Investigating the role of the glycogen debranching enzyme AGL in bladder cancer g

研究糖原脱支酶 AGL 在膀胱癌中的作用

• 批准号: 8783980
• 负责人: Carolyn Ritterson Lew
• 金额: $ 5.19万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-20 至 2016-02-08
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8783980
• 关键词: Address; Affect; American; Behavior; Bladder; Bladder Neoplasm; Bladder Urothelium; Breeding; CD44 gene; Cancer cell line; Cells; Cessation of life; Chemicals; Complement; Data; Dependency; Deposition; Development; Diagnosis; Disease; Disease model; Distant; Drug Targeting; Embryo; Enzymes; Experimental Models; Funding; Future; Gene Deletion; Gene Expression; Genes; Genetic Transcription; Genome; Glycogen; Glycogen Debranching Enzyme; Glycogen Phosphorylase; Glycogen Storage Disease; Glycogen Storage Disease Type III; Goals; Growth; HMMR gene; Health; Hepatomegaly; Human; Human Genome; Hyaluronic Acid; Hyperlipidemia; Hypoglycemia; In Vitro; Incidence; Knock-out; Knockout Mice; Knowledge; Lead; Libraries; Longevity; Malignant Neoplasms; Malignant neoplasm of urinary bladder; Mass Spectrum Analysis; Mediating; Medicare; Modeling; Monitor; Mus; Mutation; Neoplasm Metastasis; Nodal; Organ; Outcome; Pathway interactions; Patients; Phenocopy; Phenotype; Play; Population; Predisposition; Primary Neoplasm; Proteins; Publishing; RNA Interference; Regulation; Relative (related person); Research Personnel; Role; Signal Transduction; Simian virus 40; Testing; Therapeutic; Time; Tissues; Transferase; Tumor Suppressor Proteins; United States; United States National Institutes of Health; Urinary system; Urothelium; Visceral; Woman; Work; alpha-glucanotransferase; anticancer research; cancer cell; cancer diagnosis; carcinogenesis; chemical genetics; cost; genetic inducer; genome-wide; glucosidase; glycogenolysis; in vivo; inhibitor/antagonist; mRNA Expression; men; mouse model; mutant; new therapeutic target; novel; novel therapeutics; outcome forecast; overexpression; promoter; protein complex; protein expression; public health relevance; receptor; research study; small hairpin RNA; symposium; therapeutic target; transcription factor; tumor; tumor growth; tumor xenograft; tumorigenesis

DESCRIPTION (provided by applicant): Bladder cancer is the 4th most common cancer in men, and the 10th most common in women. In the United States, 72,570 new cases and 15,210 deaths are estimated in 2013. The cost per patient to treat this cancer (diagnosis to death) is ~$150,000, the greatest of any cancer in the medicare population. However, relative to its impact on human health, bladder cancer research is underfunded. Furthermore, little is known regarding the functional determinants of bladder cancer growth. Our lab developed a functional genomewide screen using an shRNA library directed towards the entire human genome to identify suppressors of bladder tumor growth. We identified and validated the glycogen debranching enzyme amylo-alpha-1, 6-glucosidase, 4-alpha- glucanotransferase (AGL) as one such tumor growth suppressor. AGL expression is also inversely associated with prognosis of patients with bladder cancer. While AGL is well studied in the context of glycogen storage disease type III (GSDIII), this is the first instance demonstrating a role for AGL in cancer. Surprisingly, we found that the enzymatic activity of AGL is not necessary for its tumor suppressor function. AGL mutants lacking this activity still suppress proliferation of bladder cancer cells in vitro and in vivo. An important goal is determining how AGL mediates its tumor suppressor function and using this knowledge for therapeutic purposes. Because loss of AGL leads to enhanced tumor growth, genes with expression enhanced by AGL depletion may drive this phenotype. Therefore, we investigated changes in gene expression upon AGL loss in bladder cancer cells. Hyaluronic Acid (HA) Synthase 2 (HAS2) was one of the top upregulated genes in cells with AGL depletion. HAS2 and its product HA have known functions in promoting cancer in several tissues, including bladder. Importantly, depletion of HAS2 blocked tumor growth driven by AGL loss, suggesting HAS2 is a driver of tumor growth upon AGL loss. The first aim of this proposal will address 1) how HAS2 expression is regulated by AGL using HAS2 promoter analysis, 2) if pharmacologically blocking HAS2 activity or the action of HA on human cancer cells in vitro and in vivo can abrogate the growth promoting effect of reduced AGL expression, and 3) finding novel proteins that complex with AGL to elucidate the pathway(s) that lead from AGL to the control of HAS2 expression. Even with the known role of AGL loss in glycogen storage disease, there is no published mouse model with a knockout (KO) of the AGL gene. This model is critically important for the study of the role of AGL in spontaneous bladder carcinogenesis and progression. We have generated mice heterozygous for an AGL KO construct which, upon breeding, should result in such a KO mouse. The second aim of this proposal focuses on characterizing this AGL KO mouse and using it to investigate the impact of AGL loss on the initiation and progression of bladder cancer tumors in mice. Chemical and genetic inducers of bladder cancer will be used in these mice and in wild type littermates. Primary tumor formation and metastasis will be monitored and compared in AGL KOs versus wild type controls.

