The role of notch pathway in multiple myeloma progression toward IL-6 independence

Introduction Multiple myeloma (MM) is a heamotologic malignancy characterized by proliferation of neoplastic plasma cells in the bone marrow (BM). Initially, myeloma cells strictly depend on BM, which supports tumor progression through adhesion molecules and soluble mediators as interleukin-6 (IL-6). Later, at the stage of plasma cell leukemia, MM cells acquire mutations resulting in proliferation indipendent from environmental factors such as IL-6. Recently, Notch signaling has been shown to be upregulated during MM progression and to positively regulate cell proliferation, drug resistance and BM infiltration. The aim of this study is to evaluate if Notch signalling plays a role in the acquirement of IL-6 independence. Methods The human MM cell lines CMA03, INA-6 and XG1 were maintained in RPMI-1640 medium supplemented with IL-6 (respectivetly at 10, 2.5 and 1ng/ml). CMA03/06, OPM2 and U266 were maintened in the same condition withouth IL-6. The murine fibroblasts NIH3T3 were used as BMSC mimic and maintained in DMEM. Viable cells were counted by trypan blue exclusion assay. Notch inhibition was obtained by using the ɣ-secretase inhibitor DAPT at 50mM, soluble Jagged1 was used at 5mg/ml. qRT-PCR reactions were performed by Maxima™ SYBR GreenqPCR Master Mix. Silencing of Jagged1 and 2 was obtained by transient expression of specific siRNAs (Select RNAiTM siRNA system, Invitrogen). Results The global expression analysis of the MM model of IL-6 independence acquisistion represented by CMA03 and CMA03/06 cell line (Verdelli et al. Genes Chromosomes Cancer, 2014), indicated that Notch pathway activation may contribute to the IL-6 independence in MM by inducing proliferative signals. Accordingly, we showed here that the activation of Notch signaling, induced by stimulation with soluble Jagged1 ligand, partially rescued IL-6 dependency in XG1 cells. Otherwise, Notch signaling inhibition obtained with DAPT in three different IL-6-indipendent MM cell lines (CMA03/06, OPM2 and U266) resulted in a significant decrease of cell growth which could be reverted by IL-6. This confirms that Notch and IL-6 are complementary in activating MM cell proliferation. Of note, Notch withdrawal induced by Jagged1/2 silencing, decreased IL-6 expression in OPM2 and U266 cell lines. This suggests that Notch-directed IL-6 regulation might have a biological significance in those MM cell lines which express high IL-6 levels. More frequently, BM stromal cells represent the main source of IL-6 for those MM cells which do not display an autonomous production. Results from co-culture systems indicate that surface Jagged expressed on MM cell lines induced Notch-directed IL-6 production in stromal cells. This effect was reverted by silencing Jagged1/2 in MM cells. Conclusions These results suggest that Notch pathway activation may contribute to the transition from IL-6-dependent to IL-6-independent cell growth, and that its inhibition may result in decreased cell proliferation.