| BACKGROUND AND OBJECTIVE:Colorectal cancer(CRC)is one of the most commonly digestive malignant tumors,and the morbidity and cancer-related mortality are among the upper third of all tumors.In recent years,with the change of the people’s lifestyle and diet,the incidence of CRC has increased in the world,and age at oneset is becoming younger and younger.Metastasis is the leading cause of death in CRC.Metastatic tumors could be found in 40-50%patients at initial diagnosis and treatment.Liver is a major target organ of the metastatic colorectal cancer.CRC patients with liver metastasis have poor prognosis.It is estimated that over 50%of patients diagnosed with CRC will die due to complications related to metastasis.Carcinogenesis of CRC is a process with multistep,multistage,multiple gene mutations,accompanied by activation of oncogenes,inactivation of tumor suppressor genes,and aberrances of apoptosis-regulating genes and DNA repair gene.However,at the molecular genetic level,there is no single gene directly responsible for the regulation of tumor cell proliferation and metastasis.The molecular mechanism of invasion and metastasis is also a multistep complex biological process,involving interaction between cancer cells and host.Decline in cell adhesion,enhancement of cell movement capacity,changes in the cytoskeleton,EMT,changes in the tumor microenvironment,inflammatory factors,angiogenesis and the dysregulation of the secretory vesicles of tumor cell and mesenchymal cell.For these reasons,it is necessary to further explore the mechanisms of the occurrence and development of CRC.in particular the mechanism of invasion and metastasis,which would help todevelop effective treatments,improve the quality of CRC patients,and promote the patient’s 10-year survival rate.The cAMP responsive element binding protein 5(CREB5)was identified as a nuclear matrix-associated protein.Mapping to human chromosome 7p15.1,the CREB5 gene encodes a protein that is located in the nuclear matrix and has a broad tissue expression profile in human.Which CREB5 has been identified as a trans-activitor with a zinc-finger at the N-terminus and bZIP DNA-binding domains at the C-terminus.The bZIP domain contains DNA-binding domains,protein-protein interaction region and leucine zipper domain.CREB5 specifically binds to CRE as a homodimer or a heterodimer with c-Jun or CRE-BP1,and functions as a CRE-dependent trans-activator.CREB5 contains alpha,beta,gamma,and delta splicing variants that are highly conserved in many kinds of species including mouse,zebra fish,chimpanzee and canine.In our previous study,we found that CREB5 expression was significantly higher in CRC tissues and metastatic carcinoma in lymph node than in paired normal tissue.However,there is little known about the relationship between CREB5 and colorectal cancer,particularly the relationship between CREB5 and invasion and metastasis process,as well as the regulation mechanisms of CREB5.Therefore,in this study we will detect CREB5 expression in CRC,illustrate the function and associated molecular mechanism of CREB5 in the invasion and metastasis of CRC,and explore regulating mechanisms of CREB5.METHODS:1.Oncomine database and GEO database were used to analyse the expression and clinical prognosis of CREB5 in CRC and other malignant cancer.2.Immunohistochemistry(IHC).immunoblotting(western blot)and Real-time quantitative PCR(RT-qPCR)were used to detect CREB5 expression in paraffin-embedded and fresh CRC samples:and then the relationship between CREB5 expression and clinical pathology parameters,including tumor differentiation,stage,metastasis and clinical prognosis,was analysed.3.The colorectal caner cell lines with CREB5 overexpression and knockdown were constructed.Cell-independent proliferation was measured by softagar colony formation assay.The invasive and migratory abilities were measured by Matrigel-coated Boyden chamber assays.Angiogenesis was measured by Human umbilical vein endothelial cells(HUVEC)tube formation assay.Chicken chorioallantoic membrane(CAM)assay and Xenografted tumor model.IHC and HE staining were performed in CRC cells of CREB5 overexpression or inhibition to identified the function of CREB5 in CRC.4.GEO database were used to analyse the enrichment of related signaling pathways in CRC patiens with high expression of CREB5.GO analysis was performed to explore the function of CREB5.5.Western blot and RT-qPCR were used to detect possible targets of