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.

[1]  K. Koretz,et al.  Expression of APO‐1 (CD95), a member of the NGF/TNF receptor superfamily, in normal and neoplastic colon epithelium , 1994, International journal of cancer.

[2]  Arul M. Chinnaiyan,et al.  FADD, a novel death domain-containing protein, interacts with the death domain of fas and initiates apoptosis , 1995, Cell.

[3]  David Wallach,et al.  Involvement of MACH, a Novel MORT1/FADD-Interacting Protease, in Fas/APO-1- and TNF Receptor–Induced Cell Death , 1996, Cell.

[4]  D. Goeddel,et al.  Casper is a FADD- and caspase-related inducer of apoptosis. , 1997, Immunity.

[5]  Margot Thome,et al.  Inhibition of death receptor signals by cellular FLIP , 1997, Nature.

[6]  R. Gentz,et al.  I-FLICE, a Novel Inhibitor of Tumor Necrosis Factor Receptor-1- and CD-95-induced Apoptosis* , 1997, The Journal of Biological Chemistry.

[7]  S. Chen,et al.  CLARP, a death effector domain-containing protein interacts with caspase-8 and regulates apoptosis. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[8]  S. Srinivasula,et al.  FLAME-1, a Novel FADD-like Anti-apoptotic Molecule That Regulates Fas/TNFR1-induced Apoptosis* , 1997, The Journal of Biological Chemistry.

[9]  Y. Goltsev,et al.  CASH, a Novel Caspase Homologue with Death Effector Domains* , 1997, The Journal of Biological Chemistry.

[10]  J. Bertin,et al.  Death-effector Filaments: Novel Cytoplasmic Structures that Recruit Caspases and Trigger Apoptosis , 1998, The Journal of cell biology.

[11]  T. Griffith,et al.  TRAIL: a molecule with multiple receptors and control mechanisms. , 1998, Current opinion in immunology.

[12]  Thomas A. Luger,et al.  Ultraviolet Light Induces Apoptosis via Direct Activation of CD95 (Fas/APO-1) Independently of Its Ligand CD95L , 1998, The Journal of cell biology.

[13]  S. Nagata,et al.  Fas ligand-induced apoptosis. , 1999, Annual review of genetics.

[14]  Ingo Schmitz,et al.  The Role of c-FLIP in Modulation of CD95-induced Apoptosis* , 1999, The Journal of Biological Chemistry.

[15]  E. Solary,et al.  Fas Ligand-independent, FADD-mediated Activation of the Fas Death Pathway by Anticancer Drugs* , 1999, The Journal of Biological Chemistry.

[16]  D. Goeddel,et al.  Requirement for Casper (c-FLIP) in regulation of death receptor-induced apoptosis and embryonic development. , 2000, Immunity.

[17]  R M Siegel,et al.  A domain in TNF receptors that mediates ligand-independent receptor assembly and signaling. , 2000, Science.

[18]  S. Fulda,et al.  Metabolic inhibitors sensitize for CD95 (APO-1/Fas)-induced apoptosis by down-regulating Fas-associated death domain-like interleukin 1-converting enzyme inhibitory protein expression. , 2000, Cancer research.

[19]  P. Krammer,et al.  FLICE-Inhibitory Proteins: Regulators of Death Receptor-Mediated Apoptosis , 2001, Molecular and Cellular Biology.

[20]  S. Chi,et al.  Increased expression of cFLIP(L) in colonic adenocarcinoma. , 2001, The Journal of pathology.

[21]  C. F. Bennett,et al.  A Constitutive Cytoprotective Pathway Protects Endothelial Cells from Lipopolysaccharide-induced Apoptosis* , 2001, The Journal of Biological Chemistry.

[22]  Min Goo Lee,et al.  Increased expression of cFLIPL in colonic adenocarcinoma , 2001 .

[23]  U. McDermott,et al.  The role of thymidylate synthase induction in modulating p53-regulated gene expression in response to 5-fluorouracil and antifolates. , 2002, Cancer research.

