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Identification of c-FLIPL and c-FLIPS as critical regulators of death receptor-induced apoptosis in pancreatic cancer cells
  1. Christian Haag1,
  2. Dominic Stadel1,
  3. Shaoxia Zhou2,
  4. Max G Bachem2,
  5. Peter Möller3,
  6. Klaus-Michael Debatin1,
  7. Simone Fulda4
  1. 1University Children's Hospital, Ulm, Germany
  2. 2Department of Clinical Chemistry, Ulm University, Germany
  3. 3Institute of Pathology, Ulm University, Germany
  4. 4Institute for Experimental Cancer Research in Pediatrics, Goethe-University Frankfurt, Germany
  1. Correspondence to Dr Simone Fulda, Institute for Experimental Cancer Research in Pediatrics, Goethe-University Frankfurt, Komturstr. 3a, D-60528 Frankfurt am Main, Germany; simone.fulda{at}kgu.de

Abstract

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-FLIPL and c-FLIPS 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-FLIPL and c-FLIPS 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-FLIPL 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-FLIPL or c-FLIPS 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.

  • Apoptosis
  • TRAIL
  • c-FLIP
  • pancreatic cancer
  • apoptosis
  • pancreatic cancer

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Footnotes

  • Funding This work has been partially supported by grants from the Deutsche Forschungsgemeinschaft, the Deutsche Krebshilfe, the EU (ApopTrain, APO-SYS) and IAP6/18 (to SF).

  • Competing interests None.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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