Identification of c-FLIP(L) and c-FLIP(S) as critical regulators of death receptor-induced apoptosis in pancreatic cancer cells.
BACKGROUND
Evasion of apoptosis is a hallmark of pancreatic cancer. However, the underlying mechanisms are still only partly understood and may involve antiapoptotic proteins such as c-FLIP. Here, the role of c-FLIP in the regulation of death receptor-mediated apoptosis in pancreatic cancer was investigated.


METHODS
Expression of c-FLIP(L) and c-FLIP(S) was analysed in primary pancreatic carcinoma samples, pancreatic carcinoma cell lines and primary tumour cells together with its function as a regulator of death receptor-induced apoptosis by knockdown and overexpression studies and through modulation by chemotherapeutics.


RESULTS
c-FLIP is expressed in pancreatic intraepithelial neoplasm (PanIN) lesions and in pancreatic ductal adenocarcinomas, whereas normal pancreatic ducts were consistently negative for c-FLIP. Simultaneous downregulation of c-FLIP(L) and c-FLIP(S) as well as individual knockdown of either isoform by RNA interference significantly enhances TRAIL (tumour necrosis factor-related apoptosis-inducing ligand)- and CD95-induced caspase activation and caspase-dependent apoptosis. Also, pretreatment with chemotherapeutic drugs--that is, 5-fluorouracil (5-FU), cisplatin or gemcitabine--downregulates c-FLIP and renders cells sensitive to death receptor-triggered apoptosis. Similarly, primary cultured pancreatic cancer cells are primed for TRAIL-induced apoptosis by pre-exposure to 5-FU or cisplatin. Mechanistic studies revealed that 5-FU-mediated suppression of c-FLIP results in increased TRAIL-induced recruitment and activation of caspase-8 at the death-inducing signalling complex (DISC), leading to caspase-3 activation and caspase-dependent cell death. Overexpression of c-FLIP(L) rescues cells from 5-FU- or cisplatin-mediated sensitisation for TRAIL-induced apoptosis, indicating that c-FLIP suppression is a key event in this chemotherapy-mediated sensitisation to TRAIL. Further, concomitant neutralisation of c-FLIP and XIAP acts in concert to potentiate TRAIL-induced apoptosis.


CONCLUSIONS
Both the long and the short isoform of the antiapoptotic protein c-FLIP are critical regulators of death receptor-induced apoptosis in pancreatic carcinoma cells and are suppressed by chemotherapeutics. Targeting either c-FLIP(L) or c-FLIP(S) is sufficient to promote death receptor-induced apoptosis in pancreatic carcinoma cells. These findings have important implications for the design of TRAIL-based combination protocols in pancreatic cancer.
Haag
Christian
C
University Children's Hospital, Ulm, Germany.
Stadel
Dominic
D
Zhou
Shaoxia
S
Bachem
Max G
MG
Möller
Peter
P
Debatin
Klaus-Michael
KM
Fulda
Simone
S
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002945:Cisplatin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D005472:Fluorouracil / Q000494:pharmacology; D055785:Gene Knockdown Techniques; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010179:Pancreas / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000378:metabolism / Q000473:pathology*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D053218:Receptors, Death Domain / Q000502:physiology*; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000494:pharmacology / Q000502:physiology; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000502:physiology
D000964:Antimetabolites, Antineoplastic; D000970:Antineoplastic Agents; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; C491901:FAS protein, human; D009363:Neoplasm Proteins; D020033:Protein Isoforms; D053218:Receptors, Death Domain; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D019014:fas Receptor; D002945:Cisplatin; D005472:Fluorouracil