The Role of Integrin Affinity Modulation during Tumor Angiogenesis

项目摘要： 病理性血管生成，肿瘤发生过程中异常增殖和新血管形成 在“血管生成开关”中达到高潮，由此小的无血管肿瘤进入快速生长驱动状态 疾病进展和转移。肿瘤微环境中的血管内皮细胞 对分泌的血管内皮生长因子（VEGF）作出反应， (ECM)在新生血管萌芽的最初阶段。整合素家族调节新生血管 通过调节内皮细胞与ECM的粘附相互作用， 细胞间连接和基底膜。整合素的药理学阻断具有 在临床前模型中证明了有效的抗肿瘤作用，但这类抑制剂的临床试验 未能显示III期疗效，表明需要更好地理解整合素信号传导。一 整联蛋白值得注意的特性是它们对细胞外配体的亲和力通过“由内而外” 发信号。Talin是一种细胞骨架衔接蛋白，在两个不同的位点与β-整联蛋白胞质尾区结合， 导致构象变化，由此胞外结构域延伸到ECM中以接合配体。 我们以前曾发表过，表达血小板特异性talin 1突变体（talin 1 L325 R）的小鼠， 其活化整联蛋白的能力在血小板整联蛋白活化中表现出严重缺陷，导致出血 素质为了验证中心假设，即血管生成需要talin依赖的整合素激活 (Aim#1），我们已经产生了可诱导的内皮细胞特异性talinlL 325 R敲入小鼠（图1）。诱导 talin 1 L325 R在出生后发育中的表达导致早期死亡和视网膜广泛缺损。 血管生成（图2）。有趣的是，使用B16/F10鼠黑色素瘤模型的初步工作显示， talin 1 L325 R小鼠中原发性肿瘤大小的减少（图3），而talin 1 L325 R和talin 1 WT小鼠 在预先存在的脉管系统中没有表现出可观察到的缺陷（图5），表明talin的潜在特异性。 依赖于整合素激活新生血管发育。我们建议测试塔林的要求- 介导的整合素活化用于原发性肿瘤生长和肿瘤血管生成。奇怪的是，几个实验室 已经报道了整合素信号促进VEGF/VEGFR 2信号，这是一种负责 实体瘤进展过程中的血管生成。最近的研究表明，刺激内皮细胞， VEGF诱导增强VEGF信号传导的αvβ3-VEGFR 2大分子复合物。在目标2中，我们将 测试VEGF介导的体内血管生成过程中整合素活化的需要，以及整合素是否 活化对于下游VEGF/VEGFR 2信号转导是必需的。这项工作将提供重要 深入了解血管生成中内皮细胞由内而外的整合素信号传导之间的关系， talin依赖的inside-out信号传导在整合素和VEGF信号传导之间的串扰中的作用。

Project Summary: Pathological angiogenesis, the aberrant proliferation and formation of new blood vessels, during tumorigenesis culminates in an “angiogenic-switch” whereby small, avascular tumors enter a state of rapid growth driving disease progression and metastasis. Endothelial cells lining blood vessels in the tumor microenvironment respond to secreted vascular endothelial growth factor (VEGF) and navigate through the extracellular matrix (ECM) during the initial steps of sprouting angiogenesis. The integrin family of proteins modulate new vessel growth by regulating endothelial cell adhesive interactions with the ECM, neighboring endothelial cells at intercellular junctions, and with the basement membrane. Pharmacological blockade of integrins has demonstrated potent anti-tumorigenic effects in pre-clinical models, but clinical trials of this class of inhibitors have failed to show phase-III efficacy demonstrating a need for a greater understanding of integrin signaling. A noteworthy property of integrins is that their affinity for extracellular ligands is regulated through “inside-out” signaling. Talin, a cytoskeletal adaptor protein, binds the β-integrin cytoplasmic tail at two distinct sites, resulting in a conformational change whereby the extracellular domain extends into the ECM to engage ligand. We have previously published that mice expressing a platelet-specific talin1 mutant (talin1 L325R) defective in its capacity to activate integrins exhibit severe defects in platelet integrin activation that lead to a bleeding diathesis. To test the central hypothesis that talin-dependent integrin activation is required for angiogenesis (Aim #1), we have generated inducible endothelial cell-specific talin1 L325R knock-in mice (Fig 1). Induction of talin1 L325R expression during postnatal development caused early lethality and extensive defects in retinal angiogenesis (Fig 2). Interestingly, preliminary work using a B16/F10 murine melanoma model showed reductions in primary tumor size in talin1 L325R mice (Fig 3) while both talin1 L325R and talin1 WT mice exhibited no observable defects in pre-existing vasculature (Fig 5) suggesting the potential specificity of talin- dependent integrin activation for nascent vessel development. We propose to test the requirement of talin- mediated integrin activation for primary tumor growth and tumor angiogenesis. Curiously, several laboratories have reported integrin signaling promotes VEGF/VEGFR2 signaling, a central pathway responsible for angiogenesis during solid-tumor progression. Recent work suggests that stimulation of endothelial cells with VEGF induces an αvβ3-VEGFR2 macromolecular complex that potentiates VEGF signaling. In Aim 2, we will test the requirement of integrin activation during VEGF-mediated angiogenesis in vivo and whether integrin activation is necessary for downstream VEGF/VEGFR2 signal transduction. This work will provide important insight into the relationship between endothelial cell inside-out integrin signaling in angiogenesis and examine the role of talin-dependent inside-out signaling in cross-talk between integrins and VEGF signaling.