c-FLIP: a key regulator of colorectal cancer cell death.
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.
Wilson
Timothy R
TR
Drug Resistance Group, Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, Northern Ireland.
McLaughlin
Kirsty M
KM
McEwan
Miranda
M
Sakai
Hidekazu
H
Rogers
Katherine M A
KM
Redmond
Kelly M
KM
Johnston
Patrick G
PG
Longley
Daniel B
DB
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015179:Colorectal Neoplasms / Q000378:metabolism*; D053200:Fas-Associated Death Domain Protein / Q000378:metabolism; D005434:Flow Cytometry; D020868:Gene Silencing; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism
D000970:Antineoplastic Agents; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D053200:Fas-Associated Death Domain Protein; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand