Searching for Novel Genes in Heparan Sulfate Biosynthesis

描述（由申请人提供）：硫酸肝素蛋白聚糖（HSPGs）含有一个或多个硫酸肝素链共价连接到蛋白质核心。HSPGs定位于细胞表面和细胞外基质中。由于其强烈的负电荷，硫酸肝素链结合并调节介导细胞增殖的各种蛋白质配体之间的相互作用，包括生长因子、形态因子、其他细胞外基质蛋白、酶和酶抑制剂。HSPGs与细胞生长和增殖有关，在癌细胞和肿瘤中容易错误表达。既往研究表明，硫酸肝素链的结构（硫酸基团和醛酸外显体的数目和排列）和HSPGs的表达水平影响肿瘤的生长。本提案的总体目标是发现和表征除编码已知硫酸肝素生物合成酶的基因外的新基因，其表达影响硫酸肝素介导的癌症进展调节网络。为了实现这一目标，我们计划：(1)开发一种高通量，全基因组，短发夹RNA筛选，检测参与硫酸肝素生物合成的基因；(2)鉴定和表征候选基因，以阻止肿瘤细胞的生长和生存能力。筛选试验将采用基于毒素的筛选，以前用于确定硫酸肝素的小分子抑制剂。我们计划寻找基因，当它们的表达减少或消除时，将赋予对硫酸肝素依赖性细胞毒素的抗性。一个全基因组shRNA文库将用于敲低HeLa（宫颈癌）细胞中的基因表达。敲除后，我们将检测细胞活力，并鉴定那些赋予肝素硫酸依赖毒素抗性的候选基因。分析硫酸肝素的结构和结合特性，以及核心蛋白的表达分析。此外，我们将确定表达候选shrna的肿瘤细胞在软琼脂中形成集落并诱导动物肿瘤形成的能力。本提案中概述的工作的综合结果将使我们更好地了解硫酸肝素的结构和功能在肿瘤发生中的作用。我们发现的这些因子可以揭示抗癌治疗的新靶点，并引导我们找到操纵硫酸肝素及其在其他细胞类型中的活性的方法。

DESCRIPTION (provided by applicant): Heparan sulfate proteoglycans (HSPGs) contain one or more heparan sulfate chains covalently linked to a protein core. HSPGs are localized to the cell surface and in the extracellular matrix. Due to their strong negative charge, the heparan sulfate chains bind to and modulate interactions between various protein ligands that mediate cell proliferation, including growth factors, morphogens, other extracellular matrix proteins, enzymes, and enzyme inhibitors. HSPGs have been implicated in cell growth and proliferation and tend to be mis-expressed in cancer cells and tumors. Previous studies have indicated that structure of the heparan sulfate chains (the number and arrangement of sulfate groups and uronic acid epimers) and the level of expression of HSPGs can affect tumor growth. The overall goal of this proposal is to uncover and characterize novel genes other than those encoding known heparan sulfate biosynthetic enzymes, whose expression influences heparan sulfate-mediated regulatory networks in cancer progression. To accomplish this goal, we plan to: (1) develop a high-throughput, genome-wide, short-hairpin RNA screen that detects genes involved in heparan sulfate biosynthesis, and (2) identify and characterize candidate genes for their capacity to block tumor cell growth and viability. A screening assay will be designed employing toxin-based screens used previously to identify small molecule inhibitors of heparan sulfate. We plan to search for genes, that when their expression is reduced or eliminated, will confer resistance to heparan sulfate-dependent cytotoxins. A genome-wide shRNA library will be used to knock down gene expression in HeLa (cervical carcinoma) cells. Following knockdown, we will assay for cell viability and identify those candidate genes that conferred resistance to the heparan sulfate-dependent toxin. Analysis of heparan sulfate structure and binding properties, as well as expression analysis of the core proteins will follow. Additionally, we will determine the ability of tumor cells expressing the candidate shRNAs to form colonies in soft agar and induce the formation of tumors in animals. The combined results from the work outlined in this proposal will give us a better understanding of the role heparan sulfate structure and function plays in tumorigenesis. The factors we identify could reveal novel targets for anti-cancer therapies, as well as lead us to methods to manipulate heparan sulfate and its activities in other cell types.