A137
VEGFR1/PKCα signaling controls melanoma vasculogenic mimicry and is not dependent on VEGFR2 kinase
Amalia Vartanian, Eugenia Stepanova, Galina Gatsina, Irina Grigorieva, Anatoly Baryshnikov, Michail Lichinitser
Russian Cancer Research Center, Moscow, Russian Federation
Background
VEGFR2 is a major mediator in tumour vascularization. VEGFR1 is thought to act as negative regulator of VEGFR2, as VEGFR1 intracellular domain is dispensible in EC. Whether it has its own important roles in other cell system is poorly understood. We have recently shown that VEGF-A, a ligand specific for the two receptors, is essential for organization of tumour cells into capillary-like structures (CLS), a phenomen also known as vasculogenic mimicry (VM, A.Vartanian et al., Mel. Res, 2007).Here we show the involvement of VEGFR1 and PKCα in melanoma VM and demonstrate that VEGFR2 kinase is dispensible in melanoma VM control.
Results
Transfection of cells Mel Il, Mel Cher, Mel P with VEGFR1 si RNA completely disrupts Matrigel-induced CLS formation. On the other hand, VEGFR2 kinase specific inhibitor (PTKI II) doesn't influence CLS formation indicating that VEGFR1 signaling pathway is involved. It is known that PKC can be activated through VEGFR1 and that its various isozymes are involved in the control of cell adhesion and cytoskeletal rearrangement, which are likely to be involved in CLS formation. Thus we further analysed PKC involvement in melanoma VM. PKC activation by phorbol ester at the time of seeding cells on Matrigel significantly speeded up the CLS fromation (4 h vs 7 h). Only PKCα and δ were expressed in melanoma cells. Pretreatment with selective PKCα and δ inhibitors (Ro 32-0432 and Rottlerin, respectively) blocked CLS formation. However, addition of Ro 04-3204 but not Rottlerin caused dramatic rearrangement of actin fibers and completely destroyed previously formed CLS. Thus, continuous PKCα activity is needed to maintain CLS. Finally, transfection of cells with PKCα siRNA abrogated CLS formation but PKCδ si RNA had no effect, suggesing that PKCα is the main player in melanoma VM. The protein level of PKCα was not change during 10 h of Matrigel adhesion.
Conclusion
The data reveal a VEGFR1/PKCα pathway, which controls CLS formation through changes in the cytoskeleton, cell motility, and adhesion. Its signaling is not dependent on VEGFR2 kinase and therefore is likely to go solely though VEGFR1. This evidence identifies VEGFR1 and PKCα as possible theraupetic targets to interfere with VM
