Understanding the AGL metastasis suppressor for therapeutic gain

 描述（由申请人提供）：膀胱癌每年夺去 15,000 人的生命，由于治疗进展甚少，因此需要创新。该项目发现了膀胱肿瘤生长和转移的抑制因子，并表明 AGL（一种参与糖原分解的酶）和 RhoGDI2（多种 GTP 酶的抑制剂）的低表达与癌症复发和死亡相关。该延续项目的目标是了解 AGL 水平降低如何导致侵袭性膀胱癌，并将这些知识转化为新疗法。我们发现，AGL 较低的膀胱癌细胞表达透明质酸 (HA) 合酶 2 (HAS2) 及其细胞外产物 HA 的水平升高，并激活了已知的肿瘤进展驱动因素 RhoC 和 Rac1。我们证明 RhoC/Rac1 活性被 RhoGDI2 抑制，将两种转移抑制剂的作用联系起来。 AGL 低的细胞还表现出糖酵解、葡萄糖输入和自噬增加。当葡萄糖输入或自噬被阻断时，低 AGL 肿瘤细胞比 AGL 水平较高的细胞显示出更大的体外生长减少，暴露了这些侵袭性细胞的脆弱性。这些数据支持一个创新假设：低 AGL 的肿瘤细胞通过产生 HA、激活其受体 CD44 和 RHAMM 以及下游效应子（如 RhoC 和 Rac1）来产生生长和转移信号。为了实现这些信号，需要通过增强葡萄糖输入和自噬来提供足够的能量。三个具体目标将检验这一假设。 1) 评估 HA 信号传导对低 AGL 膀胱癌侵袭性的影响。我们将确定 HA 是否通过其受体 CD44 和 RHAMM 以及受 RhoGDI2 抑制的效应器 RhoC 和 Rac1 驱动肿瘤进展。实验将在人类异种移植物中使用 RNAi 和受体抑制。 2) 确定AGL信号网络在膀胱癌发生和进展中的作用。我们将评估一大组注释的人类肿瘤中肿瘤抑制基因和 AGL 信号网络成分的独立预测价值，以确定预测性多基因生物标志物特征和可能的新治疗靶点。我们将在化学致癌模型中使用完全和条件性（膀胱尿路上皮）AGL 基因敲除小鼠，该模型会产生自发内脏转移，以测试缺乏 AGL 是否会使尿路上皮更容易致癌并导致肿瘤更具侵袭性。 3）AGL低表达的膀胱癌的靶分子脆弱性。在这里，我们测试阻断葡萄糖输入和/或自噬是否会减少体内膀胱肿瘤的生长。我们将在临床前/临床测试中使用 shRNA 和药物制剂。我们将筛选低 AGL 细胞中代谢和自噬基因的 shRNA 文库，以发现侵袭性肿瘤表型所必需的新基因，并期望其中一些将成为治疗靶点。我们将评估 HA 信号传导和能量产生途径的联合抑制，期望这将对肿瘤进展产生协同有害影响。影响：该项目将为未来生物标志物驱动的个性化临床试验奠定基础，这些临床试验针对膀胱癌的关键漏洞，同时告诉我们 AGL 如何在癌症中发挥其意想不到的作用。

 DESCRIPTION (provided by applicant): Bladder cancer kills 15,000 annually and because of few therapeutic advances there is a need for innovation. This project has discovered suppressors of bladder tumor growth and metastasis and shown that low expression of AGL, an enzyme involved in glycogenolysis, and RhoGDI2, an inhibitor of multiple GTPases, is associated with cancer recurrence and death. The Goal of this continuation project is to understand how reduced AGL levels result in aggressive bladder cancer and to translate this knowledge into novel therapies. We found bladder cancer cells with low AGL express elevated levels of Hyaluronic Acid (HA) synthase 2 (HAS2), and its extracellular product HA, and have activated RhoC and Rac1, known drivers of tumor progression. We demonstrated RhoC/Rac1 activity is suppressed by RhoGDI2, linking the actions of the two metastasis suppressors. Cells with low AGL also exhibit increased glycolysis, glucose import and autophagy. When either glucose import or autophagy are blocked, low AGL tumor cells show greater reduction of in vitro growth than cells with higher AGL levels, exposing a vulnerability of these aggressive cells. These data support an innovative Hypothesis: Tumor cells with low AGL generate growth and metastatic signals via production of HA, activation of its receptors CD44 and RHAMM, and downstream effectors such as RhoC and Rac1. For these signals to be actualized, sufficient energy needs to be provided via enhanced glucose import and autophagy. Three Specific Aims will test this hypothesis. 1) Evaluate impact of HA signaling on aggressiveness of bladder cancers with low AGL. We will determine if HA drives tumor progression via its receptors CD44 and RHAMM and effectors RhoC and Rac1 that are inhibited by RhoGDI2. Experiments will use RNAi and receptor inhibition in human xenografts. 2) Determine role of the AGL signaling network in bladder cancer development and progression. We will evaluate the independent predictive value of tumor suppressor genes and components of the AGL signaling network in a large panel of annotated human tumors, to define a predictive multigene biomarker signature and possible new therapeutic targets. We will use full and conditional (bladder urothelium) AGL knockout mice in a chemical carcinogenesis model that generates spontaneous visceral metastases to test whether lack of AGL makes urothelium more susceptible to carcinogenesis and resulting tumors more aggressive. 3) Target molecular vulnerabilities of bladder cancers with low AGL expression. Here we test whether blocking glucose import and/or autophagy reduces in vivo bladder tumor growth. We will use shRNA and pharmacologic agents already in preclinical/clinical testing. We will screen shRNA libraries to metabolic and autophagy genes in cells with low AGL to discover novel genes essential to the aggressive tumor phenotype, with the expectation that some of these will become therapeutic targets. We will evaluate combined inhibition of HA signaling and energy production pathways with the expectation this will have synergistic detrimental effects on tumor progression. Impact: This project will lay the foundation for future biomarker-driven personalized clinical trials that target critical vulnerabilities in bldder cancer while informing us on how AGL executes its unanticipated role in cancer.