Cholesterol Rich-Domains Regulate VEGFR-1 (FLT-1) Expression and Signaling in Acute Leukemia Cells

Vascular endothelial growth factor receptors -1 (FLT-1) and -2 (KDR) are expressed by subsets of acute and chronic leukemias, where they signal in paracrine and/or autocrine manner to induce cell survival, proliferation and migration. We have previously shown that acute lymphocytic leukemia migration in response to VEGF via FLT-1 modulates the onset of extramedullary disease, and thus has clinical predictive value (Fragoso et al, Blood 2006). Acute leukemia cell (AML) migration, induced by PlGF/VEGF activation of FLT-1 results in the formation of actin membrane protrusions with concomitant increased ERK1/2 and P38 phosphorylation and activation of Rho-GTPases (Casalou et al, 2007). Since we have found an in vitro association of FLT-1 with caveolin-1, actin and HSp90, we hypothesised that cholesterol-rich domains might regulate FLT-1 mediated survival, proliferation or migration of acute myeloid (AML) and lymphoid (ALL) leukemias. First we found by FACS and RQ-PCR that FLT-1 expression is up-regulated by increased cholesterol/HDL levels in vitro. As shown by sucrose gradient fractionation and western blotting, PlGF/VEGF stimulation of AML cells results in re-localization of FLT-1 to cholesterol-rich domains. Accordingly, FLT-1 localization within cholesterol-rich domains is abrogated by exposing leukemia cells to b-methyl-cyclodextrin (MbCD) which removes intracellular cholesterol. Additionally, FLT-1 phosphorylation is abolished by treatment of AML cells with MbCD or Nystatin, an inhibitor of lipid raft endocytosis. Functionally, AML cells exposure to high levels of total cholesterol/HDL for 24 hours exerted a protective effect from actinomycin D-induced apoptosis and promoted PlGF/VEGF-induced AML migration in transwell migration assays. Together, these results show that on subsets of acute leukemias cholesterol/HDL cellular-content regulates FLT-1 expression and signalling, resulting in decreased apoptosis and induction of cell migration. In vivo, we show that cholesterol-rich diet significantly increases bone marrow VEGF levels in mice; inoculation of FLT-1 expressing acute leukemias into mice fed with cholesterolrich diet significantly accelerated disease progression and worsened disease outcome. Taken together, our data show the molecular basis by which cellular and systemic cholesterol regulates VEGF and VEGFR-1 signalling on subsets of acute leukemias, modulating cell migration and survival and thereby regulating disease progression.