Angiogenesis and Tumor Growth

我们重点关注了4个相关领域。1)我们继续探索Notch1和Notch4的内皮特异性膜结合配体Delta4 （Dll4）作为内皮细胞功能调节剂的作用。Dll4在发育中的内皮细胞中选择性表达，是正常血管发育所必需的。出生后，Dll4在血管生成内皮中表达，特别是在肿瘤血管中。我们生成过表达Dll4蛋白的原代内皮细胞，发现Dll4在VEGF-A作用下降低内皮细胞的增殖和迁移反应。我们发现，在过表达dll4的内皮细胞中，VEGF受体2和神经肽-1 （neuropilin-1, Npn-1）表达降低是导致VEGF- a生物反应降低的因素。与Dll4通过Notch信号传导一致，我们发现在过表达Dll4的内皮细胞中显著诱导转录因子HEY2的表达，并且γ分泌酶抑制剂显著重建了被Dll4抑制的内皮细胞增殖。因此，这些研究已经确定Notch配体Dll4是VEGF-A生物活性的选择性抑制剂，可下调VEGF-A主要信号受体VEGFR-2和共受体Npn-1。在利用临床前癌症模型的其他实验中，我们探索了利用Dll4作为内皮细胞中Notch信号的激活剂来抑制血管生成和肿瘤生长的可能性。在小鼠异种和同基因肿瘤模型中，我们已经证明Dll4可以显著减少肿瘤血管生成和淋巴源性肿瘤的生长。对Dll4抗肿瘤作用机制的研究表明，这些作用至少部分归因于肿瘤微环境和肿瘤血管中的Notch激活，导致VEGFR2表达减少和肿瘤血液灌注减少。我们观察到，许多实验性癌症和其他类型的肿瘤对肿瘤血管中Dll4/Notch信号的抑制作用没有反应，这就提出了有关其潜在机制的重要问题。2. 为了阐明潜在的潜在机制，我们的研究重点是表征肿瘤相关Dll4/其他Notch配体的作用，以及肿瘤相关Notch受体的作用。在这些实验中，我们发现了Dll4/Notch/TGF-b1信号网络，将肿瘤浸润性骨髓细胞与实验性肺癌进展联系起来。我们发现，被肿瘤源性细胞因子CCL2和M-CSF吸引到肿瘤微环境的髓系细胞表达Notch配体Dll4水平升高，从而激活肿瘤细胞中的Notch信号并放大肿瘤固有的Notch激活。肿瘤细胞中Dll4/Notch信号的升高放大了tgf -b诱导的pSMAD2/3信号，这是维持tgf -b诱导的肿瘤细胞生长所必需的。相反，Notch阻断可减少TGF-b信号传导并限制肺癌的进展。为了证实这些发现，我们发现人类头颈部鳞状癌临床标本中的肿瘤和邻近正常组织提供了TGF-b/Notch串扰有助于肿瘤进展的证据。因此，这个髓细胞-癌信号网络揭示了肿瘤微环境和肿瘤生长之间的一种新的机制联系，这种联系以Notch信号为中心，但不涉及内皮细胞/肿瘤相互作用。3)我们继续研究ephrinB配体及其EphB受体如何在新形成的血管中协调内皮/内皮/周细胞组装。EphrinB配体是表面结合的；因此b型Eph/Ephrin相互作用中的受体-配体相互作用涉及邻近细胞。B ephrin除了激活其同源EphB受体外，当受体通过“反向信号传导”参与时，B ephrin还可以作为信号分子发挥作用。Eph受体是酪氨酸激酶与其膜锚定的ephrin配体相互作用。在我们之前的研究中，我们已经证明内皮细胞中Eph B受体的信号传导对血管结构的组装至关重要。我们现在已经研究了Eph/ephrin信号在内皮细胞存活调节中的潜在作用。我们发现，沉默EphrinB的表达或酪氨酸磷酸化缺陷突变体EphrinB的表达（包含所有酪氨酸残基的替代，阻止尾部磷酸化，并作为内源性WT ephrin的显性阴性抑制剂）会导致内皮细胞死亡。这种结果不能通过添加外源性VEGFA或FGF2来阻止。生化和遗传学实验表明，JNK3/MAPK10介导了这种死亡，EphrinB2酪氨酸磷酸化依赖的信号通路是MAPK10/JNK3表达的调节剂。因此，JNK3的沉默可以防止EphrinB信号缺陷的内皮细胞的细胞死亡。与这些结果一致的是，EphrinB2基因缺陷小鼠的视网膜血管系统经历与JNK3激活相关的细胞死亡，JNK3缺陷小鼠表现出与EphrinB2信号缺陷相对应的眼部血管缺陷。这些结果为支持EphrinB作为抑制血管生成的治疗靶点的作用提供了额外的证据。4)我们对信号蛋白6a （Sema6A）在血管内皮中的潜在活性进行了早期观察。我们现在发现跨膜Sema6A在内皮细胞中表达，并响应外源性和内源性VEGF，通过调节VEGFR2信号调节内皮细胞的生存和生长，并通过自分泌VEGFR信号维持内皮细胞的活力。Sema6A在原代内皮细胞中的沉默会促进细胞死亡，而外源性VEGF- a或FGF2无法挽救细胞死亡，这是由于内源性VEGF促生存信号的丧失。具有Sema6A零突变的小鼠在与内皮细胞死亡增加相关的玻璃体血管复杂性和视网膜血管发育异常减少方面表现出显著缺陷。与对照组相比，sema6a缺失的成年小鼠表现出肿瘤、基质细胞和脉络膜血管生成的减少。在这些研究之前，Sema6A已知在神经系统发育中起重要作用。我们现在已经发现它也调节血管发育和成人血管生成。

We have focused on 4 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 neuropilin-1 (Npn-1) expression as the factors responsible for reduced biological responses to VEGF-A in Dll4-overexpressing endothelial cells. 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 inhibitory effects of Dll4/Notch signaling in the tumor vasculature, raising important questions relative to the underlying mechanisms. 2. To clarify potential underlying mechanisms, our studies are focused on characterizing the role of tumor-associated Dll4/other Notch ligands, and the roles of tumor-associated Notch receptors. In these experiments, we have uncovered a network of Dll4/Notch/TGF-b1 signals that links tumor-infiltrating myeloid cells to experimental lung cancer progression. We find that myeloid cells that were attracted to the tumor microenvironment by the tumor-derived cytokines CCL2 and M-CSF expressed increased levels of the Notch ligand Dll4, thereby activating Notch signaling in the tumor cells and amplifying tumor-intrinsic Notch activation. Heightened Dll4/Notch signaling in the tumor cells magnified TGF-b-induced pSMAD2/3 signaling and was required to sustain TGF-b-induced tumor cell growth. Conversely, Notch blockade reduced TGF-b signaling and limited lung carcinoma tumor progression. Corroborating these findings, we found that tumor and adjacent normal tissue in clinical specimens of human head and neck squamous carcinoma provide evidence that TGF-b/Notch crosstalk contributes to tumor progression. Thus, this myeloid cell-carcinoma signaling network uncovers a novel mechanistic link between the tumor microenvironment and tumor growth that is centered on Notch signaling, but does not involve endothelium/tumor interaction. 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. This outcome cannot be 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, and JNK3-deficient mice display ocular vascular defects that mirror those of EphrinB2 signaling deficiency. 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. 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.