The Function And Molecular Mechanism Of ILT4 In Promoting NSCLC Progression

BackgroundLung cancer is one of the most common malignancies in the world and is also the leading reason of cancer deaths worldwide, causing 1.4 million deaths annually. Non-small cell lung cancer (NSCLC) is the primary histological type of lung cancer, accounting for about 85% of the disease. Although gradual improvements in survival have been achieved, this advance has not matched those seen in other common malignancies, partly as a result of NSCLC patients often presenting at an advanced stage. It is therefore essential to further elucidate the underlying molecular mechanisms of NSCLC progression and metastasis, and to develop novel therapeutic approaches.The immunoglobulin-like transcript (ILT) family is a group of cell-surface protein receptors preferentially expressed on myeloid lineage cells, and is 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 recruit protein tyrosine phosphatase SHP-1 to inhibit myeloid cell activation. In addition, several studies indicate that inhibitory receptors ILT2, ILT3 and ILT4 are frequently up-regulated in certain myeloid malignant tumors and correlate with adverse prognosis. ILT2 is found to be expressed in some T-cell lymphomas, and ILT2 expressing Se’zary cells are resistant to CD3 monoclonal antibody induced cell death. ILT3 and ILT4 expression represent the phenotypic abnormality in chronic lymphocytic leukemia (CLL) B cells, and ILT3 expression is more common in CLL patients with lymphoid tissue involvement. A recent study has demonstrated that ILT4 and its mouse ortholog paired Ig-like receptor (PIRB) promote haematopoietic stem cells (HSCs) repopulation and acute myeloid leukemia (AML) development.Interestingly, ILT4, as well as other inhibitory receptors ILT2 and ILT3, has also been detected in a few non-myeloid malignant tumors cells. In vitro, high expression of both ILT2 and ILT3 in gastric cancer cells inhibit cycotoxic activity of NK cells; and in primary human gastric cancer tissues, ILT2 correlates with poor cell differentiation and large tumor. ILT3 expression is significantly associated with ovarian tumor progression in laying hen model of spontaneous ovarian cancer. High ILT4 expression in NSCLC and breast cancer cells is associated with lymph node metastasis and less tumor infiltrated lymphocytes (TILs). Recently, it is reported that higher ILT4 was demonstrated in more aggressive pancreatic ductal carcinoma cell lines. All these results suggest that the aberrant expression of inhibitory ILTs including ILT4 may play an important role in tumor progression. However, the exact function of ILT4 in cancer malignancy, especially in non-myeloid malignant tumors, and the underlying molecular mechanism have been poorly understood.AimsIn previous study, our group has demonstrated the high expression of ILT4 in NSCLC cell lines and tissues. In this study, we aimed to investigate the role of ILT4 in promoting NSCLC progression. By manipulating ILT4 expression in NSCLC cells, we detected the role of ILT4 in promoting cell proliferation and motility in vitro, as well as tumor growth and metastasis in vivo, and elucidate the possible mechanisms underlying the progression-promoting effect by ILT4 in NSCLC.Methods1. The expression levels of ILT4 in different cancer cell lines were tested by Real Time RT-PCR and Western blot. ILT4 vector was formed using full length human ILT4 cDNA linked with the Pez-1v105 vector to induce ILT4 over-expression in the cultured cells. ILT4 vector was transfected into the cells using X-treme GENE HP Reagents according to the manufacturer’s instructions. Cells transfected with Pez-lv105 vector served as negative control. Human ILT4 linked with pLenti6.3 vector was used to induce ILT4 over-expressing in the cell lines. The stable cell lines were selected by blasticidin. Human ILT4 shRNA linked with Lentivirus pGLV/Hl/GFP vector was used to induce ILT4-silence in the cell lines. Cells transfected with Lentivirus pGLV/H1/GFP-NC vector served as negative control. Stable cell lines were selected by incubating on puromycin containing medium for 48h after transfection. The resulting stably transfected cell lines were collected after 6 weeks. ILT4 shRNA sequences are:5’-GAAGAAGAACACCCACAATGC-3’; shNC, 5’-GTTCTCCGAACGTGTCACGT-3’.2. MTT and plate colony formation were used to measure the effect of ILT4 on the cell growth in vitro; apoptosis analysis were tested by FACS to detect the effect of ILT4 on the cell apoptosis in vitro; transwell migration and invasion assays were used to investigate the effect of ILT4 on the cell motility in vitro.3. Proliferation assays in vivo were applied to examine the effect of ILT4 on the tumorigenicity and tumor growth