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

描述（由申请人提供）：膀胱癌是男性第四大常见癌症，女性第十大常见癌症。在美国，2013年估计有72570例新病例和15210例死亡。每位患者治疗这种癌症（从诊断到死亡）的费用约为15万美元，是医保人群中所有癌症中最高的。然而，相对于其对人类健康的影响，膀胱癌的研究资金不足。此外，对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全基因组筛选，使用shRNA文库直接针对整个人类基因组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- α - 1,6 -葡萄糖苷酶，4- α -葡聚糖转移酶（AGL）是一种这样的肿瘤生长抑制因子。AGL的表达也与膀胱癌患者的预后呈负相关。虽然AGL在III型糖原储存病（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的酶活性并不是其肿瘤抑制功能所必需的。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，并将这一知识用于治疗目的。由于AGL缺失导致肿瘤生长增强，因此AGL缺失导致表达增强的基因可能驱动这种表型。因此，我们研究了膀胱癌细胞AGL丢失时基因表达的变化。透明质酸（HA）合成酶2 （HAS2）是AGL缺失细胞中表达上调最多的基因之一。已知HAS2及其产物HA在包括膀胱在内的几种组织中促进癌症的发生。重要的是，HAS2的缺失阻断了AGL缺失驱动的肿瘤生长，这表明HAS2是AGL缺失后肿瘤生长的驱动因素。本提案的第一个目标将解决1)利用HAS2启动子分析AGL如何调节HAS2表达，2)在体外和体内是否通过药物阻断HAS2活性或HA对人类癌细胞的作用可以消除AGL表达减少的促生长作用，以及3)寻找与AGL复合物的新蛋白来阐明从AGL到控制HAS2表达的途径。即使已知AGL基因缺失在糖原蓄积性疾病中的作用，也没有已发表的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。我们已经为AGL KO构建产生了杂合的小鼠，经过育种，应该会产生这样的KO小鼠。本研究的第二个目的是对AGL KO小鼠进行表征，并利用AGL缺失对小鼠膀胱癌肿瘤发生和发展的影响。膀胱癌的化学和遗传诱导剂将用于这些小鼠和野生型幼崽。将监测原发肿瘤的形成和转移，并将AGL KOs与野生型对照进行比较。

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.