Molecular Mechanism And Clinical Significance Of STING Signaling Pathway Regulating Myelogenous Inhibitory Cells In

BackgroundNasopharyngeal carcinoma (NPC), having the highest incidence in southern China, is consistently associated with Epstein-Barr virus (EBV). Chemo-radiotherapy is the mainstream treatment for NPC, but recurrence and metastasis still pose a tough challenge for oncologists. For patients with locally advanced NPC, the recurrence rate at 5 years is higher than 40%, while the median overall survival of patients with refractory disease or metastatic disease, is less than 1 year. Immune checkpoint inhibitors such as PD-1 antibody, adoptive cellular therapy such as EBV specific T cells, have shown promising efficacy in NPC. However, the response rate of these strategies remain unsatisfactory. Suppressive infiltrating cells, regulatory cytokines and the whole inhibitory immune network in the tumor microenvironment (TME), are proposed to be the main factor contributing to immune evasion and treatment failure.The differentiation of myeloid lineage cells is perturbed in the TME and results in the expansion of tumor promoting myeloid cell populations, mostly myeloid-derived suppressor cells (MDSCs) and tumor associated macrophages. Our previous studies discovered that compared with healthy donors, the numbers of MDSCs increased significantly in peripheral blood and tumor tissues in NPC patients, leading to tumor progression and metastasis.Stimulator of interferon genes (STING; also known as ERIS, MYPS or MITA), encoded by TMEM173, is a versatile adaptor protein involved in cytosolic DNA sensing and plays a vital role in host innate immune response. Following recognition of aberrant DNA species released from a number of invading pathogens, including DNA viruses, retroviruses and certain bacteria, STING signaling is activated and induces the transcription of type Ⅰ interferons (IFNs) and numerous pro-inflammatory cytokines. Recently, activation of STING signaling by tumor-derived DNA in the tumor-residing antigen-presenting cells (APCs), especially dendritic cells and macrophages, has been shown to be essential in promoting both spontaneous and treatment induced anti-tumor immune response.ObjectiveTo examine the functional status of STING signaling in NPC neoplastic cells and its impact on the differentiation of the infiltrating myeloid cells.Materials and Methods1. Examine the functional status of STING signaling in NPC STING expression in clinical samples (tumor tissues, serum samples) and cell lines (TW03, C666, CNE2, NP69), was examined using Western Blot, the enzyme-linked immunosorbent assay (ELSIA) and immunohistochemistry (IHC). HSV-1 was used to stimulate the STING signaling in cell lines.2. Analysis the impact of STING signaling on MDSC inductionBy combining siRNA silencing, point mutation and gene transfection, we analyzed the impact of STING signaling on MDSC induction. The number, the surface markers, as well as the T cell proliferation inhibiting capacity of MDSC, were measured.3. Screen and validate the microRNAs that directly targeting STINGFollowing prediction by the bioinformatic softwares, STING transcription and expression were compared between tumor cell lines over-expressing the specific microRNA and the control group. Luciferase reporter assay, was employed to validate the prediction.4. Explore the clinical significance of STING signalingSTING expression in tumor tissues from NPC patients, were correlated with common clinicopathological parameters, with its prognostic significance of STING signaling explored.Results1. STING signaling were deregulated in NPCCompared with NP69, STING expression and STING signaling were down-regulated, before and after HSV-1 stimulation. In NPC patients, STING expression in tumor tissues was significantly lower than that of adjacent non-cancerous tissues (n=22, p<0.001) and serum STING concentration in NPC patients was also significantly lower that of healthy donors (p=0.025).2. Deregulation of STING signaling contributed to MDSC inductionDeregulating STING signaling in TW03 cell lines by silencing STING translation using siRNA or by blocking STING phosphorylation through point mutation, led to a significant increase of MDSC induction and a significant improvement of the suppression capacity of tumor-induced MDSCs on T cell proliferation. Conversely, over-expressing STING in TW03 cell lines, resulted in decreased MDSC expansion and T cell proliferation inhibiting capacity. Furthermore, STING expression was negatively correlated with CD33 expression in NPC patients’ tumor tissues (n=50, p<0.001).3. STING is a direct target of miR-24MiR-24, miR-891a,miR-106a, miR-20a, and miR-1908, were previously found to be over-expressed in NPC, of which miR-24 was predicted to directly targeting STNG. Using qPCR, Western blot and luciferase reporter assay, we demonstrated that miR-24 can directly bound to 3’UTR of STING and negatively affected its expression.4. STING expression was correlated with patient’s recurrence-free survivalIn NPC patients, STING expression in tumor tissues was found to be significantly correlated with serum STING concentration (p<0.0001), baseline plasma EBV DNA load (p=0.041), and recurrence-free survival (p=0.041).ConclusionSTING signaling was deregulated in NPC, partly by miR-24, which led to the expansion of MDSC in the tumor microenvironment and negatively affected patient’s prognosis.