in vivo. Stably transfected cell lines ILT4/H1650, shILT4/A549 and the corresponding control cells (2×106) were injected into the left flank of the mice. Tumor size was calculated and tumor weight was measured.4. Metastasis assays in vivo were applied to examine the effect of ILT4 on the tumor metastasis in vivo. Stably transfected cell lines ILT4/H1650, shILT4/A549 and the corresponding control cells (5×105) were injected into the tail vein of the mice. The number of metastasis tumor nodes was counted.5. The phosphorylation of three parallel signal transduction modules of MAPK signaling, including JNK, p38 and ERK, were detected after manipulating ILT4 expression. ERK inhibitor U0126 was used to confirm the effect of ERK signaling in ILT4-induced tumor malignant behavior.6. The expression level of VEGF-C was detected after manipulating ILT4 expression and adding ERK inhibitor U0126. siRNA VEGF-C was used to confirm the effect of VEGF-C in ILT4-induced tumor malignant behavior.7. Samples of paraffin-embedded tissue sections and clinicopathological features were obtained to detect the expression of ILT4 and VEGF-C. The correlation between ILT4 and VEGF-C and clinicopathologic characteristics was also confirmed. Survival curves were drawn using the Kaplan-Meier method and compared by means of the log-rank test.8. The expression level of B7-H3 was detected after manipulating ILT4 expression. PI3K and mTOR inhibitor were used to confirm the effect of PI3K/AKT/mTOR signaling in ILT4-induced B7-H3 expression.9. Samples of paraffin-embedded tissue sections and clinicopathological features were obtained to detect the expression of ILT4 and B7-H3. The correlation between ILT4 and B7-H3 and clinicopathologic characteristics was confirmed. Moreover, the correlation between ILT4 and B7-H3 and the number of TILs (tumor infiltrating lymphocytes) was also confirmed. Survival curves were drawn using the Kaplan-Meier method and compared by means of the log-rank test.Results1. ILT4 drives NSCLC cell proliferation and motility in vitroTo assess the functional role of ILT4 expression in NSCLC cells, we determined ILT4 expression in NSCLC cell lines (A549, H1299, H226, H1975 and H1650). And then, H1650 and H1975 cells with endogenous low ILT4 expression were selected to be transfected with ILT4 vector, leading to significant up-regulation of ILT4. Over-expression of ILT4 promoted the proliferation the number and size of formed colonies in H1650 and H1975 cells. As well, the migration and invasion in H1650 and H1975 cells transfected with ILT4 vector were increased. Next, we established the stable ILT4 silencing cell line shILT4/A549 and empty vector-transfected control cell lines NC/A549 for further studies. Silence of ILT4 reduced cell proliferation and colony formation. Meanwhile, the cell migration and invasion were significantly decreased in shILT4/A549 cell. Interestingly, anti-ILT4 blocking antibody treatment did not affect the expression of VEGF-C.In vitro over-expression and knockdown experiments, the apoptosis assay showed that both over-expression and inhibiting ILT4 did not influence cell apoptosis in H1650, H1975, H226 and A549 cell lines.2. ILT4 drives NSCLC tumor growth and metastasis in vivoTo address whether ILT4 promotes tumor growth in vivo, stable transfected ILT4/H1650 and shILT4/A549 cells and their corresponding control cells were implanted in left flank of the mice to form ectopic tumors. The tumor volumes were much massive in mice with injection of ILT4/H1650 cells and smaller in mice with injection of shILT4/A549 cells at the designated time points, compared with the corresponding control cells respectively. At day 36, compared with control group, the tumor weight in ILT4/H1650 group was increased and that in shILT4/A549 group was significantly decreased. Next, to evaluate whether ILT4 promotes distant metastasis in vivo, mice were injected with ILT4/H1650 and shILT4/A549 and the corresponding control cells via the tail vein, respectively. At day 42, the number of lung macrometastases was much more in ILT4/H1650-treated mice and much less in shILT4/A549-treated mice, compared to control groups.3. ILT4 induces malignant phenotype of NSCLC cells through activating ERK signaling pathwayFunctional annotation displayed the change of multiple genes related to MAP kinase (MAPK) phosphatase activity in ILT4 over-expressing H1650 and H1975 cells. We then detected the phosphorylation of three parallel signal transduction modules, including JNK, p38, and ERK, in MAPK signaling and found the phosphorylation of ERK1/2 was significantly enhanced in ILT4 over-expressing NSCLC cells and decreased in ILT4 knockdown cells.We next investigated whether ILT4 promotes cell malignant phenotype through activating