The Regulalory Mechanisms Of The Ring Finger Protein RNF6 In Multiple Myeloma

Part Ⅰ:RNF6 promotes multiple myeloma cell proliferationObjective:RNF6 is a ubiquitin ligase of the ring finger families.Recent studies have found that RNF6 plays an important role in many cancer types,including prostate cancer,colorectal cancer,breast cancer and leukemia.However,its significance in multiple myeloma(MM),the second common hematological malignancy,has not been studied.In the preliminary study,we found that RNF6 is highly expressed in myeloma cells and it increases the protein level of glucocorticoid receptor GR,the receptor of dexamethasone(DEX),a major drug in the treatment of MM,but its specific functions and mechanisms are yet to know.Therefore,this part mainly focuses on the regulatory roles of RNF6 in MM cell proliferation in association with GR.Methods:1.The public database "Oncomine" was retrieved and anlysed for the expression of RNF6 in MM patients and cell lines.MTT colorimetry was used to evalute the proliferation of MM cells in the presence of RNF6.2.The correlation between RNF6 protein and the GR protein in MM cells was analyzed by Western Blot(WB).Meanwhile,MM cells treated with DEX,doxorubicin(DOX),and 5-amino-8-hydroxyquinoline(5AHQ)were subjected to analyze the protein level of RNF6 and GR.3.Lentiviruses carrying RNF6 or shRNF6(pLVX-RNF6 or shRNF6)was used to infect MM cell lines to overexpress or knock down RNF6,and Western Blot was used to analyze the changes of GR protein level in cells,or immunoprecipitation/immunoblotting(IP/IB)assays were used to analyze the effect of RNF6 on the ubiquitination levels of GR.4.The effect of RNF6 overexpression on the sublocalization of GR was analyzed following nuclear and plasma separation and Western Blot;The luciferase reporter assay was used to measure the influence of RNF6 on the GR transcriptional activity.RT-PCR and Western Blot were used to evaluate the mRNA levels and protein levels of GR downstream genes.5.The effects of RNF6 on the AKT/mTOR/GSK3β signaling pathway was detected by WB following plasmid transfection or lentiviral infection.6.The effect of DEX on the binding of RNF6 to GR was detected by co-immunoprecipitation(Co-IP);Western Blot and MTT assay were used to analyze the effects of RNF6 on the drug sensitivity of MM cells.Results:1.RNF6 was highly expressed in patients of MM compared with normal bone marrow samples based on the datasets retrieved from Oncomine.Moreover,its expression level gradually increased following the progression of MM.The high expression of RNF6 was confirmed in MM cell lines.When RNF6 was knocked down,MM cell proliferation was slowed down.2.There was a positive correlation between the expression levels of RNF6 and GR in MM cells.In the drug-inhibition assay,we found that DEX,DOX and 5AHQ down-regulated the protein levels of RNF6 and GR in a dose-dependent manner.3.RNF6 increased the endogenous GR protein level in MM cells.Morevover,it significantly prevents the decrease of GR protein induced by DEX.To our expection,RNF6 as a ubiquitin ligase dramatically modulates the ubiquitination levels of GR in both tool cells and MM cells.4.RNF6 promoted the nuclear translocalization of GR induced by DEX.Consistently,RNF6 promoted pGRE-luc activity in a dose-dependent and RING-domain-dependent manner,suggesting that the ubiquitin ligase activity is critical for RNF6 in its modulation on GR stability and function.In accordance with this finding,RNF6 up-regulated the mRNA levels of BCL2L1 and Mcl-1,two typical downstream genes of GR.Furthermore,the WB assay showed that RNF6 upregulated the protein levels of Bcl-xL and Mcl-1.5.To our surprise,RNF6 was found to up-regulate the p-Akt/p-mTOR/p-GSK3βprotein levels dependent on the presence of the RING domain.6.DEX interfered with the interaction between RNF6 and GR,while overexpression of RNF6 partially reduced the drug sensitivity of MM cells to dexamethasone.Summary:RNF6 is highly expressed in both primary MM patients and cell lines.RNF6 significantly promotes the proliferation of MM cells.RNF6 is positively correlated with GR expression in MM cells,both proteins could be downregulated by anti-MM drugs.RNF6 can stabilize the GR protein levels in MM cells,induces K63 ubiquitination of GR and promotes the nuclear localization of GR and up-regulates its transcriptional activity.Furthermore,RNF6 up-regulates the protein levels of p-AKT,p-mTOR,p-GSK3β,thus participating in the regulation of the AKT/mTOR signaling pathway.In addition,RNF6 can competitively bind GR protein with DEX,thus antagonizing DEX-induced apoptosis of MM cells.Part Ⅱ:The mechanism of the deubiquitinase USP 7 stabilizes RNF6Objective:Among the six components(Ub,E1,E2,E3,Proteasome and DUB)of the ubiquitin-proteasome system,the ubiquitination of most proteins depending on exogenous E3 ligase.But some proteins such as the RING finger family ligases could undergo