MEK1/2 inhibitors and 17AAG synergize to kill human hepatoma cells in vitro via suppression of c-FLIP-s levels and activation of the extrinsic apoptosis pathway

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Prior studies have noted that inhibitors of MEK1/2 enhanced geldanamycin lethality in malignant hematopoietic cells by promoting mitochondrial dysfunction. The present studies focused on defining the mechanism(s) by which these agents altered cell survival in hepatoma cells in vitro. MEK1/2 inhibitors (PD184352; AZD6244 (ARRY-142886)) interacted in a synergistic manner with geldanamycins (17AAG, 17DMAG) to kill hepatoma cells that correlated with inactivation of ERK1/2 and AKT and activation of p38 MAPK. Treatment of hepatoma cells with MEK1/2 inhibitors and 17AAG correlated with reduced expression of BCL-XL and c-FLIP-s and caused a p38 MAPK-dependent activation of BAX and BAK. Inhibition of caspase 8 or over-expression of c-FLIP-s or BCL-XL abolished cell killing by MEK1/2 inhibitors and 17AAG exposure. Expression of activated MEK1 and activated AKT prevented MEK1/2 inhibitor and 17AAG -induced suppression of c-FLIP-s and BCL-XL levels and blocked cell killing. Treatment of hepatoma cells with MEK1/2 inhibitors and 17AAG also caused a p38 MAPK-dependent association of caspase 8 with CD95 (FAS receptor), and inhibition of p38 MAPK or knock down of BID, FADD or CD95 expression suppressed MEK1/2 inhibitor and 17AAG lethality. Treatment of hepatoma cells with MEK1/2 inhibitors and 17AAG caused a p38 MAPK-dependent plasma membrane clustering of CD95 without altering the levels or cleavage of FAS ligand. Collectively, our data demonstrate that treatment of hepatoma cells with MEK1/2 inhibitors and 17AAG induces activation of the extrinsic pathway and that suppression of c-FLIP-s expression is crucial in transduction of the pro-apoptotic signal from CD95 to promote mitochondrial dysfunction and hepatoma cell death.