The targeted therapeutics sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I evaluation. In this study we determined how CD95 is activated by treatment with this drug combination. Low doses of sorafenib and vorinostat but not the individual drugs rapidly increased ROS, Ca2+ and ceramide levels in GI tumor cells. The production of ROS was reduced in Rho zero cells. Quenching ROS blocked drug-induced CD95 surface localization and apoptosis. ROS generation, CD95 activation and cell killing was also blocked by quenching of induced Ca2+ levels or by inhibition of PP2A. Inhibition of acidic sphingomyelinase or de novo ceramide generation blocked the induction of ROS however combined inhibition of both acidic sphingomyelinase and de novo ceramide generation was required to block the induction of Ca2+. Quenching of ROS did not impact on drug-induced ceramide/dihydro-ceramide levels whereas quenching of Ca2+ reduced the ceramide increase. Sorafenib and vorinostat treatment radiosensitized liver and pancreatic cancer cells, an effect that was suppressed by quenching ROS or knock down of LASS6. Further, sorafenib and vorinostat treatment suppressed the growth of pancreatic tumors in vivo. Our findings demonstrate that induction of cytosolic Ca2+ by sorafenib and vorinostat is a primary event that elevates dihydroceramide levels, each essential steps in ROS generation that promotes CD95 activation.

Sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and the present studies have determined individually how sorafenib and vorinostat contribute to CD95 activation. Sorafenib (3-6 μM) promoted a dose-dependent increase in Src Y416, ERBB1 Y845 and CD95 Y232/Y291 phosphorylation, and Src Y527 dephosphorylation. Low levels of sorafenib (3 μM) –induced CD95 tyrosine phosphorylation did not promote surface localization whereas sorafenib (6 μM), or sorafenib (3 μM) and vorinostat (500 nM) treatment promoted higher levels of CD95 phosphorylation that correlated with DISC formation, receptor surface localization and autophagy. CD95 (Y232F, Y291F) was not tyrosine phosphorylated and was unable to plasma membrane localize or induce autophagy. Knock down / knock out of Src family kinases abolished sorafenib –induced: CD95 tyrosine phosphorylation; DISC formation; and the induction of cell death and autophagy. Knock down of PDGFRβ enhanced Src Y416 and CD95 tyrosine phosphorylation that correlated with elevated CD95 plasma membrane levels and autophagy, and with a reduced ability of sorafenib to promote CD95 membrane localization. Vorinostat increased ROS levels; and in a delayed NFκB-dependent fashion, those of FAS ligand and CD95. Neutralization of FAS-L did not alter the initial rapid drug-induced activation of CD95 however, neutralization of FAS-L reduced sorafenib + vorinostat toxicity by ~50%. Thus sorafenib contributes to CD95 activation by promoting receptor tyrosine phosphorylation whereas vorinostat contributes to CD95 activation via initial facilitation of ROS generation and subsequently of FAS-L expression.

The present studies determined in greater detail the molecular mechanisms upstream of the CD95 death receptor by which geldanamycin HSP90 inhibitors and MEK1/2 inhibitors interact to kill carcinoma cells. MEK1/2 inhibition enhanced 17AAG toxicity that was suppressed in cells deleted for mutant active RAS which were non-tumorigenic but was magnified in isogenic tumorigenic cells expressing H-RAS V12 or K-RAS D13. MEK1/2 inhibitor and 17AAG treatment increased intracellular Ca2+ levels and reduced GRP78/BiP expression in a Ca2+ -dependent manner. GRP78/BiP over-expression, however, also suppressed drug-induced intracellular Ca2+ levels. MEK1/2 inhibitor and 17AAG treatment increased ROS levels that were blocked by quenching Ca2+ or over-expression of GRP78/BiP. MEK1/2 inhibitor and 17AAG treatment activated CD95 and inhibition of ceramide synthesis; ROS or Ca2+ quenching blocked CD95 activation. In SW620 cells that are patient matched to SW480 cells, MEK1/2 inhibitor and 17AAG toxicity was significantly reduced that correlated with a lack of CD95 activation and lower expression of ceramide synthase 6 (LASS6). Over-expression of LASS6 in SW620 cells enhanced drug-induced CD95 activation and enhanced tumor cell killing. Inhibition of ceramide signaling abolished drug-induced ROS generation but not drug-induced cytosolic Ca2+ levels. Thus treatment of tumor cells with MEK1/2 inhibitor and 17AAG induces cytosolic Ca2+ and loss of GRP78/BiP function, leading to de novo ceramide synthesis pathway activation that plays a key role in ROS generation and CD95 activation.