autoubiquitination.This part will focus on(1)RNF6 ubiquitination and(2)identification of the deubiquitinase of RNF6.Methods:1.MM cell lines were treated with various inhibitors of proteolysis including chloroquine and bortezomib and MG132 to analyze the degradation pathway of RNF6 protein.The ubiquitination levels of RNF6 was measured by the IP/IB assays following plasmid transfections.2.The liquid chromotography(LC)/tandem mass spectrometry(MS/MS)was applied to identify the ubiquitination-association enzymes of RNF6.The IP/IB assays were used to analyze the interaction among RNF6,USP7 and USP9X.3.After plasmid transfection or lentivirus infection,IP/IB assays were used to analyze the ubiquitination level of RNF6 in the presence of USP7.Deletion mutation was used to construct the truncates of USP7.Co-transfection was used to analyze the effects of USP7 truncates on the ubiquitination levels of RNF6 and to identify the key functional domains of USP7.4.WB assay was used to detect RNF6 protein level in the presence of USP7.A CHX chase assay was applied to measure the on the half-life of RNF6 in the presence of USP7.5.The USP7 small-molecule inhibitor P5091 was used to verify the effects of USP7 on RNF6 ubiquitinaiton,stability,as well as MM cell proliferation and apoptosis.Results:1.The RNF6 protein levels were markedly increased by the proteosomal inhibitors bortezomib and MG 132 in a dose-dependent manner but not by the lysosomal inhibitor chloroquine.Moreover,MG132 significantly increased the ubiquitination levels of RNF6,which indicates that RNF6 is regulated by the ubiquitin-proteasomal pathway.Furthermore,the full-length RNF6 protein could ubiquitinate the RING domain deficient protein(ΔRING);On the contrary,when the RING domain was deleted,the ubiquitination ability of RNF6 was lost.These findings suggested that RNF6 may undergo autoubiquitination and the RING domain is critical for this activity.2.By using the LC-MS/MS strategy,we found that two deubiquitinases,USP7 and USP9X,were present in both the RNF6 and RNF6ARING immunoprecipitates.Subsequent cellular assays found that RNF6,USP7 and USP9X co-existed in the Co-IP in HEK293T cells.Furthermore,endogenous binding of USP9X and RNF6 was detected in MM cells after immunoprecipition of endogenous USP7 protein.These findings indicated that USP7/USP9X may collaborate in modulating RNF6 ubiquitinaiton and stability.3.USP7 significantly inhibited the ubiquitination levels of RNF6 in a dose-dependent manner.Moreover,USP7 significantly inhibited the K48-chain ubiquitinaiton of RNF6 at both endogenous and exogenous contexts.Furthermore,the TRAF domain was critical for USP7 to prevent the ubiquitination of RNF6 after the assays in the co-transfections of RNF6 and a series of USP7 truncates.4.USP7 up-regulated the protein level of RNF6 in a time-dependent and dose-dependent manner,but had no effect on its mRNA expression.Moreover,the CHX chase assay showed that overexpression of USP7 significantly inhibited the degradation rate of RNF6.All these findings thus collectively suggest that USP7 stabilizes RNF6 by preventing its ubiquitination and degradation.5.The small molecule inhibitor of USP7,P5091,significantly inhibited the protein level of RNF6 in a dose-dependent manner.Consistent with this finding,P5091 can significantly increase the K48-chain ubiquitination levels of endogenous RNF6 in MM cells.Furthermore,P5091 accelerated the degradation rate of RNF6 as shown in the CHX chase assaysSummary:RNF6 protein is regulated by the ubiquitin-proteasome pathway.As a RING type E3 ubiquitin ligase,RNF6 can undergo auto-ubiquitination dependent on the RING domain.The LC-MS/MS strategy identified that USP7 interacts with RNF6.As a deubiquitinase,USP7 prevents the K48-linked ubiquitination and degradation of RNF6.Consistent with this finding,inhibition of USP7 by a small molecule compound significantly induced the K48-chain ubiquitination levels of RNF6,thus leading to RNF6 degradation and MM cell apoptosis.ConclusionsRNF6 is a member of the RING type E3 ubiquitin ligases,which can promote the malignant proliferation of a variety of tumors by modulating specific substrate proteins ubiquitination and stability,but its roles in MM have not been reported.In the present study,we found that:(1)RNF6 is highly expressed in MM cells and promotes MM cell proliferation.(2)RNF6 binds to GR and mediates its K63-chain ubiquitination that leads to stabilized GR.(3)RNF6 increased GR transcriptional activity and increased the expression of prosurvival genes of Bcl-xL and Mcl-1,which confers to MM cell resistance to DEX.(4)RNF6 undergoes autoubiquitination dependent on its RING domain.(5)The LC-MS/MS identified that USP7 interacts with RNF6.(6)RNF6 ubiquitination could be prevented by the deubiquitinase USP7.(7)Inhibition of USP7 leads to RNF6 degradation and MM cell apoptosis.(8)The USP7/RNF6 axis could be a novel drug target for the treatment of MM...