Pathological angiogenesis, the aberrant proliferation and formation of new blood vessels, during tumorigenesis culminates in an ?angiogenic-switch? whereby small, avascular tumors enter a state of rapid growth driving disease progression and metastasis. Endothelial cells lining blood vessels in the tumor microenvironment respond to secreted vascular endothelial growth factor (VEGF) and navigate through the extracellular matrix (ECM) during the initial steps of sprouting angiogenesis. The integrin family of proteins modulate new vessel growth by regulating endothelial cell adhesive interactions with the ECM, neighboring endothelial cells at intercellular junctions, and with the basement membrane. Pharmacological blockade of integrins has demonstrated potent anti-tumorigenic effects in pre-clinical models, but clinical trials of this class of inhibitors have failed to show phase-III efficacy demonstrating a need for a greater understanding of integrin signaling. A noteworthy property of integrins is that their affinity for extracellular ligands is regulated through ?inside-out? signaling. Talin, a cytoskeletal adaptor protein, binds the ?-integrin cytoplasmic tail at two distinct sites, resulting in a conformational change whereby the extracellular domain extends into the ECM to engage ligand. We have previously published that mice expressing a platelet-specific talin1 mutant (talin1 L325R) defective in its capacity to activate integrins exhibit severe defects in platelet integrin activation that lead to a bleeding diathesis. To test the central hypothesis that talin-dependent integrin activation is required for angiogenesis (Aim #1), we have generated inducible endothelial cell-specific talin1 L325R knock-in mice (Fig 1). Induction of talin1 L325R expression during postnatal development caused early lethality and extensive defects in retinal angiogenesis (Fig 2). Interestingly, preliminary work using a B16/F10 murine melanoma model showed reductions in primary tumor size in talin1 L325R mice (Fig 3) while both talin1 L325R and talin1 WT mice exhibited no observable defects in pre-existing vasculature (Fig 5) suggesting the potential specificity of talin- dependent integrin activation for nascent vessel development. We propose to test the requirement of talin- mediated integrin activation for primary tumor growth and tumor angiogenesis. Curiously, several laboratories have reported integrin signaling promotes VEGF/VEGFR2 signaling, a central pathway responsible for angiogenesis during solid-tumor progression. Recent work suggests that stimulation of endothelial cells with VEGF induces an ?v?3-VEGFR2 macromolecular complex that potentiates VEGF signaling.
In Aim 2, we will test the requirement of integrin activation during VEGF-mediated angiogenesis in vivo and whether integrin activation is necessary for downstream VEGF/VEGFR2 signal transduction. This work will provide important insight into the relationship between endothelial cell inside-out integrin signaling in angiogenesis and examine the role of talin-dependent inside-out signaling in cross-talk between integrins and VEGF signaling.
Blood vessels are lined with endothelial cells which direct blood vessel formation during development and tumor progression. Endothelial cell adhesion regulated by the integrin family of proteins offer a favorable target during new blood vessel formation in cancer, but clinical limitations persist due to incomplete investigation of the molecular mechanisms underlying this process. Our work will test the requirement of a key step in the regulation of integrin activation exclusively during aberrant blood vessel growth using a novel mouse model which may have important implications in the future design of anti-angiogenic therapies.
