c-FLIP inhibits chemotherapy-induced colorectal cancer cell death

c-FLIP inhibits caspase 8 activation and apoptosis mediated by death receptors such as Fas and DR5. We studied the effect of c-FLIP on the apoptotic response to chemotherapies used in colorectal cancer (CRC) (5-fluorouracil, oxaliplatin and irinotecan). Simultaneous downregulation of both c-FLIP splice forms c-FLIPL and c-FLIPS with siRNA synergistically enhanced chemotherapy-induced apoptosis in p53 wild-type (HCT116p53+/+, RKO), null (HCT116p53−/−) and mutant (H630) CRC cell lines. Furthermore, overexpression of c-FLIPL, but not c-FLIPS, potently inhibited apoptosis induced by chemotherapy in HCT116p53+/+ cells, suggesting that c-FLIPL was the more important splice form in mediating chemoresistance. In support of this, siRNA specifically targeted against c-FLIPL synergistically enhanced chemotherapy-induced apoptosis in a manner similar to the siRNA targeted against both splice forms. Inhibition of caspase 8 blocked the enhanced apoptosis induced by c-FLIP-targeted (FT) siRNA and chemotherapy. Furthermore, we found that downregulating cell surface DR5, but not Fas, also inhibited apoptosis induced by FT siRNA and chemotherapy. Interestingly, these effects were not dependent on activation of DR5 by its ligand TRAIL. These results indicate that c-FLIP inhibits TRAIL-independent, DR5- and caspase 8-dependent apoptosis in response to chemotherapy in CRC cells. Moreover, targeting c-FLIP in combination with existing chemotherapies may have therapeutic potential for the treatment of CRC.

[1]  F. Lévi,et al.  Phase III multicenter randomized trial of oxaliplatin added to chronomodulated fluorouracil-leucovorin as first-line treatment of metastatic colorectal cancer. , 2000, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

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

[3]  A. Gazdar,et al.  Role of MRIT/cFLIP in Protection Against Chemotherapy-Induced Apoptosis , 2002, Cancer biology & therapy.

[4]  T. Mosmann.  Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. , 1983, Journal of immunological methods.

[5]  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.

[6]  R. James,et al.  Irinotecan combined with fluorouracil compared with fluorouracil alone as first-line treatment for metastatic colorectal cancer: a multicentre randomised trial , 2000, The Lancet.

[7]  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.

[8]  S. Park,et al.  Alternative splicing variants of c-FLIP transduce the differential signal through the Raf or TRAF2 in TNF-induced cell proliferation. , 2001, Biochemical and biophysical research communications.

[9]  A. Thorburn,et al.  Recruitment of TRADD, FADD, and caspase 8 to double-stranded RNA-triggered death inducing signaling complexes (dsRNA-DISCs) , 2005, Apoptosis.

[10]  S. Fulda,et al.  Herpes simplex virus thymidine kinase/ganciclovir-induced apoptosis involves ligand-independent death receptor aggregation and activation of caspases. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

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

[12]  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.

[13]  J. Tschopp,et al.  N-Terminal Fragment of c-FLIP(L) Processed by Caspase 8 Specifically Interacts with TRAF2 and Induces Activation of the NF-κB Signaling Pathway , 2004, Molecular and Cellular Biology.

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

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

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

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

[18]  G. Stassi,et al.  IL-4 Protects Tumor Cells from Anti-CD95 and Chemotherapeutic Agents via Up-Regulation of Antiapoptotic Proteins1 , 2004, The Journal of Immunology.

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

[20]  T. Chou,et al.  Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. , 1984, Advances in enzyme regulation.

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

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

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

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

[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]  Hong-gang Yu,et al.  Overexpression of cellular FLICE-inhibitory protein (FLIP) in gastric adenocarcinoma. , 2004, Clinical science.

[27]  P. Johnston,et al.  5-Fluorouracil: mechanisms of action and clinical strategies , 2003, Nature Reviews Cancer.

[28]  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.

[29]  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.

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

[31]  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 .

[32]  C. Chao,et al.  Up-regulation of FLIP in cisplatin-selected HeLa cells causes cross-resistance to CD95/Fas death signalling. , 2003, The Biochemical journal.

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

[34]  D. Seol,et al.  Upregulation of FLIPS by Akt, a possible inhibition mechanism of TRAIL‐induced apoptosis in human gastric cancers , 2003, Cancer science.

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