Clinical Significance And Mechanistic Studies Of GPC5on Invasion And Metastasis Of Non-Small Cell Lung Cancer

Background and Objective:Non-small cell lung cancer (NSCLC) is one of the most malignant and highly metastatic cancers worldwide, which has high morbidity and mortality. To date, although a large amount of research has covered the mechanism of invasion and metastasis of lung cancer, the molecular mechanism in each step is still not fully elucidated. Therefore, it is important to search novel gene and molecule associated with lung cancer invasion and metastasis, find its correlation with lung cancer metastasis and prognosis and elucidate its molecular mechanism in lung cancer metastasis to develop more effective diagnosis and treatment modalities to improve the survival rate in lung cancer patients.Glypican5(GPC5) is a member of glypicans (GPCs), and might play an important role in regulating cancer progression. Alterations at the GPC5locus are a common event in lung cancer and other tumor types, including lymphomas, breast cancers, and neurological tumors. More recently, genetic variations at13q31.3have been reported to modulate GPC5expression and may contribute to the development of lung cancer in never smokers. GPC5may be a potential lung cancer suppressor gene. However, thus far, no studies have fully elucidated the clinical features and potential functions of GPC5in NSCLC patients. So there is a practical significance on the study of GPC5differential expression and biological function. It will contribute to a better understanding of lung cancer development, invasion and metastasis. In the present study, we aimed to investigate the characteristics of GPC5expression in NSCLC patients and the relationship between GPC5expression and patient diagnosis and prognosis, study the relationship between GPC5expression with inhibition of NSCLC invasion and metastasis in vitro and in vivo and preliminary explore the mechanism that GPC5inhibits invasion and metastasis in NSCLC.Methods:GPC5, E-cadherin and MMP-9(matrix metal proteinase-9) mRNA expression was examined by real-time PCR in198paired freshly resected NSCLC specimens and their adjacent normal lung tissues which were collected from the Department of Thoracic Surgery at the Cancer Hospital of Jiangsu Province. The data were further analyzed according to clinicopathologic characteristics and GPC5immunohistochemical (IHC) staining. We determined the GPC5expression in a normal human bronchial epithelial cell line (BEAS-2B) and NSCLC cell lines (SK-MES-1, A549, H1650, H1975, and SPC-A-1) by real-time RT-PCR and western blot analyses. We chose three human lung cancer cell lines to construct the GPC5-overexpressing cell lines induced by lentivirus. The A549and H1975cells are human lung AC cell lines. The A549cells came from a smoker, while the H1975cells came from a non-smoker. The third cell line was SK-MES-1, which is a human lung SCC cell line. Lentivirus-induced GPC5overexpression was used in NSCLC cell lines for in vitro analyses of functions including migration, invasion, and proliferation by a wound healing assay, a transwell invasion assay and CCK-8proliferation assay, as well as the cell cycle by PI-staining cell cycle analysis through flow cytometry. A wound healing assay and a transwell invasion assay were performed to evaluate the migration and invasion capacities of the three cells lines. The expression level of the well-known metastasis suppressor gene E-cadherin and the metastasis-related gene MMP9were detected by real time PCR in three lentivirus-induced GPC5overexpression cells and normal cells. We injected A549cells or GPC5-A549cells through the tail vein of nude mice to establish the passive hepatic metastasis model of lung cancer cells in nude mice in vivo, and studied metastatic ability of A549cells influenced by the GPC5in vivo through this model. The expression levels of E-cadherin、Snail、Slug, Twist�'�N-cadherin were compared between A549cells transfected with GPC5siRNA plasmids and A549cells transfected with empty vectors in vitro and in vivo. A transwell invasion assay were performed to compare metastatic ability of GPC5-A549transfected with Twist siRNA plasmids, A549cells transfected with GPC5siRNA plasmids and A549cells transfected with