Role of JAG1 and JAG2 ligands in promoting the angiogenic switch in multiple myeloma

Multiple myeloma(MM) is an incurable hematological malignancy that develops in the bone marrow (BM) and displays “angiogenic switch” during its progression. It has been demonstrated that an increase in BM angiogenesis correlates and with adverse prognosis and with metastatization. Another feature of MM cells is the aberrant activation of Notch pathway triggered by the overexpressed JAG1 and JAG2 (J1/2) ligands. This overexpression promotes reciprocal support between MM cells and BM microenvironment. Thus, Jag-induced Notch activation may occur in nearby healthy cells including BM stromal cells (BMSCs) and osteoclasts and leads to the instauration of protective mechanisms inducing MM cell survival, proliferation and drug resistance. While the outcome on tumor vessels formation still need to be clarified. This work aims to study the role of MM-derived Jag1/2 in stimulating endothelium and in promoting a pro-angiogenic behavior in BMSC. We used RPMI8226 and U266 cell lines as models for MM; primary human pulmonary artery endothelial cells (HPAECs) as ECs model; HS5 cell line stably expressing GFP as BMSCs. JAG1/2 were silenced in MM cells (MMJAG1/2KD) using short interfering RNAs. We studied the outcome of the stimulation of MM-derived JAG1/2 in modulating EC adhesion on fibronectin, motility and tube organization on Matrigel by analyzing the effect of a direct cell-cell contact and the contribution of the conditioned media (CM). Secondly, we focused our attention on MM-BMSC co-culture system to study if JAG1/2 can induce a pro-angiogenic behavior in BMSC stimulating the release of VEGF. Direct contact and CM from MM cells improves ECs adhesion, motility and organization into a grid of tube-like structures. These effects are significantly reduced when using MMJAG1/2KD cells indicating that MM-derived JAG1/2 play a key role in the modulation of ECs behavior and the promotion of malignant phenotype. MM can upregulate the amount of BMSC-derived VEGF and promote BMSC pro-angiogenic potential, but MMJAG1/2KD cells lost this ability. Our results indicate a novel contribution of MM-derived JAG1/2 in promoting the formation of new vessels stimulating the activation of the Notch signaling pathway in the same tumor cells and in the tumoral microenvironment. The involvement of Notch-Jag axis in the angiogenic switch of MM suggests that a novel therapy targeted to interrupt this molecular interaction could contrast the support of neo vessel formation to MM progression.