Comparative proteomics analysis identifies Cdc42-Cdc42BPA signaling as prognostic biomarker and therapeutic target for colon cancer invasion

Eight rounds of selection of cancer cells invading through matrigel-coated chamber was performed to obtain highly invasive colon cancer sublines HCT116-I8 and RKO-I8. Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) technology was used to identify the differently expressed proteins and the proteomics data was analyzed by ingenuity pathway analysis (IPA). PAK1-PBD immunoprecipitation combined with Western blot were carried out to determine Cdc42 activity, and qRT-PCR and Western blot were used to determine gene expression. The functional role of Cdc42BPA and Cdc42 pathway in colon cancer invasion was studied by loss-of-function experiments including pharmacological blockade, siRNA knockdown, chamber invasion, and WST-1 assays. Human colon cancer tissue microarray was analyzed by immunohistochemistry for overexpression of Cdc42BPA and its correlation with clinicopathological parameters and patient survival outcomes.