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 6

MiR-16 directly regulates the expression of IKKα/β complex (A and B) Real-time PCR analysis revealing mRNA expression of IKKα and -β complex (A) in MΦ isolated from MM patients (n = 3), and U-937 cell line (B), each transfected with ds miR-16 or Scr control for 48 hours.

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MiR-16 directly regulates the expression of IKKα/β complex (A and B) Rea...
(C) Western blot analysis showing protein downregulation of IKKα and -β by miR-16 in U-937 (left panel) and HS-5 cells (right panel) as compared with Scr control after 48 hours of treatment; GAPDH was used as loading control. (D and E) Real-time PCR showing mRNA expression of IKKα (D) and -β (E) in differentiated MΦ isolated from WT or miR-15a/16-1–/– mouse spleens (n = 4/group; the RNA from each 2 mice/group were pooled for quality purposes and a triplicate PCR reading was done for each pool). In D and E, error bars represent standard deviation. (F) Western blot analysis showing protein levels of IKKβ and IKKα in monocytes/MΦ CD11b fractions isolated from WT or miR-15a/16-1–/– mice spleens (n = 2 mice, WT; n = 3 mice, miR-15a/16-1–/–). β-Actin is used as loading control. (G) Real-time PCR showing mRNA downregulation of IKKα expression in MM cell lines (MM.1S and NCI-H929) transfected with ds-miR–16 as compared with Scr control for 48 hours. (H) Western blot analysis showing protein downregulation of IKKα and -β by miR-16 in 3 MM cell lines (U266, MM.1S, and NCI-H929); GAPDH was used as loading control. (I) Luciferase reporter assay revealed direct downregulation of IKKβ 3′UTR transcriptional activity by miR-16. pGL4.11 luciferase vector containing the 3′UTR of IKKβ was transfected in U266 or HS-5 for 18 hours, followed by a second transfection with ds-miR–16 or Scr control for an additional 12 hours. Transfection efficiency was controlled by cotransfection with TK promoter-Renilla vector. Data are presented as percent of Scr control. When not otherwise specified in the legend, the reported P values were calculated using 2-tailed unpaired t test. Each of the reported experiments was performed in triplicate. For C and H, the data presented are representative of 2 independent experiments.