Searching for Novel Genes in Heparan Sulfate Biosynthesis

说明(申请人提供)：硫酸乙酰肝素蛋白多糖(HSPG)含有一个或多个与蛋白质核心共价连接的硫酸乙酰肝素链。HSPG定位于细胞表面和细胞外基质中。由于其强大的负电荷，硫酸乙酰肝素链结合并调节各种蛋白质配体之间的相互作用，这些蛋白质配体介导细胞增殖，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，在癌细胞和肿瘤中往往错误表达。以往的研究表明，硫酸乙酰肝素链的结构(硫酸盐基团和糖醛酸同分异构体的数量和排列)以及HSPGs的表达水平可以影响肿瘤的生长。这项建议的总体目标是发现和表征除编码已知的硫酸乙酰肝素生物合成酶的基因外的新基因，这些酶的表达影响硫酸乙酰肝素在癌症进展中介导的调控网络。为了实现这一目标，我们计划：(1)开发一种高通量、全基因组、短发夹状RNA筛选，以检测与硫酸乙酰肝素生物合成有关的基因；以及(2)鉴定和表征候选基因，以确定它们阻止肿瘤细胞生长和存活的能力。将设计一种筛选试验，使用以前用于鉴定硫酸乙酰肝素小分子抑制剂的基于毒素的筛选。我们计划寻找基因，当它们的表达减少或消除时，会对依赖于硫酸肝素的细胞毒素产生抵抗力。一个全基因组的shRNA文库将被用来抑制宫颈癌(HeLa)细胞的基因表达。在基因敲除后，我们将检测细胞活性，并确定那些对硫酸乙酰肝素依赖毒素产生抗性的候选基因。接下来将分析硫酸乙酰肝素的结构和结合特性，以及核心蛋白的表达分析。此外，我们还将确定表达候选shRNAs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。这项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所起的作用。我们确定的因素可能会揭示抗癌治疗的新靶点，并引导我们找到操纵硫酸乙酰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.