We evaluated the effects of FRS2 knockdown using shRNA in pancreatic cancer cell lines to determine the dependency of cell viability on FGF signalling. Compared 17-AAG cost with the empty vector counterpart, FRS2�� expression was mostly abrogated by shRNA1 or shRNA2 in L3.6PL, Panc4.30 and AsPC1 cells (Figure 1A). FRS2��-targeting shRNAs induced marked decrease of phosphorylated AKT or ERK, with a decrease in Mcl-1 and cleaved Bid expression. These changes were accompanied by increased cell death compared with empty vector counterparts (Figure 1B; P<0.05), suggesting the dependence of L3.6PL, Panc4.30 and AsPC1 on FGFR signalling, and that the AKT and ERK pathways may have a functional role in FRS2�� shRNA-induced cell death.

Figure 1 Inhibition of FGFR signalling by FRS2�� knockdown exerted pro-apoptotic effects in pancreatic cancer cell lines, and was mediated via Akt/Mcl-1 axis. (A) Effect of FRS2��-targeting shRNAs on FGFR downstream signalling pathways in L3.6PL, … Dovitinib treatment exerted significant pro-apoptotic effect in pancreatic cancer cell lines with heightened FGFR signalling activation We next evaluated the feasibility of targeting FGFR signalling in pancreatic cancer using dovitinib, a potent pan-FGFR small molecule inhibitor. Dovitinib is also a potent inhibitor of PDGFR�� and VEGFR2, though Dey et al (2010) previously demonstrated that the major effects of dovitinib were primarily related to FGFR blockade. The dose�Cresponse effect of dovitinib was evaluated in a panel of six human pancreatic cancer cell lines (L3.6PL, Panc4.30, AsPC1, Panc2.13, SU86.

86 and Panc02.03). In Figure 2A, pancreatic cancer cells were treated with increasing concentrations of dovitinib (0�C10��M) for 3 days. Using 10��M as a cutoff, Panc2.13, SU86.86 and Panc02.03 were considered as resistant (IC50 not identified), and L3.6PL, Panc4.30 and AsPC1 sensitive to dovitinib treatment (IC50<10��M). The expression of FGFR1�C4 was determined in Figure 2B and were not significantly different between dovitinib-sensitive and �Cresistant cell lines (Supplementary Figure S1). We evaluated the status of apoptotic markers in Figure 2C and observed marked mitochondrial-mediated apoptosis with cleavage of caspase 3 and PARP in sensitive cell lines compared with resistant cell lines. Figure 2 Dovitinib's pro-apoptotic effect was related to FGFR signalling inhibition and more pronounced in pancreatic cancers with elevated p-FRS2/FRS2 ratio. (A) Dose�Cresponse of the in vitro anti-cancer effects of dovitinib in six Cilengitide pancreatic cancer cell … The expression of signalling proteins downstream to FGFRs of sensitive cell lines were then compared with resistant cells to elucidate the underlying mechanisms of dovitinib’s pro-apoptotic effect.