A Mechanism Of Interleukin 7/Interleukin 7 Receptor Induce Lymphangiogenesis And Metastasis In Lung Cancer

ObjectiveInterleukin 7(IL-7) could induce the development and proliferation of haematopoietic cells and malignancies,including some forms of leukaemia and lymphoma.However,little was known about its involvement in solid tumours, including lung cancer.Some malignant cells,such as chronic lymphoblastic leukaemia cells,Burkitt's lymphoma cells and colonic cancer cells were also capable of producing IL-7.Other solid tumours could express the IL-7 gene,including oesophageal,renal, head and neck squamous cell carcinoma,and Warthin's tumour of the parotid gland.In addition,expression of IL-7 and IL-7R in breast cancer tissues has been shown recently, and IL-7R was positively correlated with tumours that had metastasised to the regional lymph nodes.Following the binding of IL-7R to its ligand,a series of intracellular phosphorylation events occurred,such as the activation of the Janus kinases(JAK-1 and JAK-3),phosphoinositide 3 kinase(PI3K),and the signal transducers and activators of transcription(STAT-5).13 It was therefore tempting to speculate that certain unidentified downstream gene(s) of IL-7 may have a role in tumour metastasis.In this study,we studied the relationship between IL-7 and their impact on lung cancer patients' outcomes.To explore the mechanism of IL-7/IL-7R inducing lymphangiogenesis and metastasis in lung cancer,we researched it in vivo and in vitro.MATERIALS AND METHODS1.Patients and Specimens.A total of 100 cases of NSCLC were obtained from the 1^st January 1980 to the 31^st December 2005 at the First Affiliated Hospital of China Medical University,Shenyang, China.The turnout tissues in this study were from patients who had NSCLC proved by pathological diagnosis without distant metastasis.None of the 100 cases had received radiation therapy or chemotherapy before surgery.The TNM staging system of the UICC(1997) was used to classify the specimens.2.Immunohistochemistry and Quantitation of Blood and Lymphatic Vessel Densities.Four-micron thick sections were prepared from the paraffin-embedded tissues. Immunostaining was performed by the streptavidin-peroxidase(S-P) method (UltrasensitiveTM MaiXin,Fuzhou,China).The primary antibodies were anti-IL-7, anti-IL-7R,anti-VEGF-D(1:100,1:100,1:150),anti-D2-40 and anti-CD34 antibodies. The peroxidase reaction was developed with DAB.For negative control,the primary antibodies were replaced by non-immune serum.All the samples were evaluated by 2 independent pathologists.Intensity of immunohistochemical staining:-,negative;+,focal expression＜5%of cancer tissues; ++,focal expression in 5-20%of cancer tissues;and +++,diffuse expression＞20%of cancer tissues.The section with ++ and +++ staining of IL-7,IL-7R or VEGF-D was classified as high expression and section with - and + staining was assigned as low expression.The evaluation criteria used for determination of blood and lymphatic vessel staining as follows:yellow-brown stained endothelial cells with band or fissure-like isolated or clustered structures or with tubular lumen were counted as a single blood or lymphatic vessel.Within each section,we selected 3 tumour areas with the highest density of distinctly highlighted microvessels and lymphatic vessels("hot spot") when observed under low-power fields.Next,the average number of CD34- or D2-40-1abeled tubular lumens was counted under high-power fields[34].Microvessel density(MVD) = mean(CD34-1abeled tubular lumen number—D2-40-1abeled tubular lumen number);LVD = mean D2-40-1abeled tubular lumen.If the difference between the numbers counted by the 2 pathologists was more than 10%,the lumens were recounted and a consensus between each observer was reached.3.Cell Culture.Human lung cancer cell lines A549 and SPC-A1(adenocarcinoma),NCI-H460 and LH7(large carcinoma),and SK-MES-1(squamous carcinoma) were maintained in Dulbcco's Modifed Eagle Medium(DMEM) supplemented with 10%fetal bovine serum(Gibco,USA)4.RNA Isolation and Reverse Transcriptase-PCR.Total RNA was isolated using TRIZOL(Invitrogen,USA) according to the manufacturer's instructions.The PCR products were amplified with 30 PCR cycles(1 min at 95℃;1 min at 53℃,1 min at 72℃),and visualized by ethidium bromide staining after agarose gel electrophoresis.5.Western Blot Analysis.Total protein was extracted with lysis buffer(150mM NaCl,1%v/v NP-40,0.1% v/v SDS,2μg/ml aprotinin,1mM PMSF),and 60μg of protein lysates were separated on a 12%v/v SDS-polyacrylamide electrophoresis gel,transferred to Polyvinylidene Fluoride(PVDF) membranes.Proteins were visualized with horse-radish peroxidase-conjugated goat anti-rabbit and anti-mouse IgG(Zhongshan,Beijing,China) followed by DAB.Subsequently,densitometric analyses of the bands were performed.6.Chromatin Immunoprecipitation(CHIP).We performed the ChIP assay according to the instructions of the ChIP assay kit (Upstate,USA).The procedure included DNA-protein cross-linking in chromatin, shearing DNA into smaller fragments,immunoprecipitation with anti-c-Jun antibody (negative control with normal rabbit IgG),followed by PCR identification of associated DNA sequences.7.Co-Immunoprecipitation(CoIP).The protein was extracted with cellular lysis buffer.Equal amounts of protein were incubated with c-Jun specific antibody immobilized onto protein G-bead for 1 h at 4℃with gentle rotation.Beads were washed extensively with lysis buffer,boiled,and microcentrifuged.Proteins