Angiogenesis and Tumor Growth

血管生成和肿瘤生长

基本信息

批准号：
10487194
负责人：
Giovanna Tosato
金额：
$ 85.57万
依托单位：
DIVISION OF CLINICAL SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/10487194
关键词：
2019-nCoV ACE2 Acquired Immunodeficiency Syndrome Activator Appliances Angiogenesis Inhibition Angiogenesis Inhibitors Angiogenic Factor Angiopoietin-2 Area Biochemical Biochemical Genetics Blood Cells Blood Vessels Cancer Cell Growth Cancer Model Cell Death Cell Survival Cell physiology Cells Cessation of life Characteristics Clinic Contact Inhibition DLEC1 gene Defect Development Dominant-Negative Mutation Endothelial Cells Endothelial Growth Factors Receptor Endothelium Eph Family Receptors Ephrin B Receptor Ephrins Epigenetic Process Experimental Models FGF2 gene Generations Genetic Goals Growth Hemangiosarcoma Hematopoiesis Herpesviridae Heterogeneity Human Human Herpesvirus 4 Human Herpesvirus 8 In Vitro Individual Infection Intestines Investigation KDR gene Kaposi Sarcoma Knowledge Ligands Link Literature MAPK10 gene Maintenance Malignant - descriptor Malignant Neoplasms Mediating Mediator of activation protein Membrane Metabolic Mouse Cell Line Mus NF-kappa B Neoplasm Metastasis Neuropilin-1 Nuclear Nutrient Outcome Oxygen PTPN11 gene Pathway interactions Pericytes Pharmaceutical Preparations Phenotype Phosphorylation Physiological Proteins Receptor Signaling Regulation Research Resistance Role Signal Transduction Signaling Molecule Skin Source Starvation Stress Surface TEK gene TIE-2 Receptor TNF gene TNFSF15 gene Tail Testing Tissues Transcription Coactivator Tumor Angiogenesis Tumor Expansion Tumor Suppressor Proteins Tyrosine Tyrosine Phosphorylation VEGFA gene Vascular Endothelial Growth Factors Vascular Endothelium Verteporfin angiogenesis antitumor effect base cancer therapy chemokine cytokine density experimental study in vivo inhibitor/antagonist interest lymphoid neoplasm mouse development mouse model mutant neoplastic cell new therapeutic target notch protein pre-clinical prevent receptor response selective expression stem cell niche targeted cancer therapy therapeutic target treatment response tumor tumor growth tumor microenvironment tumor progression tumorigenesis vessel regression wound healing

