Potential of a dominant-negative FGF mutant as a therapeutic in cancer

显性失活 FGF 突变体作为癌症治疗的潜力

• 批准号: 8015202
• 负责人: YOSHIKAZU TAKADA
• 金额: $ 30.84万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8015202
• 关键词: Address; Affect; Binding; Binding Sites; Breast Cancer Model; Cell Proliferation; Cell surface; Cells; Colon Carcinoma; Complex; Cytoplasmic Tail; Defect; Development; Disseminated Malignant Neoplasm; Dominant-Negative Mutation; Endothelial Cells; Engineering; Event; Extracellular Matrix; FGF1 gene; Fibroblast Growth Factor; Fibroblast Growth Factor Receptors; Fibroblasts; Genetically Engineered Mouse; Goals; Growth; Growth Factor Interaction; Health; Hematopoietic; Heparin; In Vitro; Inflammation; Integrin Binding; Integrins; Kinetics; Lead; Lesion; MAPK3 gene; Malignant Neoplasms; Mammary gland; Modeling; Molecular Weight; Mouse Mammary Tumor Virus; Mus; Noninfiltrating Intraductal Carcinoma; Phosphorylation; Pilot Projects; Play; Premalignant; Protein Isoforms; Recruitment Activity; Relative (related person); Role; Screening procedure; Signal Transduction; Specificity; Stromal Cells; Testing; Therapeutic; Tissues; Transgenic Mice; Tumor Angiogenesis; Tyrosine Phosphorylation; angiogenesis; cell motility; cell type; design; in vivo; malignant breast neoplasm; meetings; mouse model; mutant; neoplastic cell; new therapeutic target; novel therapeutics; poly(2-hydroxyethyl methacrylate)-polyamine graft copolymer; research study; surface coating; tumor growth; tumor initiation; tumor progression; tumorigenesis; virtual

DESCRIPTION (provided by applicant): We discovered that integrin av¿3 directly binds to FGF1. We developed an FGF1 mutant (Arg-50 to Glu, R50E) that is defective in integrin binding but still binds to FGF-receptor or heparin. R50E was defective in inducing FGF signaling while it binds to heparin or FGFR, suggesting that integrin binding to FGF1 is required for FGF signaling (Mori et al., 2008). Notably, R50E blocked cell proliferation and migration induced by wt FGF1 (dominant-negative effect). Furthermore, R50E markedly suppressed the growth of DLD-1 colon cancer, Met-1 highly metastatic breast cancer, and MIN-O pre-cancer lesions in vivo in pilot studies. We identified several critical defects in R50E that may be directly related to its inhibitory action: 1) wt FGF1 induced the FGFR-FGF-integrin ternary complex, but R50E did not; 2) wt FGF1 induced Tyr phosphorylation of the integrin 23 cytoplasmic domain, but R50E did not; and 3) wt FGF1 induced sustained ERK1/2 activation, but R50E did not. We thus proposed a model in which integrins are recruited to the FGFR-FGF complex through the direct binding to FGF, and this will lead to Tyr phosphorylation of ¿3, and ERK1/2 activation. R50E does not recruit av¿3 to the FGF-FGFR complex, and thereby the subsequent signaling events are blocked. OBJECTIVES: Our goal is to analyze the role of integrins in FGF signaling, to re-evaluate the current models of FGF signaling using R50E, and to establish that FGF-integrin interaction is a novel therapeutic target in cancer and angiogenesis. SPECIFIC AIMS: SA #1) Identify the role of the direct binding of integrins to FGF in FGF signaling. a) Identify the relative contribution of integrin-FGF interaction and integrin-ECM interaction to FGF signaling using cells that are suspended on poly-HEMA-coated surface. b) Analyze the role of direct binding of integrins to FGF1 in FGF signaling using newly identified candidate small-molecular-weight FGF1 antagonists. c) Identify integrins that are involved in FGF signaling in hematopoietic cells that do not express av¿3. d) Analyze the specificity of R50E to FGFR isoforms and kinetics of inhibition. SA #2) Test the hypothesis that R50E suppresses tumor growth in breast cancer by suppressing tumor initiation, progression, and angiogenesis using a genetically engineered mouse breast cancer model. a) Analyze the effect of R50E on FGF signaling in Met-1 cancer in vitro. b) Analyze the effect of R50E on tumor progression, angiogenesis, and microenvironment in vivo using Met-1. c) Analyze the effect of R50E on the tumorigenesis of precancerous MIN-O lesion in vivo. d) Analyze the effect of R50E on the initiation and progression of tumor and tumor angiogenesis in vivo using a genetically engineered mouse model of breast cancer, (Tg(MMTV-PyV-mT)). EXPECTED RESULTS: The proposed experiments will identify the role of integrins in FGF signaling, evaluate the potential of R50E to tumor initiation and development in vivo, and identify target cell types for R50E in vivo. The information obtained will enhance our understanding of FGF signaling and integrin-FGFR crosstalk and will help in designing a new therapeutic strategy for cancer and inflammation. PUBLIC HEALTH RELEVANCE: We discovered that FGF1 binds to integrins, and developed a mutant of FGF1 that does not bind to integrins. We found that the mutant is not only defective in inducing signals but also suppress FGF signaling induced by wt FGF1 in a dominant-negative fashion. We will address the hypothesis that the direct binding of FGF to integrins is required for FGF signaling, and analyze the inhibitory effect of dominant-negative mutant on tumor initiation, progression, and angiogenesis in transgenic mouse model of cancer.

