The Function And Molecular Mechanism Of ILT3 In Promoting NSCLC Progression

BackgroundLung cancer is the leading cause of cancer-related death worldwide, about 85%of which is non-small cell lung cancer (NSCLC). Although gradual improvements in therapy have been achieved, survival of NSCLC patients is still poor, partly as a result of those patients often presenting at an advanced stage. It is therefore essential to further uncover the underlying molecular mechanisms of NSCLC progression and metastasis to develop novel therapeutic targets.Progression and metastasis of tumor is a delicate multi-steps biological process involving many factors. Enhanced proliferation, survival, migration and invasion ability of tumor cells, immune escape and angiogenesis, which are activated and maintained by various cytokines, are all key processes in the procession of tumor progression and metastasis. Growing studies show that immuno-regulatory molecules are highly expressed in cancer cells and can enhance the proliferation and motility of tumor cells through non-immunological mechanisms.Immunoglobulin-like transcript (ILT) family is a group of immunoglobulin superfamily molecules, which are all located on chromosome 19. According to their functional properties, ILT family members are divided into activating receptors (ILT1, ILT6, ILT7 and ILT8) and inhibitory receptors (ILT2, ILT3, ILT4 and ILT5). Inhibitory receptors possess long cytoplasmic tails that contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which can recruit protein tyrosine phosphatase to suppress myeloid cell activation. Our previous study has demonstrated ILT4 plays an important role in NSCLC progression and metastasis.ILT3 belongs to ILT inhibitory receptors subfamily, preferentially expressed on mononuclear cells and dendritic cells (DCs). At present, studies about ILT3 mainly focus on its function on antigen-presenting cells (APC) to induce immune tolerance. But for expression of ILT3 in tumor cells and its effects on tumor cells, much less studies have been reported. ILT3 expression represents the phenotypic abnormality in human chronic lymphocytic leukemia (CLL) B cells, and positive ILT3 expression is more common in CLL patients with lymphoid tissue involvement. Similar with ILT4, ILT3 has been detected in a few non-myeloid malignant tumors. High expression of ILT3 was detected in human gastric cancer cells and laying hen model of spontaneous ovarian cancer. Soluble ILT3 could inhibit tumor allograft rejection in humanized SCID mice and T cell responses in cancer patients.Our preliminary study found higher ILT3 expression was detected in NSCLC tissues than in peritumoral normal tissues, and ILT3 expression in tumor cells was associated with less tumor infiltrated lymphocytes (TILs).The aberrant expression of ILT3 in NSCLC suggests it may play an important role in NSCLC progression. However, the exact function of ILT3 in NSCLC, especially for the non-immune effect, and its underlying molecular mechanisms have been poorly understood. AimsTo investigate the role of ILT3 in NSCLC progression, we examined the expressionof ILT3 in NSCLC tumor tissues and peritumoral normal lung tissues by immunohistochemical (IHC) staining and real-time PCR and analyzed the correlations of ILT3 expression with clinicopathological factors and overall survival of the patients. By manipulating ILT3 expression in NSCLC cells, we detected the impact of ILT3 in cell proliferation migration and invasion in vitro and in vivo, and elucidated the potential mechanisms underlying the tumor-promoting effect by ILT3 in NSCLC. Methods1. Using real-time PCR to detect the mRNA expression of ILT3 in fresh NSCLCtumor tissues and paired peritumoral normal lung tissues, and then analyzing the difference of ILT3 expression between NSCLC tumor tissues and peritumoral normal lung tissues.2. Samples of paraffin-embedded NSCLC tissues and clinicopathological features were obtained to detect the expression of ILT3 by immunohistochemistry and evaluate the correlations between ILT3 expression and clinicopathological characteristics. Survival curve was drawn using the Kaplan-Meier method and compared by