Expression Of Tissue Factor In Non Small Cell Lung Cancer And Effects And Mechanisms Of Inhibition Of Tissue Factor On Lung Adenocarcinoma

Tissue factor (TF) is a 263 amino acid transmembrane glycoprotein, the gene located on chromosome 1p21-1 p22, the total length of 12.4kb, with 6 exons and 5 introns. The transcribed mRNA is 2.1kb, and the final translation modification of the TF at the cell surface is a molecular weight of 47kd single chain transmembrane glycoprotein, which belongs to the classⅡcytokine receptor family. TF consists of a soluble amino-terminal extracellular domain (Serine 1 to Glutamic acid 219), a transmembrane domain (Isoleucine 220 to Leucine 242) and an intracellular carboxyl-terminal (Histidine 243 to Serine 263). The extracellular region of coagulation factorⅦ/Ⅶa (FⅦ/Ⅶa) receptor binds to FⅦ, activates FⅦand forms TF-FVIIa complex, which plays a key role in initiating the extrinsic coagulation pathway.According to whether binding the membrane, TF is divided into the soluble and membrane-bound forms, and according to whether combined with procoagulant particles in the blood, TF is separated into microparticles bearing TF (TF-MPs, which have procoagulant activity) and non-particulate bound TF. Recently, some scholars have found that monocytes express the full length TF (flTF) and alternatively spliced TF (asTF). AsTF is soluble due to the lack of the exon 5 encoded transmembrane domain, and the clotting activity is weak than the former, but in angiogenesis and tumor invasion may play an important role. Under physiological circumstances, TF in circulation is very low, and normal cells including vascular endothelial cells and blood mononuclear cells do not express TF, except by external stimuli such as injury, inflammation, etc. But many studies have found that the abnormally high expression of TF-MPs can be detected in the blood of cancer patients, which may be released from the surface of tumor cells and shed into the blood; cancer patients regardless of their tumor or normal tissue cells are the overexpression of TF, which not only increases the incidence of thrombotic events in patients with cancer, but also is involved in the malignant progression of tumors. The studies of breast cancer, colon cancer, pancreatic cancer and glioma have shown evidence of TF expression in tumor and tumor-related clinical and pathological features and survival were negatively correlated. Coagulation pathway and non coagulation pathway, in which TF is involved, promote tumor growth and metastasis.The signal transduction pathway in is not completely clear, but now the main view display that protease-activated receptors (PARs) family mediates signal transduction (PARs are G protein-coupled receptors, include PAR-1, PAR-2, PAR-3 and PAR-4 four subtypes). Coagulation pathway:(1) TF and FVII form the activated compound:the TF-FⅦa complex, which activates the tumor cell surface PAR-2, then the TF-FⅦa-FXa complex can activate PAR-1 and PAR-2. First, through the ERK1/2 (p44/42) MAPK, p38 MAPK and JNK signal pathways, TF promote vascular endothelial growth factor(VEGF) expression and inhibit the expression of the anti-angiogenic proteins such as thrombospondin (thrombospondin-1, TSP-1); secondly FVIIa can increase the concentration of intracellular Ca2+, then activate protein kinase C (PKC) to combine the actin-binding protein 280(ABP-280) with the cytoplasmic tail region of TF, the activation of focal adhesion kinases (FAK) by up-regulation of the MAPK signaling result in the elevation of tumor cell adhesion and migration; (2) TF initiates the extrinsic coagulation pathway and generates thrombin, which binds to PAR1, PAR3 and PAR4, activates the MARK signaling pathway, and upregulates the tumor growth factors such as:VEGF, VEGFR, bFGF and MMP-2 and so on. Non-coagulation pathway:three serine residues of intracellular tail region of TF are likely to be phosphorylated by intracellular PKC, resulting in the expression of the factors of tumor growth.Every year, about 1.6 million new patients of lung cancer are diagnosed, while nearly 1.4 million cases of lung cancer deaths. Lung cancer is the leading morbidity and mortality of cancer worldwide. Non-small cell lung cancer (NSCLC), which includes adenocarcinoma and squamous cell carcinoma accounts for about 85% of the total cases of lung cancer. Treatment of NSCLC in the past 10 years has made tremendous progress, from a simple treatment surgery and chemotherapy to the current individual treatment strategies, especially for the molecular biological mechanisms of lung