[24]  J. Tschopp,et al.  The Long Form of FLIP Is an Activator of Caspase-8 at the Fas Death-inducing Signaling Complex* , 2002, The Journal of Biological Chemistry.

[25]  N. Mitsiades,et al.  Regulation of Apo2L/tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in thyroid carcinoma cells. , 2002, The American journal of pathology.

[26]  G. Gores,et al.  cFLIP-L Inhibits p38 MAPK Activation , 2003, Journal of Biological Chemistry.

[27]  W. Roa,et al.  Calcium/Calmodulin-dependent Protein Kinase II Regulation of c-FLIP Expression and Phosphorylation in Modulation of Fas-mediated Signaling in Malignant Glioma Cells* , 2003, The Journal of Biological Chemistry.

[28]  Sang Gyun Kim,et al.  Transforming Growth Factor-β1 Induces Apoptosis through Fas Ligand-independent Activation of the Fas Death Pathway in Human Gastric SNU-620 Carcinoma Cells , 2003 .

[29]  J. Crocker,et al.  Expression of the cellular FLICE-inhibitory protein (c-FLIP) protects Hodgkin's lymphoma cells from autonomous Fas-mediated death. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[30]  T. Hickish,et al.  Oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment for colon cancer. , 2004, The New England journal of medicine.

[31]  E. Solary,et al.  Redistribution of CD95, DR4 and DR5 in rafts accounts for the synergistic toxicity of resveratrol and death receptor ligands in colon carcinoma cells , 2004, Oncogene.

[32]  R. Gniadecki.  Depletion of membrane cholesterol causes ligand-independent activation of Fas and apoptosis. , 2004, Biochemical and biophysical research communications.

[33]  B. Kay,et al.  Fas-associated Protein with Death Domain (FADD)-independent Recruitment of c-FLIPL to Death Receptor 5* , 2004, Journal of Biological Chemistry.

[34]  W. El-Deiry,et al.  Inducible Silencing of KILLER/DR5 In vivo Promotes Bioluminescent Colon Tumor Xenograft Growth and Confers Resistance to Chemotherapeutic Agent 5-Fluorouracil , 2004, Cancer Research.

[35]  J. Pablos,et al.  Intracellular Regulation of Fas-Induced Apoptosis in Human Fibroblasts by Extracellular Factors and Cycloheximide 1 , 2004, The Journal of Immunology.

[36]  H. Stein,et al.  c-FLIP Mediates Resistance of Hodgkin/Reed-Sternberg Cells to Death Receptor–induced Apoptosis , 2004, The Journal of experimental medicine.

[37]  D. Lawrence,et al.  Selective Knockdown of the Long Variant of Cellular FLICE Inhibitory Protein Augments Death Receptor-mediated Caspase-8 Activation and Apoptosis* , 2005, Journal of Biological Chemistry.

[38]  H. Schulman,et al.  Inhibition of CaMKII-mediated c-FLIP expression sensitizes malignant melanoma cells to TRAIL-induced apoptosis. , 2005, Experimental cell research.

[39]  T. Wilson,et al.  Chemotherapy and TRAIL-mediated colon cancer cell death: the roles of p53, TRAIL receptors, and c-FLIP , 2005, Molecular Cancer Therapeutics.

[40]  H. Hollema,et al.  Expression of tumour necrosis factor-related apoptosis-inducing ligand death receptors in sporadic and hereditary colorectal tumours: potential targets for apoptosis induction. , 2005, European journal of cancer.

[41]  F. Desmots,et al.  Cyclooxygenase-2 inhibition sensitizes human colon carcinoma cells to TRAIL-induced apoptosis through clustering of DR5 and concentrating death-inducing signaling complex components into ceramide-enriched caveolae. , 2005, Cancer research.

[42]  W L Allen,et al.  c-FLIP inhibits chemotherapy-induced colorectal cancer cell death , 2006, Oncogene.

[43]  Wendy L. Allen,et al.  Cellular FLICE-inhibitory protein regulates chemotherapy-induced apoptosis in breast cancer cells , 2007, Molecular Cancer Therapeutics.