| Epithelial-mesenchymal transition process plays a critical role in tumorigenesis and distant metastasis through which tumor cells abandon their epithelial phenotypes and gain mesenchymal phenotypes.Undergoing this process,tumor cells become more invasive and act as cancer stem-like cells.Snail is a key regulator of epithelial-mesenchymal transition and plays an important role in tumor progression and metastasis.It facilitates epithelial-mesenchymal transition via transactivation of mesenchymal markers and transinhibition of epithelial markers.Hence it is very important to understand the mechanism that controlling the Snail expression.Snail is rapidly degraded in the cells and its protein level is critically controlled.The proteolysis of Snail is mediated by the ubiquitin-proteasome system.We are interested in the posttranslational regulation of Snail,especially the reversible ubiquitylation regulation.E3 ligases and deubiquitinating enzymes(DUBs)are responsible for the precise regulation of Snail deubiquitylation and degradation.So far,Several E3 ligases have been characterized to promote Snail ubiquitylation and degradation.However,its reverse process-deubiquitylation of Snail remains largely unknown.Besides,the epithelial-mesenchymal transition transcription factor Snail is associated with distant metastasis and poor prognosis of esophageal squamous cell carcinoma(ESCC)patients.It is noteworthy to identify specific deubiquitinating enzymes mediating Snail degradation in esophageal squamous cell carcinoma.Given the diversity and complexity of protein ubiquitylation regulation,we performed a mass spectrometry to examine the interaction between Snail and deubiquitinating enzyme(s).Subsequently,the deubiquitinating enzyme PSMD14 was identified to physically interact with Snail and target Snail for deubiquitylation and stabilization.PSMD14 overexpression dramatically extends the half-life of Snail and decreases its ubiqutylation in esophageal squamous cell carcinoma cells.Furthermore,knockdown of PSMD14 significantly blocks Snail-induced epithelial-mesenchymal transition and then suppresses tumor cell migration and invasion in vitro and tumor metastasis in vivo.In addition,the high expression level of PSMD14 predicts poor prognosis for esophageal cancer patients and there is a positive correlation between PSMD14 and Snail expression in clinical tissues.These findings suggest PSMD14 as a bona fide deubiquitinating enzyme to regulate Snail at the post-translational level and promote tumor metastasis in esophageal squamous cell carcinoma.On the other hand,we performed a deubiquitinating enzymes cDNA library screening and identified OTUB1 as a novel DUB that stabilizes Snail through preventing its ubiquitylation and proteasomal degradation.The interaction and co-localization of OTUB1 and Snail were then examined by co-immunoprecipitation assay and immunofluorescence assay.Functionally,OTUB1 facilitates epithelial-mesenchymal transition process and metastasis of esophageal squamous cell carcinoma through promoting Snail protein stability which depends on its catalytic activity.Moreover,OTUB1 is highly expressed in esophageal squamous cell carcinoma and correlated positively with Snail expression.Higher expression of OTUB1 also predicts poor prognosis.These findings suggest that OTUB1 is another essential regulator of Snail and plays a critical role in facilitating esophageal cancer progression.In summary,we identified PSMD14 and OTUB1 as novel deubiquitinating enzymes that directly and specificly target Snail for deubiquitylation and subsequently prevent its degradation through ubiquitin-proteasome system.We further discovered that PSMD14 and OTUB1 promote Snail-induced epithelial-mesenchymal transition,cell migration,and tumor metastasis both in vitro and in vivo.These results expand our understanding of the deubiquitylation modification of Snail and provide a promising therapeutic strategy against tumor metastasis of esophageal cancer. |
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