| Epithelial-mesenchymal transition(EMT)is a process that epithelioid cells lose their differentiated epithelial-like properties and gain a mesenchymal-like phenotype,and is an essential program in the process of metastasis.As an EMT-transcription factor(EMT-TF).the well-studied function of Snail is inducing EMT progress.In many types of human cancers,Snail is found overexpressed and associated with poor prognosis and distant metastasis of cancer patients.Snail is a liable protein and degraded by ubiquitin-proteasome system.There are various E3 ligases mediating its degradation but the deubiquitinating enzyme reversed Snail degradation is not fully understood.In this study,we found USP26 is a specific deubiquitinase of Snail which could significantly increase Snail stability,and USP26 specifically interacts with Snail.USP26 binds to the Snail zinc finger domain,a region which is necessary for Snail stability and nuclear localization.USP26 interacted with Snail via its USP domain which is a conservative domain of USPs and where the catalytic sites located.Moreover,USP26 could maintain the stability of Snail by decreasing its ubiquitination.In addition,USP26-overexpressing ESCC cells showed decreased expression of epithelial marker E-cadherin and increased protein levels of mesenchymal labels N-cadherin and Vimentin,and had a stronger ability of migration and invasion.Knockdown of Snail reversed the EMT process and the effect of USP26 overexpression on promoting metastasis of ESCC cells.These observations further proved that USP26 enhanced metastasis of ESCC cells through stabilization of Snail.Finally,we examined the expression of USP26 and Snail in clinical ESCC and adjacent normal esophageal epithelial tissues.We observed a higher expression of USP26 in ESCC tissues than the normal tissues,and there was a strong positive correlation between USP26 and Snail in ESCC samples.Thus,our results suggested that USP26 performed oncogenic role in ESCC and positive role in the regulation of Snail.In summary,our study demonstrates a vital role for USP26 in ESCC metastasis by mediating Snail stability.And the accumulated Snail enhances ESCC metastasis through inducing EMT.In addition,our study provides a rationale and potential target for the treatment of human ESCC.Beta-transducin repeats-containing protein(?-TrCP)also known as BTRC and FBXW1 A,is a well-studied E3 ubiquitin ligase belonging to the Skp-Cullin-F-box(SCF)family.?-TrCP plays many critical roles in various biological processes,such as cell growth,apoptosis,migration and cell cycle,and is related with tumorigenesis,tumor metastasis and development.But in different tissue-or cellular contexts,?-TrCP plays the opposite role as a tumor suppressor or oncogene.As an E3 ubiquitin ligase,?-TrCP is also reported to be a liable protein and regulated by other E3 ligases,but the deubiquitinating enzyme reversed ?-TrCP degradation is not fully understood.In this study,we found OTUD6B is a specific deubiquitinase of ?-TrCP which could significantly increase ?-TrCP stability,and OTUD6B specifically interacts with ?-TrCP.Moreover,OTUD6B could maintain the stability of ?-TrCP by decreasing its ubiquitination.In the functional study of OTUD6B,we found Snail combined with OTUD6B and is regulated by it.Moreover,OTUD6B-overexpressing ESCC cells showed increased expression of epithelial label E-cadherin and decreased levels of mesenchymal markers Snail,N-cadherin and Vimentin.In addition,we demonstrated overexpression of OTUD6B inhibits ESCC cell migration and invasion by upregulating ?-TrCP expression.And overexpression of OTUD6B could inhibit the ability of tumorigenesis and chemoresistance in ESCC cells,decrease the expression of stemness-associated genes SOX2,NANOG,OCT4 and CK14 and increase the expression of differentiation marker CK13.Thus,we identified that OTUD6B inhibits the cancer TIC properties and metastasis of ESCC cells by suppressing the EMT process.Because of the inhibitory effect of ATRA on TIC properties and chemoresistance in various solid tumors,we detected the function of ATRA in ESCC cells.We identified ATRA represses ESCC metastasis and tumorigenesis by upregulating the protein level of OTUD6B.In addition,we found that RARa can combine with OTUD6B mRNA and inhibit its translation process.ATRA released RAR? from OTUD6B mRNA inducing its protein expression.Finally,the protein levels of OTUD6B and ?-TrCP were markedly reduced in ESCC specimens compared with the adjacent tissues,and OTUD6B protein level correlated with the ?-TrCP expression in ESCC tissues.In addition,the patients with relatively low expression of OTUD6B often bore large tumors,and had poor survival.In summary,our study demonstrates a critical regulatory mechanism of ?-TrCP in ESCC and reveals that OTUD6B is a novel DUB for ?-TrCP.Loss of OTUD6B expression promotes ESCC cell metastasis,tumorigenesis and chemoresistance.We also provide evidence that ATRA blocks ESCC cell metastasis and eliminates its TICs by targeting OTUD6B.Our results provide a potential target for the therapeutic treatment of ESCC. |
PDF Full Text Request