TIP30 Inhibits Growth And Metastasis Of Bladder Cancer

Background and Objection:Bladder urothelial cancer(BUC)is one of the most common malignancies worldwide.74%of bladder cancers are superficial at the time of diagnosis[1]About 50%of superficial bladder cancers(SBCs)recur in 6-12months and about 5%-30%progress to muscle invasive cancer that has a 60-70%5-year mortality[15].So far,valuable predictors of progression from superficial to invasive disease are limited for patients with bladder cancer except tumor stage and grade.So it is critical to find new and more effective biomarkers for bladder cancer,not only for accurate evaluation of tumor recurrence and progression,but also as a target for anticancer therapy.The human gene TIP30,also known as CC3 or HITATIP2,was first found as a suppressor of variant small cell lung carcinoma(vSCLC)[2].It has been found that TIP30 can not only inhibite proliferation and promote apoptosis[3,13],TIP30 can also inhibit angiogenesis[14]and metastasis[4].It has been demonstrated that the expression of Tip30 is down-regulated in a variety of tumors such as vSCLC[2],neuroblastoma and glioblastoma[3,5,6],metastatic breast carcinoma[4],gastric carcinoma[8],hepatocellular cancer[9,10],colorectal cancer[7],lung cancer with poor prognosis[11]and pancreatic cancer[12].TIP30 was reported to suppress tumor metastasis in various cancer cells in vitro,including lung cancer,HCC,gastric cancer,breast cancer,and colorectal carcinoma[4,7,11,16,17].The suppressive effect of TIP30 on cancer metastasis was further demonstrated in nude mice.Inhibition of TIP30 expression significantly promoted lung metastasis and angiogenesis in nude mice.TIP30 shows a positive effect in inhibiting the cancer development and progression,but its role in BUC has not been investigated before.In this study,immunohistochemistry was performed on tissue microarray which contained BUC and normal bladder mucosa to investigate the expression of TIP30 in BUC and the relation between TIP30 and clinicopathological features.And there were no studies to investigate whether the TIP 30 could affect the biological behavior in bladder cancer cells.The aim of this study is to investigate the function of TIP30 in the bladder cancer cell lines.Methods and materials1.The correlation between TIP30 expression and clinicopathological characteristics Tissues from patients with BUC were retrospectively identified from Department of Pathology,the first affiliated hospital of Wenzhou Medical university in the period of 2004?2007.All patients who were treated with transurethral resection(TUR)of the bladder tumor or partial cystectomy and histopathologically confirmed to be BUC.A total of 79 BUC patients and 15 normal bladder mucosa specimens were included in this study.TIP30 expression was examined by immunohistochemistry.The relationship of TIP30 expression with gender,tumor size,tumor stage,and histological grade were analyzed.2.The correlation between TIP30 expression and prognosis All the patients were followed up.The relationship of TIP30 expression with survival were analyzed.3.The effect of Over-expression of TIP30 on the proliferation,migration,invasion and apoptosis in bladder cancer cell.We explored the TIP30 expression level of 3 common bladder cancer cell lines.Lenti-virus expressing corresponding plasmids were applied to up-regulate expression of TIP30 by infecting bladder cancer cells.We performed CCK method to study roles of TIP30 on the growth of bladder cancer cells in Vitro.transwell cell migration assay and Matrigel cell invasion assay were applied to study the role of TIP30 on metastasis of pancreatic cancer cells in Vitro.4.The relation between TIP30 expression and EGFR path way Western bolt was performed to study the the expressions of the apoptosis related protein(bax?bcl-2?caspase-3),cell cycle related protein(P21?cyclinD1?cyclinE)and migration related protein(MMP2,6,9)in infected bladder cancer cellsStatistic analysisThe differences between groups were evaluated by Student's t-test.P<0.05 was All statistical tests were two-tailed.Curves for disease-free survival(DFS),and overall survival(OS)were drawn by the Kaplan-Meier method,and differences in the survival rates were analyzed using the log-rank test.Prognostic factors were evaluated by univariate and multivariate analyses(Cox proportional hazard regression model).P values of less than.05 were considered as statistically significant.Results1.Immunohistochemical staining of TIP30 protein was identified in the cytoplasm of both normal mucosa and UBC specimen.TIP30 expression of UBC decreased significantly compared with normal mucosa(t=-6.909,P<0.01).2.Statistic difference between the low-grade(Grade ?)and high-grade(Grade ? and?)UBC was significant(t=9.197,p<0.05).3.The TIP30 expression in muscle-invasive stage was significantly lower than that in non-muscle-invasive stage(t=9.197,P<0.05)?4.The diffence of the expression of TIP30 in primary and recurrent bladder cancer was not significant(t=2.915,P=0.005).5.Patients with low TIP 30 expression would more easily progress than that with high TIP30 expression(t=2.288,P<0.05).6.No significant differences in TIP30 expression were observed regarding to age,gender,tumor number or tumor size of UBC patients(P>0.05).7.The overall survival(OS)of patients with low TIP30 expression was significantly lower than that of patients with high TIP30 protein expression(P<0.001).But the disease free survival(DFS)of the two group showed no significant difference(P =0.94).8.In the CCK8 assay,the proliferation of T24 cells was inhibited in the over-expression group.By over-expressing the TIP30 in the bladder cancer cells,compared with the control group,the cell cycle of T24 cell line were mostly in the GO and G1 phase.Cultured T24 cell lines were stained by APC/PI with over-expression of TIP30.We found that over-expression of TIP30 could significantly promote the apoptosis of the bladder cancer cells.The percentage of the APC/PI staining in the TIP30 group was 19.16%,while the NC group and control group was 6.28%and 6.89%.9.By over-expressing the TIP30 in the bladder cancer cells,compared with the control group,theEGFR,pEGFR,p-AKT,Bcl2,,cyclin D1,cyclin E cell cycle protein was down-regulated,suggested the TIP 30 may inhibit the cell proliferation in this pathway.Conclusion:1.Decreased expression of TIP30 might be involved in carcinogenesis of UBC.2.Reduced expression of TIP30 might be correlated with the progression and prognosis of UBC.3.Over-expression of TIP30 inhibited the proliferation,migration,invasion and promoted apoptosis in bladder cancer cells.4.TIP30 plays a role of tumor suppression through EGFR/Akt way.