Expression Of Tspan5 In GC And Mechanism Of Tspan5 Regulating Cell Proliferation In Gastric Carcinoma Cell Lines

Introduction:Gastric cancer (GC) is the fourth most common cancer and one of the most common causes of cancer-related death worldwide. A total of 989,600 new stomach cancer cases and 738,000 deaths are estimated to have occurred in 2008, accounting for 8% of the total estimated cancer cases and 10% of total estimated deaths globally. Specifically, the highest incidence rates are in Eastern Asia, Eastern Europe, and South America. Although developments have been made in diagnosis and treatment, the prognosis of GC patients remain poor because of recurrence, invasion and metastasis. Moreover, the tumor-node-metastasis (TNM) classification is the most widely used staging system for prognosis of gastric cancer patients, but it is still difficult to obtain complete prognostic information. Thus, a biomarker that enables the assessment and prediction of prognosis for GC patients is urgently required.Tspan5 (tetraspanin 5, NET-4, TMS4SF9) is a member of the tetraspanin superfamily. Tetraspanins are small proteins (20-50kDa) that are characterized by four conserved transmembrane (TM) domains. TM stretches delimit two extracellular loops, a small extracellular loop and a large extracellular loop, plus a very short stretch of intracellular amino-/carboxyl-terminal tails and a short intracellular loop. Tetraspanins can form complexes by interacting with other tetraspanins or associating with different transmembrane receptors, which are known as tetraspanin-enriched microdomains (TEMs). Thirty-three members of the tetraspanin superfamily have been identified in humans, but only a few of these have been studied in mammalian cells. Several studies on several tetraspanins including TSPAN1 (NET-1), TSPAN13 (NET-6), TSPAN24 (CD151), TSPAN27 (CD82), TSPAN28 (CD81), and TSPAN29 (CD9), revealed that they were upregulated or downregulated in some types of human cancers, implying that these tetraspanins may be involved in tumorigenesis and/or tumor progression. In contrast, little is known regarding the role of Tspan5 in tumor growth and metastasis.As we all know, the malignant tumor is characterized by unlimited proliferation that is closely related to cell cycle and apoptosis obstacle. Dysregulation of cell cycle progression is believed to play an important role in the tumorigenesis. A large number of studies have shown that the incontrolable cell cycle is responsible to the occurance and development of tumor. In this paper, we aimed to investigate the clinical and functional significance of Tspan5 in GC. Furthermore, we are interested in the relationgship between Tspan5 and the changes of cell cycle-related molecules in GC. And we hope to reveal the mechanism of tumor progression and develop a novel therapeutic method and a prognostic biomarker of the disease.Objectives:1 To evaluate Tspan5 expression in clinically gastric carcinoma tissues and analyze its relationship with development and progression of GC2 To clarify definitely biological role of Tspan5 in gastric cancer cells3 To study the effect and mechanisms of Tspan5 on cell-cycle regulation in gastric cancer cells in vitro and in vivoMethods:1 Immunohistochemical staining was used to evaluate Tspan5 expression in 114 cases clinically gastric carcinoma tissues and adjacent normal tissues, and analyze its relationship with development and progression of GC.2 Based on gene cloning techniques, the Tspan5 fragment was subcloned into the retroviral packaging plasmid plncx2-GFP-IRES. After identification by restriction enzymes digestion and DNA sequencing, plncx2-GFP-IRES/Tspan5 and pVSV-G were transfected into GP2-293 cells for retrovirus packaging. The AGS and MKN45 cells infected by aforementioned retrovirus to obtain stable recombinant cell lines with G418 selection. Then identification by western blotting, the recombinant cells were cultured and cryopreserved at liquid nitrogen.3 CCK-8 assay and colony formation assay were performed to estimate the role of Tspan5 on cell proliferation in vitro in gastric carcinoma cell lines (AGS and MKN45). Nude mice subcutaneously tumor forming assay was conducted to observe the tumor forming ability of Tspan5 in GC.4 Cell cycle assay was performed to investigate the effect of Tspan5 on cell-cycle