Background
Multiple myeloma (MM) is a malignant plasma cells (PC) disorder accounting for approximately 10% of hematologic cancers. Even though advanced chemotherapeutic regimens have increased the median time of survival to 5 years after diagnosis, myeloma remains incurable. Once immortalized, the survival and proliferation of myeloma cells strictly depend on a complex interaction with the bone marrow (BM) microenvironment, which is mediated both by adhesion molecules and production of several cytokines, especially interleukin-6 (IL-6). Following MM progression, at the stage of plasma cell leukemia, the malignant PC acquires autonomous proliferative ability, becomes indipendent on growth factors like IL-6 and is no longer confined in the BM.
Several recent evidences point to a possible role for Notch signaling in mediating critical events in MM progression. The Notch pathway is highly conserved and plays a crucial role in cell-fate decision, tissue patterning and morphogenesis. Recently, Notch receptors and ligands have been shown to be upregulated during MM progression and their signaling positively regulates cell proliferation, drug resistance and BM infiltration.
Aims
The ability of Notch signaling to regulate proliferation and survival pathways (i.e. NF-kB, AKT, Myc, and the same IL-6) prompted us to study if its up-regulation during MM progression may play a role in the acquirement of IL-6 independence. To this end we used two opposite approaches. Specifically, we verified if Notch signaling upregulation in IL-6 dependent cell lines promotes their independence and assessed if, upon Notch inihibition, IL-6 independent MM cell lines lost self-sufficient proliferation.
Methods
Cell culture and cell growth analysis: HMCL CMA03, INA-6 and XG-1 were maintained in complete RPMI-1640 medium supplemented with 10% V/V FBS and IL-6 10, 2.5 or 1 ng/mL, respectivetly. OPM2, CMA03/06 and U266 cell lines were cultured in the same conditions without IL-6 addition. The number and viability of cells were assessed by means of trypan blue exclusion assay. The Notch inihibitor, DAPT, was added to the medium at the final concentration of 50mM. Soluble Jagged1 was used at 5mg/mL.
Flow cytometry analysis: Apoptosys analysis was perfomed by AnnexinV-FITC/Propidium Iodide staining. Cell cycle analysis was performed by Propidium Iodide staining.
Real time-PCR: Quantitative PCR reactions were carried out using the Maxima\u2122 SYBR Green/ROX qPCR Master Mix.
Results
To evaluate if Notch pathway upregulation is involved in the development of IL-6 independence in MM cells, we activated the Notch signaling in three MM cell lines, CMA03, INA-6 and XG-1, strictly dependent
on IL-6. At this purpose, MM cells were cultured with the soluble form of the Notch ligand Jagged1. We demonstrated that Jagged1 stimulation partially rescued the reduced cell growth due to IL-6 withdrawal.
On the other hand, three different IL-6 independent cell lines, CMA03/06, OPM2 and U266, treated with a gamma-secretase inhibitor (DAPT) which causes Notch pathway blockade, displayed a significant decrease in cell growth. Remarkably, this effect could be reverted by the addition of IL-6 in the culture medium. The mechanisms underlying Notch-IL-6 crosstalking was partially investigated. Preliminary results indicate that Notch signalling is required for MM cell autonomous IL-6 production.
Summay/Conclusion
The present results suggest that Notch pathway activation may contribute to the transition from IL-6-dependent to IL-6-independent MM cell growth. Furthermore, the inhibition of the Notch patwhay may lead to a decrease in MM cells proliferation in part due to the reduction of IL-6 expression.
Even though studies are necessary to identify further mechanisms of IL-6 independence possibly involving other Notch downstrem pathways, these preliminary results support the rationale for a Notch-directed approach in plasma cell dyscrasias
