The Study Of Foxp3 In Lung Cancer And Its Regulation By TLR4

Lung cancer is the highest morbidity and mortality of malignant tumors and its 5-year survival rate is less than 16%. The incidence of lung cancer in China is the first in the world. According to histological type of lung cancer, lung cancer is divided into two categories: small cell lung cancer (small-cell lung cancer, SCLC) and non-small cell lung cancer (non small-cell lung cancer, NSCLC). NSCLC accounts for about 80% of lung cancer,most of these patients are already in advanced stage when they receive first treatment.The chemotherapy sensitivity to NSCLC is very poor, and the mortality of NSCLC is very high.Recurrence and metastasis are the critical factors in determining prognosis of patients with NSCLC,so studying the molecular mechanism of invasion and metastasis,looking for the target to reverse or prevent the invasion and metastasis is an important goal to improve the survival of lung cancer patients.Foxp3 is a member of forkhead/winged-helix transcription factor family.It has been identified as the master regulator for the development and function of CD4+CD25+Treg cells(regulatory T cells,Tregs).Tregs which were found in 1999 were a group T cell subsets with immune suppression and played an important role in the process of maintaining tolerance.Tregs have been associated with the tumorigenesis and development of tumor.It has been reported that the levels of Tregs and Foxp3 expression were increased in peripheral blood and tumor specimens in patients with various types of cancer.Recent papers described the expression of Foxp3 not only in Tregs,but also in tumor cells.Since Hinz et al reported the expression of Foxp3 in human pancreatic carcinoma cells,other studies about the expression of Foxp3 in human breast cancer cells, prostate cancer cells have also been reported. However, the role of Foxp3 in tumor cells were not the same, even in the same tumor. It proposed a possible that Foxp3 in tumor cells may mimic Tregs contributing to tumor immune escape.Up to now, the functional studies about Foxp3 in lung cancer cells have not been reported.Toll-like receptors (TLRs) are a family of pattern recognition receptors. Now, It has been identified 13 types of TLRs in which TLR4 was studied first. TLR4 was mainly expressed in immune cells and considered an important link between innate and adaptive immunity by means of the induction of signaling cascades through recogniting endogenous and exogenous pathogens.Recent studies found that TLR4 was also expressed in many tumor cell lines and tumor tissues.Tumor cells activated by TLR4 signals may release cytokines and thus resulted in a cytokine profile that was associated with immune tolerance of the tumor microenvironment contributing to cancer progression.It has been reported that Tregs have been associated with TLR4.Exposure of Tregs to the TLR4 ligand LPS induced up-regulation of several activation markers and enhanced their proliferation and suppressive function.Foxp3 is a key regulatory gene for the development and function of Tregs.Therefore,we hypothesized that TLR4 signaling pathway may be involved in the regulation of Foxp3 expression on tumor cells.Based on the above background, we proposed the following questions:whether lung cancer cells express Foxp3? If lung cancer cells express Foxp3, what is its role in lung cancer cells? what is the mechanism of its function and regulation. In order to investigate the role of Foxp3 in lung cancer cells and possible mechanism and regulation, we chose human NSCLC specimens and mouse Lewis lung cancer (LLC) cell lines for subject investigated, from the following two areas:1. The expression and clinical significance of Foxp3 and TLR4 in NSCLCTo investigate the possible role of Foxp3 and TLR4 expression in NSCLC, we analyzed the expression of Foxp3 and TLR4 in 53 cases NSCLC specimens and 15 cases normal lung tissue by immunohistochemical staining. The results showed:(1) The expression of Foxp3 in NSCLC tumor cells were significantly high,the positive rate was 54.7%. Foxp3 staining was localized in the cytoplasm, cytoplasm/nuclear, membrane and nuclear. The expression of Foxp3 in normal lung tissue were significantly low, the positive rate was 20%.Foxp3 staining were localized in the cytoplasm of lung bronchial epithelial cells. There was statistically significant between tumor group and normal group (P<0.01). The expression of TLR4 in NSCLC tumor specimens were significantly high, the positive rate was 73.6%. TLR4 staining was localized in the cytoplasm and membrane. The expression of TLR4 in normal lung tissue were significantly low, the positive rate was 26.7%.TLR4 staining were localized in the cytoplasm of lung bronchial epithelial cells. There was statistically significant between tumor group and normal group (P<0.01). (2) Expression of Foxp3 in tumors were closely associated with lymph node metastasis and TNM stage. The positive rate of Foxp3 was significantly higher in lymph node-positive group (76.2%) than that of lymph node-negative group (39.4%) (P<0.05).The positive rate of Foxp3 was significantly higher in TNMⅡ+Ⅲstage (69.2%) than that of TNM I stage (40.7%) (P<0.05),while no association was found with patient'age,tumor size, histological types and tumor differentiation (P>0.05). Expression of TLR4 in tumors were closely associated with tumor differentiation.The positive rate of TLR4 was significantly higher in poor differentiation group (90.0%) than that of well-moderate differentiation group (63.6%) (P< 0.05), while no association were found with patient'age,tumor size, histological types,lymph node metastasis and TNM stage (P>0.05). (3) The expression of Foxp3 in tumor cells were positively associated with TLR4 (r=0.401, P<0.01).The results indicated that the expression of Foxp3 and TLR4 were associated with malignant biological behavior of NSCLC.Foxp3 and TLR4 may have some internal relationship and play cooperative role in the tumorigenesis and development of NSCLC.2. The role of Foxp3 in mouse LLC cell linesAfter founding the role of Foxp3 in lung cancer cells, we chose mouse LLC cell lines for subject investigated to further explore the possible mechanism of Foxp3 invovled in the tumorigenesis and development of NSCLC.