Proteomic analysis of colonic myofibroblasts and effect on colon cancer cell proliferation

Background. The stromal microenvironment influences many steps of tumor progression through the elaboration of signals from myofibroblasts. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway transduces signals initiated by growth factors and is involved in colonic epithelial proliferation. The purpose of this study was to determine (1) the influence of myofibroblasts on colon cancer cell proliferation and PI3K activity, and (2) the protein alterations associated with myofibroblasts derived from polyp versus normal margins. Methods. Myofibroblasts were derived from polyps and corresponding normal mucosa. Myofibroblasts were cocultured with colon cancer cells HT29 stably transfected with green fluorescent protein and KM20 cells. Proliferation was quantitated by green fluorescent protein count and cytokeratin enzyme-linked immunosorbent assay. HT29 cells were incubated with conditioned medium from myofibroblasts, and the effect on proliferation and PI3K activity was determined by 5-bromo 2-deoxyuridine incorporation and AN kinase assay, respectively. Protein profiles were obtained by SELDI-TOF MS analysis. Results. In coculture experiments, all myofibroblasts significantly enhanced HT29 and KM20 cell proliferation. However, polyp myofibroblasts enhanced proliferation of the cancer cells to a greater extent than normal myofibroblasts. Conditioned medium from all myofibroblasts stimulated AN kinase activity. SELDI-TOF MS profiles showed more than 40 protein peaks for each isolate. One protein was differentially expressed in polyps versus normal cells. Conclusions. Utilizing a novel proleomic approach, we identify distinct protein profiles in myofibroblasts of polyps compared with stromal cells of normal mucosa. Moreover, myofibroblasts can stimulate indirectly PI3K activity and enhance colon cancer cell proliferation. These findings suggest that targeted therapy to signaling pathways in myofibroblasts may be useful in colorectal cancer chemoprevention and possible treatment.