Autocrine Notch receptor 3 signaling stimulates the proliferation and enhances the osteoclastogenic potential of multiple myeloma cells

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Medicine and Health Sciences


Introduction. Multiple myeloma (MM) is a hematological cancer characterized by the expansion and accumulation of monoclonal malignant plasma cells in the bone marrow (BM). Interactions between MM cells and cells of the bone/BM niche play a critical role in MM onset and progression by promoting tumor growth, bone destruction, and resistance to therapy. We found that, upon contact with osteocytes, MM cells exhibit increased Notch signaling, a rapid upregulation of Notch receptor (NR) 3 expression, and increased proliferation rates. However, whether NR3 signaling contributes to MM cell proliferation, communication with osteocytes, and resistance to therapy is unknown. Objective. The goal of this study was to characterize the effects of the genetic deletion of NR3 in 5TGM1 MM cells on MM cell proliferation, viability, and response to anti-MM agents. Methods. Murine 5TGM1 MM cells were transduced with lentiviral particles containing shRNA-scramble (control) or shRNAs targeting NR3 and were selected with puromycin. 5TGM1 cells transduced with shRNA-NR3 clone 1 exhibited a 50-60% decrease in NR3 mRNA expression and were selected for follow up studies. To study the consequences of NR3 silencing in MM cell function we used MTT and Trypan Blue exclusion assays, and RT-qPCR. Results. NR3 expression was undetectable in MM cells transduced with shRNA-NR3, whereas the expression of NR1 and NR2 remained unchanged compared to control cells. In addition, NR3 silencing significantly decreased the mRNA expression of Notch target genes and the pro-osteoclastogenic cytokine Rankl. NR3-silenced cells displayed a 15%, 25%, and 35% reduction in cell proliferation/number after 24h, 48h, and 72h of culture compared to control cells. These effects were associated with modest increases in the number of NR3-silenced dead cells (2% vs 10%, control vs shNR3, respectively). Next, we evaluated the response to treatment with a gamma-secretase inhibitor (GSI) and bortezomib (BTZ), anti-MM agents, in both control and NR3- silenced cells. 72h treatment with GSI induced a 20-25% decrease in cell number and a 2-fold increase in cell death in both control and NR3-silenced cells. Treatment with GSI reduced the expression of Notch target genes, cyclin D1, and Rankl similarly in both control and NR3-silenced cells. In contrast, GSI did not affect the expression of NR1, 2 and 3. BTZ equally decreased (20-25%) the number/proliferation of control and NR3-silenced cells, an effect accompanied by a 2-fold increase in cell death in both cell lines. Conclusions. Our findings support that autocrine NR3 signaling in MM cells drives proliferation, favors survival, and enhances their osteoclastogenic potential by increasing Rankl expression. NR3 silencing did not alter the anti-MM efficacy of BTZ, suggesting that other NRs may contribute to Notch-mediated drug resistance in MM. Future studies will analyze the role of NR3 signaling in the communication between MM cells and osteocytes.


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