Targeting CD47 and TNFAIP8 By Mir-155 Overcomes Drug Resistance and Inhibits Tumor Growth through Induction of Phagocytosis and Apoptosis in Multiple Myeloma

CD47, an integrin-associated receptor, is highly expressed in multiple myeloma (MM). CD47 on the surface of MM cells interacts with signal regulatory protein-a (SIRPa) on macrophages and activates "don't eat me" signaling pathway inhibiting the phagocytosis of CD47-expressing MM cells by macrophages. We have shown that overexpression of CD47 is associated with shorter progression free and overall survival in patients with MM. However, the molecular mechanisms underlying CD47 overexpression and its role in drug resistance of MM remain undefined.

To explore the role of CD47 in drug resistance of MM, we evaluated its expression level in drug resistant MM cells (8226-R5 and MM.1R) and identified that CD47 mRNA and protein were significantly upregulated in drug resistant MM cells compared with their parental lines (8226 and MM.1S, respectively). Analysis of the MM datasets also revealed that CD47 expression was significantly upregulated in relapsed MM compared with newly diagnosed MM and normal donors. Using miRNA target scan algorithm, we identified miR-155 as a regulator of CD47. miR-155 was downregulated in drug resistant MM cells compared with their parental lines and its downregulation was associated with advanced stages of MM disease. In addition, datasets analysis indicated a significant negative correlation between CD47 and miR-155 level in MM patients. We further demonstrated that miR-155 directly targeted CD47-3'UTR using luciferase assay. Moreover, overexpression of miR-155 markedly decreased CD47 protein level in MM resistant cells. FACS analysis of CD47 stained MM cells also revealed that cell surface expression of CD47 was decreased after miR-155 overexpression.

Furthermore, we showed that targeting of CD47 by miR-155 abrogated the protection against phagocytosis and promoted the phagocytosis of MM cells by macrophages resulting in inhibition of MM cells growth. To explore whether CD47 overexpression could attenuate the enhancing effect of miR-155 on phagocytosis of MM cells, we performed functional rescue assay by overexpressing CD47 in 8226-R5 and MM.1R cells and co-transfecting with miR-155 mimics or scrambled miRNA. CD47 overexpression abolished the phagocytosis of MM cells by macrophages confirming that miR-155 activated "eat me" signal in MM cells by targeting CD47.

On the other hand, we demonstrated that beside promoting phagocytosis, miR-155 overexpression induced apoptosis through targeting of TNFAIP8, a negative mediator of apoptosis, and re-sensitized the resistant MM cells to bortezomib (BTZ). Analysis of the MM patient's pair dataset revealed that TNFAIP8 was significantly upregulated in relapse vs newly diagnosed stages of the disease. We also identified a significant negative correlation between TNFAIP8 and miR-155 expression level in MM patients. Moreover, TNFAIP8 level was remarkably upregulated in drug resistant cells compared to their parental cells. The luciferase assay using wild-type and mutant 3'UTR of TNFAIP8 confirmed that miR-155 directly targeted TNFAIP8. Transfection of MM resistant cells with miR-155 mimics could also reduce the expression level of TNFAIP8 and induce apoptosis in drug resistant MM cells.

In order to translate our findings to a therapeutic model, we next stablished a xenograft model and investigated the effect of miR-155 on tumorigenesis of MM resistant cells in vivo. Administration of miR-155 mimics significantly suppressed the tumor growth in combination with BTZ. The miR-155 or BTZ alone moderately prolonged survival compared to control treatment. However, the combination treatment of miR-155 mimics and BTZ significantly extended the overall survival. In addition, TNFAIP8 and CD47 levels were decreased in miR-155 overexpressing groups compared with controls, indicating in vivo targeting of these genes by miR-155. IHC analysis of xenograft tumor sections showed that combination of miR-155 mimics and BTZ treatment resulted in a decrease in the proliferation index (Ki67) and an increase in the apoptotic index (Tunnel), compared to either BTZ or miR-155 mimics alone. Collectively, these results support that regulatory interaction between miR-155 and its targets, CD47 and TNFIP8, provides the rationale that restoration of miR-155 may serve as a promising therapeutic approach by promoting of phagocytosis and inducing of apoptosis at the same time in patients with refractory/relapsed MM.