Ceramide synthase 6 modulates TRAIL sensitivity and nuclear translocation of active caspase-3 in colon cancer cells

Oncogene (2009) 28, 1132–1141 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE Ceramide synthase 6 modulates TRAIL sensitivity and nuclear translocation of active caspase-3 in colon cancer cells S White-Gilbertson1, T Mullen2, C Senkal3, P Lu1, B Ogretmen3, L Obeid2,4 and C Voelkel-Johnson1 1 Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC, USA; 2Department of Medicine, Medical University of South Carolina, Charleston, SC, USA; 3Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA and 4Ralph H Johnson Veterans Affairs Medical Center, Charleston, SC, USA We have previously shown that the death receptor ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces an increase of intracellular C16-ceramide in sensitive SW480 but not in resistant SW620 cells. Resistance in SW620 cells was overcome by exogenous ceramide, leading us to propose that defective ceramide signaling contributes to TRAIL resistance. In this study we found that the increase in C16-ceramide in SW480 cells was inhibited by fumonisin B1, an inhibitor of ceramide synthases (CerS). Protein analysis revealed that TRAILresistant SW620 cells expressed lower levels of ceramide synthase 6 (CerS6, also known as longevity assurance homologue 6), which prompted us to investigate the effect of CerS6 modulation on TRAIL phenotype. RNAi against CerS6 resulted in a specific and significant decrease of the C16-ceramide species, which was sufficient to inhibit TRAIL-induced apoptosis. In cells with decreased levels of CerS6, caspase-3 was activated but failed to translocate into the nucleus. CerS6 localized primarily to the perinuclear region, suggesting this enzyme may be important in regulation of nuclear permeability. Moderate elevation in CerS6 expression was sufficient to reverse TRAIL resistance in SW620 cells. These results suggest that modulation of CerS6 expression may constitute a new therapeutic strategy to alter apoptotic susceptibility. Oncogene (2009) 28, 1132–1141; doi:10.1038/onc.2008.468; published online 12 January 2009 Keywords: TRAIL; ceramide; apoptosis; synthase; longevity assurance homologue ceramide Correspondence: Dr C Voelkel-Johnson, Department of Microbiology and Immunology, Medical University of South Carolina, PO Box 250504, 173 Ashley Ave, Charleston, SC 29403, USA. E-mail: Received 21 July 2008; revised 3 November 2008; accepted 23 November 2008; published online 12 January 2009 Introduction TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a death receptor ligand of the TNF superfamily that can selectively kill cancer cells without toxicity toward normal cells (Wiley et al., 1995; de Jong et al., 2001). Systemic administration of recombinant TRAIL has recently been deemed safe in a Phase I clinical trial (Ashkenazi and Herbst, 2008). Unfortunately, not all malignant cells are susceptible to the apoptotic effects of TRAIL and new strategies to enhance TRAIL-mediated killing of tumor cells are an area of intense investigation. Identification of mechanisms that lead to resistance has a critical twofold purpose: (1) to facilitate stratification of tumors likely to respond to TRAIL therapy and (2) to develop therapeutic strategies that overcome TRAIL resistance. TRAIL induces apoptosis by binding to agonistic TRAIL receptors (DR4/TRAIL-R1 and/or DR5/ TRAIL-R2) followed by activation of initiator caspases and subsequent (mitochondria-dependent or -independent) activation of effector caspases-3 and -7 (MacFarlane, 2003; Koschny et al., 2007). During apoptosis, caspase-3 translocates to the nucleus to cleave targets such as poly (ADP-ribose) polymerase (PARP) (Wilson, 1998; Widlak and Garrard, 2005). Numerous proteins such as cFLIP, antiapoptotic members of the Bcl-2 family and IAPs can negatively regulate the apoptotic signal at or upstream of caspase-3 activation (Wiley et al., 1995; Dohi et al., 2004; Peter, 2004; Sharpe et al., 2004). Sphingolipids have also been shown to influence apoptotic responses. The sphingolipid ceramide in particular has been associated with antiproliferative responses, such as growth arrest, senescence, differentiation and apoptosis (Ogretmen and Hannun, 2004). Data obtained by liquid chromatography–mass spectroscopy (LC/MS), which allows study of specific ceramide species, suggest that generation of C16-ceramide is specifically involved in apoptotic signaling (Thomas et al., 1999; Kroesen et al., 2003). We have recently shown that the isogenic colon cancer cell lines SW480 and SW620 are sensitive and resistant to TRAIL, respectively (Voelkel-Johnson et al., 2005). Our data indicated that differences in TRAIL sensitivity may be TRAIL and CerS6 S White-Gilbertson et al 1133 related to differences in sphingolipid metabolism. TRAIL-sensitive SW480 cells had higher basal levels of C16-ceramide, which further increased in response to TRAIL. We also demonstrated that exogenous C6-ceramide, which is most likely metabolized to C16ceramide (Ogretmen et al., 2002), sensitized resistant SW620 cells to TRAIL-induced apoptosis but failed to further enhance TRAIL sensitivity in SW480 cells, suggesting that in these cells sufficient endogenous ceramide was available to achieve a maximal apoptotic response. We hypothesized that exogenous ceramide corrects a defect in sphingolipid metabolism present only in resistant and not in sensitive cells (VoelkelJohnson et al., 2005). Here we extended our previous study and identified ceramide synthase 6 (CerS6, also known as longevity assurance homologue 6/LASS6), which preferentially generates C16-ceramide, as a novel protein that can influence TRAIL susceptibility. RNAi against CerS6 resulted in a specific decrease in intracellular C16-ceramide and protected SW480 cells against TRAIL-mediated apoptosis while increasing CerS6 expression sensitized SW620 cells to TRAIL. Downregulation of CerS6 did not interfere with caspase activation but appeared to inhibit translocation of activated caspase-3 into the nucleus. Our data suggest that CerS6 may regulate events at the nuclear membrane and allow late-stage apoptotic signaling, exemplified by activated caspase-3, to proceed into the nucleus. This finding posits that CerS6 holds a novel position in the apoptotic pathway. Results TRAIL-induced increases in C16-ceramide are accompanied by decreases in sphingosine and can be inhibited by fumonisin B1 We have previously shown that clinically relevant concentrations of TRAIL selectively increase intracellular levels of C16-ceramide in sensitive SW480 but not resistant SW620 cells (Kelley et al., 2001; VoelkelJohnson et al., 2005). In this study, we initially expanded the time course to determine whether changes in C16ceramide can be detected at later time points in resistant cells. As shown in Figure 1a and Table 1, even 22 h after TRAIL treatment, intrac (...truncated)