Silencing of BDNF Expression by shRNA Down-Regulates Stroma-Derived VEGF Secretion and Inhibits In Vivo Multiple Myeloma Growth and Angiogenesis in the Bone Marrow Milieu.

Bone marrow (BM) neovascularization and vascular endothelial growth factor (VEGF) expression in multiple myeloma (MM) correlate with disease progression and poor prognosis. Brain derived neurotrophic factor (BDNF) is highly expressed by malignant plasma cells isolated from MM patients. Recently, BDNF is identified as an endothelial survival factor to induce angiogenesis and increase levels of VEGFR in tyrosine receptor kinase B (TrkB)-positive endothelial cells. In addition, BDNF participates in promoting vessel formation by human umbilical vein endothelial cells. High levels of BDNF are detected in the serum of patients with active MM and comparable VEGF levels. Since BDNF and its specific receptor-TrkB are expressed by stromal cells within the bone marrow microenvironment, we sought to evaluate the involvement of BDNF-TrkB axis in myeloma-marrow stroma interaction and its effects on BM neovascularization by regulating VEGF expression. Methods and results: BM stromal cells (BMSCs) isolated from MM patients (n=11) and control health donors (n=8) were confirmed to express BDNF high-affinity specific receptor TrkB by real-time RT-PCR and immunoblotting. Stimulation of BMSCs with exogenous BDNF induced a time- and dose- dependent increase in VEGF secretion (1.8- to 3.3- fold, p<0.01), which was completely abolished by K252α, an inhibitor of TrkB. Western blot analysis demonstrated that exposing BMSCs with BDNF for 30 min at varying concentration from 25 ng/mL to 150 ng/mL leaded to a dose-dependent phosphorylation of signal transducers and activators of transcription 3 (STAT3). Nuclear translocation and DNA-binding of STAT3 was further confirmed by Electrophoretic mobility shift assay (EMSA), BDNF induced a 2.6-fold increase of STAT3/DNA complex in BMSCs (100 ng/mL, 60min). Inhibition of STAT3, a downstream signaling molecule of TrKB by AG490 eliminated stimulating effects of BDNF on BMSCs as well. Meanwhile, RPMI 8226 cells were transfected with lentiviral based shBDNF-pGCSIL-GFP or control vector. Pure population of transfected RPMI 8226 cells were firstly isolated by Fluorescence Activated Cell Sorting (FACS), and then specific BDNF knockdown was double verified by real-time RT-PCR and western blot. BMSCs were co-cultured with BDNF shRNA RPMI 8226 cells in a non-contact transwell system. VEGF concentration in supernatants of BMSCs and BDNF shRNA RPMI 8226 cell co-cultures were significantly decreased when compared with control group, in which BMSCs were cocultured with control RPMI 8226 cells (p<0.05). STAT3 phosphorylation was also inhibited in BMSCs co-cultured with BDNF shRNA RPMI 8226 cells(p<0.01). Furthermore, BDNF shRNA RPMI 8226 cells were inoculated subcutaneously with BMSCs in irradiated nude mice.The tumor volume was measured every three days by in-vivo optical imaging and capliper. In addition, Microvessel density (MVD) was assessed by immunohistochemical analysis for CD31 expression. In vivo results demonstrated that silencing of MM derived BDNF expression significantly inhibited angiogenesis and VEGF expression in the BM microenvironment. Moreover, suppression of BDNF inhibited MM tumor growth and prolonged survival in mice.

Our studies demonstrate that BDNF, as a potential stimulator of angiogenesis, contributes to MM tumorgenesis; it mediates stromal–MM cell interactions via selective activation of specific tyrosine kinase receptors TrkB and downstream signal transducer STAT3 in regulating VEGF secretion.

No relevant conflicts of interest to declare.