Caspase-3 enhances lung metastasis and cell migration in a protease-independent mechanism through the ERK pathway Yu-Jung Cheng 1 , Chien-hsin Lee 1 , Yu-Ping Lin 2 , Jyun-Yuan Huang 2 , Chung-Chen Su 2 , Wen-Tsan Chang 3 and Bei-Chang Yang 1,2,4 * 1 Department of Microbiology and Immunology, National Cheng Kung University, Tainan 70428, Taiwan 2 Institute of Basic Medical Sciences, National Cheng Kung University, Tainan 70428, Taiwan 3 Department of Biochemistry, National Cheng Kung University, Tainan 70428, Taiwan 4 College of Medicine and Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University, Tainan 70428, Taiwan Caspase-3 is known as a cysteine protease that primarily executes the cell death program. However, some tumors express higher lev- els of caspase-3 in positive correlation with malignancy. Here, we showed that caspase-3 can promote tumor metastasis in a prote- ase-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 invasive- ness and metastasis ability of A549 cells that express high level of caspase-3. Moreover, caspase-3 induced ERK activation. Altera- tion 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 for- mation by blockage of the ceramide synthase or acid sphingomye- linase activity resulted in significant reduction of ERK phospho- rylation and cell migration. In summary, caspase-3 induces ERK activation through a ceramide-dependant, protease activity-inde- pendent 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 mi- tochondria 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, melano- mas 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 cas- pase-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 im- portant 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. 11–14 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 onco- protein (Raf, PI3K or RalGEF) mutants grow rapidly, but only the cells containing Ras mutant (V12S35) which activates ERK path- ways can form metastatic lesions in the lung. 17 The motility and in vitro invasiveness of K-ras-activated colon carcinoma cells depend on active ERK signaling. The evidences show that cell migration on a two-dimensional substrate or in three-dimensional matrices were severely hindered by MEK inhibitors U0126 and PD184352. 18 Thus, the activity of ERK may be a major driving force behind the highly metastatic behavior. 19,20 Particularly in the breast cancer cell line MCF-7, ERK activation is associated with increased cell migration. 21 These findings have prompted us to investigate the possible contribution of caspase-3 in ERK activation. To elucidate the role of caspase-3 during tumor progression, we introduced the caspase-3 gene into MCF-7 breast cancer cells, which are caspase-3-deficient and show low metastatic ability. 22 In addition, siRNA strategy was used to suppress the caspase-3 gene in A549 lung adenocarcinoma cells. Cell migration and lung metastasis of those cells showing various levels of caspase-3 were evaluated. A positive correlation of caspase-3 expression with ERK phosphorylation, cell migration and lung metastasis was observed. Our data thus describe a novel function of caspase-3 involving in tumor malignancy. Results Modulation of caspase-3 in MCF-7 and A549 The caspase-3 was reconstituted in MCF-7 cells by transfection with pN1-C3 plasmid, which encodes the full-length human cas- pase-3 cDNA. The stable cell line, MCF-7/C3, expressing a high level of caspase-3, was established (Fig. 1a). The caspase-3 gene in A549 cells was knocked down by transfection of siC3 plasmid encoding for caspase-3 siRNA. The stable cell line A549-siC3 expressed low level of caspase-3 (Fig. 1b). MCF-7/C3 and A549- siC3 did not show obvious changes in morphology and prolifera- tion (data not shown). Although MCF-7/C3 cells had a minute amount of auto-cleaved caspase-3 protein fragments, their sponta- neous apoptosis rate was less than 10%. Cisplatin (32lM) in- creased the apoptosis rate of MCF-7/C3 cells to 30% in 72 hr (Fig. 1a). Conversely, transfection of siC3 plasmid increased the resistance of A549 cells to cisplatin treatment (Fig. 1b). Cisplatin (16 lM) induced 28% of the control A549 cells to undergo apo- ptosis in 24 hr. In contrast, the apoptosis of A549-siC3 cells after cisplatin treatment was around 18%. Caspase-3 enhances lung metastasis of A549 and MCF-7 cells Tumor metastasis was evaluated using an experimental lung metastasis model (Fig. 1c). When injected into nude mice via the tail vein, MCF-7 cells did not form any tumor foci in the lungs, which is in agreement with previous findings that MCF-7 cells have little metastatic ability. Interestingly, three of the 10 mice Grant sponsor: National Science Council of Taiwan; Grant number: NSC 96-2320-B-006-021-MY3. *Correspondence to: Department of Microbiology and Immunology, National Cheng Kung University Medical College, Tainan 70428, Taiwan. Fax: 1886-6-208-2705. E-mail: y1357@mail.ncku.edu.tw Received 1 October 2007; Accepted after revision 15 February 2008 DOI 10.1002/ijc.23592 Published online 11 July 2008 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 123, 1278–1285 (2008) ' 2008 Wiley-Liss, Inc. Publication of the International Union Against Cancer