A Study Of The Impact Of ILT4-HLA-G Co-expression On NSCLC Patient’s Prognosis And The Underlying Mechanisms

[Background]Lung cancer, especially non-small cell lung cancer(NSCLC) which accounts for about 85%of all lung cancers, is the most common malignant tumor in the world and remains the leading cause of malignancy-related mortality worldwide. Thus, it is essential to further understand the molecular mechanisms underlying the progression and metastasis of NSCLC, which may be helpful to identify more novel therapeutic targets and improve therapeutic effects of NSCLC. Immunoglobulin-like transcript 4 (ILT4), belonging to immunoglobulin-like transcripts family, is one of the most characterized immune inhibitory receptors. It has been shown to induce inhibitory signaling via immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic tail. In previous study, we first reported ILT4 could be induced in NSCLC and was associated with clinic-pathological factors. Recently, we found that both immunoregulation and non-immunoregulation could be involved in the progression of NSCLC induced by ILT4. HLA-G, a non-classical class I gene of the human MHC-I antigens, plays an important role in inducing immune tolerance. HLA-G expresses in carcinoma and acts as a regulator of immune escape. Notable, studies found that both lesion HLA-G expression and plasma sHLA-G are related to the advanced disease stage of NSCLC patients. From the above we can see that both ILT4 and its ligand HLA-G were expressed in NSCLC cancer cells and involved in NSCLC progression. Thus we speculated that there may be an interaction between ILT4 and HLA-G, and the interaction may involve in molecular mechanisms of cancer development. To our knowledge, the co-expression of ILT4/HLA-G and their interaction in solid tumors is still a blank. In this paper, we aimed to evaluate co-expression of ILT4/HLA-G in primary human NSCLC tissues and NSCLC cell lines respectively, to determine the relationship of ILT4/HLA-G co-expression with clinic-pathological parameters and survival outcomes of patients, further to analyze the regulation mechanism underlying them. This may play important role in elevating ing the therapeutic effective rate, prolonging patients’ disease free survival, and improving patients’ prognosis.[Objective]Detect the co-expression of ILT4 and HLA-G in NSCLC cell lines and tissues. Analyze the impact of ILT4-HLA-G co-expression on the patient’s prognosis. Analyze the interaction between ILT4 and HLA-G expression in NSCLC. Explore the underlying mechanisms of ILT4-HLA-G co-expression in NSCLC progression.[Methods]1. A total of 81 primary human NSCLC specimens were collected to detect the expression of ILT4 and HLA-G by immunohistochemical analysis. Then analyze the association between ILT4 and HLA-G expression and the relationship between ILT4-HLA-G co-expression and clinic-pathological factors of NSCLC patients.2. Collect clinical medical records of 81 patients and examine the prognosis significance of ILT4-HLA-G co-expression in NSCLC patients, overall survival (OS) curves were constructed using the Kaplan-Meier method by SPSS version 18.0.3. Detect the expressions of ILT4 and HLA-G in 5 NSCLC cell lines, including A549, H1650, H226, H1299 and H1975, at protein level by Western Blot analysis, as well as at mRNA level by RT-PCR.4. Detect the effect of ILT4 expression on HLA-G in NSCLC cells. ILT4 shRNA (shILT-4) and ILT4 expression plasmid (ILT4-vecter) were transfected into ILT4 high-expression cell line (A549 cells) and ILT4 low-expression cell line (H1650 cells), respectively. Then the expression of HLA-G was assayed at both mRNA and protein levels by RT-PCR and Western Blot analysis respectively.5. Add HLA-G fusion protein with different concentration (50ng/ml, 100ng/ml, 200ng/ml, and 500ng/ml) into H1650 cells. Then the expression of ILT4 was assayed at both mRNA and protein levels by RT-PCR and Western Blot analysis respectively. The expression of signal transduction module, including phospho-AKT, phospho-ERK1/2 and phospho-NF-κ B, were assayed at protein levels by Western Blot analysis.6. After H1650 cells had been cultured with anti-ILT4 antibody, add 200ng/ml HLA-G fusion protein to these cells. Then detect the expression of phospho-ERKl/2 at protein levels by Western Blot analysis.[Results]1. Co-expression of ILT4 and HLA-G in primary human NSCLC tissues was found and ILT4/HLA-G co-expression is associated with clinic-pathological factors of NSCLC patients. There was a significant association between ILT4 and HLA-G expression (P≤0.0001, R=0.596). ILT4+/HLA-G+ was more significantly associated with regional lymph node involvement (p=0.001) and advanced stages (p=0.006) than other three groups (ILT4-/HLA-G-group, ILT4+/HLA-G-group and ILT4-/HLA-G+ group). ILT4+/HLA-G+ was also related to histological type degree (p=0.042) and cellular differentiation (p=0.013) comparing to ILT4-/HLA-G-group.2. ILT4/HLA-G co-expression was associated with overall survival of patients. Overall survival (OS) curves were constructed using the Kaplan-Meier method. The OS of patients with ILT4+/HLA-G+ was much lower than that of groups with ILT4-/HLA-G-(p=0.048) and ILT4+/HLA-G-(p=0.021). However, the difference of OS between patients with ILT4+/HLA-G+ and ILT4-/HLA-G+ cases was not statistically significant (p=0.254).3. Co-expression of ILT4 and HLA-G in NSCLC cells lines was found. Both ILT4 and HLA-G were detected in all 5 NSCLC cell lines including A549, HI650, H226, H1299 and H1975. What’s more, the expression levels of ILT4 and HLA-G in those 5 cell lines were relatively coherent.4. Interference or overexpression of ILT4 affects HLA-G expression in NSCLC. When ILT4 expression was reduced by shILT-4, HLA-G expression showed an accompanying decrease. Similarly, the over-expression of ILT4 caused a significant increase of HLA-G expression.5. HLA-G fusion protein up-regulates ILT4 expression in NSCLC cells. ILT4 expression was significantly up-regulated by HLA-G fusion protein both in mRNA and protein levels. HLA-G fusion protein up-regulated ILT4 expression in a concentration dependent manner from 50 ng/ml to 200ng/ml.6. The stimulation of HLA-G fusion protein activates ERK signaling through binding with ILT4. Phospho-ERK 1/2 was significantly enhanced in those H1650 cells stimulated by HLA-G fusion protein. And the up-regulation can be neutralized by anti-ILT4 blocking antibody.[Conclusion]In this study, we detected the expression of ILT4 and HLA-G in 81 tumor specimens from primary NSCLC patients, and we found co-expression of ILT4/HLA-G was significantly associated with regional lymph node involvement, advanced stages and the overall survival of patients. In NSCLC cell lines, HLA-G expression increased/decreased accordingly when ILT4 was up/down regulated, and ILT4 expression increased in a concentration dependent manner via the stimulation of HLA-G fusion protein. Interestingly, HLA-G fusion protein could also up-regulate the phospho-ERK1/2 expression, which means the activation of extracellular signal-regulated kinase (ERK) signaling. All in all, our results indicate that the ILT4-HLA-G interaction might play an important role in NSCLC progression. Identification of ILT4 and HLA-G expression may provide an indicator to predict prognosis and guide prevention and treatment of NSCLC.