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Cholangiocarcinoma is a highly fatal tumor arising from intra or extra-hepatic biliary epithelium. Studies from our group and others have indicated that Fas-mediated apoptosis may provide a promising therapeutic target for this lethal tumor. We have demonstrated that CaM-antagonists induce apoptosis in cholangiocarcinoma cells in vitro through a Fas-related apoptotic mechanism (Am J Pathol. 1999 Jul; 155(1):193-203). Increased expression of c-FLIP, an anti-apoptotic protein and caspase 8 homolgue, is associated with the resistance of cholangiocarcinoma cells to Fas-induced apoptosis (Am J Pathol. 2006 Nov; 169(5):1833-42). Accordingly, we investigated the molecular interaction between CaM and FLIP and its role in regulating Fas-mediated apoptosis.

A direct interaction of CaM and Fas that is regulated during Fas and CaM-antagonist-induced apoptosis has been reported by our group (J Biol Chem. 2004 Feb 13; 279(7):5661-6). Initiation of Fas-mediated apoptosis is marked by the formation Death Inducing Signaling Complex (DISC). Thus, we characterized the interaction of CaM with proteins recruited into the Fas-induced DISC, including FADD, caspase-8 and FLIP, in cholangiocarcinoma cells. A Ca++-dependent interaction between CaM and FLIP, but not FADD or Caspase-8, was demonstrated by co-immunoprecipitation, immunofluorescence and protein pull-down assays. Also over expression of c-FLIP in highly Fas-sensitive cholangiocarcinoma cell line decreased the apoptotic sensitivity of this cell line to Fas-induced apoptosis. Recruitment of FLIP as well as CaM was found to be increased in the Fas-induced DISC over 60 min. Further, a 1.3-fold increase (n=4, p=0.02) in CaM/FLIP binding was observed after Fas stimulation for 30 minutes which returned to baseline after 60 min, and correlated with a Fas-induced increase of intracellular Ca++ that peaked at 30 minutes followed by a gradual decrease over 60 minutes.

The direct binding between CaM and FLIP was confirmed with purified recombinant FLIP that bound to CaM-sepharose beads. Binding studies of CaM with various FLIP mutants revealed that a specific CaM binding motif was present in the FLIP region from amino acids 195-260.

In summary, we have characterized a Ca++-dependent direct binding between CaM and c-FLIP that is regulated in response to Fas stimulation in cholangiocarcinoma cells. Understanding the biology of CaM-FLIP binding in regulating Fas-mediated apoptosis may provide a novel therapeutic target for lethal tumors like cholangiocarcinoma.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA