Ral in human bladder cancer progression

描述（由申请人提供）：背景和意义：40%的“浅表性”（非肌肉浸润性）膀胱癌患者在随访期间发展为“浸润性”危及生命的疾病形式。在临床研究中，表皮生长因子受体（EGFR）的过度表达，Ha-Ras突变和肿瘤抑制基因PTEN的丢失与这种表型肿瘤转变有关。然而，这些基因有效触发或促进侵入过程的确切分子途径尚不完全清楚。我们最初的R29假设EGFR信号增强膀胱肿瘤的体外运动和体内侵袭，并打算确定EGFR在这一过程中使用的信号通路。自1997年9月R29的资助以来，我们进行了以下重要观察，这些观察支持了原始假设并解决了原始申请的目的：1）EGFR和Ras抑制降低了浸润性膀胱癌细胞的运动性; 2）EGF通过PI 3 K刺激非浸润性细胞的运动性，这需要Rho和Ras效应子Rat的活性; 3）在非侵袭性细胞中，基线RalA活性低，而侵袭性细胞具有组成性较高的活化; 4）侵袭性细胞具有低水平的RhoGDI 2表达。该基因的重建导致RalA而不是RhoA的运动性和活性降低，表明该基因可能是第一个被鉴定为起侵袭抑制剂作用的RalGDI; 5）在具有失活PTEN的侵袭细胞中通过PTEN重建抑制PI 3 K活性，导致体内原位侵袭的抑制和RhoA活性的降低。由于Ral和RhoGDI 2的整体生物学尚不清楚，但可能对调节膀胱癌患者的肿瘤侵袭至关重要，因此我们提出了EGF通过Ral激活介导膀胱肿瘤侵袭的指导假设。我们将用从基础生物化学到临床肿瘤学的技术矩阵来测试这一假设，以解决人类膀胱癌中的Ral生物学。其中包括：1）具有不同侵袭能力的独特配对的人膀胱癌细胞系; 2）允许肿瘤侵袭的体外研究的新型器官型膀胱模型; 3）评估候选分子对体内膀胱癌侵袭的作用的原位测定; 4）具有适当遗传和表型谱的转基因和敲除小鼠; 5）具有病理学和临床上良好表征的冷冻标本的人体组织库。具体目标：1）在器官型、鼠原位和人肿瘤研究中确定Ral在膀胱癌侵袭中的作用和病理生物学;以及它们对细胞内Ral定位和膀胱癌浸润的影响; 3）在体外和体内确定与Ral相关的蛋白复合物，包括在人类癌症中发现的那些。结论：这些特定目标的完成将提供调节膀胱癌体内侵袭的信号通路的生物学相关分子信息，并导致诊断和预后工具的合理开发，预测侵袭性疾病的发展和治疗，以干扰浅表性膀胱癌患者的这一过程。

DESCRIPTION (provided by applicant): Background and Significance: 40% of patients presenting with "superficial" (non-muscle-invasive) bladder cancer develop the "invasive" life-threatening form of the disease during follow up. In clinical studies, overexpression of Epidermal Growth Factor Receptor (EGFR), Ha-Ras mutation and loss of tumor suppressor gene PTEN have been associated with this phenotypic tumor transition. However, the exact molecular pathway by which these genes effectively trigger or facilitate the invasive process is incompletely understood. Our original R29 hypothesized that EGFR signaling enhances bladder tumor motility in vitro and invasion in vivo and intended to determine the signaling pathways used by EGFR in this process. Since funding of the R29 in 9/97, we have made the following important observations which support the original hypothesis and address the aims of the original application: 1) EGFR and Ras inhibition diminished the motility of invasive bladder cancer cells; 2) EGF stimulates motility in non-invasive cells via PI3K and this requires activity of Rho and Ras effector Rat; 3) In non-invasive cells, baseline RalA activity is low while invasive cells have constitutively higher activation; 4) Invasive cells have low levels of RhoGDI2 expression. Reconstitution of this gene leads to diminished motility and activity of RalA but not RhoA suggesting this gene may be the first RalGDI identified to function as an invasion suppressor; 5) Inhibition of PI3K activity via PTEN reconstitution in invasive cells with inactive PTEN, results in an inhibition of orthotopic invasion in vivo and a decrease in RhoA activity. Since the overall biology of both Ral and RhoGDI2 is poorly understood, but might be critical for regulating tumor invasion in patients with bladder cancer, we propose the Guiding Hypothesis that EGF mediates bladder tumor invasion via Ral activation. We will test this hypothesis with a matrix of technologies ranging from basic biochemistry to clinical oncology to address Ral biology in human bladder cancer. These include: 1) unique paired human bladder cancer cell lines with different invasive abilities; 2) a novel organotypic bladder model allowing in vitro study of tumor invasion; 3) an orthotopic assay evaluating the effects of candidate molecules on in vivo bladder cancer invasion; 4) transgenic and knockout mice with appropriate genetic and phenotypic profiles; 5) a human tissue bank with pathologically and clinically well characterized frozen specimens. Specific Aims: 1) Determine the role and pathobiology of Ral in bladder cancer invasion in organotypic, murine orthotopie and human tumor studies; 2) Determine the regulators of Ral activation (RhoGDI2, etc..) and their effect on intracellular Ral localization and bladder cancer nfigration and invasion; 3) Determine the protein complexes associated with Ral in vitro and in vivo, including those found in human cancer. Conclusion: Completion of these specific aims will provide biologically relevant molecular information on the signaling pathways regulating bladder cancer invasion in vivo and lead to the rational development of diagnostic and prognostic tools predicting the development of invasive disease and therapies to interfere with this process in patients with superficial bladder cancer.