Function of miR-146a-5p in Tumor Cells As a Regulatory Switch between Cell Death and Angiogenesis: Macrophage Therapy Revisited.

Elina Simanovich,Michal A Rahat,Maya M Rahat,Vera Brod

doi:10.3389/fimmu.2017.01931

Abstract

Tumors survive and progress by evading killing mechanisms of the immune system, and by generating a tumor microenvironment (TME) that reprograms macrophages in situ to produce factors that support tumor growth, angiogenesis, and metastasis. We have previously shown that by blocking the translation of the enzyme inducible nitric oxide synthase (iNOS), miR-146a-5p inhibits nitric oxide (NO) production in a mouse renal carcinoma cell line (RENCA), thereby endowing RENCA cells with resistance to macrophage-induced cell death. Here, we expand these findings to the mouse colon carcinoma CT26 cell line and demonstrate that neutralizing miR-146a-5p’s activity by transfecting both RENCA and CT26 cells with its antagomir restored iNOS expression and NO production and enhanced susceptibility to macrophage-induced cell death (by 48 and 25%, respectively, p < 0.001). Moreover, miR-146a-5p suppression simultaneously inhibited the expression of the pro-angiogenic protein EMMPRIN (threefolds, p < 0.001), leading to reduced MMP-9 and vascular endothelial growth factor secretion (twofolds and threefolds, respectively, p < 0.05), and reduced angiogenesis, as estimated by in vitro tube formation and scratch assays. When we injected tumors with pro-inflammatory-stimulated RAW 264.7 macrophages together with i.v. injection of the miR-146a-5p antagomir, we found inhibited tumor growth (sixfolds, p < 0.001) and angiogenesis (twofolds, p < 0.01), and increased apoptosis (twofolds, p < 0.01). This combination therapy increased nitrites and reduced TGFβ concentrations in tumor lysates, alleviated immune suppression, and allowed enhanced infiltration of cytotoxic CD8+ T cells. Thus, miR-146a-5p functions as a control switch between angiogenesis and cell death, and its neutralization can manipulate the crosstalk between tumor cells and macrophages and profoundly change the TME. This strategy can be therapeutically utilized in combination with the macrophage therapy approach to induce the immune system to successfully attack the tumor, and should be further explored as a new therapy for the treatment of cancer.

Highlights

By secreting a myriad of chemoattractants and growth factors, tumor cells actively recruit macrophages into the tumor mass and reprogram them in situ to produce elevated levels of growth factors, pro-angiogenic factors, and anti-inflammatory cytokines that collectively promote tumor growth and metastasis and mediate evasion of immune recognition [1,2,3,4].One of the hallmarks of pro-inflammatory macrophages or M1-activated macrophages is the high expression of the enzyme inducible nitric oxide synthase that generates high amounts of the cytotoxic molecule nitric oxide (NO), as well as other cytotoxic molecules (e.g., TNFα) that serve as a killing mechanism [5]
Since inducible nitric oxide synthase (iNOS) mRNA was increased in all three cell types, but protein expression was not, we reasoned that a post-transcriptional regulation of iNOS exists in CT26 and renal carcinoma cell line (RENCA) cells, but not in TRAMP-C2 cells
The expression of iNOS is inversely correlated with miR146a-5p expression in the three tumor cells, and EMMPRIN expression does not correlate to the stimulation or to miR-146a-5p expression, probably as it is already maximally expressed

Summary

Introduction

By secreting a myriad of chemoattractants and growth factors, tumor cells actively recruit macrophages into the tumor mass and reprogram them in situ to produce elevated levels of growth factors, pro-angiogenic factors, and anti-inflammatory cytokines that collectively promote tumor growth and metastasis and mediate evasion of immune recognition [1,2,3,4].One of the hallmarks of pro-inflammatory macrophages or M1-activated macrophages is the high expression of the enzyme inducible nitric oxide synthase (iNOS) that generates high amounts of the cytotoxic molecule nitric oxide (NO), as well as other cytotoxic molecules (e.g., TNFα) that serve as a killing mechanism [5]. By secreting a myriad of chemoattractants and growth factors, tumor cells actively recruit macrophages into the tumor mass and reprogram them in situ to produce elevated levels of growth factors, pro-angiogenic factors, and anti-inflammatory cytokines that collectively promote tumor growth and metastasis and mediate evasion of immune recognition [1,2,3,4]. Tumor-associated macrophages and myeloid-derived suppressor cells, both of which are M2-like activated, secrete low levels of NO that are pro-angiogenic and immunosuppressive [7, 8]. Tumor cells can produce low amounts of NO [9], it has been demonstrated that in some types of tumors, tumor cells of higher grade and stage as well as metastatic cells tend to reduce or completely lose their iNOS expression in order to resist immune killing [10]. We have recently demonstrated that in the mouse renal cell carcinoma cell line RENCA, a specific microRNA molecule—miR-146a-5p— mediates the translational inhibition of iNOS [11]

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Conclusion