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

描述(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为Glu，R50E)，该突变体在整合素结合方面存在缺陷，但仍能与成纤维细胞生长因子受体或肝素结合。当R50E与肝素或FGFR结合时，它在诱导成纤维细胞生长因子信号转导方面存在缺陷，这表明整合素与FGF1结合是成纤维细胞生长因子信号转导所必需的(Mori等人，2008年)。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，在初步研究中，R50E显著抑制了DLD-1结肠癌、Met-1高转移乳腺癌和Min-O癌前病变的体内生长。我们发现R50E的几个关键缺陷可能直接与其抑制作用有关：1)wt FGF1诱导FGFR-FGF-整合素三元复合体，但R50E不；2)wt FGF1诱导整合素23细胞质结构域的Tyr磷酸化，但R50E不；以及3)wt FGF1诱导持续的ERK1/2激活，但R50E不。因此，我们提出了一个模型，在这个模型中，整合素通过与成纤维细胞生长因子直接结合而被招募到FGFR-成纤维细胞生长因子复合体中，这将导致Tyr磷酸化和ERK1/2激活。R50E不会向FGFR复合体募集av？3，从而阻断了后续的信号事件。目的：分析整合素在成纤维细胞生长因子信号转导中的作用，利用R50E对现有的成纤维细胞生长因子信号转导模型进行重新评价，确定成纤维细胞生长因子-整合素相互作用是肿瘤和血管生成治疗的新靶点。具体目的：SA#1)确定整合素与成纤维细胞生长因子直接结合在成纤维细胞生长因子信号转导中的作用。A)利用悬浮在聚HEMA涂层表面的细胞，确定整合素-成纤维细胞生长因子相互作用和整合素-细胞外基质相互作用对成纤维细胞生长因子信号转导的相对贡献。B)使用新发现的候选小分子FGF1拮抗剂分析整合素与FGF1的直接结合在成纤维细胞生长因子信号转导中的作用。3)分析R50E对FGFR亚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测试R50E通过抑制肿瘤的启动、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肿瘤成纤维细胞生长因子信号转导的影响。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Min-O癌前病变的体内致癌作用。D)利用乳腺癌基因工程小鼠模型(Tg(MMTV-PYV-Mt))分析R50E在体内对肿瘤发生发展和肿瘤血管生成的影响。预期结果：拟议的实验将确定整合素在成纤维细胞生长因子信号转导中的作用，评估R50E在体内肿瘤发生和发展中的潜力，并确定R50E在体内的靶细胞类型。所获得的信息将加强我们对成纤维细胞生长因子信号和整合素-FGFR串扰的理解，并将有助于设计新的癌症和炎症治疗策略。公共卫生相关性：我们发现FGF1与整合素结合，并开发了一种不与整合素结合的FGF1突变体。我们发现，该突变体不仅在信号诱导方面存在缺陷，而且以显性-负性方式抑制wt FGF1诱导的成纤维细胞生长因子信号。我们将提出一种假设，即成纤维细胞生长因子与整合素的直接结合是成纤维细胞生长因子信号传导所必需的，并在转基因小鼠肿瘤模型中分析显性-阴性突变对肿瘤的发生、发展和血管生成的抑制作用。

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.