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: 7653318
• 负责人: YOSHIKAZU TAKADA
• 金额: $ 31.68万
• 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/7653318
• 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; FGF1 gene; Fibroblast Growth Factor; Fibroblast Growth Factor Receptors; Fibroblasts; Genetically Engineered Mouse; Goals; Growth; Growth Factor Interaction; Hematopoietic; Heparin; In Vitro; Inflammation; Integrin Binding; Integrins; Kinetics; Lead; Lesion; Malignant Neoplasms; Mammary gland; Modeling; Molecular Weight; Morus; 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 type; design; in vivo; malignant breast neoplasm; meetings; migration; mouse model; mutant; neoplastic cell; new therapeutic target; novel therapeutics; poly(2-hydroxyethyl methacrylate)-polyamine graft copolymer; public health relevance; 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av¿3直接与FGF1结合。我们开发了一种 FGF1 突变体（Arg-50 至 Glu，R50E），该突变体在整合素结合方面存在缺陷，但仍与 FGF 受体或肝素结合。 R50E 在与肝素或 FGFR 结合时诱导 FGF 信号传导存在缺陷，这表明整合素与 FGF1 的结合是 FGF 信号传导所必需的 (Mori et al., 2008)。值得注意的是，R50E 阻断了 wt FGF1 诱导的细胞增殖和迁移（显性负效应）。此外，在体内试验研究中，R50E 显着抑制了 DLD-1 结肠癌、Met-1 高转移性乳腺癌和 MIN-O 癌前病变的生长。我们发现了 R50E 中的几个关键缺陷，可能与其抑制作用直接相关：1）wt FGF1 诱导 FGFR-FGF-整合素三元复合物，但 R50E 没有； 2) wt FGF1 诱导整合素 23 胞质结构域的 Tyr 磷酸化，但 R50E 没有； 3) wt FGF1 诱导持续的 ERK1/2 激活，但 R50E 没有。因此，我们提出了一个模型，其中整合素通过直接结合 FGF 被招募到 FGFR-FGF 复合物中，这将导致 ¿3 的 Tyr 磷酸化和 ERK1/2 激活。 R50E 不会将 av¿3 募集到 FGF-FGFR 复合物中，从而阻断后续信号传导事件。目的：我们的目标是分析整合素在 FGF 信号传导中的作用，重新评估当前使用 R50E 的 FGF 信号传导模型，并确定 FGF-整合素相互作用是癌症和血管生成的新治疗靶点。具体目标：SA #1) 确定整合素与 FGF 直接结合在 FGF 信号转导中的作用。 a) 使用悬浮在聚 HEMA 涂层表面的细胞，确定整合素-FGF 相互作用和整合素-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 FGF1 拮抗剂分析整合素与 FGF1 直接结合在 FGF 信号传导中的作用。 c) 鉴定不表达 av¿3 的造血细胞中参与 FGF 信号传导的整合素。 d) 分析 R50E 对 FGFR 同工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 R50E 通过抑制肿瘤发生、进展和血管生成来抑制乳腺癌肿瘤生长的假设。 a) 体外分析 R50E 对 Met-1 癌症中 FGF 信号传导的影响。 b) 使用 Met-1 分析 R50E 对肿瘤进展、血管生成和体内微环境的影响。 c) 分析R50E对体内MIN-O癌前病变肿瘤发生的影响。 d) 使用乳腺癌基因工程小鼠模型 (Tg(MMTV-PyV-mT)) 分析 R50E 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：拟议的实验将确定整合素在 FGF 信号传导中的作用，评估 R50E 对体内肿瘤发生和发展的潜力，并确定体内 R50E 的靶细胞类型。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，并将有助于设计新的癌症和炎症治疗策略。公共健康相关性：我们发现 FGF1 与整合素结合，并开发了不与整合素结合的 FGF1 突变体。我们发现该突变体不仅在诱导信号方面存在缺陷，而且还以显性失活方式抑制 wt FGF1 诱导的 FGF 信号传导。我们将提出 FGF 信号传导需要 FGF 与整合素直接结合的假设，并分析显性失活突变体对转基因小鼠癌症模型中肿瘤发生、进展和血管生成的抑制作用。