Involvement of NOTCH signaling in multiple myeloma angiogenesis and progression

Background: Multiple Myeloma (MM) is a hematopoietic malignancy characterized by proliferation of malignant plasmacells (PCs) within the bone marrow (BM) usually preceded by a premalignant phase called Monoclonal Gammopathies of Undetermined Significance (MGUS). Interactions between myeloma PCs and BM stromal cells facilitate tumor progression and angiogenesis. Notch family is composed of a group of proteins which act as transmembrane receptors and as regulators of gene transcription after their cleavage by -secretase. Mammals express four Notch receptors (Notch1-2-3-4) which bind five ligands (DLL1-3-4 and Jagged1-2). Aim of our study is to investigate the role of Notch pathway in MM angiogenesis and progression. Materials and Methods: Real-time RT PCR, Western blotting and immunofluorescence were performed to assess Notch and its ligands expression in MM endothelial cells (MMECs) and MGECs (ECs of patients with MGUS). Functional in vitro assay (Matrigel assay, adhesion, chemotaxis, wound-healing, cell proliferation, apoptosis, and ELISA) were conducted in MMECs and RPMI-8226 to study Notch inhibition effects after Notch2 silencing and -secretase treatment. Results: MMECs showed an higher expression and activation of Notch2 compared to MGECs and this higher activation in MMECs correlates whit an increased angiogenesis. Notch2 knock-down, through siRNA, reduced MMECs abilities and also their proliferation. Furthermore myeloma PCs line RPMI-8226 showed an over-expression of Notch ligands Jagged1, Jagged2 and DLL1 allowing mutual interactions between MMECs and RPMI- 8226 and Notch pathway activation, as demonstrated by co-colture experiments with or without transwell. Inhibition of Notch signaling using the -secretase inhibitor MK-0752, which prevents Notch cleavage, affected MMECs abilities in co-culture and alone. The block of Notch cleavage reduced ECs chemotaxis, migration, adhesion and furthermore the ability to form capillary structure on Matrigel. Moreover the treatment decreased the production of pro-angiogenic cytokines and of cell-cycle regulators in MMECs and in RPMI-8226 respectively. Conclusion: Our results show that Notch pathway is involved in MM progression supporting BM angiogenesis through an active interaction between myeloma PCs and MMECs. The inhibition of Notch signaling affected MMECs angiogenic abilities, highlighting the idea that this pathway could became a new target in MM therapy to counteract BM angiogenesis.