Angiogenesis and Tumor Growth

血管生成和肿瘤生长

• 批准号: 8554045
• 负责人: Giovanna Tosato
• 金额: $ 65.09万
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8554045
• 关键词: Adult; Affect; Angiogenesis Inhibition; Angiogenesis Inhibitors; Angiogenic Factor; Area; Binding; Biochemical; Biochemical Genetics; Biological; Blood Vessels; Cancer Model; Carcinoma; Cell Adhesion; Cell Death; Cell Proliferation; Cell Survival; Cell physiology; Cell surface; Cells; Cessation of life; Clinic; Conflict (Psychology); Defect; Development; Dominant-Negative Mutation; Drug Delivery Systems; Endothelial Cells; Endothelium; Eph Family Receptors; Ephrin B Receptor; Ephrins; Exhibits; FGF2 gene; Goals; Heparin Binding; Hybrids; In Vitro; Investigation; Knockout Mice; Knowledge; Ligand Binding; Ligands; MAPK10 gene; Malignant Neoplasms; Mediating; Membrane; Modeling; Mus; Myeloid Cells; Names; Neurons; Neuropilin-1; Nucleotides; Oligonucleotides; Outcome; Pericytes; Pharmaceutical Preparations; Phosphorylation; Play; Polysaccharides; Protein Family; Receptor Signaling; Regulation; Resistance; Retinal; Retinal Neovascularization; Role; Semaphorin-3; Semaphorin-3A; Semaphorins; Signal Transduction; Signaling Molecule; Structure; Surface; Tail; Testing; Therapeutic; Tissues; Tumor Angiogenesis; Tumor-Derived; Tyrosine; Tyrosine Phosphorylation; VEGFA gene; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factors; Vascular Endothelium; angiogenesis; autocrine; base; blood perfusion; cancer therapy; cell growth; choroidal angiogenesis; delta protein; gamma secretase; in vivo Model; inhibitor/antagonist; lymphoid neoplasm; matrigel; mutant; neoplastic cell; nervous system development; notch protein; null mutation; overexpression; pre-clinical; prevent; receptor; reconstitution; research study; response; retina blood vessel structure; selective expression; sensor; therapeutic target; tool; transcription factor; tumor; tumor growth

We have focused on 3 related areas. 1) We have continued to explore the role of Delta4 (Dll4), an endothelial-specific membrane-bound ligand for Notch1 and Notch4, as a regulator of endothelial cell function. Dll4 is selectively expressed in the developing endothelium and is required for normal vascular development. Post-natally, Dll4 is expressed in the angiogenic endothelium, particularly in the tumor vasculature. We generated primary endothelial cells overexpressing Dll4 protein, and found that Dll4 reduces endothelial cell proliferative and migratory responses in response to VEGF-A. We identified reduced VEGF receptor 2 and Npn-1 expression in Dll4-overexpressing endothelial cells as responsible for reduced biological responses to VEGF-A. Consistent with Dll4 signaling through Notch, we found that expression of the transcription factor HEY2 was significantly induced in Dll4-overexpressing endothelial cells, and a gamma secretase inhibitor significantly reconstituted endothelial cell proliferation inhibited by Dll4. Thus, these studies have identified the Notch ligand Dll4 as a selective inhibitor of VEGF-A biologic activities down-regulating the principal VEGF-A signaling receptor, VEGFR-2 and co-receptor Npn-1. In additional experiments utilizing pre-clinical cancer models, we have explored the possibility of utilizing Dll4 as an activator of Notch signaling in endothelial cells to inhibit angiogenesis and tumor growth. In xenogeneic and syngeneic tumor models established in mice, we have documented that Dll4 can markedly reduce tumor angiogenesis and the growth of tumors of lymphoid origin. Studies of the mechanisms for the anti-tumor effects of Dll4 have shown that these are attributable at least in part, to Notch activation in the tumor microenvironment and in the tumor vasculature resulting in reduced VEGFR2 expression and reduced tumor blood perfusion. We have observed that a number of experimental carcinomas and other tumor types are unresponsive to the inhibitor effects of Dll4/Notch signaling in the tumor vasculature. Current studies are focused on characterizing the role of tumor-associated Dll4, and other Notch ligands, as well as the roles of tumor-associated Notch receptors. In these experiments, we are analyzing the effects of Notch ligand expression by tumor cells on angiogenic sprouting of tumor vessels. In addition, we are exploring the pro-angiogenic effects of Notch activation in the tumor cells induced by environmentally-expressed Notch ligands. Conversely, we are analyzing the pro-angiogenic effects of Notch signaling in tumor-infiltrating myeloid cells induced by tumor-derived Notch ligands. Our goal is to define the mechanisms of responsiveness and resistance to anti-tumor therapeutic approaches based on Dll4/Notch signaling in the tumor vasculature. 