Notch pathway and Inteleukin-6 cooperate to support multiple myeloma cell proliferation

Introduction The Notch pathway has a key role in multiple myeloma (MM) progression by positively regulating cell proliferation, drug resistance and bone marrow (BM) infiltration through the overexpression of both receptors (Notch1 and 2) and ligands (Jag1-2). MM cells are strictly dependent on the BM niche, that supports tumor growth and progression through adhesion molecules and soluble mediators, as interleukin-6 (IL6). MM cells initially depend on IL6 mainly produced by BM stromal cells (BMSCs) and later may acquire independence and/or the ability to autonomously produce IL6. Here, we investigated the cooperation between the Notch pathway and IL6 signaling in the promotion of MM cells proliferation. Material and methods Notch signaling modulation was induced in MM cell lines and primary MM cells as follows: upregulation by 5mg/mL soluble Jag1; down-regulation by 50μM DAPT or Jag1-2 RNA interference (RNAi). qPCR was performed using Maxima™ SYBR Green Master Mix. Absolute cells count and evaluation of IL6 protein expression was achieved by flow cytometry. Immunohistochemistry (IHC) for HES6, IL6 and Ig light chain was performed on BM biopsies at different stages of MM. Results By modulating Notch activity in MM cell lines, we demonstrated that dysregulated Notch proliferative signal can substitute IL6 stimulation. Indeed, upon Notch withdrawal, IL6-indipendent cell lines became dependent on IL6 for their proliferation. On the opposite, Notch activation in IL6-dependent cell lines, may stimulate proliferation in the absence of IL6. Also, Notch signaling may support MM cell proliferation by promoting IL6 autonomous expression. Indeed, IL6 autonomous expression in U266 cells is dependent upon Notch signaling and IHC study confirmed an association between Notch activation and IL6 immunoreactivity in MM cells. We focused on BMSCs since they are the most important source of IL6. We showed, by IHC staining, that mielomatous nests induced IL6 expression in nearby non-neoplastic cells and, by co-culture systems of BMSCs and MM cells, that MM cell-derived Jag1-2 ligands activate Notch in BMSCs promoting IL6 secretion and MM cell proliferation. These results were confirmed by Notch inhibition approaches. Conclusion Notch signaling in MM cells and in surrounding BMSCs promotes MM cell growth by boosting IL6 production. These results support the rationale for a Notch-directed approach in MM therapy and suggest that Jag ligands are promising molecular targets.