miRNA-21 inhibition enhances RANTES and IP-10 release in MCF-7 via PIAS3 and STAT3 signalling and causes increased lymphocyte migration

Highlights

• miRNA-21 negatively regulates chemoattractant release and lymphocyte migration.

• PIAS3 is a direct target of miRNA-21 in MCF-7 cells.

• miRNA-21-STAT3 pathway is involved in tumour immunoresistance.

• New property of miRNA-21 in regulating immune cell recruitment as an oncomiR.

Abstract

MicroRNAs (miRNAs) are a class of small endogenous gene regulators that have been implicated in various developmental and pathological processes. However, the precise identities and functions of miRNAs involved in antitumor immunity are not yet well understood. miRNA-21 is an oncogenic miRNA that can be detected in various tumours. In this study, we report that a miRNA-21 inhibitor enhances the release of chemoattractants RANTES and IP-10 in the MCF-7 breast cancer cell line and results in increased lymphocyte migration. Thus, miRNA-21 is a potential therapeutic target for cancer immunotherapy. We further demonstrated that PIAS3, a protein inhibitor of activated STAT3, is a target of miRNA-21 in MCF-7. Thus, miRNA-21 is a novel miRNA regulating immune cell recruitment, which acts at least in part via its inhibition of PIAS3 expression and oncogenic STAT3 signalling in tumour cells.

Introduction

Cancer immunotherapy has developed into a first-line therapeutic regimen, particularly in cancers refractory to chemotherapy. However, immune-mediated cancer regression preferentially occurs in the minority of patients who have a T cell-inflamed tumour environment [1], suggesting that infiltration of immune effectors into tumour locations is required for tumour eradication. Thus, a pre-existing T cell inflamed tumour microenvironment may be predictive of clinical outcome to immunotherapy [2], [3]. Previous studies have demonstrated that infiltration of CD8+ effector T cells in inflamed tissues depend on adhesion molecules and specific chemokines such as RANTES and IP-10 [4], [5]. Several intracellular signalling pathways, including signal transducer and activator of transcription 3 (STAT3), Notch, β-catenin and PI3K, regulate the expression of inflammatory cytokines and chemokines [6], [7], [8], [9]. Correlations amongst these signalling pathways, together with various inflammatory mediators of adhesion molecules, cytokines and chemokines, form a complex immune regulatory network. The absence of chemokines may present a major barrier to cancer immunotherapy in a subset of patients. Further investigation of the molecular mechanisms underlying immune cell infiltration and recruitment to tumours is urgently required to design new therapeutic strategies and improve clinical outcomes for cancer immunotherapy.

MicroRNAs (miRNAs) are small, endogenous, noncoding RNAs that target the 3′-untranslated region (3′-UTR) of specific mRNAs and result in degradation or repression of translation [10]. miRNAs regulate important cellular processes such as cell differentiation, proliferation, apoptosis and tumourigenesis. miRNAs are also involved in cancer-related inflammatory responses, and certain miRNAs display important modulation of the migration and immunogenicity of immune cells. Mark et al. have shown that induced miRNA-146 expression negatively regulates IL-8 and RANTES release in human alveolar epithelial cells [11]. They further demonstrated that divergent intracellular signalling pathways are involved in the regulation of chemokine production by miRNA-146 [12]. miRNA-125b has been reported to regulate the expression of cytokines and chemokines by targeting tumour necrosis factor α (TNF-α) transcription in mouse Raw 264.7 macrophages [13].

miRNA-21 is an oncogenic miRNA that has been reported to regulate tumour cell proliferation, tumourigenicity, migration and invasion, apoptosis and chemoresistance by repressing expression of various target molecules [14], [15], [16], [17], [18]. STAT3 has recently been predicted via bio-informatics as another target of miRNA-21 [19]. Xiong et al. [20] demonstrated that PIAS3, a protein inhibitor of activated STAT3, is a direct target of miRNA-21 and contributes to the oncogenic function of miRNA-21 in myeloma cells. STAT3 is constitutively activated in many types of human malignancies and is capable of inducing a large number of genes that are crucial for cancer inflammation [21], [22]. Blocking STAT3 signalling in tumour cells leads to the production of multiple chemoattractants that induce migration of various immune cells [23]. Based on these findings, we tested whether miRNA-21 regulates release of chemokines and affects lymphocyte migration. We present here the first evidence that downregulation of miRNA-21 increases chemokine release in MCF-7 human breast cancer cells in vitro and results in increased lymphocyte migration, partially due to targeting PIAS3, which regulates the STAT3 signalling pathway. These results represent a novel mechanism in immunoresistance.