CREB5.6.Western blot and RT-qPCR were used to verify the regulation effect of CREB5 on MET as well as the downstream of MET signaling including AKT and MAPK signaling pathways.Luciferase reporter assay was carried out to measure the activity of MET promoter.Chromatin immunoprecipitation(ChIP)assays were applied to determine the binding sites of CREB5 with MET promoter.7.Cell-independent proliferation was measured by softagar colony formation assay.The invasive and migratory abilities were measured by Matrigel-coated Boyden chamber assays.Angiogenesis was measured by Human umbilical vein endothelial cells(HUVEC)tube formation assay.Chicken chorioallantoic membrane(CAM)assay and Xenografted tumor model.IHC and HE staining were performed in CRC cells of CREB5 overexpression or inhibition together with overexpression or inhibition of MET.8.Westernblot.RT-qPCR and IHC were performed to determine the expression of CREB5,MET,MMP9,IL6,and VEGF in CRC tissues and their paired adjacent normal tissue.The correlationships between CREB5 expression and that of MET,MMP9,IL6 and VEGF expression were analyzed using statistical methods.RESULTS:1.The expression and clinical prognosis of CREB5 in CRC and other malignant cancers.1)The expression of CREB5 and clinical prognosis in CRC in the public databaseThe results from Oncomine database analysis showed that 17 CRC databases met the inclusion criteria,including 8 database of CREB5-downregulation and 9 database of CREB5-upregulation.It should be noted that downregulation of CREB5 was showed in a large sample database,the TCGA database(878 samples).The results from GSEA suggested that cancer related gene set,"KEGG_PATHWAY IN CANCER"and CRC related gene set,"KEGG_COLORECTAL CANCER",were upregulated in CRC patients with high expression of CREB5(GSE17538 ES=0.49.P<0.001;GSE35896 ES=0.45,P<0.001).Kaplan-Meier Survival analysis of GSE17538 dataset revealed that the 5-year overall survival rate of CRC patients with high expression of CREB5 was significantly lower than that in CRC patients with low expression of CREB5(Log-Rank.P<0.05).2)The expression of CREB5 in CRC tissueThe results from western blot and RT-qPCR suggested that the protein andmRNA levels of CREB5 in CRC tissue were higher than that in paired normal colon tissue.The results from IHC also showed that CREB5 positive expression mainly localized in nucleus,marked by yellow-brown.The expression of CREB5 in CRC tissue was higher than that in paired normal colon tissue.3)The relationship between CREB5 expression and clinicopathological parametersThere were no significant differences of CREB5 expression in different age groups and different gender groups(P>0.05);but there were significant differences of CREB5 expression in Dukes stage.TNM stage,metastasis and prognosis(P<0.05),and there was a positive correlation between CREB5 and these clinicopathological parameters.Kaplan-Meier Survival analysis revealed that the 5-year overall survival rate of CRC patients with high expression of CREB5 was significantly lower than that in CRC patients with low expression of CREB5(Log-Rank,P<0.05),and 5-year overall survival rate declined with the increasing of CREB5 expression.2.The function of CREB5 in the proliferation,invasion and metastasis of CRC1)The construction of the CREB5 overexpression and knockdown Lentiviral vectors,and the establishment of stable expression cell linesMolecular cloning techniques were used to construct the CREB5 overexpression and knockdown Lentiviral vectors.Sequencing results show that the insertion sequence was completely correct with no mutation,loss,or frameshift.Then,Lentiviral vectors were used to establish stable expression cell lines,and the results from western blot showed that stable expression cell lines were successfully established.2)The effects of CREB5 overexpression and knockdown on proliferation in CRC cellsThe results from softagar colony formation assay indicated that the colony numbers of CRC cells with CREB5 overexpression increased(P<0.05),but the colony numbers of CRC cells with CREB5 knockdown decreased(P<0.05).3)The effects of CREB5 overexpression and knockdown on invasion and migration in CRC cellsWestern blot results showed that the expression of the epithelial marker of EMT,E-cadherin decreased,and the expression of the mesenchymal marker,Vimentin increased in HT29 and SW480 with CREB5 overexpression;however,the expression of E-cadherin and Vimentin showed opposite results in HCT116 and