cancer targeted therapy. Many researches have been focused on the role of epidermal growth factor receptor (EGFR) pathway, and the EGFR inhibitor has made a great benefit in patients with an EGFR mutation. Therefore, looking for molecular targets for cancer therapy is a current research focus. However, there are few studies on the expression of TF in NSCLC; and the research about whether silencing the TF expression can inhibit proliferation and metastasis of lung adenocarcinoma, promote apoptosis, and related mechanisms is reported yet. Therefore, through this study on TF in lung adenocarcinoma help to identify a new molecular target for lung cancer and improve the treatment of NSCLC.In this study, we first collected 73 cases of non-small cell lung cancer and 14 cases of benign lesions of the lung specimens, and using western blot and immunohistochemistry to detect the protein expression of TF. We found the TF expression was higher in NSCLC than in tissue of benign pulmonary disease; higher in lung adenocarcinoma than squamous cell carcinoma. Therefore, in vitro, we suppressed in the expression of TF in lung adenocarcinoma cell line A549 using RNA interference (RNAi) technology to investigate the malignant behaviors of lung adenocarcinoma and its possible molecular mechanisms. Further in vivo study, we verified the role of TF in lung adenocarcinoma using nude mice xenograft model of A549 cells. Consequently, we identified whether the specific silence of TF could inhibit the malignant behavior of lung adenocarcinoma at the level of cell and animal, in order to provide the theoretical basis for treatment of lung cancer with targeting TF in the future.Ⅰ. Specimens The expression and clinical signification of tissue factor in non-small cell lung cancer Research purpose:To study the expression of tissue factor in non-small cell lung cancer (NSCLC) and discuss the relation between tissue factor and the clinical signification of NSCLC.Research methods:1.73 cases of NSCLC tissue and cancer tissue (from cancer tissue edge about 2 cm) and 14 cases of surgical specimens of patients with benign lung diseases as control group were collected from January 2009 to March 2011 in department of general thoracic surgery of Tongji hospital. Every specimen was cut into two pieces, of which one was frozen in liquid nitrogen then stored in -80℃refrigerator, and another was fixed in 4% neutral formalin and embedded in paraffin for hematoxylin-eosin (HE) staining and immunohistochemistry staining;2. The TF expression of 73 cases of NSCLC specimens and 14 cases of benign pulmonary lesions were detected using Western blot;3. Immunohistochemical method was used to detect the expression of TF in 73 cases of NSCLC specimens and 14 benign lesions of the lung specimens.Research results:1. Western blot results showed that the TF expression was higher in NSCLC than in benign pulmonary lesions (P<0.01), higher in lung carcinoma tissue than in adjacent normal (P<0.05 or P<0.01), higher in lung adenocarcinoma than in squamous cell carcinoma (P<0.01), and higher expression in the lower degree of differentiation (P<0.01);2. The positive staining rate of TF in NSCLC tissue by Immunohistochemistry detection (n=73,100%) was significantly higher than in the control group of lung benign tumor (n=5, 35.7%) (P<0.01). The high expression rate in lung adenocarcinoma (n=24,63.2%) was significantly higher than in lung squamous cell carcinoma (n= 13,37.1%) (P<0.05), higher in poorly differentiated NSCLC than high-grade NSCLC (P<0.05), in late (Ⅲ＋Ⅳ) stage was higher than in early (Ⅰ+Ⅱ) stage (P<0.05).Ⅱ. In vitro study The effects of inhibition of the tissue factor expression on lung adenocarcinoma in vitroResearch purpose:To evaluate the effects of the inhibition of the TF expression on malignant biological behavior of lung adenocarcinoma and its possible molecular mechanism.Research methods:1. Lung adenocarcinoma cell line A549 was cultured in RPMI 1640 medium containing 10% FBS, 100U/ml penicillin and 100ug/ml streptomycin, and was placed in an incubator with 5% CO2 and 37℃. Cells were used in the experiments in the logarithmic growth phase;2. The siRNA specific for silencing TF (TF-siRNA) was synthesized chemically, using Lipofectamine reagent lipofectamine 2000 transfected into A549 cells. The concentrations of siRNA were 25 nM,50nM and 100nM.24h,48h,72h and 96h after transfection, cells were harvested for further testing. Negative group (Mock) was transfected with randomized siRNA, and control group (Control) was non-treatment group;3. Fluorescent siRNA was