BACKGROUND
Multiple myeloma (MM) is an hematologic tumor caused by the accumulation of malignant plasma cells in the bone marrow (BM). The dysregulated expression of two Notch ligands, Jagged1 and Jagged2, hyperactivates the Notch pathway both in MM cells and in BM stromal cell (BMSC). Several Notch downstream mediators are involved in MM cell survival and proliferation, i.e. IL6, SDF1\u3b1, CXCR4, NF-kB, VEGF and IGF.
Although treatments with new drugs, such as alkylating agents, proteasome inhibitors and immunomodulatory agents, increased patients\u2019survival, MM remains incurable, principally due to the development of endogenous or BM-mediated drug resistance (DR). Therefore it is crucial to find new therapeutic targets.
The aim of this study was to investigate the role of Notch signaling in endogenous and BMSC-promoted DR in MM.
MATERIAL AND METHODS
U266 and OPM2 cell lines were maintained in complete RPMI-1640 medium. The BMSC lines NIH3T3 (murine) and HS5 (human) were maintained in complete DMEM medium. MM cells were cultured alone or on a BMSC monolayer for 24h, and subsequently treated with Mitoxantrone, Bortezomib, Melphalan or the vehicle in the presence or the absence of the CXCR4 antagonist AMD3100 for additional 24 hours. BMSCs were previously stained with PKH26 (Sigma-Aldrich) to discriminate them from MM by flow cytometry. Apoptosis was determined by Annexin V-FITC staining.
qRT-PCR reactions were carried out in a 7500 Fast Real-time PCR system (Applied Biosystems) using the Maxima\u2122 SYBR Green/ROX qPCR Master Mix (ThermoScientific Inc) using murine or human primer sets to discriminate the source of the RNA molecules.
Silencing of Jagged1 and 2 was obtained by transient expression of specific siRNAs (Stealth Select RNAi siRNA system, Life Technologies).
RESULTS
The possible role of Notch withdrawal in DR was investigated by silencing Jagged 1 and 2 ligands in MM cell lines OPM-2 and U266. Results showed an increased sensitivity to Bortezomib,
Mitoxantrone and Melphalan associated to a decrease in the expression of SDF1\u3b1, CXCR4, Bcl-XL, Bcl-2, Survivin and ABCC1.
When co-cultured with murine and human BMSCs, MM cells showed increased DR due to: i) increased expression of anti-apoptotic genes in MM cells, i.e. Bcl-XL, Bcl-2, Survivin and ABCC1; ii) BMSC release of soluble mediators relevant to MM cells, i.e. SDF1\u3b1 and VEGF. We suggest that these effects may be driven by the reciprocal activation of Notch signaling observed in both cell types and consistently we demonstrated that DR may be significantly reduced by silencing Jagged1 and 2 in MM cells.
Finally, the evidence that CXCR4 blockade significantly reduced MM cells resistance to Bortezomib induced by BMCSs, indicates that CXCR4/SDF1\u3b1 chemokine axis is a key mediator of Notch in MM-associated DR.
CONCLUSION
The evidence that Jagged1/2 silencing affects endogenous and BMSC-induced DR in MM cells supports the use of a Jagged-targeted approach in MM therapy alone or in combination with common drugs
