The Mechanism Of WDR5-CARM1-Notch2 Axis Promoting Cancer Cell Proliferation

Glioblastoma and Neuroblastoma are the most common solid tumors in nervous system.Approximately 80%of glioblastoma patients are over 60 years old,while most of neuroblastoma patients are under 18 months.Patients with glioblastoma and neuroblastoma have a very poor prognosis,even after surgery,radiotherapy and chemotherapy.Therefore,it is necessary to identify new and useful biomarkers for the molecular therapy of glioblastoma and neuroblastoma.The WD repeat protein family contains dozens of members,which regulate various cellular processes,such as cell cycle,apoptosis and autophagy.The members of WD repeat protein family are considered as candidate targets of some diseases,such as childhood obesity syndrome,osteoclasts and bone loss and cancer.WDR5 is a component of MLL-containing histone H3K4methyltransferase complex and has been shown to associate with the progression of leukemia.In addition,WDR5 regulates gene transcription by forming protein complexes with different transcription factors,such as Myc,MLK1 and OCT4.Both C-Myc and N-Myc belong to Myc oncogene family,so they have some similarities.As transcription factors,both of them recognize E-Box sequences（CACGCG or CACGTG）at target gene promoters.To date,many downstream genes of Myc have been demonstrated and they are involved in numerous cellular processes,such as glucose metabolism,apoptosis and DNA damage and etc.But there remain some new downstream genes of Myc to be explored.In this study,we found that WDR5 promoted the proliferation and clonal formation of glioblastoma and neuroblastoma cells.The data from databases and western blot assay showed that CARM1 is a downstream gene of WDR5-Myc axis.In addition,we observed that WDR5 promoted the binding of Myc to CARM1 promoter by interacting with Myc and inducing histone 3 lysine 4trimethylation（H3K4me3）.Dual luciferase reporter system indicated that Myc binds to the upstream region（-520--515）before transcription start site（TSS）of CARM1 promoter.These findings suggest a novel regulatory model for the proliferation and tumorigenesis of glioblastoma and neuroblastoma by WDR5-Myc axis.Coactivator-associated arginine methyltransferase 1（CARM1）,also known as arginine methyltransferase 4（PRMT4）,was originally identified as a coactivator for steroid hormone receptors.CARM1 is involved in pre-m RNA splicing,and the regulation of cell proliferation and differentiation.CARM1 is overexpressed in breast cancers,particularly in triple-negative breast tumors,and its overexpression positively correlates with poor prognosis,suggesting that it plays an oncogenic role in human cancer.To determine the possible function of CARM1 in gastric cancer,we depleted CARM1 using sh RNA and found that it inhibited cell proliferation and clonal formation.CARM1 is a transcription cofactor and was found to activate Notch2 by promoting H3R17me2 of the Notch2 promoter.In addition,we identified CARM1-interacting proteins using mass spectrometry.The results revealed that Nup54 promotes CARM1 nuclear transport by binding to CARM1.Furthermore,Notch2 was identified as a CARM1 substrate.The methylation of Notch2 at three sites（R1786,R1838 and R2047）promotes the interaction between its intracellular domain（N2ICD）and MAML1.The CARM1-mediated methylation of Notch2 promotes cell proliferation,clonal formation and gastric tumor growth.Our data shed light on the importance of CARM1 for the regulation of the Notch2signaling pathway and tumorigenicity.The main results are shown as below:1.WDR5 is a potential oncogene in glioblastoma and neuroblastoma developmentHere,R2:genomics analysis and visualization platform was used to analyze WDR5 expression in glioblastoma and neuroblastoma.The data from four different databases indicated that WDR5 is highly expressed in glioblastoma and neuroblastoma.In addition,the data from the Tumor Glioma-Kawaguchi-50 and the Tumor Neuroblastoma-public-Versteeg-88 demonstrated that high expression of WDR5 correlates with poor patient prognosis.We examined protein expression of WDR5 in four glioblastoma（A172,LN229,U251 MG and U87 MG）and neuroblastoma cell lines（SK-N-AS,BE（2）-C,SK-N-DZ and SHEP1）,the results revealed that WDR5 is commonly expressed in glioblastoma and