CUL4B Promotes Invasion And Metastasis Of Gastric Cancer And Prostate Cancer By Transcriptionally Repressing MiRNAs

Invasion and metastasis are the major features of malignant tumors that are responsible for 90%of cancer-related deaths.Tumor invasion and metastasis is a complex,multi-factor regulation of the dynamic process,which involves a number of complex and redundant pathways.The activation of these pathways mediate tumor invasion at the primary site,survival and arrest in the bloodstream,and progressive outgrowth at a distant site.Understanding these pathways and their dynamic interactions will help identify promising molecular targets for cancer therapy and key obstacles to their clinical development.Cullin 4B(CUL4B),a scaffold protein that assembles Cullin 4-Ring E3 ligases(CRL4)complexes.To date,CRLs are the largest known class of unbiquffin ligases and participate in a variety of physiologically and developmentally controlled processes such as cell cycle progression,signal transduction,DNA damage response and chromatin remodeling.Recently,CUL4B has been reported to be overexpressed in a variety of solid malignancies including esophageal cancer,lung cancer,colon cancer,cancer,cervical cancer and hepatocellular carcinoma.Colony formation and tumor xenograft assays confirmed that CUL4B might be a novel oncogene.CUL4B acts as a scaffold assembly for DDB1,ROC1 and substrate receptors to form Cullin4B-RING-based E3 ubiquitin ligases(CRL4B).We and others have demonstrated that CRL4B catalyzed H2AK119 monoubiquitination and coordinated with PRC2,SUV39H1/HP1/DNMT and SIN3A/histone deacetylase(HDAC)complexes.Furthermore,CRL4B also negatively regulated tumor suppressors including P16,PTEN.Wnt antagonists and IGFBP3 to promote tumorigenesis.However.the research of CUL4B in tumor development and progression is still in its infancy.What is the molecular mechanism of CUL4B in cancer invasion and metastasis remains poorly understood.Gastric cancer(GC)is among the most threatening malignancies with a high incidence and metastasis rate.Using Gene Expression Omnibus(GEO)and The Cancer Genome Atlas(TCGA)datasets,we showed that CUL4B mRNA levels in GC tissues were significantly higher than those in the normal gastric tissues.On the other hand,our research group is mainly focused on the mechanism of prostate cancer(PCa)invasion and metastasis and prognosis markers for PCa screening.Preliminary findings of our research group indicated that SOX4 was an independent and unfavorable prognostic indicator in Chinese PCa patients.In addition.ERG-SOX4 interaction promotes Epithelial-MesenchymalTransitionin(EMT)and migration in PCa cells.More importantly,we discovered that CUL4B was significantly correlated with SOX4 expression in PCa cohort.Above all,we can speculate that CUL4B may be an oncogene in GC and PCa.Therefore,we systematically explore the role and molecular mechanism of CUL4B in agressive progression of GC and PCa.In the current study,we utilized bioinformatics analysis,clinical tissue samples.cell model and xenograft model to clarify the role and molecular mechanism of CUL4B in PCa and GC invasion and metastasis.This will provide new ideas for clinical treatment of PCa and GC.Part I CUL4B promotes gastric cancer invasion and metastasis by regulating miR-125a and its target gene HER2CUL4B has been reported to be overexpressed in several types of solid malignancies and contributes to epigenetic silencing of tumor suppressors.However,the biological significance and underlying molecular mechanisms of CUL4B in GC remain largely unknown.Therefore,we explored the functional and mechanisms of CUL4B in clinical specimens,cells and xenograft tumors of GC.The results were as follows:1.CUL4B is overexpressed and associated with poor prognosis in gastric cancer.Immunohistochemistry(IHC)was firstly used to examine CUL4B expression in a cohort of 154 Chinese GC patients.The expression of CUL4B was further analyzed in publicly-accessible GC datasets(TCGA and GEO).We found that CUL4B was significantly overexpressed in GC tissues and its overexpression was correlated with poor prognosis.Next,we systematically characterized the relationship between CUL4B overexpression and clinicopathological parameters and the common molecular aberrations in GC.CUL4B expression was found to be significantly correlated with EGFR and HER2.distant metastasis,lymph node metastasis and tumor invasion depth.2.CUL4B regulates GC cell growth and tumorigenesis.To determine the oncogenic activity of CUL4B in GC.we established overexpression and silencing of CUL4B in MKN45 and BGC823cells.EdU.MTS and colony formation assays demonstrated that CUL4B knockdown slightly reduced cell proliferation,whereas CUL4B overexpression displayed the opposite results.Xenograft assay further confirmed that the tumors from MKN45-shCUL4B cells grew less rapidly at the implantation site than those of control cells.3.CUL4B promotes GC invasion and metastasis in vitro and in vivo.Scratch assay and transwell assay demonstrated