HCT15 with CREB5 knockdown.The results from transwell migration assay showed that the migratory CRC cell with CREB5 overexpression increased markedly compared with the control group(P<0.05),but the migratory CRC cell with CREB5 knockdown decreased dramatically(P<0.05).The results from scratch wound healing assay displayed that the speed of wound healing of CRC cells with CREB5 overexpression was faster than that in the control group.The speed of wound healing of CRC cells with CREB5 knockdown was slower than that in the control group.4)The effects of conditioned media from CREB5 overexpression and knockdown cells on angiogenesis in vivo and in vitroThe results of HUVEC tube formation assay and CAM assays exhibited that silencing CREB5 inhibited,whereas overexpressing CREB5 strongly provoked.the abilities of CRC cells to induce tube formation and migration of HUVECs(P<0.01)and the formation of second-and third-order vessels in the CAM assay..5)The effects of CREB5 overexpression and knockdown on metastatic potential of CRC cells in vivoThe results of Orthotopic implantation assay showed that CRC cells with CREB5 overexpression significantly promoted liver metastasis(P<0.01);however,the formation of metastatic foci showed opposite results in CRC cells with CREB5 knockdown(P<0.05).3.The molecular mechanism of CREB5 in the acceleration of progression,invasion and metastasis in CRC1)The bioinformatic analysis of signaling pathway regulation in CRC with high CREB5 expression in the public databaseThe results of GSEA assay suggested that multiple malignant proliferation and metastasis pathway-related genes set were enriched significantly,including TGF-beta,MTOR,WNT and Notch signaling pathway.HGF/MET signaling pathways,"MID_MET PATHWAY" was upregulated in CRC tissues with high CREB5 expression in GSE17538(P<0.001).2)The regulation of HGF/MET signaling pathway in CRC with high CREB5 expressionWestern blot and RT-qPCR revealed that stable expression of CREB5 dramatically increased the expression of the total MET(P<0.001)and phosphorylated MET as well as its downstream signaling molecules K-Ras,Rafl.phosphorylated ERK and AKT in SW480 and HT29 cells,whereas downregulation of CREB5 attenuated the expression of MET(P=0.003,P=0.002),phosphorylated MET.K-Ras.Raf1.phosphorylated ERK and AKT in HCT15 and HCT116 cells.Moreover,we also observed that the expression of Snail was increased in CREB5-overexpressing cells and conversely decreased in CREB5-silenced cells.Additionally,we verified the effect of CREB5 on upregulation of MET expression by transiently transfecting cells with a CREB5 expression vector.MET expression was increased in a dose-dependent manner at both the translational and transcriptional levels.3)The analysis of association between CREB5 and MET promoterWe performed a luciferase reporter assay to investigate whether CREB5 could increase MET promoter activity as a transcription factor.A 1.5-kb fragment of the full-length MET promoter region was subcloned into a luciferase vector.The MET promoter activity was increased by cotransfection with a CREB5 expression vector in SW480 cells but decreased in HCT116 cells expressing CREB5 shRNA in a dose-dependent manner,compared with empty vectors.To determine the effective region of MET promoter that was associated with CREB5,MET promoter truncations were transfected into S W480 cells expressing either CREB5 shRNA or an empty vector.The luciferase activity was increased in cells carrying full-length MET promoter and truncations-1568,-500,-1250～+49 bp upstream of the transcription start site as well as-1800bp to-1201 bp,but not in cells carrying truncations-1568 to-1251 or-1250bp to-501 bp bp.Knockdown of CREB5 expression by cotransfection of CREB5 shRNA significantly decreased MET promoter activity.Furthermore,we performed ChIP assays and identified that the-223 bp to-68 bp regions of the MET promoter was the binding site of CREB5.Taken together,this data identifies MET as a direct transcriptional target of CREB5.4)The regulation of the invasive phenotype of CREB5-overexpressing CRC cells after inhibition of METTo analyze the functional relationship between CREB5 and MET,we knocked down MET using two MET siRNAs in CREB5-overexpressing cells.Silencing of MET significantly decreased the expression of phosphorylated MET,Akt and ERK,and also reduced the expression of Snail.Similar result was obtained with MET inhibitor(Crizotinib)treatment.5)The regulation of the cell-independent