描述（由申请人提供）：我们发现整合素α v β 3直接与FGF 1结合。我们开发了一种FGF 1突变体（Arg-50至Glu，R50 E），其在整合素结合方面有缺陷，但仍与FGF受体或肝素结合。R50 E在诱导FGF信号传导方面有缺陷，而它与肝素或FGFR结合，表明整联蛋白与FGF 1的结合是FGF信号传导所需的（Mori等人，2008年）。值得注意的是，R50 E阻断了由wt FGF 1诱导的细胞增殖和迁移（显性负效应）。此外，在初步研究中，R50 E在体内显著抑制DLD-1结肠癌、Met-1高转移性乳腺癌和MIN-O癌前病变的生长。我们鉴定了R50 E中可能与其抑制作用直接相关的几个关键缺陷：1）wt FGF 1诱导FGFR-FGF-整联蛋白三元复合物，但R50 E不诱导; 2）wt FGF 1诱导整联蛋白23胞质结构域的Tyr磷酸化，但R50 E不诱导;和3）wt FGF 1诱导持续的ERK 1/2活化，但R50 E不诱导。因此，我们提出了一个模型，其中整合素通过直接结合FGF被募集到FGFR-FGF复合物中，这将导致酪氨酸磷酸化和ERK 1/2激活。R50 E不会将av 3募集到FGF-FGFR复合物中，因此随后的信号传导事件被阻断。目的：我们的目标是分析整联蛋白在FGF信号传导中的作用，使用R50 E重新评估FGF信号传导的当前模型，并建立FGF-整联蛋白相互作用是癌症和血管生成的新治疗靶点。具体目的：SA #1）鉴定整联蛋白与FGF的直接结合在FGF信号传导中的作用。a）使用悬浮在聚HEMA包被的表面上的细胞鉴定整联蛋白-FGF相互作用和整联蛋白-ECM相互作用对FGF信号传导的相对贡献。B）使用新鉴定的候选小分子量FGF 1拮抗剂分析整联蛋白与FGF 1的直接结合在FGF信号传导中的作用。c）鉴定在不表达α v β 3的造血细胞中参与FGF信号传导的整合素。d）分析R50 E对FGFR同种型的特异性和抑制动力学。SA#2）使用基因工程小鼠乳腺癌模型检验R50 E通过抑制肿瘤起始、进展和血管生成来抑制乳腺癌中的肿瘤生长的假设。a）在体外分析R50 E对Met-1癌症中FGF信号传导的影响。B）使用Met-1分析R50 E对肿瘤进展、血管生成和体内微环境的影响。c）分析R50 E对癌前病变MIN-O体内成瘤的影响。d）使用乳腺癌的基因工程小鼠模型（Tg（MMTV-PyV-mT）），分析R50 E对体内肿瘤和肿瘤血管生成的起始和进展的影响。预期结果：拟进行的实验将确定整联蛋白在FGF信号传导中的作用，评估R50 E在体内肿瘤发生和发展的潜力，并确定R50 E在体内的靶细胞类型。所获得的信息将增强我们对FGF信号传导和整合素-FGFR串扰的理解，并将有助于设计癌症和炎症的新治疗策略。公共卫生相关性：我们发现FGF 1与整合素结合，并开发了一种不与整合素结合的FGF 1突变体。我们发现该突变体不仅在诱导信号方面有缺陷，而且以显性负性方式抑制wt FGF 1诱导的FGF信号传导。我们将解决的假设，FGF的直接结合整合素所需的FGF信号，并分析显性负突变体对肿瘤的发生，发展和肿瘤的转基因小鼠模型中的血管生成的抑制作用。