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Principal Investigator: Giovanna Tosato
Organization: DIVISION OF CLINICAL SCIENCES - NCI
Fiscal Year: 2021
Award: $855,728
Funding agency: National Cancer Institute
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.
Terms: <(TNF)-α><2019 novel corona virus><2019 novel coronavirus><2019-nCoV><A5 Antigen><ACE2><AIDS><Acquired Immune Deficiency><Acquired Immune Deficiency Syndrome><Acquired Immuno-Deficiency Syndrome><Acquired Immunodeficiency Syndrome><Acquired Immunologic Deficiency Syndrome><Activator Appliances><Activator Orthodontic Appliances><Adventitial Cell><Ang-2><Ang2><Angiogenesis Antagonists><Angiogenesis Blockers><Angiogenesis Factor><Angiogenesis Inhibition><Angiogenesis Inhibitors><Angiogenetic Antagonists><Angiogenetic Inhibitors><Angiogenic Antagonists><Angiogenic Factor><Angiogenic Inhibition><Angiogenic Inhibitors><Angiopoietin 1 Receptor><Angiopoietin Receptor Tie-2><Angiopoietin-2><Angiosarcoma><Angiostatic Agents><Anti-Angiogenetic Agents><Anti-Angiogenic Agents><Anti-Angiogenic Drugs><Antiangiogenesis Agents><Antiangiogenic Agents><Antiangiogenic Drugs><Antimorphic mutation><Area><BPD verteporfin><BPD-MA><Basic Fibroblast Growth Factor><Basic Fibroblast Growth Factor Gene><Benzoporphyrin Derivative Monoacid Ring A><Biochemical><Biochemical Genetics><Blood Cells><Blood Vessels><Body Tissues><Burkitt Herpesvirus><Burkitt Lymphoma Virus><C-Jun Kinase 3><CD202B Antigen><COVID-19 virus><COVID19 virus><Cachectin><Cancer Cell Growth><Cancer Model><Cancer Treatment><CancerModel><Cancers><Cell Body><Cell Communication and Signaling><Cell Death><Cell Function><Cell Process><Cell Signaling><Cell Survival><Cell Viability><Cell physiology><Cells><Cellular Function><Cellular Physiology><Cellular Process><Cessation of life><Characteristics><Chemotactic Cytokines><Clinic><CoV-2><CoV2><Contact Inhibition><DLC1><DLEC1><DLEC1 gene><Death><Defect><Development><Dominant Negative><Dominant-Negative Mutant><Dominant-Negative Mutation><Drugs><E-B Virus><EB virus><EBV><Endothelial Cells><Endothelial Growth Factors Receptor><Endothelial TEK Tyrosine Kinase><Endothelium><Eph Family Receptors><Eph Receptor Ligands><Eph Receptor Tyrosine Kinase><Eph Receptors><EphB Receptors><Ephrin B Receptor><Ephrin Receptors><Ephrins><Epigenetic><Epigenetic Change><Epigenetic Mechanism><Epigenetic Process><Epithelial-Specific Protein Receptor Tyrosine Kinase TIE-2><Epstein Barr Virus><Experimental Models><F56><FGF-2><FGF2><FGF2 gene><FGFB><FLK1><Fibroblast Growth Factor 2><Fibroblast Growth Factor 2 Gene><Function Activator><Generalized Growth><Generations><Genetic><Goals><Growth><HBGF-2><HHV-4><HHV-8><HHV4><HHV8><Hemangiosarcoma><Hematopoiesis><Hematopoietic Cellular Control Mechanisms><Heparin-Binding Growth Factor 2><Heparin-Binding Growth Factor Class II><Herpesviridae><Herpesviruses><Heterogeneity><Homologous Chemotactic Cytokines><Human><Human Herpesvirus 4><Human Herpesvirus 8><Immunoglobulin Enhancer-Binding Protein><In Vitro><Individual><Infection><Infectious Mononucleosis Virus><Intercrines><Intestinal><Intestines><Intracellular Communication and Signaling><Investigation><JNK3><KDR gene><KSHV><Kaposi Sarcoma><Kaposi Sarcoma-Associated Herpes Virus><Kaposi Sarcoma-Associated Herpesvirus><Kaposi sarcoma associated virus><Kaposi sarcoma herpes virus><Kaposi's Sarcoma><Kaposi's sarcoma (KS)-associated herpesvirus><Knowledge><Ligands><Link><Literature><Lymphocytic Neoplasm><Lymphocytic Tumor><Lymphocytic and Plasma Cell Neoplasm><Lymphocytic and Plasma Cell Tumour><Lymphocytic and Plasmacytic Neoplasm><Lymphoid Tumor><Lymphoid and Plasma Cell Tumour><Lymphoid and Plasmacytic Neoplasm><Lymphoid and Plasmacytic Tumour><MAP Kinase 10><MAPK10><MAPK10 gene><Macrophage-Derived TNF><Maintenance><Malignant><Malignant - descriptor><Malignant Neoplasm Therapy><Malignant Neoplasm Treatment><Malignant Neoplasms><Malignant Tumor><Malignant hemangioendothelioma><Mediating><Mediator><Mediator of Activation><Mediator of activation protein><Medication><Membrane><Metabolic><Metastasis><Metastasize><Metastatic Lesion><Metastatic Mass><Metastatic Neoplasm><Metastatic Tumor><Mice><Mice Mammals><Mitogen-Activated Protein Kinase 10><Modern Man><Monocyte-Derived TNF><Mouse Cell Line><Multiple Hemorrhagic Sarcoma><Murine><Mus><NF-kB><NF-kappa B><NF-kappaB><NFKB><NRP1 Protein><Neoplasm Metastasis><Neovascularization