The Role Of TIM-4 In IL-6 Promoted NSCLC Metastasis And The Related Mechanism

Background:Lung cancer is the predominant cause of cancer deaths in both men and women. While lung cancer is heterogeneous in cell types, which is generally divided into two major subtypes:small cell lung carcinomas (SCLCs) and non-small cell lung carcinomas (NSCLCs), with the latter comprising 85% of all lung cancer cases. Despite decades of research and clinical trials on different therapeutic interventions, the treatment outcome of lung cancer remains unsatisfactory.IL-6 level has been suggested as a prognostic marker for survival in advanced NSCLC patients treated with chemotherapy. Nevertheless, in analysis of patient sera or cell lines, inconsistent results were obtained. IL-6 level is higher in the sera of NSCLC patients than those of healthy controls, which is relative low in NSCLC cell lines. Recent studies suggest that IL-6 can induce EMT to play a role in promoting the migration and invasion of NSCLC and is expected to be a candidate prognostic indicator for NSCLC. At present, IL-6 antibody to treat NSCLC has entered phase ? clinical trial. However, the mechanism of IL-6 promoting NSCLC progression need to be further investigated.T cell immunoglobulin and mucin domain protein-4 (TIM-4),which is expressed on myeloid cells including dendritic cells (DCs) and macrophages from spleen,lymph nodes, orperitoneal cavity, serves as a critical sensor for controlling the functions of naive and activated T cells. TIM-4 differentially regulates T cell homeostasis by inhibiting naive T cells during the induction phase of an immune response and enhancing T cell responses at the effector phase. In addition, TIM-4 plays a critical role in phagocytosis of apoptotic cells by APCs through interaction with hosphatidylserine (PS). Although these studies verify the role of TIM-4 in the regulation of immune homeostasis by controlling APC functions, it remains unknown how TIM-4 impacts host immunity within tumor microenvironments. In particular, it is critical to evaluate the role of TIM-4 expressed on myeloid cells of distinct tissues including tumors in regulating immunity and tolerance.The pro-inflammatory cytokine interleukin-6 (IL-6) represents a keystone cytokine in infection, cancer and inflammation. Recent studies suggest that high expression of IL-6 is associated with poor survival of lung cancer patients. TIM-4 is a novel cell-surface glycoprotein belonging to TIM family. Recently, we identify TIM-4 as a new player in non-small cell lung cancer (NSCLC) progression. However, the role of TIM-4 in IL-6 promoted NSCLC migration and invasion remains unclear.PART ONEThe effects of IL-6 on TIM-4 expression in NSCLC cells and related mechanismObjective:This study aims to investigate the relationship between IL-6 and TIM-4 in migration and invasion of NSCLC cellsMethods & Results:1. IL-6 promotes expression of TIM-4 of lung cancer cell:IL-6 was used to stimulate A549 or NCI-H1975 cells, and TIM-4 mRNA and protein were detected by RT-PCR and Western blotting. We found that IL-6 induced TIM-4 mRNA and protein expression in lung cancer cells in a time and dose dependent manner.2. IL-6 promotes expression of TIM-4 of lung cancer cell depending on NF-?B signal transduction pathways:NF-?B or Stat3 inhibitor and IL-6 was used to stimulate A549 or NCI-H1975 cells, Pp65 and TIM-4 protein were detected by western blotting. We found that IL-6 induced TIM-4 protein expression depending on NF-?B signal transduction pathways but not Stat3 pathway in lung cancer cells.Conclusions:TIM-4 promotes expression of TIM-4 of lung cancer cell depending on NF-?B signal transduction pathway.PART TWOTIM-4 promotes migration and invasion of NSCLC cells and related mechanismObjective:This study aims to investigate the role of TIM-4 in driving migration and invasion of NSCLC cells.Methods & Results:1.TIM-4 promotes lung cancer cell migration and invasion.Wound healing assay was performed to show the effects of TIM-4 overexpression on migration of A549 and NCI-H1975 cells. A scratch wound was made on cell surface and cells were photographed at 0 h and 24 h or 48h. Representative pictures are shown.Effects of TIM-4 over expression on migration and invasive abilities of A549 cells. The migrated and invasived cells were photographed. We found that TIM-4 promotes lung cancer cell migration and invasion.2. TIM-4 overexpression promotes tumor invasion of lung cancer cells in vivo. Mice were injected with 2.5×106/ml A549-luc control cells or A549-luc-TIM-4 cells by cauda vein. At the end of the experiment, mice were photographed. We found that TIM-4 overexpression promotes tumor invasion of lung cancer cells in vivo.3.TIM-4 promotes expression of EMT markers. A549 and NCI-H1975 cells were transfected with pcDNA3-TIM-4 or control vector plasmid DNA, and expression of EMT markers and TIM-4 expression were detected by qRT-PCR and western blot. The results showed that TIM-4 overexpression increased expression of N-cadherin, Vimentin and Slug, but decreased E-cadherin expression..4. TIM-4 induces EMT depending on N-linked glycosylation. Mutated plasmid TIM-4(N291Q) with N-glycosylation mutation of TIM-4 was constructed and identified by enzyme digestin and sequencing. Wild type TIM-4 and mutated TIM-4(N291Q) plasmid were transfected into A549 or NCI-H1975 cells.48h later, western blot was performed to detect EMT related markers. Actin was used as an internal loading control. We found that N-glycosylation mutation reversed TIM-4 induced EMT progression.Conclusions:TIM-4 promotes cell migration and invasion of lung cancer through inducing EMT depending on N-linked glycosylationPART THREEThe role of TIM-4 in IL-6 promoted NSCLC metastasisObjective:This study aims to investigate the role of TIM-4 in driving migration and invasion of NSCLC cells in response to IL-6.Methods & Results:1. IL-6 promoted invasion and EMT of NSCLC cells. IL-6 was used to stimulate A549 cells, and the lung cancer cell invasion was assayed by transwell and EMT markers were detected by western blot. In accordance with the references, we found that IL-6 stimulation promoted invasion of NSCLC cells and EMT progression.2. TIM-4 involved in IL-6 promoted migration and invasion of NSCLC cells. Letivirus containing TIM-4 shRNA or control were used to infect A549 or NCI-H1975 cells with or without the stimulation of IL-6. The lung cancer cell migration and invasion was assayed by transwell and wound healing assay. The results showed that TIM-4 knockdown inhibited the migration and invasion of NSCLC cells. IL-6 stimulation promoted migration and invasion of NSCLC cells, while TIM-4 knockdown reversed this effect..3. TIM-4 involved in IL-6 promoted EMT of NSCLC cells. A549 cells were infected with letivirus containing TIM-4 shRNA or control and stimulated with IL-6 for 24h. qPCR was performed to analyze the EMT-related genes. The results showed that IL-6 stimulation promoted EMT progression, while TIM-4 knockdown reversed this effect.Conclusions:These data indicate that TIM-4 plays a key role in IL-6 promoted migration and invasion of NSCLC. TIM-4 blocking might be a new treatment option in the therapy of metastases of lung cancer.