Searching for Novel Genes in Heparan Sulfate Biosynthesis

性状（由申请方提供）：硫酸乙酰肝素蛋白聚糖（HSPG）含有一条或多条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。由于其强负电荷，硫酸乙酰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，所述蛋白质配体包括生长因子、形态发生素、其他细胞外基质蛋白、酶和酶抑制剂。HSPG与细胞生长和增殖有关，并且倾向于在癌细胞和肿瘤中错误表达。以往的研究表明，硫酸乙酰肝素链的结构（硫酸基团和糖醛酸差向异构体的数量和排列）和HSPGs的表达水平可以影响肿瘤的生长。该提案的总体目标是发现和表征除了那些编码已知硫酸乙酰肝素生物合成酶的新基因，其表达影响硫酸乙酰肝素介导的癌症进展调控网络。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RNA筛选，检测硫酸乙酰肝素生物合成中涉及的基因，（2）识别和表征候选基因的能力，以阻止肿瘤细胞的生长和活力。筛选试验将采用先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来设计。我们计划寻找基因，当它们的表达减少或消除时，将赋予对硫酸乙酰肝素依赖性细胞毒素的抗性。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，我们将测定细胞活力，并确定那些赋予硫酸乙酰肝素依赖性毒素抗性的候选基因。硫酸乙酰肝素的结构和结合特性的分析，以及核心蛋白的表达分析将遵循。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乙酰肝素结构和功能在肿瘤发生中的作用。我们确定的因素可以揭示抗癌疗法的新靶点，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。 公共卫生相关性：本文概述的研究旨在揭示和表征其表达影响硫酸乙酰肝素介导的肿瘤生长调控网络的新基因。更好地了解硫酸乙酰肝素的结构和功能，以及确定参与确定其生物活性的因素，可以揭示抗癌治疗的新靶点。此外，潜在的发现也可能使我们深入了解硫酸乙酰肝素可以影响人类病理学中许多其他细胞过程的机制。

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. PUBLIC HEALTH RELEVANCE: The studies outlined here seek to uncover and characterize novel genes whose expression influences heparan sulfate-mediated regulatory networks in tumor growth. A better understanding of heparan sulfate structure and function as well as identification of factors involved in determining its biological activities could reveal novel targets for anti-cancer therapies. In addition, the potential findings might also give us insight into the mechanisms by which heparan sulfate can influence many other cellular processes that go awry in human pathologies.