The Research For Regulation Mechanism Of DDR2 Prompting Gastric Cancer Progression

BackgroundGastric cancer is one of the most common tumors in the world,and the incidence and mortality of gastric cancer is the second highest in China due to diet,food storage habit and Hp high susceptibility,which is significantly higher than the world average.Over the years,the five-year survival rate after treatment of gastric cancer has been less than 20%.The high mortality of gastric cancer is due to its invasion and metastasis,and its recurrence after treatment.It has become a serious national health problem.In spite of improvements in early detection and diagnosis rate along with progress of clinical diagnosis technology and tumor basic research for gastric cancer,the fundamental situation of high mortality has not changed.Therefore,to clarify the mechanism of metastasis is the focus of its clinical and basic research.Discoidin Domain Receptors(DDRs)belong to the superfamily of tyrosine kinases,which are characterized by their ability to respond to collagen signaling,which is one of the major components of extracellular matrix(ECM)in almost all cell types.In cancers,dysregulaiton of ECM-induced signaling disrupts normal tissue structure and promotes cancer cells migration and invasion.Besides their structural role as scaffolding proteins,DDRs can also trigger cell signaling events through intiating specific cell surface receptors,and to enhance the malignant infiltration of cancer cells.Recently,it has been reported that DDRs can influence the biological activity of cancer cells through posttranslational modification or site-directed mutagenesis of specific domains,such as involved in the survival of tumor cells mediated by p53,and induce a variety of signals of tumor cellsmatrix adhesion,matrix remodeling,and EMT for distant colonization or invasion.DDR2 as a member of the DDRs family is less than DDR1 in cancer-related studies.Especially,few studies have been performed on the expression,mechanism and clinical value of DDR2 in gastric cancer.In the present study,we plan to conduct a preliminary study on DDR2,which is of great significance to reveal the pathophysiological changes in gastric cancer and to improve the clinical therapeutic outcomes.AimsIn this study,we aimed to determine DDR2 expression levels in GC cell lines and tissue samples,to analyze their clinical relevance,and to investigate the effects of DDR2 on gastric cancer cells in vivo and in vitro as well as their underlying mechanisms.Methods1.Real time PCR and Western blot were used to detect the expression of DDR2 mRNA and protein in different differentiation gastric cancer cells(MKN28,MGC803,SGC-7901,BGC823,AGS,MKN-45)and normal gastric mucosa cell GES.The expression on DDR2 mRNA and protein was assayed by the same methods in 45 cases of gastric cancer and adjacent normal tissues.Meanwhile,DDR2 expression was disclosed by immunohistochemical staining in gastric cancer tissues,and the correlation between its levels and patients' clinicopathological features were statistically analyzed.2.To investigate the effects of DDR2 on malignant phenotype of gastric cancer cells,siRNAs,interference and expression vectors for DDR2 were constructed and transfected into gastric cancer cells with different DDR2 expression.At the same time,overexpression of DDR2 cells was also induced by collagen type I stimulation.The proliferation and migration of overexpressed or lowexpressed DDR2 gastric cancer cells were observed by MTT,healing and transwell chamber experiments.BALB/c mice(8-10w)were randomly divided into subcutaneous tumorigenesis groups.After 40 days,mice were sacrificed and subcutaneous tumors were dissected.The tumors volume and weight were determined to observe the effect of DDR2 expression on tumorigenicity.Meanwhile,we constructed gastric cancer xenografts in situ in nude mice,combined with imaging technology,to evaluate the effect of DDR2 on tumor growth in vivo.3.To explore the possible mechanism of DDR2 on gastric cancer cells,Immunohistochemical staining was used to detect the expression of EMT markers,E-cadherin,N-cadherin and Vimentin,in 45 cases of gastric cancerous tissues.And to score in combination with DDR2 staining results(intensity scores: 0 = no staining,1 = weak intensity,2 = moderate intensity