The Function And Potential Mechanisms Of Immunoglobulin-like Transcript 4-regulated Angiogenesis In Non-small Cell Lung Cancer

BackgroundLung cancer is the leading cause of cancer incidence and mortality worldwide Non-small cell lung cancer(NSCLC)is the most important type of lung cancer,accounting for 80-85%of all lung cancers.NSCLC is characterized by insidious onset,mild symptoms and advanced disease stages at diagnosis.In recent years,with the development of various treatments such as surgery,chemotherapy,radiotherapy,targeted therapy and immunotherapy,the prognosis of NSCLC has been greatly improved,but its overall 5-year survival rate remains no more than 20%.Local recurrence and metastasis are the main factors affecting the therapeutic effect and prognosisAs is known to all,tumor growth and metastasis rely on the formation of new blood vessels to provide energy and metastasis pathways,so anti-angiogenesis therapy has been used as an important approach for NSCLC.At present,the therapeutic strategies for anti-tumor angiogenesis include monoclonal antibodies against VEGF/VEGFR pathway,multi-target small molecule TKI,endostatin and so on.However,the clinical benefit is usually transient and modest due to therapeutic resistance induced by complex pro-angiogenic mechanisms in the tumour microenvironment,such as metabolic symbiosis and activation of bypass signalings in tumor cells and stroma cells.Moreover,the multi-target anti-angiogenic drugs have serious toxicity due to their blockade to multiple pathways.Therefore,to explore the molecular mechanism of NSCLC metastasis,discover effective therapeutic targets,develop specific targeted drugs are important directions to inhibit tumor metastasis and improve clinical benefitsImmunoglobulin-like transcripts(ILTs),also known as leukocyte immunoglobulin-like receptors(LIR or LILR),or monocyte/macrophage immunoglobulin-like receptors(monocyte/macrophage immunoglobulin-like receptors,MIR),is found in the gene cluster of chromosomal region 19q13.4.ILTs(LILRs)are highly homologous in the extracellular region and have different sequences in the intracellular region.They contain 11 homologous stimulatory or inhibitory receptors of the leukocyte receptor complex,which are characterized by cytoplasmic immunoreceptor tyrosine-based activation motif(ITAM)and immunoreceptor tyrosine-based inhibitory motif(ITIM)respectively.Therefore,in 2001,ILTs(LILRs)were divided into two subfamilies:the activated receptor LILRA(leucocyte immunoglobulin-like receptor subfamily A)and the inhibitory receptor LILRB(leucocyte immunoglobulin-like receptor subfamily B).LILRA subfamily includes LILRA1-6,while the inhibitory LILRB subfamily includes LILRB1-5.Immunoglobulin-like transcript(ILT)4,also known as lymphocyte immunoglobulin-like receptor B(LILRB)2,monocyte/macrophage immunoglobulin-like receptor 10(MIR-10),or CD85d,is a classic immunosuppressive molecule.As a type I transmembrane protein,ILT4 processes three immunoreceptor tyrosine-based inhibitory motifs(ITIMs)in the cytoplasm,which transduce inhibitory signals upon binding with different ligands.ILT4 was initially identified in myeloid cells including dendritic cells,macrophages,neutrophils and platelets.Recently,it is reported to be expressed in activated CD4+T cells as well.ILT4 in these cells inhibits the maturation and antigen presentation of dendritic cells,induces M2-like polarization of macrophages and myeloid-derived suppressor cells(MDSCs),impairs the phagocytic function of neutrophils,drives Th2 differentiation of CD4+T cells,and inhibits platelet activation,which negatively orchestrates the immune response.In addition to immune cells,ILT4 is also abundant in tumor cells of most malignancies including NSCLC,colorectal cancer,breast cancer,endometrial cancer and primary hepatocellular carcinoma.We and other groups discovered that tumor cell-derived ILT4 drives malignant tumor biologies including proliferation,invasion and metastasis,induces M2-like tumor-associated macrophages(TAMs)recruitment,T cell aging and immune cells infiltration.Our research team discovered in the early stage that tumor ILT4 could up-regulate vascular endothelial growth factor A(VEGF-A)and matrix metalloproteinase 9(MMP9)through gene chips.VEGF-A is a powerful mitogen for vascular endothelial cells and a classic factor regulating angiogenesis;MMP9 has also been proven to promote the decomposition of vascular basement membrane,induce endothelial cell migration,and promote the formation of new blood vessels.Therefore,we speculate that ILT4 may be closely related to the regulation of tumor angiogenesis.Since the current research at home and abroad on ILT4 in tumor angiogenesis is still unclear,considering the limitations of current NSCLC anti-angiogenesis therapy,and our findings from gene chips analysis,here we focused on the effect of ILT4 on NSCLC angiogenesis.Moreover,the underlying mechanisms for ILT4-related angiogenesis was also explored using both in vivo and in vitro studies.Our findings would bring new breakthroughs in the research of ILT4 in NSCLC anti-angiogenesisObjectives1.To verify the expression of ILT4 in NSCLC tissues and cell lines by immunohistochemical(IHC)staining,qPCR