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The manuscripts by Park et al.1 and Zhang et al.2 were initially planned as studies to understand the regulation of cell survival in transformed cells treated with sorafenib and vorinostat, and in primary hepatocytes treated with a bile acid+MEK1/2 inhibitor. In both cell systems we discovered that the toxicity of sorafenib and vorinostat or bile acid+MEK1/2 inhibitor exposure depended on the generation of ceramide and the ligand-independent activation of the CD95 death receptor, with subsequent activation of pro-caspase 8. We noted, however, in these systems that, in parallel with death receptor–induced activation of the extrinsic pathway, CD95 signaling also promoted increased phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eIF2α, increased expression of ATG5, and increased processing of LC3 and vesicularization of a GFP-LC3 construct. The knockdown of ATG5 expression blocked GFP-LC3 vesicularization and enhanced cell killing. Thus ceramide-CD95 signaling promoted cell death via activation of pro-caspase 8 and cell survival via autophagy. PERK was shown to signal in a switch-hitting fashion; PERK promoted CD95-DISC formation and an eIF2α-dependent reduction in c-FLIP-s levels that were essential for cell killing to proceed, but in parallel it also promoted autophagy that was protective. The death receptor-induced apoptosis and autophagy occur proximal to the receptor rather than the mitochondrion, and the relative flow of death receptor signaling into either pathway may determine cell fate. Finally, death receptor induced apoptosis and autophagy could be potential targets for therapeutic intervention.
In the United States, hepatoma, renal and pancreatic carcinomas have five-year survival rates of less than 10%, 5–10%, and less than 5%, respectively.3 Melanoma that has spread beyond the radial growth phase (≥ stage III) has a mean five-year survival rate of >20%.4 Non-small cell lung cancer has a statistical survival rate that also approaches that of hepatoma.3 These statistics emphasize the need to develop novel therapies against these lethal malignancies.
The Raf/mitogen-activated protein kinase (MAPK) kinase 1/2 (MEK1/2)/extracellular signal–regulated kinase 1/2 (ERK1/2) pathway is frequently dysregulated in neoplastic transformation.5–13 In view of the importance of the RAF-MEK1/2-ERK1/2 pathway in neoplastic cell survival, inhibitors have been developed that have entered clinical trials, including sorafenib (Bay 43-9006, Nexavar®; a Raf kinase inhibitor) and AZD6244 (a MEK1/2 inhibitor).14,15 Sorafenib is a multi-kinase inhibitor that was originally developed as an inhibitor of Raf-1, but which was subsequently shown to inhibit multiple other kinases, including class III tyrosine kinase receptors such as platelet-derived growth factor, vascular endothelial growth factor receptors 1 and 2, c-Kit and FLT3.16 Anti-tumor effects of sorafenib in renal cell carcinoma and in hepatoma have been ascribed to anti-angiogenic actions of this agent through inhibition of the growth factor receptors.17–19 However, several groups, including ours, have shown in vitro that sorafenib kills human leukemia cells at concentrations below the maximum achievable dose (Cmax) of 15–20 μM, through a mechanism involving downregulation of the anti-apoptotic BCL-2 family member MCL-1.20,21 In these studies sorafenib-mediated MCL-1 downregulation occurs through a translational rather than a transcriptional or post-translational process that is mediated by endoplasmic reticulum (ER) stress signaling.22,23 In a recently published study we also noted that sorafenib lethality as a single agent, and when combined with the histone deacetylase inhibitor (HDACI) vorinostat, is not simply due to loss of ERK1/2 activity.24 This suggests that the previously observed anti-tumor effects of sorafenib are mediated by a combination of inhibition of Raf family kinases and the ERK1/2 pathway, receptor tyrosine kinases that signal angiogenesis, and the induction of ER stress signaling.
