BACKGROUND Calponin 3(CNN3)is an actin-binding protein expressed in smooth muscle and non-smooth muscle cells.It is required for cytoskeletal rearrangement and wound healing.AIM To dissect the role of CNN3 in carcinog...BACKGROUND Calponin 3(CNN3)is an actin-binding protein expressed in smooth muscle and non-smooth muscle cells.It is required for cytoskeletal rearrangement and wound healing.AIM To dissect the role of CNN3 in carcinogenesis with a focus on colon cancer.METHODS A total of 20 cancer cell lines(8 breast,11 colon,and HeLa cervical cancer cell as a positive control for mesenchymal phenotype)and 57 formalin-fixed,paraffinembedded sections from archived sporadic colorectal carcinomas were included in this study.CNN3 expression analysis by western blot or immunohistochemistry was followed by functional analyses.The CNN3 gene was silenced by specific small interfering RNA(commonly known as siRNA),followed by confirmation of the silencing efficiency by western blotting.Then,the silenced cells and control siRNA-transfected cells were analyzed for changes in epithelial and mesenchymal markers,invasion,and response to 5-fluoruracil treatment.We also performed proteomics analysis using a phospho-kinase array-based panel of 45 proteins.RESULTS CNN3 showed positive expression in 6/8 breast and 9/11 colon cancer lines and in HeLa cells.Interestingly,the colorectal adenocarcinoma line SW480 was negative,while the cell line developed from its matching lymph node metastasis(SW620)was positive for CNN3.CNN3 expression was fairly consistent with the metastatic phenotype in colon cancer because it was absent in one other colon cell line from a primary site and expressed in all others.We selected SW620 for subsequent functional analyses.CNN3-silenced SW620 cells showed a reduction in collagen invasion and loss of mesenchymal markers.CNN3 silencing caused an increase in the SW620 colon cancer cell sensitivity to 5-fluorouracil.Phosphokinase array-based proteomics analysis showed that CNN3 silencing in SW620 reduced extracellular signal-regulated kinase,β-Catenin,mutant p53,c-Jun,and heat shock protein 60 activities but increased that of checkpoint kinase 2.CNN3 was expressed in 20/57(35%)colon cancer cases as shown by immunohistochemist展开更多
文摘BACKGROUND Calponin 3(CNN3)is an actin-binding protein expressed in smooth muscle and non-smooth muscle cells.It is required for cytoskeletal rearrangement and wound healing.AIM To dissect the role of CNN3 in carcinogenesis with a focus on colon cancer.METHODS A total of 20 cancer cell lines(8 breast,11 colon,and HeLa cervical cancer cell as a positive control for mesenchymal phenotype)and 57 formalin-fixed,paraffinembedded sections from archived sporadic colorectal carcinomas were included in this study.CNN3 expression analysis by western blot or immunohistochemistry was followed by functional analyses.The CNN3 gene was silenced by specific small interfering RNA(commonly known as siRNA),followed by confirmation of the silencing efficiency by western blotting.Then,the silenced cells and control siRNA-transfected cells were analyzed for changes in epithelial and mesenchymal markers,invasion,and response to 5-fluoruracil treatment.We also performed proteomics analysis using a phospho-kinase array-based panel of 45 proteins.RESULTS CNN3 showed positive expression in 6/8 breast and 9/11 colon cancer lines and in HeLa cells.Interestingly,the colorectal adenocarcinoma line SW480 was negative,while the cell line developed from its matching lymph node metastasis(SW620)was positive for CNN3.CNN3 expression was fairly consistent with the metastatic phenotype in colon cancer because it was absent in one other colon cell line from a primary site and expressed in all others.We selected SW620 for subsequent functional analyses.CNN3-silenced SW620 cells showed a reduction in collagen invasion and loss of mesenchymal markers.CNN3 silencing caused an increase in the SW620 colon cancer cell sensitivity to 5-fluorouracil.Phosphokinase array-based proteomics analysis showed that CNN3 silencing in SW620 reduced extracellular signal-regulated kinase,β-Catenin,mutant p53,c-Jun,and heat shock protein 60 activities but increased that of checkpoint kinase 2.CNN3 was expressed in 20/57(35%)colon cancer cases as shown by immunohistochemist
