Tumor suppressor miR-34a targets PD-L1 and functions as a potential immunotherapeutic target in acute myeloid leukemia
Introduction
The mainstay of the adaptive immune system is the antigen presentation of processed peptides by antigen presenting cells (APC) [1], [2], and recognition of the T cell receptor of a peptide presented on MHC molecules of an APC provides the first signal [3]. However, the optimal activation of a T lymphocyte requires a second signal provided by co-stimulatory molecules, which are normally balanced with inhibitory molecules [4].
The type I transmembrane protein PD-L1 (also called B7-H1 and CD274) is one of the recently identified T lymphocyte inhibitory molecules [5], [6]. PD-L1 is expressed on APCs and delivers an inhibitory signal to its receptor, programmed death-1 (PD-1) in T cells, and the formation of the PD-1/PD-L1 complex leads to both inhibition and induction of apoptosis in effector T lymphocytes [7], [8]. In recent years, the aberrant expression of PD-L1 in tumor tissues has been reported in various cancers [5], [9], [10] and is associated with high-risk prognostic factors [11]. There is also evidence that in solid tumors PD-L1 expression is upregulated in response to the common inflammatory cytokines such as IFN-γ, TNF-α, IL-1 [12], and common chemopreventive agents. In breast cancer cells, tumor cells can thus escape from adaptive immunity by increasing their surface expression of inhibitory PD-L1, through chemotherapeutic agent mediated PD-L1-specific T cell apoptosis [13]. Therefore, there is a potential link between chemotherapy and immunoresistance. Although considerable evidence supports the involvement of the PD-1/PD-L1 pathway in the negative regulation of many adaptive immune responses, the regulation of the transcriptional activity of PD-L1 is not yet well known.
MicroRNAs (miRNAs, or miRs) are a class of small (18–25 nucleotides) noncoding RNA molecules that modulate gene expression post-transcriptionally [14], [15]. MicroRNAs bind to the 3′-untranslated region (3′UTR) of messenger RNAs (mRNAs) of a target gene resulting in mRNA cleavage and/or translational suppression, and play a pivotal role in the regulation of many biological functions [16], [17]. Increasing evidences reveal that miRNAs could perform either as an oncogene or tumor suppressor in tumorigenesis [18], [19], [20].
Although most studies of microRNAs have been focused on its function as a tumor oncogene or a tumor suppressor gene, recent evidence has also shown that microRNAs play important roles in the regulation of host immune responses [21]. Several members of microRNAs have been shown to translationally regulate a TLR signaling cascade [22], [23]. Xu et al. [24] demonstrated the down-regulation of the miR-29 family in a broad spectrum of solid tumors. Moreover, this down-regulation was inversely correlated with the high protein expression of an immune modulator protein B7-H3, and it was further found that the interaction between miR-29 and B7-H3 was direct, that there is a conserved miR-29 binding site in B7-H3 3′UTR. Recent reports confirmed that the phosphatase and tensin homolog/phosphatidylinositol-3-kinase (PTEN/PI3K) pathway [25], [26], miR-513 [27], and miR-570 [28] played important roles in the transcriptional regulation of PD-L1 cell surface expression. Nevertheless, whether other miRNAs are involved in the transcriptional regulation of PD-L1 expression remains unclear.
miR-34a, which has been studied extensively in solid tumors, is known to be down-regulated in chronic lymphocytic leukemia [29], colorectal cancer [30], lung cancer [31], and several other types of cancer [32], [33]. In work described here, we show that miR-34a is down-regulated in AML. Moreover, this down-regulation is inversely correlated with high protein expression of PD-L1. Transfection of miR-34a reduces PD-L1 expression and reduces IFN-γ-induced PD-L1 expression; in contrast, an inhibitor to miR-34a induces PD-L1 protein expression. We also showed that miR-34a is capable of targeting a predicted site in the PD-L1 3′UTR, resulting in transcriptional repression. miR-34a transfection or PD-L1 blocking antibody reduces INF-γ induced PD-L1-associated apoptosis of cocultured T cells, and there is a positive feedback between PD-L1 expression and AKT activation. Thus, a novel miR-34a-mediated regulation of PD-L1 expression has been identified in AML cells, and miR-34a may play a role in chemotherapeutic agent induced immune-resistance, and immune-based therapy of human AML.
Section snippets
Patient samples
Aspirated bone marrow (BM) specimens from thirteen patients with newly diagnosed AML and five healthy donors with no evidence of hematologic neoplasia used as reference controls were collected and evaluated at the Department of Hematology of Tangdu Hospital of the Fourth Military Medical University (Xi'an, Shaanxi, China) between May 2012 and July 2012. For the use of these clinical materials for the purpose of research, all patients provided written informed consent, and approval from the
Overexpression of PD-L1 in AML patients
PD-L1 expression levels in AML patients and healthy donor samples were examined by means of quantitative real-time RT-PCR. As shown in Fig. 1A, all (100%) of the 44 samples expressed PD-L1 ranging from 9.0 × 10− 1 to 3.9 × 101 (PD-L1 expression level was normalized to β-actin), However, a very low level of PD-L1 expression was detected in 5/5 healthy donor samples. The expression of miR-34a ranged from 0.6 × 10− 1 to 2.4 × 100 (Fig. 1B, miR-34a expression level was normalized to U6).
Expression of miR-34a and PD-L1 was reversely correlated in human leukemia
When compared with
Discussion
The key findings in this report are the following: 1) the expression profile of PD-L1 and miR-34a is reversely correlated in AML samples; 2) transcriptional repression of PD-L1 by miR-34a exists in human HL-60 leukemia cells; and 3) IFN-γ and As2O3 altered the expression of PD-L1, and overexpression of miR-34a reversed IFN-γ and As2O3-induced expression of PD-L1 and consequently influenced PD-L1 specific-T cell apoptosis. These data indicate that miR-34a targets PD-L1 expression and is involved
Conclusion
All together, the present study offers a new insight into a novel relationship between miR-34a and PD-L1 expression in AML patients. Our data indicate that the expression profile between PD-L1 and miR-34a was reversely correlated; miR-34a directly targets the 3′UTR of PD-L1; IFN-γ and As2O3-induced PD-L1 expression involves relief of miR-34a-mediated translational repression in human HL-60 cells; more importantly, PD-L1 specific T cell apoptosis was reduced as well following miR-34a
Conflict of interest
The authors have no financial conflict of interest.
Acknowledgments
This study was supported by a grant from the National Natural Science Foundation of China (no. 81201775). We thank everyone from the Department of Medical Laboratory and Research Center in Tangdu Hospital, Fourth Military Medical University for their sincere help and technical support; and we also thank everyone from the Department of Hematology in Tangdu Hospital, Fourth Military Medical University for offering bone marrow samples.
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Xi Wang and Jinge Li contributed equally to this work.