Ubiquitination And Proteasomal Regulation Of Tumor Suppressor Gene EAF2/U19 Protein Stability And Its Effect On Apoptosis In Prostate Cancer Cells

Prostate cancer(PCa)is one of common cancers in middle and aged men and being a serious threat to the healthy of men.The incidence of prostate cancer was the second one in all male malignant tumors(the first was lung cancer)and representing the number of 14%in all male cancer patients.The incidence rate of prostate cancer ranked first of male malignancy in more than past 10 consecutive years in American and European countries and the mortality ranked second.The incidence of prostate cancer ranks first in all American male malignancies,accounting for 29%of the incidence in all male cancer.The incidence of prostate cancer grows rapidly in China in recent years.Prostate cancer is being the cancer with the highest incidence in male genitourinary cancer in Chinese men.World Health Organization classification scheme classified prostate cancer into androgen-dependent prostate cancer and androgen-independent prostate cancer based on the state of androgen blood circulation.Hormone-independent prostate cancer includes androgen-independent prostate cancer(AIPC)and hormone-refractory prostate cancer(HRPC).Hormone-independent prostate cancer is still the difficulty of the current clinical treatment.For the etiology of prostate cancer,the prevailing view is that the formation and development of the cancer is related to activation of oncogenes and inactivation or deletion of tumor suppressor genes.Tumor suppressor genes also known as anti-cancer genes are a class genes which can inhibit cancer cells proliferation,invasion and metastasis.These genes products could be found in normal cells,have different function to oncogene,play an important role in cell development,regulation of cell growth and differentiation.Under some unnormal situation,Tumor suppressor genes are suppressed or lost and their tumor suppressor roles would be weaken or even eliminated,being an important factors of cancer development and one maker of prognosis.Prostate cancer(PCa)is one of the common cancers in middle and aged men which is a serious threat to the health of men.The incidence of prostate cancer was the second one in all male malignant tumors(the first one was lung cancer)and representing the number of 14%in all male cancer patients.The incidence rate of prostate cancer ranked first of male malignancy in more than past 10 consecutive years in American and European countries and the mortality ranked second.The incidence of prostate cancer ranks first in all American male malignancies,accounting for 29%of the incidence in all male cancer.The incidence of prostate cancer grows rapidly in China in recent years.Prostate cancer is being the cancer with the highest incidence in male genitourinary cancer in Chinese men.World Health Organization classification scheme classified prostate cancer into androgen-dependent prostate cancer and androgen-independent prostate cancer based on the state of androgen in blood circulation.Hormone-independent prostate cancer includes androgen-independent prostate cancer(AIPC)and hormone-refractory prostate cancer(HRPC).Hormone-independent prostate cancer is still the difficulty of current clinical treatment.For the etiology of prostate cancer,the prevailing view is that the formation and development of the cancer is related to activation of oncogenes and inactivation or deletion of tumor suppressor genes.Tumor suppressor genes also known as anti-cancer genes are a class of genes which can inhibit cancer cells proliferation,invasion and metastasis.These genes products could be found in normal cells,which have different function to oncogene,play an important role in cell development,regulation of cell growth and differentiation.Under some unnormal situation,tumor suppressor genes are being suppressed or lost,then their tumor suppressor roles would be weaken or even eliminated.It was an important factor of cancer development and a maker of prognosis of cancer patients.Protein ubiquitination is a common form of post-translational modifications of protein.Substrate could be modified into ubiquitinated protein and then be degraded,thereby regulating signal response.Ubiquitin-dependent protein degradation pathway can selectively degrade proteins within the cells.It is an important non-lysosomal protein degradation pathway.More and more evidences showed that the ubiquitin proteasome signaling pathway disorders was associated with tumorigenesis.The development of cancer usually associated with the unnormal expression of oncogenes or tumor suppressor gene.Some cancers resulted from the stability of oncogene protein and growth factor or instability of the tumor suppressor gene products.Some oncogenes or tumor suppressor genes products usually were degraded by the proteasome.If an exception occurs during the degradation process,it might induce malignant transformation.Some unstable tumor suppressor genes protein,such as P27 had been shown to closely correlate with protein ubiquitin proteasome system and degradation.Deubiquitination might stabilize tumor suppressor genes protein,thus inhibiting tumorigenesis.Understanding ubiquitin proteasome pathway and deubiquitination process played an important role in tumor development had opened new areas and ideas for the prevention and treatment of cancers.Human up-regulated gene 19(U19)/ELL-associated factor 2(EAF2)is an androgen up-regulated gene in the rat prostate and as a binding partner for eleven-nineteen lysine-rich leukemia(ELL),making it the second member of the EAF family of proteins.EAF2 shows significant sequence homology to the original family member,EAF1.EAF2 was a potential tumor suppressor.Advanced human prostate cancer specimens displayed EAF2 down-regulation,allelic loss,promoter hypermethylation,and possibly homozygous deletion.Transfected EAF2 to prostate cancer cell lines induced apoptosis and inhibited the growth of xenograft prostate tumor models.EAF2/U19 inhibited xenograft prostate tumor growth and was down regulated in prostate cancer cell lines.Moreover,a high frequency of loss of heterozygosity(LOH,82.6%)and homozygous deletion(8%)was noted in high-grade human prostate cancers.These findings linked EAF2/U19 with two major human cancers—prostate cancer and acute myeloid leukemia.EAF2 knockout mice developed lung adenocarcinoma,hepatocellular carcinoma,B-cell lymphoma,and high-grade prostatic intraepithelial neoplasia(PIN),confirming its tumor suppressive activity under physiological conditions.EAF2 played an important role in prostate cancer occurrence and development as tumor suppressor genes.Did EAF2 protein degrade via the ubiquitin proteasome system?If EAF2 protein degraded via the ubiquitin proteasome system,which lysines were its ubiquitination sites located on EAF2 protein?Did other proteins affect EAF2 protein stability?Did EAF2 protein stability affect its tumor suppressor gene function in prostate cancer?How did EAF2 protein degrade and be ubiqtuitinated in prostate cancer?Did EAF2 protein level relate to prognosis of prostate cancer patients?Many questions about the relationship between EAF2 protein and prostate cancer need to be solved.This study addressed the relationship between the ubiquitin proteasome degradation pathway of EAF2 protein and cell apoptosis,as well as EAF2 ubiquitin proteasome pathway in prostate cancer.According to the preliminary result of our lab,EAF2 protein was induced and up-regulated to an expression peak by androgen and then the protein level subsequently decreased.These results showed that EAF2 protein almost could not be detected without treatment with R1881 in LNCaP cells.But when LNCaP cells were treated with R1881 for 24 hours,EAF2 protein would be induced and then it would degrade in 96 hours.C4-2 cells had basic expression of EAF2 protein.When C4-2 cells were treated with R1881 for 24 hours,EAF2 protein would be significantly up-regulated and then it degraded in 72 hours.EAF2 protein could be induced by R1881 in LNCaP and C4-2 cells and then its protein level decrease after 24 hours.These results showed that EAF2 was an unstable protein.LNCaP and C4-2 cells were treated with R1881 for 24 hours then treated with proteasome inhibitor MG132 and CHX for 18 hours,EAF2 protein almost degraded in the CHX group when new protein production was inhibited.But EAF2 protein level was stable when the cells treated with MG 132 and CHX when proteasome pathway was inhibited by MG 132.LNCaP cells were treated with R1881 for 24 hours then treated with MG 132 for 18 hours,EAF2 protein level increased significantly compared with untreated group.EAF2 protein level increasing as MG 132 concentration increasing showed that EAF2 protein degradation pathway was inhibited resulting in protein degradation decreased and protein level increased.Endogenous EAF2 protein level decreased to about 1/10 in 24 hours and almost degraded in 48 hours when C4-2 cells were treated with CHX to inhibit new protein production.These results showed that EAF2 protein degradation was related with proteasome degradation pathway.Previous results indicating