regulation in AGS and MKN45 cells by flow cytometry analysis. Meanwhile, cell cycle proteins Cyclin D1, CDK4, p15, p27, E2F1 and pRB were detected by western blotting and immunohistochemical staining.5 Cell migration assays and Wound healing assays was used to analyze the change of migration ability of over-expressing Tspan5 in AGS and MKN45 cells compared with the control ones in vitro.Results:1 In 114 cases of clinical specimens, the result of immunohistochemical staining showed that Tspan5 was low-expressed in gastric cancer tissues and high expression in the adjacent tissues. Based on staining scores, we divided the cohort patients into low expression group (N=55) and high expression group (N=59) respectively to investigate if the expression level of Tspan5 is associated with clinicopathological features. Interestingly, decreased expression of Tspan5 was significantly associated with tumour size, tumour invasive depth, lymph node metastasis and TNM stage. In addition, low expression of TSPAN5 predicted poor survivals in patients with GC. Tspan5 acted as an independent favourable prognostic factor for GC2 Retroviral packaging vector plncx2-GFP-IRES/Tspan5 was successfully constructed, confirmed by restriction enzymes digestion, and sequencing, and could correctly package Tspan5 over-expressed retrovirus with pVSV-G in GP2-293 cells. The recombinant GC cells obtained from AGS and MKN45 cells infected by aforementioned retrovirus, were verified successful after identification by Western blotting.3 CCK-8 assay showed the proliferation of AGS and MKN45 recombinant cells with over-expressing Tspan5 were significantly suppressed compared with the control one. It was found by colony formation assay that over-expressing Tspan5 could inhibit clone forming in AGS and MKN45 recombinant cells. Nude mice subcutaneously tumor forming assay verified that Tspan5 functions as a tumor suppressor to inhibit tumour growth in vivo.4 We also further demonstrated that Tspan5 regulated cell cycle transition from G1-S phase by increasing p15/p27 and decreasing Cyclin D1/CDK4/pRB/E2F1 in vitro and in vivo.5 We reconstituted CDK4 in Tspan5-overexpressing tumour cells by transfecting the pCMV-Myc-CDK4 construct and confirmed the upregulation of CDK4 in tumor cells by Western blotting. After 48 hours, we assessed cell proliferation in vitro by using CCK-8 assays. For either AGS or MNK45 cell lines, the proliferation of Tspan5-CDK4 cells was significantly increased as compared to both Tspan5-Control and Tspan5 controls. We then reconstituted Cyclin Dl in Tspan5-overexpressing tumor cells by transfection with the pENTER-Cyclin Dl expression vector. Similarly, the proliferation of Tspan5-Cyclin D1 cells was significantly increased as compared to either Tspan5-Control or Tspan5 cells for both AGS and MKN4 cell lines. Together, the results suggest that reconstitution of either CDK4 or Cyclin D1 rescues the inhibitory phenotype produced by Tspan5, supporting further that Tspan5 suppresses the tumour growth of GC by control of the cell cycle transition from G1-S phase via decreasing the expression of CDK4 and Cyclin D1.6 Chamber migration (transwell) assays showed that Tspan5 dramatically inhibited the migration of AGS and MKN45 toward the bottom chamber. Consistent with the chamber results, similar results were also obtained from wound healing assays for either AGS or MKN45 cells. Thus, the results suggest that Tspan5 may act as a tumor suppressor to control tumour growth and progression of GC in vitro.Conclusion:Tspan5 is downregulated in tumour tissue and inversely correlated with clinicopathological features and overall survival of GC patients. Increased expression of Tspan5 serves as an independent favourable prognostic factor for predicting the outcome of GC patients. Tspan5 decreases cell proliferation and colony formation in vitro and suppresses the xenograft growth of GC in vivo. Thus, Tspan5 functions as a tumour suppressor in stomach to control the tumour growth of GC. Mechanistically, Tspan5 suppresses the tumour growth through regulating the cell cycle transition from G1-S phase by increasing the expression of p15 and p27 and decreasing the expression of Cyclin D1, CDK4, pRB and E2F1. Our findings supported the idea that Tspan5 may become a potential prognostic marker and may serve as a therapeutic target.