that CUL4B promoted migration and invasion of MKN45 and BGC823 cells.Using subcutaneous and intravenous tumor models,we demonstrated that the majority of xenograft tumors of the shCUL4B group were well encapsulated without muscle invasion and decreased lung metastasis.It is well documented that EMT enables cancer cells with invasive and metastatic properties and plays critical roles in tumor progression.Here,we showed that MKN45 cells with CUL4B depleted appeared round,with a more epithelial morphology,whereas CUL4B overexpressed cells acquired a dispersed,spindle-shaped morphology.Immunofluorescence,RT-qPCR and western-blot assay showed that CUL4B knockdown increased the expression levels of epithelial markers(E-cadherin and Claudin-1).but decreased the levels of mesenchymal markers(N-cadherin and vimentin)in MKN45 and BGC823 cells.4.CUL4B is a direct target of miR-101.Three algorithms including TargetScan,PicTar,and miRanda.showed that the seed sequences of miR-101,miR-204,miR-217 and miR-133 were complementary to the 3'UTR of CUL4B.Western-blot revealed that only miR-101 significantly altered the CUL4B protein expression.Notably,the relative luciferase activity of the wild-type construct of CUL4B 3'UTR was reduced by miR-101 mimics but increased by miR-101 inhibitors.By contrast,such effects were not observed in which miR-101 binding site was mutated.5.CUL4B was positively corralted with HER2 at protein level.To further elucidate the mechanism by which CUL4B regulates cell invasion and metastasis in GC,we conducted gene expression profiling of CUL4B-depletion(si4B)vs control(NC)in MKN45 cells.955 genes with 554 down-regulated and 401 up-regulated were identified in CUL4B knockdown MKN45 cells.Gene Set Enrichment Analysis(GSEA)was then carried out and we found that HER2,EIF4E and VEGF-A gene sets were significantly enriched in CUL4B down-regulated MKN45 cells.A positive relationship between CUL4B and HER2 was found by western-blot and immunofluorescence assay.Furthermore,the association of CUL4B with HER2 was identified in xenograft tumors and GC cases.However,no correlation of CUL4B and HER2 was identified at mRNA level.Taken together,we hypothesized that CUL4B regulated HER2 expression via post-transcriptional level.6.CUL4B positively regulates HER2 expression by transcription repressing miR-125a.Venn analysis was conducted using miRNA-seq data and miRNAs predicated by TargetScan and miRanda,and three candidate miRNAs(miR-125a,miR-1254 and miR-193a)were obtained.Combining previous reports and RT-qPCR data,miR-125a was chosen for further investigation.RT-qPCR analysis showed that both pri-miR-125a,pre-miR-125a and miR-125a expressions were significantly increased following CUL4B knockdown.but these levels were dramatically decreased by CUL4B overexpression.ChIP assay revealed that antibody against CUL4B efficiently immunoprecipitated the S2 region of miR-125a promoter.In addition.a reverse association between CUL4B and miR-125a was found in GC clinical specimens.Western-blot results revealed that miR-125a mimics reduced HER2 protein expression and miR-125a inhibitor resulted in an increased amount of HER2 proteins.Notably,the relative luciferase activity of the wild-type construct of HER23'UTR was inhibited by miR-125a mimics but increased by miR-125a inhibitor.7.miR-125a is involved in CUL4B-induecd HER2 expression and GC migration.As assayed by western-blot.CUL4B overexpression significantly induced HER2 expression,whereas miR-125a mimics in these cells could significantly block this increase.Rescue experiments demonstrate that miR-125a reconstitution partially blocked CUL4B-induced cell migration.8.HER2 inhibitors partially reverse CUL4B-induced EMT and metastasis.HER2 inhibition by Herceptin or siHER2 could partially reverse the EMT induced by CUL4B in MKN45 cells.To expand our in vitro observations,we utilized subcutaneous and intravenous tumor xenografts model for further investigation.The tumor xenografts were treated with Herceptin(20mg/Kg)or PBS as control after the tumors had formed.As shown,HER2 inhibition could partially block CUL4B significantly induced tumor growth and metastasis in xenografts.In conclusion,we showed that CUL4B positively regulated HER2 expression by transcriptionly repressing miR-125a in GC.Inhibition of HER2 disrupted the CUL4B-miR-125a-HER2-PI3K/AKT axis,resulting in suppression of CUL4B-induced EMT and metastasis in vitro and in vivo.Part ?CUL4B promotes prostate cancer invasion and metastasis by regulating miR-204 and its target gene SOX4Prostate cancer(PCa)is a leading cause of cancer-related death of men in western world.The poor prognosis of PCa is mainly attributable to the high rate of tumor recurrence or metastasis.which contributes to around 90%of cancer-related mortality.Therefore,there is an urgent need to elucidate the molecular mechanism underlying PCa progression,recurrence and metastasis.This study focuses on the correlation between the "CUL4B-miR-204-SOX4 axis" and the aggressive progression