2) We have explored the role of neuropilin-1 (Npn1) as a receptor shared by heparin-binding forms of vascular endothelial growth factor (VEGF) and class 3 semaphorins, protein families that regulate endothelial and neuronal function, respectively. Previous studies have shown that ligand binding to Npn1 dictates the choice of signal transduction; plexins tranduce semaphorin signaling and VEGF receptors transduce VEGF signaling. We have now examined the mechanisms underlying Npn1 binding to VEGF or Sema3A, and how the engagement of Npn1 by Sema3A affects endothelial cell function. We have identified Sema3A as an inhibitor of endothelial cell adhesion, survival and proliferation and formation of vascular-like structures. Furthermore, we have found that Npn1-binding forms of VEGF block all these activities of Sema3A. VEGF-A can compete with Sema3A for endothelial cell binding, and can promote Npn-1 internalization from the cell surface. Biochemical analysis of VEGF-A binding to endothelial cells revealed that Npn1 internalization requires ligand bridging of Npn1 and VEGF receptors, and that Sema3A can promote Npn1 internalization, but requires a significantly higher concentration than VEGF-A. Thus, our results unveil an essential role for Npn1 as a sensor and priority setter for endothelial cell responses to conflicting signals. In additional studies, we have explored the possibility of targeting Npn1 for internalization as a tool to regulate endothelial cell responses to VEGF. In so doing, we have identified a group of polysaccharides, oligonucleotides, and other hybrid molecules that can induce Npn1 internalization and can thus serve as inhibitors of angiogenesis. We have named these compounds as "internalization inducers". Such internalization-inducing compounds could be useful as therapeutics to reduce angiogenesis. One such synthetic compound,an oligoguanosine nucleotide, has shown clear efficacy both in vitro and in an in vivo model of retinal neovascularization. 3) We have continued investigations on how ephrinB ligands and their EphB receptors orchestrate endothelial/endothelial/pericyte assembly in newly-formed vessels. EphrinB ligands are surface-bound; thus receptor-ligand interactions in the B-type Eph/Ephrin interactions involve adjacent cells. In addition to activating their cognate EphB receptors, B Ephrins can function as signaling molecules when engaged by the receptor through "reverse signaling". Eph receptors are tyrosine kinases interacting with their membrane-anchored ephrin ligands. In our previous studies, we have demonstrated that signaling by Eph B receptors in endothelial cells is critical to assembly into vascular structures. We have now investigated the potential role of Eph/ephrin signaling in the regulation of endothelial cells survival. We have found that silencing EphrinB expression or expression of a tyrosine-phosphorylation-deficient mutant EphrinB (contains substitutions of all tyrosine residues that prevent tail phosphorylation and acts as a dominant-negative inhibitor of endogenous WT ephrin) causes endothelial cell death. Such outcome cannot e prevented by the addition of exogenous VEGFA or FGF2. Biochemical and genetic experiments have revealed that such death is mediated by JNK3/MAPK10, and that EphrinB2 tyrosine phosphorylation-dependent signaling serves as a modulator of MAPK10/JNK3 expression. Thus, the silencing of JNK3 prevents cell death in endothelial cells, which are EphrinB signaling-deficient. Consistent with these results, the retinal vasculature in mice genetically-deficient of EphrinB2 undergoes cell death in association with JNK3 activation. These results provide additional evidence supporting a role for EphrinB as a therapeutic target for inhibition of angiogenesis. 4)We have pursued earlier observations on the potential activities of semaphorin6A (Sema6A) in the vascular endothelium. We now found that transmembrane Sema6A is expressed in endothelial cells, and regulates endothelial cell survival and growth by modulating VEGFR2 signaling in response to exogenous and endogenous VEGF, which contributes to maintain endothelial cell viability by autocrine VEGFR signaling. The silencing of Sema6A in primary endothelial cells promotes cell death that is not rescued by exogenous VEGF-A or FGF2, attributable to the loss of pro-survival signaling from endogenous VEGF. Analyses of mouse tissues demonstrate that Sema6A is expressed in angiogenic and remodeling vessels. Mice with null mutations of Sema6A exhibit significant defects in hyaloid vessels complexity associated with increased endothelial cell death, and in retinal vessels development that is abnormally reduced. Adult Sema6A-null mice exhibit reduced tumor, Matrigel and choroidal angiogenesis compared to controls. Prior to these studies, Sema6A was known to play important roles in development of the nervous system. We have now discovered that it also regulates vascular development and adult angiogenesis.

