Ral in human bladder cancer progression

背景和意义：40%的“浅表性”（非肌肉侵袭性）膀胱癌患者在随访期间发展为“侵袭性”危及生命的疾病。在临床研究中，表皮生长因子受体（EGFR）的过表达、Ha-Ras突变和肿瘤抑制基因PTEN的缺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袭过程的确切分子途径尚不完全清楚。我们最初的R29假设EGFR信号增强膀胱肿瘤在体外的运动和体内的侵袭，并试图确定EGFR在这一过程中使用的信号通路。自1997年9月资助R29以来，我们已经做了以下重要的观察，这些观察支持了最初的假设，并解决了最初应用的目标：1)EGFR和Ras抑制降低了侵袭性膀胱癌细胞的运动；2) EGF通过PI3K刺激非侵袭性细胞的运动，这需要Rho和Ras效应大鼠的活性；3)在非侵袭性细胞中，RalA的基线活性较低，而侵袭性细胞则具有较高的活性；4)侵袭细胞中RhoGDI2表达水平较低。该基因的重组导致RalA的活力和活性降低，但RhoA却没有，这表明该基因可能是第一个被鉴定为具有入侵抑制功能的RalGDI；5) PTEN失活的侵袭细胞通过PTEN重构抑制PI3K活性，导致体内原位侵袭受到抑制，RhoA活性降低。由于对Ral和RhoGDI2的整体生物学知之甚少，但可能对调节膀胱癌患者的肿瘤侵袭至关重要，我们提出EGF通过Ral激活介导膀胱肿瘤侵袭的指导性假设。我们将通过从基础生物化学到临床肿瘤学的一系列技术来验证这一假设，以解决人类膀胱癌的Ral生物学问题。这包括：1)具有不同侵袭能力的独特配对人类膀胱癌细胞系；2)一种新的器官型膀胱模型，可用于肿瘤侵袭的体外研究；3)原位实验评估候选分子对体内膀胱癌侵袭的影响；4)具有适当遗传和表型特征的转基因和基因敲除小鼠；5)具有病理和临床特征的冷冻标本的人体组织库。具体目的：1)在器官型、小鼠原位和人类肿瘤研究中确定Ral在膀胱癌侵袭中的作用和病理生物学；2)确定Ral激活的调节因子（RhoGDI2等）及其对细胞内Ral定位和膀胱癌浸润侵袭的影响；3)测定体内和体外与Ral相关的蛋白复合物，包括在人类癌症中发现的蛋白复合物。结论：这些特定目标的完成将为体内调控膀胱癌侵袭的信号通路提供生物学相关的分子信息，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干预浅表性膀胱癌患者这一过程的治疗。

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.