Wnt7a Activates Canonical Wnt Signaling,Promotes Bladder Cancer Cell Invasion

Urinary bladder cancer(UBC)is one of the most common malignancies in urological system and it ranks the eighth position in cancer-related mortality in men in USA.Low-grade,non-muscle-invasive tumors accounts for 75%of newly diagnosed UBC cases,among which 15-20%of these patients advance towards muscle invasive bladder cancers(MIBCs;≥stage T2).Unfortunately,Most of MIBC will develop to metastasis,with five-year survival<50%.So characterizing the molecular events that results in tumor invasion and metastasis is very important.The Wnt family,consisting of 19 secreted glycoproteins,regulates many important cellular processes,such as cell proliferation,differentiation and cell fate specification.Wnt family members bind to the Frizzleds(Fzds),which are members of the seven transmembrane family of proteins,and then their associated co-receptors stimulates complex network events,which are either dependent on canonical pathway(dependent on β-catenin)or non-canonical pathways(independent of β-catenin).Although it has been detected frequent genetic alterations of the Wnt pathway in different cancers,somatic mutations of Wnt pathway are seldom reported in UBCs,except the connection between single nucleotide polymorphisms of Wnt-associated genes and UBC risk.While combined Wnt and AKT pathway constitutively activating in murine urothelial cells synergistically results in UBC formation,indicting the significance of the Wnt pathway during bladder carcinogenesis.However,so far it is still unknown which components in Wnt pathway may play an important part in UBC invasion and metastasis.In this study,we generated two subpopulations of 5637 UBC cells,one with high invasiveness(5637 HMI)and the other with low invasiveness(5637 NMI).By proteomic analysis,we identified that Wnt7a protein expression is higher in 5637 HMI cells than that in 5637 NMI cells and we characterized that overexpressed Wnt7a in UBC predicts unfavorable prognosis.So we knocked down Wnt7a in 5637 HMI and T24 cells,which reduced UBC invasion and β-catenin protein expression and EMT related genes and ECM related genes expression.Besides,we used recombinant Wnt7a protein to treat 5637 NMI and J82 cells,which is contrary to former.Moreover,our results showed Wnt7a activated canonical β-catenin signaling in UBC cells and increased Wnt7a expression was associated with nuclear β-catenin in UBC samples by TOP/FOPflash luciferase assays.Wnt7a depletion suppressed matrix metalloproteinase 10(MMP10)expression,and Wnt7a overexpression increased MMP10 promoter activity through two TCF/LEF promoter sites,confirming that Wnt7a activates canonical Wnt/β-catenin pathway to induce MMP10 expression.Finally,we found both mRNA and protein expression levels of Wnt7a were significantly increased in UBC samples compared with adjacent normal tissues.Importantly,we found that UBC patients with high Wnt7a mRNA level have shorter overall survival time than those with low Wnt7a level from TCGA database.We confirmed that there was less cytosolic Wnt7a staining and strong membraneβ-catenin staining in the urothelium at earlier invasive stage,whereas there were stronger cytosolic Wnt7a staining and more nuclear staining of β-catenin in the invasive UBC section,which supporting the notion that Wnt7a overexpression is associated with the activation of Wnt/β-catenin pathway in UBC samples.In addition,MMP10 mRNA level was significantly increased in UBC tissues compared with normal tissues,and MMP10 mRNA level was also positively correlated with that of Wnt7a.Our results have identified an axis that controls UBC invasion through canonical Wnt/β-catenin signaling,which may offer prognostic and therapeutic opportunities.