Brain-Derived Neurotrophic Factor Induces Proliferation, Migration and VEGF Secretion in Human Multiple Myeloma Cells Via Activation of MEK-ERK and PI3K/Akt Signaling.

Multiple myeloma (MM) is an incurable form of cancer characterized by accumulation of malignant plasma cells in the bone marrow. Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin superfamily that is overexpressed in patients with MM. Recent data indicate that in addition to its neuropoietic actions, BDNF is crucially involved in the angiogenesis and may be developed as a new angiogenic factor. Our previous study has defined a functional role of BDNF activation in MM cell proliferation and migration. In this study, we investigated the ability of BDNF activation to regulate VEGF levels in MM cells and the involvement of the different signaling cascades in the proliferation, migration and the stimulation of VEGF production. Myeloma cell lines, RPMI 8226, U266, KM3, and freshly isolated multiple myeloma cells from bone marrow aspirates of patients were exposed to different concentrations of BDNF for 48 hours. Using ELISA analysis, we here demonstrated the increased expression and secretion of VEGF by myeloma cell lines and sorted primary myeloma cells stimulated with BDNF. In the presence of 100 ng/ml BDNF, median VEGF concentrations in the conditioned media increased by 46% over controls in RPMI 8226(P <0.05), by 21% in U266 (P >0.05), by 69% in KM3 (P <0.01), and by 81% in patient MM cells (P <0.01). The results of RT-PCR assays are consistent with these data. To characterize downstream signaling molecules, we first assessed the activation states of ERK1/2 and Akt by western blot. In time-course experiments, specific phosphorylation of ERK1/2 was observed in RPMI 8226, U266 and KM3. ERK phosphorylation was inhibited by pretreatment with MEK-1 inhibitor PD98059. However, treatment with PI3K inhibitor LY249002 or wortmannin had no effect on ERK phosphorylation. BDNF also induced phosphorylation of Akt signaling pathway. Notably, this induction of phosphorylation was inhibited by pretreatment with the LY294002 but not by PD98059. These results show that activation of ERK and Akt pathways occur separately from each other without any cross talk in MM. To further determine the specific signaling pathways involved in the BDNF-induced tumor cell proliferation and migration, a pharmacological approach was taken. Proliferation induced by BDNF was blocked by 50 μM PD98059 significantly as determined by [³H]thymidine incorporation assays. However, the PI3K inhibitor (LY294002 and wortmannin) had little effect on BDNF-induced MM proliferation. In contrast, using transwell migration assay, we demonstrate that migration of RPMI 8226 cells induced by BDNF was inhibited by means of 50 μM LY294002 but not by PD98059. Furthermore, treatment with PI3K inhibitors LY294002 inhibited the secretion of VEGF, whereas the MEK-1 inhibitor PD98059 had no significant effect on BDNF-induced VEGF expression and secretion. Taken together, these data indicate that BDNF-induced proliferation requires activation of MEK, whereas BDNF-controlled migration and VEGF expression require activation of PI3K. These provide the framework for novel therapeutic strategies targeting BDNF signaling to inhibit proliferation and progressive disease in MM.