ERK signal pathway. ERK1/2 inhibitor U0126 was used to treat ILT4 over-expressing cells. The proliferation, migration and invasion ability of those cells were significantly suppressed.4. ILT4 induces malignant phenotype of NSCLC cells through VEGF-CThe functional annotation also displayed the change of multiple genes related to growth factor activity in ILT4 over-expressing HI650 cells. And hot map showed a cluster of growth factor genes including VEGF-C were up-regulated in those cells. Next, we confirmed the mRNA and protein levels of VEGF-C were elevated in ILT4 over-expressing H1650 and H1975 cells, and decreased in ILT4 over-expressing A549 and H226 cells. However, anti-ILT4 blocking antibody treatment did not affect the expression of VEGF-C.In addition, we examined ERK signal participated in ILT4-induced VEGF-C expression. After treated with ERK1/2 inhibitor U0126, the protein expression of VEGF-C in the H1650 cells transfected with ILT4 vector.Since VEGF-C is crucial for NSCLC cell growth and metastasis, we investigated whether ILT4 enhances NSCLC cell malignant phenotypes via up-regulating VEGF-C expression. ILT4 over-expressing H1650 cells were transfected with si VEGF-C, leading to the down-regulation of VEGF-C. And then the phenotypic changes of cell proliferation, migration and invasion were analyzed. Silence of VEGF-C expression inhibited the migratory and invasive behaviors of ILT4 over-expressing H1650 cells. However, VEGF-C down-regulation in those cells had no effect on cell proliferation.5. ILT4 and its co-expression with VEGF-C in human NSCLC tissuesILT4 expressing tissues accounted for 45.7% (48/105) of the whole samples. Positive ILT4 expression in cancer cells was correlated with worse cell differentiation (p=0.038), regional lymph node involvement (p=0.04), advanced stages (p=0.013) and age of more than 60 years (p=0.044). In addition, we observed the expression pattern of ILT4 was consistent with that of VEGF-C (p=0.024). Kaplan-Meier analysis showed that the overall survival (OS) of ILT4 and VEGF-C expressing group was lower than the corresponding negative group, respectively (ILT4,p=0.035; VEGF-C,p=0.038). In addition, the OS of patients with ILT4+VEGF-C+was much lower than that of group with ILT4-/VEGF-C-(p=0.005).6. ILT4 up-regulated B7-H3 expression through PI3K/AKT/mTOR signaling but not ERK signalingB7-H3 expression was significantly increased in ILT4 over-expressing H1650 and H1975 cells at mRNA and protein levels. A549 and H226 were used for loss of function transfection assay; the expression of B7-H3 was consequently down-regulated when ILT4 expression was knockdowned. We attempted to assess the signaling linking ILT4 to B7-H3 up-regulation. The results showed that ILT4 activated ERK and PI3K/AKT/mTOR signaling. Furthermore, treatment with PI3K specific inhibitor LY294002 or mTOR inhibitor rapamycin led to a significant decrease of B7-H3 expression in ILT4 over-expressing H1650 and H1975 cells. However, treatment with ERK specific inhibitor U0126 could not influence B7-H3 expression.7. Co-expression of ILT4 and B7-H3 in primary human NSCLC tissuesThere was a significant association between ILT4 and B7-H3 expression. Higher level of ILT4 was observed in B7-H3 positive group compared to the corresponding negative expression group (p=0.021). Higher level of ILT4 was also observed in pAKT positive expression group compared to the pAKT negative expression group (p=0.163); However, there was not a significant correlation between them. The mean TILs in ILT4 or B7-H3 positive group was significantly lower than that in the corresponding negative group. And the mean number of TILs in positive expression of both ILT4 and B7-H3 group was smallest (p=0.007). Kaplan-Meier analysis also showed that the OS of patients with positive expression of both ILT4 and B7-H3 was the lower than that of group with negative expressions of both ILT4 and B7-H3 (p=0.029).Conclusion1. ILT4 plays an important role in promoting tumor growth and metastasis in vitro and in vivo. Moreover, ILT4 can function as a useful biomarker to predict the prognosis of NSCLC patients. Therefore, targeting ILT4 may be a feasible and effective approach for NSCLC treatment.2. ILT4 up-regulate VEGF-C expression via ERK signaling, and inhibition either ERK or VEGF-C attenuated ILT4-induced malignant behavior in NSCLC. Therefore, activation of ILT4-ERK-VEGF-C axis may mediate tumor progression.3. ILT4 up-regulate B7-H3 expression via PI3K/AKT/mTOR signaling in NSCLC. And the mean number of TILs in positive expression of both ILT4 and B7-H3 group was smaller than the other groups. Therefore, the activation of ILT4-PI3K/AKT/mTOR-B7-H3 axis might be involved in tumor immune evasion, leading to NSCLC progression.