Molecular mechanisms of activation of tyrosine kinase receptors in angiogenesis
Final Report Abstract
During the funding period we have shown that the activation of two angiogenic tyrosine kinase receptors (RTKs), c-Met and VEGFR-2, by their ligands HGF and VEGF-A, require the function of the same co-receptor CD44v6. On the one hand the ectodomain of CD44v6 binds to the ligands and “presents” them to the authentic receptors. On the other hand the cytoplasmic domain of CD44v6 is required for downstream signaling by binding to ERM proteins (Ezrin, Radixin, Moiesin) that in turn recruit the cytoskeleton to the cell membrane. The specific amino acids in the CD44v6 ectodomain that account for the co-receptor function are EWQ in rat, GWQ in mouse and RWH in human Peptides comprising these amino acids, the smallest being a 5mer, inhibit completely the activation of the c-Met and VEGFR-2 receptors. Furthermore, these peptides strongly impaired angiogenesis in in vitro and in vivo assays. These peptides do not only inhibit angiogenesis but also block tumour metastasis in a syngeneic rat pancreas carcinoma system and in an orthotopic model of a human pancreas tumour. Most strikingly, even already established metastases regressed upon treatment with the peptides suggesting that the peptides might be powerful tools for fighting metastasizing tumours. In haematopoietic stem cells CD44 is necessary for homing to the bone marrow niche by binding to hyaluronan (HA), that also triggers the settlement and the migration of the stem cells towards the cytokine CXCL-12 (also called SDF1-α). This suggests that CD44 and CXCR4, the chemokine receptor for CXCL-12, collaborate. Moreover CXCR4 appears to be essential for metastatic spreading of tumour cells to CXCL-12 expressing organs and CXCL-12 can promote angiogenesis HA can either stimulate angiogenesis when it is degraded to small fragments (sHA) or counteract this stimulation as the native high molecular weight form (nHA). We studied a possible collaboration between CD44 and CXCR4 and the impact nHA and sHA might have on this collaboration in the second funding period. In several systems we have demonstrated that the signaling induced by SDF1α is strongly induced by nHA but is repressed by sHA. Similarly, in several angiogenic assays the pro-angiogenic effect of SDF1α was increased by nHA and blocked by sHA. These effects seem to be mediated by CD44 since antibodies that block the CD44-HA interaction repress the effect of HA on CXCR4 activation A collaboration is further supported by the identification of a complex between CD44 and CXCR4 upon SDF1α treatment.
