Cellular Cholesterol Modulates VEGFR-1 Function on Acute Leukemia Cells.

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. Following our recent report on the role of VEGFR-1 (FLT-1) in ALL (Fragoso R et al, 2006), in the present study we analyzed 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 polymerized 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 by impairing the formation of lipid rafts/caveolae using drugs that either sequester (nystatin) or deplete (methyl-β-ciclodextrin) total cholesterol. Accordingly, co-treatment of leukemia cells with nystatin or MβCD 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. Given that leukemia cells have an increased rate of cholesterol up-take we sought to understand if increased cholesterol levels affected FLT-1 function in leukemia cells. Cholesterol repletion in leukemia cells enhanced leukemia cells migration in response to VEGF/PlGF (about 3 folds). This significant increase was associated with an increase in FLT-1 protein expression that, very interestingly, was particularly concentrated intracellulary in the cytoplasm. At this time we are trying to understand if this increase in FLT-1 expression after cholesterol repletion is associated with increase protein translation or impairment in proteasome activity. Finally, our preliminary in vivo experiments using Nod-Scid mice subjected (n=3) or not (n=3) to high fat diet (that results in increased cholesterol levels in the BM and in the spleen), showed this metabolic condition worsens disease symptoms and significantly decreases mouse survival. These results reveal for the first time some of the molecular mechanisms involved in FLT-1-mediated leukemia migration, namely the involvement of cholesterol metabolism, which may be crucial for new therapeutics delineation.