项目摘要

We have focused on 4 related areas. 1) Our previous studies have identified a critical 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 have found that Dll4 is a negative regulator of angiogenesis, as it functions as a selective inhibitor of VEGF-A by down-regulating the principal VEGF-A signaling receptor, VEGFR-2 and co-receptor neuropilin-1 (Npn-1). In pre-clinical cancer models, we have documented that Dll4 can markedly reduce tumor angiogenesis and the growth of tumors of lymphoid origin by reducing VEGF-A responses in the tumor vascular endothelium. In related experiments, we have begun to explore the role of the Notch ligand JAG2 in angiogenesis. To this end, we have developed and continue to perfect a new mouse model of JAG2-deficiency and explored the potential contribution of Notch-dependent and Notch-independent pathways in endothelial cell function and angiogenesis. Preliminary results provide strong evidence for a role of Jag2 in the maintenance of the endothelial intestinal stem cell niche. 2) We have continued investigations on how ephrinB ligands and their EphB receptors orchestrate endothelial/endothelial/pericyte assembly in the vasculature. EphrinB ligands are surface-bound; receptor-ligand interactions in the B-type Eph/Ephrin interactions involve adjacent cells (trans) or can occur in the same cell (cis). In addition to activating their cognate EphB receptors, B-type 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. We have 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 signaling, 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 that are EphrinB signaling-deficient. Consistent with these results, the hyaloid vasculature in mice genetically-deficient of EphrinB2 undergoes increased 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 evidence supporting a role for EphrinB signaling as an endothelial pro-survival pathway and a therapeutic target for inhibition of angiogenesis. Based on this evidence, we have further explored the possibility of targeting EphrinB2 signaling in the tumor vasculature to induce vessel regression and promote tumor cell starvation of collapse. 3) Pursuing this observation, we have explored different approaches to block EphrinB2-derived pro-survival signals in the vasculature. We have identified the phosphatase SHP2 as an essential mediator of EphrinB2 prosurvival functions in endothelial cells. Further, we have identified the SHP2 allosteric inhibitor, SHP099 as a potent inhibitor of phospho-EphrinB2-STAT signaling and a selective inducer of endothelial cell death in vitro and in vivo. We have characterized the signaling consequences of SHP2 inhibition in endothelial cells and exploited this information to selectively target the tumor vasculature rather than the tumor cells. Furthermore, we have identified endothelial phosphorylation of TIE2 (receptor for Angiopoietins 1 and 2) as a source of endothelial EphrinB2 phosphorylation in cis. Based on this information we have analyzed the combined anti-angiogenic activity of SHP2 and TIE2 blockade using SHP099 in combination with the peptibody AMG386; 4) In earlier observations we have linked the loss of the tumor-suppressor protein DLC1 with increased survival in primary endothelial cells under conditions of stress. We now discovered that DLC1 is a critical regulator of cell contact inhibition of proliferation in primary human endothelial cells, promoting cell death when the cells reach high density. DLC1 depletion confers a pro-survival phenotype to confluent, but not sparse endothelial cells, attributable to increased NF-kB activation associated with increased tumor necrosis factor alpha-induced protein 3 (TNFAIP3/A20) signaling. Consistent with a role of DLC1 depletion in endothelial cell tumorigenesis, we found that DLC1 is abnormally low and TNFAIP3/A20 is abnormally high in human angiosarcoma. Experimental treatment with the NF-kB inhibitor Tanespimycin/17-AAG significantly reduced angiosarcoma tumor growth in mouse. These results identify DLC1 as a previously unrecognized regulator of endothelial cell contact inhibition of proliferation that is depleted in angiosarcoma, and provide evidence supporting the targeting of NF-kB for the treatment of angiosarcoma where DLC1 is lost. 5) Based on the observation that the tumor suppressor DLC1 protein (our results) and the transcriptional co-activator YAP (literature) regulate cell-contact inhibition of growth, we have explored biochemical interactions between DLC1 and YAP. We found that DLC1 is a regulator of YAP and that the transcriptional co-activator function of YAP are required for the loss of cell-contact inhibition manifested by DLC1-deficient endothelial cells. If YAP is depleted from endothelial cells, DLC1-depleted endothelial cells stop growing when confluent and fail to pile-up. These results in vitro were corroborated by the observation that angiosarcoma tissues contain a significant proportion of DLC1-negative malignant endothelial cells where YAP is nuclear and active. This is not the case in the normal skin vasculature adjacent to the tumor. Verteporfin, an inhibitor of YAP, displayed a clear anti-tumor effect in an experimental model of angiosarcoma in mice. 6) Additional ongoing experiments on the role of DLC1 in endothelial cells have prompted the generation of an endothelial-specific inducible DLC1-deficient mouse cell line. We are currently evaluating the role of such deficiency in different contexts, including mouse development, physiologic endothelial cell functions in hematopoiesis, wound healing and cancer.

