MiR-16 regulates crosstalk in NF-κB tolerogenic inflammatory signaling between myeloma cells and bone marrow macrophages
Jihane Khalife, … , Enrico Caserta, Flavia Pichiorri
Jihane Khalife, … , Enrico Caserta, Flavia Pichiorri
Published October 8, 2019
Citation Information: JCI Insight. 2019;4(21):e129348. https://doi.org/10.1172/jci.insight.129348.
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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 3

Extracellular miR-16 impairs MM-EV–induced M2-MΦ.

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Extracellular miR-16 impairs MM-EV–induced M2-MΦ. (A) Representative ima...
(A) Representative images captured by light microscopy showing PB-M isolated from a MM patient differentiated to M2-MΦ in the presence of the matched BM acellular fraction (matched BM-ac) (right panel), in contrast to undifferentiated PB-M (UI) (left panel). (B) qPCR showing decreased expression of miR-16 in PB-M isolated from MM patient and differentiated to M2-MΦ in the presence of the matched BM acellular fraction (PB-M + BM-ac), as well as in total MΦ isolated from the BM of the same patient (MM-BM-MΦ), as compared with undifferentiated PB-M using samples obtained from n = 3 MM patients. Values represent the mean ± SD; P values were calculated using ordinary 1-way ANOVA multicomparison. (C) Representative images captured by light microscopy showing differentiation of PB-M obtained from a HD incubated with EV isolated from the BM-acellular fraction of a MM patient (BM-ac) (left panel) or EV-depleted BM-ac (right panel) (n = 4 patients; see Supplemental Figure 2B). (D) Representative images showing phagocytosis of latex beads coated with GFP fluorescently labeled IgG antibody by PB-M differentiated to M2-MΦ when incubated with EV isolated from the BM-ac of a MM patient (+) (right panel), whereas no phagocytosis was observed when PB-M were incubated with the EV-depleted BM-ac (–) (left panel). (E) Flow cytometric analysis showing percent increase in expression of M2-MΦ surface marker (CD163) on PB-M treated with EV isolated from the conditioned media of a MM cell line, NCI-H929, for 7 days (EV) (upper right panel) and compared with cells incubated with EV-depleted acellular fraction (Ctl) (upper left panel). The same effect was seen using EV isolated from another MM cell line (MM.1S) (lower panel). Gating strategy was set using IgG anti-PE antibody isotype control. (F) Flow cytometric analysis showing percent surface expression of CD163 on PB-M differentiated with EV isolated from the conditioned media of MM.1S cells and concomitantly incubated with either ds-miR–16 (middle right panel), ds-miR–223 (lower right panel), or scramble control (Scr) (upper right panel). CD163 percent surface expression on undifferentiated cells (UI) incubated with the microRNAs cited above are also shown (left panels). Gating strategy was set using IgG anti-PE antibody isotype control. (G) Representative images captured by light microscopy showing impairment of PB-M differentiation to M2-MΦ in the presence of ds-miR–16. PB-M were incubated with the EV isolated from MM.1S, along with ds miR-16 (right panel) or Scr control (left panel) for 7 days. All magnifications ×40.