and Western blot assays;to determine the impact of ILT4 on the prognosis of lung cancer patients using both the follow-up data of patient survival and the survival information of GEO database;2.To determine the role of ILT4 on angiogenesis by testing the microvascular density in NSCLC tissues using IHC analysis,and by exploring the effect of ILT4 on the HUVECs migration and tubule formation;3.To clarify the underlying mechanisms for ILT4-regulated angiogenesis by detecting ILT4-induced alterations in proangiogenic factors,signaling pathways and alterations after treatment with neutralizing antibodies and corresponding inhibitors:4.To further explore the influence of ILT4 on angiogenesis and possible molecular mechanisms in vivo by establishing transplantation tumor models using ILT4 overexpressing/knockdown tumor cells in nude mice.Material and methods1.The expression of ILT4 in NSCLC cells and normal lung epithelial cells was detected by qPCR and Western blot,to determine the expression of ILT4 in NSCLC cells and normal bronchial epithelial cells2.The expression of ILT4 in NSCLC tissues and adjacent normal tissues was detected by immunohistochemistry,and the correlation between ILT4 expression and clinicopathological parameters was analyzed;the survival status of patients was followed up,and the survival curve was drawn using Kaplan Meier method to analyze the influence of ILT4 on the overall survival of NSCLC patients;Meanwhile,Kaplan-Meier plotter online survival analysis system was used to analyze the effect of ILT4 expression on survival3.The correlation between ILT4 and CD34 was statistically analyzed based on GEPIA database and our patient cohort by IHC.The relationship between ILT4/CD34 co-expression and clinicopathological parameters was also analyzed4.Lentiviruses carrying the ILT4 overexpression vector were used to upregulate ILT4 levels in H1650 and A549 cell lines who showed low ILT4 expression;the lentivirus carrying ILT4 short hairpin RNA(shRNA)was used to downregulate ILT4 levels in H1975 and H1299 cells which had high endogenous ILT4 expression.Then the medium from these cells were collected as conditioned medium(CM)for HUVECs culture.qPCR and Western blot were used to verify the transfection effect.Transwell cell migration assay and HUVECs tubule formation assay were used to detect the effect of ILT4 on the migration and angiogenesis of vascular endothelial cells5.Gene chip technology showed that up-regulation of ILT4 can induce the up-regulation of the expression of VEGF-A and MMP9.The NSCLC tissues were analyzed by immunohistochemistry to verify the correlation between ILT4 and the expression of VEGF-A and MMP9 in NSCLC tissues.The expression of ILT4 was regulated bidirectionally by transfection.qPCR and Western blot were used to verify the regulatory effect of ILT4 on VEGF-A and MMP9 at the cell level6.H1650 and A549 cells were first transfected with ILT4-overexpression plasmid.Then VEGF-A or MMP9 neutralizing antibodies were used to pretreat these H1650 and A549 cells.Afterwards,conditioned medium from these cells was collected to culture HUVECs.Transwell cell migration assay and tubule formation assay of HUVECs were used to observe the effect of conditioned medium on the migration and vascularization of HUVECs7.ILT4-regulated signaling pathways were first screened by mRNA microarrays and then confirmed by Western blot in ILT4-overexpressed/-downregulated NSCLC cells.8.After overexpression of ILT4 in H1650 and A549 cell lines,ERK and p38 inhibitors were added to block ERK and p38 signaling pathways respectively Western blot was used to detect the expression of VEGF-A and MMP9 in these cells.Transwell cell migration assay and HUVECs tubule formation assay were determined to clarify the role of ERK signaling in ILT4-regulated HUVEC vascularization.9.Stably transfected cell lines A549 LV-ILT4,H1299 LV-shILT4 and the corresponding control cells were injected into the right flanks of the mice.The tumor weight was measured and the volume was calculated.IHC was undertaken to check the correlation between ILT4 and the expression of CD34,VEGF-A,MMP9,ERK and pERK.Results1.ILT4 is highly expressed in NSCLC cells lines and NSCLC tumor tissuesILT4 expression is higher in NSCLC cells compared to normal bronchial epithelial cell line BEAS-2B using qPCR and western blot assay.IHC showed that ILT4 was much higher in NSCLC tumor tissues(57.7%)compared with that in adjacent normal tissues(4.4%)2.Enriched ILT4 expression in NSCLC tissues predicted poor patient outcome.A total of 90 NSCLC tissues and corresponding adjacent normal lung tissues were collected and sectioned for IHC staining of ILT4.We analyzed the correlation of ILT4 levels with clinicopathological features including tumor size,lymph node involvement,TNM stages and OS.We found that high ILT4 level predicted positive lymph node metastasis(p=0.001),advanced TNM stages(p=0.0104)and longer OS(p=0.0419).Similarly,we analyzed the relationship between the expression of ILT4 and the OS?FS of lung cancer patients.OS and FP curves were generated and obtained from the Kaplan-Meier plotter which includes the information from GEO database.Affymetrix ID:207697 x at showed that