transfected into A549 cells, then fluorescence microscopy and flow cytometry were used to detect the transfection efficiency and the silencing effects of TF was analysed by western blot and semi-quantitative reverse transcriptase PCR; 4.24h,48h,72h and 96h after TF-siRNA transfection, the proliferation of A549 cells were detected by the MTT assay;5.48h after TF-siRNA transfected into A549 cells, the colony-forming ability of A549 was evaluated by the colony-formation assay;6.48h after TF-siRNA transfected into A549 cells, the migration of lung adenocarcinoma was observed by wound healing assay;7.48h after TF-siRNA transfected into A549 cells, the invasion and migration capacity of lung adenocarcinoma were tested by the transwell chamber;8.48h after TF-siRNA transfected into A549 cells, cell apoptosis was detected by flow cytometry using AnnexinV-FITC and PI staining kit;9.48h after TF-siRNA transfected into A549 cells, cells were collected to extract total cellular protein, the protein expression of PI3K, P-AKT, total AKT, P-ERK, total ERK, VEGF, MMP-2, MMP-9 andβ-actin were detected by Western blot.Research results:1. TF-siRNA can effectively transfected into A549 cells, cells were observed under a fluorescence microscope 48h after 100nM FAM fluorescence siRNA transfection and flow cytometry 6h after 100nM FAM fluorescence siRNA transfection, the transfection efficiency were detected up to 85%;2.48h after TF-siRNA transfected A549 cells, the protein and mRNA expression of TF, which were detected by western blot and semi-quantitative RT-PCR, were significantly decreased compared with the negative control group,25nM group (P<0.05),50nM group (P<0.01) and 100nM group (P<0.01) in a dose-dependent manner;3.48h after TF-siRNA transfected A549 cells, the numbers of colony formation in 50nM and 100nM TF-siRNA groups were (205.0±13.2) and (132.3±11.7), which were significantly less than in the negative control group (490±19.1), and the differences were statistically significant (P<0.01);4.48h after TF-siRNA transfected A549 cells, the wound healing assay showed that the migration indexes of 50nM and 100nM TF-siRNA groups were (0.35±0.06) and (0.23±0.09)respectively,, which was significantly smaller than that of the negative control group (0.61±0.05), and these differences were statistically significant (P<0.01). Using transwell chamber, the migration cells per field in 50nM and 100nM TF-siRNA groups were (111.0±10.6) and (75.7±11.0) respectively, and compared with the control group (202.7±7.1), the migration cells was significantly decreased (P<0.01);5.48h after TF-siRNA transfected A549 cells,50nM and 100nM TF-siRNA groups per field displayed the numbers of passed through the Magtrigel-coated membranes cells were respectively (76.7±7.6) and (40.0±5.0), whereas the negative control group was (155.3±8.6), and there was statistical significant differences (P<0.01);6.48h after TF-siRNA transfected A549 cells, apoptosis was detected by flow cytometry and the results showed that the apoptosis rates in 25nM,50nM and 100nM TF-siRNA groups were (7.62±1.20)%, (10.51±1.36)% and (17.36±1.59)%, in which 50nM and 100nM groups were significantly increased in a dose-dependent manner compared with the control group (5.77±0.96)% (P<0.01);7.48h after TF-siRNA transfected A549 cells, Western blot results suggested that the inhibition of TF expression resulted in inhibiting the PI3K/AKT, ERK MAPK signaling pathway and attenuating the expression of VEGF and MMP-2/-9 compared with the negative control group,and there were statistical differences(P<0.05 or P<0.01).ⅢIn Vivo Study The effects of the inhibition of the tissue factor expression on lung adenocarcinoma in vivo Research purpose:To investigate the effects of inhibition of the expression of TF in the nude mice xenograft model on the growth of lung carcinoma.Research methods:1. Lung adenocarcinoma cell line A549 was cultured in RPMI 1640 medium containing 10% FBS, 100U/ml penicillin and 100ug/ml streptomycin, and was placed in an incubator with 5% CO2 and 37℃. Optimize the cell performance, the best cells is used in the experiment;2.15 female BALB/c nu/nu mice were given a one-week adaptation period, then were randomly divided into three groups, and each group had 5 mice.1×107 cells of lung adenocarcinoma cell line A549 were injected subcutaneously on the right side of the back of nude mice. After 14 days the human lung adenocarcinoma xenograft, tumor size can be reached 50-00 mm3, and then according to the groups, the intervention were performed. Three groups were:(1) control