neuroblastoma.To explore the WDR5 function in glioblastoma and neuroblastoma,WDR5 was knocked down using sh RNA.MTT assay revealed that the proliferation of LN229 and BE（2）-C cells was decreased with WDR5 downregulation.Furthermore,we found that fewer and smaller colonies were produced in WDR5 silencing groups by performing soft agar assay.These results demonstrate that WDR5 may play an oncogene role in glioblastoma and neuroblastoma.2.WDR5-Myc axis up-regulates CARM1 expressionIt has been reported that WDR5 is a transcriptional co-factor of Myc,but the downstream genes of WDR5-Myc axis are still to explore.To explore the downstream genes of WDR5-Myc axis,we analyzed the correlation between two genes using R2 platform and found that the expression of CARM1 was positively correlated with the expression WDR5-Myc complex.In addition,m RNA and protein expression levels of CARM1 were significantly reduced after Myc or WDR5 knocked down.Based on these results,we speculated that both Myc and WDR5 could regulate the expression of CARM1 at transcriptional level.To test this hypothesis,we performed Ch IP assay and the result showed that both Myc and WDR5 could bind with the promoter of CARM1.To determine the binding region of Myc and WDR5,dual-luciferase reporter system was performed.The results of dual-luciferase reporter system demonstrate that Myc recognize and bind to the E-Box that is located on the upstream region（-520--515）before transcription start site of CARM1 promoter.In order to test whether WDR5 is necessary for Myc regulation CARM1,we selected A172 and SHEP1 cells with relatively low expression levels of Myc and WDR5,and overexpressed Myc in these two cell lines.Both m RNA and protein expression of CARM1 was upregulated in Myc overexpressing groups,but inhibited by WDR5 silencing.Moreover,Ch IP assay was performed after WDR5 knocked down and indicated that the binding of Myc and H3K4me3 to the promoter of CARM1 were decreased significantly in WDR5 silencing groups.These data determine that WDR5is an indispensable factor in Myc regulation CARM1.3.CARM1 is highly expressed in human gastric cancer and promotes gastric cancer cell proliferationTo determine whether CARM1 is dysregulated in gastric cancer,we used the Oncomine database to analyze CARM1 expression in gastric cancer and revealed that CARM1 expression is upregulated in gastric cancer.Similarly,the m RNA and protein levels are obviously higher in gastric cancer cell lines than normal gastric tissue cell line.The data form Kaplan-Meier plotter showed that a high level of CARM1 expression was associated with poor prognosis.Next,CARM1 was knocked down using sh RNA.The results of MTT assay,Brd U assay and soft agar assay suggest that cell proliferation of gastric cancer were inhibited by CARM1 silencing.Collectively,these results indicate that CARM1 may function as an oncogene in gastric cancer.4.Nup54 interacts with CARM1 and promotes its nuclear transportionTo explore CARM1 binding proteins,immunoprecipitation assay and mass spectrometry were performed.The results revealed that CARM1 interact with Nup54.To explore the influence of Nup54 on CARM1 protein,Nup54 was knocked down using sh RNA.There was no obvious change in the expression of CARM1 after Nup54 knockdown,indicating that Nup54 does not affect the stability of the CARM1 protein.The Nups complex has been shown to participate in protein nuclear transport.CARM1 is a transcriptional coactivator located in the nucleus.Based on previous reports,we speculated that Nup54 may be involved in the nuclear localization of CARM1.To test this hypothesis,we extracted nuclear and cytoplasmic proteins and found that CARM1 was retained in the cytoplasm after Nup54 knockdown.In addition,the results of immunofluorescence assay showed that the CARM1 fluorescence signal was present in the cytoplasm of Nup54-silenced cells.These results suggest that Nup54 promotes the nuclear localization of CARM1.5.CARM1 regulates the Notch2 signaling pathway by promoting H3R17me2 of the Notch2 promoterTo further understand the mechanism by which CARM1 regulates gastric cancer,we analyzed several major proliferation pathways and found that the expression of Notch2 pathway genes was markedly altered in CARM1-deficient cells.Because the m RNA expression