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 2

MiR-16 is downregulated in the BM-MΦ of MM patients (A) Cytokine array showing, under stimulated conditions (i.e., in the presence of single-stranded RNA–mir-25 (ssRNA–miR-25), which stimulates TLR-7 and -8, the levels of NF-κB–induced, M2-associated cytokines (IL-6, IL-8, TNF-α, and VEGF) secreted by CD14+ macrophages (BM-MΦ), and CD14– cells (BM-CD14 neg.) isolated from the BM of MM patients (n = 3).

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MiR-16 is downregulated in the BM-MΦ of MM patients (A) Cytokine array s...
Data represent mean values ± SD. Cells were incubated with ssRNA–miR-25 for 24 hours prior to detection of cytokines. Cytokine levels were measured in pg/mL using a multiplex cytokine assay. (B) qPCR showing mRNA expression levels of IL-6, IL-8, and TNF-α in BM-MΦ isolated from MM patients (MM) or cancer-free donors (HD) (n = 4/group). P values were calculated using 2-tailed multiple t test. (C) qPCR showing decreased miR-16 expression in BM-MΦ isolated from MM patients as compared with that isolated from HD (n = 7/group). (D) Real-time PCR showing decreased expression of miR-16 in BM-MΦ compared with that in monocytes isolated from the peripheral blood (PB-M) of the same MM patients (n = 3). Data are presented as 2-ΔCT values. (E) Representative images captured by light microscopy showing PB-M differentiated to M2-like MΦ (M2-MΦ) with macrophage CSF (M-CSF) treatment for 7 days (right panel) as compared with undifferentiated PB-M (UI) (left panel). Magnification ×40. (F) Flow cytometric analysis showing phagocytosis of M2-MΦ in vitro. Phagocytosis assay was performed using latex beads coated with GFP fluorescently labeled IgG antibody. The engulfed fluorescent beads were detected by flow cytometry. Differentiated PB-M to M2-MΦ in the presence of M-CSF showed 84% GFP+ cells in contrast to UI cells that were only 4.63%. (G–I) Real-time PCR showing increases in CD163 (G) and IRF4 (H) mRNA expression, as well as a decrease in miR-16 expression (I) in M2-MΦ as compared with UI. CD163 and IRF4 levels were presented as fold changes over UI controls. miR-16 levels were presented as 2-ΔCT values (n = 3 patients). Data reported in C, D, and G–I represent the mean ± SD; P values were calculated using a 2-tailed unpaired t test.