Evaluation Of The Oncogenic Role Of CDK5 In Modulating The Malignant Development Of Colorectal Cancer

Colorectal cancer(CRC)is one of the most common malignant tumor both in China and the world.CRC showed a high morbidity and mortality ranked both fifth in our country1.As a malignant tumor with complex developing process,the CRC carcinogenesis is a multistep and multi-factorial process related to various genetic and epigenetic alterations and the pathogenic mechanism of CRC has not been fully understood2-3.Thus,the present hot topic in the fIeld of CRC mainly foucused on pathogenesis of CRC.We had identified several proteins closely correlated the malignant progress of CRC using the proteomics technology of isobaric tags for relative and absolute quantitation(ITRAQ)in our previous study4.We found that cyclin-dependent kinase 5(CDK5)was abnormal expression on CRC.After tracing more literature about CDK5,we found that CDK5 was a key regulator of cell survival,proliferation and migration,indicating that CDK5 was of great worth for further research in CRC.Thus,we targeted CDK5 as potential tumor promotor of CRC and developed intensive study about the role of CDK5 in CRC.Methods:1.Evaluation of the expression level of CDK5 and its clinicopathological significance of CDK5 in CRC(1).Immunohistochemistry staining,western blot analysis and a dataset of CRC patients' gene expression profiles from TCGA were used for analyse the expression level of CDK5 and its activator protein,p35 between normal colon tissue and CRC tissue.(2).Tissue microarray along with several statistical methods including pearson's chi-square,cox proportional hazard model and kaplan-meier method were used for evaluating the clinicopathological significance of CDK5 in CRC.2.Investigation of the biological function of CDK5 in CRC cells.(1).In vitro cell biological experiments:To investigate the biological function of CDK5 in CRC cells,first we established stable CDK5 silencing and CDK5 overexpressing CRC cell lines.Then,CCK8 proliferation assay,colony-formation assay,soft agar assay cell cycle analysis and cell migration and invasion assays of transwell chambers were used for further research.(2).In vivo cell biological experiments:The lung metastasis models of nude mice were built by inj ecting the indicated stable cell lines into the tail vein.3.Whole-genome expression microarray experiments were performed to further understand the underlying molecular mechanisms of CDK5 mediating carcinogenesis in CRCTotal RNA was isolated from three replicate samples of the stable cell lines HCT116/Scramble and HCT116/ShCDK5.Agilent Whole Human Genome Oligo Microarray(4×44K)was used for transcriptome analysis.To assess the molecular signaling pathway change after knockdown of CDK5,data analysis,including canonical pathway and gene network interaction,was performed by the Ingenuity Pathway Analysis(IPA).4.Evaluation of the molecular mechanism between CDK5 and ERK5-AP-1 signaling axis and their role played in CRC carcinogenesis.To investigate whether CDK5 was involed in the regulation of ERK5-AP-1 signaling axis,we performed related experiments including co-immunoprecipitation,in vitro kinase assay,electrophoretic mobility shift assay(EMSA),western blot analysis and cell immunofluorescence assay for further research.Besides,colony-formation assay,xenograft tumor model and clinical CRC samples were used for evaluating the role of CDK5-ERK5-AP-1 signaling played in CRC carcinogenesis.Results:1.CDK5 and p35 were both upregulated in CRC;Western blotting showed that the expression of CDK5 and p35 protein was significantly upregulated in the 10 CRC tissues compared with their adjacent normal intestine epithelial tissues.Besides,CDK5 protein was detected in 85 of the 89(95.5%)cases of CRC tissue samples with 49(55.1%)cases displaying high CDK5 expression and 40(44.9%)cases showing a relatively low CDK5 expression.2.CDK5 overexpression was associated with progression and poor survival of CRC:Pearson's chi-square tests revealed that the expression of CDK5 was significantly correlated with AJCC(P=0.01),tumor differentiation(P=0.029),tumor size(P=0.001)and nodal metastasis(P=0.027).Cox regression analysis showed that CDK5 expression(HR:2.428,95%CI:1.089-5.413;P=0.030)was considered as the independent risk predictor for poor overall survival.Notably,Kaplan-Meier survival analysis revealed that patients with high CDK5 expression had poorer overall survival than patients with low CDK5 expression(44 versus 54 months,P=0.001).3.CDK5 overexpression promoted the proliferation and metastasis ability ofCRC cells,while CDK5 knocking down performed the opposite effect.4.Knocking down CDK5 induced differential expression of genes involved in cellular proliferation,survival,movement and cellular transcription of DNA.Also,knocking down CDK5 significantly repressed several key carcinoma molecular pathways,with the ERK5 signaling pathway mostly inhibited.5.CDK5 activated the ERK5-AP-1 signaling axis via regulating the phosphorylation level of ERK5 at Thr732.The CDK5-ERK5-AP-1 signaling axis could promote CRC carcinogenesis both in vitro and in vivo experiments.6.Expression level of CDK5-ERK5-AP-1 showed a high correlation in clinical CRC samples.Conclusion:In conclusion,our findings help provide evidence that elevated CDK5 in human CRC has an important role in the acquisition of the progression as well as poor prognosis of the tumor.Furthermore,we also identified the CDK5-ERK5-AP-1 axis as a potential oncogenic pathway in CRC.This study not only further elucidates the precise role of CDK5 in the pathogenesis of CRC but also provides new insight into the biological basis of cancer progression and may help to contribute to more effective therapeutic strategies for CRC.