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 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 the roles of tumor-associated Dll4/other Notch ligands and 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, which 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, 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 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) In related experiments, we have begun to explore the role of the Notch ligand JAG2 in angiogenesis. To this end, we have developed 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 intestinal stem cell pool within the crypts. 4) 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 EphB 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 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. 5) 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. 6) 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 have now explored the conditions under which DLC1 is a physiological regulator of endothelial cell survival and defined the biochemical pathways underlying these effects. We reasoned that understanding how endothelial cell survive under adverse circumstances might help design strategies to overcome such defense and induce cell death. Angiosarcomas and Kapok's sarcomas, malignancies that arise from the endothelium, display reduced DLC1 expression and related signaling deficiencies, which likely contribute to endothelial tumorigenesis.
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