HDACIs represent a class of agents that act by blocking histone de-acetylation, thereby modifying chromatin structure and gene transcription. HDACs, along with histone acetyl-transferases, reciprocally regulate the acetylation status of the positively charged NH2-terminal histone tails of nucleosomes, allowing chromatin to assume a more open conformation, which favors transcription.25 However, HDACIs also induce acetylation of other non-histone targets, actions that may have pleiotropic biological consequences, including inhibition of HSP90 function, induction of oxidative injury and upregulation of death receptor expression.26–28 With respect to combinatorial drug studies with a multi-kinase inhibitor such as sorafenib, HDACIs are of interest in that they also down-regulate multiple oncogenic kinases by interfering with HSP90 function, leading to proteasomal degradation of these proteins. Vorinostat (suberoylanilide hydroxamic acid, SAHA, Zolinza™) is an FDA approved hydroxamic acid HDACI that has shown preliminary pre-clinical evidence of activity in hepatoma and other malignancies with a Cmax of ~9 μM.29–31 Sorafenib and vorinostat interact to kill in renal, pancreatic, and hepatocellular carcinoma cells via activation of the CD95 extrinsic apoptotic pathway, and reduced expression of c-FLIP-s.24 The observation that sorafenib and vorinostat exposure causes a ligand-independent activation of CD95 to promote cell death is of particular interest to our laboratories because our prior studies show that bile acids such as deoxycholic acid (DCA) can promote ligand-independent activation of CD95 that played a role in DCA-induced cell killing and DCA-induced regulation of enzymes which control cholesterol 7 α-hydroxylase expression.2,32–34 In one study we discovered that expression of the cyclin-dependent kinase inhibitor (CDKI) p21Cip-1/WAF1/mda6 (p21) in primary hepatocytes enhances bile acid toxicity, and argue that this effect, too, is CD95-dependent.2,34 The studies in Park et al. and Zhang et al. were primarily designed to understand in greater molecular detail how sorafenib and vorinostat, and DCA in the presence of the CDKIs p21 or p27Kip-1, respectively, regulate cell survival through signaling by CD95.1,2
The results of the studies in Park et al. indicate that low concentrations of sorafenib and vorinostat interact in a synergistic manner to kill hepatoma, renal, pancreatic, and non-small cell lung carcinoma cell types, and malignant melanoma cells, in vitro. In this study, the lethality of the drug combination regimen is blocked by inhibition of CD95 function and abolished by overexpression of c-FLIP-s. Of particular note, neither as a single agent nor when combined with vorinostat, do we observe sorafenib lethality correlating with expression of mutated active B-Raf in melanoma cells. In the studies by Zhang et al., we discovered that in addition to p21, the related CDK inhibitor family member p27 also promotes bile acid–induced apoptosis. Increased expression of p21 or p27 enhances p53 levels which correlate with reduced expression of MDM2; reduced ubiquitination of p53; and reduced association of MDM2 with p53. In cells overexpressing p21, p53 is predominantly localized in the nucleus, and treatment of vector control cells with bile acid causes p53 to become localized in the nucleus. The increased expression or nuclear localization of p53 correlates with increased expression of multiple pro-apoptotic gene products including BAX, NOXA, PUMA and CD95. The knockdown of BAX, NOXA and PUMA significantly reduces the enhancement in cell killing caused by p21 and p27 overexpression.
Activation of CD95 promotes the formation of several associated complexes of proteins with the receptor on the intracellular side of the plasma membrane. The most widely recognized of these complexes is the “DISC” in which the FAS-associated death domain (FADD) protein associates with CD95 and with pro-caspase 8 (and pro-caspase 10), leading to the auto-catalytic cleavage of pro-caspases.35 The actions of c-FLIP-s and c-FLIP-l can prevent caspase activation, although c-FLIP-l has in some studies been argued to facilitate caspase 8 activation.35,36 Pyo et al. argue that ATG5 can interact with FADD to modulate survival after interferon γ exposure and it is known that the RNA-activated kinase, PKR, can induce cell killing that is often mediated via a FADD-caspase 8 pathway.37,38 The findings in Park et al., demonstrate that caspase 8, PERK, ATG5 and Grp78/BiP all associate with sorafenib and vorinostat–activated CD95 and that the interaction is dependent on FADD. PERK activation is CD95-dependent. Similarly, the findings in Zhang et al., demonstrate that caspase 8 and ATG5 associates with DCA+MEK1/2 inhibitor–activated CD95. PERK activation is also CD95-dependent. In both cell systems, treatment of cells with stress-inducing drugs promotes GFP-LC3 vesicularization; and the knockdown or knockout of acidic sphingomyelinase, CD95 or ATG5 abolishes the induction of GFP-LC3 vesicle formation.