means of the log-rank test.3. The expression of ILT3 in six NSCLC cancer cell lines was tested by real-time PCR and Western blot to determine their endogenous expression levels. ILT3 vector was formed using human full length ILT3 cDNA linked with pEZ-Lv105 vector to achieve ILT3 over-expression in H1650 and H1299 cells. And cells transfected with empty pEZ-Lv105 vector served as negative control. Lentivirus vectors produced by 293T cells were infected into H1650 cells to establish stable transfection cell lines. Human ILT3 shRNA linked with pGIPZ-lentivirus vector was used to induce ILT3-silence in A549 and H226 cell lines. Cells transfected with pGIPZ-lentivirus vector served as negative control.4. MTT assay was used to determine the effect of ILT3 on NSCLC cell proliferation in vitro; AnnexinV-FITC/PI analyses were tested by flow cytometry to detect the effect of ILT3 on cell apoptosis in vitro; transwell migration and invasion assays were applied to measure the effect of ILT3 on cell motility and invasiveness in vitro.5. Tail vein injection model was established to examine the effect of ILT3 on tumor metastasis in vivo. Stably transfected cell line ILT3/H1650 and the negative control cells (1×106) were injected into the tail vein of nude mice. The number of metastatic tumor nodes was counted.6. The phosphorylation of signal transduction modules of MAPK signaling, including ERK JNK, and p38 and PI3K-Akt signaling were detected after manipulating ILT3 expression. ERK signaling inhibitor U0126 and PI3K-Akt signaling inhibitor LY294002 were applied to confirm the effect of ERK and PI3K-Akt signaling in ILT3-mediated tumor malignant behavior.7. The expression levels of biomarkers for EMT were detected by western blotting after manipulating ILT3 expression.8. The expression levels of angiogenesis-related molecules (VEGF-A and FGF1) were detected after manipulating ILT3 expression. ERK signaling inhibitor U0126 and PI3K-Akt signaling inhibitor LY294002 were used to determine the key signaling in the regulation. Culture supernatants of ILT3/H1650, shILT3/A549 and the corresponding control cells were collected to investigate the effect of ILT3 on human umbilical vascular endothelial cells (HUVEC).9. FGF1 is identified as a candidate cancer biomarker, however there is no study focused on the expression and function of FGF1 in NSCLC so far. Samples of paraffin-embedded NSCLC tissues and clinicopathological features were obtained to detect the expression of FGF1 by immunohistochemistry and evaluate the correlations between FGF1 expression and clinicopathological parameters. Survival curve was drawn using the Kaplan-Meier method and compared by means of the log-rank test.Results1. Expression of ILT3 in NSCLC tissues was significantly higher than in peritumoral normal lung tissues and associated with poor prognosis of NSCLC patients.The mRNA expression level of ILT3 in 20/28 cases of NSCLC tissues was significantly higher than that in paired peritumoral normal lung tissues. Consistent with this result, IHC staining showed high expression of ILT3 was detected in 66.4% (75/113) primary NSCLC tissues. High ILT3 expression in cancer cells was correlated with larger primary tumor size (p=0.026), worse cell differentiation (p=0.039), vascular invasion (p=0.027) and advanced TNM stages (p=0.018). The intratumoral microvessel density (MVD) in ILT3-high group was higher than in ILT3-low group. Kaplan-Meier analysis showed that the survival rate of ILT3-high group was significantly lower than that of ILT3-low group.2. ILT3 promoted NSCLC cell migration and invasion in vitroTo determine the functional role of ILT3 in NSCLC cells, we assessed ILT3 expression level in six NSCLC cell lines (A549, H226, H520, H1299, H1650 and H1975). And then, H1650 and H1299 cells with low endogenous ILT3 expression were selected to be transfected with ILT3 vector, leading to significant over-expression of ILT3. Up-regulation of ILT3 promoted the migration and invasion of H1650 and H1299 cells, but has no obvious effect on cell proliferation and apoptosis. Next, we interfered the expression of ILT3 in A549 and H226 cells with relative high endogenous 1LT3 