| With development of society and economy,morbidity and mortality of malignant tumor increase.Surgery,chemotherapy and radiotherapy are conventional reatments,which can not cure the most cancers,not mention to advanced stage of caners.The effects of conventional treatments are disappointed,and can not meet the needs of patients. Immunotherapy is the most possible to cure the tumors,especially advanced malignant tumor. Nowadays, Immunotherapy is the auxiliary to conventional treatments,because of limitation of specificity and numbers of tumor antigen,then,the most important thing is to find specific tumor antigen.Testes-specific protease50(TSP50) is a testis-specific gene that encodes a protein, which is homologous to serine proteases. Normally, TSP50protein is specifically expressed in the spermatocytes of testes, but abnormally activated and expressed in breast cancer. Currently, TSP50is considered as a member of cancer-testis antigens (CTAs), which include almost140members, such as Melanoma Antigen-Encoding Gene-1(MAGE-1), Cancer/testis antigen cancer-associated gene (CAGE), and Opa Interacting Protein5(OIP5), and so on. These proteins are expressed in various types of human cancers including gastric cancer and may serve as tumor markers for clinical prognosis or targets for therapeutic approaches. In this regard, MAGE-1protein is a predictive marker of poor prognosis in differentiated advanced gastric cancer patients Nakamura et al. revealed that OIP5might be a novel immunotherapy target for patients with gastric cancer. However, the expression of TSP50protein in gastric cancer and its diagnostic and/or prognostic significance has not been elucidated.In this study, the expression of TSP50protein was examined in a large number of human gastric cancer specimens and its clinicopathological and prognositc significance was also assessed.Methods:Immunohistochemistry (IHC) analysis of TSP50was performed on a tissue microarray (TMA) containing334primary gastric cancers and20adjacent non-tumor tissues. Western blot was carried out to confirm the expression of TSP50in3pairs of gastric cancers and adjacent non-tumor tissues.Results:IHC analysis revealed high expression of TSP50in57.2%human gastric cancer samples (191out of334). However, it was poorly expressed in all of the20adjacent non-tumor tissues. This was confirmed by western blot, which showed significantly higher levels of TSP50expression in gastric cancer tissues than adjacent non-tumor tissues. A significant association was found between high levels of TSP50and clinicopathological characteristics including junior age at surgery (P=0.001), later TNM stage (P=0.000), and present lymphnode metastases (P=0.003). The survival rate of gastric cancer patients with high expression of TSP50was significantly shorter than those with low levels of TSP50(P=0.021). Multivariate Cox regression analysis indicated that TSP50overexpression was an independent prognostic factor for gastric cancer patients (P=0.017).Conclusions:Our data demonstrate that elevated TSP50protein expression could be a potential predictor of poor prognosis in gastric cancer patients. Gastric cancer is the second most common cancer in the world, causing nearly one million deaths annually.Although the incidence of gastric cancer has decreased, it still remains among the leading causes of death from cancer in China. Despite major advances in diagnosis and treatment in the past few decades, gastric cancer remains a major clinical challenge.Prognostic factors for survival are useful in the management of gastric cancer. Many molecular markers, such as HER2, E-cadherin, Caveolinl, and so on, have been evaluated as candidate prognostic factors in gastric cancer. However, the prognosis for gastric cancer patients still stays poor and many prognostic factors which can effectively predict the prognosis of gastric cancer patients have not investigated.TMEM16A(transmembrane protein16A), also known as ANO1, DOG1or TAOS2, is an established sensitive biomarker for the diagnosis of gastrointestinal stromal tumors (GISTs). It is a member of the Anoctamin family of membrane proteins, which consists of ten components (known as TMEM16A-K or ANO1-10). Moreover, it has been proved to be a calcium-activated chloride channel, and contribute to many important physiologic functions. The coding sequence of TMEM16A is located within the11q13region, which is one of the most frequently amplified chromosomal regions in human cancers, such as head and neck squamous cell carcinoma, breast cancer and gastric cancer. Furthermore, TMEM16A protein is overexpressed in many tumor types including head and neck squamous cell carcinoma, esophageal cancers, breast cancer and prostate cancer, and it plays a vital role in the tumorigenesis and migration of some cancers. However, the expression of TMEM16A in gastric cancer, its prognostic significance and whether it is required for tumor development and migration has not been elucidated.In the present study, we examined the expression of TMEM16A protein in gastric cancer cell lines and tissues by Western blot analysis, and in gastric cancer tissues microarray(TMA) by immunohistochemistry.The clinical and prognostic significance of TMEM16A expression in gastric cancer was evaluated. Additionally, scratch wound assay and transwell matrix penetration assay are employed to assess the role of knockdown TMEM16A in the proliferation, migration and invasion of gastric cancer cell lines.Methods:Immunohistochemistry (IHC) analysis of TMEM16A was performed on a tissue microarray (TMA) containing367primary gastric cancers and20adjacent non-tumor tissues. Additionally,30matched lymphnode metastatic lesions and5whole-mount section tissues were recruited for this study. Western blot was carried out to confirm the expression of TMEM16A in4pairs of gastric cancers and adjacent non-tumor tissues.Furthermore, scratch wound assay and transwell matrix penetration assay are employed to assess the role of knockdown TMEM16A in the proliferation, migration and invasion of gastric cancer cell lines.Results:IHC analysis revealed strong expression of TMEM16A in66.4%human gastric cancer samples and lymphnode metastasis lesions,while weak expressed in adjacent non-tumor mucosal tissues.Consistent with this result, immunohistochemistry for whole-mount sention and western blot analysis displayed a similar finding in gastric cancer and adjacent non-tumor tissues. By univariate survival analyses using Kaplan-Meier method and log-rank test, the impact of TMEM16A on patient survival was analyzed and we found that elevated expression of TMEM16A was closely associated with poor overall survival in both testing set(P=0.022) and overall patients(P=0.018). Furthermore,scratch wound assay and transwell matrix penetration assay showed that silencing of TMEM16A can inhibit AGS cells to migrate and invade.Conclusions:Our data demonstrate that elevated TSP50protein expression could be a potential predictor of poor prognosis in gastric cancer patients. The most important features of malignant tumors are invasion and metastasis, which contribute to the majority of death because of cancer. Epithelial mesenchymal transitions(EMT) plays vital role in invasion and metastasis of tumors, which is represent of reduction or decrease of E-cadherin. In the previous study we found that TMEM16A is closely relationship with the invasion and metastasis of gastric cancer. To further investigate the underlying mechanism of TMEM16A in the invasion of gastric cell, the relationship between TMEM16A and E-Cadherin, a well-known marker regulating cadherin-based adhesion, was evaluated by immunohistochemistry and Western blot.Then, we speculated that whether or not TMEM16A and E-cadherin are relevant.First, E-Cadherin protein was detected by immunohistochemistry in gastric cancer TMA. High E-Cadherin expression was detected in34.1%(125of367) gastric cancer tissues according to the cutoff point generated by ROC curve analysis. Phi and Cramers V correlation analysis indicated that a significant negative correlation between expression of TMEM16A and E-Cadherin (r=-0.330, P=0.000). Moreover, similar negative relation was investigated in lymph node metastases lesions and adjacent non-tumor mucosa tissues.Then, we down-regulated TMEM16A expression in AGS cells transfected with TMEM16A shRNA for48hours, protein levels of E-Cadherin was significantly increased compare with the control group by Western blot analysis. |
PDF Full Text Request