Inhibitors><Neuropilin-1><Non-Receptor Type 11 Protein Tyrosine Phosphatase><Npn-1 Protein><Nuclear><Nuclear Factor kappa B><Nuclear Transcription Factor NF-kB><Nutrient><O element><O2 element><Oncogenesis><Outcome><Oxygen><PRKM10><PTP-2 enzyme><PTP2C><PTPN11><PTPN11 gene><Pathway interactions><Pericapillary Cell><Pericytes><Peripheral Blood Cell><Perivascular Cell><Pharmaceutic Preparations><Pharmaceutical Preparations><Phenotype><Phosphorylation><Physiologic><Physiological><Prostate Epithelial Cell Growth Factor><Protein Phosphorylation><Protein Tyrosine Phosphatase 2C><Protein-Tyrosine Phosphatase 2C><Proteins><Receptor Protein><Receptor Signaling><Regulation><Research><Resistance><Role><Rouget Cells><SARS corona virus 2><SARS-CoV-2><SARS-CoV2><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><SHP2><SHP2 Phosphatase><SHPTP2><SIS cytokines><Secondary Neoplasm><Secondary Tumor><Sema III Receptor><Semaphorin III Receptor><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome related corona virus 2><Shp-2 tyrosine phosphatase><Signal Transduction><Signal Transduction Systems><Signaling><Signaling Molecule><Skin><Source><Starvation><Stress><Subcellular Process><Surface><TEK Tyrosine Kinase><TEK gene><TIE-2><TIE-2 Receptor><TIE-2 Receptor Tyrosine Kinase><TIE-2-RTK><TIE2><TIE2 Tyrosine Kinase><TL1><TNF><TNF A><TNF Alpha><TNF Ligand-Related Molecule 1><TNF gene><TNF-α><TNF15><TNFA><TNFSF15><TNFSF15 gene><TNFα><Tail><Tek Receptor><Testing><Tie2 Receptor><Tissue Growth><Tissues><Transcription Activator><Transcription Coactivator><Transcription Factor Coactivator><Transcription Factor NF-kB><Transcriptional Activator><Transcriptional Activator/Coactivator><Transcriptional Coactivator><Tumor Angiogenesis><Tumor Cell><Tumor Expansion><Tumor Necrosis Factor><Tumor Necrosis Factor Ligand Superfamily Member 15><Tumor Necrosis Factor-alpha><Tumor Suppressor Proteins><Tunica Interna Endothelial Cell Kinase><Tyrosine><Tyrosine Phosphatase SHP2><Tyrosine Phosphorylation><Tyrosine-Protein Kinase Receptor TEK><Tyrosine-Protein Kinase Receptor TIE-2><VEGF><VEGF Receptors><VEGFA><VEGFA gene><VEGFR><VEGFR-2><VEGFR2><VEGFs><VEGI><VMCM><VMCM1><VPF Receptor><Vascular Endothelial Cell Growth Factor 165 Receptor><Vascular Endothelial Cell Growth Factor Receptor><Vascular Endothelial Growth Factor A><Vascular Endothelial Growth Factor Receptor 2><Vascular Endothelial Growth Factors><Vascular Endothelial Growth Inhibitor><Vascular Endothelium><Vascular Permeability Factor Receptor><Vasculotropin><Verteporfin><Virus-HHV8><Visudyne><Wound Repair><Wuhan coronavirus><angiogenesis><angiotensin converting enzyme 2><angiotensin converting enzyme II><anti-cancer therapy><anti-tumor effect><antiangiogenic><anticancer therapy><antitumor effect><bFGF><base><benzoporphyrin D><biological signal transduction><blood cell formation><bowel><cancer metastasis><cancer microenvironment><cancer progression><cancer therapy><cancer-directed therapy><chemoattractant cytokine><chemokine><coronavirus disease 2019 virus><cytokine><density><developmental><drug/agent><experiment><experimental research><experimental study><hCoV19><herpes virus><in vivo><inhibitor><inhibitor/antagonist><interest><kaposi's sarcoma herpesvirus><kaposi's sarcoma-associated human herpesvirus><kappa B Enhancer Binding Protein><lymphoid neoplasm><malignancy><membrane structure><mouse development><mouse model><murine model><mutant><nCoV2><necrocytosis><neoplasm progression><neoplasm/cancer><neoplastic cell><neoplastic progression><new drug target><new druggable target><new pharmacotherapy target><new therapeutic target><new therapy target><notch><notch protein><notch receptors><novel drug target><novel druggable target><novel pharmacotherapy target><novel therapeutic target><novel therapy target><nuclear factor kappa beta><ontogeny><p140 TEK><pathway><pre-clinical><preclinical><prevent><preventing><receptor><resistant><response><response to treatment><selective expression><selectively expressed><social role><stem cell niche><targeted cancer therapy><therapeutic response><therapeutic target><transcription co-activator><transcriptional co-activator><treatment response><tumor><tumor cell metastasis><tumor growth><tumor microenvironment><tumor progression><tumor suppressor><tumorigenesis><vascular><vascular regression><verteporphin><vessel regression><wound healing><wound resolution>