Atorvastatin enhances bone marrow endothelial cell function in corticosteroid-resistant immune thrombocytopenia patients

Key Points Impaired BM EPCs were found in corticosteroid-resistant ITP patients. Atorvastatin improved BM EPC quantity and function, representing a novel therapy approach for corticosteroid-resistant ITP patients.

Arachidonic acid drives amoeboid transendothelial invasion through ligand independent activation of EphA2.

221 Background: We have previously shown that the omega-6 poly unsaturated fatty acid arachidonic acid is a potent stimulator of prostate epithelial transendothelial migration across the bone marrow endothelium and can completely restore the invasive stimulatory capacity of adipocyte depleted human bone marrow stroma. Arachidonic acid induces of EphA2 ligand independent signaling via pAkt with subsequent downstream activation of FAK and Src. Here we present further characterization of the arachidonic acid induced EphA2 pathway. Methods: Transendothelial migration of PC-3 cells across the bone marrow endothelial cell line BMEC was followed by time lapse microscopy and by SEM with or without amoeboid or mesenchymal inhibitors. Western blot analysis of the PC-3 cells or their lipid raft (LR) components post AA stimulation was conducted and signaling pathways validated by siRNA knockdown. Results: Initial studies demonstrated a requirement of PC-3 cells to undergo a mesenchymal to amoeboid switch for transendothelial migration towards bone marrow stroma. The mesenchymal to amoeboid switch was also induced by direct treatment with arachidonic acid as characterized by induction of the Rho/Rock pathway and phosphorylation of myosin light chain. Knockdown of EphA2 by siRNA blocked the induction of the mesenchymal to amoeboid switch and prevented transendothelial invasion. Conclusions: Malignant prostate epithelial cells undergo a switch from mesenchymal to amoeboid movement in response to arachidonic acid in order to cross the bone marrow endothelial barrier.

Individual Rac GTPases Mediate Aspects of Prostate Cancer Cell and Bone Marrow Endothelial Cell Interactions

The Rho GTPases organize the actin cytoskeleton and are involved in cancer metastasis. Previously, we demonstrated that RhoC GTPase was required for PC-3 prostate cancer cell invasion. Targeted down-regulation of RhoC led to sustained activation of Rac1 GTPase and morphological, molecular and phenotypic changes reminiscent of epithelial to mesenchymal transition. We also reported that Rac1 is required for PC-3 cell diapedesis across a bone marrow endothelial cell layer. In the current study, we queried whether Rac3 and RhoG GTPases also have a role in prostate tumor cell diapedesis. Using specific siRNAs we demonstrate roles for each protein in PC-3 and C4-2 cell adhesion and diapedesis. We have shown that the chemokine CCL2 induces tumor cell diapedesis via Rac1 activation. Here we find that RhoG partially contributes to CCL2-induced tumor cell diapedesis. We also find that Rac1 GTPase mediates tight binding of prostate cancer cells to bone marrow endothelial cells and promotes retraction of endothelial cells required for tumor cell diapedesis. Finally, Rac1 leads to β1 integrin activation, suggesting a mechanism that Rac1 can mediate tight binding with endothelial cells. Together, our data suggest that Rac1 GTPase is key mediator of prostate cancer cell-bone marrow endothelial cell interactions.