我们专注于3个相关领域。 1）我们继续探索Delta4（DLL4）的作用，Delta4（DLL4）是Notch1和Notch4的内皮特异性膜结合的配体，作为内皮细胞功能的调节剂。 DLL4在发育中的内皮中有选择地表达，是正常血管发育所必需的。 在产后，DLL4在血管生成内皮中表达，尤其是在肿瘤脉管系统中。 我们产生了过表达DLL4蛋白的原发性内皮细胞，发现DLL4响应于VEGF-A响应于VEGF-A，可降低内皮细胞增殖和迁移反应。 我们确定在过表达DLL4的内皮细胞中降低的VEGF受体2和NPN-1表达是负责降低对VEGF-A的生物学反应。 与DLL4信号通过Notch一致，我们发现转录因子HEY2的表达在表达DLL4的内皮细胞中显着诱导，而DLL4抑制的γ泌尿酶抑制剂可显着重新组成的内皮细胞增殖。 因此，这些研究已将Notch配体DLL4确定为VEGF-A生物学活性的选择性抑制剂，从而下调了主VEGF-A信号受体，VEGFR-2和共受体NPN-1。 在利用临床前癌症模型的其他实验中，我们探索了将DLL4用作内皮细胞中Notch信号传导激活因子抑制血管生成和肿瘤生长的可能性。 在小鼠中建立的异构和合成性肿瘤模型中，我们已经证明DLL4可以显着降低肿瘤血管生成和淋巴样肿瘤的生长。 DLL4抗肿瘤作用机制的研究表明，这些机制至少部分归因于肿瘤微环境的缺口激活以及肿瘤脉管系统的凹口激活，从而导致VEGFR2表达降低和肿瘤血液灌注降低。我们已经观察到，许多实验性癌和其他肿瘤类型对DLL4/Notch信号在肿瘤脉管系统中的抑制剂效应没有反应。当前的研究集中在表征与肿瘤相关的DLL4和其他缺口配体以及与肿瘤相关的Notch受体的作用的作用。在这些实验中，我们正在分析肿瘤细胞的Notch配体表达对肿瘤血管血管生成的影响。 此外，我们正在探索Notch活化在环境表达的Notch配体诱导的肿瘤细胞中的促血管生成作用。 相反，我们正在分析Notch信号传导在肿瘤渗透的髓样细胞中由肿瘤衍生的Notch配体诱导的髓样细胞的促血管生成作用。 我们的目标是定义基于肿瘤脉管系统中DLL4/Notch信号的反应能力和抗抗肿瘤治疗方法的机制。 2）我们探索了神经蛋白-1（NPN1）作为血管内皮生长因子（VEGF）和3类闪光蛋白的受体，分别调节内皮和神经元功能的蛋白质家族的作用。 先前的研究表明，配体与NPN1结合决定了信号转导的选择。 plexins tranduce semaphorin信号传导和VEGF受体传递VEGF信号传导。 现在，我们已经检查了NPN1与VEGF或SEMA3A结合的机制，以及SEMA3A对NPN1的参与如何影响内皮细胞功能。 我们已经将SEMA3A确定为内皮细胞粘附，生存和增殖以及血管状结构的抑制剂。 此外，我们发现VEGF的NPN1结合形式阻止了SEMA3A的所有这些活动。 VEGF-A可以与SEMA3A竞争内皮细胞结合，并可以促进细胞表面的NPN-1内在化。 