The Effect And Mechanism Of Discoidin Domain Receptor 2 Promoting The Invasion And Metastasis Of Colorectal Cancer

Objectives: Colorectal cancer(CRC)is the third most common malignant tumor and also the second leading cause of tumor-related mortality worldwide.Tumor metastasis is one of the main reasons for the high mortality of CRC,and the 5-year overall survival(OS)rate of patients with advanced stage CRC is only 10-18%.In addition to KRAS,BRAF and MEK,discoidin domain receptor 2(DDR2)and its roles in cancers have also drawn attention of researchers.DDR2 is a unique receptor tyrosine kinase that takes a variety of collagens as ligands.Studies have reported that DDR2 is highly expressed in both stromal cells and tumor cells of various tumors such as lung cancer,breast cancer,and gastric cancer,and is involved in the processes of proliferation,differentiation,epithelial mesenchymal transition(EMT),migration,invasion and metastasis of tumor cells.However,the effect of DDR2 and its underlying regulatory mechanism on CRC have not been fully understood.This study aims to explore the role and mechanism of DDR2 on the biological characteristics of CRC,especially on the invasion and metastasis.Methods: We analyzed the data of CRC patients from public databases and performed the immunohistochemistry(IHC)staining of the tissue microarray of CRC patients to verify the correlation between the expression of DDR2 and the prognosis of patients.The pooled DDR2-knockdown RKO cell line was constructed by lentivirus-mediated CRISPR/Cas9 method,and DDR2-overexpressing HCT116,SW480,and DLD-1 stable cell lines were established using lentivirus-mediated plasmid transfection.Besides,DDR2 inhibition in MC38,SW480,and HCT8 cells were achieved by Dasatinib treatment,which is a multi-kinase inhibitor including DDR2.In in vitro experiments,proliferation assay was detected by CCK8 method,and migration as well as invasion ability were verified by transwell chambers.Additionally,the effect of DDR2 on tumor growth,invasion and metastasis of CRC in vivo was verified by subcutaneous xenograft nude mouse and tail vein injection-based metastasis models.Finally,the changes of EMT-related proteins(E-cadherin and vimentin)and core components of AKT/GSK-3β/Slug signaling pathway were detected by western blot.Results: By analyzing the data from the GEPIA2 and Progno Scan public databases,high DDR2 expression level correlated with poor survival in CRC patients.DDR2-targeted IHC staining on tissue microarrays collected from 94 CRC patients showed that DDR2 was positively expressed in 48.9%(46/94)of tumor tissues;however,only 4.7%(4/86)of matched normal adjacent tissues were DDR2-positive.Patients with high DDR2 expression had a trend toward poor overall survival(P=0.53).In in vitro experiments,although knockdown or overexpression of DDR2 in CRC cells had no significant effect on the proliferation,enhanced expression of DDR2 significantly promoted the migration and invasion of CRC.Inversely,downregulation of DDR2 and application of Dasatinib inhibited their migration and invasion ability.In vivo,overexpression of DDR2 substantially promoted the growth and local invasion ability of subcutaneous xenografts,while knockdown of DDR2 significantly decreased the lung metastatic capacity of CRC.Mechanically,DDR2 overexpression promoted EMT,especially significantly reducing the expression of E-cadherin and increasing the expression of vimentin.Conversely,knockdown of DDR2 increased E-cadherin and decreased vimentin expression.Additionally,upregulated DDR2 induced the phosphorylation of AKT and GSK-3β,thereby increasing the expression of Slug proteins.Conclusions: In CRC,DDR2 expression was higher in tumors than that in matched normal tissues.Patients with high expression of DDR2 tended to have a poor prognosis.DDR2 did not directly increase cell proliferation,but promoted the invasion and metastasis of CRC through regulating the process of EMT by activating the AKT/GSK-3β/Slug signaling pathway.In conclusion,DDR2 promotes the progression of CRC and may become one of the therapeutic targets for inhibiting the invasion and metastasis of CRC in the future.