expression by small hairpin RNA (shRNA). Silence of ILT3 weakened cell migration and invasion.3. ILT3 drived NSCLC metastasis in vivoTo address whether ILT3 promotes tumor metastasis in vivo, stably transfected ILT3/H1650 and negative control cells were injected into the tail vein of nude mice. At day 42, the number of lung metastatic nodules was much more in ILT3/H1650-treated mice, compared to control group.4. ILT3 promoted NSCLC cell migration and invasion through activating ERK and PI3K-Akt signaling pathwaysWe detected the activation of MAPK signal transduction modules, including ERK, JNK and p38 and PI3K-Akt signaling and found the phosphorylation of ERK1/2 and Akt were significantly enhanced in ILT3 over-expressing NSCLC cells and decreased in ILT3 knockdown cells.We next wondered whether 1LT3 promoted cell migration and invasion through activating ERK or P13K-Akt signal pathway. ERKl/2 inhibitor U0126 and PI3K-Akt inhibitor LY294002 were used to treat ILT3 over-expressing HI650 cells. The migration and invasion ability of those cells were significantly suppressed by ERK1/2 inhibitor U0126 or PI3K-Akt inhibitor LY294002.5. ILT3 induced incomplete EMT in NSCLC cells.EMT represents a phenotypic conversion by which epithelial cells lose their polarity and cohesiveness and acquire migratory features characteristic of fibroblasts, which could enhance cell motility Western blot results demonstrated ILT3 could up-regulate mesenchymal marker vimentin but had no obvious effect on epithelial marker E-cadherin, meanwhile, silence of ILT3 also confirmed the effect of ILT3 on vimentin.6. ILT3 up-regulated VEGF-A and FGF1 expression via ERK and PI3K-Akt signaling pathway and promoted tube formation of HUVECIHC results suggested ILT3 was associated with NSCLC progression and angiogenesis. Then we investigated whether ILT3 could regulate the expression of canonical angiogenesis related factors, VEGFA and FGF1. The protein levels of VEGF-A and FGF1 were significantly elevated in ILT3 over-expressing HI650 cells, and decreased in ILT3 knock-down A549 cells. Next, we examined whether ERK or PI3K-Akt signal participated in ILT3-induced VEGF-A and FGFl expression. The up-regulations of VEGF-A and FGF1 in the H1650 cells transfected with ILT3 vector were blocked by ERK1/2 inhibitor U0126 or PI3K-Akt inhibitor LY294002.In addition, we collected the culture supernatants of ILT3/H1650, shILT3/A549 and the corresponding control cells to culture HUVEC. Tube formation assay revealed supernatant from ILT3 over-expressing cells could enhance the capillary formation of HUVEC, while ILT3 knockdown group formed less capillary than control group. Besides, western blot results demonstrated that supernatant from ILT3 over-expressing group could activate the ERK and PI3K-Akt signaling in HUVEC.7. Clinicopathological significance of FGF1 in NSCLCFGF1 was frequently overexpressed in NSCLC and its overexpression was correlated with larger primary tumor size, SQCC histological type, vascular invasion. In addition, FGF1 expression was correlated with intratumoral microvessel density in both SQCC and adenocarcinoma subgroups. Moreover, NSCLC patients with high FGF1 expression had a significantly lower overall survival rate, compared with those with low FG1 expression. Furthermore, subgroup analyses showed that FGF1 expression was associated with poor prognosis in lung SQCC, but not in adenocarcinoma.Conclusions1. ILT3 is overexpressed in NSCLC and plays an important role in promoting tumor metastasis. Moreover, ILT3 could function as a useful biomarker to predict the prognosis of NSCLC patients. Therefore, ILT3 may be a candidate target for NSCLC treatment.2. ILT3 up-regulates VEGF-A and FGF1 expression via ERK and PI3K-Akt signaling and promotes the tube formation of HUVEC. Therefore, ILT3 may mediate tumor progression by promoting angiogenesis.3. FGF1 is frequently overexpressed in NSCLC and its overexpression is correlated with larger primary tumor size, SQCC histological type, vascular invasion and higher intratumoral MVD. Subgroup analyses showed that FGF1 expression was associated with poor prognosis in lung SQCC, but not in adenocarcinoma.