Caspase-3 is known as a cysteine protease that primarily executes the cell death program. However, some tumors express higher levels of caspase-3 in positive correlation with malignancy. Here, we showed that caspase-3 can promote tumor metastasis in a protease-independent mechanism. Ectopic expression of caspase-3 enhanced lung metastasis and cell motility of caspase-3 deficient MCF-7 cells. By contrast, caspase-3 siRNA reduced the invasiveness and metastasis ability of A549 cells that express high level of caspase-3. Moreover, caspase-3 induced ERK activation. Alteration of caspase-3 by introducing non-processable mutation at its cleavage site or treatment of caspase-3 inhibitor did not diminish the caspase-3-associated increases in ERK phosphorylation and cell migration. Confocal microscopy study showed that caspase-3 was not physically associated with ERK. Inhibiting ceramide formation by blockage of the ceramide synthase or acid sphingomyelinase activity resulted in significant reduction of ERK phosphorylation and cell migration. In summary, caspase-3 induces ERK activation through a ceramide-dependant, protease activity-independent mechanism, which represents a novel role of caspase-3 in tumor metastasis. ' 2008 Wiley-Liss, Inc.Key words: caspase-3; extracellular signal-regulated kinase; metastasis Stimuli, such as death receptor ligation, cytotoxic stress and mitochondria injury, lead to cleavage and activation of the full length caspase-3 by granzyme B, caspase-8 and caspase-9. Subsequently, the activated caspase-3 executes proteolytic events required for programmed cell death. 1,2 It is proposed that the elimination of cancer cells through the apoptosis pathway either by stimulating the expression of caspase-3 or by activating its protease activity can serve as a common strategy in cancer therapies. 3 However, contradictory results exist. For instance, gastric cancer cells express higher levels of both procaspase-3 and activated-caspase-3 as compared to normal tissues. 4,5 Some breast cancers, melanomas and leiomyomas have an elevated caspase-3 expression along with an increase in tumor progression and metastasis. 6-8 Caspase-3 also plays a critical role in erythroid maturation 9 and osteogenic differentiation. 10 The potential tumor promoting effects of caspase-3, thus, constrain the application of caspase-3 in antitumor therapies. Currently, how caspase-3 affects tumor malignancy and differentiation remains unknown.Migration of cancer cells into the surrounding tissue is an important step for metastasis. Mitogen activated protein kinases (MAPKs) play a major role in basal membrane degradation, cell migration and survival of invasion cells in a new environment. [11][12][13][14] Among the MAPK family, extracellular signal-regulated kinases 1 and 2 (ERK1/2), located at the membrane periphery, are required for focal adhesion disassembly, cell spreading and motility. 15,16 Tumor-explanted cells carrying the effectors domain of Ras oncoprotein (Raf, PI3K or RalGEF) mutants grow rapidly, but only the cells c...