Nur77 Induces The Ubiquitin-mediated Degradation Of ID1

Nuclear receptor Nur77 is an orphan receptor encoded by an immediate early gene.It has complex and extensive biological functions in cancer development,including cancer cell proliferation,apoptosis,metabolism and metastasis as well as angiogenesis,which render it as an attractive drug target for cancer treatment.The dysregulation of inhibitor of differentiation(ID1)protein expression is closely related to cancer development,and manipulation of ID1 expression is an effective approach for the treatment of many diseases including of cancer.Here,we report that the nuclear receptor Nur77 could regulate the protein stability of ID1 in colon cancer cells independent of its transcriptional activity.We found that overexpression of Nur77 decreased ID1 protein level while downregulation of Nur77 increased ID 1 expression in colon cancer cells.Thus,the expressions of Nur77 and ID1 were inversely correlated.Our qRT-PCR results showed that the mRNA level of ID1 was not significantly affected by Nur77 expression,indicating that Nur77 post-transcriptionally regulated ID1 expression.Smurf2 is an E3 ligase that promotes ubiquitin-mediated degradation of ID1.Here,we found that both Nur77 and Smurf2 significantly shortened the biological half-life of ID1 protein,further indicating that Nur77 acted at ID1 protein level.We treated colon cancer cells with proteasomal inhibitor MG 132,PI3K/AKT pathway inhibitor Wortmannin and the autophagy inhibitor Chloroquine and Bafilomycin in HCT116 colon cancer cells to detect ID1 degradation pathway promoted by Nur77.The results showed that only the proteasomal inhibitor MG 132 prevented Nur77-induced ID1 degradation.In the mechanism study,we found that Nur77 in colon cancer cells bound to ID1,thereby enhancing the interaction between ID1 and Smurf2.Further studies showed that knockout of Nur77 in SW620 colon cancer cells significantly attenuated the ubiquitination of ED1.Thus,Nur77 promoted Smurf2 bound to and ubiquitinated ID1,leading to ID 1 degradation in a proteasomal pathway.Celastrol is a traditional natural medicine with prominent activities and extensive functions.It regulates various cell signaling pathways to exert its potent anti-inflammatory and anti-cancer activities.Our group previously reported that Nur77 binds to Celastrol.In this study,we found that Celastrol dramatically induced ID1 degradation in a Nur77 dependent manner.Ubiquitination assay showed that Celastrol also potently enhanced ID1 ubiquitination.Together,our results showed that Nur77 downregulated ID1 protein level through inducing Smurf2-mediated ID1 ubiquitination and degradation in colon cancer cells.We also revealed that Celastrol promoted ubiquitination and degradation of ID1 in a Nur77-dependent manner.Thus,our study suggested that Nur77 was of great potential as a drug target for treating colon cancers.