and 3 = strong intensity.Proportion scores: 0 = 0–10%,1 = 11–50%,2 = 51–80% and 3 = >80%),and to evaluate the association between DDR2 and EMT expression.Western blot and immunofluorescence staining were used to observe the effect of overexpression or interference of DDR2 on the expression of EMT markers in gastric cancer cells.Next,we examined the effect of down-regulation of DDR2 expression on the phosphorylation of key proteins in classical pathways that may cause EMT alterations in gastric cancer cells by Western blot.Finally,the concrete pathways of DDR2 influencing EMT were detected by different inhibitors of signal molecules and conditioned culture.Results1.We found that DDR2 mRNA and protein levels were higher in GC cell lines versus normal control,and in poorly differentiated GC cells(AGS and MKN45)versus high or middle differentiated GC cells.Next,we assayed DDR2 mRNA level in 45 GC samples,the results showed significant upregulation of DDR2 mRNA expression in GC tissues compared with adjacent normal tissues(2.43 ± 1.97 Vs 1.19 ± 0.68;P=0.038).Analysis of the 12 paired samples showed that DDR2 mRNA was also higher in cancer tissues than in matched non-cancer tissues(P=0.02).We analyzed DDR2 expression in GC specimens using immunohistochemistry.The results revealed that high DDR2 expression was found in 25 GC samples(55.56%)and low expression was found in 20 GC samples(44.44%).DDR2 overexpression was significantly correlated with T stage(P=0.028).The high expression of DDR2 was associated with tumor type,and the expression in intestinal-type GC was higher compared with other types(P=0.045).2.To further evaluate the functional roles of DDR2 in GC cells,we had respectively suppressed DDR2 in GC cell lines(AGS and MKN45)that has higher level of DDR2 protein,and overexpressed DDR2 in gastric cancer cell lines(AGS and MKN45)with lower DDR2 level.MTT assay showed that up /down-regulation of DDR2 expression significantly promote or inhibit the proliferation of gastric cancer cells.Healing and invasion experiments found that up /down-regulation of DDR2 expression could significantly promote or inhibit gastric cancer cell migration and invasion.In particular,the effect of up-regulation of DDR2 can be enhanced by stimulation of collagen I.Tumorigenesis experiment results were consistent with the in vivo study.The tumor volume(MKN28 P<0.001,MGC803 P=0.019)and weight(MKN28 P=0.014,MGC803 P<0.001)of DDR2 overexpression group were significantly higher than those of the control group.On the contrary,interference with DDR2 expression significantly inhibited tumorigenesis in vivo,tumor weight(AGS P=0.019,MKN45 P=0.016)and volume(AGS P=0.01,MKN45 P<0.001)were significantly smaller than the control group.In situ xenografts experiments showed that overexpression of DDR2 enhanced tumor formation,and the average signals were significantly higher than those infected with control.3.We analyzed the correlations between DDR2 and EMT-related proteins(E-cadherin,N-cadherin and Vimentin)in GC tissues.Spearman rank correlation analysis showed that high expression of DDR2 was significantly associated with increased N-cadherin and Vimentin expression(r = 0.28,P=0.006,and r=0.328,P=0.028,respectively)and reduced E-cadherin expression(r =-0.3328,P=0.023).We also found that when DDR2 was suppressed,E-cadherin expression was increased,and the expression of N-cadherin and Vimentin was significantly reduced in GC cells.These data suggest that DDR2 may be an important regulator of EMT in GC tissues.The results of signal screening showed that the expression of DDR2 in GC cells affected ERK or AKT signaling pathway,and AKT was more significant,but had little effect on Src and GSK-3? signal.Next,inhibitors and conditioned cultures found that DDR2 showed AKT phosphorylation through mTORC2 rather than mTORC1,ultimately affecting the EMT process in GC cells.Conclusions1.DDR2 is highly expressed in GC cell lines and tissues,and correlated with clinical stage and pathologic type.2.DDR2 promotes proliferation,migration and invasion of gastric cancer cells.This effect depends partly on its binding to collagen.3.DDR2 plays an important role in the migration and invasion of gastric cancer cells by stimulating the formation of mTORC2 complex with collagen,activating AKT signaling,and leading to EMT process.