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.

我们专注于4个相关领域。 1）我们以前的研究确定了Delta4（DLL4）的关键作用，Delta4是Notch1和Notch4的内皮特异性膜结合的配体，是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。我们发现DLL4是血管生成的阴性调节剂，因为它通过下调主VEGF-A信号受体，VEGFR-2和共受体神经蛋白神经蛋白-1（NPN-1）来充当VEGF-A的选择性抑制剂（NPN-1）。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来起源肿瘤的生长。在相关的实验中，我们开始探讨Notch配体JAG2在血管生成中的作用。为此，我们已经开发并继续完善了一种新的JAG2缺陷的小鼠模型，并探讨了Notch依赖性和与Notch独立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在维持内皮肠道干细胞生态位维持中的作用提供了有力的证据。 2）我们一直在调查ephrinb配体及其EPHB受体如何在脉管系统中编排内皮/内皮/周细胞组件。 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），也可以发生在同一细胞（CIS）中。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型以Ephrins可以用作信号分子。 EPH受体是酪氨酸激酶与膜锚定的埃弗林配体相互作用。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。通过添加外源性VEGFA或FGF2，不能预防这种结果。生化和遗传实验表明，这种死亡是由JNK3/MAPK10信号传导介导的，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Ephrinb信号缺陷的内皮细胞中的细胞死亡。与这些结果一致，以弗林布2的遗传缺陷型的透明脉管系统与JNK3激活相关的细胞死亡增加，而JNK3缺陷的小鼠表现出眼部血管缺陷，反映了ephrinb2信号缺乏症的细胞缺陷。这些结果提供了支持ephrinb信号传导作用的证据。基于这些证据，我们进一步探讨了靶向肿瘤脉管系统中ephrinb2信号传导的可能性，以诱导血管退化并促进肿瘤细胞塌陷的饥饿。 3）追求这一观察结果，我们探索了不同的方法来阻止脉管系统中源自Ephrinb2衍生的亲寿使信号。我们已经将磷酸酶SHP2鉴定为内皮细胞中埃菲林布2肌作用的必不可少的介体。此外，我们已经将SHP2变构抑制剂SHP099鉴定为磷酸化磷-Ephrinb2-Stat信号传导的有效抑制剂，并且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抑制作用的信号传导后果，并利用了这些信息，以选择性地靶向肿瘤脉管系统而不是肿瘤细胞。此外，我们已经确定了TIE2（血管生成素1和2的受体）的内皮磷酸化是CIS中内皮埃菲林B2磷酸化的来源。基于这些信息，我们分析了SHP2和TIE2封闭的抗血管生成活性，并结合了peptibody AMG386。 4）在较早的观察中，我们将肿瘤抑制剂蛋白DLC1的丧失与在压力条件下的原代内皮细胞中的存活增加联系起来。我们现在发现，DLC1是原代人内皮细胞增殖的关键调节剂，当细胞达到高密度时促进细胞死亡。 DLC1的耗竭将促生存的表型赋予汇合，但不是稀疏的内皮细胞，这是由于与肿瘤坏死因子α诱导的蛋白3（TNFAIP3/A20）信号有关的NF-KB激活增加而造成的。与DLC1耗竭在内皮细胞肿瘤发生中的作用一致，我们发现DLC1异常低，而TNFAIP3/A20在人类肉瘤中异常高。 NF-KB抑制剂tanespimycin/17-AAG的实验治疗显着降低了小鼠的血管肉瘤肿瘤的生长。这些结果将DLC1确定为先前未识别的内皮细胞接触抑制抑制在血管肉瘤中耗尽的增殖的调节剂，并提供了支持NF-KB靶向损失DLC1的血管肉瘤的证据。 5）基于观察结果，即肿瘤抑制DLC1蛋白（我们的结果）和转录共激活剂YAP（文献）调节细胞接触抑制生长的抑制作用，我们探索了DLC1和YAP之间的生化相互作用。我们发现DLC1是YAP的调节剂，并且YAP的转录共激活因子函数是由DLC1缺陷型内皮细胞表现出的细胞触觉抑制作用所必需的。如果YAP从内皮细胞中耗尽，则DLC1耗尽的内皮细胞在汇合时停止生长并且无法堆积。通过观察结果，这些结果在体外得到了证实，即血管肉瘤组织包含YAP核和活性的大量DLC1阴性恶性内皮细胞。在肿瘤附近的正常皮肤脉管系统中，情况并非如此。 YAP的抑制剂Verteporfin在小鼠的血管肉瘤实验模型中表现出明显的抗肿瘤作用。 6）关于DLC1在内皮细胞中的作用的额外正在进行的实验促使内皮特异性诱导型DLC1缺陷小鼠细胞系产生。我们目前正在评估这种缺乏在不同情况下的作用，包括小鼠发育，造血性内皮细胞功能，伤口愈合和癌症。