MicroRNA-92 Expression in CD133⁺ Melanoma Stem Cells Regulates Immunosuppression in the Tumor Microenvironment via Integrin-Dependent Activation of TGFβ.

Abstract

In addition to being refractory to treatment, melanoma cancer stem cells (CSC) are known to suppress host antitumor immunity, the underlying mechanisms of which need further elucidation. In this study, we established a novel role for miR-92 and its associated gene networks in immunosuppression. CSCs were isolated from the B16-F10 murine melanoma cell line based on expression of the putative CSC marker CD133 (Prominin-1). CD133⁺ cells were functionally distinct from CD133^- cells and showed increased proliferation <i>in vitro</i> and enhanced tumorigenesis <i>in vivo</i>. CD133⁺ CSCs also exhibited a greater capacity to recruit immunosuppressive cell types during tumor formation, including FoxP3⁺ Tregs, myeloid-derived suppressor cells (MDSC), and M2 macrophages. Using microarray technology, we identified several miRs that were significantly downregulated in CD133⁺ cells compared with CD133^- cells, including miR-92. Decreased expression of miR-92 in CSCs led to higher expression of target molecules integrin α_V and α₅ subunits, which, in turn, enhanced TGFβ activation, as evidenced by increased phosphorylation of SMAD2. CD133⁺ cells transfected with miR-92a mimic and injected <i>in vivo</i> showed significantly decreased tumor burden, which was associated with reduced immunosuppressive phenotype intratumorally. Using The Cancer Genome Atlas database of patients with melanoma, we also noted a positive correlation between integrin α₅ and TGFβ1 expression levels and an inverse association between miR-92 expression and integrin alpha subunit expression. Collectively, this study suggests that a miR-92-driven signaling axis involving integrin activation of TGFβ in CSCs promotes enhanced tumorigenesis through induction of intratumoral immunosuppression. SIGNIFICANCE: CD133⁺ cells play an active role in suppressing melanoma antitumor immunity by modulating miR-92, which increases influx of immunosuppressive cells and TGFβ1 expression.