Ral in human bladder cancer progression

Ral 在人膀胱癌进展中的作用

• 批准号: 7600785
• 负责人: DAN THEODORESCU
• 金额: $ 3.81万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7600785
• 关键词: 1-Phosphatidylinositol 3-Kinase; Actins; Address; Adhesions; Affect; Anus; Back; Basement membrane; Biochemistry; Biological; Biological Assay; Biology; Bladder; Bladder Neoplasm; Bladder Urothelium; Cancer cell line; Cell Line; Cell membrane; Cells; Cellular Morphology; Clinical Oncology; Clinical Research; Complex; Connective Tissue; Cytoskeleton; Data; Development; Diagnostic; Disease; EGF gene; EGFR Protein Overexpression; Endopeptidases; Epidermal Growth Factor Receptor; Epithelial; Family; Fibroblasts; Foundations; Freezing; Funding; Future; GTP-Binding Proteins; Genes; Genetic; Growth Factor; Guanine Nucleotide Dissociation Inhibitors; Guanosine Triphosphate Phosphohydrolases; HRAS gene; Human; In Vitro; Incidence; Invaded; Invasive; Knockout Mice; Laboratories; Lead; Life; Malignant Neoplasms; Malignant neoplasm of urinary bladder; Mediating; Membrane; Modeling; Molecular; Molecular Weight; Mucous Membrane; Mus; Muscle; Mutate; Mutation; PTEN gene; Pathway interactions; Patients; Peptide Hydrolases; Phenotype; Phosphotransferases; Process; Production; Protein Overexpression; Proteins; RRAS2 gene; Range; Rattus; Receptor Activation; Receptor Signaling; Regulation; Research Personnel; Risk; Role; Role playing therapy; Running; Second Messenger Systems; Sequence Homology; Signal Pathway; Signal Transduction; Specimen; Stimulus; Stress Fibers; Technology; Testing; Time; Tissue Banks; Tissues; Transgenic Animals; Transgenic Organisms; Transport Vesicles; Tumor Cell Invasion; Tumor Suppressor Genes; United States; base; bladder Carcinoma; cancer cell; cell growth; cell motility; follow-up; human RRAS2 protein; human tissue; in vivo; mRNA Expression; member; migration; novel; p120 GTPase Activating Protein; phospholipase C gamma; polymerization; prognostic; programs; ras GTPase-Activating Proteins; receptor; reconstitution; response; rho; second messenger; tool; tumor; tumor progression

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.

背景和意义：有40％的患者出现“表面”（非肌肉侵入性）膀胱癌发展 随访期间，疾病的“侵入性”威胁生命形式。在临床研究中，表皮生长因子的过表达 受体（EGFR），HA-RAS突变和抑制肿瘤基因PTEN的丧失与该表型肿瘤有关 过渡。但是，这些基因有效触发或促进侵入性过程的确切分子途径是 不完全理解。我们最初的R29假设EGFR信号在体外增强了膀胱肿瘤运动性，并且 体内入侵，旨在确定EGFR在此过程中使用的信号传导途径。自从9/97的R29资金以来 我们做出了以下重要观察，这些观察支持原始假设并解决了原始的目的 应用：1）EGFR和RAS抑制减少了浸润性膀胱癌细胞的运动； 2）EGF刺激运动性 通过PI3K非侵入性细胞，这需要Rho和Ras效应大鼠的活性。 3）在非侵入性细胞中，基线RALA活性为 低侵入性细胞具有组成性更高的激活。 4）侵入性细胞的RHOGDI2表达水平较低。 该基因的重建导致Rala的运动性和活性减少，但没有提示该基因可能是第一个基因 ralgdi被确定为入侵抑制器； 5）通过侵入性细胞中的PTEN重构抑制PI3K活性 随着无效的PTEN，导致抑制体内原位浸润和RhoA活性的降低。自从整体 RAL和RHOGDI2的生物学知识很少，但对于调节膀胱患者的肿瘤侵袭至关重要 癌症，我们提出了一个指导假设，即EGF通过RAL激活介导膀胱肿瘤侵袭。我们将测试这个 假设具有从基本生物化学到临床肿瘤学的技术矩阵，以解决人类的RAL生物学 膀胱癌。其中包括：1）具有不同侵入性能力的独特成对的人膀胱癌细胞； 2）小说 器官膀胱模型允许体外研究肿瘤侵袭； 3）评估候选人影响的原位测定 体内膀胱癌入侵的分子； 4）具有适当遗传和表型特征的转基因和敲除小鼠； 5） 具有病理和临床表征良好的冷冻标本的人体组织库。具体目的：1）确定角色 RAL在膀胱癌入侵的病理生物学中，在器官，鼠矫形和人类肿瘤研究中； 2）确定 RAL激活的调节剂（RHOGDI2等）及其对细胞内RAL定位和膀胱癌的影响 入侵； 3）确定与体外和体内RAL相关的蛋白质复合物，包括在人类癌症中发现的蛋白质复合物。 结论：这些特定目标的完成将提供有关信号通路的生物学相关分子信息 调节体内膀胱癌入侵，并导致诊断和预后工具的合理发展，以预测 侵入性疾病和疗法的发展，以干扰浅膀胱癌患者。