group(Control), no treatment; (2) negative control group(Mock),50ug randomized siRNA were intratumoral injected every 5 days, a total of 6 times; (3) SiTF group,50ug TF-siRNA were intratumoral injected every 5 days, a total of 6 times. The tumor diameters were measured 2 times a week with a caliper, The tumor volume (mm3) was calculated according to the following formula:volume (V, mm3)= diameter (L, mm)×[short diameter (W, mm)]2/2. All the animal treatments were in accordance with the guidelines of experimental animal center of Tongji Hospital and the normative standards for laboratory animal of Chinese Academy of sciences;3. Mice were sacrificed humanely at the end of the experiment after 5 times of treatment; the right subcutaneous tumors were removed, weighed and photographed. Each tumor were cut into two parts, of which one was frozen in liquid nitrogen then stored in -80℃refrigerator, and another was fixed in 4% neutral formalin and embedded in paraffin;4.The expression of TF in tumor tissue were detected by Western blot and immunohistochemistry staining.Research results:1.14 days after xenotransplantation of A549 cells into nude mice, the tumor sizes on mice's right back of Control group, Mock group and SiTF group were (95.15±22.97) mm3, (95.42±39.31) mm3 and (88.52±28.38) mm3 respectively, and the differences were not statistically significant in groups (P>0.05). SiRNAs were began to injected into the mice on 14 days after xenotransplantation of A549 cells.18 days,22 days,26 days,30 days,34 days,38 days, and 42 days after xenotransplantation of A549 cells, the tumor size of Control group were:(136.22±27.46) mm3, (229.28±45.34) mm3, (351.74±76.38) mm3, (428.81±91.96)mm3,(517.98±106.04)mm3,(562.73±173.30)mm3 and(601.02±84.72) mm3; Mock group (147.05±37.97) mm3, (242.43±60.49) mm3, (319.04±75.26) mm3, (389.31±93.84)mm3,(471.26±117.57)mm3,(502.56±22.64)mm3 and(513.81±112.57) mm3; SiTF group (103.22±30.42) mm3, (108.77±32.68) mm3, (121.32±42.83) mm3, (140.69±53.33) mm3, (156.16±57.32) mm3, (176.07±66.79) mm3 and (209.56±97.56) mm3. respectively.22 days after xenotransplantation of A549 cells that is 8 days after the injection of TF-siRNA into the mice, the tumor growth of SiTF group was significantly slower than Control group and Mock group, and the differences were statistically significant (P<0.01);2. All the mice were sacrificed on the 42nd day after xenotransplantation of A549 cells, and the weight of resected tumors in three groups were Control group (0.59±0.18)g, Mock group (0.52±0.12)g and SiTF group (0.21±0.10)g. Compared with the weight of tumor in Mock group that in SiTF group was significantly reduced, and the differences were statistically significant (P<0.01);3. The expression of TF in SiTF group detected by western blot and immunohistochemistry was significantly decreased compared with Mock group, and the difference was statistically significant (P<0.01).Statistical analysisAll data were analyzed by SPSS 12.0 statistical software. Enumeration data were expressed as frequency or composition ratio, and the Chi-square test were used to evaluate the differences between two groups. Values were present as mean±standard deviation (x±SD). Analysis of variance (ANOVA) was used to compare group differences. Student's t test was used to assess whether differences in the values of two groups were statistically significant. The level for statistical differences was set at P< 0.05.Conclusions1. TF was not only overexpressed in NSCLC, but also correlated with the clinical and pathological features of NSCLC, the rate of high expression of TF in lung adenocarcinoma was significantly higher than that in squamous cell carcinoma, and higher in poorly differentiated NSCLC than high-grade NSCLC, in late (Ⅲ+Ⅳ) stage was higher than in early (Ⅰ+Ⅱ)stage.2. In vitro, inhibition of the expression of TF in lung adenocarcinoma cells can inhibit cell proliferation and colony formation ability, and promote apoptosis of lung cancer cell.3. In vitro, inhibition of the expression of TF in lung adenocarcinoma cells can attenuate migration and invasion of lung cancer cell4. In vitro, inhibition of the expression of TF in lung adenocarcinoma cells caused decreased expression of VEGF and MMP-2/-9, and PI3K/AKT, ERK MAPK signaling pathway may be involved in the role of TF in lung cancer.5. In xenograft studies, the results demonstrated that inhibition of the expression of TF in lung cancer cells may inhibit tumor growth.6. This study of TF provided a theoretical basis for a new molecular therapeutic target and a new treatment of lung cancer.