Role And Mechanism Of C5aR In Non-small Cell Lung Cancer Invasion And Metastasis

Lung cancer is one of the major causes of cancer related mortality worldwide. Non-small-cell lung cancer (NSCLC) accounts for nearly85%of lung cancer cases. Despite the improvement in diagnostic approaches and introduction of new therapeutic agents these years, the5-year survival rate of lung cancer remains low. The high recurrence rates and early metastasis of lung cancer imply the importance and urgency of exploring new prognostic markers to screen patients for unfavorable prognosis.C5aR, also know as CD88, is a G-protein-coupled receptor. It localizes to the chromosome19q13.3-19q13.4. C5aR was initially recognized to be expressed in myeloid cells, and subsquently many non-myeloid-derived cells are also found to express C5aR. C5aR is well known as its functions in many inflammatory diseases, however, its role in tumorigenesis has not been completely recognized. The role of C5aR in tumor was first described in human hepatocellular carcinoma cell line HepG2. Buchner RR et al. found hepatocellular carcinoma overexpressed C5aR, while normal hepatocyte expressed little. Rencently, Hidetoshi Nitta et al. reported enhancement of human cancer cell motility and invasiveness by anaphylatoxin C5a via aberrantly expressed C5a-receptor. However its role in NSCLC is not clear and the mechanism of C5aR in tumor invasion is still need to be elucidated.In the present study, we investigated the expression of C5aR in NSCLC and their adjacent nontumorous tissues. Then we explored its role and mechanism in non-small cell lung cancer cell invasion and motility.PART ONE:C5aR is overexpressed in NSCLC To assess the biological function of C5aR in NSCLC, we first examined its expression in non-small cell lung cancer cell lines and tissues. Five non-small cell lung cancer cell lines (A549, H460, H1355,95-C and95-D) and a normal bronchial epithelial cell line (16HBE) were examined the expression of C5aR at the mRNA level. Six primary non-small cell lung cancer tissues and their adjacent nontumorous tissues were analyzed for C5aR expression by using western blot. C5aR was siginificantly overexpressed in NSCLC cell lines compared to the normal bronchial epithelial cell line. C5aR was overexpressed in NSCLC tissues than their adjacent nontumorous tissues.These findings indicated C5aR was overexpressed in NSCLC. C5aR may play an important role in NSCLC which need to be further elucidated.PART TWO:Down-regulation of C5aR impaired the invasion and metastasis of NSCLC cellsIn order to investigate the biological function of C5aR in non-small cell lung cancer, three pairs of siRNAs targeted to three different sites in C5aR mRNA were constructed. Then we packaged these siRNAs with Lipofectamine and transfeeted the mixture into A549cells. SiRNA-C5aR-3was validated for the most efficient interference of C5aR. Then we performed Cell Counting Kit-8assay and cell clone formation to analyze cell proliferation. Transwell assay and scratch assay were performed to determine whether C5aR has a crucial role in cell migration and invasion. We observed a significant decrease in cell migration in A549cells transfected with SiRNA-C5aR compared with those transfected with the SiRNA-control. Down-regulation of C5aR impared cell proliferation and cell clone formation (p<0.05)In addition, A549cells (1×106) transfected with negative control and siRNA-C5aR were implanted subcutaneously into either posterior flank of the same nude mice. Tumor growth was monitored every week. After40days, A549cells transfected with siRNA-C5aR exhibited a marked reduction in tumor size compared to the control group, suggesting that knowdown of C5aR expression lead to siginificant inhabition of tumor growth in vivo.The morphology of A549cells transfected with siRNA-C5aR presented the typical cobblestone-like appearance of normal epithelium instead of spindle-like, fibroblastic morphology. E-cadherin expression was up-regulated and Vimentin expression was down-regulated.The results of this study indicated C5aR promoted invasion and motility of NSCLC cell line. In vitro, knockdown of C5aR expression impaired cell invasion, motility and proliferation. In vivo, the down-regulation of C5aR can inhabit tumor formation. C5aR played an important role in EMT of NSCLC.PART THREE:The clinicopathological significance of C5aR expression in primary non-small cell lung cancerIn this part of research, we analyzed the correlation between C5aR expression and other clinicopathological features in208primary non-small cell lung cancer tissues. We detected the C5aR expression in208primary non-small cell lung cancer tissues by tissue miroassay (TAM) and immunohistochemistry. We evaluated the relation between C5aR expression and patients’ outcome by Kaplan-Meier and Cox analysis. High expression of C5aR accounted for53.4%specimens from patients with NSCLC (111of208patients). The expression of C5aR significantly correlated with lymph node metastasis of NSCLC patients (p=0.012). Interestingly, we found that C5aR expression levels were significantly higher in tissues of squamous cell carcinoma than tissues of adenocarcinomas (p=0.002). However, other clinicopathological features, including age, gender, smoking status, tumor stage, tumor size, and tumor differentiation were not directly associated with the expression of C5aR.During the follow up,109patients died of recurrence or metastasis of disease. The5-year overall survival rate for all patients was47.6%. The5-year overall survival rate for patients with high CD88expression was significant lower than patients with low C5aR expression (p=0.001). As C5aR expression level was different between squamous cell carcinoma and adenocarcinomas, we performed a subgroup analysis by pathological subtype. In squamous cell carcinoma and adenocarcinomas the association remained significant with p value0.019and0.049respectively. Univariate analysis revealed that tumor size (≥3cm), lymph node metastasis, high TNM stage, poor differentiation and high C5aR expression were associated with shorter OS. In multivariate analysis, tumor size, lymph node metastasis, tumor stage and C5aR expression were identified as independent prognostic factors in patients’ overall survival. These results indicated C5aR expression correlated with lymph node metastasis and predicted poor prognosis of NSCLC.PART FOUR:The mechanism of C5aR in tumor invasion and metastasis.It had been reported that C5aR delayed neutrophils apoptosis by phosphatidylinositol3-kinase-signaling pathway. C5a increased the release of MMPs and promoted tumor cell motility. C5aR was also reported to actived MAPK pathway. MAPK pathway had been reported play a pivotal role in EMT of many tumors.There for we investigated AKT、ERK、p38and MMPs to elucidated the mechanism of C5aR in lung cancer invasion.C5aR delayed A549apoptosis by phosphatidylinositol3-kinase-signaling pathway and promoted the release of MMP-2and MMP-9. C5aR only actived ERK and ERK pathway might participate in the process of C5aR induced EMT of lung cancer.