MiR-16 regulates crosstalk in NF-κB tolerogenic inflammatory signaling between myeloma cells and bone marrow macrophages
Jihane Khalife, Jayeeta Ghose, Marianna Martella, Domenico Viola, Alberto Rocci, Estelle Troadec, Cesar Terrazas, Abhay R. Satoskar, Emine Gulsen Gunes, Ada Dona, James F. Sanchez, P. Leif Bergsagel, Marta Chesi, Alex Pozhitkov, Steven Rosen, Guido Marcucci, Jonathan J. Keats, Craig C. Hofmeister, Amrita Krishnan, Enrico Caserta, Flavia Pichiorri
Jihane Khalife, Jayeeta Ghose, Marianna Martella, Domenico Viola, Alberto Rocci, Estelle Troadec, Cesar Terrazas, Abhay R. Satoskar, Emine Gulsen Gunes, Ada Dona, James F. Sanchez, P. Leif Bergsagel, Marta Chesi, Alex Pozhitkov, Steven Rosen, Guido Marcucci, Jonathan J. Keats, Craig C. Hofmeister, Amrita Krishnan, Enrico Caserta, Flavia Pichiorri
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Research Article Hematology Oncology

MiR-16 regulates crosstalk in NF-κB tolerogenic inflammatory signaling between myeloma cells and bone marrow macrophages

Abstract

High levels of circulating miR-16 in the serum of multiple myeloma (MM) patients are independently associated with longer survival. Although the tumor suppressor function of intracellular miR-16 in MM plasma cells (PCs) has been elucidated, its extracellular role in maintaining a nonsupportive cancer microenvironment has not been fully explored. Here, we show that miR-16 is abundantly released by MM cells through extracellular vesicles (EVs) and that differences in its intracellular expression as associated with chromosome 13 deletion (Del13) are correlated to extracellular miR-16 levels. We also demonstrate that EVs isolated from MM patients and from the conditioned media of MM-PCs carrying Del13 more strongly differentiate circulating monocytes to M2-tumor supportive macrophages (TAMs), compared with MM-PCs without this chromosomal aberration. Mechanistically, our data show that miR-16 directly targets the IKKα/β complex of the NF-κB canonical pathway, which is critical not only in supporting MM cell growth, but also in polarizing macrophages toward an M2 phenotype. By using a miR–15a-16-1–KO mouse model, we found that loss of the miR-16 cluster supports polarization to M2 macrophages. Finally, we demonstrate the therapeutic benefit of miR-16 overexpression in potentiating the anti-MM activity by a proteasome inhibitor in the presence of MM-resident bone marrow TAM.

Authors

Jihane Khalife, Jayeeta Ghose, Marianna Martella, Domenico Viola, Alberto Rocci, Estelle Troadec, Cesar Terrazas, Abhay R. Satoskar, Emine Gulsen Gunes, Ada Dona, James F. Sanchez, P. Leif Bergsagel, Marta Chesi, Alex Pozhitkov, Steven Rosen, Guido Marcucci, Jonathan J. Keats, Craig C. Hofmeister, Amrita Krishnan, Enrico Caserta, Flavia Pichiorri

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Figure 7

36.

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36. MiR-16 increases MM cell sensitivity to bortezomib by impairing NF-κ...
MiR-16 increases MM cell sensitivity to bortezomib by impairing NF-κB signaling. (A) Western blot analysis showing decrease in P65 nuclear protein in NCI-H929 upon transfection with ds-miR–16 or Scr Control for 48 hours. Histone Deacetylase 1 (HDAC1) was used as nuclear loading control. (B) Single cell flow cytometric analysis showing decrease in nuclear p65 protein expression in MM cells upon transfection with ds-miR–16 as compared with Scr control (upper panel). Percent of normalized P65 frequency in 1 × 104 cells transfected with ds-miR–16 or Scr control are shown (lower panel). (C) Luciferase reporter assay showing decrease in NF-κB transcriptional activity in U-937 cells by miR-16 as compared with Scr control. (D) Parallel to C, except that luciferase activity was assessed in HS-5 cells, no stimulation was induced, and an additional transfection with pGL3 vector containing a trimer of mutated NF-κB binding sites sequence (mut 3×NF-κB) was performed. Data are presented as percent of WT 3×NF-κB Scr control. (E) Cytokine array showing concentrations of cytokines/chemokines IL-6, IL-8, TNF-α, and INF-γ released by BM-MΦ isolated from MM patient upon treatment with ds-miR–16 or Scr sequences encapsulated in liposomes for 48 hours. (F) Luciferase reporter assay showing decrease in NF-κB transcriptional activity in U266 upon transfection with miR-16 as compared with Scr control. Cells were transfected with pGL3 luciferase vector containing a trimer of consensus WT NF-κB binding sites for 6 hours and were then treated with 5 nM bortezomib (bortez), 10 μM BAY11-7082, or vehicle control (veh) for 12 hours. Data are presented as percent of Scr veh control. (G) Representative flowsight showing apoptosis analysis by Annexin-V/PI double staining. Annexin-V/PI staining was performed on MM.1S cells upon transfection with miR-16 or Scr sequences and treated with bortez 2.5 nM or veh DMSO control for 48 hours. The percentages of Annexin-V–positive cells are shown in the upper left quadrants. (H) Luciferase assay showing percent of viable MM.1S GFP/Luc+ cells in suspension upon treatment with bortez or DMSO veh control, cocultured with BM-MΦ isolated from a patient for 48 hours. (I) Luciferase assay showing percentage of viable MM.1S GFP/Luc+ cells in suspension upon treatment with bortez or DMSO veh control cocultured with BM-MΦ isolated from a MM patient and treated with ds-miR–16 or Scr sequences for 48 hours. Values represent the mean ± SD for each experiment performed in triplicate. When not otherwise specified in the legend, the reported P values were calculated using 2-tailed unpaired t test.