VEGF Regulates Leukemia Migration Via FLT-1, Involving Pi3 Kinase, RhoA and Rac1 Activation and Lipid Rafts Formation.

Vascular endothelial growth factor (VEGF) and its receptors play a crucial role in malignancy and in disease, regulating the survival, proliferation, and migration of several cell types, such as endothelium and also leukemia cells. We previously demonstrated crucial roles for VEGFR-2 in acute myeloid leukemia, where its blockade showed clinical potential in murine models, by affecting leukemia survival and proliferation. In the present study we focused on a different VEGF receptor, FLT-1, and studied the molecular mechanisms whereby it modulates acute leukemia cell migration in response to VEGF/Placental Growth Factor (PLGF). First, we observed the formation of cell protrusions on ALL cells after VEGF/PLGF stimulation, with evidence for polimerized actin and FLT-1 co-localization (as determined by phalloidin, immunofluorescence staining, and confocal microscopy). Western blot analysis revealed that PLGF/VEGF stimulation resulted in increased RhoA and Rac1 GTPases expression. Co-treatment with LY200942 significantly decreased RhoA and Rac1 induction and cell migration by PLGF/VEGF, demonstrating this effect is modulated via Pi3 kinase. Next, we investigated the mechanisms whereby FLT-1 and actin co-localize at the cell “leading edge” (protrusions), after VEGF/PLGF stimulation, and the relevance of such co-localization for cell migration. We addressed this question using a drug (nystantin) that impairs the formation of lipid rafts, cholesterol and sphingolipid rich membrane domains that modulate the interaction of several proteins (Rho GTPases) with downstream effectors, an event essential to cell migration. Accordingly, co-treatment of leukemia cells with nystantin and PLGF/VEGF blocked cell migration, an effect that was associated with a decrease in FLT-1 polarization and co-localization with actin filaments. Instead, FLT-1 was now found mostly in the cell cytosol (possibly undergoing lysosomal degradation). Taken together, we hypothesize that FLT-1 localization in lipid raft-rich membrane domains allows interaction with the actin cytoskeleton and downstream effectors, resulting in cell migration. Our data reveal for the first time some of the molecular mechanisms involved in VEGF-mediated leukemia migration, which may be crucial for determining the onset of extramedullary disease (the exit of leukemia cells from the bone marrow).