Abstract 4776: Calmodulin modulates Fas-induced survival signals in pancreatic cancer cells

Abstract

Abstract Pancreatic cancer remains a devastating malignancy with poor prognosis, which is largely resistant to current therapies. Death receptor-initiated apoptotic pathways have been implicated both in pancreatic cancer pathogenesis and therapy. To understand the resistance of pancreatic tumor to Fas death receptor-induced apoptosis, we investigated the molecular mechanisms of Fas-activated survival signaling in pancreatic cancer cells. Using lentivirus-mediated RNA interfering strategy, we generated pancreatic cancer cell lines MiaPaCa-2 and BxPc-3 with stable knockdown of the Fas associated protein with death domain (FADD), the adaptor that mediates downstream signaling upon Fas activation. Knockdown of FADD rendered these Fas-sensitive pancreatic cells resistant to apoptosis induced by the Fas agonist antibody, CH-11. Consistently, CH-11 induced recruitment of caspase-8 into the Fas-activated death inducing signaling complex was blocked in FADD knockdown cells, which was associated with no activation of caspase-8 and caspase-3. By contrast, CH-11 promoted the survival of the FADD knockdown MiaPaCa2 and BxPc3 cells in a concentration-dependant manner, as measured by MTS assay. Increased phosphorylation of ERK was demonstrated in the FADD knockdown cells in response to CH-11 treatment. The pharmacological inhibitor of ERK, PD98059, abrogated CH-11-promoted cell survival in FADD knockdown MiaPaCa-2 and BxPC-3 cells. Furthermore, increased phosphorylation of Src was demonstrated in FADD knockdown cells in response to CH-11. The Fas-induced activation of Src was blocked by the specific Src inhibitor, PP2, which further inhibited the Fas-induced activation of ERK and cell survival of the FADD knockdown cells. To elucidate the mechanisms underlying Fas-induced Src activation, we analyzed Fas-associated proteins by immunoprecipitaion of Fas in the FADD knockdown cells. Increased Src and phosphorylated Src was indentified in the Fas-associated protein complex activation by CH-11. Concomitantly, increased recruitment of calmodulin (CaM) into the Fas-associated protein complex was demonstrated as well. Trifluoperazine, a CaM antagonist, inhibited the Fas-induced recruitment of CaM, Src and phosphorylated Src. Consistently, trifluoperazine inhibited Fas-induced activation of Src and blocked Fas-promoted cell survival in the FADD knockdown cells. These results demonstrate that Fas induces FADD-independent survival signaling in pancreatic cancer cells via CaM-mediated activation of Src-ERK signaling pathways. Understanding the molecular mechanisms responsible for the resistance of pancreatic cells to apoptosis induced by Fas-death receptor signaling may provide molecular insights into designing novel therapies to treat pancreatic tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4776. doi:10.1158/1538-7445.AM2011-4776