high ILT4 group had a lower OS than low ILT4 group but did not reach the significance(p=0.056).High ILT4 group had a significant lower FP than low ILT4 group(p=0.022)3.ILT4 expression was positively correlated with MVD in NSCLC tissues.The positive relationship between ILT4 and CD34 was verified using the GEPIA database.Moreover,our patient cohort proved that ILT4 expression was positively related with CD34 expression(p=0.0058).CD34 is a classic marker for vascular endothelial cells in tissues.Additionally,the combined ILT4 expression and high MVD predicted lymph node metastasis(p=0.0466)and high proportion of patients with adenocarcinoma(p=0.012).4.ILT4 promoted HUVECs migration and tube formation in vitroCM from ILT4-overexpressed cells elevated the migration of HUVECs Meanwhile,the tubule formation ability of HUVECs was significantly increased by CM from ILT4-overexpressed H1650 and A549 cells.Similarly,CM from ILT4-downregulated H1975 and H1299 cells restricted the migration and tubule formation of HUVECs.5.ILT4 enhanced the VEGF-A and MMP9 expression in NSCLCmRNA microarray analysis showed that ILT4 overexpression heighted the expression of an array of proangiogenic factors including VEGF-A and MMP9.Then,our pathological continuous section showed that the tumor tissues with high ILT4 expression had a relatively high expression of VEGF-A and MMP9.At the same time,we proved that ILT4 overexpression increased VEGF-A and MMP9 expression in both mRNA and protein levels in H1650 and A549 cells.In contrast,knockdown of ILT4 remarkedly decreased the VEGF-A and MMP9 expression in H1975 and H1299 cells.6.ILT4 participated in HUVECs migration and tubule formation of HUVECs by regulating the expression of VEGF-A and MMP9 in NSCLC cellsAfter being modified by ILT4 overexpression,H1650 and A549 cell lines with endogenous low ILT4 expression were added with VEGF-A neutralizing antibody or MMP9 neutralizing antibody.The conditioned medium was collected to culture HUVECs.The results showed that the migration ability of HUVECs was significantly reduced after blocking the expression of VEGF-A and MMP9;and the tubule forming ability of HUVECs was significantly decreased.7.ILT4 could activate the ERK and p38 signaling pathway.Gene chip analysis showed that up-regulation of ILT4 could induce the expression of multiple genes in MAPK signaling pathway.MAPK signaling pathway mainly includes ERK pathway,JNK pathway and p38 signaling pathway.Western blot analysis indicated that after overexpression of ILT4 in H1650 and A549 cells,the levels of pERK and pp38 were increased,and when the expression of ILT4 was down-regulated in H1975 and H1299 cells,the expression levels of pERK andpp38 were significantly decreased.8.ILT4 induced the expression of VEGF-A and MMP9 through ERK signaling pathway.H1650 and A549 cells with endogenous low expression of ILT4 were modified with ILT4,and then incubated with ERK inhibitor U0126 or p38 pathway inhibitor sb 203580(20 ? M)for 48 hours.The conditioned medium was collected.Western blot showed that the expression of VEGF-A and MMP9 in the group with ERK inhibitor U0126 decreased significantly,but did not change in the group with p38 pathway inhibitor.9.ILT4 regulated vascular endothelial migration and tubule formation of HUVECs via ERK signaling pathway.HUVECs were cultured in conditioned medium.Transwell migration assay showed that the migration ability of HUVECs was significantly decreased in the conditioned medium treated with ILT4 overexpression of lung cancer cells with ERK signaling pathway inhibited.Tubule formation assay confirmed that the tubulogenesis of HUVECs was significantly decreased in the conditioned medium treated with ILT4 overexpression of lung cancer cells with ERK signaling pathway inhibited.These results suggested that ILT4 induces the expression of VEGF-A and MMP9 through ERK signaling pathway,and then regulates vascular endothelial migration and tubule formation of HUVECs.10.ILT4 drove NSCLC tumor growth and angiogenesis.To determine whether ILT4 promote tumor growth in vivo,stably transfected cell lines A549 LV-ILT4,H1299 LV-shILT4 and the corresponding control cells were injected into the right back of the mice.The result suggested that NSCLC volumes were more massive in ILT4 overexpression group than in the control group.The tumor weight was increased in ILT4 overexpression group compared with control group.Mice injected with H1299 LV-shILT4 had the opposite results.To explore the effects of ILT4 on tumor angiogenesis in vivo,stable transfected A549 LV-ILT4,H1299 LV-shILT4 cells and their corresponding cells were implanted in the right back of the mice.Mice injected with A549 LV-ILT4 cells significantly increased the MVD,VEGF-A/MMP9,pERK expression compared with the control conditions.Mice injected with H1299 LV-shILT4 cells showed the opposite results.These results supported the role of ILT4 in angiogenesis in vivoConclusions1.ILT4 is highly expressed in NSCLC cells,which predicts poor patient outcome and increases microvessel density2.ILT4 activated ERK signaling pathway and subsequently upregulated the expression of VEGF-A and MMP9,which is responsible for tumor angiogenesis in vitro and in vivo.