Includes bibliographical references (leaves 50-53). ; In this study, we attempt to simultaneously target the major hallmarks of colorectal cancer (CRC), the MAPK pathway and the urokinase plasminogen activator system using a dual-selective, Urokinase-activated, recombinant anthrax lethal toxin (PrAgU2/LF). Colorectal cancer is the third most common cancer type in the world, and the second leading cause of death in the United States according to the American cancer society. Therefore, the need for alternative approaches is essential in order to overcome the difficulties faced with traditional therapies. PrAgU2/LF is composed of 2 components the binding moiety PrAgU2 and the catalytic moiety LF. PrAgU2 was obtained by replacing the furin cleavage sequence on PrAg with a urokinase- specific cleavage sequence. LF is a metalloprotease that cleaves and inactivates MEKs and hence inhibits the MAPK pathway. We assessed the cytotoxic effects and mechanisms of PrAgU2/LF on a panel of 9 human colorectal cancer cell lines.PrAg/LF, PrAg/FP59 and PrAgU2/FP59 were used as controls for the different components of this system. PrAgU2/LF was not significantly cytotoxic to any of the cell lines tested, which were also resistant to PrAg/LF, indicating resistance to the inhibition of the MAPK pathway. This was further confirmed by resistance of these cells to the MEK1/2 inhibitor U0126. Notably, the majority of cell lines showed high levels of phospho-MEK1/2 but, surprisingly, no phospho-ERK1/2. Furthermore, targeting both the MAPK pathway and the PI3-kinase/Akt pathway also failed to induce cytotoxicity in colon cancer cell lines. In order to assess the possibility of targeting the uPA/uPAR system alone, we determined expression levels of uPAR and tested the sensitivity of CRC cells to PrAgU2/FP59, a urokinase activated, MAPK-independent version of the toxin. The vast majority of cell lines tested expressed uPAR and were sensitive to PrAgU2/FP59, indicating the possibility for targeting this protease system in CRC. ; 1 hard copy: ...