of Notch2 was decreased significantly after CARM1 silencing,we hypothesized that CARM1 may participate in the transcriptional regulation of Notch2.To test this hypothesis,we performed a Ch IP assay.The results showed that CARM1 directly binds to the Notch2 promoter.As a transcriptional coactivator,CARM1 promotes the binding of transcription factors to promoters by upregulating the H3R17me2 and H3R26me2 of the target gene promoter region.Thus,we examined H3R17me2 and H3R26me2 levels in CARM1-silenced cells and found that both H3R17me2 and H3R26me2 levels were decreased after CARM1 knockdown.Based on our previous results,a Ch IP assay was performed using anti-H3R17me2 and anti-H3R26me2 antibodies in the control and CARM1-deficient cells.After CARM1 knockdown,the level of H3R17me2 but not H3R26me2 was decreased significantly on the Notch2 promoter.These data suggest that CARM1regulates the Notch2 signaling pathway by promoting H3R17me2 of the Notch2 promoter.6.Notch2 is identified as a CARM1 substrateThe results of immunoprecipitation assay and mass spectrometry indicated that CARM1interact with Notch2.In addition,bioinformatics analyses revealed that the EVH1 domain of CARM1 is necessary for substrate recognition and methylation.Thus,to discern whether Notch2 is a CARM1 substrate,we generated CARM1 constructs and performed immunoprecipitation assay.The results showed that Notch2 coprecipitates with the EVH1 domain of CARM1 and is a candidate CARM1 substrates.Notch2 is proteolytically cleaved by metalloproteases andγ-secretase,releasing a Notch2extracellular domain（N2ECD）and a Notch2 intracellular domain（N2ICD）.Next,we constructed a MYC-tagged N2ICD overexpression plasmid to test the arginine methylation level of N2ICD in CARM1-silenced cells.Western blot analyses revealed that the arginine methylation level of N2ICD was decreased in CARM1-silenced cells compared with control cells.Taken together,these findings suggest that Notch2 is methylated by CARM1 in gastric cancer cells.To determine which domain of N2ICD is associated with CARM1,we constructed four N2ICD MYC-tagged domain.Then,Flag-CARM1 and MYC-N2ICD plasmids were cotransfected in HEK293FT cells.The result of immunoprecipitation suggested that the N2ICD PEST domain mediates the interaction with CARM1.To explore the N2ICD domain methylation,we constructed three additional MYC-tagged N2ICD domain plasmids fused with the PEST domain.A western blot assay using an anti-ADMA antibody indicated that the AR domain of N2ICD is methylated by CARM1.Using an online tool to predict arginine methylation sites and referencing published literature,four putative arginine methylation sites were identified in the N2ICD AR domain.We mutated these four putative arginine methylation sites to lysine（K）.And found that R1786K,R1838K and R2047K,but not R1787K,mutations decreased the methylated level of N2ICD.To explore whether CARM1also methylates other N2ICD sites,a triple mutant（R1786K,R1838K and R2047K）plasmid was constructed.The arginine methylation level of the triple mutant(N2ICD^TM)was not affected by CARM1 silencing.Overall,these results indicate that CARM1 methylates the AR domain of N2ICD at R1786,R1838 and R2047.7.N2ICD methylation promotes its interaction with MAML1 and the progression of gastric cancerAfter translocating to the nucleus,N2ICD first interacts with mastermind-like protein 1（MAML1）through its AR domain and forms a transcriptional complex with RBPjk.Thus,based on our previous data,we hypothesized that the arginine methylation of N2ICD may regulate the interaction between MAML1 and N2ICD.To test this hypothesis,we performed immunoprecipitation assays using wild-type（WT）N2ICD(N2ICD^WT)and triple mutant N2ICD(N2ICD^TM).The results revealed that the interaction between MAML1 and N2ICD^TM was weaker than that between MAML1 and N2ICD^WT.In addition,the results of MTT assay and soft agar assay determined that cell proliferation and clonal formation of gastric cancer were partially recused by rescue with N2ICD^WTbut not N2ICD^TMafter Notch2 downregulation.At the same time,mouse xenograft got the similar results of vitro experiments.These results suggest that N2ICD methylation regulates its interaction with MAML1and promotes cell proliferation,clonal formation and gastric tumor growth.