VEGF-A与内皮细胞结合的生化分析表明，NPN1内部化需要对NPN1和VEGF受体进行配体桥接，并且SEMA3A可以促进NPN1内在化，但需要比VEGF-A更高的浓度。 因此，我们的结果揭示了NPN1作为传感器和优先设置器的重要作用，用于内皮细胞对冲突信号的响应。 在其他研究中，我们探索了将NPN1靶向内部化的可能性，作为调节内皮细胞对VEGF的反应的工具。 这样一来，我们已经确定了一组可以诱导NPN1内在化的多糖，寡核苷酸和其他混合分子，因此可以用作血管生成的抑制剂。 我们将这些化合物称为“内部化诱导剂”。 这种内在化诱导化合物可以用作减少血管生成的治疗剂。 一种这样的合成化合物，一种寡素核苷核苷酸，在体外和视网膜新血管形成模型中都表现出明显的功效。 3）我们一直在调查ephrinb配体及其EPHB受体如何在新形成的血管中编排内皮/内皮/周细胞组件。 ephrinb配体是表面结合的。因此，B型EPH/Ephrin相互作用中的受体配体相互作用涉及相邻的细胞。 除了激活其同源EPHB受体外，当受体通过“反向信号传导”接合时，B源自bephrins还可以充当信号分子。 EPH受体是酪氨酸激酶与膜锚定的埃弗林配体相互作用。 在我们以前的研究中，我们已经证明，EPH B受体在内皮细胞中的信号传导对于组装成血管结构至关重要。现在，我们研究了EPH/Ephrin信号传导在调节内皮细胞存活中的潜在作用。我们发现，沉默的埃菲林表达或表达酪氨酸 - 磷酸化缺陷型突变体ephrinb（包含所有抑制所有酪氨酸残基的取代，这些酪氨酸残基可防止尾磷酸化并作为内源性WT Ephrin的主导性抑制剂）导致内皮细胞死亡。 通过添加外源VEGFA或FGF2，不能阻止这种结果。生化和遗传实验表明，这种死亡是由JNK3/MAPK10介导的，Ephrinb2酪氨酸磷酸化依赖性信号传导是MAPK10/JNK3表达的调节剂。 因此，JNK3的沉默阻止了内皮细胞中的细胞死亡，而内皮细胞是ephrinb信号缺乏的。与这些结果一致，埃菲林B2遗传缺陷的小鼠视网膜脉管系统与JNK3激活有关。 这些结果提供了支持Ephrinb作为抑制血管生成的治疗靶标的作用的其他证据。 4）我们已经对Semaphorin6a（Sema6a）在血管内皮细胞中的潜在活动进行了早期观察。现在，我们发现跨膜SEMA6A在内皮细胞中表达，并通过调节vegfr2信号传导对外源性和内源性VEGF调节内皮细胞的存活和生长，这有助于通过自动分泌VEGFR信号来维持内皮细胞的生存能力。 SEMA6A在原代内皮细胞中的沉默促进了未被外源性VEGF-A或FGF2挽救的细胞死亡，这归因于内源性VEGF的促生物性信号传导的丧失。 小鼠组织的分析表明，SEMA6A在血管生成和重塑血管中表达。 SEMA6A无效突变的小鼠在与内皮细胞死亡增加以及视网膜血管发育异常降低有关的透明血管复杂性中表现出明显的缺陷。 与对照组相比，成年SEMA6A无效小鼠表现出肿瘤，基质和脉络膜血管生成的减少。在进行这些研究之前，已知SEMA6A在神经系统的发展中起着重要作用。 现在，我们发现它也调节血管发育和成人血管生成。