Objectives: Metastasis is the main cause of treatment failure and tumor associated death in cancer patients.Elucidated the molecular mechanism of colorectal cancer metastasis will be beneficial to provide potential therapeutic targets and improve the prognosis of patients.Our research groups previously founded that CEMIP(cell migration inducing protein)promoted colorectal cancer metastasis.Unfortunately,the mechanisms of colorectal cancer metastasis remain to be elucidated.Therefore,our research would explore the specific mechanisms of colorectal cancer metastasis by CEMIP.Methods: To elucidate the latent molecular mechanisms involved in CEMIP-mediated colorectal cancer metastasis,Co-IP(co-immunoprecipitation)and mass-spectrometric peptide sequencing was conducted to identify CEMIP-interacting proteins in cells and then the most valuable protein,GRAF1(GTPase regulator associated with focal adhesion kinase-1),was identified.CEMIP had negative effects on the expression of GRAF1 in colon cancer cells which were examined by PCR(polymerase chain reaction)and Western blot.The expression levels of CEMIP and GRAF1 in human colon cancer tissues were evaluated by immunohistochemical techniques,and the correlation between CEMIP and GRAF1 expression was explored by correlation analysis.The E3 ubiquitin ligase-Mi B1(Mind Bomb-1)regulating GRAF1 degradation was investigated and verified by database analysis and Co-IP experiment.Co IP and Western blot experiments were used to explore the relationship between the three proteins.CDC42(Cell division control protein 42)and MAPK(mitogen-activated protein)and Epithelial mesenchymal transition(EMT)related protein which were the downstream of GRAF1 were determined by Western blot assay.The effects of CEMIP,MIB1,GRAF1 and CDC42 on invasion and metastasis of human colon cancer cells were investigated by Transwell assay in vitro and splenohepatic metastasis model in nude mice.The specific molecular mechanism of CEMIP promoting GRAF1 degradation and then tumor metastasis was investigated by Co-IP,Western blot,immunohistochemistry,immunofluorescence,and proximity ligation assay experiments.Results: In the current study,it is identified that CEMIP interacts with GRAF1,and the combination of high-CEMIP and low-GRAF1 predicts poor survival of patients.Mechanistically,it is elucidated that CEMIP interacts with the SH3 domain of GRAF1 through the 295-819 aa domain,and negatively regulates the stability of GRAF1.Moreover,MIB1 is founded to be an E3 ubiquitin ligase for GRAF1,not for CEMIP.Importantly,CEMIP acts as a scaffold protein in bridging MIB1 and GRAF1,promoting the binding between MIB1 and GRAF1,which is critical to GRAF1 degradation and CEMIPmediated CRC metastasis.Furthermore,it is found that CEMIP activates CDC42/MAPK pathway-regulated EMT by enhancing the degradation of GRAF1,which is indispensable to CEMIP-mediated migration and invasion of CRC cells.Subsequently,CDC42 inhibitor ZCL278 suppresses CEMIP-mediated CRC metastasis in vitro and in vivo.Conclusions: In conclusion,this study found that CEMIP mediated ubiquitination and degradation of GRAF1 by acting on the SH3 domain of GRAF1,a cancer suppressor protein.Further studies found that CEMIP acted as a scaffold protein,promoting the bridge between E3 ubiquitin-ligase MIB1 and GRAF1,leading the degradation of GRAF1,and then activating downstream CDC42/MAPK signaling pathway and inducing EMT,and finally promoting the invasion and migration of colon cancer cells.Importantly,in vitro and in vivo studies further demonstrated that interfering with CDC42 significantly inhibited CEMIP’s ability to induce colon cancer cell metastasis.Collectively,CEMIP promotes CRC metastasis through GRAF1/CDC42/MAPK pathway-regulated EMT and suggest that CDC42 inhibitor could be a novel therapeutic strategy for CEMIP-mediated CRC metastasis. |