(1) Foxp3 expression in LLC cells and its eukaryotic expression vector and identification of Foxp3 over-expressionFirst, we detected the expression of Foxp3 in LLC cells using RT-PCR and immunocytochemistry. Then we constructed recombinant plasmid of pcDNA3.1-Foxp3 and transiently transfected it into LLC cells. The over-expression of Foxp3 were identified by RT-PCR, immunocytochemistry and Western blot.The results showed that LLC cells expressed Foxp3 mRNA and protein and the recombinant plasmid pcDNA3.1-Foxp3 was successfully constructed. The expression of Foxp3 mRNA and protein in pcDNA3.1-Foxp3-LLC group were significantly higher than that of pcDNA3.1-LLC group and LLC group (P<0.05), indicating that Foxp3 was over-expressed in pcDNA3.1-Foxp3 transfected LLC cells and those transfected cells could be used for further experiment.(2) Effects of Foxp3 over-expression on immune function in LLC cellsTo study the effect of Foxp3 in LLC cells on the proliferation of T lymphocytes through mimicking Tregs function then contributing to tumor immune escape, the proliferation of T lymphocytes were detected by lymphocyte transformation test. The expression of TGF-β1 and IL-10 mRNA and protein were detected by RT-PCR and ELISA.The results showed that the inhibitory rate of T cell proliferation in pcDNA3.1-Foxp3-LLC group were significantly higher than that of LLC group and pcDNA3.1-LLC group (P<0.05). The mRNA and protein expression of TGF-(31 and IL-10 were significantly increased in pcDNA3.1-Foxp3-LLC group than that of LLC group and pcDNA3.1-LLC group (P<0.05). The results indicated that Foxp3 in LLC cells could inhibit the proliferation of T lymphocytes mimicking Tregs, one of its inhibition mechanisms may be through up-regulating the expression of TGF-β1 and IL-10 achieved.(3) Effects of Foxp3 over-expression on sensitivity to chemotherapeutic agents and related mechanism in LLC cellsTo study the possible effect of Foxp3 on sensitivity to chemotherapeutic agents, Foxp3 over-expressed LLC cells were treated by different concentrations (0,5.10.20,40μg/ml) chemotherapeutic agents including adriamycin (ADM), mitomycin C (MMC) and cisplatin (DDP). The expression of mdr 1 and mdr 3 mRNA were detected by RT-PCR and P-gp expression were detected by immunocytochemistry and FCM. The results showed that the growth inhibition rate of pcDNA3.1-Foxp3-LLC group were significantly lower than that of LLC group and pcDNA3.1-LLC group under ADM and MMC treatment at 20μg/ml and 40μg/ml (P<0.05),and IC50 were increased in pcDNA3.1-Foxp3-LLC group. The sensitivity to DDP were not changed significantly (P>0.05). The expression of mdr 1 mRNA and P-gp were significantly increased in pcDNA3.1-Foxp3-LLC group than that of LLC group and pcDNA3.1-LLC group (P<0.05), while the expression of mdr 3 were not changed significantly(P>0.05). The results indicated that Foxp3 in LLC cells may reduce the sensitivity to ADM and MMC through up-regluating the expression of P-gp then promoting tumor progression.3. The regulation of Foxp3 expression by TLR4 in LLC cellsTo further study of specific relations between TLR4 and Foxp3 in LLC cells, we chose LPS as exogenous ligands activating TLR4 signaling pathway. The expression of Foxp3 was detected by RT-PCR and FCM after activating and blockingTLR4 signaling pathway. The expression of TLR4 protein were detected by FCM after LPS stimulation.The results showed that the expression of Foxp3 in LPS stimulation group were significantly up-regulated than that of No stimulation group (P<0.05). The expression of Foxp3 was significantly decreased in blocking group than that of no blocking group (P<0.05). The expression of TLR4 were significantly up-regulated after LPS stimulation (P<0.05).The results indicated that the TLR4 signaling pathway may be involved in the regulation of Foxp3 expression in LLC cells, and TLR4 may be the upstream signaling molecules of Foxp3.Comprehensive study on, we come to the following conclusions:1. The over-expression of Foxp3 and TLR4 in tumor cells were associated with malignant biological behavior of human NSCLC. TLR4 may be involved in the regulation of Foxp3 expression. TLR4 may up-regulate Foxp3 expression and play cooperative role in the tumorigenesis and development of NSCLC.2. Foxp3 in lung cancer cells may inhibit the proliferation of T lymphocytes mimicking Tregs function by up-regulating the expression of inhibitory molecules TGF-β1 and IL-10 then contributing to tumor immune escape.3. Foxp3 in lung cancer cells may reduce the sensitivity to ADM and MMC by up-regulating the expression of P-gp then contributing to tumor progression.This study first investigated the role of Foxp3 in lung cancer cells, provided relevant mechanisms about Foxp3 involving in tumorigenesis and development, found the regulation of Foxp3 expression by TLR4.The results provided new insights for tumor invasion and metastasis mechanisms of lung cancer cells, and provided new targets for clinical immunotherapy of lung cancer.