EAF2 protein degradation was closely related with proteasome pathway,in vivo ubiquitination co-immunoprecipitation was used to verify if EAF2 protein was ubiquitinated.HEK293 cells and prostate cancer C4-2 cells,pCMV-myc-EAF2 and pEGFP-EAF2 plasmids were used in this experiment.According to the co-IP results,myc-EAF2 and GFP-EAF2 showed significant diffusion bands which indicated the ubiquitination of EAF2 protein after MG132 treatment in the presence of Ub plasmids in HEK293 cells.This also happened for myc-EAF2 in C4-2 cells after MG132 treatment.These results indicated the different labels had no significant effect on EAF2 protein ubiquitination.Also,EAF2 protein ubiquitination was happened not only in prostate cancer C4-2 cell lines,but also in non-prostate cancer cells,HEK293 cells.There are 17 lysines on EAF2 protein.Since it was still very difficult to know the exact ubiquitination sites of a protein using the prediction software,we mutated each single lysine site of EAF2 to test its function.The single point mutation of each lysine to arginine of EAF2 was mutated by using QuikChangeTM II XL Site-Directed Mutagenesis Kit.Totally,we constructed 17 lysine-to-arginine single-point mutant plasmids on myc-EAF2 plasmid.To test whether the single point mutation(K-R)will influent the stability of the protein EAF2,myc-EAF2-WT and each mutation plasmids were transfected into the C4-2 cells.MG132 was given to the cells after 24 hours and cells were collected for future test after 18 hours.Compared to the untreated group,EAF2 level was significantly increased after MG 132 treatment in myc-EAF2-WT transfected cells.While for the cells transfected with EAF2-K39R,EAF2-K81R,EAF2-K85R and EAF2-K111R single point mutation plasmids,MG 132 treatment did not influent myc-EAF2 protein levels compared with cells transfected with myc-EAF2-WT plasmid.These results indicated us that the mutation of these lysine sites resulted in the decrease of EAF2 protein ubiquitination.That means these sites had close relationship with EAF2 protein ubiquitination.Further we constructed myc-EAF2-K39-85-111R,myc-EAF2-K39-81-111R and myc-EAF2-K39-81-85-111R multi-point mutant plasmids.CHX treatment experiments showed most of myc-EAF2-WT protein was degraded in 20 hours,while the half-life of single point mutation myc-EAF2-K81R protein was prolonged and the protein still maintained in a certain level even at 40 hours.Compared to WT,the three mutations myc-EAF2-K39-85-111R protein had longer half-life but the degradation rate was almost consistent with K81R,illustrating the effect of the three point mutations on the EAF2 protein degradation was not much different with K81R single point mutation.Compared to K81R and K39-85-111R mutations,mutant protein K39-81-85-111R EAF2 had longer half-life.Thus,in these four ubiquitination site mutant amino acid residues,K81R had the greatest impact on the EAF2 degradation rate.We further tested the mutate EAF2 protein degradation levels by co-IP experiment.Mutant EAF2 protein ubiquitination level were decreased differently compared with WT EAF2 protein when 39,81,85,111 lysines amino acid residues were mutated.Single point mutation K81R EAF2 protein ubiquitination level decreased significantly compared with WT EAF2.Multi-point mutation EAF2-K39-81-111R and EAF2-K39-81-85-111R could be detected in very small amounts EAF2 ubiquitinated proteins,indicating that multi-point mutation EAF2 protein ubiquitination almost being suppressed.Siahl,member of Siah family,had been identified as E3 ligase of ELL2,a member of SEC complex,by prof.Qiang Zhou’s Lab.Siah family have two members,Siah1 and Siah2.We speculated Siah1 or Siah2 might be involved EAF2 protein degradation since the importance of EAF2 protein in SEC complex.The results showed that EAF2 protein level decreased significantly when EAF2 was co-expressed with Siah1 or Siah2,especially with Siah2.This indicated that EAF2 protein stability would decrease when Siah2 protein exited.EAF2 protein level was increased significantly when EAF2 co-expressed with Siah2 for 24 hours and then treated with proteasome inhibitor MG 132,indicating MG 132 could block the effect of Siah2 to the degradation of EAF2 protein.Co-IP was used to verify whether there was a direct interaction in siah2 and EAF2 protein and provide further evidence of subsequent verification siah2 protein as E3 ligase of EAF2.IP results showed that only HA-Siah2 could pull down EAF2 protein but not HA-Siahl when HA-Siah2 or HA-siahl were co-expressed with EAF2,indicating Siah2 protein had direct interaction with EAF2.To further determine whether siah2 was related with EAF2 protein ubiquitination process,ubiquitination co-IP was used to observe whether Siah2 cloud change EAF2 protein ubiqutination level when Siah2 was co-expressed with EAF2.The short exposure time result showed that siahl and Siah2 could enhance the level of EAF2 protein ubiquitination in MG132 treatment group,while Siah2 effect was stronger than siah1.The long exposure time result showed that Siahl and Siah2 could enhance EAF2 protein ubiquitination level in MG 132 untreated group,while siah2 effect was stronger than siahl,indicating that Siah2 still significantly enhanced EAF2 protein ubiquitination level even without the accumulation effect of MG132 when siah2 was co-expressed with EAF2.In addition,the Co-IP results showed that only HA-siah2 protein could be pulled down by GFP-EAF2 protein,and no obvious band could be found when Siahl was co-expressed with EAF2.These results indicated that EAF2 and siah2 had direct interaction from other side.All these results indicated that siah2 could increase EAF2 protein ubiquitination level and siah2 and EAF2 had direct interaction with each other.Further experiments would be done to verify if siah2 was the E3 ligase of EAF2.Transcription elongation complex(SEC)composed of ELL 1/2/3,EAF1/2,AFF1/2/3/4 and others proteins,which played an important role in RNA transcription and replication process.EAF2,as an important protein of SEC,its protein stability was important to SEC.Previous studies showed that EAF2 protein could be stabilized by ELL1.Did ELL2 have same effect on EAF2 protein stability?Why ELL1 could stabilize EAF2 protein?When ELL1 or ELL2 was co-expressed with EAF2 for 24 hours and then treated with MG 132 for 18 hours,results showed that EAF2 protein almost degraded in 18 hours when EAF2 expressed alone,but EAF2 protein stability was increased significantly and there was almost no difference between the groups of treated with or without MG132 when ELL1 was co-expressed with EAF2.But EAF2 protein stability didn’t change significantly when ELL2 was co-expressed with EAF2.From the above results showed EAF2 protein stability was increased significantly and EAF2 protein degradation pathway was inhibited when ELL1 was co-expressed with EAF2.Whether the ubiquitination sites of EAF2 protein were blocked by ELL1 when they bind to each other?Ubiquitination co-IP results showed that EAF2 protein almost lost ubiquitination modification compared with the groups of treated with or without MG132 when ELL1 was co-expressed with EAF2 and that EAF2 protein ubiquitination sites were blocked by ELL1.While EAF2 ubiquitination level did not change significantly when ELL2 was co-expressed with EAF2.We already knew that the protein binding region of ELL1 and EAF2 located on 1-113 amino acid residences of EAF2 protein.Combining the above results,39,81,85 and 111 lysines residences on EAF2 protein sequence were identified as EAF2 protein ubiquitination sties,which were located within the binding region of ELL1 with EAF2 protein.These ubiquitination sites would be blocked by ELL1 and it would lead to EAF2 protein lost its ubiquitination process when ELL1 binding to EAF2.The results of ELL1 stabilizing EAF2 protein and identification of EAF2 ubiqtuination sites were consistent.All EAF2 preponderant ubiquitination sites were blocked by ELL 1 when ELL1 binding to EAF2 protein.The binding of ELL1 to EAF2 would block the process of EAF2 protein ubiquitination.It was important for SEC to keep its function as a complex.Meanwhile,EAF2 might be another key protein which regulated SEC function besides ELL2.EAF2 was an important protein of SEC complex,its stability was very necessary for the whole complex.How would mutant EAF2 ubiquitination sites influent the binding ability of ELL1 and EAF2 protein?Co-IP result showed that GFP-ELL1 could pull down myc-EAF2-WT or myc-EAF2-K39-81-85-111R mutant protein when ELL1 was co-expressed with EAF2-WT or EAF2 multi-mutant.This indicated that the mutant of EAF2 ubiquitination sites did not affect the binding ability of ELL 1 and EAF2.According to the previous literatures,some specific ubiquitination sites might be associated with the cytoplasmic-nuclear translocation of protein.Therefore,fluorescence was used to test the location of these mutate ubiquitination sites EAF2 protein in prostate cancer cells.GFP-EAF2-WT,GFP-EAF2-K39R,GFP-EAF2-K81R and GFP-EAF2-K85R even located in the cytoplasmic and nuclear of C4-2 cells,but GFP-EAF2-K111R only located in the nuclear of the cells.This indicated K111R single point mutation affected