of PCa,which will open new avenues for the design of effective diagnostic,prognostic and therapeutic strategies for better clinical management of PCa.In this section,the following results were obtained:1.CUL4B is up-regulated and highly correlates with aggressive progression in PCa.IHC and bioinformatics analysis demonstrated that CUL4B was frequently up-regulated in PCa patients at mRNA and protein level.Furthermore,CUL4B expression was found to be significantly correlated with Gleason score,clinical stage and distant metastasis.Kaplan-Meier survival analysis showed that PCa patients with CUL4B overexpression displayed a lower biochemical recurrence-free survival rate.2.CUL4B promotes PCa cell proliferation.MTS and EdU assays showed that CUL4B down-regulation significantly decreased cell proliferation whereas CUL4B overexpression promoted cell growth,when compared with negative controls.Similarly,silencing of CUL4B resulted in the reduction of the clonality in VCaP cells;whereas,overexpressing CUL4B led to opposite effects.The xenograft experiment further confirmed that CUL4B knockdown could suppress PCa growth in vivo.3.CUL4B promotes EMT in PCa cells.In the present study,we demonstrate that knockdown of CUL4B by siRNA significantly suppressed the migration and invasion in both VCaP and 22RV1 cells,whereas overexpressing CUL4B had the opposite effects.We next sought to examine whether CUL4B promotes PCa migration and invasion is mediated by EMT.As assayed by western-blot and RT-qPCR.the depletion of CUL4B resulted in an increase in E-cadherin protein expression but decreases in N-cadherin and vimentin expression in VCaP cells.Similar results were observed by immunofluorescence.4.Identification of SOX4 as a downstream target gene of CUL4B in PCa.Preliminary research findings of our research group have defined an important role for SOX4 in the progression of PCa by orchestrating EMT.More importantly,SOX4 may serve as a prognostic marker for PCa patients.In addition,CUL4B has been reported to overexpressed in various solid tumor,such as esophagus cancer and cervical cancer,which plays an oncogenic role.This indicated that there might be an association between CUL4B and SOX4.As expected,CUL4B silencing in VCaP and 22RV1 cells led to a marked depression in SOX4 expression levels.In contrast,SOX4 expression levels were significantly increased in these cells with CUL4B overexpression.In consistent with these data,immunofluorescence analysis verified the co-expression of CUL4B and SOX4 in VCaP cells.We next examined the protein expression of CUL4B and SOX4 in human PCa cases by IHC.It showed that the protein levels of CUL4B and SOX4 were much higher in PCa cases with Gleason score 8-10 than those with Gleason score 6-7,indicating a positive association of CUL4B with SOX4 in PCa.5.CUL4B positively regulated Wnt/?-catenin pathway by activating SOX4 expression.It is well known that Wnt/?-catenin pathway is closely related to tumor development and progression.Our data demonstrated that the level of ?-catenin and CyclinDl was significantly reduced in CUL4B-depleted VCaP cells.On the other hand,ectopic expression of CUL4B led to significantly increased expression of(3-catenin and CyclinDl.Notably,depletion of SOX4 could partially inhibit CUL4B-induced increase of ?-catenin and CyclinD1 in VCaP cells.6.CUL4B induces SOX4 expression through modulating miR-204 level.The above data indicated that CUL4B regulates SOX4 expression at protein level.However.CUL4B knockdown has no effect on SOX4 mRNA level.Taken together.these results indicated that CUL4B regulated SOX4 expression via post-transcriptional level.Combing target prediction algorithms(TargetScan.miRDB.PicTar and RNA22)and RT-qPCR analysis,miR-204 was chosen for further investigation.ChIP assay confirmed that CUL4B could bind to the promoter of miR-204.Western-blot results revealed that miR-204 mimics reduced SOX4 protein expression and miR-204 inhibitor resulted in an increased amount of SOX4 protein in VCaP and 22RV1 cells.Luciferase reporter assay demonstrated that the relative luciferase activity of the wildtype construct of pmirGLO-SOX4 3'UTR was attenuated by miR-204 mimics,whereas such effects of miR204 on luciferase activity were not observed with the mutant construct of pmirGLO-SOX4 3'UTR.7.MiR-204 is involved in CUL4B-induced cell invasion and migration.As assayed by western-blot,scratch assay and transwell assays,CUL4B overexpression promotes EMT,invasion and migration of PCa cells.Importantly,miR-204 mimics partially reversed these phenotype changes induced by CUL4B.Collectively,CUL4B plays critical role in driving PCa invasion and progress.CUL4B contributes to PCa invasion and metastasis by activating SOX4 through repressing miR-204.Significantly,miR-204 could partially block CUL4B-miR-204-SOX4 induced PCa invasion and metastasis.Our findings have enriched the knowledge of the molecular mechanisms underlying PCa aggressive progression and provide potential targets for future therapeutic invention.