MicroRNA-92 expression in CD133+ melanoma stem cells regulates immunosuppression in the tumor microenvironment through integrin-dependent TGF-β activation

Abstract

Abstract The existence of cancer stem cells (CSC) accounts for the high degree of chemoresistance and heterogeneity characterizing refractory melanomas. Yet, the degree to which CSCs modulate immune cells in the tumor microenvironment has yet to be revealed. The present study aims to establish a novel role for miR-92 and its associated gene networks in immunomodulation. We employed syngeneic mouse models utilizing B16-F10 melanoma cells to observe primary tumor and metastatic growth. CSCs were isolated from the parental line based on expression of the putative CSC marker, CD133. CD133+ cells were functionally distinct from CD133− cells with CD133+ cells showing increased proliferation in vitro and enhanced tumorigenesis when administered subcutaneously. Our data indicated that compared to CD133− cells, CD133+ CSCs had a greater capacity to recruit immunosuppressive cell types during tumor formation. Using microarray technology in order to expose disparities in microRNA expression between CSC and non-CSC compartments, we identified several miRs that were significantly downregulated in CD133+ cells including miR-92. We hypothesized that lower levels of miR-92 in CSCs led to higher expression of integrin subunits as predicted by gene alignment software and confirmed using qRT-PCR. Flow cytometry analysis of dissociated tumors demonstrated that tumors initiated by CD133+ cells displayed significantly higher levels of TGF-β compared to CD133− cells. Additionally, qRT-PCR for TGF-β associated genes confirmed a significant increase in expression in CD133+ cells. These data suggest that CSCs efficiently induce intratumoral immunosuppression which is potentially regulated by a miR-92 driven axis involving integrin activation of TGF-β.