were detected with c-Fos antibody by Western blot analysis.8.Cell Migration and Invasion Assays.For the migration assay,5×10⁴ cells were trypsinized,washed,resuspended in serum-free DMEM,and placed in the top portion of the chamber.The lower compartment of the chamber contained 10%v/v FBS as a chemo-attractant.The chambers were incubated for 6 h,then cells on the membrane were washed with PBS, and fixed in 100%methanol,stained with hematoxylin,photographed,and counted.For the invasion ability assay,pre-cooled serum-free DMEM was mixed with Matrigel(BD Biosciences,USA)(1:7 dilution).The upper compartments were filled with 100μl of the mixture,and the Matrigel was allowed to solidify at room temperature for 3 h. Other procedures followed the migration protocol(BD Biosciences,USA).The chambers were incubated for 24 h.9.In vitro cell growth assays.Cells were plated in a 96-well plate and MTT was added to each well.After incubation for 4h,the medium was replaced with dimethyl sulfoxide.The OD value of each well was measured with a test wavelength of 490nm.10.In vivo Nude mice model of lung cancer.24(4 weeks old) mice were inoculated with 2×10⁷ A549 human lung carcinoma cells under mouse dorsal skin.The mice were divided into four groups of six by random.After 1 weeks,each group were injected into the tail vein with 100ul complex once a week.First group:PBS;second group:IL-7(20mg/ml);third group:anti-IL-7R antibody(20mg/ml);fourth group:IL-7(20mg/ml)+ anti-IL-7R antibody(20mg/ml). After 10 weeks,the mice were killed.11.Statistical Analysis.The statistical package SPSS13.0(SPSS incorporated,Chicago) was used for all analysis.Values of P＜0.05 were considered statistically significant.RESULTS1.Expression of IL-7/IL-7R Correlate with variable expression levels.Immunohistochemical analysis of 100 NSCLC specimens revealed that the overexpression rates of IL-7 and IL-7R were 61%and 62%,respectively;VEGF-D overexpression rates 59%.IL-7 and IL-7R expression level were correlated respectively with the stage of lung cancer(P = 0.001,P = 0.007),lymph node metastases(P＜0.001,P＜0.001).IL-7 and IL-TR expression level were not correlated with age,gender,the histological type, and differentiation of the cancer.IL-7 and IL-7R expression level were correlated with the expression level of VEGF-D(P＜0.001,P = 0.002) respectively.IL-7,IL-7R,and VEGF-D expression level were correlated respectively with LVD(P = 0.003,P= 0.041,and P＜0.001),but they were not correlated respectively with MVD.Clinically, compared to the NSCLC with low expression of IL-7,tumours with high expressions of IL-7,IL-7R,and VEGF-D were more p shorter survival(P＜0.05).2.IL-7/IL-7R Increase the Expression of VEGF-D in Lung Cancer Cell Lines.Treatment of IL-7R⁺ lung cancer A549,SPC-A1,LH7,and SK-MES-1 cells with recombinant human IL-7 increased the expression of VEGF-D,while the expression of VEGF-C were not affected.Interestingly,the expression of VEGF-D were not affected in IL-7R- lung large carcinoma H460 cells after IL-7 stimulation.We then detected the expression of VEGF-D of the five cells after blocking IL-7R.Blockage of IL-7R in A549,SPC-A1,LH7,and SK-MES-1 cells decreased the expression of VEGF-D, which were not affected by the IL-7.However,blocking IL-7R in H460 cells did not affect the expression of VEGF-D.3.IL-7/IL-7R Induce VEGF-D via c-Fos/c-Jun Pathway.Incubation of A549 cells with IL-7 increased the expression of c-Fos and c-Jun protein,while blocking IL-7R with sc-662 decreased the expressions of c-Fos,c-Jun, and p-c-Jun.We then inhibited the activity of c-Fos/c-Jun with a specific AP-1 inhibitor SP600125.The analyses showed that the expressions of VEGF-D were decreased significantly after treatment with SP600125.These results imply that IL-7/IL-7R up-regulate VEGF-D via c-Fos/c-Jun.4.IL-7/IL-7R Lead to the Formation of c-Fos/c-Jun Heterodimer.Using CoIP approach,we found that c-Fos/c-Jun heterodimer was increased after incubation with the IL-7,and blocking IL-7R with sc-662 decreased the dimer formation.5.IL-7/IL-7R Enhance the DNA Binding Activity of AP-1 to the Promoter of VEGF-D.CHIP analysis demonstrated that AP-1 could bind to VEGF-D promoter.Then we detected AP-1 DNA binding activity in A549 cell after incubation with either the IL-7 with or without sc-662.IL-7 enhanced AP-1 binding to VEGF-D promoter,while blocking IL-7R reduced AP-1 binding to VEGF-D promoter.6.IL-7 promote the proliferation of the cells.The results of the MTT assay showed that the proliferation of the A549 cells was significantly higher with IL-7 than without IL-7.7.In vivo Nude mice model of lung cancer assays.It demonstrated that,in vivo,IL-7 and its receptor IL-7R,are able to induce VEGF-D gene expression via AP1(c-Fos/c-Jun)-dependent pathway to upregulate lymphangiogenesis and lymphatic node metastasis.Conclusions1.IL-7/IL-7R high expression level were positive correlated with the stage, lymphatic node metastasis,and lower survival of lung cancer.In IL-7/IL-7R and VEGF-D high expression groups LVD were higher than in low expression groups.2.In human lung cancer cells,this study demonstrated that IL-7 and its receptor IL-7R,are able to induce VEGF-D gene expression via AP1(c-Fos/c-Jun)-dependent pathway.3.In vivo Nude mice model of lung cancer assays demonstrated that,in vivo,IL-7 and its receptor IL-7R,are able to induce VEGF-D gene expression via AP1 (c-Fos/c-Jun)-dependent pathway to upregulate lymphangiogenesis and lymphatic node metastasis.