empty vectors.Results:In general, we found that the mean normalized GPC5mRNA expression was lower in both the SCC and AC tissues than that in the matched adjacent normal lung tissues (average decrease was18.33-and8.75-fold, respectively, p<0.001), which indicated that GPC5might be a NSCLC suppressor gene. Additionally, GPC5expression in the SCC subgroup was also significantly lower than that in the AC subgroup (p<0.001).GPC5expression in the tumor tissues in the Tla,b group was significantly higher than that in the T2a,b group (p=0.046), although no statistical significance was found between the T2a,b and T3,4groups. More importantly, GPC5expression in the lymph node metastasis group (stage N1-2) was remarkably lower than that in the non-metastasis group (stage NO)(p=0.002). Similar differences were also observed in both the AC and SCC sub groups (p=0.011and p=0.032, respectively).Lower GPC5mRNA expression was also correlated with poorer pathologic differentiation (p<0.001). However, there was no significant difference in the GPC5expression between smokers and non-smokers (p=0.054). In addition, we divided these patients into four sub groups:SCC in smokers (SCC/S), AC in non-smokers (AC/Non), AC in smokers (AC/S), and SCC in non-smokers (SCC/Non). We found that GPC5expression in the SCC/S group was significantly lower than that in the AC/Non group (p=0.013).Both GPC5mRNA and protein expression levels in BEAS-2B were higher than those in the NSCLC cell lines, especially in the SK-MES-1, H1975, and SPC-A-1cell lines.All three cell lines that overexpressed GPC5had remarkably suppressed migrating length and invasion rates (p<0.001). In addition, GPC5overexpression altered the expression level of the well-known metastasis suppressor gene E-cadherin and the metastasis-related gene MMP9. Compared with negative control cells, GPC5-overexpressing cells had upregulated E-cadherin and MMP9expression levels.When compared with negative control cells, the GPC5-overexpressing cells proliferated at a significantly lower rate. The cell cycle analysis indicated that the GPC5-overexpressing AC cell lines (A549and H1975) had a larger G1phase population increase than their respective non-treated control cells (76.37%vs.62.55%and80.39%vs.57.03%, respectively). The GPC5-SK-MES-1SCC cells had a larger S phase population increase than the non-treated control cells (51%vs.31.5%). Altogether, GPC5overexpression significantly suppresses NSCLC cell line proliferation as well as induces cell cycle arrest in the G1or S phase.We established the passive metastasis model of lung cancer cells in nude mice in vivo, and found that GPC5could inhibit metastasis of lung cancer. As compared with the NC-A549group, the metastatic nodes in livers were significantly lower in GPC5-A549group. The survival rate of nude mice injected with overexpression GPC5in A549cells by tail vein was larger than that injected with NC-A549cells (negative control A549cells, A549cells transfected with empty vector). A log-rank test showed significant difference between NC-A549group and GPC5-A549groups (p<0.01). The metastatic ability of A549cells was inhibited obviously by overexpression GPC5.Both E-cadherin protein and mRNA expression levels were significantly up-regulated while the protein and mRNA expressions levels of N-cadherin, Snail, Twist and Slug was down-regulated in A549cells with overexpression GPC5compared with the NC-A549cells in vitro and in vivo, but there is no statistically significant in the mRNA expression level of Slug (p>0.05). Our rescue assay showed that overexpreess GPC5could inhibit A549cells migration, and the metastatic ability of A549cells with overexpression GPC5was counterbalanced by overexpression Twist.Conclusions:The present study suggests that the expression level of GPC5is significantly downregulated in NSCLC, and that a low expression level indicates the possibility of lymph node metastasis. Both in vivo and in vitro analyses show that a downregulation of GPC5is associated with metastasis of NSCLC. GPC5inhibit metastasis of NSCLC by inhibiting Twist-mediated epithelial mesenchymal transition (EMT) process. Thus, GPC5can be a novel metastasis suppressor gene in NSCLC and may be a potential biomarker that predicts NSCLC metastasis and survival.