c-FLIP: A Key Regulator of Colorectal Cancer Cell Death

Abstract

c-FLIP is an inhibitor of apoptosis mediated by the death receptors Fas, DR4, and DR5 and is expressed as long (c-FLIP L ) and short (c-FLIP S ) splice forms. We found that small interfering RNA (siRNA)-mediated silencing of c-FLIP induced spontaneous apoptosis in a panel of p53 wild-type, mutant, and null colorectal cancer cell lines and that this apoptosis was mediated by caspase-8 and Fas-associated death domain. Further analyses indicated the involvement of DR5 and/or Fas (but not DR4) in regulating apoptosis induced by c-FLIP siRNA. Interestingly, these effects were not dependent on activation of DR5 or Fas by their ligands tumor necrosis factor-related apoptosis-inducing ligand and FasL. Overexpression of c-FLIP L , but not c-FLIP S , significantly decreased spontaneous and chemotherapy-induced apoptosis in HCT116 cells. Further analyses with splice form-specific siRNAs indicated that c-FLIP L was the more important splice form in regulating apoptosis in HCT116, H630, and LoVo cells, although specific knockdown of c-FLIP S induced more apoptosis in the HT29 cell line. Importantly, intratumoral delivery of c-FLIP-targeted siRNA duplexes induced apoptosis and inhibited the growth of HCT116 xenografts in BALB/c severe combined immunodeficient mice. In addition, the growth of c-FLIP L -overexpressing colorectal cancer xenografts was more rapid than control xenografts, an effect that was significantly enhanced in the presence of chemotherapy. These results indicate that c-FLIP inhibits spontaneous death ligand-independent, death receptor-mediated apoptosis in colorectal cancer cells and that targeting c-FLIP may have therapeutic potential for the treatment of colorectal cancer.

Combined inhibition of FLIP and XIAP induces Bax-independent apoptosis in type II colorectal cancer cells

“…In addition, we found that FLIP promotes resistance to chemotherapy, as FLIP gene silencing enhanced chemotherapy-induced cell death in vitro, and FLIP L overexpression conferred resistance to chemotherapeutic agents both in vitro and in vivo (Longley et al, 2006;Wilson et al, 2007). We and others have found that FLIP overexpression renders cells resistant to death ligands (Jonsson et al, 2003;Longley et al, 2006), whereas FLIP downregulation increases sensitivity (Yeh et al, 2000;Mathas et al, 2004;Galligan et al, 2005;Longley et al, 2006).…”

“…FLIP has been found to be overexpressed in many human cancers (Ryu et al, 2001;Zhou et al, 2004;Valnet-Rabier et al, 2005), and has emerged through our work and that of others, as a potential anti-cancer drug target (Hyer et al, 2005;Wilson et al, 2007). Furthermore, XIAP has been found to be overexpressed in several cancers, and small molecule and antisense approaches to target XIAP are in development (Hu et al, 2003;LaCasse et al, 2006).…”

“…We have demonstrated previously that silencing FLIP induces apoptosis in a panel of colorectal cancer cell lines in a manner that is dependent on caspase 8 and DR5, but that appears to be independent of DR5 ligation by TRAIL (Wilson et al, 2007). Furthermore, we found that injecting FLIP Cells were re-transfected 24 h later with 1 nM SC or FT siRNA and co-treated with 5 ng/ml TRAIL for a further 24 h. The statistical significance of differences in apoptosis were calculated using two-way ANOVA; * represents the difference between SC, FT, XT and FT þ XT groups (Po0.05); # represents the difference between SC, SC þ TRAIL, XT and XT þ TRAIL groups (Po0.05); a represents the difference between SC, SC þ TRAIL, FT and FT þ TRAIL groups (P ¼ 0.03); b represents the difference between SC, SC þ TRAIL, FT þ XT and FT þ XT þ TRAIL groups (P ¼ 0.007).…”

“…siRNA transfections FLIP-targeted and the non-silencing control siRNAs (Dharmacon, Lafayette, CO, USA) were described earlier (Wilson et al, 2007). Pre-designed siRNAs were purchased for Bid (SMARTpool, Dharmacon), XIAP (Cell Signaling Technology), caspase 9 (Qiagen, Germantown, MD, USA and Imgenex, San Diego, CA, USA) and Smac (Qiagen).…”

“…We have shown previously that FLIP gene silencing can induce apoptosis in colorectal cancer cells and sensitize them to TRAIL-and chemotherapy-induced apoptosis (Longley et al, 2006;Wilson et al, 2007). It has been suggested that chemotherapy-induced DR5 upregulation sensitizes type II colorectal cancer cells to TRAIL when mito-chondrial-mediated apoptosis is blocked by converting them into type I cells (Wang and El-Deiry, 2003).…”