描述（由申请人提供）：膀胱癌是男性中第4大常见癌症，女性中第10大常见癌症。在美国，2013年估计有72，570例新病例和15，210例死亡。治疗这种癌症（诊断至死亡）的每位患者的费用约为15万美元，是医疗保险人群中最高的癌症。然而，相对于其对人类健康的影响，膀胱癌研究资金不足。此外，关于膀胱癌生长的功能决定因素知之甚少。我们的实验室开发了一种功能性全基因组筛选，使用针对整个人类基因组的shRNA文库来识别膀胱肿瘤生长的抑制因子。我们鉴定并验证了糖原脱支酶淀粉-α-1，6-葡糖苷酶，4-α-葡聚糖转移酶（AGL）作为一种这样的肿瘤生长抑制剂。AGL表达与膀胱癌患者的预后也呈负相关。虽然AGL在糖原累积病III型（GSDIII）的背景下得到了很好的研究，但这是第一次证明AGL在癌症中的作用。令人惊讶的是，我们发现AGL的酶活性对于其肿瘤抑制功能不是必需的。缺乏这种活性的AGL突变体在体外和体内仍然抑制膀胱癌细胞的增殖。一个重要的目标是确定AGL如何介导其肿瘤抑制功能，并将此知识用于治疗目的。由于AGL的缺失导致肿瘤生长增强，因此通过AGL缺失增强表达的基因可能驱动这种表型。因此，我们研究了膀胱癌细胞中AGL缺失后基因表达的变化。透明质酸（HA）合成酶2（HAS 2）是AGL缺失细胞中上调最多的基因之一。已知HAS 2及其产物HA在促进包括膀胱在内的多种组织中的癌症方面具有功能。重要的是，HAS 2的耗尽阻断了由AGL损失驱动的肿瘤生长，表明HAS 2是AGL损失后肿瘤生长的驱动因素。该提议的第一个目的将解决1）使用HAS 2启动子分析，HAS 2表达如何被AGL调节，2）如果在体外和体内阻断HAS 2活性或HA对人癌细胞的作用可以消除降低的AGL表达的生长促进作用，和3）发现与AGL复合的新蛋白，以阐明从AGL到HAS 2表达控制的途径。即使已知AGL损失在糖原累积病中的作用，也没有公开的AGL基因敲除（KO）的小鼠模型。该模型对于研究AGL在自发性膀胱癌发生和发展中的作用至关重要。我们已经产生了AGL KO构建体杂合的小鼠，其在繁殖时应该产生这样的KO小鼠。该提案的第二个目的集中在表征这种AGL KO小鼠，并使用它来研究AGL缺失对小鼠膀胱癌肿瘤的发生和进展的影响。膀胱癌的化学和遗传诱导剂将用于这些小鼠和野生型同窝仔。将在AGL科斯与野生型对照中监测和比较原发性肿瘤形成和转移。