The Role And Mechanism Of CD44v6 On Chemotherapy Resistance And Its Expression In Human Colorectal Cancer

Background&ObjectiveChemotherapy is an effective treatment of colorectal cancer.Chemotherapy resistance is the main reason of failure of chemotherapy.CD44v6,a marker of colorectal cancer stem cells,play an important role on tumorigenesis,progression and metastasis.However,the role of CD44v6 in the chemotherapy resistance of colorectal cancer is unclear,and its molecular mechanism is still unclear.In this study,we investigated the role of CD44v6 on the chemotherapy resistance of human colorectal cancer and the related molecular mechanisms in colorectal cancer cell lines.Moreover,the correlation between CD44v6 and clinical prognosis and intratumor heterogeneity were studied in colorectal cancer tissues.MethodsThe experiment is divided into three chapters.In Chapter 1,we constructed a stable low-expressed cell model and a stable over-expressed model of CD44v6,and observed the effects of CD44v6 on the drug tolerance,cell proliferation and apoptosis in the presence of 5-FU.Then the common mechanisms of chemotherapy resistance were screened.In Chapter 2,through retrospective analysis of clinical and pathological data of colorectal cancer,immunohistochemistry of tissue microarray was used to evaluate CD44v6,ABCG2 and Beclinl,respectively,to investigate the relationship between CD44v6,ABCG2,Beclinl and clinical pathological data.In Chapter 3,the intratumoral heterogeneity of CD44v6 expression in rectal cancer was analyzed by immunohistochemistry,real-time fluorescence quantitative PCR and immunofluorescence double staining.ResultsChapter 1:chemotherapy could increase the expression of CD44v6 in colorectal cancer.Over expression of CD44v6 could reduce the sensitivity of colorectal cancer cells to chemotherapeutic drugs,and be able to maintain drug resistance.The relative expression levels of ABCB1 and ABCG2 mRNA cells in CD44v6 over-expressed cells were lower than that of the control group in the presence or absence of 5-FU.Compared with control group,the relative expression level of BECN1 mRNA had no significant difference in the absence of 5-FU,but was significantly higher in the presence of 5-FU.Overexpression of CD44v6 can enhance autophagy of SW480 cells in the presence of 5-FU.After autophagy was blocked,the drug sensitivity of CD44v6 overexpressing cells was increased.5-FU could increase the expression of E-cadherin in colon cancer cells and decrease the expression of Vimentin.However,in CD44v6 overexpressing cells,the expression level of E-cadherin was lower than that of the control group in the presence or absence of 5-FU,but the expression level of Vimentin was higher than that of the control group.These results suggested that CD44v6 could promote the epithelial mesenchymal transition of SW480 cells and inhibited the 5-FU induced epithelial mesenchymal transition.Under 5-FU treatment,the levels of Erkl/2 and Akt phosphorylation in CD44v6 overexpressing cells were significantly higher than those in the control group.These results suggested that 5-FU could activate Erkl/2 and Akt related signaling pathways in SW480 cells,and overexpression of CD44v6 could increase the level of activation,thereby promoting the survival of cells in the drug.In the second chapter,CD44v6,ABCG2 and Beclinl were expressed in different degrees in colorectal cancer tissues by immunohistochemistry of tissue microarray.Survival analysis showed that the expression levels of CD44v6,ABCG2 and Beclinl were not correlated with OS and DFS.Through the correlation analysis of CD44v6,ABCG2 and Beclinl by MOD values,there was a significant positive correlation between CD44v6 and ABCG2,there was a significant positive correlation between CD44v6 and Beclinl,and there was no significant correlation between ABCG2 and Beclinl.However,the correlations between CD44v6 and ABCG2,Beclinl were weak,which might be no significance.In the third chapter,the expression level of CD44v6 was successively increased in rectal cancer adjacent tissues,primary tumor and positive lymph nodes.In the primary tumor,CD44v6 could be both expressed in the cytomembrane and cytoplasm,and a few of them were only expressed in one location.The expression level of CD44v6 in rectal cancer adjacent tissues was significantly higher than that in the cancer center.Similar expression level and expression pattern of CD44v6 had different activation effects on downstream p-Erk1/2 and p-Akt related signaling pathways.Conclusions1 Chemotherapy drugs could induce the expression of CD44v6.In the presence of chemotherapy drugs,CD44v6 can activate autophagy to acquire drug resistance.Other possible mechanisms of resistance included epithelial mesenchymal,altered drug pump,and activation of p-Erk1/2 and p-Akt signaling pathways.2 Survival analyses showed that the expression levels of CD44v6,ABCG2 and Beclinl were not correlated with OS and DFS.3 The expression of intratumoral heterogeneity of CD44v6 existed in rectal cancer.It could be described as location heterogeneity,spatial heterogeneity and functional heterogeneity.