The Role of Integrin Affinity Modulation during Tumor Angiogenesis

项目总结： 病理性血管生成，即肿瘤形成过程中新血管的异常增殖和形成 最终导致“血管生成开关”，小的、无血管的肿瘤进入快速生长驱动状态 疾病的进展和转移。肿瘤微环境中的血管内皮细胞 响应分泌的血管内皮生长因子(VEGF)并在细胞外基质中导航 (ECM)在萌发血管生成的初始步骤中。整合素家族蛋白对新血管的调控 通过调节内皮细胞与ECM的黏附相互作用而生长，邻近的内皮细胞在 细胞间连接，并与基底膜连接。整合素的药理阻断有 在临床前模型中显示了强大的抗肿瘤作用，但这类抑制剂的临床试验 未能显示出III期疗效，表明需要更多地了解整合素信号转导。一个 整合素值得注意的特性是，它们与细胞外配体的亲和力是由内向外调节的。 发信号。Talin是一种细胞骨架接头蛋白，它在两个不同的位置与β-整合素细胞质尾巴结合， 导致构象变化，由此细胞外结构域延伸到ECM内与配体结合。 我们之前已经发表过，表达血小板特异性talin1突变体(Talin1 L325R)的小鼠在 其激活整合素的能力显示出血小板整合素激活的严重缺陷，从而导致出血 素质。为了验证塔林依赖的整合素激活是血管生成所必需的这一中心假设 (目的#1)，我们已经获得了可诱导的内皮细胞特异性talin1L325R敲入小鼠(图1)。诱导 Talin1 L325R在出生后发育过程中的表达导致视网膜早期死亡和广泛的缺陷 血管生成(图2)。有趣的是，使用B16/F10小鼠黑色素瘤模型的初步工作显示 Talin1 L325R小鼠和talin1 WT小鼠的原发肿瘤体积缩小(图3) 在先前存在的血管系统中没有表现出明显的缺陷(图5)，这表明Talin的潜在特异性- 依赖整合素激活促进新生血管发育。我们建议测试达林的要求- 介导的整合素激活与原发肿瘤生长和肿瘤血管生成。奇怪的是，几个实验室 已经报道整合素信号促进血管内皮生长因子/血管内皮生长因子R2信号，这是一条负责 实体瘤进展过程中的血管生成。最近的研究表明，对血管内皮细胞的刺激 血管内皮生长因子诱导α-v-β-3-VEGFR2大分子复合体，增强血管内皮生长因子信号转导。在目标2中，我们将 血管内皮生长因子介导的体内血管生成对整合素活化的要求及整合素 VEGF/VEGFR2下游信号转导通路的激活是必需的。这项工作将提供重要的 血管生成中内皮细胞内向外整合素信号转导与检测的关系 Talin依赖的内向外信号在整合素和血管内皮细胞生长因子信号之间的相互作用。

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.