Thus, using the stress-inducing agents in our studies, we note that activation of CD95 generates both survival and cytotoxic signals, with the relative outcome of signaling being pro-apoptotic provided that no impediment is placed upon the migration of the toxic cell signal away from the death receptor. In the drug/bile acid treatment systems discussed in this manuscript, autophagy represents a survival signal, whereas in other systems recently examined by our laboratory, autophagic signaling was noted to play an active role in promoting cell death.39,40 In these other systems, using OSU-03012 or GST-MDA-7, the induction of autophagy followed by cell killing is independent of CD95, caspase 8 or c-FLIP-s function suggesting that CD95-dependent and independent stimulated forms of autophagy coexist whilst still utilizing proteins such as ATG5 and Beclin 1 to promote vesicle formation.
One obvious difference comparing the induction of autophagy between the drug-induced forms of autophagy discussed in the last paragraph is the apparent involvement of the protein c-FLIP-s; with vorinostat and sorafenib or DCA+MEK1/2 inhibitor-induced killing we note that sustained c-FLIP-s expression results in blockade of BID cleavage, and in the studies using vorinostat and sorafenib we find that sustained c-FLIP-s expression reduces the amount of pro-caspase 8 co-immunoprecipitating with CD95 and causes the levels of autophagy to increase. These findings suggest that in the presence of high levels of c-FLIP-s, activation of a death receptor may not simply be negated by overexpression of c-FLIP-s; activation of a death receptor may be actively subverted by c-FLIP-s towards an autophagic survival signal that could have the potential to improve tumor cell viability in response to other stresses. The “DISC” complex containing caspase 8 has been known for many years; and in protein kinase signal transduction in both yeast and mammalian biology, the concept of a multi-protein “signalosome” containing many protein kinases docked to a transducing protein has also been proposed and established for a decade. The discovery that multiple proteins which regulate endoplasmic reticulum stress signaling and autophagy also form a complex with an activated death receptor and its associated death domain protein expands this concept. Inhibition of autophagy under these circumstances may represent an attractive target for further cancer therapeutic intervention.
This work was funded by the following grants: to P.D. from PHS grants (R01-DK52825, P01-CA104177, R01-CA108520); and to S.G. from PHS grants (R01 CA63753; R01 CA77141) and a Leukemia Society of America grant 6405 97. The measurement of ceramide was performed by the MUSC Lipidomics core funded by grant C06-RR018823. These studies were also funded in part by The Jimmy V Foundation. P.D. is the holder of the Universal Inc. Professorship in Signal Transduction Research.
Addendum to: Zhang G, Park MA, Mitchell C, Walker T, Hamed H, Studer E, Graf M, Rahmani M, Gupta S, Hylemon PB, Fisher PB, Grant S, Dent P. Multiple cyclin kinase inhibitors promote bile acid -induced apoptosis and autophagy in primary hepatocytes via p53-CD95-dependent signaling. J Biol Chem 2008; 283:24343–58.
Park MA, Zhang G, Martin AP, Hamed H, Mitchell C, Hylemon PB, Graf M, Rahmani M, Ryan K, Liu X, Spiegel S, Norris J, Fisher PB, Grant S, Dent P. Vorinostat and sorafenib increase ER stress, autophagy and apoptosis via ceramide-dependent CD95 and PERK activation. Cancer Biol Ther 2008; In press, PMID: pending.