to respond growth and angiogenic stimuli. This hypothesis will be tested by altering heparan sulfate synthesis in tumor cells and model organisms. The novel molecular target in this proposal is the biosynthetic pathway for heparan sulfate. Blocking or reducing the expression of specific genes in the pathway by genetic strategies is needed to validate specific enzymatic targets for eventual pharmacologic intervention. Screening technologies are in place to identify compounds derived from natural products and synthetic libraries. To achieve the long-term goal of developing novel chemotherapeutic agents, we have the following specific aims: 1. Validate the heparan sulfate biosynthetic pathway as a target for cancer drug therapy. Previous studies of Chinese hamster ovary cells suggest that ablation of heparan sulfate formation prevents tumor formation in athymic mice. To extend these studies to more common tumor lines, heparan sulfate biosynthesis will be altered genetically using classic mutagenesis, anti-sense methods, new RNA interference procedures, and chimeraplasty (RNA/DNA hybrids). These studies will focus on EXT1 and NDST1 since these enzymes are responsible for the polymerization of the polysaccharide chain and initiation of all downstream modification reactions. 2. Identify new targets in the heparan sulfate biosynthetic pathway. Although many of the genes that encode the biosynthetic enzymes for heparan sulfate assembly have been identified, several critical components have not. To characterize the role of these other components and to study their role in tumor formation, new mutants of CHO cells will be isolated in the C5 epimerase and EXT2, a subunit of the co- polymerase complex. RNAi methods recently developed for Drosophila tissue culture cells will be employed to determine the function of D-Ext2, D-epimerase, and developed for Drosophila tissue culture cells will be employed to determine the function of D-Ext2, D-epimerase, and D-Ext1 in heparan sulfate biosynthesis. 3. Identify and characterize inhibitors of heparan sulfate biosynthesis. A high throughput screening method was been developed to identify compounds that inhibit heparan sulfate biosynthesis in cultured cells. To date, over 60,000 samples have been screened from the Natural Products Branch of the Developmental Therapeutics Program of the NCI and active extracts have been identified. Parallel screening of synthetic libraries is planned. Assays for screening drug candidates using sensitized genetic backgroups in Drosophila will be established and inhibition of PTEN-mediated overgrowth will be measured. The molecular target of active compounds will be examined by characterizing intermediates that accumulate in cells or tissues after drug treatment. Identification of active components coupled with large scale synthesis would allow us to test if the compounds have anti-tumor activity in mice.

肿瘤的生长取决于多种分泌的生长因子和血管生成因子。细胞对许多这些因素的反应依赖于细胞表面硫酸乙酰肝素蛋白聚糖的存在，其作为多种信号传导途径的共同受体。越来越多的证据表明，抑制硫酸乙酰肝素生物合成可能会阻碍肿瘤细胞响应生长和血管生成刺激的能力。该假设将通过改变肿瘤细胞和模型生物体中硫酸乙酰肝素的合成来检验。该提案中的新分子靶点是硫酸乙酰肝素的生物合成途径。需要通过遗传策略阻断或减少该途径中特定基因的表达，以验证最终药理干预的特定酶靶标。筛选技术已到位，可以识别源自天然产物和合成库的化合物。为了实现开发新型化疗药物的长期目标，我们有以下具体目标： 1. 验证硫酸乙酰肝素生物合成途径作为癌症药物治疗的靶点。先前对中国仓鼠卵巢细胞的研究表明，消除硫酸乙酰肝素的形成可以防止无胸腺小鼠的肿瘤形成。为了将这些研究扩展到更常见的肿瘤系，硫酸乙酰肝素的生物合成将使用经典的诱变、反义方法、新的 RNA 干扰程序和嵌合成形术（RNA/DNA 杂合体）进行基因改变。这些研究将集中于 EXT1 和 NDST1，因为这些酶负责多糖链的聚合和所有下游修饰反应的引发。 2. 确定硫酸乙酰肝素生物合成途径中的新靶点。尽管编码用于硫酸乙酰肝素组装的生物合成酶的许多基因已被鉴定，但一些关键成分尚未鉴定。为了表征这些其他成分的作用并研究它们在肿瘤形成中的作用，将在 C5 差向异构酶和 EXT2（共聚酶复合物的一个亚基）中分离出 CHO 细胞的新突变体。最近为果蝇组织培养细胞开发的 RNAi 方法将用于确定 D-Ext2、D-差向异构酶的功能，为果蝇组织培养细胞开发的 RNAi 方法将用于确定 D-Ext2、D-差向异构酶和 D 的功能-Ext1 硫酸乙酰肝素生物合成。 3. 鉴定并表征硫酸乙酰肝素生物合成的抑制剂。开发了一种高通量筛选方法来鉴定抑制培养细胞中硫酸乙酰肝素生物合成的化合物。迄今为止，已从 NCI 发展治疗计划天然产物部门筛选了 60,000 多个样品，并鉴定出了活性提取物。计划对合成文库进行平行筛选。将建立使用果蝇致敏遗传背景群筛选候选药物的测定法，并测量对 PTEN 介导的过度生长的抑制。通过表征药物治疗后在细胞或组织中积累的中间体来检查活性化合物的分子靶标。活性成分的鉴定与大规模合成相结合将使我们能够测试这些化合物是否在小鼠中具有抗肿瘤活性。