the translocation function of EAF2 protein and when EAf2 lost 111 lysine ubiquitination it would gather together in the nuclear of C4-2 cells.Whether did the ubiquitination state of EAF2 protein affect the binding of ELL I and EAF2?According to previous results of our laboratory,ELL1 and EAF2 would bind together and become fluorescent particles when ELL 1 and EAF2 were co-expressed in prostate cancer cells,indicating that the combination of these two proteins in the nuclear would have its corresponding biological function.We could analyzed if EAF2 ubiquitination sites mutations affect the location of the complex of EAF2 and ELL1 by observing the expression and location of GFP-EAF2-WT or EAF2-K39-81-85-111R multi-point mutant plasmids and RFP-ELL1 plasmid in prostate cancer C4-2 cells.Results showed that the nuclear location and the amount the complex particles did not change significantly when GFP-EAF2-WT or EAF2-K39-81-85-111R mutant protein binding to RFP-ELL1.EAF2 was an unstable protein and mainly degraded by the ubiquitination system,therefore,the ubiquitination system was very important for EAF2 biological function.How did ubiquitination system affect EAF2 pro-apoptotic function?Through previous experiments,we determined that K81R had a greater impact on EAF2 protein half-life.In order to avoid the unknown impact of multiple ubiquitination sites on EAF2 protein function,K81R was used to analyze the apoptotic function of mutant EAF2 protein.Commonly used apoptotic marker caspase3 and PARP were chosen as apoptosis marker in this experiment.Total PARP protein was significantly decreased and cl-caspase-3 expression was significantly increased compared with the groups of EAF2-K81R with GFP-Vector and GFP-EAF2-WT when they transfected HEK 293 cells and C4-2 cells for 72 hours.EAF2-K81R mutant EAF2 protein was stable and its pro-apoptotic function was increased compared with EAF2-WT when EAF2 protein 81st amino acid lysine residence was mutated to arsine.Since EAF2 ubiquitination sites K81R mutation could increase EAF2 pro-apoptotic effect,intracellular apoptotic genes Bcl-2,Bax and Survivin were observed and detected after transfection of mutant EAF2-K81R.Physiological function of Bcl-2 protein was mainly to inhibit apoptosis and prolong cell life without affecting the cell cycle and differentiation.Bax protein had pro-apoptotic function.Survivin was a new member of the apoptosis inhibitor protein family,closely related to the differentiation,proliferation,invasion and metastasis of tumor cells.The results showed that expression of Bcl-2 and Survivin were significantly decreased,while the pro-apoptotic gene Bax was significantly upregulated compared with the groups of EAF2-K81R with GFP-Vector and GFP-EAF2-WT when they transfected HEK 293 cells and C4-2 cells for 72 hours.Cell began to be apoptotic after transfection of EAF2 and EAF2-K81R,especially EAF-K81R.Result showed EAF2 protein ubiquitination sites K81R mutation enhanced its pro-apoptotic function.In summary,this study validated EAF2 was a androgen-induced unstable protein,and its degradation was via the ubiquitin-proteasome system.EAF2 protein 39,81,85 and 111 lysine were identified as its ubiquitination sites related with EAF2 protein stability by lysine mutation method and found K81R was one of its preponderant ubiquitination sites.111 lysine also was found related with the nuclear localization of EAF2 protein and EAF2 ubiquitination sites mutant did not affect the binding of EAF2 and ELL1 and their complex’s nuclear localization.Siah2 was found that it was closely related with EAF2 protein stability and they had direct interaction.Siah2 increased EAF2 protein degradation when they were co-expressed.Ubiquitination IP showed that Siah2 could significantly ubiquitination level of EAF2 protein.While ELL1 could block EAF2 protein ubiquitination process and stabilize EAF2 protein by binding to the region of EAF2 protein which EAF2 ubiquitination sites located.Ubiquitination sites mutated EAF2 protein pro-apoptotic effect was significantly increased.The total PARP protein was significantly reduced,while cl-caspase-3 expression was significantly upregulated,expression of apoptosis inhibitor gene Bcl-2 and Survivin was significantly decreased and pro-apoptotic gene Bax was significantly increased after transfection of K81R for 72h.This study further confirmed EAF2 was a tumor suppressor gene,and its ubiquitination sites mutation could enhance its tumor suppressor function by promoting its pro-apoptotic function.EAF2 might be a potential treatment target for prostate cancer.