The Function And Mechanism Study Of CDK15 In Promoting The Occurrence And Development Of Colorectal Cancer Through Phosphorylating PAK4

Background and ObjectivesColorectal cancer(CRC)is the most common tumor in the world,ranking the third in morbidity and second in mortality among all cancers.The incidence and fatality rate of CRC in our country are showing an upward trend,and this trend is developing rapidly especially among people younger than 50.Results from molecular studies have indicated that CRC initiation and progression are complex processes involving in accumulation of abnormal expression of multiple genes.The different accumulations of abnormal expressed genes in different individuals have led to poor effects for many patients receiving traditional treatment.Although with the development of modern biotechnology and technology,some targeted drugs have achieved good results in the treatment of clinical colorectal cancer,but with the progression of colorectal cancer and accumulated genetic differences of individuals patients eventually became low sensibility and resistant to targeted drugs.So far,few effective therapeutic targets have been identified.Therefore,the elucidation of mechanism-based biomolecules,as well as identification of novel therapeutic targets for CRC are the top priorities in the field.CDKs(cyclin-dependent kinases)are a group of serine/threonine protein kinases,which are important regulators for cell activities.Abnormal cell cycle leads to the occurrence and development of tumors.Almost all tumor cells have a variety of abnormal regulation of CDK.There have reports that CDKs play a critical role in CRC progression.CDK15,as a newly discovered member,has a high degree of homology with CDK14 and CDK16.Only a few studies have shown that the function of CDK15 is closely related to tumors,but its specific function and mechanism in colorectal cancer are still unclear.In this study,we will explore the expression of CDK15 in colorectal cancer,study the role of CDK15 in the development of colorectal cancer in vitro and in vivo,clarify the detail molecular mechanism of CDK15 in colorectal cancer and provide a theoretical basis for exploration of clinical therapeutic targets in colorectal cancer.Methods(1)Explore the expression and clinicopathological significance of CDK15 in human colorectal cancer(CRC).CDK15 protein level was determined by Western blot and immunohist ochemistry(IHC)in CRC cell lines,patients’ tumor tissues and commercial tissue microarray;Kaplan-Meier analysis and Chi-squared test were performed to evaluate clinicopathological significance of CDK15 in human CRC.(2)Study the function of CDK15 in human colorectal cancer(CRC).CRC cells with overexpression or knockdown of CDK15 were established and MTT,anchorage-independent growth were performed to examine biological function of CDK15 in CRC cells;Effect of CDK15 on the tumorigenesis was confirmed in Patient-derived-xenograft(CDX)models derived from CDK15 stable knockdown CRC cells;Patient-derived-xenograft(PDX)models with high CDK15 protein level were intratumorally injected with shCDK15-lentivirus to verify CDK15 function in human CRC in vivo.(3)Investigate the effect of Cdk15 knockout on primary colorectal tumor induced by AOM/DSS in mice.AOM/DSS induced CRC model in Cdk15 knockout mice was developed;The role of Cdk15 in the occurrence and development of primary colorectal tumors was further verified by observing the pathological process in mice and detecting the tumor formation in the colorectal section of mice.(4)Explore the detail mechanism of CDK15 in colorectal cancer.Pull down assay and LC-MS/MS analysis were conducted to identify the possible protein which can interact with CDK15;Then the targeted protein which interacted with CDK15 was confirmed by exogenous and endogenous coimmunoprecipitation assay;In vitro kinase assay was performed to detect the phosphorylation of CDK15 on the target protein;The phosphorylated sites in target proteins were predicted by Netphos3.0 software followed by site-specific mutagenesis assay;Finally,kinase assay was performed between specific site-mutation target protein and CDK15 to verify the amino acid sites phosphorylated by CDK15.(5)Investigate the function of PAK4 and its S291 site in colorectal cancer.CRC cells with PAK4 silencing were established.MTT and anchorageindependent assay were used to detect the biological function of PAK4 in colorectal cancer.PAK4 overexpressed CRC cell lines with different mutation states(wild type,inactive,activate)at S291 residue were established,then MTT and anchorageindependent growth assays were performed to examine the function of S291 site in CRC.(6)Explore the role of PAK4 on oncogenic function of CDK15 in colorectal cancer.CDK15 overexpression and PAK4 silencing cell model,CDK15 overexpression plus PAK4 inhibitor cell model were conducted;MTT and anchorage-independent growth assays were used to determine whether PAK4 mediates the oncogenic function of CDK15 in colorectal cancer;PDX mice models with high level of CDK15 and PAK4 protein were intraperitoneally injected with inhibitor of PAK4 to further confirm PAK4 function in the colorectal cancer in vivo.Results(1)CDK15 was highly expressed in human CRC cells,patients’ tumor tissues.High level CDK15 protein predicted poor prognosis and shorter overall survival in CRC patients.(2)CDK15 silencing suppressed cell proliferation and anchorage-independent growth of CRC cells and inhibit the tumorigenic ability of CRC cells in CDX model;On the contrary,overexpression of CDK15 promoted cell proliferation and transformation of CRC cells;In PDX mice model,CDK15 knockdown significantly inhibited tumor growth,and β-catenin/c-Myc,MEK1/2-ERK1/2 signaling were decreased in PDX tumors after CDK15 silencing.(3)Cdk15 knockout significantly alleviated AOM/DSS-induced pathological damage and suppressed the initiation and progression of primary colorectal tumors in mice.(4)PAK4 was identified as a potential target of CDK15 through pull down assay and LC-MS/MS analysis;Exogenous and endogenous co-immunoprecipitation assay indicated that CDK15 could bind with PAK4;CDK15 could phosphorylate PAK4 at S291 residue;Motif containing S291 in PAK4 matches the consensus motif of CDK15 phosphorylated substrates and is highly evolutionarily conserved.(5)PAK4 knockdown inhibited the proliferation and anchorage-independent growth of CRC cells;Inactivated S291 site of PAK4 also inhibited the proliferation and anchorage-independent growth of CRC cells;On the contrary,activated S291 site of PAK4 promoted the proliferation and anchorage-independent growth of CRC cells.(6)Knockdown or inhibition of PAK4 could attenuated the pro-proliferation effect caused by CDK15 overexpression in CRC cells;In PDX models,PAK4 inhibitor significantly suppressed tumor growth in mice,and β-catenin/c-Myc,MEK/ERK signaling were decreased in PDX tumors.ConclusionsCDK15 acts as an oncogene in colorectal cancer and high CDK15 protein level suggests a poor prognosis in CRC patients.CDK15 phosphorylates PAK4 at the S291 residue and actives β-catenin/c-Myc and MEK/ERK signaling pathway,thereby promotes proliferation of CRC cells.Therefore,CDK15 and PAK4 could be potential targets for the treatment and prevention of colorectal cancer.