proliferation of CREB5-overexpressing CRC cells after inhibition of METThe results of softagar colony formation assay showed that,the colony numbers of CRC cells with CREB5 overexpression increased(P<0.01).but the colony numbers of CRC cells with both CREB5-overexpression and MET-silence changed little compared with empty vectors(P>0.05).6)The regulation of the invasion ability of CREB5-overexpressing CRC cells after inhibition of METThe results of Matrigel-coated Boyden chamber assays and wound healing assays exhibited that the invasive and migratory abilities were increased in cells with CREB5 overexpression(P<0.01),but declined again by CREB5 overexpression and MET silence(P<0.01).7)The regulation of the angiogenesis in vivo and in vitro of CREB5-overexpressing CRC cells after inhibition of METThe results of HUVEC tube formation assay and CAM assays exhibited that overexpressing CREB5 provoked the abilities of CRC cells to induce tube formation and migration of HUVECs(P<0.01),and the formation of second-and third-order vessels in the CAM assay.However,silencing MET in CREB5-overexpressing cells could weaken the effect of CREB5 on angiogenesis.8)The regulation of the metastatic potential in vivo of CREB5-overexpressing CRC cells after inhibition of METThe results of Orthotopic implantation assay showed that CREB5 significantly promoted liver metastasis(P<0.01);however,silencing of MET by shRNAs or MET inhibitor treatment significantly weaken the formation of metastatic foci by CREB5-overexpression cells(P<0.05).9)CREB5-MET signaling pathway regulated Wnt pathwayThe results of GSEA suggested that Wnt_beta-catenin signaling pathway was upregulated in CRC with high CREB5 expression,and pathway-related genes were enriched significantly(P<0.05).The results of dual luciferase reporter gene assay of TOP/FOP showed that,the activity of Wnt signaling pathway increased in SW480 and HT29 with CREB5 overexpression compared with the control group;by contrast,the activity of Wnt signaling pathway decreased in HCT116 and HCT15 with CREB5 knockdown compared with the control group.Moreover,the results of westernblot showed that the expression of CyclinDlincreased in SW480 and HT29 with CREB5 overexpression compared with the control group;but the expression of CyclinDl showed opposite results in HCT116 and HCT15 with CREB5 knockdown.The expression of p27 was downregulated in SW480 and HT29 with CREB5 overexpression,but upregulated in HCT116 and HCT15 with EPB49 knockdown.4.The clininal relevance of CREB5 and MET in CRC patients1)The expression of CREB5 and MET in tissue chip from CRC patientsAnalysis of 78 CRC tissue chip using IHC anlalysis showed that 85.9%(65/78)of CRC specimens were of CREB5 high expression,among which 61 CRC specimens are high expression of MET.These results indicated that CREB5 expression was consistently correlated with the expression of MET(p<0.001).2)The expression of CREB5 and MET in freshly collected CRC biopsiesThe results of westernblot analyses indicated that CREB5 and MET were upregulated significantly in the 10 tumor samples examined,compared to the paired adjacent noncancerous tissues from the same patients.3)The correlation of CREB5 and MET in freshly collected CRC biopsiesThe results of RT-qPCR and correlation studies analysis demonstrated that CREB5 expression was correlated with the expression of MET(r=0.895.P<0.05)at mRNA level.4)The correlation of CREB5,MMP9,IL6 and VEGF in freshly collected CRC biopsiesThe results of RT-qPCR and correlation analysis denonstrated that CREB5 expression was positively correlated with the expression levels of MMP9(r=0.636,P<0.05),IL6(r=0.648,P<0.05)and VEGF(r=0.721,P<0.05).CONCLUSION:1.The expression of CREB5 is upregulated in CRC.There is a positive correlation between CREB5 and clinicopathological parameters,including Dukes stage,TNM stage,metastasis and prognosis.2.CREB5 upregulation binds to the specific region of MET promoter and thus activates HGF/MET signaling pathway,leading to the promotion of CRC progression,invasion and metastasis.3.CREB5 expression is positively correlated with the expression of MET,MMP9,IL6 and VEGF in CRC.INNOVATION:1.The first research about the function role of CREB5 in CRC cancer;2.CREB5 binds to the specific region of MET promoter and thus activates HGF/MET signaling pathway.3.CREB5 might act as a key role in the metastasis signaling network of CRC cancer. |
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