我们重点关注 4 个相关领域。 1) 我们之前的研究已经确定了 Delta4 (Dll4)（Notch1 和 Notch4 的内皮特异性膜结合配体）作为内皮细胞功能调节剂的关键作用。 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，特别是在肿瘤脉管系统中。我们发现 Dll4 是血管生成的负调节因子，因为它通过下调主要 VEGF-A 信号传导受体 VEGFR-2 和辅助受体 Neuropilin-1 (Npn-1) 作为 VEGF-A 的选择性抑制剂。。在临床前癌症模型中，我们已经证明Dll4可以通过减少肿瘤血管内皮中的VEGF-A反应来显着减少肿瘤血管生成和淋巴源性肿瘤的生长。在相关实验中，我们已经开始探索Notch配体JAG2在血管生成中的作用。为此，我们开发并继续完善JAG2缺陷的新小鼠模型，并探索Notch依赖性和Notch非依赖性途径在内皮细胞功能和血管生成中的潜在贡献。初步结果为 Jag2 在维持内皮肠干细胞生态位中的作用提供了强有力的证据。 2) 我们继续研究 ephrinB 配体及其 EphB 受体如何协调脉管系统中的内皮/内皮/周细胞组装。 EphrinB 配体是表面结合的； B 型 Eph/Ephrin 相互作用中的受体-配体相互作用涉及相邻细胞（反式）或可以发生在同一细胞中（顺式）。除了激活其同源 EphB 受体外，B 型肝配蛋白在通过“反向信号”与受体结合时还可以充当信号分子。 Eph 受体是酪氨酸激酶，与其膜锚定的肝配蛋白配体相互作用。我们研究了 Eph/ephrin 信号传导在内皮细胞存活调节中的潜在作用。我们发现，沉默 EphrinB 表达或酪氨酸磷酸化缺陷突变体 EphrinB（包含所有酪氨酸残基的取代，防止尾部磷酸化并充当内源性 WT ephrin 的显性失活抑制剂）的表达会导致内皮细胞死亡。添加外源 VEGFA 或 FGF2 无法阻止这一结果。生化和遗传实验表明，这种死亡是由 JNK3/MAPK10 信号传导介导的，并且 EphrinB2 酪氨酸磷酸化依赖性信号传导可作为 MAPK10/JNK3 表达的调节剂。因此，JNK3 的沉默可防止 EphrinB 信号传导缺陷的内皮细胞死亡。与这些结果一致的是，EphrinB2 基因缺陷的小鼠的玻璃体脉管系统经历了与 JNK3 激活相关的细胞死亡增加，而 JNK3 缺陷的小鼠表现出与 EphrinB2 信号传导缺陷相似的眼部血管缺陷。这些结果提供了支持 EphrinB 信号传导作为内皮促存活途径和抑制血管生成的治疗靶点的作用的证据。基于这一证据，我们进一步探讨了靶向肿瘤血管系统中的 EphrinB2 信号传导以诱导血管消退并促进肿瘤细胞饥饿崩溃的可能性。 3)根据这一观察，我们探索了不同的方法来阻断脉管系统中 EphrinB2 衍生的促生存信号。我们已经确定磷酸酶 SHP2 是内皮细胞中 EphrinB2 促生存功能的重要介质。此外，我们还鉴定出 SHP2 变构抑制剂 SHP099 是磷酸 EphrinB2-STAT 信号传导的有效抑制剂，也是体外和体内内皮细胞死亡的选择性诱导剂。我们已经表征了内皮细胞中 SHP2 抑制的信号转导结果，并利用这些信息选择性地靶向肿瘤脉管系统而不是肿瘤细胞。此外，我们还发现 TIE2（血管生成素 1 和 2 的受体）的内皮磷酸化是顺式内皮 EphrinB2 磷酸化的来源。基于这些信息，我们使用 SHP099 与肽体 AMG386 组合分析了 SHP2 和 TIE2 阻断的联合抗血管生成活性； 4) 在早期的观察中，我们将肿瘤抑制蛋白 DLC1 的缺失与原代内皮细胞在应激条件下存活率的增加联系起来。我们现在发现DLC1是原代人内皮细胞增殖的细胞接触抑制的关键调节因子，当细胞达到高密度时促进细胞死亡。 DLC1 耗竭赋予融合的内皮细胞促生存表型，但不是稀疏的内皮细胞，这归因于与肿瘤坏死因子 α 诱导蛋白 3 (TNFAIP3/A20) 信号传导增加相关的 NF-kB 激活增加。与 DLC1 缺失在内皮细胞肿瘤发生中的作用一致，我们发现在人血管肉瘤中 DLC1 异常低，TNFAIP3/A20 异常高。使用 NF-kB 抑制剂 Tanespimycin/17-AAG 进行的实验治疗显着减少了小鼠血管肉瘤的生长。这些结果确定 DLC1 是一种以前未被识别的内皮细胞接触抑制增殖调节因子，在血管肉瘤中缺失，并提供了支持靶向 NF-kB 治疗 DLC1 缺失的血管肉瘤的证据。 5）基于肿瘤抑制DLC1蛋白（我们的结果）和转录共激活因子YAP（文献）调节细胞接触抑制生长的观察，我们探索了DLC1和YAP之间的生化相互作用。我们发现 DLC1 是 YAP 的调节因子，并且 YAP 的转录共激活子功能是 DLC1 缺陷的内皮细胞表现出的细胞接触抑制丧失所必需的。如果内皮细胞中的 YAP 被耗尽，DLC1 耗尽的内皮细胞在汇合时就会停止生长并且无法堆积。血管肉瘤组织含有相当比例的 DLC1 阴性恶性内皮细胞，其中 YAP 位于细胞核且具有活性，这一观察结果证实了这些体外结果。在肿瘤附近的正常皮肤脉管系统中情况并非如此。维替泊芬是一种 YAP 抑制剂，在小鼠血管肉瘤实验模型中显示出明显的抗肿瘤作用。 6) 关于 DLC1 在内皮细胞中的作用的其他正在进行的实验促进了内皮特异性可诱导 DLC1 缺陷小鼠细胞系的产生。我们目前正在评估这种缺陷在不同情况下的作用，包括小鼠发育、造血、伤口愈合和癌症中的生理内皮细胞功能。